Polysaccharide-protein conjugate polysaccharide-protein for inducing immune response and protection against staphylococcus aureus infection, methods of conjugate obtaining (versions), conjugate-containing composition and methods of inducing immune response and prevention of staphylococcus aureus infection

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed group of inventions relates to field of immunology and deals with polysaccharide-protein conjugate, composition, containing such conjugate, method of inducing immune response against Staphylococcus aureus in subject, methods of obtaining such conjugate and method of reducing or prevention of Staphylococcus aureus infection. Characterised conjugate contains capsular Staphylococcus aureus. Said conjugate contains capsular polysaccharide, conjugated with protein carrier Said polysaccharide represents polysaccharide of serotype 5 or serotype 8, has molecular weight between 70 kDa and 800 kDa and degree of O-acetylation between 75 and 100%. Methods of obtaining charactersed comjugate include covalent conjugation of capsular polysaccharides with protein carrier by chemical method with application of either 1,1-carbonyl-di-1,2,4-triazole (CDT) or 3-(2-pyridylthio)propionylhydraside (PDPH).

EFFECT: claimed solutions make it possible to obtain vaccine preparations against infections caused by Staphylococcus aureus with higher immunogenicity.

29 cl, 11 dwg, 25 tbl, 20 ex

 

Cross references to related applications

This application claims priority to provisional applications U.S. patent No. 61/219143 and 61/219151 with a filing date of June 22, 2009, a full description of which is incorporated by reference in their entirety.

The scope of the invention

This invention in General relates to immunogenic compositions of conjugates of capsular polysaccharides of Staphylococcus aureus serotypes 5 and 8, and methods for their preparation and use.

Prior art

Humans are the natural reservoir of gram-positive Staphylococcus aureus. For example, S.aureus can colonize the skin, nasal cavity and throat either permanently or temporarily, without causing disease. The S.aureus infection can range from mild skin infections to endocarditis, osteomyelitis, bacteremia and sepsis. S.aureus also causes the majority of nosocomial infections, and its prevalence in local outbreaks of infections is increasing. Moreover, in 2005, methicillin-resistant S.aureus infection (MRSA) in the United States were established in the 31.8 per 100,000 individuals, including 16650 deaths in 2005 (Klevens et al. (2007) J.Am.Med. Assoc. 298:1763-1771). Subsequently, the disease may occur in individuals with a weakened immune system due to gaps in immune barriers, such as occur during surgery, the installation of permanent catheters or other devices, if the injury or wound is the second.

S.aureus produces a large number of intra - and intercellular antigens, including numerous toxins and enzymes. Of particular interest are the capsular polysaccharides of certain serotypes S.aureus (see Karakawa & Vann, "Capsular polysaccharides of Staphylococcus aureus" in Weinstein & Fields, eds. Seminars in Infectious Diseases IV, Bacterial Vaccines. (New York, NY, Thieme Stratton; 1982. pp.285-293), especially capsular polysaccharides of serotypes 5 and 8. Epidemiological studies on a large number of strains of S.aureus isolated from individuals, showed that 70% to 80% belong to the capsular polysaccharide serotype 5 or 8 (Arbeit et al. (1984) Diagn. Environ. Infect. Dis. 2:85-91). Unfortunately, these capsular polysaccharides are poor immunogenum.

Staphylococcal infections and diseases has increased dramatically in the last twenty years, as the use of intravascular devices and invasive procedures. This increase morbidity causes even more anxiety due to the parallel growth of antibiotic resistance; therefore, there is an urgent need for immunogenic compositions for the prevention of staphylococcal infections and diseases.

A BRIEF DESCRIPTION of the INVENTION

The present invention is directed to immunogenic conjugates containing capsular polysaccharide S.aureus serotype 5 or 8, conjugated to a protein carrier, and to methods of obtaining such conjugate is. Capsular polysaccharides S.aureus serotype 5 or 8 can be obtained directly from the bacteria using the methods of selection, well-known experts, they can be obtained by using synthetic methods, or they can be obtained by recombinant image using genetic engineering methods known in the art. In addition, in the present invention, methods of inducing an immune response against the bacteria Staphylococcus, ways of prevention of the disease caused by the bacterium Staphylococcus, and methods of reducing the severity of at least one symptom of the disease caused by infection by the bacterium Staphylococcus.

In one of the embodiments of this invention includes immunogenic conjugate polysaccharide-protein containing the selected capsular polysaccharide of Staphylococcus aureus serotype 5 or 8, conjugated to protein carrier, where the polysaccharide has a molecular weight between 20 kDa and 1000 kDa. In some embodiments of the immunogenic conjugate has a molecular weight between 200 kDa and 5000 kDa. In one of the embodiments of the polysaccharide part of the immunogenic conjugate has a range of molecular masses between 70 kDa and 300 kDa. In one of the embodiments of the immunogenic conjugate has a range of molecular weights between 500 and 2500 kDa kDa.

In one of the embodiments of the capsular polysaccharide serotype 5 or 8 has a degree of O-acetylation equal to 10-100%. In one of the in the of leweni the degree of O-acetylation is 50-100%. In one of the embodiments of the degree of O-acetylation is 75-100%. In one of the embodiments of the immunogenic conjugate generates an antibody that is functional when measuring for killing bacteria or model of efficiency on the animal or in the analysis opsonophagocytosis killing.

In one of the embodiments of the protein carrier immunogenic conjugate contains CRM197. In one of the embodiments of the CRM197covalently linked to the polysaccharide by urethane links, amide linkages, or both of them. In one embodiments the molar ratio of lysine, conjugated to CRM197may be from about 10:1 to about 25:1. In one embodiments, the conjugate contains a single covalent bond between CRM197and the polysaccharide at least every 5-10 duplicate sharidny units of the polysaccharide. In one of the embodiments of the relationship between protein carrier and polysaccharide takes place once every 5 repeating units of the polysaccharide.

In one of the embodiments of the immunogenic conjugate containing CRM197contains from 5 to 22 lysine or from 8 to 15 lysine covalently associated with the polysaccharide. In one of the embodiments of the immunogenic conjugate containing CRM197contains from 5 to 23 lysine or from 8 to 12 lysine covalently associated with the polysaccharide.

In one of the embodiments of the immunogenic conjugate contain what it polysaccharide type 5 or 8, which O-acetiminophen for 10-100%. In one of the embodiments of the immunogenic conjugate contains polysaccharide type 5 or 8, O-acetiminophen by 50-100%. In one of the embodiments of the immunogenic conjugate contains polysaccharide type 5 or 8, O-acetiminophen at 75-100%. In some embodiments of the immunogenic composition can be used to generate antibodies that are functional in the model of efficiency on the animal or in the analysis opsonophagocytosis killing.

In one of the embodiments of the immunogenic conjugate contains less than about 30% of free polysaccharide type 5 or 8 in comparison with the total amount of the polysaccharide type 5 or 8.

In one of the embodiments of the immunogenic conjugate contains less than about 20% of free polysaccharide type 5 or 8 in comparison with the total amount of the polysaccharide type 5 or 8.

In one of the embodiments of this invention includes an immunogenic composition comprising the immunogenic conjugate, as described here, and at least one adjuvant, diluent or carrier.

The adjuvant may be an adjuvant based on aluminum, such as one or more than one of aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one of the embodiments of the adjuvant contains aluminum phosphate.

In one of the embodiments of the immunogenic composition contains less than about 30% of free polysaccharide is IPA 5 or 8 in comparison with the total amount of the polysaccharide type 5 or 8.

In one of the embodiments of the immunogenic composition contains less than about 20% of free polysaccharide type 5 or 8 in comparison with the total amount of the polysaccharide type 5 or 8.

In one of the embodiments of this invention includes a method of inducing immune response against the conjugate of the capsular polysaccharide of Staphylococcus aureus serotype 5 or 8 for a subject, comprising the introduction of this subject immunologically effective amount of the immunogenic composition, as described here.

In one of the embodiments of this invention includes a method of obtaining immunogenic conjugate polysaccharide-protein containing the selected capsular polysaccharide of Staphylococcus aureus serotype 5 or 8, conjugated with protein a media, comprising the stage of: communicating the selected capsular polysaccharide S.aureus serotype 5 or 8 reagent 1,1-carbonyl-di-(1,2,4-triazole) (CDT) in an organic solvent to obtain an activated polysaccharide serotype 5 or 8; and the interaction of the activated polysaccharide serotype 5 or 8 with protein carrier in an organic solvent to obtain conjugate polysaccharide serotype 5 or 8 with protein carrier.

In one of the embodiments of the method of activation of the capsular polysaccharide of Staphylococcus aureus serotype 5 or 8 further includes a lyophilization selected polysaccharide serotype 5 or 8 and re-suspension of life yserowanego polysaccharide in an organic solvent. In one of the embodiments resuspending polysaccharide activate and then directly subjected to interaction with protein carrier. In one of the embodiments of the activated selected polysaccharide serotype 5 or 8 emit before interaction with protein carrier. In one of the embodiments selected activated polysaccharide serotype 5 or 8 lyophilizer obtaining dried activated the selected polysaccharide serotype 5 or 8 before the interaction of the polysaccharide with protein carrier. In one of the embodiments of the method of obtaining the selected conjugate polysaccharide with protein carrier includes a step of freeze-drying a protein carrier to obtain lyophilized protein carrier before interacting protein carrier with a polysaccharide. In one of the embodiments of the method of obtaining the selected conjugate polysaccharide with protein carrier includes a step resuspendable dried activated the selected polysaccharide serotype 5 or 8 and lyophilized protein carrier in an organic solvent as part of the interaction of the activated selected polysaccharide serotype 5 or 8 with protein carrier.

In one of the embodiments of the method of obtaining a conjugate selected capsular polysaccharide S.aureus type 5 or 8 with protein carrier includes a step of dilution of the reaction mixtures and activated polysaccharide and protein carrier and maintain a pH of from about 8.8 to about 9.2 deaths for at least 4 hours at a temperature of from about 20°to about 26°C.

In one embodiments, the reaction mixture is activated capsular polysaccharide S.aureus type 5 or 8 and the protein carrier support at pH about 9.0 in for at least 4 hours at a temperature of about 23°C.

In one of the embodiments of the method of obtaining the selected capsular polysaccharide S.aureus type 5 or 8 with protein carrier includes a step of allocating conjugate selected polysaccharide serotype 5 or 8 with the protein after it is received. In one of the embodiments of the organic solvent used in the method of obtaining a conjugate selected capsular polysaccharide S.aureus type 5 or 8 with protein carrier is a polar aprotic solvent. In one embodiments, the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO). In one of the embodiments of the method of obtaining a conjugate selected polysaccharide with protein carrier organic solvent represents DMSO.

In one of the embodiments of the method of obtaining a conjugate selected capsular polysaccharide S.aureus type 5 with protein carrier includes a step of bringing the water concentration in the reaction mixture containing capsular polysaccharide of type 5 and CDT in an organic solvent, to about 0.1 to 0.3%. In one embodiments the concentration of water in the reaction mixture containing capsular polysaccharide of type 5 of the CDT in an organic solvent, brought to approximately 0.2%.

In one of the embodiments of the phase of activation of the selected capsular polysaccharide S.aureus type 5 includes the interaction of the polysaccharide with the number of CDT, which is approximately 20-fold molar excess relative to the amount of polysaccharide present in the reaction mixture containing capsular polysaccharide of type 5 and CDT in an organic solvent.

In one of the embodiments of the method of obtaining a conjugate selected capsular polysaccharide S.aureus type 8 with protein carrier includes a step of determining water content in the reaction mixture containing capsular polysaccharide type 8. In one of the embodiments CDT add to the reaction mixture to activate the polysaccharide in an amount approximately equal to the number of CDT, which is equimolar to the amount of water present in the reaction mixture containing capsular polysaccharide type 8 and CDT in an organic solvent.

In one of the embodiments CDT add to the reaction mixture to activate the polysaccharide in such quantity that it is in a molar ratio of about 0.5:1, compared with the amount of water present in the reaction mixture containing capsular polysaccharide type 8 and CDT in an organic solvent. In one of the embodiments CDT add to the reaction mixture to activate the polysaccharide in such quantity that it is in a molar ratio of about 0.75:1 compared to the amount of water present in the reaction mixture containing capsular polysaccharide type 8 and CDT in an organic solvent.

In one of the embodiments of the method, which includes the extraction of the activated polysaccharide includes a step of diafiltration.

In one of the embodiments of the method, which includes the lyophilization of the protein carrier, the carrier protein before lyophilization is subjected to diafiltration against NaCl, and the mass ratio of NaCl/protein carrier was adjusted to about 0.5 to 1.5. In one embodiments the ratio of NaCl and the protein carrier is about 1.

In one of the embodiments of the carrier protein used in the method of obtaining a conjugate selected capsular polysaccharide S.aureus type 5 or 8 and protein media, CRM contains197.

In one of the embodiments of the CRM197used in the production method of conjugate selected capsular polysaccharide S.aureus type 5 or 8 with protein carrier, is subjected to the interaction with the activated polysaccharide serotype 5 or 8 in a mass ratio of about 1:1.

In one of the embodiments of the method of obtaining a conjugate selected capsular polysaccharide S.aureus type 5 or 8 with protein carrier includes a step of mixing the capsular polysaccharide of type 5 or 8 with imidazole or triazole before mixing with CDT in an organic solvent.

In one of the embodiments of the method of obtaining conjugate selected capsular polysaccharide S.aureus type 5 or 8 with protein carrier includes a step of hydrolysis of conjugate polysaccharide serotype 5 or 8 with protein carrier to remove unreacted activation groups.

In one of the embodiments of this invention, a method for obtaining immunogenic conjugate containing the selected capsular polysaccharide of Staphylococcus aureus serotype 5 or 8, conjugated with protein a media, comprising the stage of: communicating capsular polysaccharide S.aureus serotype 5 or 8 with 3-(2-pyridyldithio)-propionitrile (PDPH) and a carbodiimide in an organic solvent to obtain a polysaccharide associated with PDPH; interaction of polysaccharide associated with PDPH, with a reducing agent to obtain an activated polysaccharide; selection of the activated polysaccharide serotype 5 or 8 to obtain the selected activated polysaccharide serotype 5 or 8; providing activated protein media; interaction of selected activated polysaccharide serotype 5 or 8 with activated protein carrier with obtaining conjugate polysaccharide serotype 5 or 8 with protein carrier; whereby get immunogenic conjugate containing the selected capsular polysaccharide S.aureus serotype 5 or 8, conjugated to a protein carrier. In one of the embodiments of the activated protein carrier emit before the interaction of activated protein carrier with the activated polysaccharide.

In one of the embodiments of the phase selection activated protein carrier further includes libfile is the situation highlighted activated polysaccharide serotype 5 or 8 with getting dried activated polysaccharide serotype 5 or 8.

In one of the embodiments brooksyne acid comprises N-hydroxysuccinimidyl ether bromoxynil acid (BAANS).

In one of the embodiments of the method of obtaining a conjugate of the capsular polysaccharide serotype 8 with protein carrier, which use PDPH, involves the use of an organic solvent, which is a polar aprotic solvent. In one embodiments, the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone and hexamethylphosphoramide (NMRA). In one of the embodiments of the organic solvent is a sulfoxide (DMSO).

In one of the embodiments of carbodiimide used in the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH is a 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC).

In one of the embodiments of the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH and EDAC, includes a step of interaction capsular polysaccharide serotype 5 or 8 with PDPH and EDAC organics mass ratio of polysaccharide:PDPH:EDAC about 1:5:3.

In one of the embodiments of the reducing agent used in the method of producing capsular conjugate polisher is Yes serotype 5 or 8 with protein carrier, which uses PDPH and EDAC, is a dithiothreitol (DTT).

In one of the embodiments of the activation of the protein carrier in the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH and EDAC, includes the interaction of the protein carrier with bromoxynil acid.

In one of the embodiments of the stage of selection of the activated polysaccharide serotype 5 or 8 in the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH and EDAC includes diafiltration.

In one of the embodiments of the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH and EDAC, includes a step of hydrolysis of conjugate polysaccharide serotype 5 and 8 with protein carrier to remove unreacted activation groups. In one of the embodiments of the phase hydrolysis of conjugate polysaccharide serotype 5 and 8 with protein carrier includes adding hydrochloride group probably facilitates.

In one of the embodiments of the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH and EDAC additionally includes a selection of immunogenic conjugate containing the selected capsular polysaccharide S.aureus serotype 5 or 8, conjugated to protein carrier.

In one of the embodiments of separation is their conjugate polysaccharide serotype 5 and 8 with protein carrier includes diafiltration.

In one of the embodiments of the carrier protein used in the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH and EDAC, CRM contains197.

In one of the embodiments of the CRM197in the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with CRM197to use PDPH and EDAC add mass ratio of CRM197molecule capsular polysaccharide of about 1:1.

In one of the embodiments of the activated capsular polysaccharide of type 5 or 8 used in the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH and EDAC, has a size of from about 50 kDa to about 500 kDa.

In one of the embodiments of the immunogenic conjugate obtained by the method of obtaining a conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, which use PDPH and EDAC, has a size between about between 400 kDa and about 5000 kDa.

In one of the embodiments of this invention proposed immunogenic composition comprising a conjugate of the capsular polysaccharide of type 5 or 8 with protein carrier, obtained by any method described here.

In one of the embodiments of this invention proposed immunogenic composition comprising a conjugate of the capsular polysaccharide of type 5 or 8 with protein carrier is, get any by the way described here, and at least one adjuvant, diluent or carrier. In one of the embodiments of the immunogenic compositions contain adjuvant based on aluminum, which can be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one of the embodiments described herein immunogenic compositions contain adjuvant, which represents a phosphate of aluminum.

Described here immunogenic composition can contain less than 30% and less than 20% of free polysaccharide type 5 or 8 in comparison with the total amount of the polysaccharide type 5 or 8. Described here immunogenic compositions can be stored in water or in a buffer with low ionic strength with a neutral pH.

In one of the embodiments of this invention, a method for reducing or preventing staphylococcal infections, diseases or conditions associated with bacteria Staphylococcus, subject, comprising the stage of introduction of therapeutic or prophylactic amount of the immunogenic composition, as described here, to this subject. In one of the embodiments of the infection, disease or condition selected from the group consisting of invasion of Staphylococcus aureus sepsis and carriage.

In one of the embodiments of this invention, a method for reducing or preventing staphylococcal detail the functions of the subject, passing a surgical procedure, comprising the stage of introduction of a prophylactically effective amount of the immunogenic composition, as described here, this subject before the surgical procedure.

In one of the embodiments of the method according to this invention includes replacement CDT on CDI.

In one of the embodiments of this invention proposed a capsular polysaccharide of Staphylococcus aureus type 5 or 8, having a molecular weight of between 50 kDa and 800 kDa, covalently linked to a protein carrier, where the combined molecular weight of the polysaccharide covalently linked to a protein carrier is between about 400 kDa and 5000 kDa.

In one embodiments the polysaccharide covalently linked to a protein carrier, contains polysaccharide part, which has a range of molecular masses between 70 kDa and 300 kDa. In one embodiments the polysaccharide covalently linked to a protein carrier, has a range of molecular weights between 500 and 2500 kDa kDa.

In one of the embodiments of the part, which is a protein carrier, polysaccharide covalently linked to a protein carrier, CRM contains197. In one of the embodiments of the CRM197covalently linked to the polysaccharide by urethane links, amide linkages, or both of them. In some embodiments the molar ratio of conjugated lysine to CRM197is from about 10:1 to the example is about 25:1. In some embodiments, the polysaccharide covalently linked to a protein carrier comprises at least one covalent bond with CRM197at least every 5-10 duplicate sharidny units of the polysaccharide. In some embodiments, the polysaccharide covalently linked to a protein carrier comprises at least one connection between CRM197and a polysaccharide, which accounts for repeated every 5 sharidny units of the polysaccharide. In some embodiments CRM197part of the polysaccharide covalently linked to CRM197contains from 5 to 22 lysine covalently associated with the polysaccharide. In some embodiments CRM197part of the polysaccharide covalently linked to CRM197contains from 5 to 23 lysine covalently associated with the polysaccharide. In some embodiments CRM197part of the polysaccharide covalently linked to a protein carrier contains from 8 to 15 lysine covalently associated with the polysaccharide. In some embodiments CRM197part of the polysaccharide covalently linked to a protein carrier contains from 8 to 12 lysine covalently associated with the polysaccharide.

In one of the embodiments of this invention proposed immunogenic composition comprising the polysaccharide S.aureus type 5 or 8, covalently linked to a protein carrier, as it is described here, and at least one adjuvant, will dilute the ü, or the media.

In one of the embodiments of this invention, a method for introduction of immunogenic compositions containing polysaccharide S.aureus type 5 or 8, covalently linked to a protein carrier, as he described herein, subject to receipt of the immune response, as described here.

In one of the embodiments of this invention, a method for selection of a polysaccharide with a molecular mass between 20 kDa and 1000 kDa.

In one of the embodiments of this invention proposed an antibody generated capsular polysaccharide, immunogenic conjugate or immunogenic composition of the present invention.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

The present invention will be better understood, and other features, aspects and advantages than those mentioned above will become apparent when reviewing the following detailed description. This detailed description refer to the following graphic materials.

Figure 1 shows a repeating polysaccharide structure of the capsular polysaccharide S.aureus serotype 8 (N-acetylaminofluorene acid designated as ManNAca, N-acetyl-L-fucosamine designated as L-FucNAc, and N-acetyl-D-fucosamine designated as D-FucNAc).

Figa shows analysis of the fractions for ion-exchange chromatography (Q-Sepharose) capsular polysaccharide S.aureus serotype 8 (analysis of O-acetyl) and tahaawi acid analysis phosphate); Figb shows analysis of the fractions for ion-exchange chromatography (Q-Sepharose) capsular polysaccharide S.aureus serotype 8 way double immunodiffusion.

Figa shows the effect of pH (3, 5, 4, or 5) at 95°C to reduce the molecular weight capsular polysaccharide S.aureus serotype 8 during the heat treatment; Figb shows the effect of temperature (55°C, 75°C or 95°C) at pH 3.5 to reduce the molecular weight capsular polysaccharide S.aureus serotype 8 during the heat treatment.

Figure 4 shows the molecular weight of purified capsular polysaccharide S.aureus serotype 8 in comparison with the capsular polysaccharide serotype 5 with time during heating at pH 3.5 and 4.5, respectively, and 95°C.

Figure 5 shows the increased survival of mice that received the conjugate of the capsular polysaccharide serotype 8 with CRM197(diamonds) when compared with controls who received AlPO4(circles).

6 shows a repeating polysaccharide structure of the polysaccharide S.aureus serotype 5 (N-acetylaminofluorene acid designated as ManNAcA, N-acetyl-L-fucosamine designated as L-FucNAc, and N-acetyl-D-fucosamine designated as D-FucNAcA).

Figa shows analysis of the fractions for ion-exchange chromatography (Q-Sepharose) polysaccharide S.aureus serotype 5 (analysis of O-acetyl) and tahaawi acid (analysis phosphate); Figb shows analysis of the fractions is ri ion-exchange chromatography (Q-Sepharose) polysaccharide S.aureus serotype 5 way double immunodiffusion.

Figa shows the effect of pH (3.5-inch, 4 or 5) at 95°C to reduce the molecular weight capsular polysaccharide S.aureus serotype 5 by heat treatment; Figb shows the effect of temperature (55°C, 75°C or 95°C) at pH 3.5 to reduce the molecular weight capsular polysaccharide S.aureus serotype 5 by heat treatment.

Fig.9 shows the weakening of pyelonephritis in mice that received the conjugate polysaccharide serotype 5 with CRM197compared with controls who received phosphate buffered saline (PBS) (shaded area represents the treated mice).

Figure 10 shows the colony forming units (CFU)isolated from the kidney after infection with S. aureus PFESA0266 mice vaccinated with high molecular weight (HMW) CP5-CRM, low molecular weight (LMW) CP5-CRM or control PP5-CRM.

11 shows a comparison of the titles of PR (geometric average) of the sera obtained from mice vaccinated with different compositions polysaccharide conjugate (high molecular weight (HMW) CP5-CRM, low molecular weight (LMW) CP5-CRM). The group consisted of 5-9 mice.

DETAILED description of the INVENTION

Overview

The present invention relates to the immunogenic conjugates containing capsular polysaccharides of S. aureus serotype 5 or 8 conjugated to protein carriers, and to methods for their preparation and use. New signs immuno the n conjugates of this invention include profiles of molecular weight polysaccharides and derived conjugates, the ratio of conjugated lysine to protein carrier CRM197and the number of lysine covalently linked to the polysaccharide, the number of covalent bonds between the protein carrier and polysaccharides as a function of repeating units of the polysaccharide and the relative amount of free polysaccharide in comparison with the total polysaccharide. The term "free polysaccharide" when used here refers to a polysaccharide that is not anywhereman with protein carrier, but, nevertheless, present in the conjugate composition.

Methods of obtaining immunogenic conjugates of this invention include covalent conjugation of capsular polysaccharides to protein carriers by chemical conjugation with the participation of CDI (1,1-carbonyldiimidazole), CDT (1,1-carbol-di-1,2,4-triazole) or PDPH (3-(2-pyridyldithio)-propenylidene). CDI-specific conjugation SR. Using CDI/CDT is the result of one-or zero-carbon linker between the capsular polysaccharide and the protein carrier, while the use of PDPH is the result of a covalent thioester linkage between the capsular polysaccharide and the protein carrier.

Additional cross-linkers for-SH (etiolirovannye CF) K-NH2relations include, without limiting them to, the following: sulfo-LC-SMPT; sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)-tol the amido]hexanoate)); sulfo-KMUS (N-[k-maleimidomethyl]-sulfosuccinimidyl ether); sulfo-LC-SPDP (sulfosuccinimidyl-6-(3'-[2-pyridyldithio]-propionamido)hexanoate), which is cleaved thiol; sulfo-SMPB (sulfosuccinimidyl-4-[p-maleimidomethyl]butyrate); sulfo-fairs are forthcoming-Siab (N-sulfosuccinimidyl[4-iodates]aminobenzoate); sulfo-EMCS ([N-o-maleimidopropionamide]-sulfosuccinimidyl ether); EMSA (N-e-maleimidomethyl acid); sulfo-SMCC (sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate); sulfo-MBS (m-maleimidomethyl-N-hydroxycarbonylmethyl ether); sulfo-GMBS (N-[g-maleimidomethyl]sulfosuccinimidyl ether); VMR (N-β-maleimidomethyl acid); 2-immunotherapy hydrochloride; 3-(2-pyridyldithio)propionic acid N-Succinimidyl ester; 3-multimediaphoto acid N-Succinimidyl ester; 4-maleimidomethyl acid N-Succinimidyl ether; SMPT (4-succinimidylester-methyl-a-[2-pyridyldithio]toluene)); LC-SMCC (Succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-aminocaproate]); KMUA (N-k-maleimidomethyl acid); LC-SPDP (Succinimidyl-6-(3-[pyridyldithio]-propionamido)hexanoate); SMPH (Succinimidyl-6-[β-maleimidopropionamide]hexanoate); SMPB (Succinimidyl-4-[p-maleimidomethyl]butyrate); Fairs are forthcoming-Siab (N-Succinimidyl-[4-iodates]-aminobenzoate); EMCS ([N-o-maleimidopropionamide]-operations ether); SMCC (Succinimidyl-4-[N-maleimidomethyl]qi is logican-1-carboxylate); MBS (m-maleimidomethyl-N-hydroxysuccinimidyl ether); SBAP (Succinimidyl-3-[bromoacetamide]propionate); BMPS (N-[β-multimediafiles]operations ether); AMAS (N-(a-maleinizatsiei)operations ether); SIA (N-succinimidylester) and N-Succinimidyl-(4-iodates)-aminobenzoate.

Agents can also cross link, using cross-linkers from groups,- SH groups-HE. Such cross-linkers include, without limitation, RMR (N-[p-maleimidomethyl]isocyanate).

The compositions and methods described herein are useful in many applications. For example, the conjugates can be used in obtaining the conjugate immunogenic compositions to protect recipients from S.aureus infections. Alternatively, various conjugates can be used to obtain antibodies against bacterial capsular polysaccharides, which can then be used in research and clinical laboratory tests, such as the detection and serotyping of bacteria. Such antibodies can also be used to create passive immunity in the subject. In some embodiments the antibodies obtained against bacterial polysaccharides are functional or models of efficiency on the animal or in the analysis opsonophagocytosis killing.

Unless otherwise noted, all used here is the technical and scientific terminateur the same value, as understood by ordinary specialist in the field to which this invention relates. Although any methods and substances are the same as those described here, or equivalent, can be used in the practice or testing of the present invention, the preferred methods and materials are disclosed here. In the description of the embodiments and the formula of this invention will be used certain terminology in accordance with the definitions listed below. Using here the singular include references to the plural, unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more than one method and/or stage of the type described here and/or becoming obvious to the ordinary person skilled in the art upon reading this description, and so on.

When used here "approximately" means within a statistically significant range of values, such as the specified concentration range, time frame, molecular weight, temperature or pH. This range can be within the order of magnitude in typical cases within 20%, more typically still within 10%, and even more typically within 5% of the given value or range. Possible variations covered by the term "about"should depend on the specific the ow of the system under investigation, and they can easily assess an ordinary person skilled in the art. Whenever in this description of the specified range, each integer in this range are also provided as embodiments of this invention.

It is noted that in this description, terms such as "contains", "contains", "containing", "cover", "cover", and the like can have the meaning attributed to them in the patent laws of the United States, for example, they can mean "includes", "comprised", "comprising" and the like. Such terms refer to the introduction of specific ingredients or sets of ingredients without the exclusion of any other ingredients. Terms such as "consisting essentially of" and "consists essentially of"have the meanings assigned to them in the patent laws of the United States, for example, they allow the inclusion of additional ingredients or steps which are not new or belittle the main characteristics of this invention, that is, they exclude additional unspecified ingredients or stage that belittle new or basic characteristics of this invention, and they eliminate the ingredients or the stage of the prior art, such as documents in the art cited here or is included here by reference, especially insofar as the purpose of this document is definitely the tion of incarnations, which are patentable, for example, new, non-obvious, the invention relative to the prior art, for example regarding the documents cited here or is included here by reference. As well as the terms "comprises" and "comprising" shall have the meaning ascribed to them in the patent laws of the United States; namely, they are closed terms of type. Accordingly, these terms refer to the inclusion of a specific ingredient or set of ingredients and the exclusion of all other ingredients.

Immunogenic conjugates

As described above, the present invention relates to the immunogenic conjugates containing capsular polysaccharides S.aureus serotype 5 or 8 conjugated to protein carriers. In one of the embodiments of this invention proposed immunogenic conjugates containing capsular polysaccharide S.aureus serotype 5 or 8, conjugated with a molecule or protein carrier having one or more of the following characteristics: the polysaccharide has a molecular mass of between 50 kDa and 700 kDa; immunogenic conjugate has a molecular weight between 500 and 2500 kDa kDa; and the conjugate contains less than about 30% of free polysaccharide relative to the total polysaccharide. In some embodiments, the polysaccharide has a molecular weight between 20 kDa and 1000 kDa. In some embodiments of the immunogenic to nyugat has a molecular weight between 200 kDa and 5000 kDa. In other embodiments, the conjugate contains less than about 25%, about 20%, about 15%, about 10% or about 5% of free polysaccharide relative to the total polysaccharide.

"Conjugates" when used here contain capsular polysaccharide, usually with the desired range of molecular masses, and a carrier protein and the capsular polysaccharide anywhereman with protein carrier. The conjugates can contain or not to contain a certain amount of free capsular polysaccharide. When used here "free capsular polysaccharide" refers to the capsular polysaccharide, which ecovalence associated (i.e. ecovalence bound, adsorbed or trapped "in" or "with") with the conjugate of the capsular polysaccharide to a protein carrier. The terms "free capsular polysaccharide", "free polysaccharide" and "free sugar" can be used interchangeably, and are intended to convey the same meaning.

Regardless of the nature of the molecules of the medium, it can be konjugierte with capsular polysaccharide, either directly or through a linker. When using this "conjugality", "conjugated" and "conjugation" refers to the process by which bacterial capsular polysaccharide covalently attached to a molecule of the media. Conjugation increases immunodeficiency is hennoste bacterial capsular polysaccharide. The conjugation can be performed in accordance with the methods described below or by other methods known in this field.

Molecular weight capsular polysaccharide S.aureus should be considered for use in immunogenic compositions. High molecular weight capsular polysaccharides are able to induce specific immune responses or antibody-based test due to the higher valency of epitopes present on the antigenic surface. Selection of high molecular weight capsular polysaccharides" are designed for use in the compositions and methods of the present invention. In one of the embodiments of this invention may be isolated and purified high molecular weight capsular polysaccharide serotype 5 or 8 within the range of molecular masses ranging from 20 kDa to 1000 kDa. In one of the embodiments of this invention may be isolated and purified high molecular weight capsular polysaccharide serotype 5 or 8 within the range of molecular masses from 50 kDa to 700 kDa. In one of the embodiments of this invention may be isolated and purified high molecular weight capsular polysaccharide serotype 5 or 8 within the range of molecular masses from 50 kDa to 300 kDa. In one of the embodiments may be isolated and purified high molecular weight capsular polysaccharide serotype 5 or 8 within the range of molecular masses from 70 kDa to 300 kDa. In one of the embodiments can be the ü isolated and purified high molecular weight capsular polysaccharide serotype 5 or 8 within the range of molecular weights from 90 kDa to 250 kDa. In one of the embodiments may be isolated and purified high molecular weight capsular polysaccharide serotype 5 or 8 within the range of molecular weights from 90 kDa to 150 kDa. In one of the embodiments may be isolated and purified high molecular weight capsular polysaccharide serotype 5 or 8 within the range of molecular weights from 90 kDa to 120 kDa. In one of the embodiments may be isolated and purified high molecular weight capsular polysaccharide serotype 5 or 8 within the range of molecular masses of 80 kDa to 120 kDa. Other ranges of high molecular weight capsular polysaccharide serotype 5 or 8, which can be isolated and purified by the methods of this invention include from 70 kDa to 100 kDa in molecular weight; 70 kDa to 110 kDa in molecular weight; 70 kDa to 120 kDa in molecular weight; 70 kDa to 130 kDa in molecular weight; 70 kDa to 140 kDa in molecular weight; 70 kDa to 150 kDa in molecular weight; 70 kDa to 160 kDa in molecular weight; 80 kDa to 110 kDa molecular weight; 80 kDa to 120 kDa in molecular weight; 80 kDa to 130 kDa in molecular weight; 80 kDa to 140 kDa in molecular weight; 80 kDa to 150 kDa in molecular weight; 80 kDa to 160 kDa in molecular weight; from 90 kDa to 110 kDa in molecular weight; from 90 kDa to 120 kDa in molecular weight; from 90 kDa to 130 kDa in molecular weight; from 90 kDa to 140 kDa in molecular the weight; from 90 kDa to 150 kDa in molecular weight; from 90 kDa to 160 kDa in molecular weight; from 100 kDa to 120 kDa in molecular weight; from 100 kDa to 130 kDa in molecular weight; from 100 kDa to 140 kDa in molecular weight; from 100 kDa to 150 kDa in molecular weight; from 100 kDa to 160 kDa in molecular weight; and similar desired ranges of molecular weights. Any integer within any specified range is provided as an embodiment of this invention.

In one embodiments, the conjugate has a molecular weight of between about 50 kDa and about 5000 kDa molecular weight. In one embodiments, the conjugate has a molecular weight between about 200 kDa and about 5000 kDa molecular weight. In one of the embodiments of the immunogenic conjugate has a molecular weight between about 500 kDa and about 2500 kDa. In one of the embodiments of the immunogenic conjugate has a molecular weight between about 500 kDa and about 2500 kDa. In one of the embodiments of the immunogenic conjugate has a molecular weight between about 600 kDa and about 2800 kDa. In one of the embodiments of the immunogenic conjugate has a molecular weight between about 700 kDa and about 2700 kDa. In one of the embodiments of the immunogenic conjugate has a molecular weight between about 1000 kDa and about 2000 kDa; between about 1800 kDa and about 2500 kDa; between about 1100 kDa and about 2200 is Yes; between about 1900 kDa and about 2700 kDa; between about 1200 kDa and about 2400 kDa; between about 1700 kDa and about 2600 kDa; between about 1300 kDa and about 2600 kDa; between about 1600 kDa and about 3000 kDa. Any integer within any specified range is provided as an embodiment of this invention

When used here "immunogenic" refers to the ability of the antigen (or epitope of the antigen), such as bacterial capsular polysaccharide or conjugate immunogenic composition comprising the antigen to induce an immune response in the host, such as a mammal, either humoral or cellular, or both. Accordingly, "immunogenic conjugate" or "conjugate" when used here means any immunogenic conjugate containing the antigen or antigenic determinant (epitope) of a bacterial capsular polysaccharide conjugated to a carrier molecule, which can be used to induce an immune response. Immunogenic conjugate can be used for sensitization of the host by the presentation of antigen in Association with molecules of the major histocompatibility complex (MHC) on the cell surface. In addition, you can generate antigen-specific T-cells or antibodies to protect the immunized host in the future. Immunogenic conjugates, that is they way can protect the owner from one or more than one symptom associated with infection by bacteria, or can protect the owner from loss due to infection by bacteria associated with this capsular polysaccharide. Immunogenic conjugates can also be used to obtain polyclonal or monoclonal antibodies that can be used to give passive immunity to a subject. Immunogenic conjugates can also be used to generate antibodies that are functional when measuring for killing bacteria in any model of efficiency on the animal or in the analysis opsonophagocytosis killing.

"Antibody" is an immunoglobulin molecule capable of specific binding to a target, such as carbohydrate, polynucleotide, lipid, polypeptide, etc. through at least one site recognition of antigens localized in the variable regions of immunoglobulin molecules. When you use here, unless the context indicates otherwise, the term is intended to encompass not only intact polyclonal or monoclonal antibodies, but also engineered antibodies (e.g., chimeric, humanitariannet and/or derivateservlet to change effector functions, stability and other biological activities) and fragments thereof (such as Fab, Fb', F(ab')2, Fv), single-chain (ScFv) and domain antibodies, including shark and camel antibodies, and fused proteins containing part, representing the antibody, multivalent antibodies, multispecific antibodies (for example, bispecific antibodies so long as they exhibit the desired biological activity) and fragments of antibodies, which are described here, and any other modified configuration of the immunoglobulin molecules that contain the site of antigen recognition. The antibody includes the antibody of any class such as IgG, IgA or IgM (or its subclass), and the antibody does not necessarily belong to any particular class. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be attributed to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, IgA1 and IgA2 in humans. The constant domains of the heavy chain, which correspond to the different classes of immunoglobulins are designated as alpha, Delta, Epsilon, gamma and mu, respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known.

"Fragments of the antibodies contain only a portion of an intact antibody, and this part of prepact the tion retains at least one function, preferably most of the functions or all functions normally associated with this part when it is present in an intact antibody.

The term "antigen" in General refers to the biological molecule, usually a protein, peptide, polysaccharide or conjugate in immunogenic compositions or immunogenic substance that may stimulate the production of antibodies or T-cell responses, or both in an animal, including compositions that are injected animal or absorbed by the animal. The immune response can be generated for a molecule or on different parts of the molecule (e.g., epitope or hapten). This term can be used in relation to an individual molecule or a homogeneous or heterogeneous population of antigenic molecules. The antigen recognized by antibodies, T-cell receptors or other elements of a specific humoral and/or cellular immunity. "Antigen" also includes all related antigenic epitopes. Epitopes of a given antigen can be identified by using any number of methods of epitope mapping are well known in this field. See, for example, an epitope Mapping Protocols in Methods in Molecular Biology, Vol.66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. for Example, linear epitopes can be determined, for example, the simultaneous synthesis of large numbers of peptides on a solid is odaiko, moreover, these peptides correspond to the parts of the protein molecule, and the interaction of the peptides with antibodies while, when the peptides are still attached to the substrate. Such techniques are known in this field and are described, for example, in U.S. patent No. 4708871; Geysen et al. (1984) Proc. Natl. Acad Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715; each of which is incorporated here by reference, as if it were set forth in its entirety. Similarly, conformational epitopes can be identified by determining spatial conformation of amino acids, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, an epitope Mapping Protocols above. Moreover, for the purposes of the present invention the antigen can also be used in relation to a protein which includes modifications to the native sequence, as deletions, insertions and substitutions (generally conservative in nature, but they may not be conservative) insofar as the protein retains the ability to induce an immunological response. These modifications may be deliberate, as by site-directed mutagenesis or by using specific synthetic methods or genetic engineering approach, or random, such as when mutations in the host, which produces antigens. Moreover, the antigen can be produced to obtain or provide the best of the microbe, for example, bacteria, or it can represent the whole body. Similarly, oligonucleotide or polynucleotide, which expresses the antigen for immunization of nucleic acid, is also included in this definition. Also included are synthetic antigens, such as polyepitope flanking epitopes and other recombinant or synthetically derived antigens (Bergmann et al. (1993) Eur. J.Immunol. 23:2777 2781; Bergmann et al. (1996) J.Immunol. 157:3242-3249; Suhrbier (1997) Immunol. Cell Biol. 75:402 408; Gardner et al. (1998) 12 th World AIDS Conference, Geneva, Switzerland, Jun. 28 to Jul. 3,1998).

"Protective" immune response refers to the ability of the immunogenic composition to induce an immune response or a humoral or cellular, or both, which serve to protect the subject from infection. Provided protection need not be absolute, that is, the infection does not necessarily have to be totally prevented or eradicated, if there is a statistically significant improvement when compared with a control population of subjects, such as infected animals, which have not entered the vaccine or immunogenic composition. Protection may be limited to reduce the severity or speed the onset of symptoms of infection. In General, a "protective immune response" should include the induction of increased levels of antibodies specific to a particular antigen of at least 50% of the subjects, which includes all the I some levels are measurable functional or antibody-based test responses to each antigen. In private situations "protective immune response" may include the induction of a twofold increase in antibody levels or fourfold increase levels of antibodies specific to a particular antigen, at least 50% of the subjects, which includes some level of measurable functional or antibody-based test responses to each antigen. In certain embodiments of the protective immune response correlate opsonizing antibodies. Thus, a protective immune response can be analyzed by measuring the percentage reduction in the number of bacteria in the analysis of opsonophagocytosis, for example, as described below. Preferably there is a decrease in the number of bacteria by at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more than 95%. "Immunogenic amount" of a particular conjugate in the composition is typically dosed on the basis of the total polysaccharide conjugated and unconjugated, this conjugate. For example, a conjugate of the capsular polysaccharide serotype 5 or 8 with 20% of free polysaccharide has at the dose of 100 μg to have about 80 μg of conjugated polysaccharide and about 20 μg unconjugated polysaccharide. When calculating the dose of the conjugate contribution of protein in the conjugate usually do not take into account. The number of conjugate can vary depending on the serotype of Staphylococcus. Each dose should contain from 0.1 to 100 μg for the of Ishida, in particular from 0.1 to 10 μg, and more specifically, from 1 to 10 mcg.

The term "subject" refers to a mammal, bird, fish, reptile or any other animal. The term "subject" also includes people. The term "subject" also includes animals. Non-limiting examples of animals include dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, Guinea pigs, ferrets, birds, snakes, lizards, fish, turtles and frogs. The term "subject" also includes farm animals. Non-limiting examples of farm animals include Alpaca, bison, camel, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese and turkeys.

As shown in figure 1, capsular polysaccharides S.aureus serotype 5 and 8 have the following structure:

serotype 5

[→4)-β-D-ManNAcA-(1→4)-3-0-Ac-α-L-FucNAc-(1→3)-β-DFucNAc-(1→]n

and serotype 8

[→3)-4-O-Ac-β-D-ManNAcA-(1→3)-α-L-FucNAc-(1→3)-β-DFucNAc-(1→]n,

see, Jones (2005) Carbohydr. Res. 340:1097-1106. Capsular polysaccharide serotype 8 has the same trisaccharide repeating unit which has a capsular polysaccharide serotype 5; however, they differ on the links between sugars and sites of O-acetylation, which causes serologically distinct profiles of immunoreactivity (Fournier et al. (1984) Infect. Immun. 5:87-93; and Moreau et al. (1990) Carbohydr. Res. 201:285-297). Therefore, the capsular polysaccharides of serotypes 8 and 5 are relatively complex carbohydrates, which are water-soluble, usually acidic, and previously it was thought that they have a molecular mass of approximately 25 kDa (Fattom (1990) Infect. Immun. 58, 2367-2374).

In some embodiments of the capsular polysaccharides of serotypes 5 and/or 8 according to this invention, O-azetilirovanny. In some embodiments, the degree of O-acetylation of the capsular polysaccharide or oligosaccharide of type 5 is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In some embodiments, the degree of O-acetylation of the capsular polysaccharide or oligosaccharide of type 8 is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In some embodiments, the degree of O-acetylation of the capsular polysaccharides or oligosaccharides of the type 5 and type 8 is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%.

The degree of O-acetylation of the polysaccharide or oligosaccharide can be determined by any method known in this field, for example, proton NMR (Lemercinier and Jones 1996, Carbohydrate Research 296; 83-96, Jones and Lemercinier 2002, J Pharmaceutical and Biochemical Analysis 30; 1233-1247, WO 05/033148 or WO 00/56357). Another commonly used method is described in Hestrin (1949) J.Biol. Chem. 180; 249-261.

In some embodiments of the capsular polysaccharides of serotypes 5 and/or according to this invention is used to produce antibodies, which are functional when measuring for killing bacteria in a model of efficiency on animals or analysis opsonophagocytosis killing, which demonstrates that antibodies to kill the bacteria. Functional destruction may not be demonstrated using analysis, which tracks only the generation of antibodies, which is not an indicator of the importance of O-acetylation for efficiency.

Capsular polysaccharides, such as related to the serotype 5 or 8, can be obtained directly from the bacteria using the methods of isolation, known to the ordinary person skilled in the art. See, for example, Fournier et al. (1984), above; Fournier et al. (1987) Ann. Inst. Pasteur/Environ. 138:561-567; publication of patent application U.S. No. 2007/0141077; publication of international patent application WO 00/56357; each of which is incorporated here by reference as if presented in full. In addition, they can be obtained by using synthetic methods. Moreover, the capsular polysaccharide serotype 5 or 8 can be obtained by recombinant, using methods of genetic engineering, also known specialist in this field (see Sau et al. (1997) Microbiology 143:2395-2405; U.S. patent No. 6027925; each of which is incorporated here by reference as if presented in full).

One of the strains of S.aureus, which can be used to obtain from the military capsular polysaccharide serotype 8, is S.aureus R2 PFESA0286. This strain was selected flow cytometry with rabbit antibodies against polysaccharide serotype 8 after cultivation S.aureus PFESA0286 (American type culture collection (American Type Culture Collection; Manassas, VA; the access number of ATSS 49525) in a modified broth Franz. During flow cytometry observed two populations, R1 and R2. R1 and R2 were purified and ricultural. R2 gave capsular polysaccharide serotype 8. Analysis of flow cytometry showed homogeneous fluorescence intensity. As such, R2 would be chosen to produce capsular polysaccharide serotype 8.

One of the strains of S.aureus, which you can use to obtain the selected capsular polysaccharide serotype 5 is S.aureus PFESA0266. This strain produces capsular polysaccharide serotype 5 during growth, and the production reaches its peak when the cells are in stationary phase. To obtain the corresponding polysaccharides can be used other strains of S.aureus type 5 or type 8, which receive either from the collections of stable crops, or from clinical specimens.

Another component of the immunogenic conjugate according to this invention is a molecule or a protein carrier, which anywhereman bacterial capsular polysaccharide. The term "protein carrier" or "protein carrier" refers to any protein molecule, colorwoman to konjugierte with the antigen (such as capsular polysaccharides), against which the desired immune response. Conjugation with a carrier may enhance the immunogenicity of the antigen. The conjugation can be performed by standard methods. Preferred proteins as carriers for antigens include toxins, toxoids or any mutant cross-reactive material (CRM) of the toxin from the causative agent of tetanus, diphtheria, pertussis, Pseudomonas, E.coli, Staphylococcus and Streptococcus. In one particularly preferred embodiments, the carrier is diphtheria toxoid CRM197derived from strain C7 .diphtheriae (p197), which produces a protein CRM197. This strain has ATSS room access 53281. A method of obtaining a CRM197described in U.S. patent No. 5614382, which is included here by reference as if he was fully described. Alternatively, you can use a fragment or an epitope of the protein carrier or another immunogenic protein. For example, hapenny antigen may be associated with T-cell epitope of a bacterial toxin, toxoid or CRM. Cm. application for U.S. patent No. 150688, filing date February 1, 1988, entitled "Synthetic peptides Representing a T-CeIl an epitope as a Carrier Molecule For Conjugate Vaccines", included here by reference as if she was fully described. Other suitable media include proteins inactivated bacterial toxins such as cholera toxoid (for example as described in international patent application W 2004/083251), .coll LT, E. coli ST, and exotoxin a from Pseudomonas aeruginosa. You can also use proteins of the bacterial outer membrane, such as complex proteins of the outer membrane (OMRS), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), protein adhesion of pneumococci (PsaA) or protein D of Haemophilus influenzae. Can also be used as protein carriers other proteins, such as ovalbumin, hemocyanin sea plate (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD).

Accordingly, in one of the embodiments of the protein carrier in the immunogenic conjugate according to this invention is CRM197and this CRM197covalently linked to the capsular polysaccharide urethane bond, amide bond or both parties. In some embodiments the protein carrier in the immunogenic conjugate according to this invention is CRM197and this CRM197covalently linked to the capsular polysaccharide thioester bond. The number of lysine residues in the protein carrier, which becomes conjugated with capsular polysaccharide, can be described as range conjugated lysine. For example, this immunogenic composition CRM197may contain from 5 to 15 lysine 39, covalently associated with capsular polysaccharide. Another way of expressing this parameters is and is that from 12% to 40% lysine CRM197covalently linked to the capsular polysaccharide. For example, this immunogenic composition CRM197may contain from 18 to 22 lysine 39, covalently associated with capsular polysaccharide. Another way of expressing this option is that from 40% to 60% lysine CRM197covalently linked to the capsular polysaccharide. In some embodiments CRM197contains from 5 to 15 lysine 39, covalently linked with SR. Another way of expressing this option is that from 12% to 40% lysine CRM197covalently linked to SR. In some embodiments CRM197contains from 18 to 22 lysine 39, covalently linked with SR. Another way of expressing this option is that from 40% to 60% lysine CRM197covalently linked to SR.

As discussed above, the number of lysine residues in the protein carrier, kongugirovannom with capsular polysaccharide, can be described as range conjugated lysine, which can be expressed as the molar ratio. For example, the molar ratio of conjugated lysine to CRM197in SR the immunogenic conjugate can be between about 18:1 and about 22:1. In one embodiments, the range of molar ratio of conjugated lysine to CRM197in SR the immunogenic conjugate can be between about 15:1 and p is IMEMO 25:1. In one embodiments, the range of molar ratio of conjugated lysine to CRM197in SR the immunogenic conjugate can be between about 14:1 and about 20:1, about 12:1 and about 18:1; about 10:1 and about 16:1; about 8:1 and about 14:1, about 6:1 and about 12:1; about 4:1 and about 10:1; about 20:1 and about 26:1; about 22:1 28:1; about 24:1 and about 30:1; about 26:1 and about 32:1; about 28:1 and 34:1, about 30:1 and about 36:1; about 5:1 and about 10:1; about 5:1 and about 20:1; about 10:1 and about 20:1 or about 10:1 and about 30:1. Also the molar ratio of conjugated lysine to CRM197in SR the immunogenic conjugate can be between about 3:1 and 25:1. In one embodiments, the range of molar ratio of conjugated lysine to CRM197in SR the immunogenic conjugate can be between about 5:1 and about 20:1. In one embodiments, the range of molar ratio of conjugated lysine to CRM197in SR the immunogenic conjugate can be between about 4:1 and about 20:1, about 6:1 and about 20:1, about 7:1 and about 20:1; about 8:1 and about 20:1; about 10:1 and about 20:1, about 11:1 and about 20:1, about 12:1 and about 20:1, about 13:1 and about 20:1, about 14:1 and about 20:1, about 15:1 and about 20:1, about 16:1 and about 20:1; about 17:1 is about 20:1; about 18:1 and about 20:1; about 5:1 and about 18:1, about 7:1 and about 16:1 or about 9:1 and about 14:1.

Another way of expressing the number of lysine residues in the protein carrier conjugated with capsular polysaccharide may be a range conjugated lysine. For example, in this SR the immunogenic conjugate CRM197may contain from 5 to 15 lysine 39, covalently associated with capsular polysaccharide. Alternatively, this parameter can be expressed in percent. For example, in this SR the immunogenic conjugate percentage of conjugated lysine can be between 10% and 50%. In some embodiments, from 20% to 50% lysine can be covalently linked to SR. Alternatively, from 30% to 50% lysine CRM197can be covalently linked to SR; from 10% to 40% lysine CRM197; from 10% to 30% lysine CRM197; from 20% to 40% lysine CRM197; from 25% to 40% lysine CRM197; from 30% to 40% lysine CRM197; from 10% to 30% lysine CRM197; from 15% to 30% lysine CRM197; from 20% to 30% lysine CRM197; from 25% to 30% lysine CRM197; from 10% to 15% lysine CRM197or from 10% to 12% lysine CRM197covalently linked to SR. Also, in this SR the immunogenic conjugate CRM197may contain from 18 to 22 lysine 39, covalently associated with capsular polysaccharide. Alternatively, this parameter can be expressed in percent. For example, in the data the m SR the immunogenic conjugate percentage of conjugated lysine can be between 40% and 60%. In some embodiments, from 40% to 60% lysine can be covalently linked to SR. Alternatively, from 30% to 50% lysine CRM197can be covalently linked to SR; from 20% to 40% lysine CRM197; 10% to 30% lysine CRM197; from 50% to 70% lysine CRM197; from 35% to 65% lysine CRM197; from 30% to 60% lysine CRM197; from 25% to 55% lysine CRM197; from 20% to 50% lysine CRM197; from 15% to 45% lysine CRM197; from 10% to 40% lysine CRM197; from 40% to 70% lysine CRM197; or from 45% to 75% lysine CRM197covalently linked to SR.

Frequency attach chain capsular polysaccharide to a lysine on a molecule of carrier is yet another parameter to characterize the conjugates of capsular polysaccharides. For example, in one of the embodiments of the at least one covalent bond between CRM197and the polysaccharide takes place for at least every 5-10 sharidny repeating unit of the capsular polysaccharide. In yet another embodiment with at least one covalent bond between CRM197and capsular polysaccharide for every 5-10 sharidny repeating units; every 2-7 sharidny repeating units, each of 3-8 sharidny repeating units; each 4-9 sharidny repeating units; each 6-11 sharidny repeating units; each of 7-12 sharidny repeating units; each 8-13 sharidny beeps repeated the units; every 9-14 sharidny repeating units; every 10-15 sharidny repeating units; every 2-6 sharidny repeating units, each 3-7 sharidny repeating units; every 4-8 sharidny repeating units; every 6-10 sharidny repeating units; every 7-11 sharidny repeating units; every 8-12 sharidny repeating units; each 9-13 sharidny repeating units; every 10-14 sharidny repeating units; every 10-20 sharidny repeating units or every 5-10 sharidny repeating unit of the capsular polysaccharide. In yet another embodiment at least one relationship between CRM197and capsular polysaccharide has a place for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 sharidny repeating unit of the capsular polysaccharide.

In one of the embodiments of this invention proposed immunogenic composition comprising any immunogenic conjugate containing capsular polysaccharide S.aureus serotype 5 or 8, conjugated to protein carrier described above.

The term "immunogenic composition" refers to any pharmaceutical composition comprising the antigen, for example a microorganism or a component that can be used to induce an immune response in the subject. Immunogenic compositions of the present invention can be used for the protection or treatment of the person, susceptible S.aureus, through the introduction of immunogenic compositions systemic, transdermal by or through the mucous membranes, or can be used for the preparation of polyclonal or monoclonal antibodies that can be used to give passive immunity to another entity. The introduction may include an injection into a muscle, into the abdominal cavity, in the skin or under the skin or through the mucous membranes in the oral/alimentary, respiratory or urinary track. In one of the embodiments of intranasal introduction used for the treatment or prevention of nasopharyngeal carriage of S.aureus and thereby to weaken the infection at its earliest stage. Immunogenic compositions can also be used to generate antibodies that are functional in the measurement on the death of the bacteria either in the model of efficiency on the animal, or analysis opsonophagocytosis killing.

The optimal number of components for a specific immunogenic compositions can be clarified by standard studies involving observation of appropriate immune responses in subjects. After the initial vaccination, subjects may receive one or several booster immunizations through appropriate intervals.

Immunogenic compositions of the present invention can also include one Il is more of the following antigens: ClfA, ClfB, SdrC, SdrD, SdrE, MntC/SitC-binding protein saliva, IsdB, IsdA, Orra, DItA, HtsA, LtaS, SdrH, SrtA, SpA, SB1, alpha-hemolysin (hia), beta-hemolysin, fibronectin-binding protein A (fnbA), coagulase, map, leukocidin Panton-Valentine (pvl), gamma-toxin (nlg), ica, immunodominant ABC Transporter, RAP, autolysin, laminin receptors, IsaA/PisA, IsaB/PisB, SPOIIIE, SsaA, EbpS, SasF, SasH, EFB (FIB), FnbB, Npase, HEB, bone SIAL-bonded protein II, the predecessor of aureolin (AUR)/Seppl, Cna, TSST-1, mecA, dPNAG, GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin binding protein, HarA, enterotoxin a, enterotoxin, enterotoxin CI and new autolysin.

In one of the embodiments of the immunogenic compositions of this invention optionally contain at least one adjuvant, buffer, cryoprotector, salt, divalent cation, a non-ionic detergent, an inhibitor of free radical oxidation, diluent or carrier. In one of the embodiments of the adjuvant in an immunogenic composition according to this invention is an adjuvant based on aluminum. In one embodiments the adjuvant is an adjuvant on the basis of aluminum selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one embodiments the adjuvant is an aluminum phosphate.

Adjuvant is a substance that enhances the immune response when introduced together with the immunogen or antigen. It was shown that immuno is delirous activity and have, thus, it can be similarly useful adjuvants or similar, many cytokines or lymphokines, including, without limitation, interleukin 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, for example, U.S. patent№5723127), 13, 14, 15, 16, 17 and 18 (and its mutant forms); interferons-α, β and γ; the factor, colony stimulating granulocyte/macrophage (GM-CSF) (see, for example, U.S. patent No. 5078996 and the access number in ADS 39900); factor, colony stimulating macrophages (M-CSF); factor, colony stimulating granulocyte (G-CSF), and the factors a and p of tumor necrosis. More other adjuvants that are useful in immunogenic compositions described herein include chemokines, including, without limiting them, MCP-1, Ì1Ð-1α, Ì1Ð-1β and RANTES; adhesion molecules, such as selectin, such as L-selectin, P-selectin and E-selectin; mezinarodni molecules such as CD34, GIyCAM-1 and MadCAM-1, a member of the family of integrins, such as LFA-1, ILA-1, Mac-1 and R; member of the immunoglobulin superfamily, such as the RESOURCES, ICAM, such as ICAM-1, ICAM-2 and ICAM-3, CD2, and LFA-3; co-stimulatory molecules such as B7-1, B7-2, CD40 and CD40L; growth factors, including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1 and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Fit, Apo-1, p55, VVSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR, KILLER, TRAIL-R2, TR1CK2 and DR6; and caspase, including ICE.

Suitable adjuvants used to enhance the immune response may optionally include, without limiting them, MPL™ (3-O-describeany monophosphorylated A, Corixa; Hamilton, MT), which is described in U.S. patent No. 4912094. Also suitable for use as adjuvants are synthetic analogues of lipid a or connection aminoalkylphosphonic (AGP), or derivatives or analogs which are available from Corixa, as well as those that are disclosed in U.S. patent No. 6113918. One such AGP is 2-[(R)-3-tetradecanoate-tetradecanoyl]-ethyl-2-deoxy-4-O-phosphono-3-O-[(R)-3-deletion-oxitetraciclina]-2-[(R)-3-tetradecanoylphorbol-amino]-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529). This adjuvant 529 prepared in aqueous form (AF) or in the form of a stable emulsion (SE).

More other adjuvants include muramylpeptide, such as N-acetyl-muramyl-L-threonyl-O-isoglutamine (thr-MDP), N-acetyl-norbornyl-L-alanine-2-(1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphonic-hydroxy)-ethylamine (MTP-PE); emulsions of the type oil-in-water, such as MF59 (U.S. patent No. 6299884) (containing 5% squalene, 0.5% Polysorbate 80 and 0.5% Span 85 (possibly containing various amounts of MTP-PE)), prepared in the form of submicron particles using microfluidizer such to the to microfluidizer Model HOY (Microfluidics, Newton, MA)and SAF (containing 10% squalene, 0.4% Polysorbate 80.5% polymer with plutonomy blocks L121, and thr-MDP, either microfluidized in submicron emulsion or processed vortex shaker to obtain emulsions with a large particle size); incomplete adjuvant's adjuvant (IFA); aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate; Empigen (Amphigen); Avidin (Avridine); L121/squalene, D-lactide-polylactide/glycoside; plutonomies polyols; killed Bordetella cells; saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, MA), described in U.S. patent No. 5057540, Iscomatrix® (CSL Limited, ParkviIIe, Australia), described in U.S. patent No. 5254339, and immunostimulating complexes (ISCOMS); Mycobacterium tuberculosis; bacterial lipopolysaccharides; synthetic polynucleotide, such as oligonucleotides containing a CpG motif (for example, U.S. patent No. 6207646); IC-31 (Intercell AG, Vienna, Austria), described in European patent No. 1296713 and 1326634; pertussis toxin (RT) or its mutants, cholera toxin or its mutants (for example, U.S. patent No. 7285281, 7332174, 7361355 and 7384640); or thermolabile toxin E. coli (LT) or its mutant, particularly LT-K63, LT-R72 include (for example, U.S. patent No. 6149919, 7115730 and 7291588).

The immunogenic composition may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means a carrier approved by the Federal government is whether other regulatory bodies, or listed in the U.S. Pharmacopoeia or other generally accepted Pharmacopoeia for use in animals, including humans, mammals, non-human beings. The term "carrier" refers to a diluent, adjuvant, excipient or media, which administered the pharmaceutical composition. As liquid carriers, particularly for injectable solutions, you can use water, saline solutions and aqueous dextrose and glycerol. Examples of suitable pharmaceutical carriers are disclosed in "Remington''s Pharmaceutical Sciences" by E. W. Martin. The drug must correspond to the method of administration.

Immunogenic compositions of the present invention can optionally contain one or more additional "immunomodulators", which are agents that stimulate or alter the immune system so that there is either strengthening or weakening of the humoral and/or cellular immunity. In one of the embodiments of the provided activation of humoral and/or cell-mediated component of the immune system. The specific examples of immunomodulators include, for example, adjuvant or cytokine, or Iscomatrix® (CSL Limited, Parkville, Australia), described in U.S. patent No. 5254339 among other things. Non-limiting examples of adjuvants that can be used in immunogenic compositions of the present invention, on the receive adjuvant system RIBI (Ribi Inc.; Hamilton, MT), alum, mineral gels such as gel aluminum hydroxide emulsion of the type oil-in-water emulsion of the type water-in-oil, such as, for example, complete and incomplete adjuvants's adjuvant, and the block copolymer (CytRx; Atlanta, GA), QS-21 (Cambridge Biotech Inc.; Cambridge, MA), SAF-M (Chiron; Emeryville, CA), adjuvant Amphigen®, saponin, Quil a or another faction saponins, monophosphorylated and lipid-amine adjuvant Avidin. Non-limiting examples of emulsions of the type oil-in-water, useful in immunogenic compositions according to this invention include modified drugs SEAM62 and SEAM 1/2. Modified SEAM62 is an emulsion of the type oil-in-water containing 5% (vol./about.) squalene (Sigma), 1% (vol./about.) detergent Span® 85 (ICI Surfactants), and 0.7% (vol./about.) detergent Polysorbate 80 (ICI Surfactants), and 2.5% (vol./about.) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol and 0.5% (vol./about.) lecithin. Modified SEAM 1/2 is an emulsion of the type oil-in-water containing 5% (vol./about.) squalene, 1% (vol./about.) detergent Span® 85, of 0.7% (vol./about.) detergent Polysorbate 80, and 2.5% (vol./about.) ethanol, 100 μg/ml Quil a and 50 μg/ml cholesterol. Other "immunomodulators"that can be included in the immunogenic composition include, for example, one or more than one interleukin, interferon, or other known cytokines or chemokines. In one of the embodiments of the adjuvant may be a derivative of cyclodextrin or polyanionic polymer is such as those that described in U.S. patent No. 6165995 and 6610310, respectively. You have to understand that immunomodulator and/or adjuvant for use will depend on the subject, which you need to enter immunogenic composition, the method of injection and the number of injections that need to be done.

Immunogenic compositions according to this invention can, in addition to the aggregate conjugates staphylococcal capsular polysaccharide with protein, optionally contain one or more than one preservative FDA (Federal office for food and medical products) requires that biological products in mnogochasovykh bottles (multi-dose) only a few exceptions contained a preservative. Vaccine products containing preservatives include vaccines containing chloride benzene (anthrax), 2-Phenoxyethanol (DTaP, Nera, Lyme disease, Polio (poliomyelitis) (dropped)), phenol (Pneumo (pneumonia), Typhoid (fever) (parenteral), Vaccinia (smallpox)and thimerosal (DTaP, DT, Td, HepB, Hib, influenza, JE, meningitis (Mening), pneumonia (Pneumo), rabies (Rabies)). Preservatives approved for use in injectable drugs include, for example, chlorbutanol, m-cresol, methylparaben, propylparaben, 2-Phenoxyethanol, chloride benzene, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, and the nitrate of finalstate.

The drugs on this from the retenu can optionally contain one or more than one buffer, salt, a divalent cation, a non-ionic detergent, cryoprotector, such as sugar, and an antioxidant, such as a scavenger of free radicals or chelating agent, or any multiple combination. The selection of any component, for example, a chelating agent may determine the desirability or undesirability of another component (e.g., absorber). The finished composition is prepared for administration must be sterile and/or pyrogen-free. An experienced specialist can empirically determine which combinations of these and other components must be optimal for inclusion in containing preservative immunogenic composition according to this invention, depending on many different factors, such as specific required conditions of storage and administration.

In certain embodiments the composition according to this invention, which is compatible with parenteral introduction, contains one or more than one physiologically acceptable buffer, selected from the following, but not limited by them: Tris (trimethylamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate. In certain embodiments the composition is buffered in the pH range from about 6.0 to about to 9.0, preferably from about 6.4 to about 7,4.

In certain embodiments it may be desirable to adjust the pH of the immunogenic composition is or preparation according to this invention. The pH value of the preparation according to this invention can be adjusted using standard techniques in the field. The pH value of the product may be adjusted so that it was between a 3.0 and 8.0. In certain embodiments the pH of the product may be or may be adjusted to values between 3,0 and 6,0, of 4.0 and 6.0 or 5.0 and 8.0. In other embodiments, the pH of the drug may be or may be adjusted to about a 3.0, about 3.5, about 4.0, about 4.5, about 5.0 and about 5.5, about 5.8 to about 6.0 and about 6.5 to about 7.0 and about 7.5, or about to 8.0. In certain embodiments the pH may be or may be adjusted in the range from 4.5 to 7.5, or from 4.5 to 6.5, from 5.0 to 5.4, from 5.4 to 5.5, 5.5 to 5.6, from 5.6 to 5.7, from 5.7 to 5.8, from about 5.8 to 5.9, 5.9 to 6.0, from 6.0 to 6.1, from 6.1 to 6.2, from 6.2 to 6.3, from 6.3 to 6.5, 6.5 to 7.0, from 7.0 to 7.5 or 7.5 to 8.0. In a specific embodiment, the pH of the drug is approximately 5.8.

In certain embodiments, the preparation according to this invention, which is compatible with parenteral introduction, contains one or more than one bivalent cation, including, without limiting them, MgCl2, CaCl2and MnCl2in a concentration in the range from about 0.1 mm to about 10 mm, and up to about 5 mm is preferred.

In certain embodiments, the preparation according to this invention, which are the two which is compatible with parenteral introduction, contains one or more than one salt, including, without limitation, sodium chloride, potassium chloride, sodium sulfate and potassium sulfate present in ionic strength, which is physiologically acceptable to the subject of parenteral and which is included in the final concentration to achieve a selected ionic strength or osmolarity in the finished product. The final ionic strength or the osmolarity of the preparation is determined by the sum of the components (for example, ions from the buffer(data) connection(s) and buffer salts). The preferred salt, NaCl, is present in the range up to about 250 mm, and the salt concentration is chosen to complement the other components (e.g., sugars), so that the final total osmolarity of the drug was compatible with parenteral administration (e.g. intramuscular or subcutaneous injection) and should promote long-term stability of the immunogenic components of the preparation of immunogenic compositions in different temperature ranges. Free from salts drugs must withstand elevated ranges of one or more than one selected cryoprotectant to maintain the desired target levels of osmolarity.

In certain embodiments, the preparation according to this invention, which is compatible with parenteral introduction, sterilizin or more than one cryoprotector, selected, without limiting them, disaccharides (e.g. lactose, maltose, sucrose or trehalose) and polyhydroxyalkanoates (for example, dulcita, glycerol, mannitol and sorbitol).

In certain embodiments, the osmolarity of the preparation is in the range from about 200 mOsm/l to about 800 mOsm/l, and the preferred range is from about 250 mOsm/l to about 500 mOsm/l, or from about 300 mOsm/l to about 400 mOsm/L. Free from salts, the preparation can contain, for example, from about 5% to about 25% sucrose and, preferably, from about 7% to about 15% or from about 10% to about 12% sucrose. Alternative, free from salts, the preparation can contain, for example, from about 3% to about 12% sorbitol and, preferably, from about 4% to 7%, or from about 5% to about 6% sorbitol. If you add salt, such as sodium chloride, then the effective range of sucrose or sorbitol relatively reduced. These and other indicators, such as osmolarity and consideration of osmolarity, it is within the skill in this field.

In certain embodiments, the preparation according to this invention, which is compatible with parenteral introduction, contains one or more than one inhibitor of free radical oxidation and/or chelating agent. A variety of sinks of Svobodnaya and chelating agents known in the field and applicable to products and application methods, as described here. Examples include, without limitation, ethanol, EDTA (ethylenediaminetetraacetic acid), a combination of EDTA/ethanol, triethanolamine, mannitol, histidine, glycerin, sodium citrate, insidecatholic, tripolyphosphate, ascorbic acid/ascorbate, succinic acid/succinate, malic acid/malate, Desferal, EDDHA (Ethylenediamine-N,N'-bis(2-hydroxyphenylarsonic acid) and DTPA (diethylenetriaminepentaacetic acid), and various combinations of two or more than two of those agents. In certain embodiments, at least one non a scavenger of free radicals can be added in a concentration that effectively increases long-term stability of the drug. One or more than one inhibitor of free radical oxidation /chelating agent can also be added in various combinations, such as the absorber and the divalent cation. The choice of chelating agent will determine the need for scavenger.

In certain embodiments, the preparation according to this invention, which is compatible with parenteral introduction, contains one or more than one non-ionic surfactant, including, without limiting them, esters of fatty acids with polyoxyethylenesorbitan, Polysorbate-80 (Tween 80), Polysorbate-60 (Tween 60), Polysorbate-40 (Tween 40) and Polisar the at-20 (Tween 20), polyoxyethylenesorbitan esters, including, without limiting them, Brij 58, Brij 35, and others, such as Triton X-100; Triton X-114, NP40, Span 85 and floranova series of nonionic surfactants (for example, Pluronic 121), and preferred ingredients are Polysorbate-80 at a concentration of from about 0,001% to about 2% (preferably up to about 0.25 per cent) or Polysorbate-40 at a concentration of from about 0,001% to 1% (preferably up to about 0.5%).

In certain embodiments, the preparation according to this invention contains one or more than one additional stabilizer, suitable for parenteral administration, for example a reducing agent containing at least one Tilney (-SH) group (for example, cysteine, N-acetylcysteine, restored glutathione, thioglycolate sodium thiosulfate, monothioglycerol or mixtures thereof). An alternative or possibly containing preservative preparations immunogenic composition according to this invention can be further stabilized by the removal of oxygen from containers for storage, protection of the drug from light (for example, the use of glass containers of dark glass).

Contain preservative preparations immunogenic composition according to this invention may contain one or more than one pharmaceutically acceptable carrier or excipient, which is s include any excipient, which does not itself induce the immune response. Suitable excipients include, without limiting them, macromolecules, such as proteins, sugars, lactic acid, polyglycolic acid, polymers of amino acids, copolymers of amino acids, sucrose (Paoletti et al, 2001, Vaccine, 19:2118), trehalose, lactose and aggregates of lipids (such as oil droplets or liposomes). Such carriers are well known to the specialist. Pharmaceutically acceptable excipients are discussed, for example, in Gennaro, 2000, Remington: The Science and Practice of Pharmaceutics, 20thedition, ISBN:0683306472.

The compositions of this invention can be liofilizirovannami or may be in aqueous form, i.e. in the form of solutions or suspensions. Liquid preparations can preferably be entered directly from their Packed form, and they, therefore, are ideal for injection without the need for dissolution in the aqueous environment, as is otherwise required for freeze-dried compositions according to this invention.

Direct delivery of immunogenic compositions of the present invention to a subject can be accomplished by parenteral administration (intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, by or in the interstitial space of a tissue); or by rectal, oral, vaginal, local, transdermal, by intranasal, ocular, aural, pulmonary or other is the introduction through the mucous membranes. In the preferred embodiment of the parenteral administration is an intramuscular injection, for example, in the thigh or upper arm of the subject. The injection can be done with a needle (for example, hypodermal needle), but you can use needleless injection. Typical intramuscular dose of 0.5 ml of the compositions of this invention can be obtained in various forms, for example in the form of injection or in the form of liquid solutions or suspensions. In certain embodiments the composition can be prepared in the form of a powder or spray for pulmonary administration, for example in the inhaler. In other embodiments the composition can be prepared in the form of a suppository or pessary, or for nose, ear or eye of the introduction of, for example, in the form of a spray, drops, gel or powder.

The amount of conjugate to each dose of the immunogenic composition is chosen as the amount that induces a protective immune response without significant side effects. This number can vary depending on staphylococcal serotype. Each dose contains from 0.1 to 100 µg of polysaccharide, in particular from 0.1 to 10 μg, and more specifically from 1 to 5 micrograms. The optimal number of components for a specific immunogenic compositions can be clarified by standard studies involving observation of appropriate immune responses in subjects. And after the initial vaccination, subjects may receive one or several booster immunizations through appropriate intervals.

Packaging and dosage forms

Immunogenic compositions according to this invention can be packaged in single dose form or in mnogochasovoj form (for example, 2 doses 4 doses or more). For parenteral multi-dose forms vials are typical, but not necessarily more desirable than syringes. Suitable mnogorazovye formats include, without limitation, by them, as follows: from 2 to 10 doses per container at 0.1-2 ml per dose. In certain embodiments, the dose is a dose of 0.5 ml. Cm., for example, international patent application WO 2007/127668, which is incorporated here by reference.

The composition can be supplied in vials or other suitable containers for storage, or they can be provided in pre-filled devices for delivery, for example in one - or multicomponent syringe, and the syringe may or may not be equipped with needles. Syringe, typically, but not necessarily, contains a single dose containing preservative immunogenic composition according to this invention, although it is also provided pre-filled syringes in many doses. Similarly, the vial may contain one dose, but may also contain multiple doses.

Effective dosage amounts can be set routine, but a typical dose of the composition for injection has a volume of 0.5 ml In certain embodied eniah dose is prepared for administration to a subject person. In certain embodiments, the dose is prepared for the introduction of an adult subject, teenager, teen, novice walk the child or the child (i.e. not older than one year), and in preferred embodiments to enter the injection.

Liquid immunogenic composition according to this invention are also suitable for dissolving other immunogenic compositions, which are presented in lyophilized form. If the immunogenic composition is intended for use for such dissolved immediately before use in this invention proposed a set with two or more bottles, with two or more ready-filled syringes or with one or more than one of each, and the contents of the syringe used for dissolving the contents of the vial before injection or Vice versa.

Alternatively, the immunogenic compositions of the present invention can be lyophilized and dissolved by using one of the many ways the freeze drying process, well known in this area, with the formation of dry, have a regular shape (e.g. spherical) particles, such as micropellet or microspheres having characteristics of particles, such as the average size in diameter, which can be selected and controlled by variation of the specific methods used to obtain them. Immunogen the basic composition can optionally contain adjuvant, which may possibly be made with a separate dry, have a regular shape (e.g. spherical) particles, such as micropellet or microspheres, or contained in them. In such embodiments of the present invention is additionally proposed a set with immunogenic composition comprising a first component that includes a stabilized dry immunogenic composition may additionally contain one or more than one preservative according to this invention, and the second component containing a sterile aqueous solution for dissolving the first component. In certain embodiments the aqueous solution contains one or more than one preservative and may possibly contain at least one adjuvant (see, for example, WO2009/109550 (included here by reference)).

In yet another embodiment of the container-parenteral multi-dose format selected from one or more than one group, consisting of, without restriction by them, standard laboratory glassware, flasks, chemical beakers, measuring cylinders, fermenters, bioreactors, tubes, pipettes, bags, cans, bottles, caps for bottles (for example, a rubber tube, screw cap), ampoules, syringes, two - and multi-chamber syringes, syringe stoppers, syringe pistons, rubber tubes, plastic tubes, glass tubes, cartridges and one is gas pens and the like. The container of the present invention is not limited by the materials used, and includes such materials as glass, metals (e.g. steel, stainless steel, aluminum, etc) and polymers (e.g., thermoplastics, elastomers, thermoplastic elastomers). In a particular embodiment of the container of this format is a glass vial Shota type 1 volume of 5 ml with butyl tube. The specialist must understand that the format specified above, does not constitute an exhaustive list, but merely serves as a guide for professionals on many different formats available for the present invention. Additional formats are provided for use in the present invention, can be found in the published directory of suppliers and manufacturers of laboratory equipment, such as United States Plastic. (Lima, OH), VWR.

Methods of obtaining immunogenic conjugates

The present invention also includes methods of obtaining described here immunogenic conjugates. Methods of obtaining immunogenic conjugates of this invention include covalent conjugation of capsular polysaccharides to protein carriers by means of the conjugation chemistry involving CDI (1,1-carbonyldiimidazole), CDT (1,1-carbol-di-1,2,4-triazole) or PDPH (3-(2-pyridyldithio)-propenylidene).

Accordingly, the water of the embodiments of this invention, a method is proposed based on the CDT to obtain immunogenic conjugate, containing capsular polysaccharide S.aureus serotype 5 or 8, conjugated with protein a media, comprising the stage of: (a) obtaining compounds of capsular polysaccharide S.aureus serotype 5 or 8 with imidazole or triazole with getting compounded polysaccharide; (b) interaction compounded polysaccharide with CDT in an organic solvent containing from about 0.1% to about 0.3% (wt./about.) water, with the production of activated capsular polysaccharide serotype 5 or 8; (C) purifying the activated capsular polysaccharide serotype 5 or 8 with obtaining purified activated capsular polysaccharide serotype 5 or 8; (d) interaction of purified activated capsular polysaccharide serotype 5 or 8 with protein carrier in an organic solvent to obtain the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier; and (d) hydrolysis of the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier to remove unreacted activation groups; by which are immunogenic conjugate containing capsular polysaccharide S.aureus serotype 5 or 8 conjugated with protein carrier. In one of the embodiments of the prior stage (g) is purified activated capsular polysaccharide serotype 5 or 8 compounding with protein carrier.

In one of embodiments e is th invention is proposed another method of obtaining immunogenic conjugate, containing capsular polysaccharide S.aureus serotype 5 or 8, conjugated to protein carrier, based on the CDT, which includes stages: (a) compounding capsular polysaccharide S.aureus serotype 5 or 8 with imidazole or triazole with getting compounded polysaccharide; (b) interaction compounded polysaccharide with CDT in an organic solvent containing from about 0.1% to about 0.3% (wt./about.) water, with the production of activated capsular polysaccharide serotype 5 or 8; (C) interaction of activated capsular polysaccharide serotype 5 or 8 with protein carrier in an organic solvent to obtain the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier, and (g) hydrolysis of the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier to remove unreacted activation groups; by which are immunogenic conjugate containing capsular polysaccharide S.aureus serotype 5 or 8, conjugated to protein carrier.

In one of the embodiments of the organic solvent in the methods of obtaining immunogenic conjugate based on the CDT is a polar aprotic solvent. In one of the embodiments of the organic solvent is a polar aprotic solvent selected from the group consisting of dimethyl sulfoxide (MSO), of dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone and hexamethylphosphoramide (NMRA). In one of the embodiments of the organic solvent is a DMSO.

In one of the embodiments of the phase interaction compounded polysaccharide with CDT in the methods of obtaining immunogenic conjugate based on CDT includes providing approximately 20-fold molar excess CDT compared with the polysaccharide.

In one of the embodiments of the cleaning stage activated capsular polysaccharide serotype 5 or 8 ways to get immunogenic conjugate based on CDT includes diafiltration.

In one of the embodiments of the protein carrier in the methods of obtaining immunogenic conjugate based on the CDT is a CRM197. In one of the embodiments of the activated capsular polysaccharide serotype 5 or 8 ways to get immunogenic conjugate is subjected to interaction with CRM197mass ratio of about 1:1.

In one of the embodiments of the phase hydrolysis of conjugate polysaccharide serotype 5 or 8 with protein carrier to remove unreacted activation groups in ways of producing immunogenic conjugate based on CDT includes dilution buffer and maintaining a pH of from about 8.8 to about 9.2 deaths for at least 4 hours at a temperature of from about 20°to about 26°C. In one of the embodiments of tadia hydrolysis of the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier includes dilution buffer and maintaining a pH of about 9.0 in for at least 4 hours at a temperature of about 23°C.

In one of the embodiments of the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier obtained in accordance with methods of obtaining immunogenic conjugate based on the CDT, is subjected to cleaning. In one of the embodiments of the purification of the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier includes diafiltration.

In one of the incarnations before interaction compounded polysaccharide with CDT in the methods of obtaining immunogenic conjugate based on CDT compounded polysaccharide serotype 5 or 8 lyophilizer and resuspending. In one of the embodiments as compounded polysaccharide and protein carrier separately lyophilizer and resuspending before interaction compounded polysaccharide with CDT. In one of the embodiments of the dried compounded polysaccharide and/or liofilizirovanny protein carrier resuspended in an organic solvent. In one of the embodiments of the organic solvent is a DMSO.

In one of the incarnations before the interaction of activated capsular polysaccharide serotype 5 or 8, compounded with protein carrier, in the method of obtaining immunogenic conjugate based on CDT purified activated capsular polysaccharide serotype 5 or 8 and the protein carrier separately lyophilizer and resuspending. In the bottom of the embodiments the protein carrier is a CRM 197and before lyophilization CRM197put diafiltration against NaCl. In one of the incarnations before lyophilization CRM197put diafiltration against NaCl mass ratio of NaCl/CRM is brought to a value from about 0.5 to about 1.5.

In one of the embodiments of the present invention, a method for obtaining immunogenic conjugate containing capsular polysaccharide S.aureus serotype 5 or 8, conjugated to protein carrier, on the basis of PDPH, which includes stages: (a) interaction of capsular polysaccharide S.aureus serotype 5 or 8 with PDPH and a carbodiimide in an organic solvent to obtain PDPH-linked polysaccharide; (b) interaction of PDPH-linked polysaccharide with a reducing agent to obtain an activated polysaccharide; (C) purifying the activated capsular polysaccharide serotype 5 or 8 with obtaining purified activated capsular polysaccharide serotype 5 or 8; (d) interaction of protein media bromoxynil acid in an organic solvent to obtain activated protein carrier; (d) purification of activated protein carrier with obtaining purified activated protein carrier; (e) interaction of purified activated capsular polysaccharide serotype 5 or 8 with purified activated protein carrier of getting capsular conjugate of Polish the IDA serotype 5 or 8 with protein carrier; and (g) hydrolysis of the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier to remove unreacted activation groups; by which are immunogenic conjugate containing capsular polysaccharide S.aureus serotype 5 or 8, conjugated to protein carrier.

In one of the embodiments brooksyne acid used in the methods of obtaining immunogenic conjugate on the basis of PDPH is a N-hydroxysuccinimidyl ether bromoxynil acid (BAANS). In one of the embodiments of the protein carrier used is based on PDPH methods according to this invention, is a CRM197and BAANS add mass ratio of CRM197:BAANS from about 1:0.1 to about 1:0.5 in.

In one of the embodiments of the organic solvent-based PDPH ways of producing immunogenic conjugate is a polar aprotic solvent. In one of the embodiments of the organic solvent is a polar aprotic solvent selected from the group consisting of DMSO, DMF, dimethylacetamide, N-methyl-2-pyrrolidone, NMRA. In one of the embodiments of the organic solvent is a DMSO. In one of the embodiments of carbodiimide used based on PDPH ways of producing immunogenic conjugate, is a 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDAC). In one of the embodiments of the phase interaction capsular polysaccharide serotype 5 or 8 with PDPH and EDAC in the organic solvent includes maintaining the mass ratio of the polysaccharide:PDPH:EDAC equal to about 1:5:3.

In one of the embodiments of the reducing agent used in based on PDPH ways of producing immunogenic conjugate is a dithiothreitol (DTT).

In one of the embodiments, each cleaning stage activated capsular polysaccharide serotype 5 or 8 and protein purification media based on PDPH ways of producing immunogenic conjugate comprises diafiltration.

In one of the embodiments of the protein carrier-based PDPH ways of producing immunogenic conjugate is a CRM197. In one of the embodiments of the activated polysaccharide serotype 5 or 8 in these methods of obtaining immunogenic conjugate is subjected to interaction with CRM197mass ratio of about 1:1.

In one of the embodiments of the phase hydrolysis of conjugate polysaccharide serotype 5 or 8 with protein carrier to remove unreacted activation groups based on PDPH ways of producing immunogenic conjugate comprises adding hydrochloride group probably facilitates.

In one of the embodiments of the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier obtained in accordance with the core is private on PDPH ways of obtaining immunogenic conjugate, purify. In one of the embodiments of the purification of the conjugate of the capsular polysaccharide serotype 5 or 8 with protein carrier includes diafiltration.

In one of the incarnations before interaction of purified activated capsular polysaccharide serotype 5 or 8 with purified activated protein carrier-based PDPH ways of producing immunogenic conjugate as purified activated polysaccharide and purified activated protein carrier separately lyophilizer and resuspending. In one of the embodiments of the dried activated polysaccharide and/or dried activated protein carrier resuspended in an organic solvent. In one of the embodiments of the organic solvent is a DMSO.

When used here "lyophilization" means the method of dehydration, in which the bacterial capsular polysaccharide is frozen and ambient pressure reduces in the presence of sufficient heat to provide a sublimation of the frozen water directly from the solid phase into the gas phase. You can use any method known in this field for lyophilization of polysaccharides. See, for example, Harris & Angal (1989) "Protein Purification Methods," in: Kennedy & Cabral, eds. "Recovery Processes for Biological Materials (John Wiley & Sons; 1993); U.S. patent No. 4134214; and the publication of the international application to the PA is UNT WO 2003/086471; each of which is incorporated here by reference as if reproduced in full. Perhaps during lyophilization can be used cryoprotector, such as, for example, sucrose, glucose, lactose, trehalose, arabinose, xylose, galactose, sorbitol or mannitol.

When used here "activate" and "activation" means that the bacterial capsular polysaccharide or protein carrier modify so that it is "likely" to conjugation (i.e. at least one grouping should be done capable of covalently to contact the media molecule). For example, with regard to methods based on CDT conjugation of the present invention, the polysaccharide activate in an environment with low humidity (for example, in DMSO) with the formation of triazolinone groups with available hydroxyl and illtreating groups with carboxylic acids. Activated polysaccharides can then be subjected to interaction with protein CRM197that leads to nucleophilic substitution triazole lysine residues in CRM197and the formation of urethane linkages (for activated hydroxyl and amide linkages (for activated carboxylic acids). On the contrary, with regard to based on PDPH methods of conjugation of the present invention, as a protein carrier, and polysaccharides before conjugation is kiviruut as follows: (1) activation CRM 197includes an introduction bromoacetyl groups in protein CRM197the interaction of the amine groups with N-hydroxysuccinimidyl ether bromoxynil acid; and (2) activation of the polysaccharide includes a combination activated by carbodiimide carboxylate groups of the N-acetylaminophenol acid in the polysaccharide with a hydrazide group sulfhydryl-reactive hydrazide heterobifunctional linker PDPH, followed by reduction with DTT. Activated polysaccharides can then be subjected to interaction with activated protein carrier so that the thiols PDPH-etiolirovannyh polysaccharides interact with bromacetyl groups activated protein carrier with the formation of covalent thioester bond formed by the substitution of the bromide.

In accordance with the methods according to this invention capsular polysaccharides, proteins carriers and/or conjugates polysaccharide-protein can be purified. Can be used with any method known in this field for the purification of polysaccharides or proteins, such as concentration/diafiltration, sedimentation/elution, column chromatography and filtration through a surround filter. See, for example, Farres et al. (1996) Biotechnol. Tech. 10:375-380; Goncalves et al. B: Communicating Current Research and Educational Topics and Trends in Applied Microbiology (Antonio Mendez Vilas, ed. 1sted. Badajoz, Espanha: Formatex; 2007, pp.450-457); Tanizki et al. (1996) J.Microbiol. Methods 27:19-23; and U.S. patent No. 6146902 and publication of an application for U.S. patent No. 2008/0286838; each of which is incorporated here by reference as if reproduced in full.

When used herein, the term "isolated" or "purified" means that the substance is removed from the environment of its origin (e.g., the natural environment if it occurs in nature, or from the body of its owner, if it is recombinant, or transferred from one environment to another environment). For example, the selected polysaccharide, peptide or protein is substantially free of cellular material or other contaminating proteins from the cell source or tissue source from which derived protein or essentially free from chemical precursors or other chemicals, if it is chemically synthesized or is otherwise in the mix as part of a chemical reaction medium. In the present invention the protein or polysaccharide can be isolated from a bacterial cell or from the cell residues so that they were presented in a form suitable for receiving the immunogenic composition. The term "isolated" or "allocation" may include cleaning or purification procedures, including, for example, the methods described here purification of capsular polysaccharides. The expression "essentially free of cellular material" on which includes drugs polysaccharide/polypeptide/protein, in which the polysaccharide/polypeptide/protein is separated from cellular components of the cells from which it emit or receive recombinant method. Thus, the polypeptide/protein or polysaccharide conjugate, which is essentially free of cellular material or other compounds includes preparations of the polypeptide/protein or polysaccharide conjugate, with less than about 30%, 20%, 10%, 5%, 2,5% or 1% (by dry weight) contaminating protein, polysaccharide, or other compounds. When the polypeptide/protein obtained by recombinant means, it also preferably is essentially free of culture medium, i.e., culture medium is less than about 20%, 10%, 5%, 4%, 3%, 2% or 1% of the volume of the protein drug. When the polypeptide/protein or polysaccharide obtained by chemical synthesis, it is preferably essentially free from chemical precursors or other chemicals, that is, it is separated from chemical precursors or other chemicals which are involved in the synthesis of protein or polysaccharide. Accordingly, such preparations of the polypeptide/protein or polysaccharide have less than about 30%, 20%, 10%, 5%, 4%, 3%, 2% or 1% (by dry weight) of chemical precursors or compounds other than the polypeptide/protein or polysaccharide fragment of interest.

The immunogen is haunted conjugates, obtained by any method described here, can be stored in water or a buffer with low ionic strength and neutral pH or liofilizirovanny in the dry powder.

Methods induction of immune response and protection against infection with S.aureus

The present invention also includes methods of application described here immunogenic compositions. For example, in one of the embodiments of this invention, a method of inducing an immune response against S.aureus, including the introduction of a subject immunogenic amount of any described here immunogenic composition. In one of the embodiments of this invention, a method for protecting a subject against infection with S.aureus or way to prevent infection with S.aureus, or a method of reducing the severity, or delaying the start of the occurrence of at least one symptom associated with infection, which is caused by S.aureus, including the introduction of a subject immunogenic amount of any described here immunogenic composition. In one of the embodiments of this invention, a method for treating or preventing staphylococcal infection, disease or condition associated with Staphylococcus sp. the subject, including the stage of introducing a therapeutically or prophylactically effective amounts described herein immunogenic composition to the subject. In some embodiments the method of treating or preventing taphylococcus infection, disease or condition includes treatment of human and veterinary treatment, treatment of animal or crop treatment. In another embodiment, a method for treating or preventing staphylococcal infection, disease or condition associated with Staphylococcus sp. for a subject, comprising obtaining a preparation of polyclonal or monoclonal antibodies described here immunogenic compositions and use of the specified drug antibodies to create passive immunity in the subject. In one of the embodiments of this invention, a method for prevention of staphylococcal infection in a subject undergoing a surgical procedure, comprising the stage of introduction of a prophylactically effective amounts described herein immunogenic composition to the subject before the surgical procedure.

"Immune response" to an antigen or immunogenic composition is a development of the subject of a humoral and/or cell-mediated immune response to molecules present in the antigenic or vaccine compositions of interest. For the purposes of the present invention "humoral immune response" is mediated by antibodies of the immune response and includes the induction and generation of antibodies that recognize the antigen in the immunogenic composition according to this izopet the tion, contact him with a certain affinity, whereas cell-mediated immune response" is a response mediated by T-cells and/or other white blood cells. "Cell-mediated immune response" is called by the presentation of antigenic epitopes in Association with molecules of the major histocompatibility complex (MHC) Class I or Class II, CD 1, or with other non-classical MHC-like molecules. This activates antigen-specific T-helper cells CD4+) or cytotoxic T-lymphocytes CD8+ ("CTL"). CTL have a specificity for peptide antigens that are presented in Association with the protein encoded by classical or non-classical MHC and expressed on the cell surface. CTL helps to induce and activate intracellular degradation of intracellular microbes or lysis of cells infected with these germs. Another aspect of cellular immunity includes the antigen-specific response helper T cells. Helper T-cells function, helping to stimulate the functions and direct the activity of nonspecific effector cells against cells expressing the peptide or other antigens in Association with classical or non-classical MHC molecules on their surface. "Cell-mediated immune response" also refers to the production of cytokines, chemokine and other such molecules, produced by activated T-cells and/or other white blood cells, including those that originate from T-cells CD4+ and CD8+. The ability of a particular antigen or composition to stimulate cell-mediated immunological response may be determined by numerous analyses, such as analyses of lymphoproliferation (lymphocyte activation)assays of cytotoxic cells in CTL assays for T-lymphocytes specific for the antigen in sensitized subject, or by measuring the production of cytokines by T-cells in response to re-stimulation with antigen. Such analyses are well known in this field. See, for example, Erickson et al. (1993) J.Immunol. 151: 4189-4199; and Doe et al. (1994) Eur. J.Immunol. 24:2369-2376.

When used here "treatment" (including its variations, for example, "treat" or "treated") means any one or more of the following: (1) prevention of infection or re-infection, as with traditional vaccine, (2) reducing the severity or elimination of symptoms, and (3) the substantial or complete elimination of this pathogen or violations. Therefore, the treatment can be performed prophylactically (prior to infection) or therapeutically (after infection). In the present invention prophylactic treatment is preferred. According to one of specific embodiments of the present invention p is adlouni compositions and methods who care, including preventive and/or therapeutic immunization, the animal host from microbial infection (e.g., bacteria, such as Staphylococcus). The methods of the present invention are useful for making preventive and/or therapeutic immunity to a subject. The methods of the present invention can also be practiced on the subjects for biomedical research applications.

When used here "mammal" means any person or animal, not a person. More specifically, a mammal refers to any animal classified as a mammal, including humans, domestic and farm animals, and animals kept in zoos, sports and Pets, such as living in the house and other domesticated animal, including, without limiting them, cattle, sheep, ferrets, pigs, horses, rabbits, goats, dogs, cats and the like. Preferred animals home are dogs and cats. Preferably, the mammal is man.

The terms "immunogenic amount" and "immunologically effective amount"used interchangeably, refers to the amount of antigen or immunogenic composition sufficient to eleirovania immune response or a cellular (T-cell)or humoral the tion (b-cell or antibody-based test or) reply or both when measured by standard assays known to the expert.

The number of specific conjugate in the composition is usually calculated by the total polysaccharide, anywherefrom and unconjugated, for a given conjugate. For example, the conjugate SR with 20% of free polysaccharide should have about 80 μg of conjugated polysaccharide SR and about 20 μg unconjugated polysaccharide SR at a dose of 100 µg of polysaccharide SR. The contribution of protein in the conjugate is typically not considered when calculating the dose of the conjugate. The number of conjugate can vary depending on staphylococcal serotype. Each dose should contain from 0.1 to 100 µg of polysaccharide, specifically from 0.1 to 10 μg, and more specifically, from 1 to 10 mcg. "Immunogenic amount" of different polysaccharide components in the immunogenic composition can vary, and each can be 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 mcg, 15 mcg, 20 mcg, 30 mcg, 40 mcg, 50 mcg, 60 mcg, 70 mcg, 80 mcg, 90 μg, or about 100 μg any specific polysaccharide antigen.

"Invasive disease" S.aureus is a selection of bacteria from the area, which normally is sterile, where there are clinical signs/symptoms associated with the disease. Sterile normal areas of the body include blood, cerebrospinal fluid (CSF), plait the General liquid pericardial fluid, peritoneal fluid, joint/synovial fluid, bone, internal organs (lymph node, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, ovary) or other areas, sterile normal. Clinical condition characterizing invasive disease include bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis, septic shock and other.

The efficiency of antigen as the immunogen can be measured either by proliferation assays, or cytolysis assays, such as assays for the release of chromium to measure the ability of T cells to lyse its specific the target cell, or by measuring the levels of b-cell activity by measuring the levels of circulating antibodies specific for the antigen in the serum. The immune response can also be detected by measuring levels of antibodies specific to the antigen in the serum, which is induced after injection of antigen, and, more specifically, by measuring the ability of the antibodies induced thus to strengthen opsonophagocytic the ability of specific white blood cells, as described herein. The security level of the immune response can be measured by the effect on the immunized host antigen that was entered. For example, if the antigen against which desire is Elen immune response, is a bacterium, the level of protection induced immunogenic amount of the antigen is measured by determining the survival rate or percent mortality after exposure to bacterial cells in animals. In one of the embodiments of the amount of protection can be assessed by measuring at least one symptom associated with bacterial infection, such as fever associated with infection. The amount of each antigen in multiantigenic or multicomponent vaccine or immunogenic composition will vary with respect to each other component, and it can be determined by methods known to the expert. Such methods may include procedures for measuring the immunogenicity and/or efficacy in vivo.

In certain embodiments, the term "about" means within 20%, preferably within 10%, and more preferably within 5%.

This invention additionally proposed antibodies or antibody-based test and compositions that bind specifically and selectively with capsular polysaccharide serotype 5 or 8 or immunogenic conjugates of the present invention. In some embodiments the antibodies are formed by the introduction of the subject of the capsular polysaccharide serotype 5 or 8 or immunogenic conjugates of the present invention. Some in the lewinian in this invention proposed peeled or dedicated antibodies directed against one or more than one capsular polysaccharide serotype 5 or 8 or immunogenic conjugate according to the present invention. In some embodiments the antibodies of the present invention are functional when measuring for killing bacteria or model of efficiency on the animal, or analysis opsonophagocytosis killing. In some embodiments the antibodies according to this invention give the subject of passive immunity. The present invention additionally proposed polynucleotide molecule encoding the antibody or antibody fragment according to this invention, the cell, cell line (such as hybridoma cells or other engineered cell lines for recombinant production of antibodies) or transgenic animal to produce the antibody or antibody-based test or a composition of this invention using techniques well known to specialists in this field.

Antibodies or antibody-based test or the compositions of this invention can be used in a method of treatment or prevention of staphylococcal infection, disease or condition associated with Staphylococcus sp., the subject, including the generation of the preparation of polyclonal or monoclonal antibodies and the use of such antibodies or antibody-based test or compositions to ensure Passy the aqueous immunity in the subject. Antibodies according to this invention may also be useful for diagnostic methods, such as detecting the presence or quantifying levels SR, SR or its conjugate.

To assess the effectiveness of any described here immunogenic compositions can be used several models on the animal known in this field. For example, the following.

The passive model of sepsis in mice. Mice passively subjected to immunization intraperitoneal (I.P. Pavlova.) introduction immune IgG or monoclonal antibodies. Mice 24 hours after impact with a lethal dose of S.aureus. Bacterial dose administered intravenously (i.v. or I.P. Pavlova.), being sure that any survival can be attributed to the specific interaction of the antibody with the bacteria in vivo. The dose of bacterial contamination is defined as the dose required to achieve lethal sepsis in about 20% not immunized control mice. Statistical evaluation research survival is possible to carry out the analysis by Kaplan-Meier.

The model of active immunization and bacteria. In this model, mice are subjected to active immunization subcutaneously (s.c.) when using the target antigen at time 0, 3 and 6 weeks (or in a similar mode, known in the art) and exercise control S.aureus infection in the period of 8 weeks (or in another similar mode, known the nom specialists) intravenous or intraperitoneal route. The dose of bacterial contamination are calibrated to achieve approximately 20% survival in the control group during the 14-day period. Statistical evaluation research survival is possible to carry out the analysis by Kaplan-Meier.

The passive model of infective endocarditis. The model of passive immunization against infectious endocarditis (IE)caused by S.aureus, used previously to show that protective immunity can be induced Clfa. See, Vernachio et al. (2006) Antimicro. Agents & Chemo. 50:511-518. In this model, IE used rabbits or rats to simulate clinical infections, including Central venous catheter bacteremia and hematogenous migration in distal organs. Catheterized rabbits or rats with sterile enlargement of the aortic valve did a single intravenous injection of monoclonal or polyclonal antibodies specific to the target antigen. After 24 hours, the animal was injected intravenously heterologous staphylococcal strains or strain of MRSA. 48 hours after infection of the heart growths, kidney and blood were collected and cultured. Then determined the frequency of staphylococcal infections in the enlargement of the heart valve, kidney and blood. In one study, when animals affected or MRSE strain ADS 35984, or strain of MRSA PFESA0003 demonstrated signicant SN is weaker infection using drugs or polyclonal antibodies, either monoclonal antibodies against ClfA. Cm. above Vernachio et al.

The passive model of infective endocarditis. This model of infective endocarditis is also adapted for studies of active immunization. Rabbits or rats were immunized intramuscularly (i.m.) the target antigen and S.aureus infected within two weeks of injection.

The model of pyelonephritis. In the model of pyelonephritis mice subjected to immunization at time 0, 3 and 6 weeks (or in the same mode, known in the art) of the target antigens. On term of 8 weeks to infect animals, for example, intraperitoneally injection, for example, 1,7×108CFU (colony forming units) S.aureus PFESA0266. After 48 hours the kidneys and/or other tissues collected and cultivated. Finally, in the kidneys and/or other tissues counting colony-forming units introduced bacteria. This model evaluates the systemic dissemination of the animal.

Monitoring of functional antibody analysis by opsonophagocytosis killing

For this analysis, you can use differentiated effector cells of cell lines (e.g HL60) or polymorphically cells (PMN)isolated from human donor blood using solution LYMPHOLYTE®-poly (Cedariane laboratories limited, Ontario, Canada) according to the manufacturer's instructions. Effector cells resuspendable buffer for analysis (modificar the bathroom environment the Needle, containing 1% bovine serum albumin) at a concentration of about 2×107cells/ml and placed in an incubator at 37°C before use. S.aureus strain PFESA0266 were grown overnight on agar tablets trypsinogen the hydrolyzate of soybean. Bacterial cells were scraped, washed twice and resuspendable buffer for analysis containing 5% glycerin, and the optical density OD600=1, which is equal to the concentration of approximately 5×108CFU/ml. aliquots of 1 ml of bacterial suspension was frozen and stored at -40°C until use. Frozen bacterial suspension was thawed and brought to a concentration of 106CFU/ml in the buffer for analysis and placed on ice. The analysis was performed using sterile polypropylene tablets with 96 deep holes of 1 ml were Obtained twofold serial dilution of antibodies (50 μl) and then added 300 μl of buffer for analysis to the mixture with the antibody. Added to the tablets bacteria (50 μl) and placed on a rotary shaker at 4°C for 30 minutes. By stage opsonization was followed by adding 50 μl of human complement (final concentration 1%). Finally, the tablet was added 50 μl of effector cells (concentration of 107cells/ml) and well stirred suspension by repeated pipetting. Conducted 10-fold serial dilution of aliquots of 50 μl of the suspension in the sterilized the nom 1%solution of saponin, stirred on the vortex to minimize clumping of bacteria and were sown on agar with trypsinogen the hydrolyzate of soybean in two Parallels. Analysis tablet incubated at 37°C for 1 hour with continuous agitation on a rotary shaker. At the end of the incubation was performed 10-fold serial dilution of aliquots of 50 μl of the suspension into a sterile 1%solution of saponin, stir on vortex to minimize clumping of bacteria, and were sown in two Parallels on agar with trypsinogen the hydrolyzate of soybean. The percentage of killing was calculated by determining the ratio of the number of CFU surviving in the term of 60 minutes in the wells with bacteria, antibodies, complement and effector cells, and the number of CFU surviving in test tubes containing no antibodies, but containing bacteria, complement and effector cells. Controls containing bacteria, complement and serum, were considered for amendments to any reduction in CFU due to clumping.

Adsorption of complement

In this analysis as a source of complement, you can use human donor serum subjected to adsorption against S.aureus strains PFESA0266, PFESA0286 and PFESA0270. The S.aureus strains were grown overnight on TSA tablets when S. Cells were scraped from the tablet and resuspendable in sterile PBS. Bacterial cells were centrifuged at 10,000 rpm for 10 minutes is at 4°C and the cell sediment resuspendable in human serum adsorption. Serum incubated with bacteria on the rocking at 4°C for 30 minutes. Cells were centrifuged, serum was transferred to another test tube containing the bacteria, and the stage of adsorption was repeated again within 30 minutes. Finally, cells were centrifuged and missed the serum through the 0.2 micron filter, after which aliquots of 0.5 ml were frozen in liquid nitrogen.

Method II - Analysis of opsonophagocytosis (ORA) using cells HL-60

Cells HL-60 were subjected to differentiation in S.Romero-Steiner et al., Clin Diagn Lab Immunol 4 (4) (1997), pp.415-122. The collected cells HL-60 resuspendable buffer for analysis (modified atmosphere Needle containing 1% bovine serum albumin) at about 108cells/ml and placed in an incubator at 37°C before use. S.aureus were grown overnight on agar tablets trypsinogen the hydrolyzate of soybean. Bacterial cells were scraped, washed twice and resuspendable buffer for analysis containing 5% glycerin, and the optical density OD600=1, which corresponds to about 5×108CFU/ml. aliquots of 1 ml of bacterial suspension was frozen and stored at -40°C until use. Frozen bacterial suspension was thawed and brought to a concentration of 106CFU/ml in the buffer for analysis and placed on ice. The analysis was performed using sterile polypropylene Board is s with 96 deep wells in 1 ml Got a serial twofold dilution of monoclonal antibody (25 ál) and then to a suspension of antibody was added 150 μl of buffer for analysis. Added to the tablets bacteria (25 μl) and placed on a rotary shaker at 4°C for 30 minutes and then added 25 μl of human complement (final concentration 1%). Finally, added in tablet 25 μl of cells HL-60 (107cells/ml) and well stirred suspension by repeated pipetting. Conducted 10-fold serial dilution of aliquots of a suspension of 25 ál in sterile 1%solution of saponin were mixed on the vortex to minimize clumping of bacteria and were sown in two Parallels on agar with trypsinogen the hydrolyzate of soybean. Analysis tablet incubated at 37°C for 1 hour with constant mixing on a rotary shaker. At the end of the incubation was performed 10-fold serial dilution of aliquots of a suspension of 25 ál in sterile 1%solution of saponin were mixed on the vortex and were sown in two Parallels on agar with trypsinogen the hydrolyzate of soybean. The percentage loss was calculated by determining the ratio of the number of CFU surviving in the term of 60 minutes in the wells with bacteria, antibodies, complement and cells HL-60, and the number of CFU surviving in test tubes containing no antibodies, but containing bacteria, complement and cells HL-60. Controls containing bacteria complement and mAb, consideration for amendments to any reduction in CFU due to clumping.

The following examples are given to illustrate but not to limit.

EXAMPLES

Example 1: getting a capsular polysaccharide S.aureus serotype 8

This example describes various size ranges capsular polysaccharide S.aureus serotype 8. The structure of the repeating unit of the capsular polysaccharide S.aureus serotype 8 shown in figure 1. The described methods are effective in obtaining capsular polysaccharide serotype 8 with molecular masses varying in the range from about 20 kDa to 700 kDa. By proper choice of conditions it is possible to isolate and purify high molecular weight capsular polysaccharide serotype 8 in the range from 50 kDa to 700 kDa molecular weight. For use in immunogenic compositions can be selected and clear capsular polysaccharide serotype 8 in the range from 70 kDa to 300 kDa in molecular weight and in many desired ranges. On the basis of the growth characteristics and the amount of capsules to obtain capsular polysaccharide serotype 8 strains were used PFESA0005 or PFESA0286. It was shown that capsules isolated from strains PFESA0005 or PFESA0286 are identical.

To obtain capsular polysaccharide serotype 8 strains were grown in complex medium, comprising whom she principally of carbon source (either lactose, or sucrose, hydrolyzed soy flour as a source of nitrogen and trace amounts of metals. Strains were grown in bioreactors in 2-5 days.

Before autoclaving took a sample to test the level of staphylococcal enterotoxin B (SEB) in culture. In the presence of 0.05% Polysorbate 80 concentration of SEB in the fermentation product was 15-20 ng/ml Previous experiments showed that autoclaving the culture for 1 hour reduced the level of SEB to less than 0.1 ng/ml, which is below the detection limit set TECRA.

After diafiltration and fractionation ethanol polysaccharide was applied on a column of Q-Separate AES and suirable linear NaCl gradient as described above. Fractions were analyzed by O-acetyl analysis and test double immunodiffusion in the presence of polysaccharide serotype 5 and the analysis of phosphates in the presence of tahaawee acid (TA). The presence of polysaccharide serotype 8 was detected in the fractions from 35 to 95 (Figa-B).

To reduce contamination of tahaawee acid fraction from 35 to 75 were combined and all residual taikoubou acid was oxidized by metaperiodate sodium to allow its removal 3K-diafiltrate against distilled water.

Purification of capsular polysaccharide serotype 8, which is used to obtain the conjugates was carried out in two different with the people, based on the fact that high temperature and low pH affect the release of the capsule from the cell and reduce the molecular weight of the polysaccharide. The resulting molecular weight was dependent on time, temperature and pH of the hydrolysis step.

The characterization of the capsular polysaccharide serotype 8 was performed using the methods specified in Table 1.

Table 1
Characterization analyses of purified capsular polysaccharides S.aureus serotype 8
SpecificityAnalysis
Residual proteinColorimetric analysis at Lowry
Residual nucleic acidsScanning at 260 nm
The residual tagaeva acidColorimetric analysis of phosphate
Residual peptidoglycanHPAEC-PAD*
SizeSEC-MALLS**
CompositionHPAEC-PAD
Identity1H-NMR or cooperation is the interaction with

specific mAb
O-acetylation1H-NMR
ConcentrationMALLS-R1 or HPAEC-PAD
* high-performance anion-exchange chromatography with pulsed amperometric detection
** gel filtration, combined with detektirovaniem on scattering of laser light by many angles.

Capsular polysaccharides obtained by the methods described below, result in a clean well-described polysaccharides with low levels of contamination with proteins, nucleic acids, composition and tahaawi acid.

In the first method, after the release of the capsular polysaccharide from the cell and reduce the molecular weight of the product was treated with a mixture of enzymes (e.g., ribonuclease, deoxyribonuclease, lysozyme and protease) for the decomposition of impurities. After incubation, the remaining impurities are precipitated by adding ethanol (final concentration approximately 25%). After removal of residual ethanol solution containing capsular polysaccharide, were applied to the anion exchange column (Q-Sepharose) and LWIR the Wali linear salt gradient. The fractions containing capsular polysaccharide, were combined and treated with metaperiodate sodium. This treatment resulted in oxidative hydrolysis impurity, representing the residual taikoubou acid but had no effect on capsular polysaccharide serotype 8. This reaction mixture was suppressed by the addition of ethylene glycol. The substance was concentrated and subjected diafiltration against distilled water to remove any residual reactants and by-products.

The second method used to obtain the capsular polysaccharide without the use of enzymes for the degradation of different originating from cells of impurities. In this way after the release of the capsular polysaccharide from the cell and reduction of molecular weight hydrolysate fermentation broth was osvetleni by microfiltration and then by ultrafiltration and diafiltration. The solution was treated with activated charcoal to remove impurities. Substance after treatment with coal were treated with metaperiodate sodium for oxidation of the residual tahaawee acid and then extinguished by propylene glycol. The substance was concentrated and subjected diafiltration against distilled water to remove any residual reactants and by-products.

Drugs obtained using any of the methods gave pure capsular polysaccharides low the levels of impurities, representing proteins, nucleic acids and taikoubou acid. The described methods can be used for specific ranges of the desired high molecular weight polysaccharides by variation of the conditions of hydrolysis. Examples of capsular polysaccharide obtained is described here by the methods shown below in Table 2. Party purified capsular polysaccharide serotype 8 was of high purity, as evidenced by the absence of tahaawee acid (TA) and peptidoglycan and low level of residual protein (see Table 2). The range of lower molecular mass stretched from of 20.4 kDa to 65.1 kDa, and purified polysaccharides were highly O-acetylated (approximately 100%). Pollution levels nucleic acids were low (0.12 to 2,45%).

Table 2
Characteristics of the drugs capsular polysaccharide serotype 8
SampleTotal cleared SRMolecular weightProteinNucleic acidO-Acetyl
mgkDa (g/mol) Lowry, % of the mass.Scan at 260 nm, % of the mass.NMR, %
131027,01,20,94100
243829,02,42100
317920,40,370,12108

Selection of the capsular polysaccharides of molecular weight

Kinetic analysis demonstrated that described here ways you can get a wide range of molecular masses of capsular polysaccharides. The original bacterial cells were produced larger polysaccharides, and subsequently chose the desired range of molecular weight and then was purified by manipulation of pH and heating conditions in the stages of heat and hydrolysis.

Heat treatment of fermentation broth S.aureus is the stage between fermentation and extraction of the capsular polysaccharide. At this stage use heat to process the broth with the established pH within a certain lie is neither. Objectives of heat treatment at low pH are in the killing of cells, inactivation of enterotoxins, the release associated with cells of the polysaccharide and the reduction in molecular weight to the desired size. Among these tasks, the decrease in molecular weight was the slowest time required at this stage. Therefore, the time inevitably solved other tasks.

Heat treatment by heating. Was determined by pH and temperature conditions for breeding of different ranges of molecular weight capsular polysaccharides. For these studies used a Biolafitte fermenter 15L. Fermentation broth was transferred into a fermenter with a peristaltic pump. When the stirring speed of about 200 rpm brought the pH of the broth is concentrated sulfuric acid. Then the temperature of the broth was raised to the set value. The time of heat treatment was spotted as soon as the temperature reached the set point. When it was desired processing time, the broth was cooled to room temperature. During the process took samples to determine the concentration and molecular weight of the polysaccharide systems HPLC (high performance liquid chromatography) and SEC-MALLS, respectively. Data on the molecular weight (MW) used in the kinetic analysis. Profiles of molecular weight was determined with the tip the of time at pH 3.5, 4,0 and 5,0 (see Figa).

The kinetics of mild acid hydrolysis of the polysaccharides were determined using purified capsular polysaccharide serotype 8, obtained during this process. The purified polysaccharide solution brought with sulfuric acid until the desired experiment pH. Approximately 1.5 ml of solution was transferred into each centrifuge tube with a capacity of 15 ml test Tube was placed in an oil bath, equipped with precision temperature control system. Swabs were taken at certain intervals of time and put in a bucket with ice. At the end of the experiment, an aliquot of 1 M Tris buffer (pH 7.5) was added to the sample to bring the pH back up to about 7. The samples were analyzed on a system of SEC-MALLS. The molecular weight used in the kinetic analysis. The effect of temperature on the profile MW SR at pH 3.5 was determined over time (see Figb).

Results

As shown in Figa, lower pH was more effective in reducing the molecular weight of the polysaccharide. Molecular weight of between 300 kDa and 600 kDa can be obtained with the use of pH 5 at 95°C for a time between 15 minutes and 120 minutes. Similarly, the molecular weight between 250 kDa and 450 kDa can be obtained when using pH 4 at 95°C for a time between 15 minutes and 120 minutes. Moreover, the molecular weight between 120 kDa and 450 kDa can be obtained when used in the research Institute pH 3.5 at 95°C for a time between 15 minutes and 120 minutes.

As shown in Figb, the higher the temperature, the higher the rate of hydrolysis, and the wider the range of molecular mass polysaccharide obtained over time. The lowered temperature, 55°C in comparison To 95°C, with the same pH gives a more narrow range of molecular mass polysaccharide.

Moreover, Figure 4 shows the correlation between the molecular weight of purified SR and processing time in soft acidic (pH 3.5 at 95°C) hydrolysis. The purified polysaccharide is an end-product obtained during the extraction process, detailed above. As also shown in figure 4, increasing the time of heat treatment of S.aureus strain PFESA0005 at pH 3.5 leads to lower molecular weight SR, while reducing the time of heat treatment at pH 3.5 leads to higher molecular weight SR. The size of the capsular polysaccharide serotype 8 is in the range from about 80 kDa to about 220 kDa, depending on the length of time of heat treatment at pH 3.5. The correlation between the time of heat treatment at low pH and the amount of purified SR, as shown in figure 4, allows us to estimate the processing time required for the production of purified polysaccharide with a specific range of molecular weight.

It is important to note that, as demonstrated above, it is possible to obtain, extract, and cleaned the TB full range of molecular masses capsular polysaccharide serotype 8 from 20 kDa to 500 kDa. Therefore, these methods can be used for specific ranges of the desired high molecular weight capsular polysaccharides, such as are shown in Table 3. A relatively narrow range of molecular mass polysaccharide that is obtained when the peak molecular mass is in the range of 87 to 108 kDa kDa, is a well-characterized molecular mass range, which you can get ways described here. Particularly preferred ranges of high molecular weight polysaccharides, in the range from 70 kDa to 300 kDa or 70 kDa to 150 kDa, are useful in obtaining immunogenic compositions conjugation of capsular polysaccharide with media molecule or protein (see table 3). The conditions used to obtain the capsular polysaccharide SR, having a range of molecular weights from about 80 to 120 kDa, are as follows: 95°C, pH 3.5 for 300 minutes.

89
Table 3
Obtaining a specific range of high molecular weight capsular polysaccharide serotype 8
RunMW capsular polysaccharide serotype 8 (kDa)
198
2
3108
4108
589
6100
199
2113
3105
4100
587

Example 2: Conjugation of capsular polysaccharide serotype 8 with CRM197

This example describes the methods and analyses for charactistic used in obtaining conjugates of capsular polysaccharide S.aureus serotype 8 with CRM197. Different chemical methods of conjugation were developed for conjugation of capsular polysaccharide S.aureus serotype 8 with the protein carrier. For example, conjugation using PDPH (3-(2-pyridyldithio)-propenylidene) is the result of a covalent thioester linkage between the CF and the protein carrier. Alternatively, conjugation using CDI/CDT (1,1-carbonyldiimidazole/1,1-carbol-di-1,2,4-triazole) is the result of one-or zero-carbon linker between the CF and the protein carrier.

The conjugation of capsular the th polysaccharide serotype 8 with CRM 197using PDPH

The method of chemical conjugation using PDPH is sequential and involves activation of the polysaccharide, remove tylenol protective group, treatment of intermediate activated polysaccharide, activation and protein purification CRM197and conjugation of activated components and subsequent cleaning. After the introduction of a linker containing Tilney group in the polysaccharide and halogenoacetyl group in a protein carrier CRM197capsular polysaccharide S.aureus serotype 8 was associated with protein carrier via a thioester bond. Bromacetyl group were injected in protein CRM197by reacting amine groups with N-hydroxysuccinimidyl ether bromoxynil acid. To obtain tarirovannogo polysaccharide activated by carbodiimide carboxylate group of the N-acetylanthranilic acid in the polysaccharide was subjected to combination with the hydrazide group sulfhydryl-reactive hydrazide heterobifunctional linker 3-(2-pyridyldithio)-propionitrile (PDPH). Thiols PDPH-tarirovannogo polysaccharide obtained by reduction with DTT and purified by SEC on a column of Sephadex G25, brought into interaction with bromacetyl groups activated protein that leads to the formation of covalent thioester linkages in the bromine substituted the I between the polysaccharide and the protein carrier. Unreacted bromacetyl group would cover hydrochloride group probably facilitates (hydrochloride of 2-aminoethanethiol). Then the reaction mixture was concentrated and subjected diafiltration. The remaining disconjugacy bromacetyl group would cover hydrochloride group probably facilitates to ensure the absence of reactive bromoacetyl groups remaining after conjugation. This gave covalent bond between tilenum end group probably facilitates and acetyl group on the lysine residue after bromine substitution.

1. Milirovanie capsular polysaccharide S.aureus gray/pool 8 using PDPH. The polysaccharide is first activated by milirovanie with PDPH. The polysaccharide was mixed with their mother liquor PDPH (250 mg/ml in DMSO), uterine EDAC solution (90 mg/ml in distilled water) and mother liquor MES buffer (0.5 M, pH 4,85) to obtain the final solution of 0.1 M MES, and 2 and 4 mg polysaccharide/ml while maintaining the mass ratio of the polysaccharide: PDPH:EDAC 1:0,6:1,25. This mixture is incubated for 1 hour at room temperature and then deliberately against a 1000-fold volume of distilled H2About four times, using the device for dialysis 3500 M WCO, at a temperature between 4°C and 8°C to remove unreacted PDPH. PDPH-linked polysaccharide was treated with 0.2 M DTT and incubated at room temperature for hours or during the night when the temperature is between 4°C and 8°C. Excess DTT, as well as by-products of this interaction was separated from the activated saccharide by SEC using a resin Sephadex G25 and distilled water as the mobile phase. Fractions were analyzed using dicipline (DTDP) for tylnej groups and combined thiol-positive fraction, which was loirevalley close to the "free volume" column. Pooled fractions were analyzed using hydrazide-parahydroxybenzoic acid (RANMAN) and tests for O-acetyl to determine the degree of activation, which was expressed as the molar percentage of the repeating units containing Tilney group (molar concentration of thiols/molar concentration of repeating units). The activated polysaccharide liofilizirovanny and kept at -25°C prior to conjugation.

Results reproducibility of etiolirovaniya polysaccharide serotype 8 using PDPH shown in Table 4. The degree of activation of the polysaccharide serotype 8 was in the range of from 12% to 16%, which corresponds to a range from about one attached to the linker molecules ten repeating units of capsular polysaccharide to one molecule of linker five repeating units.

Table 4
The study is espressamente activation capsular polysaccharide serotype 8 using PDPH
PDPH-polysaccharide serotype 8Activation(% MSH/MEN)Scale mgOutput, mg (wt. -%)
1143630 (83)
2163027 (91)
4163842 (110)
5124044 (110)

2. Activation of the protein carrier. Protein carrier separately activated by bromotetradecane. CRM197was diluted to 5 mg/ml in 10 mm phosphate buffered with 0.9%NaCl, pH 7 (PBS) and then brought to 1 M fallopian solution of 0.1 M NaHCO3at pH 7.0. Was added N-hydroxysuccinimidyl ether bromoxynil acid (BAANS) in a mass ratio of CRM197:BAANS 1:0.25 mass., using the mother solution 20 mg/ml BMNS in DMSO. This reaction mixture is incubated at a temperature between 4°C and 8°C for 1 hour, then was purified using SEC on Sephadex G-25. Purified activated CRM197analyzed by the method of Lowry to determine the concentration of protein and then Rabaul is whether PBS to 5 mg/ml Sucrose was added as a cryoprotectant to 5% wt./about. and the activated protein was frozen and stored at -25°C until they are needed in conjugation

Bromotetradecane lysine residues CRM197it was very stable and led to activation of 19-25 lysine 39 available lysine (see table 5). This interaction gave high outputs activated protein.

Table 5:
The outputs and the degree of bromotetradecane CRM197
MedicationActivated lisini (n=)Scale (mg)Output (% wt.)
1242385
2203887

3193577
4223594
523 3587
62548104

3. The reaction mix. Immediately upon receipt of activated capsular polysaccharide and activated protein carrier they were combined in the reaction mixture for conjugation. Dried and etiolirovannye polysaccharide was dissolved in 0.16 M borate at pH of 8.95 and mixed with thawed bromotetradecane CRM197and distilled water to obtain the final solution of 0.1 M borate and 1 mg/ml of polysaccharide with a ratio of CRM197: polysaccharide 1:1 by weight. This mixture is incubated at room temperature for 16-24 hours. Unreacted bromacetyl groups on the protein "covered" by adding hydrochloride group probably facilitates in relation CRM197: group probably facilitates 1:2 (by weight), using the mother liquor group probably facilitates 135 mg/ml, dissolved in 0.1 M borate at pH of 8.95 and incubated for 4 hours at room temperature. The conjugate of the capsular polysaccharide from CRM197(conjugate) was purified 50-fold diafiltration against 0.9%NaCl, using polyethersulfone the 100K ultra-filter.

Studies on the reproducibility of etiolirovaniya capsular polysaccharide serotype 8 using PDPH demonstrated that the degree of activation policies is reed was in the range of 12% to 16%, that corresponds to approximately one attached to the linker molecule to ten polysaccharide repeating units of one molecule of linker five repeating units.

The conjugation of capsular polysaccharide serotype 8 with CRM197through CDI/CDT conjugation chemistry

CDI and CDT provide one-step method of conjugation, in which the polysaccharide activate in an anhydrous environment (DMSO) with the formation of the imidazole or triazolinone groups with available hydroxyl and elliminating or illtreating groups with carboxylic acids. Adding protein carrier (DMSO) leads to nucleophilic substitution of the imidazole or triazole lysine and the formation of urethane linkages (for activated hydroxyl and amide linkages (for activated carboxylic acids).

Methods of conjugation with CDI and CDT give capsular polysaccharide serotype 8, covalently linked to a protein carrier, as evidenced by the presence of sugars and protein in the fractions with gel filtration and amino acid analysis of the conjugate, covered by glycolaldehyde or hydrochloride group probably facilitates.

Summary results for several parties conjugates produced by as PDPH and CDI/CDT chemistry, for a range of sizes capsular polysaccharide serotype 8 from 20 kDa to 40 kDa, showing the and in Table 6 below. Significant differences in free capsular polysaccharide, the ratio of polysaccharide-protein and outputs conjugates obtained by these two methods of conjugation, was not. Antigenicity conjugated capsular polysaccharide serotype 8 was not altered by conjugation, as shown by the identity of the lines precipitation conjugates and native polysaccharide.

Table 6
Characterization of conjugates of capsular polysaccharide serotype 8 with CRM197obtained in two ways conjugation
MethodThe output of the polysaccharide (%)The protein yield (%)The ratio of the outputFree sugar (%)Free protein (%)Modified LisinaSize (MW or kDa (%0,3), saccharide/protein
CDI/CDT46-6254-550,8-0,922-25<17-834/57 to 60/57
PDPH34-70 61-830,6-0,915-41NO11-1674-92%

As shown above, the described methods can be used for specific ranges of the desired high molecular weight capsular polysaccharides. The authors wanted to obtain the conjugates of the pre-selected range of high molecular weight capsular polysaccharide serotype 8, which can be filtered and cleaned for use in immunogenic compositions. Table 7 summarizes the analysis of conjugates of capsular polysaccharide serotype 8 capsular polysaccharide serotype 8 molecular weight was in the range of from about 80 kDa to 120 kDa and was used way imidazole conjugation. The molecular weight of the obtained conjugates ranged from 595 kDa to 1708 kDa. The number of conjugated lysine on CRM197ranged from a high 9 to low - 3. The content of free capsular polysaccharide ranged from high (6%) to low (2%).

tr>
Table 7
Compared with the pre-selected ranges of molecular weight capsular polysaccharide serotype 8
ImplementationMW of the polymer (kDa)Output (%)Free sugars (%)Molecular weight by SEC-MALLS (kDa)Modified Lisina
1998869434
21137358413
31057937197
41008626309
5879035956

Both conjugation give capsular polysaccharide serotype 8, covalently linked to a protein carrier. Significant differences on the empty capsular polysaccharide, sootnosheniyami polysaccharide serotype 8 and protein and outputs conjugates, get these two ways, was not.

Example 3: Method using CDI/CDT in the same capacity in comparison with the complex way

As described above, methods of obtaining immunogenic conjugates of this invention include covalent conjugation of capsular polysaccharides to protein carriers using conjugation chemistry, including CDI (1,1-carbonyldiimidazole), CDT (1,1-carbol-di-1,2,4-triazole) or PDPH (hydrazide 3-(2-pyridyldithio)-propionyl). Using CDI/CDT is the result of one-or zero-carbon linker between the capsular polysaccharide and the protein carrier, while the use of PDPH is the result of a covalent thioester linkage between the capsular polysaccharide and the protein carrier.

Way on the basis of PDPH was a multistage process, which included the activation of the polysaccharide, remove tylenol protective groups on the polysaccharide purification of intermediate activated polysaccharide, activation and protein purification media and conjugation of activated components with subsequent cleaning. In this way capsular polysaccharides S.aureus serotype 8 was brought into interaction with PDPH and a carbodiimide in an aqueous solution such as 0.1 M MES, obtaining polysaccharides associated with PDPH. Polysaccharides associated with PDPH, brought into interaction with the reducing agent with the floor is rising activated polysaccharides, which is then purified. Protein carriers brought into interaction with the N-hydroxysuccinimide ester bromoxynil acid in aqueous solution with an activated protein carriers, which are then purified. Purified activated polysaccharide serotype 8 is then brought into interaction with purified activated protein carriers with obtaining conjugates polysaccharide serotype 8 with protein carrier.

On the contrary, methods based on CDI and CDT was a single-stage or two-stage processes of conjugation, in which the capsular polysaccharide activated in an anhydrous environment (i.e. DMSO) with the formation of the imidazole or triazolinone groups with available hydroxyl and elliminating or illtreating groups with carboxylic acids. Adding protein carrier (DMSO) resulted in nucleophilic substitution of the imidazole or triazole lysine and the formation of urethane linkages (for activated hydroxyl and amide linkages (for activated carboxylic acids). Respectively, were developed two methods based on the CDI or CDT: a more complex way and a simpler method, implemented in a single container. In a more complex way capsular polysaccharide S.aureus serotype 8 was compoundable with imidazole or triazole, liofilizirovanny and then subjected to interaction with CDI or CDT in the op is adicheskim solvent (such as DMSO) with an activated polysaccharide serotype 8. Activated polysaccharide serotype 8 was purified and then brought into interaction with the protein carriers in an organic solvent to obtain conjugates polysaccharide serotype 8 with protein carrier. The process is carried out in one tank was the same as the hard method, except that the activated polysaccharide serotype 8 was not cleaned prior to interaction with proteins in the media.

Integrated method with CDI/CDT

Activation of the polysaccharide. Polysaccharide serotype 8 was mixed with 10 g of triazole on 1 g of the polysaccharide serotype 8 and liofilizirovanny. The resulting mass was dissolved in DMSO at a concentration of 2.0 mg of polysaccharide serotype 8 in 1 ml. was Determined by the water content. Was added freshly prepared mother liquor CDT at a concentration of 100 mg/ml in DMSO to achieve a molar amount CDT equivalent amount of water. Alternatively, the number of added CDT can be adjusted to achieve a higher or lower degree of activation. Kept for 30 minutes at 23°C.

Purification of the activated polysaccharide serotype 8. The solution of the activated polysaccharide serotype 8 (ASR) was poured into 25 volumes of water to destroy excess CDT. Concentrated to the original volume on PES-membrane of 10 kDa at approximately 1 mg/cm3and subjected diafiltration against water with at least 10 volumes. This stage is aversely less than 4 hours. Subject diafiltration substance was mixed with 10 g of triazole on 1 g of the original polysaccharide serotype 8 and liofilizirovanny.

Getting dried CRM. CRM subjected diafiltration against a solution of 0.4% NaCl/5% sucrose at constant volume on PES-membrane of 10 kDa in at least 10 volumes. Determined concentration of protein was added in amount of buffer to diafiltration sufficient to bring the concentration of the protein to 5.0 g/l, thus bringing the mass ratio of NaCl/CRM to 0.8. CRM liofilizirovanny.

The conjugation. Activated subjected diafiltration polysaccharide serotype 8 was dissolved in DMSO at a concentration of 1 mg/ml was Added to 100 mm borate solution to achieve 2% vol./about.

CRM resuspendable at a concentration of 2 mg/ml and, when dissolution was complete, was combined with a solution ASR. Left to interact at 23°C for 20 hours. Reaction medium conjugation was poured into 24 volumes of 5 mm borate at pH 9.0 and left mixed at room temperature for 1 hour. Then brought to pH 7.5 with 0.5 M phosphate buffer at pH 6.5. Was filtered through a 5 micron filter and concentrated to the original volume on PES-membrane 300 kDa at a load of about 1 mg/cm2and subjected diafiltration against at least 10 volumes of water. The resulting concentrate was filtered through a 0,22 μm Phi is Tr and kept at 2°C-8°C.

Way with CDI/CDT in the same container.

Matrix currency CRM197. CRM197subjected diafiltration for the exchange of matrix mass of approximately 10 mm phosphate/80 mm NaCl/15% sucrose at pH 7 by 5 mm imidazole/0,72% NaCl/15 mm octyl-β-D-glucoside at pH 7. This exchange provided the removal of phosphate and sucrose, which are harmful for conjugation, and determined the content of sodium chloride, transferred in conjugation. Octyl-β-D-glucopyranosid added to prevent the formation of particles after sterile filtration.

Matrix CRM197replaced the filter in a tangential flow against 5 mm imidazole/0,72%/15 mm octyl-β-D-glucopyranosid at pH 7 through 10 dialysis volumes using 10K MWCO PES membrane with the concentration of holding approximately 4 mg/ml Typical load on the membrane was 2 g/ft2(21,5 g/m2), the target final concentration CRM197in the matrix was 6 mg/ml CRM197kept at 2°C-8°C.

Activation/Conjugation. Activation/conjugation for capsular polysaccharide S.aureus serotype 8 consisted of the following stages: (1) matrix currency CRM197; (2) the compounding of the polysaccharide; (3) freezing the shell and lyophilization CRM197and compounded polysaccharide; (4) dissolving the lyophilized polysaccharide and CRM197; (5) activation of the polysaccharide; (6) conjugation enable the cell polysaccharide with CRM 197; and (7) purification of the conjugate (dilution, diafiltrate, sterile filtration).

The polysaccharide was compoundable with 10 grams of excipient 1,2,4-triazole, 1 gram of polysaccharide. Excipient was added in powder form to the polysaccharide, and the solution was received in less than 15 minutes of stirring at ambient temperature.

Compounded polysaccharide and CRM197individually froze on the membrane using ethanol bath at -75°C. the Volume of the bottle with a capacity of 1 l was approximately 500 ml.

For dissolution of the polysaccharide DMSO was added in individual bottles for lyophilization of the polysaccharide with getting the suspension and then transferred to heat in the vessel for activation/conjugation. Added DMSO to obtain a concentration of 2 g/l Clear solution was obtained when the suspension reached approximately 45°C with stirring. The solution then was cooled to 23°C.±2°C.

For dissolution CRM197added DMSO in individual bottles for lyophilization, containing CRM197with the receipt of the suspension and then transferred to the second vessel for mixing. DMSO was added to obtain a concentration of 2 g/l Clear solution in typical cases received less than 15 minutes. From the polysaccharide solution in DMSO took samples for analysis by Karl Fischer to determine the moisture content. CDT was prepared in the form of process is and 100 mg/ml in DMSO and were added on the basis of a certain moisture content. Continuous addition of a solution CDT carried out for approximately 5 minutes at 23°C±2°C under stirring. The reaction was allowed to continue for at least 30 minutes at 23°C±2°C. From the reaction mixture was sampled to determine the level of activation (UV when 220/205 nm) and then added to 100 mm sodium borate at pH 9 with getting a 1.5%aqueous solution. This reaction solution was then stirred 30 minutes at 23°C±2°C.

For conjugation of the activated polysaccharide with CRM197added DMSO to the intended concentration of 0.8 mg/ml in the reaction medium. Then CRM197dissolved in DMSO, was added to a solution of the activated polysaccharide with stirring. This reaction mixture was stirred at least 4 hours at 23°C±2°C.

For hydrolysis of the remaining activation groups, the reaction solution was diluted 10-fold by addition of 5 mm sodium tetraborate at pH 9 with stirring. The diluted solution was passed through a 5 μm filter and concentrated to the planned concentration retention 2 g/HP Filtering in a tangential flow was performed using membranes from regenerated cellulose 300 kDa dialysis by means of a 20 volumes of 5 mm succinate, pH 7. A typical load on the membrane was 1 g/ft2(10,75 g/m2). The purified conjugate was passed through to 0.22 micron filter and stored at 2°C-8°C.

Example 4: Conjugation of capsule the aqueous polysaccharide serotype 8 through odnoimennogo and complex ways conjugation

This example shows that a pre-selected range of molecular masses of capsular polysaccharides can be used for conjugation or odnoimjonnym or comprehensive way. Bacterial cell source producing larger polysaccharides, and purification of the obtained range of molecular weights can be controlled by pH and heating during hydrolysis of Example 1 (as shown in Table 3).

In this example, chose eight parties, where the capsular polysaccharide serotype 8 had a molecular weight in the range from about 80 kDa to about 120 kDa, and conjugation of capsular polysaccharide serotype 8 was carried out using the activation of 1,1-carbonyl-di-(1,2,4-triazole), see Table 8. The molecular weight of the obtained conjugates were in the range of 595 kDa to 1708 kDa. The number of conjugated lysine on CRM ranged from a high 13 to low 3. The content of free sugars was in the range of high (11%) to low (1%).

Table 8
Conjugates of capsular polysaccharide serotype 8, obtained with capsular polysaccharides with molecular masses of 80 kDa to CD
MethodMileageMW polysaccharide (kDa) The output of the saccharide (%)Free sugar (%)MW according to SEC-MALLS (kDa)Lysine
Odnoskatnyj19886175111
28980167513
310876410735,0
41086948195,2
58985,18170810
610094,01115775
Complex1 998869434
21137358413
31057937197
41008626309
5879035956

Example 5: Evaluation of conjugated native and processed the basis of capsular polysaccharide serotype 8 in the model of bacteremia in mice

For conjugates of capsular polysaccharide evaluated the importance of O-acetyl groups present in the native capsular polysaccharide serotype 8 to conjugation, for the induction of functional or antibody-based test answers. Capsular polysaccharide serotype 8 de-O-azetilirovanie in mild alkaline conditions, and as NMR and ion chromatography (IC) confirmed the absence of O-acetyl who work in capsular polysaccharide serotype 8 in de-O-Ac-CRM. Conjugate SR de-O-Ac-CRM was obtained by conjugation of polysaccharide de-O-Ac SR with CRM by PDPH chemistry as described in Example 2.

The conjugate of the capsular polysaccharide serotype 8 unexpectedly showed the absence of acetyl groups defined by the way IC. This can be explained by differences in site structure of O-acetylation compared with other capsular polysaccharides S.aureus, which in turn can cause destruction or modification of acetyl groups in capsular polysaccharide serotype 8 during conjugation.

Model of bacteremia in mice was used to evaluate the effectiveness of the native, in comparison with the processed base, capsular polysaccharide serotype 8 conjugated to CRM197. Groups of female BALB/c mice (15/group) were vaccinated at time 0, 3 and 6 weeks using 1 µg of capsular polysaccharide serotype 8 de-O-Ac-CRM or 1 mg capsular polysaccharide serotype 8 O-Ac-CRM. The vaccine was prepared from 22 ág AlPO4. Animals were injected S.aureus PFESA0003 and counted the number of bacteria in the blood three hours later. The data showed a statistically significant (p=0,0362) reduction in bacterial CFU isolated from the blood of animals immunized with the conjugate raw native capsular polysaccharide serotype 8 in terms of t-test t-test (table 9). In animals which were immunized with a conjugate-treated bases is of capsular polysaccharide serotype 8, bacterial CFU isolated from the blood were the same as in the control group with saline solution.

Table 9
The conjugate of the capsular polysaccharide serotype 8 with CRM197reduces bacteremia, which is caused by S.aureus PFESA0003 in mice
AntigenStrain/DoseLog CFU in bloodSignificance values (p)
SalinePFESA00034,35
SR de-O-Ac-CRM1,14×108of 4.45
SR O-Ac-CRM3,930,03

Example 6: Evaluation of conjugated native and processed the basis of capsular polysaccharide serotype 8 in the model of bacteremia in mice

Conjugates of capsular polysaccharide serotype 8 was evaluated on their ability to protect mice in a model of pyelonephritis. The presence of bacteria in the blood of mice treated by intraperitoneal S.aureus was significantly decreased what about when compared with controls, which PBS was injected.

Two studies were conducted to evaluate the effectiveness of the conjugate CP8-CRM197in the model of bacteremia in mice, described above, after the introduction of S.aureus PFESA0268 (Type 8). The first study (Figure 5) showed a significant reduction in bacteremia (p=0,0308). For this study, groups of mice Swiss Webster at the age of 6-8 weeks (n=30) were subjected to active immunization by subcutaneous injection of 1 μg of the conjugate of the capsular polysaccharide serotype 8 with CRM197or saline, and both were prepared with 100 µg AlPO4at time 0, 2 and 4 weeks, after which they were introduced for the period of 6 weeks by intravenous S.aureus PFESA0268 (Type 8). Preliminary experiments on the introduction of S.aureus PFESA0268 carried out to optimize the dose of strain for the age of the mice reached after three vaccinations. Statistical evaluation of the survival studies were carried out by analysis by Kaplan-Meier.

Example 7: Opsonic activity of sera from mice immunized with the conjugates of native and chemically modified capsular polysaccharide serotype 8

Selected mouse serum from the study vaccination (n=5) with high titers of capsular polysaccharide serotype 8 were compared opsonic activity (PRA), using strain PFESA0005. Results for ORA (table 10) show that only the conjugates obtained conju is the situation of the native capsular polysaccharide serotype 8, cause in mice opsonic antibodies. It should be noted that the conjugate of the capsular polysaccharide serotype 8 de-OAc was immunogenic in mice, but the resulting antibodies were not opsonic in this analysis. Titles ORA expressed as the reciprocal dilution at which observed a 40%reduction in death.

Table 10
Opsonic activity of sera of mice immunized with conjugate CRM with native capsular polysaccharide serotype 8 in comparison with CRM conjugate with de-O-acetylated capsular polysaccharide serotype 8
Conjugate CRM with de-O-acetylated capsular polysaccharide serotype 8Conjugate CRM capsular polysaccharide serotype 8
The title of the PR, the serum at time 0 weeksCaption EO serum at term 8 weeksThe title of the PR, the serum at time 0 weeksCaption EO serum at term 8 weeks
<50<5050150
<50<50<501350
<50<50<50450
<50<50<501350
<50<50<504050

Example 8: the Death of strains of S.aureus by antisera conjugate serotype 8 it is possible to inhibit the addition of native capsular polysaccharide serotype 8

To confirm the specificity of the death, opsonophagocytic reaction was carried out in the presence of the native capsular polysaccharide serotype 8 or unrelated pneumococcal polysaccharide (Pn 14 poly) essentially as described above.

The results (table 11) showed that the presence of the native capsular polysaccharide serotype 8 in the reaction mixture inhibited opsonophagocytic death S.aureus PFESA0286 (Type 8). These results confirm that opsonophagocytosis death under the influence of immune sera mediated by antibodies specific to the capsule.

Table 11
Adding capsular polysaccharide serotype 8 inhibits opsonophagocytosis death S.aureus under attack by the receiving immune serum
MonkeyThe serum sampleTitle ORA
02D133Week 0<50
Week 84050
Week 0+20 µg SR poly<50
Week 8+20 µg SR poly<50
Week 0+20 ug Pn 14 poly<50
A4N122Week 8+20 ug Pn 14 poly4050
Week 0<50
Week 84050
Week 0+20 µg SR poly<50
Week 8+20 µg SR poly<50
Week 0+20 ug Pn 14 poly<50
Week 8+20 ug Pn 14 poly1350

CONCLUSION

All methods of chemical conjugation gave capsular polysaccharide serotype 8, covalently linked to a protein carrier CRM197. He had significant differences on these measures the resultant saccharide, value capsular polysaccharide serotype 8: protein and outputs conjugates produced by those methods.

Example 9: Getting capsular polysaccharide S.aureus serotype 5

This example describes various size ranges capsular polysaccharide S.aureus serotype 5. The structure of the repeating unit of the capsular polysaccharide S.aureus serotype 5 is shown in Fig.6. The described methods are effective in obtaining capsular polysaccharide serotype 5 with molecular masses in the range from about 20 kDa to 800 kDa. The proper choice of conditions it is possible to isolate and purify high molecular weight capsular polysaccharide serotype 5 in the range from 50 kDa to 800 kDa molecular weight. For use in immunogenic compositions capsular polysaccharide serotype 5 you can select and clear in the range from 70 kDa to 300 kDa molecular weight, from 70 kDa to 150 kDa and many other desired ranges. Strain PFESA0266 was chosen to produce capsular polysaccharide serotype 5 on the basis of the growth characteristics and quantity of the capsule.

To obtain capsular polysaccharide serotype 5 strain PFES A were grown in a complex medium consisting mainly of carbon source (lactose or sucrose, hydrolyzed soy flour as a source of nitrogen and trace amounts of metal the century The strain is grown in a bioreactor in 2-5 days.

Fermentation of strain PFESA0266 was carried out as described in detail above. While collecting the optical density OD600culture was 7,38. Culture autoclaved for 1 hour and after cooling were processed as described above to separate the cells from the substance of the supernatant. Received approximately 1 l of filtered and concentrated supernatant and cells.

Before autoclaving took samples for testing the level of staphylococcal enterotoxin B (SEB) in culture. In the presence of 0.05% Polysorbate 80 concentration of SEB in a fermentation medium was 15-20 ng/ml Previous experiments showed that autoclaving the culture for 1 hour reduces the level of SEB to less than 0.1 ng/ ml, which is below the detection limit set TECRA.

Subject diafiltration fractionated by ethanol polysaccharide was applied on a column of Q-Separate AES and suirable linear NaCl gradient as described above. Fractions were analyzed on myst group and a test double immunodiffusion in the presence of polysaccharide serotype 5 and the analysis of phosphate in the presence of tahaawee acid. The presence of polysaccharide serotype 5 were detected in the fractions 60-105 (Figa-B). To reduce contamination of tahaawee acid fraction 60-85 United and the rest of taikoubou keys, the GTC was oxidized by metaperiodate sodium, to enable deleting the 3K diafiltrate against distilled water.

Purification of capsular polysaccharide serotype 5, is used to obtain conjugates was carried out in two different ways, which is based on the fact that high temperature and low pH affect release capsules out of the cells and reduce the molecular weight of the polysaccharide. The resulting molecular mass depends on time, temperature and pH of the hydrolysis step.

The characterization of the capsular polysaccharide serotype 5 was performed using the methods listed above in Table 1.

Capsular polysaccharides obtained by the methods described below, lead to pure polysaccharides with low levels of contamination with proteins, nucleic acids, composition and tahaawi acid.

In the first method, after the release of the capsular polysaccharide from the cell and reduce the molecular weight of the product was treated with an enzyme cocktail (such as ribonuclease, desoksiribonukleaza, lysozyme and protease) for the destruction of the impurities. After incubation the remaining impurities precipitated with addition of ethanol (final concentration approximately 25%). After removal of residual ethanol solution containing capsular polysaccharide, were applied to the anion exchange column (Q-Sepharose) and suirable linear salt gradient. Faction, operasie capsular polysaccharide, were combined and treated with metaperiodate sodium. This treatment resulted in oxidative hydrolysis of the remaining impurities of tahaawee acid but had no effect on capsular polysaccharide serotype 5. The reaction mixture was suppressed by the addition of ethylene glycol. The substance was concentrated and subjected diafiltration against distilled water (dH2O) to remove any residual reactants and by-products.

The second method used to obtain the capsular polysaccharide without the use of enzymes for the destruction of various impurities, originating from the cells. In this way after the release of the capsular polysaccharide from the cell and reduction of molecular weight hydrolysate fermentation broth was osvetleni by microfiltration and ultrafiltration and diafiltration. The solution was treated with activated charcoal to remove impurities. After treatment with carbon material was treated with metaperiodate sodium for oxidation of the remaining tahaawee acid and then extinguished by propylene glycol. The substance was concentrated and subjected diafiltration against distilled H2O to remove any residual reactants and by-products.

Drugs obtained using any of the methods resulted in pure capsular polysaccharides with low levels of contamination with proteins, nucleic acid is Tami and tahaawi acid. The described methods can be used for specific ranges of the desired high molecular weight polysaccharides manipulation of the conditions of hydrolysis.

Examples of capsular polysaccharide obtained is described here by the methods shown below in Table 12. Party purified capsular polysaccharide serotype 5 was of high purity, as evidenced by the absence of tahaawee acid (TA) and composition and a low level of residual protein (see Tables 12 and 13). The range of molecular masses ranged from 132,7 kDa to 800 kDa, and purified polysaccharides were highly O-acetylated, in the range of 90%-100%, and were 100% N-acetylated. Outputs purification of capsular polysaccharide serotype 5 ranged from 39% to 63%, and the amount of purified polysaccharide serotype 5 was varied from 35 kDa to 65 kDa (see Table 12). The level of pollution of tahaawee acid (TA) was acceptable, and the levels of residual proteins and nucleic acids were also within the acceptable range. The NMR spectra of the polysaccharide serotype 5 were identical to those described in the literature.

td align="left"> Output
Table 12
Characterization of the capsular polysaccharide serotype 5 (SR)
SampleTotal cleared SRMWProteinNucleic acidO-Acetyl-licensing
mg%(kDa) (g/mol)% (mass.)(scanning at 260 nm) % (mass.)NMR (%)
1101394700,594
29148651,22,596
357863352,50,775

Table 13
Additional characterization of the capsular polysaccharide serotype 5 (SR)
SampleMW (kDa) SR (mg/ml)O-acetyl NMR (%)Identification by NMRN-acetyl-NMR (%)
1800,13,164100Test passed100
2132,71,17290Test passed100
3335,40,97590Test passed100
4366,80,86590Test passedBUT
BUT = not determined

The choice of molecular weight capsular polysaccharides. Kinetic analysis demonstrated that described here ways you can get a wide range of molecular masses of capsular polysaccharides. The source of bacterial cells produce larger polysaccharides, and the desired range of molecular masses choose and then clear manipulirovanie is m pH and heating conditions in the stages of heat and hydrolysis.

Heat treatment of fermentation broth S.aureus is a stage between fermentation and extraction of the capsular polysaccharide. At this stage of the process heat is used for processing the broth when the pH within a certain time. Purpose of heat treatment at low pH are killing cells, inactivation of enterotoxins, the release associated with cells of the polysaccharide and the reduction in molecular weight to the desired size. Among these objectives, the reduction in molecular weight was the slowest in terms of processing time required at this stage. Therefore, within the specified processing time must inevitably be achieved for other purposes.

Thermal treatment. Defined conditions of pH and temperature for different ranges of molecular weight capsular polysaccharides. For these studies used the fermenter BioIafitte15L. Fermentation broth was transferred into a fermenter with a peristaltic pump. When using the stirring speed of about 200 rpm brought the pH of the broth is concentrated sulfuric acid. Then the temperature of the broth was increased to a predetermined value. The time of heat treatment was spotted as soon as the temperature has reached a predetermined level. Upon reaching the desired processing time the broth was cooled to room temperature. In BP is me this process took samples to determine the concentration of the polysaccharide and the molecular weight systems HPLC and SEC-MALLS, respectively. Data on the molecular weight (MW) used in the kinetic analysis. Profiles of molecular weight was determined in a time-dependent at pH 3.5, 4.0 and 5.0mm. Cm. Figa.

The kinetics of mild acid hydrolysis of the polysaccharides were determined using purified capsular polysaccharide serotype 8, obtained by this method. The solution of purified polysaccharide made with sulfuric acid to pH desired for the experiment. Approximately 1.5 ml of solution was transferred into centrifuge tubes with a volume of 15 ml test Tube was placed in an oil bath, equipped with precision temperature control system. The tubes were removed at certain intervals of time and put in a bucket with ice. At the end of the experiment, an aliquot of 1 M Tris buffer (pH 7.5) was added to the sample to bring the pH back up to about 7. The samples were analyzed system SEC-MALLS. The molecular weight used in the kinetic analysis. The effect of temperature on the molecular profile of the masses SR at pH 4.5 was determined in a time-dependent (see Figb).

Results

As shown in Figa, lower pH was more effective in reducing the molecular weight of the polysaccharide. In this example, the range of molecular weights between about 300 kDa and about 600 kDa can be obtained by using pH 5, at 95°C for period of time from 15 minutes to 120 minutes (see f GU). Similarly, choosing pH 4.5 at 95°C for period of time from 15 minutes to 120 minutes, you can get the molecular weight of the polysaccharide in the range between 200 kDa and 400 kDa. In addition, choosing pH 4.0 at 95°C for period of time from 15 minutes to 120 minutes, you can get the molecular weight of the polysaccharide in the range between 120 kDa and 300 kDa.

As shown in Figb, the higher the temperature, the higher the rate of hydrolysis and a wider range of molecular mass polysaccharide get over time. In other words, the use of a lower temperature, 55°C against 95°C, with the same pH gives a more narrow range of molecular mass polysaccharide.

Moreover, Figure 4 shows the correlation between the molecular weight of purified capsular polysaccharide serotype 5 and the processing time in soft acidic (pH 4.5 at 95°C) hydrolysis. The purified polysaccharide is the final product obtained during the extraction process, detailed above. As also shown in figure 4, increasing the time of heat treatment strain S.aureus PFESA0266 at pH 4.5 resulted in capsular polysaccharide serotype 8 with lower molecular weight, whereas shorter periods of time of heat treatment at pH 4.5 resulted capsular polysaccharide serotype 5 with higher molecular weight. The size of the capsular polysaccharide serotype 5 on Odessa in the range of about 90 kDa to about 220 kDa, depending on the length of time of heat treatment at pH 4.5. The correlation between the time of heat treatment at low pH and the amount of purified capsular polysaccharide serotype 5, as shown in figure 4, allows us to estimate the processing time required for the production of purified polysaccharide with a specific range of molecular masses.

As demonstrated above, can be produced, released and cleared the full range of molecular masses capsular polysaccharide serotype 5 from 20 kDa to 500 kDa. The described methods can be used to obtain a specific range of the desired high molecular weight capsular polysaccharides, as shown in Table 14. Relatively narrow obtain the range of molecular mass polysaccharide, where the peak molecular mass is in the range of 63 kDa to 142 kDa, is well characterized by a range of molecular weights, which you can get ways described here. Especially preferred range of high molecular weight polysaccharides, ranging from 70 kDa to 300 kDa or 70 kDa to 150 kDa, is useful in obtaining immunogenic compositions by conjugation of capsular polysaccharide molecules or protein carrier. The conditions used to produce capsular polysaccharide SR, having a range of molecular weights from about 100 kDa to 140 kDa, are the following: 95°C, pH 4.5 for 15 minutes. Other combinations of pH, temperature and time will, however, also generate molecules SR with a range of molecular weights from about 100 kDa to 140 kDa.

Table 14
Obtaining a specific range of high molecular weight capsular polysaccharide serotype 5
RunMW capsular polysaccharide serotype 5 (kDa)
1142
2108
3142
4108
5BUT
6BUT
763
1072
974
1063
11BUT
BUT: not implemented

Example 10: the Conjugation of capsular polysaccharides serote the 5 with CRM 197

This example describes the methods and analyses for characterization used in obtaining conjugates of capsular polysaccharide S.aureus serotype 5 with CRM197. Different chemical methods of conjugation were developed for conjugation of capsular polysaccharide S.aureus serotype 5 with the protein carrier. For example, conjugation with PDPH (3-(2-pyridyldithio)-propenylidene) is the result of a covalent thioester linkage between the CF and the protein carrier, whereas conjugation using CDT (1,1-carbol-di-1,2,4-triazole) leads to a one-or zero-carbon linker between the capsular polysaccharide and the protein carrier.

The conjugation of capsular polysaccharide serotype 5 with CRM197by PDPH conjugation chemistry

The conjugation chemistry with PDPH is a multistage process involving activation of the polysaccharide, remove tylenol protective group, treatment of intermediate activated polysaccharide, activation and protein purification CRM197and the conjugation of activated components with subsequent cleaning. After the introduction of a linker containing Tilney group in the polysaccharide and halogenoacetyl group in a protein carrier CRM197capsular polysaccharide S.aureus serotype 5 was associated with protein carrier via a thioester bond. Bromacetyl group were injected in protein CRM197mutual is the action of the amine groups with N-hydroxysuccinimidyl ether bromoxynil acid. To obtain tarirovannogo polysaccharide activated by carbodiimide carboxylate group of the N-acetylanthranilic acid in the polysaccharide was subjected to combination with the hydrazide group sulfhydryl-reactive hydrazide heterobifunctional linker 3-(2-pyridyldithio)-propionitrile (PDPH). Thiols PDPH-tarirovannogo polysaccharide obtained by reduction with DTT and purified by SEC on a column of Sephadex G25 were subjected to interaction with bromacetyl groups activated protein that had the result of covalent thioester bond formed by substitution of bromine, between the polysaccharide and the protein carrier. Unreacted bromacetyl group would cover hydrochloride group probably facilitates (hydrochloride of 2-aminoethanethiol). Then the reaction mixture was concentrated and subjected diafiltration. The remaining disconjugacy bromacetyl group would cover hydrochloride group probably facilitates to ensure the absence of reactive bromoacetyl groups remaining after conjugation. After substitution of bromine it gave covalent bond between tilenum end group probably facilitates and acetyl group on the lysine residue.

1. Milirovanie capsular polysaccharide S.aureus cepomuna 5 with PDPH. The polysaccharide is first activated by milirovanie with PDPH, for capsular polysaccharide with whom rotia 5 polysaccharide was mixed with a freshly prepared mother liquor PDPH (250 mg/ml in DMSO), Royal EDAC solution (90 mg/ml in distilled water) and mother liquor MES buffer (0.5 M, pH 4,85) to obtain the final solution of 0.1 M MES, and 2 and 4 mg polysaccharide/ml while maintaining the mass ratio of the polysaccharide: PDPH:EDAC equal to 1:5:3. This mixture is incubated for 1 hour at room temperature and then deliberately against a 1000-fold volume of distilled H2About four times using a dialysis device 3500 M WCO at a temperature between 4°C and 8°C to remove unreacted PDPH. PDPH-linked polysaccharide was treated with 0.2 M DTT and incubated at room temperature for 3 hours or over night at a temperature between 4°C and 8°C. the Excess of DTT, as well as by-products of this interaction was separated from the activated saccharide using SEC, using the resin Sephadex G25 and distilled water as the mobile phase. Fractions were analyzed using DTDP on tirinya group and thiol-positive fraction, which was loirevalley about the empty volume of the column were combined. Pooled fractions were analyzed by RANJAN and analyses on O-acetyl to determine the degree of activation, which was expressed as the molar percentage of the repeating units containing Tilney group (molar concentration of thiols/molar concentration of repeating units). The activated polysaccharide liofilizirovanny and kept at 25°C until they are needed in conjugation.

Results reproducibility of etiolirovaniya polysaccharide serotype 5 with PDPH shown in Table 15. The degree of activation of the polysaccharide serotype 5 was in the range from 11% to 19%, which corresponds to a range from approximately one attached to the linker molecule to ten repeating units of capsular polysaccharide to one molecule of linker five repeating units.

Table 15
The study of the reproducibility of the activation of the capsular polysaccharide of zeotype 5 using PDPH
PDPH-polysaccharide serotype 5Activation(% MSH/MEN)Scale mgOutput, mg (wt. -%)
1112319,6 (85)
2133028 (93)
4193023 (77)
5153229 (90)

2. Activation of the protein carrier. Protein carrier activated by bromotetradecane separately. CRM197was diluted to 5 mg/ml 10 mm phosphate buffered with 0.9%NaCl with pH 7 (PBS) and then brought to 1 M fallopian solution of 0.1 M NaHCO3at pH 7.0. Was added N-hydroxysuccinimidyl ether bromoxynil acid (BAANS) at a ratio of CRM197:BAANS 1:0,25 (mass./mass.), using the mother liquor BAANS in DMSO (20 mg/ml of This reaction mixture is incubated at a temperature between 4°C and 8°C for 1 hour, then was purified by SEC on a Sephadex G-25. Purified activated CRM197analyzed by the method of Lowry to determine protein concentration and then diluted in PBS to 5 mg/ml Sucrose as cryoprotectant was added to 5% wt./about. and the activated protein was frozen and stored at -25°C until the emergence of the need for conjugation.

Bromotetradecane lysine residues CRM197it was very stable and led to activation of 19-25 lysine 39 available lysine (see Table 16). This interaction gave high outputs activated protein.

Table 16
The outputs and the degree of bromotetradecane CR 197
MedicationActivated lisini (n=)Scale (mg)Output (% wt.)
1242385
2203887
3193577
4223594
5233587
62548104

3. The reaction mix. Immediately upon receipt of activated capsular polysaccharide and activated protein carrier they were combined in the reaction mixture for conjugation. Dried and etiolirovannye polysaccharide was dissolved in 0.16 M borate at pH of 8.95, mixed with thawed bromotetradecane CRM197and distilled water to obtain the final solution of 0.1 M borate and 2 mg/ml capsular polysaccharide serotype 5 when appropriate is Oseni CRM 197: polysaccharide 1:1 wt./mass. This mixture is incubated at room temperature for 16 to 24 hours. Unreacted bromacetyl groups on the protein "covered" by adding hydrochloride group probably facilitates in relation CRM197: catenin 1:2 (wt./mass.) using the mother liquor group probably facilitates 135 mg/ml in 0.1 M borate at pH of 8.95 and incubated for 4 hours at room temperature. The conjugate of the capsular polysaccharide from CRM197(conjugate) was purified 50-fold diafiltration against 0.9%NaCl, using polyethersulfone the 100K ultra-filter.

Studies on the reproducibility of etiolirovaniya capsular polysaccharide serotype 5 with PDPH demonstrated that the degree of activation of the polysaccharide ranged from 11% to 19%, which corresponds to a range from approximately one attached to the linker molecule to ten polysaccharide repeating units of one molecule of linker five repeating units.

The conjugation of capsular polysaccharide serotype 5 with CRM197by CDT conjugation chemistry

CDT provides a one-step method of conjugation, in which the polysaccharide activate in an anhydrous environment (DMSO) with the formation of triazolinone groups with available hydroxyl and elliminating or illtreating groups with carboxylic acids. Appendix the s protein carrier (DMSO) leads to nucleophilic substitution triazole lysine and the formation of urethane linkages (for activated hydroxyl and amide linkages (for activated carboxylic acids). The reaction solution was diluted 10 times in water solution to prepare for cleaning the filtration tangential flow.

The way chemical conjugation using CDT gave capsular polysaccharide serotype 5, covalently linked to a protein carrier, as evidenced by the presence of the saccharide and protein in the fractions with gel filtration and amino acid analysis of the conjugate, covered by glycolaldehyde or hydrochloride group probably facilitates.

Summary results for several parties conjugates obtained by using as PDPH and CDT chemistry, for a range of sizes capsular polysaccharide serotype 5 from 20 kDa to 40 kDa are shown in Table 17 below. Significant differences in free capsular polysaccharide, the ratio of polysaccharide-protein and outputs conjugates produced by those methods of conjugation, was not. Antigenicity conjugated capsular polysaccharide serotype 5 has not been modified by conjugation, as shown by the identity of the lines precipitation conjugates and native polysaccharide.

Table 17
Characterization of conjugates of capsular polysaccharide serotype 5 with CRM197obtained in two ways chemical conjugation
Method The output of the polysaccharide (%)The protein yield (%)The ratio of outputsFree sugar (%)Free protein (%)Modified LisinaSize (MW or Kd (%<0,3), saccharide/protein)
CDT19-27350,5-0,810-40<118-22from 38/61 to 76/64
PDPH26-5240-990,4-1,023-40NVNV7.5×105to 2.3×106
NV = not detected

As shown above, the described methods can be used to obtain a specific range of the desired high molecular weight capsular polysaccharides. The authors wanted to obtain the conjugates of the pre-selected range of high molecular weight capsular polysaccharide serotype 5, which can be filtered and cleaned for use in immunogenic compositions. Table 18 summarizes the analysis of conjugates of capsule the aqueous polysaccharide serotype 5, where capsular polysaccharide serotype 5 molecular weight was in the range of from about 92 kDa to 119 kDa, and activated by triazole (CDT). The molecular weight of the obtained conjugates ranged from 1533 kDa to 2656 kDa. The number of conjugated lysine on CRM197ranged from high - 22 to low 15. The content of free capsular polysaccharide varied from a high of -18% to a low of -11%.

Table 18
Compared with the pre-selected ranges of molecular weight capsular polysaccharide serotype 5
RunMW polysaccharide (kDa)Output (%)Free sugars (%)MW according to SEC-MALLS (kDa)Modified Lisina
11216311213019
2927216153322
3 1197414265615
41156318191115

Both chemical conjugation give capsular polysaccharide serotype 5, covalently linked to a protein carrier. Significant differences on the empty capsular polysaccharide, the ratio of the capsular polysaccharide serotype 5 and protein and outputs conjugates obtained by these two methods was not.

Example 11: the Method of CDT in the same capacity in comparison with the complex way

As described above, methods of obtaining immunogenic conjugates of this invention include covalent conjugation of capsular polysaccharides to protein carriers using conjugation chemistry, including CDI (1,1-carbonyldiimidazole), CDT (1,1-carbol-di-1,2,4-triazole) or PDPH (hydrazide 3-(2-pyridyldithio)-propionyl). Using CDI/CDT is the result of one-or zero-carbon linker between the capsular polysaccharide and the protein carrier, while the use of PDPH is the result of a covalent thioester linkage between the capsular polysaccharide and the protein carrier.

Way on the basis of PDPH was a mn is ghostdini process which included the activation of the polysaccharide, remove tylenol protective groups on the polysaccharide purification of intermediate activated polysaccharide, activation and protein purification media and conjugation of activated components with subsequent cleaning. In this way capsular polysaccharides S.aureus serotype 5 was brought into interaction with PDPH and a carbodiimide in an organic solvent, such as DMSO, obtaining polysaccharides associated with PDPH. Polysaccharides associated with PDPH, brought into interaction with the reducing agent with an activated polysaccharides, which are then purified. Protein carriers brought into interaction with bromoxynil acid in an organic solvent with an activated protein carriers, which are then purified. Purified activated polysaccharide serotype 5 is then brought into interaction with purified activated protein carriers with obtaining conjugates polysaccharide serotype 5 protein carrier.

On the contrary, methods based on the CDT was a single-stage processes of conjugation, in which the capsular polysaccharide activated in an anhydrous environment (i.e. DMSO) with the formation of triazolinone groups with available hydroxyl and elliminating or illtreating groups with carboxylic acids. Adding protein carrier (MSO) resulted in nucleophilic substitution of the imidazole or triazole lysine and the formation of urethane linkages (for activated hydroxyl and amide linkages (for activated carboxylic acid), thereby providing the possibility of conjugation in one container". Respectively, were developed two methods based on CDT: a more complex way and a simpler method, implemented in a single container. In a more complex way capsular polysaccharide S.aureus serotype 5 was compoundable with imidazole or triazole, and then was subjected to interaction with CDT in an organic solvent (such as DMSO) and approximately 0.2% of the mass./about. water with an activated polysaccharide serotype 5. Activated polysaccharide serotype 5 was purified and then brought into interaction with the protein carriers in an organic solvent to obtain conjugates polysaccharide serotype 5 protein carrier. The process is carried out in one tank was the same as the hard method, except that the activated polysaccharide serotype 5 was not cleaned prior to interaction with proteins in the media.

Integrated method with CDT

Activation of capsular polysaccharide serotype 5. Capsular polysaccharide serotype 5 was mixed with 10 g of triazole on 1 g of the polysaccharide serotype 5 and liofilizirovanny. The resulting mass was dissolved in DMSO at a concentration of 2.0 mg capsular polysaccharide serotype 5 in 1 ml. Determined the content of the water and brought up to 0.2%. Was added freshly prepared mother liquor CDT at a concentration of 100 mg/ml in DMSO for achievement of the 20-fold molar excess of CDT compared with the number SR. Alternatively, the number of added CDT can be adjusted to achieve a higher or lower degree of activation. Kept for 30 minutes at 23°C.

Purification activated capsular polysaccharide serotype 5. The solution of the activated polysaccharide serotype 5 (ASR) was poured into 25 volumes of water to destroy excess CDT. Concentrated to the original volume on PES-membrane of 10 kDa at approximately 1 mg/cm2and subjected diafiltration against water with at least 10 volumes. This stage was completed in less than 4 hours. Subject diafiltration substance was mixed with 10 g of triazole on 1 g of the original polysaccharide serotype 5 and liofilizirovanny.

Getting dried CRM. CRM subjected diafiltration against a solution of 0.4% NaCl/5% sucrose at constant volume on PES-membrane of 10 kDa in at least 10 volumes. Determined concentration of protein was added in amount of buffer to diafiltration sufficient to bring the concentration of the protein to 5.0 g/l, thus bringing the mass ratio of NaCl/CRM to 0.8. CRM liofilizirovanny.

The conjugation. Activated subjected diafiltration capsular polysaccharide serotype 5 was dissolved in DMSO at a concentration of 1 mg/ml was Added to 100 mm borate solution to achieve 2% vol./about.

CRM resuspendable at a concentration of 2 mg/ml and, when dissolution was complete, the volume of Denali with a solution ASR. Left to interact at 4°C for 20 hours.

Reaction medium conjugation was poured into 24 volumes of 5 mm borate at pH 9.0 and left mixed at room temperature for 1 hour. Then brought to pH 7.5 with 0.5 M phosphate buffer at pH 6.5. Was filtered through a 5 micron filter and concentrated to the original volume on PES-membrane 300 kDa at a load of about 1 mg/cm2and subjected diafiltration against at least 10 volumes of water. The resulting concentrate was filtered through a 0,22 μm filter and stored at 2°C-8°C.

Way with CDT in one capacity

Matrix currency CRM197. CRM197subjected diafiltration for the exchange of matrix mass of approximately 10 mm phosphate/80 mm NaCl/15% sucrose at pH 7 by 5 mm imidazole/0,72% NaCl/15 mm octyl-β-D-glucopyranosid at pH 7. This exchange provided the removal of phosphate and sucrose, which are harmful for conjugation, and determined the content of sodium chloride, transferred in conjugation. Octyl-β-D-glucopyranosid added to prevent the formation of particles after sterile filtration.

Matrix CRM197replaced the filter in a tangential flow against 5 mm imidazole/0,72%/15 mm octyl-β-D-glucopyranosid at pH 7 through 10 dialysis volumes using 10K MWCO PES membrane with the concentration of holding approximately 4 mg/ml Typical load is on the membrane was 2 g/ft 2(21,5 g/m2), the target final concentration CRM197in the matrix was 6 mg/ml CRM197kept at 2°C-8°C.

Activation/Conjugation. Activation/conjugation for capsular polysaccharide S.aureus serotype 5 consisted of the following stages: (1) compounding polysaccharide; (2) freezing the shell and lyophilization CRM197and compounded polysaccharide; (3) dissolving the lyophilized polysaccharide and CRM197; (4) activation of the polysaccharide; (5) conjugation of the activated polysaccharide with CRM197; and (6) purification of the conjugate (dilution, diafiltrate, sterile filtration).

The polysaccharide was compoundable with 10 grams of excipient 1,2,4-triazole, 1 gram of polysaccharide. Excipient was added in powder form to the polysaccharide, and the solution was received in less than 15 minutes of stirring at ambient temperature.

Compounded polysaccharide and CRM197individually froze on the membrane using ethanol bath at -75°C. the Volume of the bottle with a capacity of 1 l was approximately 500 ml.

For dissolution of the polysaccharide DMSO was added in individual bottles for lyophilization of the polysaccharide with getting the suspension and then transferred to heat in the vessel for activation/conjugation. Added DMSO to obtain a concentration of 2 g/l Clear solution of the floor of the Ali after mixing for 5-10 minutes.

For dissolution CRM197added DMSO in individual bottles for lyophilization, containing CRM197with the receipt of the suspension and then transferred to the second vessel for mixing. DMSO was added to obtain a concentration of 2 g/l Clear solution in typical cases received less than 15 minutes.

From the polysaccharide solution in DMSO took samples for analysis by Karl Fischer to determine the moisture content. CDT was prepared in the form of a solution of 100 mg/ml in DMSO and added to approximately 5-fold molar excess relative to the polysaccharide type 5 (in the complex method used 20 molar equivalents CDT, whereas in odnoetazhnom method used 5 molar equivalents CDT:CP5). Continuous addition of a solution CDT carried out for approximately 5 minutes at 23°C±2°C under stirring. The reaction was allowed to continue for at least 30 minutes at 23°C±2°SIS the reaction mixture was sampled to determine the level of activation (UV when 220/205 nm) and then added to 100 mm sodium borate at pH 9 with getting a 1.5%aqueous solution. This reaction solution was then stirred 30 minutes at 23°C±2°C.

For conjugation of the activated polysaccharide with CRM197added DMSO to the intended concentration of 0.55 mg/ml in the reaction medium. Then CRM197dissolved in DMSO, was added to a solution of the activated polysaccharide with paramasivan is I. This reaction mixture was stirred 16 hours at 23°C±2°C.

The reaction solution was diluted 10 times with 5 mm sodium tetraborate at pH of 8.6 with the final diluted pH 9±0,2. The solution was stirred at 23°C±2°C for at least 4 hours. The diluted solution was passed through a 5 μm filter and concentrated to the planned concentration retention 2 g/HP Filtering in a tangential flow was performed using membranes from regenerated cellulose 300 kDa dialysis by means of a 20 volumes of 5 mm succinate, pH 7. A typical load on the membrane was 1 g/ft2(10,75 g/m2). The purified conjugate was passed through to 0.22 micron filter and stored at 2°C-8°C.

Example 12: the Conjugation of capsular polysaccharide serotype 5 by odnoimennogo and complex ways conjugation

This example shows that a pre-selected range of molecular masses of capsular polysaccharides can be used for conjugation or odnoimjonnym or comprehensive way. Bacterial cell source producing larger polysaccharides, and purification of the obtained range of molecular weights can be controlled by pH and heat in the process of hydrolysis according to Example 9. In this example, chose eight parties, where the capsular polysaccharide serotype 5 had a molecular weight in the range of about is about 90 kDa to about 140 kDa, and the conjugation was carried out using the activation-triazole (CDT) or odnoimjonnym or complex ways described above, see Table 19. The molecular weight of the obtained conjugates ranged from 1125 kDa to 2656 kDa. The number of conjugated lysine on CRM ranged from high - 22 to low 15. The content of free sugars was in the range from a high of 23% to a low of 11%.

Example 13: Conjugates of capsular polysaccharide serotype 5 sustainably provide protection in a model of pyelonephritis in mice

Conjugates of capsular polysaccharide serotype 5 were evaluated for their ability to protect mice in a model of pyelonephritis. The presence of bacteria in the blood of mice treated by intraperitoneal S.aureus was significantly reduced when compared with controls treated with PBS.

All six of the individual studies showed a significant reduction in CFU/ml in kidney immunized animals (Figure 9). When these studies were combined for meta-analysis, the overall significance of the research as a whole increased to lower than 0.0001. These data showed a steady decrease colonization of the kidneys after active immunization with a conjugate of the capsular polysaccharide.

Example 14: Conjugates of capsular polysaccharide serotype 5, obtained by different methods of chemical conjugation, protects mice against experimental Talnah infections

Research active immunization model of pyelonephritis in mice was carried out with conjugates of capsular polysaccharide serotype 5, obtained either through PDPH or CDT chemistry. These methods of conjugation of capsular polysaccharides with CRM197disclosed above. The results showed that both conjugate reduce colonization in mice when compared with false immunized animals (table 20).

Table 20
The effect of conjugation with PDPH compared with CDT on protection against infection with S.aureus in the model of pyelonephritis
No. studiesAntigensStrain/DoselogKOE/KidneySignificance
ResearchSaline + AlPO4PFESA0266of 5.53±1,90--

No. 11 µg CP5-CRM (PDPH)+AlPO42×1083,01±1,83p<0,001
Study No. 21 µg CP5-CRM (CDT)+AlPO41,67±0,23p<0,0001
Saline + AlPO4PFESA0266S6,17±1,76
1 µg CP5-CRM (PDPH)+AlPO4of 2.7×1083,06±1,69p<0,0001
1 µg CP5-CRM (CDT)+AlPO41,87±0,69p<0,0001

Example 15: Active immunization with a conjugate of the capsular polysaccharide serotype 5 protects rat model of endocarditis in rats

Carried out four studies with PDPH-conjugate CP5-CRM197. Conjugates of capsular polysaccharide serotype 5 significantly reduced the number of CFU after infection with S.aureus PFESA0266 as the heart and kidney in 2 of 3 experiments (table 21). In the third study of geometric mean titer (GMT) of anti-SR was the smallest of the data obtained in the three experiments, but only slightly lower than in the previous experiment.

Table 21
IMM is the shadowing of a conjugate of the capsular polysaccharide serotype 5 with CRM reduces SOME models of endocarditis in rats
Log highlighted SOMEsignificanceGMT
Immunogenic compositionThe infecting Strain/DoseHeartKidneyHeartKidneyTitre CF
1 µg CP5-CRMPFESA02664,34±1,78to 3.92±1,73103,000
1 µg PP5-CRMof 2.21×108SOME7,94±0,786,77±0,79p<0,001p<0,05
1 µg CP5-CRMPFESA02664,43±2,303,11±2,3351,000
Saline6,5×107SOME5,63±2,484,19±2,05 NoNo
1 µg CP5-CRMPFESA02664,01±2,493,90±1,9267,000
Saline4,0×108SOME7,52±1,386,52±1,17p<is 0.0002p<is 0.0002

Example 16: Enhanced immunogenicity of the vaccines, which represents a conjugate high-molecular SR, in mice when compared with vaccines, which represents a low-molecular weight conjugate SR

Conducted a study in a murine model of pyelonephritis to assess the immunogenicity and efficacy of different conjugates SR. Tested two drugs. The first drug consisted of high molecular weight (HMW) SR (approximately 300 kDa)conjugated to CRM197. The second product contained low molecular weight (LMW) SR (approximately 25 kDa)conjugated to CRM197. For high molecular vaccines tested three levels of doses (1, 0.1, and 0.01 μg). Low-molecular vaccine was tested at a dose of 1 µg. The negative control group was also included vaccine composed of conjugate polysaccharide is, derived from Streptococcus pneumoniae conjugated to CRM197(RR). Polysaccharides were prepared from 22 ág AlPO4at time 0, 3 and 6 weeks and S.aureus infection PFESA0266 conducted in the period of 8 weeks. 48 hours after infection was removed kidney and counting bacterial colonies. Both vaccines effectively evoked immune response and reduced SOME of S.aureus PFESA0266 in the kidneys of mice vaccinated groups 1 microgram as high-molecular and low-molecular vaccines. It depended on the dosage, as demonstrated reduced efficiency at reduced dosages of the vaccine (Figure 10). Counting CFU was not sensitive enough to detect differences in efficacy between high-molecular and low-molecular vaccines. Therefore, sera from the mice were tested by analysis of PR (opsonophagocytosis). Captions analysis opsonophagocytosis was defined as the serum dilution required to amarouchene 40% of S.aureus strain PFESA0266 in the analysis of the ORA. Improved OPA titers were observed for high molecular vaccine compared with low molecular weight drug (11).

Example 17: Conjugates of capsular polysaccharide containing high-molecular polysaccharides, show enhanced immunogenicity when compared with conjugates containing low molecular weight polysaccharides

Studies in primates, not representing the people NHP), carried out to assess the immunogenicity of drugs of different capsular conjugates. Two drugs tested in two different dosage levels (2 and 20 μg). The first drug contained high molecular weight (HMW) polysaccharide (approximately 130 kDa)conjugated to CRM197. The second product contained low-molecular-weight (HMW) polysaccharide (approximately 25 kDa)conjugated to CRM197. Groups of five primates were vaccinated with one dose of vaccine and immune titers were monitored before vaccination and two weeks after vaccination. OPA titers were defined as the serum dilution required to killing 40% of S.aureus strain PFESA0266 in the PRA analysis. Antibody titers were also tracked enzyme-linked immunosorbent assay (ELISA). Enhanced activity was observed with high molecular vaccine compared with low molecular weight drug (table 22), as evidenced by a ten-fold increase in titers of antibodies to high molecular vaccine compared with low-molecular vaccine. The proportion of ER treatment for NHP, which received high molecular vaccine was also higher (80% compared with 40%).

Table 22
Enhanced immunogenicity was observed for vaccines conjugate high-molecular polysacharide on cf is the ranking with a conjugate vaccine with low-molecular-weight polysaccharide
The dose CP5-CRM197(g) an animalThe geometric mean PD1*Share (%) ORA responders ±
HMW (125 kDa)203280
22180
LMW (25 kDa)20340
2840
* The rate of increase was calculated based on the title SR in ELISA 2 weeks after vaccination when compared with titers before vaccination. ± The percentage of responders was calculated based on the number of monkeys that have OPA titer was increased two weeks after one dose of vaccine. Each group contained 5 macaques of resursov, and vaccines have been prepared with AlPO4(250 μg/dose).

Example 18: O-polysaccharide Acetylation is important for the induction of protective or antibody-based test answers to the conjugate of the capsular polysaccharide serotype 5

To assess the importance of O-acetylation of the capsular polysaccharide serotype 5, native capsular polysaccharide de-O-azetilirovanie (dOAc) is conjugatively with CRM 197(dOAc-CRM197using PDPH chemical conjugation, as discussed above. The effectiveness of the conjugate dOAcCP-CRM197compared with CP5-CRM197in the model of pyelonephritis in mice.

Immunization with conjugates, devoid of O-acetyl groups (dOAc SR-CRM), could not reduce the number of bacterial CFU in the kidneys. These data (table 23) show that O-acetylation is important for the formation of functional antibodies against SR.

Table 23
Immunization with conjugates of de-O-acetylated capsuleneo polysacharide serotype 5 does not protect mice from colonization of the kidney
No. studiesAntigensStrain/DoseLogCFU/KidneySignificance
Study 11 µg PP5-CRMPFESA02663,89±2,24
1 µg dOAc SR-
CRM7×1084,20±1,75
1 µg CP5-CRM1,75±0,39p<0,008
Study 2SalinePFESA02665,08±1,96
1 µg dOAc CP5-
CRM2,4±108of 5.89±1,29
1 µg CP5-CRM2,93±2,11p<0,02

Example 19: acknowledgement of the importance of O-acetylation as a functional epitope capsular polysaccharide serotype 5 by ORA using monoclonal antibodies with known specificnosti

Monoclonal antibodies against capsular polysaccharide serotype 5 with specificnosti SLA+(SR-7-1), SLA+/-(SR-5-1) and SLA-(SR-6-1), were evaluated in the analysis opsonophagocytosis activity umartwiania strain PFESA0266 related to the type 5 (table 24). Monoclonal antibodies against capsular polysaccharide serotype 8 (SR-3-1 specific to SR SLA+) was used as negative control.

OA the specific anti-SR mAb SR-7-1 was oposredovany killing S.aureus PFESA0266 (table 24). Also posredovano killing strain PFESA0266 monoclonal antibody SR-5-1, which recognizes an epitope common to SR SLA+ and SR SLA-. Monoclinal antibody specific for epitopes present in the capsular SLA-polysaccharide serotype 5 was not posredovano killing strain PFESA0266. These results indicate that O-acetyl epitopes on capsular polysaccharide serotype 5 is required for the functional activity of antibodies specific to serotype 5.

Antibodies should be functional when measuring for killing bacteria in a model of efficiency on the animal or in the analysis opsonophagocytosis killing that demonstrate that antibodies to kill the bacteria. Functional killing may not be displayed when using the analysis, which tracks only the generation of antibodies, which shows the importance of O-acetylation for efficiency.

Table 24
Monoclonal antibodies specific for O-aimilianos (+) capsuleneo polysacharide of zeotype 5 and O - de-O-azetilirovanna (+/-) capsuleneo polysaccharide serotype 5 are opsonisation against S.aureus PFESA0266 (Type 5)
SR-5-1 (0-AC+/-) (µg) SR-6-1 (0-AC) (µg)SR-7-1 (0-AC+) (ug)SR-3-1 (control) (µg)
201052,5201052,5201052,5201052,5
28333021-12-5-12-531464955-18-3-13-5
Data are shown as the percentage of killing and were calculated by determining the ratio of the number of CFU surviving in the term of 60 minutes in the wells with bacteria, antibodies, complement and cells HL-60 to the number of CFU surviving in the wells without antibody, but the soda is containing bacteria, complement and cells HL-60.

Example 20: Enhancement of immunogenicity observed in conjugates SR consisting of high molecular weight polysaccharides, when compared with low-molecular-weight polysaccharides in primates, not representing the person (NHP)

Studies in primates, not representing the person (NHP), was carried out to assess the immunogenicity of drugs of different capsular conjugates. Two drugs were tested at two different dosage levels (2 and 20 μg). The first drug contained high molecular weight (HMW) polysaccharide (approximately 130 kDa)conjugated to CRM197. The second product contained low molecular weight (LMW) polysaccharide (approximately 25 kDa)conjugated to CRM197. Groups of five primates were vaccinated with one dose of vaccine and immune titers were monitored before vaccination and two weeks after vaccination. OPA titers were defined as the serum dilution required to killing 40% of S.aureus strain PFESA0266 in the PRA analysis. Antibody titers were also tracked enzyme-linked immunosorbent assay (ELISA). Enhanced activity was observed for HMW vaccine compared with LMW drug (table 25). Took place from three to ten-fold increase in antibody titers for HMW vaccine compared with LMW vaccine. The proportion of ER treatment for NHP, which received HMW vaccine was also higher (80% compared with 40%).

Table 25
Enhanced immunogenicity observed in vaccines. containing conjugate high-molecular polysaccharide, when compared with vaccine containing the low-molecular weight conjugate polysaccharide
The dose CP5-CRM197(g) an animalThe geometric mean PD1*The proportion of EO response rate (%) ±
HMW (125 kDa)203280
22180
LMW (25 kDa)20340
2840
* The rate of increase was calculated based on the title SR in ELISA 2 weeks after vaccination compared with titers before vaccination. ± The percentage of responders was calculated based on the number of monkeys that have OPA titer increased after a single dose of the vaccine within two weeks after vaccination. Each group contained 5 macaques of resursov, and VA is the care prepared with AlPO 4(250 μg/dose).

SUMMARY

Both chemical conjugation described here, give capsular polysaccharide serotype 5, covalently linked to a protein carrier CRM197. He was a significant difference in the free saccharide, the ratio of polysaccharide serotype 5: protein and outputs conjugates obtained by these two methods.

All publications and patent applications mentioned in this description, indicate the level of experts, to which this invention relates. All publications and patent applications are incorporated here by reference as if each individual publication or patent application was specifically and individually indicated as included here by reference.

While this invention has been described in some detail by way of illustrations and examples for clarity of understanding, certain changes and modifications can be done within the scope of the attached claims.

1. Conjugate polysaccharide-protein to induce immune response and protection against infection Staphylococcus aureus containing capsular polysaccharide of Staphylococcus aureus, conjugated to protein carrier, where the polysaccharide is a capsular polysaccharide serotype serotype 5 or 8, having a molecular weight of between 70 kDa and 800 kDa and a degree of O-acetylation between 75 and 100%.

2. The conjugate according to claim 1, which is the meet of molecular weight between 200 and 5000 kDa kDa, or between 500 and 2500 kDa kDa.

3. The conjugate according to claim 1, where the protein carrier is a CRM197or pneumolysin.

4. The conjugate according to claim 3, where CRM197covalently linked to the polysaccharide by urethane links, amide linkages, or both of them.

5. The conjugate according to claim 3, where the molar ratio of conjugated lysine and CRM197is from about 10:1 to about 25:1.

6. The conjugate according to claim 5, where at least one covalent bond between CRM197and the polysaccharide has at least every 5-10 sharidny repeating units of the polysaccharide or every 5 sharidny repeating units of the polysaccharide.

7. The conjugate according to any one of p-6, where CRM197contains from 5 to 25, 5 to 20, or from 10 to 25 lysine covalently associated with the polysaccharide.

8. The composition for inducing an immune response and protection against infection Staphylococcus aureus containing the conjugate according to any one of claims 1 to 7 and at least one adjuvant, diluent or carrier.

9. The composition according to claim 8, which contains less than 30%, or less than 20% of free capsular polysaccharide of Staphylococcus aureus serotype 5 or 8 in comparison with the total quantity of capsular polysaccharide of Staphylococcus aureus serotype 5 or 8.

10. Method of inducing an immune response against Staphylococcus aureus in a subject, comprising the introduction of this subject immunologically effective amount of the composition of claim 8 or 9.

p> 11. A method of obtaining a conjugate according to any one of claims 1 to 7, comprising the stage of:
a) mixing a selected capsular polysaccharide of S. aureus specified serotype with imidazole or triazole with getting compounded polysaccharide;
b) interaction of the specified compounded polysaccharide with carbonyldiimidazole (CDT) in an organic solvent to obtain an activated polysaccharide;
C) interaction of the activated polysaccharide with protein carrier in an organic solvent to obtain the conjugate of a polysaccharide to a protein carrier; and
g) allocating the specified conjugate of the capsular polysaccharide to a protein carrier.

12. The method according to claim 11 which further includes the lyophilization of selected polysaccharide and re-suspension of lyophilized polysaccharide in an organic solvent.

13. The method according to claim 11, where the activated polysaccharide isolated from the reaction medium activation before interaction with protein carrier.

14. The method according to item 13, where stage (b) includes:
1) lyophilization of the activated polysaccharide selected from getting dried activated polysaccharide selected;
2) the protein lyophilization media receiving lyophilized protein carrier; and
3) re-suspension of lyophilized activated polysaccharide selected, lyophilizer the protein data carrier in an organic solvent to obtain an activated polysaccharide selected, mixed with protein carrier.

15. The method according to claim 11 which further includes the dilution of the reaction mixture from stage (C) in the buffer maintaining the pH in the range of from about 8.8 to about 9.2 deaths, such as approximately 9,0, for at least 4 hours at a temperature of from about 20°C to about 26°C, such as about 23°C.

16. The method according to claim 11, where the stage of interaction of the polysaccharide with CDT includes determining the content of water present in the capsular polysaccharide of S. aureus serotype 8, and bringing the CDT concentration to molar ratio CDT: water of about 1:1, about 0.5:1 to about 0.75:1, in an organic solvent.

17. The method according to claim 11, where the stage of interaction of the polysaccharide of S. aureus serotype 5 with CDT includes providing approximately 20-fold molar excess CDT compared with the polysaccharide.

18. The method according to claim 11, where the mixture of polysaccharide serotype 5 and CDT in an organic solvent is brought to the water concentration between 0.1 and 0.3%, such as 0.2 percent.

19. The method according to any of § § 11-16, which further includes a conjugate hydrolysis of the polysaccharide to a protein carrier to remove unreacted activating groups.

20. A method of obtaining a conjugate according to any one of claims 1 to 7, comprising the stage of:
a) interaction of the polysaccharide of S. aureus with 3-(2-pyridyldithio)-propionitrile (PDPH) and a carbodiimide in an organic dissolve the barely obtaining polysaccharide, associated with PDPH;
b) interaction of polysaccharide associated with PDPH, with a reducing agent to obtain an activated polysaccharide;
C) selection of the activated polysaccharide to obtain the selected activated polysaccharide;
g) protein interaction media bromoxynil acid to provide activated protein carrier;
d) interaction of selected activated polysaccharide with activated protein carrier with obtaining a conjugate of a polysaccharide to a protein carrier;
(e) allocation of conjugate polysaccharide with protein carrier; and
g) hydrolysis of the specified conjugate polysaccharide with protein carrier together with the hydrochloride group probably facilitates removal of unreacted activating groups.

21. The method according to claim 20, where the activated protein carrier emit before the interaction of activated protein carrier with the activated polysaccharide.

22. The method according to claim 20 or 21, where stage (C) further includes the lyophilization selected activated polysaccharide with getting dried activated polysaccharide.

23. The method according to claim 11 or 20, where the organic solvent is a polar aprotic solvent.

24. The method according to item 23, where the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethyl is etamide, N-methyl-2-pyrrolidone and hexamethylphosphoramide (NMRA).

25. The method according to claim 20, where carbodiimide represents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (D).

26. The method according A.25, where stage interaction capsular polysaccharide with PDPH and EDAC in the organic solvent includes maintaining the mass ratio of the polysaccharide: D:D equal to about 1:5:3.

27. The method according to claim 20, where the reducing agent is a dithiothreitol (DTT).

28. The method of reduction or prevention of Staphylococcus aureus infection or disease or condition associated with Staphylococcus aureus, the subject, including the stage of introduction of immunologically effective amount of the composition of claim 8 or 9 of this entity.

29. The method according to p where the infection, disease or condition selected from the group consisting of invasive disease Staphylococcus aureus sepsis and carriage.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to molecular biology, virology, immunology and medicine. The invention presents a composition which contains a sequenced and repetitive antigen or antigen determinant array, and particularly a composition which contains an Aβ1-6-peptide-HPV conjugate. More specifically, the invention provides the composition which contains a virus-like particle and at least one coupled Aβ1-6-peptide. Also, the invention describes a method for preparing the conjugates and the sequenced and repetitive arrays respectively.

EFFECT: compositions presented in the invention may be applicable for producing vaccines intended for treating Alzheimer's disease, and as pharmaceutical agents intended for preventing or treating Alzheimer's disease and for effective immune response induction, particularly antibody responses; besides, the compositions presented in the invention are established to be applicable first of all for effective induction of autoantigenic responses.

45 cl, 35 dwg, 22 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology, molecular biology and genetic engineering. There are presented an immunogenic composition containing a mixture of staphylococcal proteins, and comprising a staphylococcal protein binding an extracellular component, and a staphylococcal transport protein, or the staphylococcal protein binding the extracellular component, and a staphylococcal virulence regulator or a toxin, or the staphylococcal transport protein and the staphylococcal virulence regulator or the toxin. There are also presented vaccines, methods of treating, using and methods for preparing a staphylococcus vaccine.

EFFECT: invention may be used in medicine for treating and preventing a staphylococcal infection.

23 cl, 8 tbl, 7 dwg, 8 ex

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