Immunogenic compositions of staphylococcus aureus antigens

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and represents a composition for inducing an immune response against Staphylococcus aureus (versions), a method for inducing the immune response against Staphylococcus aureus, and a method for eliciting passive immunity against Staphylococcus aureus in an individual. The composition contains an effective amount of the recovered capsular polysaccharide 5 S. aureus conjugated with the CRM197 carrier protein, an effective amount of the recovered capsular polysaccharide 8 S. aureus conjugated with the CRM197 carrier protein, and at least one ingredient specified in a group consisting of an effective amount of the recovered polypeptide of the clumping factor A (ClfA) of S. aureus, an effective amount of the recovered polypeptide of the clumping factor B (ClfB) of S. aureus (ClfB) and an effective amount of the recovered protein MntC of S. aureus. The capsular polysaccharide type 5 represents a capsular polysaccharide of high molecular weight from 70 to 300 kD. The capsular polysaccharide type 8 represents a capsular polysaccharide of high molecular weight from 70 to 300 kD, wherein from 5 to 23 lysines in CRM197 are conjugated. The CP5 and CP8 conjugates have the molecular weight from approximately 200 kD to approximately 5000 kD.

EFFECT: presented invention enables eliciting the improved immune response against Staphylococcus aureus.

25 cl, 29 dwg, 26 tbl, 32 ex

 

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of provisional patent application U.S. No. 61/219134, filed June 22, 2009, the entire contents of which is hereby incorporated here by reference.

The SCOPE of the INVENTION

The present invention relates to immunogenic compositions containing the polypeptides and capsular polysaccharides isolated from Staphylococcus aureus. In addition, the invention relates to methods of inducing an immune response in subjects against Staphylococcus aureus using immunogenic compositions of polypeptides of Staphylococcus aureus and capsular polysaccharides. The resulting antibodies can also be used for treatment or prevention of Staphylococcus aureus infection by passive immunotherapy.

PRIOR art

Humans are the natural reservoir of Staphylococcus aureus (S. aureus). Healthy individuals can be colonized with S. aureus on the skin, nose and throat constantly (10-35%), occasionally (20-75%) or to be in a state of nanosatellite (5-70%) without associated disease. See Vandenbergh et al., J. Clin. Micro. 37:3133-3140 (1999). Subsequently, the disease occurs when individuals with weakened immunity due to abnormalities in the immune barriers, such as during surgery, the installation of permanent catheters or other condition which the devices injuries or wounds. Resulting from S. aureus infection can cause a wide range of different diseases that range from mild skin infections to endocarditis, osteomyelitis, bacteremia, sepsis and other forms of the disease with accompanying high percentage of mortality. Great human reservoir increases the opportunity for the evolution and distribution of adapted pathogenic types of clones.

Invasive staphylococcal infections from gram-positive cocci S. aureus and S. epidermidis are of special concern due to the fact that they represent a growing public health problem worldwide. In particular, S. aureus is responsible for most hospital-acquired (nosocomial) infections, and its prevalence among community-acquired (community onset) infections is increasing. For example, the incidence of invasive methicillin-resistant S. aureus (MRSA) was estimated as of 31.8 per 100,000 people, including 18650 deaths in the United States in 2005. See Klevens, R. M. et al., JAMA, 298:1763-71 (2007).

Over the last 20 years staphylococcal disease has undergone a sharp rise, and this rise is parallel with the use of intravascular devices and invasive procedures. This increase in the frequency of the disease is a growing concern due to the parallel increase of antibiotic resistance, CL is therefore there is an urgent need for immunogenic compositions for use in vaccines or for the induction of polyclonal or monoclonal antibodies to give passive immunity as a means for the prevention or treatment of staphylococcal infections and associated diseases.

SUMMARY of the INVENTION

The present invention is directed to mnogoetajnoe or multicomponent immunogenic composition comprising at least three antigen isolated from the bacteria Staphylococcus. Antigens, which are polypeptides and polysaccharides, can be obtained, among others, directly from the bacteria using the methods of selection, well-known specialists in the field, or they can be produced using the protocols of synthesis, or they can be produced recombinante using techniques of genetic engineering, also known experts in the field, or by any combination of the above. In certain embodiments the immunogenic composition according to the invention contains three or more than three antigen selected from the selected polypeptide of clumping factor A of S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 (SR) S. aureus conjugated to protein-but what ielem, selected capsular polysaccharide type 8 (SR) S. aureus conjugated to a protein carrier, and a selected protein of S. aureus MntC. In addition, according to the present invention, methods of inducing an immune response against the bacteria Staphylococcus, preventing, reducing the severity, or delay the onset of the disease caused by the bacterium Staphylococcus, and methods of preventing, reducing the severity, or delay the start of at least one symptom of a disease caused by infection by the bacterium Staphylococcus.

Accordingly, in one embodiment according to the invention proposed immunogenic composition containing the selected polypeptide of clumping factor And S. aureus (ClfA), isolated capsular polysaccharide type 5 (SR) S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 (SR) S. aureus, conjugated to protein carrier.

In one embodiment according to the invention proposed immunogenic composition containing the selected polypeptide of clumping factor And S. aureus (ClfA), highlighted the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 (SR) S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 (SR) S. aureus, conjugated to protein carrier.

In one embodiment according to the invention proposed immunogenic composition, tereasa selected polypeptide factor clumping And S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 (SR) S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 (SR) S. aureus, conjugated to protein carrier.

In one embodiment according to the invention proposed immunogenic composition containing the selected polypeptide of clumping factor And S. aureus (ClfA), isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 (SR) S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 (SR) S. aureus, conjugated to protein carrier.

In one embodiment according to the invention proposed immunogenic composition containing the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 (SR) S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 (SR) S. aureus, conjugated to protein carrier.

In one embodiment according to the invention proposed immunogenic composition containing the selected polypeptide of clumping factor B (ClfB) S. aureus, isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 (SR) S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 (SR) S. aureus, conjugated to protein carrier.

In one embodiment the AI according to the invention proposed immunogenic composition, containing the selected polypeptide of clumping factor A (ClfA) S. aureus, isolated polypeptide of clumping factor B (ClfB) and Staphylococcus aureus isolated protein of S. aureus MntC.

In one embodiment according to the invention proposed immunogenic composition containing the isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 (SR) S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 (SR) S. aureus, conjugated to protein carrier.

In one embodiment the immunogenic composition comprises the selection of the ClfA polypeptide, where the polypeptide fragment ClfA contains fibrinogenolysis domain of ClfA. In one embodiment the fragment of the ClfA polypeptide contains fibrinogenolysis domain containing domains N1, N2 and N3 ClfA. In one embodiment the fragment of the ClfA polypeptide contains fibrinogenolysis the domain that contains the domain N2 and N3 ClfA. In one embodiment the composition containing fibrinogenolysis domain of ClfA, show weak binding to fibrinogen. In one embodiment fibrinogenolysis domain of ClfA binds to fibrinogen to a lesser extent compared with the binding of fibrinogen observed for native fibrinogenesis domain of ClfA. In one embodiment the composition containing fibrinogenolysis domain of ClfA, show weak binding to fibrinogen and have aminokislot the Yu replacement of one or more than one of Tyr 338, Tyr 256, Pro 336, Lys 389, 254 Ala and Ile 387 full of protein, containing a signal sequence. In one embodiment the composition containing fibrinogenolysis domain of ClfA, show amino acid replacements at one or more than one of Tyr 338, Tyr 256, Pro 336, Lys 389, 254 Ala and Ile 387, where the amino acid in any one or more than one of these provisions is replaced with Ala or Ser. In one embodiment the composition comprises fibrinogenolysis domain of ClfA, where Tyr at position 338 is replaced by Ala.

In one embodiment the immunogenic composition comprises a selected fragment of the polypeptide ClfB, where a fragment of the polypeptide ClfB contains fibrinogenolysis domain ClfB. In one embodiment a fragment of the polypeptide ClfB contains fibrinogenolysis domain containing domains N1, N2 and N3 ClfB. In one embodiment a fragment of the polypeptide ClfB contains fibrinogenolysis the domain that contains the domain N2 and N3 ClfB. In one embodiment the composition containing fibrinogenolysis domain ClfB, show weak binding to fibrinogen. In one embodiment fibrinogenolysis domain ClfB binds to fibrinogen to a lesser extent compared with the binding of fibrinogen observed for native fibrinogenesis domain ClfB.

In one embodiment the immunogenic composition comprises capsular polysaccharide type 5 (SR) S. aureus, which is particularly the high molecular weight polysaccharide with a molecular mass of from 20 to 1000 kDa. In one embodiment, high molecular weight polysaccharide type 5 has a molecular mass of from 50 to 300 kDa. In one embodiment, high molecular weight polysaccharide type 5 has a molecular weight of from 70 to 150 kDa.

In one embodiment the immunogenic composition comprises capsular polysaccharide type 5 S. aureus, which O-acetiminophen 10%-100%. In one embodiment the immunogenic composition comprises capsular polysaccharide type 5 S. aureus, which O-acetiminophen 50%-100%. In one embodiment the immunogenic composition comprises capsular polysaccharide type 5 S. aureus, which O-acetiminophen 75%-100%.

In one embodiment the immunogenic composition comprises capsular polysaccharide type 8 S. aureus, which is a high molecular weight polysaccharide with a molecular mass of from 20 to 1000 kDa. In one embodiment, high molecular weight polysaccharide type 8 has a molecular mass of from 50 to 300 kDa. In one embodiment, high molecular weight polysaccharide type 8 has a molecular weight of from 70 to 150 kDa.

In one embodiment the immunogenic composition comprises capsular polysaccharide type 8 S. aureus, which O-acetiminophen 10%-100%. In one embodiment the immunogenic composition comprises capsular polysaccharide type 8 S. aureus, which O-acetiminophen 50%-100%. In one embodiment the immunogenic composition comprises capsular polysaccharide type 8 S. aureus, which O-acetiminophen 75%-10%.

In one embodiment of the capsular polysaccharide 5 and/or 8 present in the immunogenic composition, anywhereman with protein carrier. In one embodiment the carrier protein is a toxoid CRM197Corynebacterium diphtheriae (C. diphtheriae).

In one embodiment of the immunogenic composition contains S. aureus MntC, which is libidinously protein. In one embodiment of the immunogenic composition contains S. aureus MntC, which is not limitirovany protein.

In one embodiment according to the invention proposed immunogenic composition as herein described, optionally containing at least one protein family of proteins with serine-aspartate repeats (Sdr) selected from the group consisting of SdrC, SdrD and SdrE.

In one embodiment according to the invention proposed immunogenic composition as herein described, optionally containing protein with surface determinant In iron (IsdB).

In each of the embodiments described here, in which the immunogenic composition comprises three or more than three of these antigens, this composition may further contain other immunogenic and/or non-immunogenic substances. In certain embodiments, each immunogenic composition may, alternatively, consist essentially of" or "consist of" three or more than three of these antigens is optionally contain one or more non-immunogenic substance, as described here in more detail.

In one embodiment according to the invention proposed immunogenic composition as herein described, optionally containing any of the following antigens: Orra, DltD, HtsA, LtaS, IsdA, IsdC, SdrF, SdrG, SdrH, SrtA, SpA, Sbi FmtB, alpha-hemolysin (hla), beta-hemolysin, fibronectine protein A (fnbA), fibronectine protein B (fnbB), coagulase, Fig, map, leukocidin Panton-Valentine (pvl), alpha-toxin and its variants, gamma-toxin (hlg) and variants ica, immunodominant ABC Transporter, Mg2+conveyor, Ni ABC Transporter, RAP, autolysin, laminin receptors, IsaA/PisA, IsaB/PisB, SPOIIIE, SsaA, EbpS, Sas A, SasF, SasH, EFB (FIB), SBI, Npase, HEB, SIAL-bonded protein II bone, the predecessor of aureolin (AUR)/Sepp1, Cna, and their fragments, such as M55, TSST-1, mecA, exopolysaccharide poly-N-acetylglucosamine (PNAG/dPNAG), GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin protein, HarA, exhibiting esxa, EsxB, enterotoxin a, enterotoxin, enterotoxin C1 and a new autolysin. In certain embodiments of the invention, when the immunogenic composition contains certain forms SR and/or SR, it may not additionally contain PNAG.

In one embodiment of the immunogenic composition further comprises adjuvant. In one embodiment of the immunogenic composition further comprises a pharmaceutically acceptable carrier.

In one embodiment of the immunogenic composition for use you need a kitchen is of vaccines in the form of the drug. In one embodiment the vaccine is used for the induction of an immune response against S. aureus in the subject. In one embodiment of the immunogenic composition is used to produce compositions of antibodies to make the subject of the passive immunity.

In one embodiment according to the invention, a method of inducing an immune response against S. aureus, including the introduction of a subject immunogenic amounts of any of these immunogenic compositions and a pharmaceutically acceptable carrier.

In one embodiment according to the invention, a method for prevention or reduction of S. aureus infection or a way to prevent or reduce the severity of at least one symptom associated with infection caused by S. aureus, and these methods include the introduction of a subject immunogenic amounts of any of these immunogenic compositions and a pharmaceutically acceptable carrier.

In one embodiment of the methods of inducing an immune response against S. aureus include the delivery of immunogenic compositions with adjuvant. In one embodiment of the methods of inducing an immune response against S. aureus for the delivery of immunogenic compositions with a pharmaceutically acceptable carrier.

In one embodiment, the immune response induced as described here immunogenic compositions, prevents or reduces disease is s or condition, associated with staphylococcal organism in the subject, or prevents or reduces one or more than one symptom associated with staphylococcal organism in the subject. In one embodiment the disease is selected from the group consisting of invasive disease caused by S. aureus sepsis and carriage.

In one embodiment of the induced immune response includes the generation of antibodies with opsonophagocytic activity (OPA) against S. aureus. In one embodiment of the induced immune response includes the generation of higher opsonophagocytic titers of specific antibodies against S. aureus, compared to the titers observed in non-immunized subjects. In one embodiment opsonophagocytic titer is at least 1:20.

In one embodiment of S. aureus, against which the induced immune response is a MRSA. In one embodiment of S. aureus, against which the induced immune response is a MSSA (sensitive to methicillin in S. aureus). In one embodiment of S. aureus, against which induced immune response, represents to the VRSA (vancomycin-resistant S. aureus). In one embodiment of S. aureus, against which the induced immune response is a VISA (S. aureus with intermediate resistance to vancomycin).

In one embodiment according to the image is the shadow, a method for prevention of staphylococcal infection in a subject, undergoing a surgical procedure involving the introduction of a subject immunologically effective amount of any of the immunogenic compositions as described here, to surgical procedures. Surgical procedure can be planned (elective) surgical procedure or unplanned (non-elective) surgical procedure. In one embodiment of the surgical procedure is a cardiothoracic surgical procedure. In one embodiment the subject is a person, home (veterinary) animal or livestock.

In one embodiment according to the invention, a method of making the subject of the passive immunity, which includes stages (1) obtaining a preparation of antibodies using immunogenic compositions according to the invention and (2) the injection of antibodies to a subject to give passive immunity.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

In Fig.1 shows the different forms of recombinant ClfA and disclosed SEQ ID NO:125, 127-129, respectively, in order of appearance.

In Fig.2 shows the stage of cloning used to construct pLP1179 for the expression of ClfA. bp - pairs(a) grounds.

In Fig.3 shows the expression vector T7ClfA(N123)Y338A, pLP1179.

In Fig.4 shows a repeating structure of polysaccharides SR and SR.

In Fig.5A and 5B shows the profiles of molecular weight CR (a) and SR (B, biogas produced at different pH liquid nutrient medium.

In Fig.6A and 6B shows the profiles of molecular weight CR (a) and SR (B) produced at different temperatures.

In Fig.7 illustrates the correlation between the molecular weight of purified SR and SR with time for mild acid hydrolysis.

In Fig.8A-8E shows the alignment of ClfA between different strains of S. aureus (SEQ ID NO:62, 64, 68, 84, 70, 104, 66, 78, 86, 88, 90, 72, 74, 76, 80, 94, 82, 92, 96, 98, 100, 102, 106 and 108, respectively, in order of appearance).

In Fig.9 shows a phylogenetic tree of ClfA.

In Fig.10A-10D shows the alignment of ClfB between different strains of S. aureus (SEQ ID NO:26, 28, 32, 18, 54, 34, 36, 30, 16, 20, 22, 24, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58 and 60, respectively, in order of appearance).

In Fig.11 shows the phylogenetic tree of ClfB.

In Fig.12 shows the alignment of the MntC between different strains of S. aureus (SEQ ID NO:2, 8, 10, 4, 6, 14 and 12, respectively, in order of appearance).

In Fig.13 demonstrated that polyclonal rabbit antibodies against ClfA reduce the number of colonies of S. aureus 659-018 in a murine model of sepsis.

In Fig.14 demonstrated that active immunization ClfA reduces colonization heart of S. aureus PFESA0003 in a rabbit model of infective endocarditis.

In Fig.15A and 15B demonstrated that immunization MntC reduces the level of S. aureus in the blood. A: strain PFESA0237 S. aureus; B: strain PFESA0266 S. aureus.

In Fig.6 illustrates that, what immunogenic composition based conjugate SR - S. aureus-CRM197consistently protection in a murine model of pyelonephritis.

In Fig.17 demonstrated that vaccination is immunogenic composition based conjugate SR - CRM197reduces mortality in a model of sepsis.

In Fig.18 shows the colony forming units (CFU) isolated from the kidney after infection with S. aureus PFESA0266, in mice vaccinated CP5-CRM with high molecular weight (HMW), CP5-CRM with low molecular weight (LMW) or control PP5-CRM.

In Fig.19 shows a comparison of the titles of PR (opsonic activity) (geometric mean (geomean) of sera obtained from mice vaccinated with different compositions of conjugate polysaccharide (CP5-CRM with high molecular weight (HMW), CP5-CRM with low molecular weight (LMW)). The group consisted of 5-9 mice.

In Fig.20 shows the titer of PR for serum Primate, non-human, to ned. 0, empty symbols) and after 2 weeks (weeks. 2, filled symbols) after vaccination with different combinations of antigens of S. aureus. 3-antigen vaccine (3Ag) consisted of three antigens, and 4-antigen vaccine (4Ag) consisted of four antigens. Each song has two conjugate WED and either 1 or 2 peptide.

DETAILED description of the INVENTION

Before will be disclosed to these methods and methodology Leche is tion, it should be understood that this invention is not limited to particular methods, and describe the experimental conditions, such as methods and conditions may vary. It should also be understood that the terminology used here is only for the purpose of describing particular embodiments and is not meant that it is restrictive.

Although in the embodiment in practice or testing of the invention can use any methods and materials similar or equivalent described herein methods and materials are now described the preferred methods and materials. All publications mentioned here are included here by reference in their entirety.

Used herein, the terms have the meanings recognized and well-known experts in this field, however, for convenience and completeness, specific terms and their meanings set forth below.

Using here the forms of the singular include references to the plural, unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more than one method and/or the stage of the type described here, and/or which will become apparent to experts in this field after reading this description, and so on.

The term "about" or "approximately" means within a statistically nacimos the interval value. This interval can be within an order of magnitude, typically within 20%, but more typically in the range of 10% and even more typically within 5% of this value or interval. Permissible variation covered by the term "about" or "approximately", depends on the investigated system, and can be easily assessed by the specialist of ordinary skill in this field. Whenever within this application are listed interval, each integer in the interval is also seen as the embodiment of the invention.

"Antibody" is an immunoglobulin molecule capable of specific binding to a target, such as carbohydrate, polynucleotide, lipid, polypeptide, and so on, through at least one site of antigen recognition, located in the variable regions of immunoglobulin molecules. It is implied that this term, as it is used here, unless the context indicates otherwise, includes not only intact polyclonal or monoclonal antibodies, but also engineered antibodies (e.g., chimeric, humanized and/or derivateservlet to change effector functions, stability and other biological activities) and fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain (ScFv) and single domain antibodies, including antibodies sharks and bludova), and fused proteins containing part, representing the antibody, multivalent antibodies, multispecific antibodies (for example, bispecific antibodies, as long as they exhibit the desired biological activity) and antibody fragments, as described here, and any other modified configuration of the immunoglobulin molecules, which contains the site of antigen recognition. The antibody includes the antibody of any class such as IgG, IgA or IgM (or a subclass), and it is not necessary that the antibody belonged to any particular class. Depending on the amino acid sequence of the constant domain of heavy chain antibodies, immunoglobulins can be assigned 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., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 in human. The constant domains of the heavy chain, which correspond to the different classes of immunoglobulins are called alpha, Delta, Epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"Fragments of the antibodies contain only a portion of an intact antibody, where this part preferably stores at least one of the functions that is usually associer the bathrooms come with this part, when it is in an intact antibody, preferably the majority of these functions or all of these functions.

The term "antigen" usually refers to a biological molecule, usually a protein, peptide, polysaccharide, lipid or conjugate, which contains at least one epitope, which can be selectively contact cognate antibody; or, in some cases, to immunogenic substance that can stimulate the production of antibodies or the responses of T cells, or both, in an animal, including compositions that are injected the animal or that the animal absorbs. Can be generated immune response to the whole molecule or one or more than one from different parts of the molecule (e.g., epitope or hapten). This term can be used for referring to an individual molecule or a homogeneous or heterogeneous population of antigenic molecules. The antigen recognized by the antibodies, receptors of T cells or other elements of a specific humoral and/or cellular immunity. The term "antigen" includes all related antigenic epitopes. Epitopes of a given antigen can be identified 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 EPI the experience can be determined by, for example, the parallel synthesis of large numbers of peptides on solid supports, the peptides corresponding to parts of the protein molecule, and carrying out the reaction of peptides with antibodies, while 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, which are all incorporated here by reference in their entirety. Similarly, conformational epitopes can be identified by determining spatial conformation of amino acids, as, for example, by x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, an epitope Mapping Protocols, above. In addition, for the purposes of the present invention, the term "antigen" can also be used in relation to a protein which includes modifications, such as deletions, insertions and substitutions (generally conservative in nature, but they can be non-conservative), the native sequence, so long as the protein retains the ability to induce an immune response. These modifications may be deliberate, such as obtained through site-directed mutagenesis, or by specific synthetic procedures, or by the approach of genetic engineering, or may be accidental, such as when the ICC is of the owner, which produce antigens. In addition, the antigen can be derived, obtained or isolated from a microbe, for example, bacteria, or can represent the whole body. Similarly, oligonucleotide or polynucleotide, which expresses the antigen, such as, for example, in applications immunization nucleic acids are also included in this definition. Also included are synthetic antigens, such as polyepitope flanking epitopes and other antigens, obtained by recombinant or fusion (Bergmann et al. (1993) Eur. J. Immunol. 23:2777 2781; Bergmann et al. (1996) J. Immunol. 157:3242 3249; Suhrbier, A. (1997) Immunol. and Cell Biol. 75:402 408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, Jun. 28 - Jul. 3, 1998).

The term "adjuvant" refers to a compound or mixture that enhances the immune response to the antigen, as described below and illustrated with examples here.

"Bacteremia" is a temporary presence of bacteria in the blood. Bacteremia may develop septicemia or sepsis, which will be considered as infection and is a constant presence of bacteria in the blood associated with clinical signs/symptoms. Not all bacteria can survive in the blood. Only those who really have a special genetic characteristics that provide this ability. Also important are the factors of the host.

Ter is in the capsular polysaccharide or polysaccharide capsule" refers to a polysaccharide capsule, which is located outside the cell wall of most isolates of staphylococci. For example, S. aureus includes a component of the cell wall, consisting of peptidoglycan complex, which makes it possible for the organism's survival under adverse osmotic conditions and also includes a unique taikoubou acid that is associated with the peptidoglycan. On the outside of the cell wall thin polysaccharide capsule covers most isolates of S. aureus. This is serologically special capsule can be used for serotyping of different isolates of S. aureus. It has been shown that many of clinically significant isolates include two types of capsules: serotype 5 (SR) and serotype 8 (SR). Patterns SR and SR shown schematically in Fig.4.

The term "conjugate" as used here, includes capsular polysaccharide, usually with the desired interval, molecular weight, and a carrier protein, where the capsular polysaccharide anywhereman with protein carrier. The conjugates may contain or may not contain some free capsular polysaccharide. The phrase "free capsular polysaccharide" as it is used here, refers to the capsular polysaccharide, which is ecovalence associated with (i.e. ecovalence associated with adsorbed on or taken, or (C) conjug is included capsular polysaccharide-protein carrier. The terms "free capsular polysaccharide", "free polysaccharide" and "free sugar" may be used interchangeably and are intended for transmission to the same value. Regardless of the nature of the molecules of the carrier, it can be conjugated with capsular polysaccharide, either directly or through a linker. The terms "conjugality", "conjugated" and "conjugation" as used here, refers to the method by which bacterial capsular polysaccharide covalently attached to the molecule-media. Conjugation enhances the immunogenicity of a bacterial capsular polysaccharide. Conjugation can be performed according to the methods described below or by methods known in this field.

As described above, the present invention relates to conjugates containing capsular polysaccharide serotype 5 (SR) S. aureus conjugated to protein carriers and conjugates containing capsular polysaccharide serotype 8 (SR) S. aureus conjugated to protein carriers. According to one embodiment of the invention proposed conjugates containing capsular polysaccharide serotype 5 S. aureus, conjugated to a protein carrier, and capsular polysaccharide serotype 8 S. aureus, conjugated to protein carrier, where: capsular polysaccharide type 5 is molecule the reduction in weight from 50 kDa to 800 kDa; capsular polysaccharide type 8 has a molecular mass of from 50 to 700 kDa; immunogenic conjugates have a molecular weight from about 1000 kDa to about 5000 kDa; and conjugates contain less than about 30% of free polysaccharide in relation to the total polysaccharide. In one embodiment, the conjugates contain less than about 25%, about 20%, about 15%, about 10% or about 5% of free polysaccharide in relation to the total polysaccharide. In one embodiment, the polysaccharide type 5 or 8 has a molecular mass of 20 kDa to 1000 kDa.

In one embodiment, the conjugate has a molecular weight of from about 50 kDa to about 5000 kDa. In one embodiment, the conjugate has a molecular weight of from about 200 kDa to about 5000 kDa. In one embodiment of the immunogenic conjugate has a molecular weight of from about 400 kDa to about 2500 kDa. In one embodiment of the immunogenic conjugate has a molecular weight of from about 500 kDa to about 2500 kDa. In one embodiment of the immunogenic conjugate has a molecular weight of from about 600 kDa to about 2800 kDa. In one embodiment of the immunogenic conjugate has a molecular weight of from about 700 kDa to about 2700 kDa. In one embodiment of the immunogenic conjugate has a molecular weight of from about 1000 kDa to about 2000 kDa, from about 1800 kDa to about 2500 kDa, from about 1100 kDa to about 2200 kDa; about what about 1900 kDa to about 2700 kDa; from about 1200 kDa to about 2400 kDa; from about 1700 kDa to about 2600 kDa; from about 1300 kDa to about 2600 kDa; from about 1600 kDa to about 3000 kDa.

Accordingly, in one embodiment the carrier protein to the immunogen conjugate according to the invention is a CRM197and CRM197covalently linked to the capsular polysaccharide via a urethane bond, amide bond, or both links. The number of lysine residues in the protein carrier, which are conjugated with capsular polysaccharide, can be described as the interval 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 option is that from 12% to 40% lysine CRM197covalently linked to the capsular polysaccharide. In some embodiments part of the CRM197polysaccharide covalently linked to CRM197contains from 5 to 22 lysine covalently associated with the polysaccharide. In some embodiments part of the CRM197polysaccharide covalently linked to CRM197contains from 5 to 23 lysine covalently associated with the polysaccharide. In some embodiments part of the CRM197polysaccharide covalently linked to a protein carrier contains from 8 to 15 lysine covalently associated with the polysaccharide. is some embodiments the part of the CRM 197polysaccharide covalently linked to a protein carrier contains from 8 to 12 lysine covalently associated with the 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 associated 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 associated 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 characterized by the interval conjugated lysine, which can be expressed as a molar ratio. For example, the molar ratio of conjugated lysine to CRM197in the immunogen conjugate SR can be from about 18:1 to about 22:1. In one embodiment the interval molar ratio of conjugated lysine to CRM197in the immunogen conjugate SR can costal is to be from about 15:1 to about 25:1. In some embodiments, the interval molar ratio of conjugated lysine to CRM197in the immunogen conjugate SR may be from about 14:1 to about 20:1; from about 12:1 to about 18:1; from about 10:1 to about 16:1; from about 8:1 to about 14:1; from about 6:1 to about 12:1; from about 4:1 to about 10:1; from about 20:1 to about 26:1; from about 22:1 to about 28:1; from about 24:1 to about 30:1; from about 26:1 to about 32:1; from about 28:1 to about 34:1; from about 30:1 to about 36:1; from about 5:1 to about 10:1; from about 5:1 to about 20:1; from about 10:1 to about 20:1 or from about 10:1 to about 30:1. Also the molar ratio of conjugated lysine to CRM197in the immunogen conjugate SR may be from about 3:1 to 25:1. In one embodiment the interval molar ratio of conjugated lysine to CRM197in the immunogen conjugate SR may be from about 5:1 to about 20:1. In one embodiment the interval molar ratio of conjugated lysine to CRM197in the immunogen conjugate SR can be from about 4:1 to about 20:1; from about 6:1 to about 20:1; from about 7:1 to about 20:1; from about 8:1 to about 20:1; from about 10:1 to about 20:1; from about 11:1 to about 20:1; from about 12:1 to about 20:1; from about 13:1 to about 20:1; from about 14:1 to about 20:1; about the about 15:1 to about 20:1; from about 16:1 to about 20:1; from about 17:1 to about 20:1; from about 18:1 to about 20:1; from about 5:1 to about 18:1; from about 7:1 to about 16:1 or from about 9:1 to about 14:1.

Another way of expressing the number of lysine residues in the protein-carrier, kongugirovannom with capsular polysaccharide may be spaced conjugated lysine. For example, given by the immunogen conjugate SR CRM197may contain from 5 to 15 lysine 39, covalently associated with capsular polysaccharide. Alternatively, this parameter can be expressed as a percentage. For example, given by the immunogen conjugate SR percentage of conjugated lysine can be from 10% to 50%. In some embodiments, from 20% to 50% lysine can be covalently linked to SR. However, 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 CRM197; or from 10% to 12% lysine CRM197covalently linked to SR. Also in this by the immunogen conjugate SR CRM197may contain from 18 to 22 lysine 39, Cova is into associated with capsular polysaccharide. Alternatively, this parameter can be expressed as a percentage. For example, given by the immunogen conjugate SR percentage of conjugated lysine can be from 40% to 60%. In some embodiments, from 40% to 60% lysine can be covalently linked to SR. However, alternatively, from 30% to 50% lysine CRM197can be covalently linked to SR; from 20% to 40% lysine CRM197; from 10% to 30% lysine CRM197; from 50% to 70% lysine CRM197; from 35% to 65% lysine CRMigy; 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 CRM197or from 45% to 75% lysine CRM197covalently linked to SR.

Frequency attach chain capsular polysaccharide to a lysine on the media molecule is another parameter for characterization of conjugates of capsular polysaccharides. For example, in one embodiment at least one covalent bond between CRM197and the polysaccharide occurs at least every 5-10 sharidny recurring units of the capsular polysaccharide. In another embodiment there is at least one covalent bond between CRM197and capsular polysaccharide on every 5-10 sharidny repeating units; every 2-7 sharidny repeating units; every 3-8 sa is arid repeating units; every 4-9 sharidny repeating units; every 6-11 sharidny repeating units; every 7-12 sharidny repeating units; every 8-13 sharidny repeating units; every 9-14 sharidny repeating units; every 10-15 sharidny repeating units; every 2-6 sharidny repeating units; every 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; every 9-13 sharidny repeating units; every 10-14 sharidny repeating units; every 10-20 sharidny repeating units; every 5-10 sharidny recurring units of the capsular polysaccharide. In another embodiment, at least one link between CRM197and capsular polysaccharide occurs on every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 sharidny recurring units of the capsular polysaccharide.

Chemical activation of polysaccharides and subsequent conjugation to a protein carrier can be achieved by traditional methods. See, for example, U.S. patent No. 4673574 and 4902506. Alternatively, you can use other methods of activation and conjugation.

The terms "a carrier protein" or "protein carrier" as used here, refers to either the protein molecule, which can be conjugated to the antigen (such as capsular polysaccharides), against which is the desired immune response. Conjugation of an antigen, such as a polysaccharide with protein carrier can make the immunogenic antigen. Carrier proteins preferably are proteins that are non-toxic and directivename, and which can be obtained in sufficient quantity and with sufficient purity. Examples of protein-carriers are toxins, toxoids or any mutant cross-reacting material (CRM197) tetanus toxin, diphtheria toxin, pertussis toxin, a toxin from Pseudomonas species, Escherichia coli, species of Staphylococcus and Streptococcus species. Carrier proteins must be susceptible to the standard methods of conjugation. In a particular embodiment of the present invention CRM197used as a protein carrier.

CRM197(Wyeth/Pfizer, Sanford, NC) is a non-toxic variant (i.e., the toxoid) of diphtheria toxin isolated from cultures of strain C7 Corynebacterium reagent grade (β197) grown in an environment based on Kazimirovich acids and yeast extract. CRM197purified by ultrafiltration, precipitation with ammonium sulfate and ion-exchange chromatography. Culture of a strain of Corynebacterium diphtheriae C7 (β197), which produces a protein CRM197was deposited in the U.S. is the oceanic type culture collection (Rockville, Maryland) and it was given the number to access ATS 53281. Other diphtheria toxoids are also suitable for use as a protein carrier.

Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (for example as described in international patent application WO2004/083251), LT (thermolabile toxin E. coli, ST (heat-stable toxin) E. coli and exotoxin a from Pseudomonas aeruginosa. You can also use bacterial outer membrane proteins, such as protein complex with the outer membrane (OMRS), porins, transferringthe proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal protein-adhesin A (PsaA), enterotoxin (toxin a) and cytotoxin (toxin B) C. difficile or protein D of Haemophilus influenzae. As protein carriers can also be used for other proteins, such as ovalbumin, hemocyanin lymph snails (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD).

After conjugation of capsular polysaccharide to a protein carrier conjugates polysaccharide-protein purified (enriched in the quantity of conjugate polysaccharide-protein) of different methods. These techniques include, for example, concentration/diafiltration, sedimentation/elution, column chromatography and the GLA is different filtering. See the examples below.

After purification of individual conjugates, you can combine them to cook in the preparation of immunogenic compositions of the present invention, which can be used, for example, in the vaccine. Preparation in the form of the preparation of immunogenic compositions of the present invention can be implemented using known in the field of methods.

It is noted that in this specification, terms such as "contains", "contains", "containing", "contains", "containing", and the like can have the meaning attributed to them in the patent law of the United States; for example, they can mean "includes", "comprised", "comprising" and the like. Such terms to include specific ingredient or set of ingredients without the exclusion of any other ingredients. Terms such as "consisting essentially of" and "consists essentially of" have the meanings ascribed to them in the patent law of the United States; for example, they allow the inclusion of additional ingredients or stages that do not detract from the new or the main characteristics of the invention, i.e., they exclude the non-listed ingredients or stage that belittle new or basic characteristics of the invention, and they eliminate the ingredients or the stage of the prior art, such as the document which you are in this area, listed here or included here by reference, especially since the purpose of this document is to define the embodiments that are patentable, for example, new, subtle, inventive, in light of the prior art, for example, compared with the documents cited herein or incorporated here by reference. And the terms "comprises" and "comprising" shall have the meaning ascribed to them in the patent law of the United States; namely, that these terms are closed (close-ended). Accordingly, these terms refer to the inclusion of a specific ingredient or set of ingredients and to the exclusion of all other ingredients.

"Conservative amino acid substitution" refers to substitution of one or more than one amino acid residue of the protein with other amino acid residues having similar physical and/or chemical properties. Substitutes for an amino acid within the sequence may be selected from other members of the class to which belongs the amino acid. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, Proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structure, are phenylalanine, tryptophan and tyrosine. The polar neutral amino acids involved in the t glycine, series, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic amino acids include aspartic acid and glutamic acid. It is expected that such changes will not affect the apparent molecular mass determined by polyacrylamide gel electrophoresis, or isoelectric point. Particularly preferred substitutions are substitution of Lys for Arg and Vice versa, so you keep a positive charge; Glu for Asp and Vice versa, so that you can maintain a negative charge; Ser for Thr so that you can preserve a free group-HE; and Gln for Asn, so that you can preserve a free group NH2.

The term "fragment" refers to proteins, in which only selected domains of a larger protein. For example, each of the proteins ClfA and ClfB contain up to 8 domains, if you enable signal sequence. Each polypeptide corresponding to the domains N1N2N3, N2N3, N1N2, N1, N2 or N3, is a fragment of ClfA or ClfB. The term "fragment" also refers to either a protein or polypeptide containing the amino acid sequence of at least 4 amino acid residues (preferably at least 10 amino acid residues, at least 15 amino acid residues, n is at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues) amino acid sequence of the parent protein or polypeptide, or nucleic acid, containing the nucleotide sequence of at least 10 base pairs, preferably at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 50 base pairs, at least 100 base pairs, at least 200 base pairs) of the nucleotide sequence in the parent nucleic acid.

The terms "functional activity of the antibodies or functional antibody", as used here, refers to an antibody that, at least, may be specific to contact the antigen. In this area known more features, and they may include additional components of the immune system which carry out clearance or killing pathoge is a, as, for example, through opsonization, ADCC (antibody-dependent cellular cytotoxicity) or cytotoxicity, mediated by complement. After binding of the antigen any subsequent functions antibodies may be mediated by the Fc region of antibodies. Analysis of opsonophagocytic activity of antibodies (PRA) represents the in vitro assays designed to measure the in vitro killing of bacteria effector cells (white blood cells), performed using Ig complement that, therefore, mimics the biological process. The binding of an antibody may also directly inhibit the biological function of antigen with which it is associated, for example, antibodies that bind ClfA, can neutralize its enzymatic function. In some embodiments, the term "functional antibody" refers to an antibody that is functional according to the measurement results for killing bacteria in an animal model of efficiency or test opsonophagocytosis killing, which demonstrates that the antibodies cause the lysis of bacteria.

Molecular weight capsular polysaccharides of S. aureus is a factor considered when used in immunogenic compositions. For example, capsular polysaccharides with high molecular weight can be able to induce certain Mannie answers antibodies due to the higher valency of epitopes, present on the antigenic surface. Selection of capsular polysaccharides with high molecular weight" is considered for use in the compositions and methods of the present invention. For example, in one embodiment of the invention considers the allocation of polysaccharides type 5 with high molecular weight, ranging in size from about 50 to about 800 kDa molecular weight. In one embodiment of the invention considers the allocation of polysaccharides type 5 with high molecular weight, ranging in size from about 20 to about 1000 kDa molecular weight. In one embodiment of the invention addresses the isolation and purification of capsular polysaccharide of type 5 of high molecular mass, ranging in size from about 50 to about 300 kDa molecular weight. In one embodiment discusses the isolation and purification of capsular polysaccharide type 5 with high molecular weight, varying in molecular weight from 70 kDa to 300 kDa. In one embodiment discusses the isolation and purification of capsular polysaccharide type 5 with high molecular weight, varying in molecular weight from 90 kDa to 250 kDa. In one embodiment discusses the isolation and purification of capsular polysaccharide type 5 with high molecular weight, varying in molecular weight from 90 kDa to 150 kDa In one embodiment discusses the isolation and purification of capsular polysaccharide type 5 with high molecular weight, ranging in molecular weight from 90 kDa to 140 kDa. In one embodiment discusses the isolation and purification of capsular polysaccharide type 5 with high molecular weight, varying in molecular weight from 80 kDa to 120 kDa. Other intervals capsular polysaccharide serotype 5 with high molecular weight that you can select and clear the way for this invention include ranges in size from about 70 kDa to about 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; from 80 kDa to 110 kDa in 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 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 cddo 150 kDa in molecular weight; from 100 kDa to 160 kDa in molecular weight; and similar desirable intervals molecular weight.

As discussed above, the molecular weight capsular polysaccharides of S. aureus is a factor considered when used in immunogenic compositions. For example, capsular polysaccharides with high molecular weight can be able to induce certain immune responses, antibodies due to the higher valency of epitopes present on the antigenic surface. In one embodiment of the invention addresses the isolation and purification of capsular polysaccharide type 8 with high molecular weight, varying in molecular weight from about 20 kDa to about 1000 kDa. In one embodiment of the invention addresses the isolation and purification of capsular polysaccharide type 8 with high molecular weight, varying in molecular weight from about 50 kDa to about 700 kDa. In one embodiment of the invention addresses the isolation and purification of capsular polysaccharide type 8 with high molecular weight, varying in molecular weight from 50 kDa to 300 kDa. In one embodiment discusses the isolation and purification of capsular polysaccharide type 8 with high molecular weight, varying in molecular weight from 70 kDa to 300 kDa. In one embodiment discusses the isolation and purification of ka is solnyh polysaccharide type 8 with high molecular weight, ranging in molecular weight from 90 kDa to 250 kDa. In one embodiment discusses the isolation and purification of capsular polysaccharide type 8 with high molecular weight, varying in molecular weight from 90 kDa to 150 kDa. In one embodiment discusses the isolation and purification of capsular polysaccharide type 8 with high molecular weight, varying in molecular weight from 90 kDa to 120 kDa. In one embodiment discusses the isolation and purification of capsular polysaccharide type 8 with high molecular weight, varying in molecular weight from 80 kDa to 120 kDa. Other intervals capsular polysaccharide serotype 8 with high molecular weight that you can select and clear the way for this invention include ranges in size from about 70 kDa to about 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; from 80 kDa to 110 kDa in 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 mole on Blarney 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 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 desirable intervals molecular weight.

"The immune response to the immunogenic composition is a development of the subject of a humoral and/or cell-mediated immune response to molecules present in the desired composition (e.g., antigen, such as a protein or polysaccharide). For the purposes of the present invention "humoral immune response" is an immune response mediated by antibodies, and includes the generation of antibodies with affinity against antigens present in the immunogenic compositions according to the invention, whereas cell-mediated immune response is an immune response mediated by T lymphocytes and/or other white blood cells. "Cell-mediated immune response causes the presentation of antigenic epitopes in Association with molecules of class I or class II major histocompatibility complex (MHC). Uh what about the activates cells antigen specific T-helper cells CD4+ cell or cytotoxic T-lymphocytes CD8+ ("CTL"). CTL have specificity against peptide or lipid antigens that are presented in Association with proteins encoded by the major histocompatibility complex (MHC) or CD1, and expressed on the surfaces of cells. CTL helps to induce and stimulate the intracellular destruction of intracellular microbes or lysis of cells infected with these germs. Another aspect of cellular immunity involves antigen specific response of T cells-helper cells. T-cell helper function, helping to stimulate the function and focus the activity of nonspecific effector cells against cells, demonstrating peptide antigens in Association with classical or non-classical MHC molecules on their surface. "Cell-mediated immune response" also refers to the production of cytokines, chemokines and other molecules produced by activated T-cells and/or other white blood cells, including cells derived from t cells, CD4+ and CD8+. The ability of a particular antigen or composition to stimulate cell-mediated immunological response may be determined by a number of analyses, such as analyses of lymphoproliferation (lymphocyte activation) assays cytotoxic cells CTL, analysis of T-lymphocytes that are specific against the antigen in sensitized subject, or measured is eaten 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., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376.

The term "immunogenic" refers to the ability of an antigen or vaccine to induce an immune response or a humoral or cell-mediated, or both.

The terms "immunogenic amount" or "immunologically effective amount" or "dose", each of which is used here interchangeably, in General, refers to the amount of antigen or immunogenic composition sufficient to produce an immune response or a cellular (T-cell) or humoral (b-cell or antibody) response, or both when measured by standard assays known to a person skilled in the field.

A number of specific conjugate in the composition is usually calculated on the basis of the total polysaccharide conjugated and unconjugated, with respect to the conjugate. For example, the conjugate SR with 20% of free polysaccharide will have about 80 μg of conjugated polysaccharide SR and about 20 μg unconjugated polysaccharide SR 100 μg dose 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 the serotype of Staphylococcus. Each dose will contain from 0.01 to 100 μg of polysaccharide in h is particular from 0.1 to 10 μg, and more specifically from 1 to 10 mcg. "Immunogenic amount" of different polysaccharide components in the immunogenic composition may be different, and each may contain 0,01 ág, 0.1 ág, of 0.25 μg, 0.5 μg, 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.

In another embodiment "immunogenic amount" of protein components in the immunogenic compositions can vary from about 10 μg to about 300 μg of each protein antigen. In a specific embodiment of an "immunogenic amount" of protein components in the immunogenic compositions can vary from about 20 μg to about 200 μg of each protein antigen. "Immunogenic amount" of different protein components in immunogenic compositions may deviate, and each contains 10 mcg, 20 mcg, 30 mcg, 40 mcg, 50 mcg, 60 mcg, 70 mcg, 80 mcg, 90 mcg, 100 mcg, 125 mcg, 150 mcg, 175 mcg, or about 200 μg any specific protein antigen.

The efficiency of antigen as the immunogen can be measured by measuring the levels of activity In cells by measuring the levels of circulating antibodies specific against the antigen in serum, with use of immunoassays, analyses thus, functional antibody assays, such as opsonic analysis in vitro, and many of the x other analyses, known in this field. Another indicator of the effectiveness of antigen as the immunogen T cells can be measured either by proliferation assays, or cytolytic tests, such as tests with radioactive chromium to measure the ability of T cells to lyse its specific the target cell. In addition, in the present invention "immunogenic amount" can be determined by measurement of the levels of antigen specific antibodies induced after injection of antigen in serum or by measuring the ability of the antibodies induced in this way, to increase opsonophagocytic capacity of specific white blood cells, as described here. The security level of the immune response can be measured by stimulation of the immunized host antigen, which was injected. For example, if the antigen to which the desired immune response, is a bacterium, the level of protection induced "immunogenic amount" of an antigen can be measured by determining the percent survival or percent mortality after infection animals bacterial cells. In one embodiment 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 of the antigens is multiantigenic or multicomponent vaccine, or immunogenic compositions will vary with respect to each other component and can be determined by methods known to the expert. Such methods include, for example, methods for measuring the immunogenicity and/or efficacy in vivo.

The term "immunogenic composition" refers to any pharmaceutical composition containing the antigen, such as a microorganism or its component, the composition can be used to invoke an immune response in the subject. Immunogenic compositions of the present invention can be used for treatment of humans, susceptible to S. aureus infection, through the introduction of immunogenic compositions through the system percutaneous path, or through the mucous. Such introduction may include injection via the intramuscular (i.m.), intraperitoneal (i.p.), intradermal (i.d.) or subcutaneous routes; application through a patch or other device for percutaneous delivery; or by insertion through the mucous membrane in the mouth/digestive tract, respiratory, or genitourinary tracts. In one embodiment for the treatment or prevention of nasopharyngeal carriage of S. aureus using intranasal introduction, thus, weakening the infection at its earliest stage. In one embodiment of the immunogenic composition can be used in the manufacture of vaccines or in the products (elicitation) polyclonal or monoclonal antibodies, which can be used for passive protection or treatment of the animal.

The optimal number of components for a specific immunogenic composition can be ascertained by standard studies involving observation of appropriate immune responses in subjects. After the initial vaccination, subjects may receive one or several booster immunizations with an adequate interval of time.

In one embodiment of the present invention immunogenic composition based S. aureus contains a fragment (N1N2N3 or combinations thereof) recombinant clumping factor And S. aureus (ClfA), isolated capsular polysaccharide type 5 conjugated to CRMigy, and selected capsular polysaccharide type 8 conjugated to CRM197. In another embodiment of the immunogenic composition based S. aureus is a sterile composition (liquid, dried, DNA vaccine, intradermal drug) fragment (N1N2N3, or a combination) of recombinant factor in the adhesion of S. aureus (ClfA), fragment (N1N2N3 or combinations thereof) recombinant clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 conjugated to CRM197and selected capsular polysaccharide type 8 conjugated to CRM197. In one embodiment of the present invention immunogenic composition based S. aureus contains a fragment (N1N2N3 or them to whom Binali) recombinant clumping factor A S. aureus (ClfA), iron-binding protein of S. aureus MntC allocated capsular polysaccharide type 5 conjugated to CRM197and selected capsular polysaccharide type 8 conjugated to CRM197. In one embodiment of the immunogenic composition based S. aureus is a sterile composition (liquid, dried, DNA vaccine, intradermal drug) fragment (N1N2N3 or combinations thereof) recombinant clumping factor A of S. aureus (ClfA), fragment (N1N2N3 or combinations thereof) recombinant clumping factor In S. aureus (ClfB), iron-binding protein of S. aureus MntC allocated capsular polysaccharide type 5 conjugated to CRM197and selected capsular polysaccharide type 8 conjugated to CRM197. In one embodiment of the present invention immunogenic composition based S. aureus contains a fragment (N1N2N3 or combinations thereof) recombinant clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 conjugated to CRM197and selected capsular polysaccharide type 8 conjugated to CRM197. In one embodiment of the present invention immunogenic composition based S. aureus contains a fragment (N1N2N3 or combinations thereof) recombinant clumping factor In S. aureus (ClfB), iron-binding protein of S. aureus MntC allocated capsular polysaccharide type 5 conjugated to CRM197and dedicated ka is solnye polysaccharide type 8, conjugated to CRM197. In one embodiment of the present invention immunogenic composition based S. aureus contains iron-binding protein of S. aureus MntC allocated capsular polysaccharide type 5 conjugated to CRM197and selected capsular polysaccharide type 8 conjugated to CRM197.

Immunogenic compositions of the present invention can optionally contain one or more than one additional "immunomodulator", which is an agent that disrupts or alters the immune system, so that there is either positive or negative regulation of humoral and/or cell-mediated immunity. In one particular embodiment, it is preferable positive regulation of humoral and/or cell-mediated branches of the immune system. The specific examples of immunomodulators include, for example, adjuvant or cytokine, or ISCOMATRIX (CSL Limited, Parkville, Australia) described, among others, in U.S. patent No. 5254339. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention, include adjuvant system RIBI (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as gel aluminium hydroxide, emulsions of the type oil-in-water, emulsion type water in oil", such as, for example, complete and incomplete adjuvants blockers, block-copolyme the (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), adjuvant AMPHIGEN®, saponin, Quil a or another faction of saponin, monophosphorylated and lipid-amine adjuvant Avridine. Non-limiting examples of emulsions of the type oil-in-water, useful in the vaccine according to the invention include modified SEAM62 SEAM and 1/2 of the composition. 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", which can be included in the vaccine include, for example, one or more than one interleukin, interferon, or other known cytokine or chemokine. In one embodiment the adjuvant may be a derivative of cyclodextrin or polyanionic polymer, such as derivatives of cyclodextrin or polyanionic polymers described in U.S. patent number 6165995 and 6610310, respectively. It should be understood that the immunomodulator and/or adjuvant to be applied, will depend on the subject, which will enter the TB vaccine or immunogenic composition, route of injection and the number of injections to be administered.

"Invasive disease caused by S. aureus, is a selection of bacteria from a normally sterile area when there are associated clinical signs/symptoms. Normally sterile areas of the body include blood, CSF (cerebrospinal fluid), pleural fluid, pericardial fluid, peritoneal fluid, joint/synovial fluid, bone, internal area in the body (lymph node, brain, heart, liver, spleen, vitreous body, kidney, pancreas, ovary) or other normally sterile sites. Clinical condition characterizing invasive disease include bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis, septic shock and other.

The term "isolated" means that the substance has been removed from its original environment (for example, the natural environment if it occurs in nature, or from the organism of the host, if it is a recombinant object, or taken from one environment and placed in another environment). For example, isolated capsular polysaccharide, protein or peptide essentially does not contain cellular material or other contaminating proteins from the cell or tissue source from which derived protein, or essentially does not contain a chemical which is a mini precursors or other chemicals, if he synthesized chemically or is otherwise in the mix as part of a chemical reaction medium. In the present invention are proteins or polysaccharides can be isolated from bacterial cells or from the decomposition products of the cells so that they are received in a form useful for the production of immunogenic compositions. The term "isolated" or "allocation" may include cleaning or purification procedures, including, for example, methods of purification of proteins or capsular polysaccharides, as described here. The phrase "essentially does not contain cellular material" includes preparations of the polypeptide/protein in which the polypeptide/protein is separated from cellular components of the cells from which it is selected or in which he recombinante produced. Thus, capsular polysaccharide, protein or peptide, which essentially does not contain cellular material includes preparations of capsular polysaccharide, protein or peptide having less than about 30%, 20%, 10%, 5%, 2,5% or 1% (by dry weight) contaminating protein or polysaccharide, or other cellular material. When recombinant production of the polypeptide/protein, it is also preferably essentially does not contain culture medium, i.e., culture medium represents less than about 20%, 10% or 5% of the volume of the protein drug. When the polypeptide/protein or polysaccharide produced by himicheskim synthesis, it is preferably essentially does not contain chemical precursors or other chemicals, i.e., 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% (by dry weight) of chemical precursors or compounds other than interest polypeptide/protein or polysaccharide fragment.

The phrase "non-conservative amino acid substitution" refers to substitution of one or more than one amino acid residue of the protein with other amino acid residues having different physical and/or chemical properties, using the characteristics defined above.

The term "pharmaceutically acceptable carrier" means a carrier approved by the regulatory authority of the Federal government, state government or other regulatory body, or listed in the U.S. Pharmacopoeia or other generally recognized Pharmacopoeia for use in animals, including humans, mammals, non-human. The term "carrier" refers to a diluent, adjuvant, excipient or filler, which is administered pharmaceutical composition. Such pharmaceutical carriers can be sterile liquids, t is Kimi as water and oil, including liquid petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol can be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor quantities of moisturizing, obamaobama, emulsifying and tabularasa agents. These compositions can take the form of solutions, suspensions, emulsions, preparations, slow release, and the like. Examples of suitable pharmaceutical carriers are described in "Remington''s Pharmaceutical Sciences" by E. W. Martin. The composition should correspond to the method of administration.

The terms "protein", "polypeptide" and "peptide" refer to a polymer of amino acid residues and are not limited by the minimum length of the product. Thus, peptides, oligopeptides, dimers, multimer and the like are included within this definition. The definition of a covered full-sized proteins, and fragments thereof. These terms also include modifications,such as deletions, insert (additions and substitutions (generally conservative in nature, but which may be non-conservative), relative to the native sequence, preferably such that the protein retains the ability to induce an immunological response in an animal, which is introduced protein. Also included postexplosion modifications, e.g. glycosylation, acetylation, limitirovanie, phosphorylation and the like.

The term "protective" immune response refers to the ability of the immunogenic composition to induce an immune response or a humoral or cell-mediated, which serves to protect the subject from infection. Protection has been provided need not be absolute, i.e., the infection does not necessarily have to be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population of subjects, for example, infected animals that did not enter the vaccine or immunogenic composition. Protection may be limited to reduce the severity or rate of appearance of symptoms of the infection. In General, a "protective immune response" will include the induction of increased levels of antibodies specific against a particular antigen, at least 50% of the subjects, including some level of measurable responses of functional antibodies for each antigen. In to the particular situations "protective immune response" may include the induction twofold increase in antibody levels or a fourfold increase in antibody levels, specific specific antigen, at least 50% of the subjects, including some level of measurable responses of functional antibodies for each antigen. In certain embodiments opsonizing antibodies correlate with the protective immune response. Thus, a protective immune response can be analyzed by measuring the percentage reduction in the number of bacteria in the analysis opsonophagocytic aktivnosti, for example, the analyses 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.

The term "recombinant" as used here simply refers to any protein, polypeptide or a cell expressing the gene of interest, which is produced by means of genetic engineering. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of recombinant polynucleotide. Proteins used in the immunogenic compositions according to the invention can be isolated from a natural source or obtained by means of genetic engineering, such as recombinant ClfA, recombinant or recombinant ClfB MntC. The term "recombinant" as it is used here, additionally describes a nucleic acid molecule, which, by virtue of its origin is placed or through manipulation, not associated with all polynucleotides or part of polynucleotide, with which it is associated in nature. The term "recombinant" as used in relation to the cell-master means the cell host, which was introduced recombinant polynucleotide.

Recombinant ClfA (rClfA) and recombinant ClfB (rClfB), as they are used here, refers to forms of ClfA or ClfB for use in immunogenic compositions according to the invention. In one embodiment rClfA is a fragment of a ClfA containing one or more than one of the N domains, for example, N1N2N3, N2N3, N2 or N3, and is referred to here as "recombinant ClfA or rClfA". In one embodiment rClfB is a fragment of ClfB, containing one or more than one of the domains N ClfB, for example, N1N2N3, N2N3, N2 or N3, and is referred to here as "recombinant ClfB or rClfB".

The term "subject" refers to a mammal, bird, fish, reptile or any other animal. The term "subject" includes humans. 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 livestock. Non-limiting examples of livestock include Alpaca. bison, verbl is Yes, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese and turkeys.

The term "treatment" as it is used here (including its variations, for example, "treat" or "treated"), refers to one or more than one of the following: (1) prevention of infection or re-infection, as in the case of traditional vaccines, (2) reduce the severity or eliminate symptoms, and (3) substantial or complete elimination of the considered pathogen or disorder. Therefore, the treatment can be carried out prophylactically (prior to infection) or therapeutically (after infection). In the present invention can be used for preventive or therapeutic treatment. According to a specific embodiment of the present invention proposed compositions and methods that treat, including preventive and/or therapeutic immunization of an animal host against microbial infection (e.g., bacteria such as bacteria of the genus Staphylococcus). The methods of the present invention are useful for giving the subject a prophylactic and/or therapeutic immunity. The methods of the present invention also can be practiced on the subjects for biomedical research applications.

The terms "vaccine" or "vaccine composition", to the E. are used interchangeably, refer to pharmaceutical compositions containing at least one immunogenic composition that induces an immune response in an animal.

General description

The present invention relates to immunogenic compositions containing at least three antigen from Staphylococcus organism such as S. aureus. Antigens can be isolated from the body with the use of biochemical methods for the extraction, or they can be produced synthetically or by recombinant methods. Antigens can be polypeptides or polysaccharides, or a combination of both. These immunogenic compositions can be used in the manufacture of vaccines to immunize subjects against infections caused by staphylococcal organism. Components suitable for use in these compositions, described in more detail below.

Staphylococcal immunogenic composition

S. aureus is the causative agent of a wide range of human diseases, ranging from superficial skin infections to life-threatening conditions such as pneumonia, sepsis and endocarditis. See Lowy N. Eng. J. Med. 339:580-532(1998). In cases of invasive disease S. aureus can be isolated from normally sterile areas of the body, including blood, cerebrospinal fluid (CSF), pleural fluid, pericardial fluid, peritoneal LM the bone, joint/synovial fluid, bone, internal region in the body (lymph node, brain, heart, liver, spleen, vitreous (vitreous fluid), kidney, pancreas, ovary) or other normally sterile areas. This can lead to life-threatening clinical condition, such as bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis and septic shock. Adults, elderly patients and patients are children most at risk of S. aureus infections.

In embodiments of the present invention described selected antigens in immunogenic compositions comprising the selected polypeptide of clumping factor And S. aureus (ClfA), isolated capsular polysaccharide type 5 S. aureus, conjugated to protein carrier, selected capsular polysaccharide type 8 S. aureus, conjugated to protein carrier allocated to the clumping factor In S. aureus (ClfB) and a recombinant protein of S. aureus MntC. Then the antigens were characterized in immunogenic compositions as a series of combinations to demonstrate that a specific combination provide immune responses that can surpass the immune responses obtained with the use of the individual components of immunogenic compositions.

Accordingly, in one combination proposed immunogenic composition containing the selected polypeptide of clumping factor And S. aureus (ClfA), leased the military capsular polysaccharide type 5 S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 S. aureus, conjugated to a protein carrier. In the second proposed combination immunogenic composition containing the selected polypeptide of clumping factor A of S. aureus (ClfA), highlighted the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 S. aureus, conjugated to a protein carrier. In the third combination proposed immunogenic composition containing the selected polypeptide of clumping factor A of S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 S. aureus, conjugated to a protein carrier. In the fourth combination of the proposed immunogenic composition containing the selected polypeptide of clumping factor A of S. aureus (ClfA), isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 S. aureus, conjugated to a protein carrier. In the fifth combination proposed immunogenic composition containing the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 S. aures, conjugated with protein carrier and selected capsular polysaccharide type 8 S. aureus, conjugated to a protein carrier. In the sixth combination proposed immunogenic composition containing the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 S. aureus, conjugated to a protein carrier. In the seventh combination proposed immunogenic composition containing the isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 S. aureus, conjugated with protein carrier and selected capsular polysaccharide type 8 S. aureus, conjugated to a protein carrier. In the eighth combination proposed immunogenic composition containing the selected polypeptide of clumping factor A of S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB) and isolated protein of S. aureus MntC. In some embodiments of the above combination additionally contain at least one of the following antigens: EkeS, DsqA, detail, KrkN, KrkN2, RkaS, RrkN, KnkA, SdrC, SdrD, SdrE, Orsa, DltD, HtsA, LtaS, IsdA, IsdB, IsdC, SdrF, SdrG, SdrH, SrtA, SpA, Sbi alpha-hemolysin (hla), beta-hemolysin, fibronectine protein A (fnbA), fibronectine protein B (fnbB), coagulase, Fig, map, leukocidin Panton-Valentine (pvl), alpha-toxin and its variants, gamma-toxin (hl) and variants ica, immunodominant ABC Transporter, Transporter Mg2+, ABC Transporter Ni, RAP, autolysin, laminin receptors, IsaA/PisA, IsaB/PisB, SPOIIIE, SsaA, EbpS, Sas A, SasF, SasH, EFB (FIB), SBI, Npase, HEB, SIAL-bonded protein II bone, the predecessor of aureolin (AUR)/Sepp1, Cna and its fragments, such as M55, TSST-1, mecA, exopolysaccharide poly-N-acetylglucosamine (PNAG/dPNAG), GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin protein, HarA, exhibiting esxa, EsxB, enterotoxin a, enterotoxin, enterotoxin C1 and a new autolysin.

Epidemiological studies of outbreaks of S. aureus indicate that the evolution of S. aureus is clonal in nature, when one clone, which became a successful genotype, spread rapidly and caused many of the infections. Therefore, evolution is considered to be clonal. Bacterial genome consists of a larger, more stable core (core) species genome and more diverse set of helper genes. See Feil et al., Nature Reviews: Microbiology 2:483-495 (2004). Major genes are universally present in all clones, and auxiliary genes are not necessarily present in any given clone. Treating S. aureus in one study using DNA microarray, representing more than 90% of the genome of S. aureus, found that 78% of the genes of the genome were common to all S. aureus, thus representing the "specific basis", and the remaining 22% are "VSP the service genes". Auxiliary genes include optional genetic material, much of which encodes virulence factors, proteins, mediating antibiotic resistance, and genes encoding proteins that are specific for interaction with the specific environment of the host. See Fitzgerald et al., PNAS 98:8821-8826 (2001); Feil et al., Nature Reviews: Microbiology 2:483-495 (2004). In General, the main genes evolve more slowly, and auxiliary genes are polymorphic. See Kuhn et al., J. Bact. 188:169-178 (2006). Therefore, appropriately selected key genes give the best antigens of the target for use in immunogenic compositions for the prevention of infection.

Expressed on the surface of antigens from the isolates causing disease, or clonal types of S. aureus provide a source of antigens capable of inducing the immune system and antibodies. At the macromolecular level (either amino acid sequence or sequences of polysaccharide) can be selected conservative form of the antigen expressed by the different isolates causing disease, to ensure a broad cross-reactivity of antibodies to those strains that may have antigenic variations vaccine target.

One important consideration for inclusion antigen described herein multiantigen immunogenic compositions I have is that demonstrated whether the antigen efficiency when introduced in the form of immunogenes composition by providing protection in one or more than one animal model of bacterial infection. There are numerous animal models for various diseases caused by S. aureus. Each of these models has advantages and disadvantages.

Clearance of bacterial infections in humans can go through opsonic killing, which serves as an intermediate after phagocytic uptake. There are many convincing examples of studies using polysaccharide antigens from gram-positive bacteria), such as the capsular polysaccharide of Streptococcus pneumoniae and capsular polysaccharide of S. aureus. See Lee et al., Crit. Rev. Micro. 29:333-349 (2003). There is less evidence opsonic activity induced by protein antigens of gram-positive bacteria). Observed uptake by phagocytes, but the direct killing was more difficult to demonstrate. It was shown that monoclonal antibodies to proteins provide protection against infection with S. aureus in animal models of infection; and mechanisms other than opsonophagocytosis killing may be responsible for the observed protection.

Induction of antibodies that have measurable functional activity, such as opsonophagocytic activity (OPA), is one in which Tatarov, is there a specific antigen useful for inclusion in immunogenic compositions of the present invention. Other indicators include the expression of antigen on the cell surface during expression in vivo when measured with the use of antigen specific antibodies or the ability of antibodies to inhibit/neutralize the function of antigen, but are not limited to this.

Species/strains

The type of any specific hospital or calling the disease strain is useful for determining the origin of the clonal relationship and monitoring the epidemiology of outbreaks. Available numerous ways of typing strains of S. aureus. Classic practical definition of bacterial species is a group of strains that are characterized by more than 70% genomic hybridization (genomic hybridization DNA-DNA DDH) and more than 97% identity gene sequence of 16S ribosomal RNA. See Vandamme et al., Environ. Rev. 60:407-438 (1996). Bacteriophagous typing (W) is a method for typing strains of S. aureus on the basis of their sensitivity to lysis by certain types of phages. See Blair et al., Bull. W. H. O. 24:771-784 (1961). This older method suffers from a lack of reproducibility between laboratories and the inability to typing 15-20% of the isolates.

Immunogenic compositions with one antigen compared to multiand the gene immunogenic compositions

The question arises as to whether the optimal immunogenic composition for protection against infection prevalent strains of S. aureus to contain a single component or multiple components. Numerous studies have shown that immunogenic compositions on the basis of a single protein or carbohydrate component can provide some protection from infection by a strain of S. aureus expressing this component, in certain animal models. Importantly, it was also demonstrated that protection against a single antigen may depend on the selected strain.

Surface proteins, such as adhesin, have been investigated as single-component vaccines. For example, in mice immunized with S. aureus ClfA, developed less severe arthritis than in mice immunized with the control protein. See Josefsson et al., J. Infect. Dis. 184:1572-1580 (2001). Fragments collagenopathies adhesin (ate) gave protection in a murine model of sepsis. See Nilsson, et al., J. Clin. Invest., 101:2640-2649 (1998). Immunization of mice with A domain ClfB could reduce nasal colonization in a murine model. See Schaffer et al., Infect. Immun. 74:2145-2153(2006).

One of the fourteen proteins sekvestirovanija iron S. aureus, known as IsdB, is investigated in the univalent immunogenic compositions for protection against S. aureus infection. This protein showed a good protective effect in mice and a good immunogen is ity in primates, not a person. See Kuklin et al., Infect. Immun. 74:2215-2223 (2006).

Because of the huge potential of S. aureus to change or substitution of different proteins to perform the same or similar functions, the optimal immunogenic composition to protect most people from most of the diseases caused by S. aureus, is multiantigen composition comprising 2 or more than 2 (for example, 3, 4, 5, and so on) antigen, properly selected and presented in an immunogenic composition. In certain embodiments the immunogenic composition according to the invention contains three or more than three antigen selected from the selected polypeptide of clumping factor A of S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 (SR) S. aureus conjugated to a protein carrier, selected capsular polysaccharide type 8 (SR) S. aureus conjugated to a protein carrier, and a selected protein of S. aureus MntC. In certain embodiments the immunogenic composition according to the invention contains four or more than four antigen selected from the selected polypeptide of clumping factor A of S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 (SR) S. aureus conjugated to a protein carrier, selected capsular polysaccharide type 8 (SR) S. aureus, conjugated to a protein carrier, and a selected protein of S. aureus MntC. In certain embodiments the immunogenic composition according to the invention contains the selected polypeptide of clumping factor A of S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 (SR) S. aureus, conjugated to protein carrier, selected capsular polysaccharide type 8 (SR) S. aureus, conjugated with protein carrier and isolated protein of S. aureus MntC as antigens.

Adjuvants

Immunogenic compositions as described herein in certain embodiments also contain one or more than one adjuvant. Adjuvant is a substance that enhances the immune response when introduced together with the immunogen or antigen. It was shown that a number of cytokines or lymphokines have together with immunomodulating activity and thus are useful as adjuvants, including interleukins 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; granulocyte-macrophage colony-stimulating factor (GM-CSF) (see, for example, U.S. patent No. 5078996 and the access number of ATSS 39900); macrophage colony-stimulating factor (M-CSF); granulocyte colony-stimulating factor (G-CSF) and factors tumor necrosis α and β, but not limited to. the moreover, other adjuvants that are useful are described here immunogenic compositions include chemokines, including without limitation, MCP-1, Ì1Ð-1α, Ì1Ð-1β and RANTES; adhesion molecules, such as selectin, for example, L-selectin, P-selectin and E-selectin; muzikpapare molecules, for example, CD34, GlyCAM-1 and MadCAM-1, a member of the family of integrins, such as LFA-1, VLA-1, Mac-1 and P150.95; member of the immunoglobulin superfamily, such as the RESOURCES, ICAM, for example, ICAM-1, ICAM-2 and ICAM-3, CD2, and LFA-3; co-stimulating 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 (platelet-derived growth factor), BL-1 and growth factor vascular endothelial; molecule receptors, including Fas, TNF receptor (tumor necrosis factor), Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRA1L-R2, TRICK2, DR6 and caspase (ICE).

Suitable adjuvants used to enhance the immune response, optionally, include, without limitation, 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 aminoalkylsilane compounds (AGP), or derivatives thereof, or analogs, which are commercially available from Corixa (Hamilton, MT), and which are described in U.S. patent No. 6113918. One such AGP performance is to place the 2-[(R)-3-tetradecanoylphorbol-amino]ethyl-2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoylphorbol]-2-[(R)-3-tetradecanoylphorbol-amino]-b-D-glucopyranosid, also known as 529 (formerly known as RC529). This adjuvant 529 prepared in the form of the drug in aqueous form (AF) or in the form of a stable emulsion (SE).

In addition, other adjuvants include muramylpeptide, such as N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylmuramyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)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 different amounts of MTP-PE), prepared in the form of submicron drug particles using microfluidizer (microfluidizer), such as microfluidizer Model HOY (Microfluidics, Newton, MA), and SAF (containing 10% squalene, 0.4% Polysorbate 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized (microfluidized) in submicron emulsion or mixed (vortexed) with the generation of the emulsion with a large particle size); incomplete adjuvant's adjuvant (IFA); aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate; antigen; avidin; L121/squalene, D-lactide-polylactide/glycoside; polyols-pluronic; mortified Bordetella; saponins such as Stimulon™ QS-21 (Antigenics, Framingham, MA), described in U.S. patent No. 5057540, ISCOMATRIX (CSL Limited, Parkville, Australia), described in U.S. patent No. 5254339, and immunostimulating complexes (ISCOMATRIX); Mycobacterium tuberculsis; 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 (PT) or its mutants, cholera toxin or its mutants (for example, U.S. patent No. 7285281, 7332174, 7361355 and 7384640); or thermo-labile toxin (LT) from E. coli or its mutant, particularly LT-K63, LT-R72 include (for example, U.S. patent No. 6149919, 7115730 and 7291588).

Antigens candidates:

ClfA: domain organization

The clumping factor A (ClfA) is a surface protein of S. aureus associated with protein binding matrix of host in the binding site of fibrinogen. ClfA is a member of a family of proteins containing carboxykinase motif LPXTG (SEQ ID NO:125), which enables covalent binding of the protein to the cell surface. ClfA also belongs to another family of proteins (the adhesive matrix molecule that recognize microbial components of the surface, or MSCRAMM), which is associated with the binding of host proteins, such as fibrinogen (link ClfA), fibronectins proteins (FnbA and FnbB), collagenase protein (Cna), and others. All these proteins have a common aminobenzene signal sequence that mediates the transport to the cell surface. MSCRAMM also include domain, And that the present is the focus of a functional area, contains the active site for binding of ligand (e.g., fibrinogen, fibronectin, elastin, keratin). After the domain And is followed by a region consisting of serine-aspartate repeats (SD repeat), which is thought crosses peptidoglycan layer. After SD retry should cross the membrane area, which includes the motif LPXTG (SEQ ID NO:125) for covalent binding of protein to the peptidoglycan. ClfA is described in U.S. patent No. 6008341.

Landscapeusa ClfA region containing N1N2N3 domain A (Fig.1) comprises amino acids 40-559. Domains N ClfA were defined as follows: N1 covers the remains of 45-220; N2 covers the remains of 229-369 and N3 covers the remains of 370-559. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). For ease of reference domains N1N2N3 can be designated as N123, similarly N2N3 can be designated as N23. It was found that in preparations of recombinant N1N2N3 domain N1 is protectchristian and easily cleaved or hydrolyzed, leaving N2N3 in the form of stable landscapebased recombinant fragment. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). Crystal structure fibrinogenesis fragment N2N3 domain And ClfA has revealed that as in N2, and N3 are dominated by antiparallel beta-strands. In addition antiparallel beta-strands of the domain N2 contains one turn of an alpha helix and two 310spiral, and N3 domain contains t and 3 10the spiral. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). The alignment of the sequence N2 and N3 reveals only 13% sequence identity and 36% similarity to the sequence according to their length. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). The topology of the domain N2 and N3 is similar to the classic styling of IgG, and it has been suggested that it represents new ways of laying IgG. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002).

The sequence ClfA

Gene protein clumping factor A, denoted by ClfA, was cloned, sequenced and analyzed in detail at the molecular level (McDevitt et al., Mot. Environ. 11:237-248 (1994); McDevitt et al., Mol. Environ. 16:895-907 (1995)). The sequence identifiers for the amino acid sequences of ClfA 111 isolates of S. aureus that cause disease are shown in Table 10. Amino acid sequence of full-ClfA wild type (including signal sequence) from strain PFESA0237 S. aureus are presented in SEQ ID NO:130. This sequence shows the tyrosine at position 338, which is replaced by alanine in the mutant form of ClfA. Full gene that encodes a ClfA wild-type strain PFESA0237 S. aureus containing region N123, the repeat region and the anchor region is presented in SEQ ID NO:131. Amino acid sequence of the mutated forms Y338A ClfA presented in SEQ ID NO:123. However, it should be noted that the substitution of tyrosine at the Ala is in, which takes place in ClfA wild type at position 338 of SEQ ID NO:130, and which is designated as Y338A presented in mutated form of ClfA in SEQ ID NO:123 in position 310. In addition, a mutated form of ClfA presented in the amino acid sequence of SEQ ID NO:123, is a Mature form ClfA without the signal sequence, thus, responsible for the difference in position of this mutation between SEQ ID NO:130 and SEQ ID NO:123.

ClfB: domain organization

ClfB is a protein of S. aureus with fibrinogenesis activity, and initiates the formation of colonies (clumps) of S. aureus in the presence of plasma. ClfB is a MSCRAMM protein and shows a typical domain organization MSCRAMM, including domain a, which is a functional area that contains the active site for binding of ligand (e.g., fibrinogen, fibronectin, elastin, keratin). After the domain And is followed by a region consisting of serine-aspartate repeats (SD repeat), which is thought crosses peptidoglycan layer. After SD retry should cross the membrane area, which includes the motif LPXTG (SEQ ID NO:125) for covalent binding of protein to the peptidoglycan. ClfB described in WO 99/27109 and U.S. patent No. 6680195.

The internal organization of the N-terminal domain And ClfB is very similar to the organization found in ClfA. Domain a consists of three N1, N2 and N3. Landscapeusa region ClfB containing N1N2N3 domain A (Fig.1) comprises amino acids 44-585. For ease of reference domains N1N2N3 can be designated as N123, similarly N2N3 can be designated as N23. Domains N ClfB were identified as follows: N1 covers the remains of 44-197; N2 covers the remains of 198-375; and N3 covers the remains of 375-585. It was found that in the crystal structure of ClfA a domain And has a unique version of immunoglobulin styling, and, by analogy, we can assume that the same thing occurs in the case of ClfB. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). Even though the organization of domains And ClfB and ClfA is similar, the sequence identity is only 26%. See Ni Eidhin et al., Mol. Environ. 30:245-257 (2002).

The sequence ClfB

The gene encoding ClfB classified as a major gene adhesion. Sequence ClfB of the 92 strains of S. aureus associated with many painful conditions, are summarized in Table 11. Additional sequences were obtained from GenBank.

Other MSCRAMMS

For use in the immunogenic compositions of the present invention can be considered other MSCRAMMS. For example, proteins with serine-aspartate repeats (Sdr), SdrC, SdrD and SdrE are related by primary sequence and structural organization of proteins ClfA and ClfB and localized on the cell surface. B is the CTL, SdrC, SdrD and SdrE are proteins associated with cell wall having a signal sequence at the N-end and motif LPXTG (SEQ ID NO:125), a hydrophobic domain and a positively charged residues at the C-end. Each also has a region R containing SD repetitions, of sufficient length so that, along with motives, to make possible the efficient expression of the field And landscapebased domain on the cell surface. By region And proteins SdrC, SdrD and SdrE, located on the cell surface, these proteins can interact with proteins in plasma, extracellular matrix or with molecules on the surface of host cells. Proteins Sdr have some limited amino acid sequence similarity with ClfA and ClfB. Like ClfA and ClfB, SdrC, SdrD and SdrE also demonstrate nationalisme the binding of the ligand protein of the extracellular matrix.

Genes sdr are closely related and are tandemly arranged. Proteins Sdr (from SdrC, SdrD, SdrE, ClfA and ClfB) typically contain the area And, where there is a highly conserved amino acid sequence, which can be used to obtain a consensus motif TYTFTDYVD (SEQ ID NO:126). This motif shows a small variation between different proteins. This variation, along with the consensus sequence motif described in U.S. patent number 6680195. In proteins Clf-Sdr this motive is vysokokonservativnykh is. The motif can be used in immunogenic compositions for imparting immunity to a wide range of bacterial infections and can also be used as antigen in obtaining monoclonal or polyclonal antibodies. This antibody can be used to give passive immunity to a wide spectrum.

Proteins Sdr differ from ClfA and ClfB the presence of from two to five additional repetitive sequences from 110-113 residues (motifs) that are located between a region R and a-scope. Each In-motif contains a consensus of CA2+-binding loop of the EF-hands, usually found in eukaryotic proteins. The structural integrity of the recombinant protein containing five repeats SdrD, as was shown by fluorescence analysis bisANS, is the CA2+dependent, causing the assumption that the EF-hands are functional. When removing the Ca2+the structure was destroyed in an expanded conformation. The original structure was restored by the addition of CA2+. The C-terminal R-domains Sdr proteins contain 132-170 residues SD. After them follow conservative, anchored in the wall region, characteristic of many surface proteins of gram-positive bacteria.

In Sdr proteins and Clf this motif is highly conserved, whereas in fibronectins MSCRAMMS, and collagenopathies the tree Can is a degenerate version. The motives of B in combination with regions R are necessary for the emergence of landscapebased domain at some distance from the cell surface. Recurring motifs are one common denominator described here subgroups of proteins with SD repetitions. These motifs are found in different quantities in the three Sdr proteins from strain PFESA0237. There are clear differences between individual motives Century the Most conservative elements are the elements located in the vicinity of regions R (B2 SdrC, B5 SdrD and B3 SdrE). They differ from the others in several sites, especially in the C-terminal half. Notable structural feature is that adjacent duplicates are always separated by a Proline residue present in the C-terminal region, but Proline never occurs between the last and repetitions In the area of R. Instead, the linker acid is characterized by short cut. These differences are evidence that the end elements have different structural or functional role in comparison with other motives Century. N-terminal motifs In SdrD and SdrE away (drifted), and there are numerous changes of amino acids, including small insertions and deletions, while the remaining inner motives are more highly conserved. Note that each of the three proteins Sdr has come about at least one motif In each type.

The C-terminal R-domains Sdr proteins contain 132-170 residues SD. Followed by conservative anchored in the wall region, characteristic of many surface proteins of gram-positive bacteria.

Other molecules SdrD candidates for use in immunogenic compositions according to the invention may originate from different species of organisms, some of which include the following SdrD of S. aureus: strain USA300 FPR3757 (access number of protein SAUSA300 0547); strain NCTC8325 (access number of protein SAOUHSC 00545); strain MW2 (the access number of protein MW0517); strain MSSA476 (access number of protein SAS0520; and strain Mu50 (access number of protein SAV0562).

Additional MSCRAMMS that can be considered for use in the immunogenic compositions of the present invention, include EkeS, DsqA, detail, KrkN, KrkN2, RkaS, RrkN and KnkA. Data MSCRAMMS described in WO 02/102829, which is hereby incorporated by reference. Additional MSCRAMMS identified no access GenBank include NP_373261.1, NP_373371.1, NP_374246.1, NP_374248.1, NP_374841.1, NP_374866.1, NP_375140.1, NP_375614.1, NP_375615.1, NP_375707.1, NP_375765.1 and NP_375773.1.

Capsular polysaccharides type 5 and type 8

Staphylococcal microorganisms capable of causing invasive disease, usually also able to produce capsular polysaccharide (CP), which encapsulates the bacteria and increases its resistance to clearance of the innate immune system of the host. CF is used to cover the bacterial cells and the protective capsule, which makes the bacteria resistant to phagocytosis and intracellular killing. Bacteria that do not have capsules, are more susceptible to phagocytosis. Capsular polysaccharides are often important virulence factor for many bacterial pathogens, including Haemophilus influenzae, Streptococcus pneumoniae and Streptococcus group C.

Capsular polysaccharide can be used for serotyping of specific types of bacteria. Typing is usually carried out by reaction with a specific anticorodal or a monoclonal antibody generated to a specific structure or unique epitope characteristic of the capsular polysaccharide. Encapsulated bacteria tend to grow in smooth colonies, whereas colonies of bacteria that have lost their capsules seem rough. Colonies with slideology appearance, known as strongly encapsulated. S. aureus types 1 and 2 are strongly encapsulated and are rarely associated with disease.

Most clinical isolates of S. aureus are encapsulated or serotype 5 or serotype 8. Capsular polysaccharides type 5 (SR) and type 8 (SR) have similar trisaccharide repeating units consisting of N-acetylaminophenol acid, N-acetyl-L-fucosamine and N-acetyl-D-fucosamine. See Fournier, J. M. et al., Infect. Immun. 45:97-93 (1984) and Moreau, M., et al. Carbohydrate Res. 201:285-297 (1990). Two CF who have the same sugar, but differ in relations sugars and sites of O-acetylation, produce serologically excellent pattern of immunoreactivity.

In some embodiments of the capsular polysaccharides of serotypes 5 and/or 8 according to the invention are O-acetylated. In some embodiments, the degree of O-acetylation of the capsular polysaccharide of type 5 or oligosaccharide 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 type 8 or oligosaccharide 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 of type 5 and type 8 or oligosaccharide 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 Biomedical Analysis 30; 1233-1247, WO 05/033148 or WO 00/56357). Another commonly used method is described Hestrin (1949) J. Biol. Chem. 180; 249-261.

In some embodiments of the capsular polysaccharides of serotypes 5 and/or 8 according to the invention are used to generate antibodies that are functional in dimensions the AI killing bacteria in an animal model of efficiency or in the analysis opsonophagocytosis killing, which demonstrates that antibodies to kill the bacteria. Such functionality may not be observed when using an analysis that tracks the generation of some of the antibodies that is not a sign of the importance of O-acetylation for efficiency.

Epidemiology capsules

The Association of specific serotypes capsules with the disease is possible through the monitoring of clinical isolates. Of the eight identified different serotypes of S. aureus (Karakawa and Vann (1982) only serotypes 1 and 2 are strongly encapsulated, and they are rarely isolated. See the Capsular Polysaccharides of Staphylococcus aureus, p.285-293, In J. B. Robbins, J. C. Hill and J. C. Sadoff (ed.), Seminars in infectious disease, vol. 4, Bacterial Vaccines. Thieme Stratton, Inc. New York). Studies have shown that approximately 85-90% of clinical isolates of S. aureus Express SR or SR (Arbeit RD, et al., Diagn. Environ. Infect. Dis. (1984) Apr;2(2):85-91; Karakawa WW, et al., J. Clin. Environ. (1985 Sep;22(3):445-7; Essawi T, et al., Trop. Med. Int. Health. (1998) Jul;3(7):576-83; Na was T, et al., J. Clin. Environ. (1998) 36(2):414-20. Most Netherway strains SR and SR genetically represent a type 5 or type 8 containing mutations in the locus SAR/8 (please contact our consulting business, Gomez et al., (2006), Mol. Environ. Feb. 59(3):948-960). Encapsulation of some strains quickly lost within several passages in vitro, due to the repressive effect of high concentrations of phosphate in the environments used in clinical diagnostics, for the production of the capsules. Also reported is the unencapsulated public isolates restore the expression of the capsule after passing through the cows. See Opdebeck, J. P. et al., J. Med. Environ. 19:275-278 (1985). Some Netherweave strains become positive in relation to the capsule under suitable growth conditions.

Structure SR and SR

Repeating unit and SR, and SR consists of 2-acetamido-2-deoxy-D-mannurone acid, 2-acetamido-2-deoxy-L-fucose and 2-acetamido-2-deoxy-D-fucose. See C. Jones et al., Carbohydr. Res. 340:1097-1106 (2005). Although SR and SR have the same composition of Sugars, it was shown that they are immunologically different. They differ in glycosidic linkages and website O-acetylation of uronic acid. Observed dependent strain partial N-acetylation of one of the FucNAc residues. See Tzianabos et al., PNAS V98: 9365(2001).

Capsular polysaccharide of S. aureus in immunogenic compositions

Molecular weight capsular polysaccharides of S. aureus is an important factor to consider when used in immunogenic compositions. Capsular polysaccharides with high molecular weight are able to induce specific immune responses antibody due to the higher valency of epitopes present on the antigenic surface. The described methods provide isolation and purification of capsular polysaccharide type 5 and type 8 with a significantly higher molecular weight than previously available.

MntC/SitC/saliva binding protein

MntC/SitC/svyazyvayus the saliva protein is a protein ABC Transporter and has homology with S. epidermidis and S. aureus. In the present invention it is marked as MntC. This protein is a 32 kDa lipoprotein and localized in the bacterial cell wall. See Sellman et al., and Cockayne et al., Infect. Immun. 66: 3767(1998). In S. epidermidis he is part of the operon, which is regulated by iron. It shows significant homology with adesanmi, including FimA S. parasanguis, and lipoproteins family of ABC-transporters with proven or putative functions of the transport of metallic iron. (See Table 12 for strains of S. aureus and sequences).

Protein of S. aureus MntC

Homolog of S. aureus MntC known as saliva binding protein, and was disclosed in U.S. patent No. 5801234, and can be included in the immunogenic composition according to the invention. The sequence of the protein homolog MntC/SitC/ saliva binding protein of S. aureus is found in GenBank under access number NP_371155 for strain Mu50. (Also known as SAV0631). The sequence identifier SEQ ID NO:119. The access number for the nucleotide sequence for the full genome of strain Mu50 - NC_002758.2 (coordinates 704988-705917).

Protein of S. epidermidis SitC

Homolog MntC/SitC/saliva binding protein of S. epidermidis known as SitC and was discovered in Sellman et al. (Sellman et al., Infect. Immun. 2005 October; 73(10): 6591-6600). The sequence of the protein homolog MntC/SitC/ saliva binding protein of S. epidermidis is in GenBank under access number YP_187886.1. (Also known as ka is SERP0290). The sequence identifier SEQ ID NO:121.

The access number for the nucleotide sequence of the full genome of strain RP62A - NCJ302976 (coordinates 293030-293959). Other molecules SitC candidates for use in immunogenic compositions according to the invention may originate from different species of organisms, some of which are listed in Table 1 below.

Table 1
ProteinExample of strainThe room access of protein
SitCS. haemolyticusJCSC1435UAE.1
SitCS. epidermidisATCC 12228AAO04002.1
SitCS. saprophyticusATCC 15305UAE.1
SitCS. xylosusDSM20267ABR57162.1
SitCS. carnosusTMCAL27186.1

Iron-binding proteins of S. aureus

Another potentially the first antigen-candidate, subject to consideration for use in immunogenic compositions according to the invention includes a surface protein of S. aureus surface determinants of iron (IsdB). This MSCRAMM was described Mazmanian et al. (Mazmanian, SK et al. Proc. Natl. Acad. Sci., USA 99:2293-2298 (2002)) and then was tested and it was shown that it is effective as a vaccine candidate in murine models of infection and immunogenicity study on rhesus monkeys conducted Kuklin, et al. (Kuklin, NA, et al. Infection and Immunity, Vol.74, No. 4, 2215-2223, (2006)). This molecule IsdB is present in different strains of S. aureus, including strains MRSA252 (access number of protein CAG40104.1); strain Newman (access number of protein BAF67312.1); strain MSSA476 (access number of protein CAG42837.1); strain Mu3 (access number of protein BAF78003.1); strain RF122 (access number of protein SA.1).

Antigens candidates:

Immunogenic compositions of the present invention can also include one or more than one of the following antigens: Orra, DltD, HtsA, LtaS, IsdA, IsdC, SdrF, SdrG, SdrH, SrtA, SpA, alpha-hemolysin Sbi (hla), beta-hemolysin, fibronectine protein A (fnbA), fibronectine protein B (fnbB), coagulase, Fig, map, leukocidin Panton-Valentine (pvl), alpha-toxin and its variants, gamma-toxin (hlg) and variants, ica, immunodominant ABC Transporter, Transporter Mg2+, ABC Transporter Ni, RAP, autolysin, laminin receptors, IsaA/PisA, IsaB/PisB, SPOIIIE, SsaA, EbpS, Sas A, SasF, SasH, EFB (FIB), SBI, Npase, EBP, SIAL-bonded protein Caste, the predecessor of aureolin (AUR)/Sepp1, Cna, and fragments thereof, such as M55, TSST-1, mecA, exopolysaccharide poly-N-acetylglucosamine (PNAG/dPNAG), GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin protein, HarA, exhibiting esxa, EsxB, enterotoxin a, enterotoxin, enterotoxin C1 and a new autolysin. In certain embodiments of the invention, when the immunogenic composition contains certain forms SR and/or SR, it may not contain additional PNAG.

Drugs immunogenic composition

In one embodiment of the immunogenic composition according to the invention additionally contain at least one adjuvant, buffer, cryoprotectant, salt, a divalent cation, a non-ionic detergent, an inhibitor of oxidation by free radicals, diluent or carrier.

Immunogenic compositions according to the invention can additionally contain one or more than one preservative addition to many staphylococcal protein antigens and conjugates of capsular polysaccharide-protein. The FDA (US food control and drugs) requires, with few exceptions, to biological products in mnogochasovykh (multi-dose) blood vessels contained a preservative. Vaccine products containing preservatives include vaccines containing chloride benzene (anthrax), 2-Phenoxyethanol (DTaP, HepA, Lyme disease, poliomyelitis (Polio (parenteral)), phenol (pneumonia (Pneumo), typhoid (Typhoid) (parenteral), smallpox (Vaccinia) 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 composition of the invention can optionally include one or more than one buffer salt, a divalent cation, a non-ionic detergent, cryoprotectant, such as sugar, antioxidant, such as an acceptor of free radicals or chelate forming agent, or any of their many combinations. The choice of any one component, for example, chelat forming agent may determine whether another component (e.g., acceptor) is desirable. The final composition is prepared in the form of the drug for administration must be sterile and/or free from pyrogens. The specialist can empirically determine which combinations of these and other components will be optimal for inclusion in immunogenic compositions according to the invention containing preservative, depending on many factors, such as specific required conditions of storage and administration.

In certain embodiment is the second composition according to the invention, which is compatible with parenteral introduction, contains one or more than one physiologically acceptable buffer is selected from Tris(tromethamine)-buffer, phosphate, acetate, borate, citrate, glycine, his-tag and succinate buffer, but not limited to. In certain embodiments the composition is buffered to a value within the range of pH 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 summing pH immunogenic composition or preparation according to the invention. the pH of the composition according to the invention can be made using standard in this field techniques. the pH of the composition can be summed up values from 3.0 to 8.0. In certain embodiments the pH of the composition can comprise, or can be summed up values from 3.0 to 6.0V; from 4.0 to 6.0, or from 5.0 to 8.0. In other embodiments the pH of the composition may comprise or may be brought about 3.0; about 3.5; about 4.0; about 4.5; about 5,0; 5.5; approximately 5.8; approximately 6,0; approximately 6.5, about 7.0 and about 7.5, or about to 8.0. In certain embodiments the pH may be or may be made to values 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 and from 5.6 to 5.7; from 5.7 to 5.8; from about 5.8 to 5.9; 5.9 to 6.0; and from 6.0 to 6.1; from 6.1 to 6.2; from 6, to 6.3; from 6.3 to 6.5; 6.5 to 7.0; 7.0 to 7.5 or 7.5 to 8.0. In a particular embodiment the pH of the composition is approximately 5.8.

In certain embodiments the composition according to the invention, which is compatible with parenteral introduction, contains one or more than one bivalent cation, including MgCl2, CaCl2and MnCl2but not limited to, in concentrations ranging from about 0.1 mm to about 10 mm, and the concentration of up to about 5 mm is preferred.

In certain embodiments the composition according to the invention, which is compatible with parenteral introduction, contains one or more than one salt, including sodium chloride, potassium chloride, sodium sulfate and potassium sulfate, but not limited to, present in ionic strength, which is physiologically acceptable to the subject, parenteral, and included in a final concentration of obtaining the selected ionic strength or osmolarity in the final composition. The final ionic strength or the osmolarity of the composition will be determined by many components (for example, ions from the buffer (s) compounds (compounds) and other non-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) is AK, to end the total osmolarity of the composition was compatible with parenteral administration (e.g. intramuscular or subcutaneous injection), and will promote long-term stability of the immunogenic components of the preparation of immunogenic compositions in different temperature intervals. In compositions that do not contain salt, will be valid for longer intervals of the contents of one or more than one selected cryoprotectant to maintain the desired final osmolarity.

In certain embodiments the composition according to the invention, which is compatible with parenteral introduction, contains one or more than one cryoprotector selected from disaccharides (e.g. lactose, maltose, sucrose or trehalose) and polyhydroxyalkanoates (for example, dulcita, glycerol, mannitol and sorbitol), but not limited to.

In certain embodiments, the osmolarity of the composition is in the range from about 200 mOsm/l to about 800 mOsm/l with a preferred range from about 250 mOsm/l to about 500 mOsm/l, or from about 300 mOsm/l to about 400 mOsm/L. Composition not containing salt, may 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. Alternatively, the composition, not the soda is containing salt, may 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 interval of sucrose or sorbitol is relatively less. These and other such osmolarity and considerations for osmolarity entirely within the training of specialists in this field.

In certain embodiments the composition according to the invention, which is compatible with parenteral introduction, contains one or more than one of the inhibitors of the oxidation of free radicals and/or chelat forming agents. In this area there are a number of acceptors of free radicals and chelat forming agents, and they are described here applies to compositions and methods of use. Examples include, but are not limited to, ethanol, EDTA (ethylenediaminetetraacetic acid), a combination of EDTA/ethanol, triethanolamine, mannitol, histidine, glycerin, sodium citrate, inositoltrifosfata, tripolyphosphate, ascorbic acid/ascorbate, succinic acid/succinate, malic acid/malate, Desferal, EDDHA (etilendiamintetrauksusnoy acid) and DTPA (diethylenetriaminepentaacetic acid) and different combinations of two or more than two of the above. In certain embodiments may be add the Yong at least one non acceptor of free radicals in a concentration which effectively increases long-term stability of the composition. You can also add one or more than one inhibitor of the oxidation of free radicals/chelate forming agent in various combinations, such as the acceptor and divalent cation. The choice chelat forming agent will determine whether or not the addition of the acceptor.

In certain embodiments the composition according to the invention, which is compatible with parenteral introduction, contains one or more than one non-ionic surfactant, including, but not limited to, esters of polyoxyethylenesorbitan and fatty acids, Polysorbate-80 (Tween 80), Polysorbate-60 (Tween 60), Polysorbate-40 (Tween 40), and Polysorbate-20 (Tween 20), alkalemia esters of polyoxyethylene, including, but not limited to, Brij 58, Brij 35, and others, such as Triton X-100; Triton X-114, NP40, Span85 and non-ionic surfactant Pluronic series (e.g., Pluronic 121), with the preferred components Polysorbate-80 at a concentration of from about 0,001% to about 2% (with a concentration of up to about 0.25% of which is preferred) or Polysorbate-40 at a concentration of from about 0,001% to about 1% (with a concentration of up to about 0.5% being preferred).

In certain embodiments the composition according to the 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). Alternative or perhaps drugs immunogenic composition according to the invention, containing a preservative, can be additionally stabilized by removing oxygen from the container for storage, protection of the drug from light (for example, through the use of containers of yellow glass).

Drugs immunogenic composition according to the invention, containing a preservative, can contain one or more than one pharmaceutically acceptable carrier or excipient, which includes any excipient, which by itself does not induce an immune response. Suitable excipients include macromolecules, such as proteins, sugars, lactic acid, polyglycolic acids, polymeric amino acids, copolymers of amino acids, sucrose (Paoletti et al, 2001, Vaccine, 19:2118), trehalose, lactose and lipid aggregates (such as oil droplets or liposomes), but are not limited to. Such carriers are well known to the skilled technician. Pharmaceutically acceptable excipients are discussed, for example, in Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20thedition, ISBN:0683306472.

The composition of the invention can be lief is lysed or be in aqueous form, i.e., in the form of solutions or suspensions. Liquid compositions mainly you can enter directly from their Packed form, and thus, they are ideal for injection without dilution in liquid medium, as in other cases, would be required for the freeze-dried compositions according to the invention.

Direct delivery of immunogenic compositions of the present invention to a subject can be performed by parenteral administration (intramuscular, intraperitoneally, intradermally, subcutaneously, intravenously or into the interstitial space of a tissue); or by rectal, oral, vaginal, local, percutaneous, intranasal, ocular, aural, pulmonary or other introduction to the mucous. In the preferred embodiment of the parenteral Vvedenie is carried out by intramuscular injection, for example, in the hip or the shoulder of the subject. Injection can be performed through a needle (for example, a needle for subcutaneous injection), but an alternative you can use needleless injection. Typical intramuscular dose of 0.5 ml of the Composition according to the invention can be prepared in various forms, for example, for 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, e.g., in the inhaler. In other embodiments to which the position can be prepared in the form of a suppository or pessary, or for nasal, aural or ocular injection, for example, as sprays, drops, gel or powder.

The optimal number of components for a specific immunogenic compositions can be installed by standard studies involving observation of appropriate immune responses in subjects. After the initial vaccination, subjects may receive one or several booster immunizations with an adequate interval of time.

Packaging and dosage forms

Immunogenic compositions according to the invention can be packaged in the form of a unit dose or mnogochasovoj form (for example, 2 doses 4 doses or more). For parenteral multi-dose forms vials typically, but not necessarily, are preferred in comparison with pre-filled syringes. Suitable mnogorazovye formats include 2-10 doses in the container of 0.1-2 ml per dose, but are not limited to this. In certain embodiments, the dose is a dose of 0.5 ml See, for example, international patent application WO2007/127668, which is incorporated here by reference.

The compositions may be presented in vials or other suitable containers for storage, or can be presented in pre-filled devices for delivering, for example, one-component or multicomponent syringe, which can be supplied with or without needles is the main The syringe is typically, but not necessarily, contains one dose of the immunogenic composition of the invention containing the preservative, although also considered mnogorazovye, pre-filled syringes. Similarly, the bottle may include one dose, but, alternatively, may include many of the doses.

Effective dosage amounts can be set in the usual way, but the typical dose of the composition for injection has a volume of 0.5 ml In certain embodiments, the dose is prepared in the form of the drug for administration to a subject person. In certain embodiments, the dose is prepared in the form of the drug for administration to a subject, an adult, teenager, adolescent, child begins to walk, or to the infant (i.e., with age not more than one year), and, in the preferred embodiments, it may be administered by injection.

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

Alternatively, the immunogenic compositions of the present invention can be liofilizirovannami and dissolved, for example, using one of the many ways of freeze drying is well known in this area, with the formation of dry particles is correct (e.g., spherical) shape, such as micropellet or microspheres having characteristics of particles, such as average diameter, which can be selected and controlled by varying the exact methods used to obtain them. Immunogenic compositions can optionally contain adjuvant, which may possibly be made with a separate dry particles is correct (e.g., spherical) shape, such as microspheres or microspheres or contained in them. In such embodiments according to the present invention is additionally proposed a set of immunogenic compositions containing the first component, which includes a stable, dry immunogenic composition may additionally contain one or more than one preservative according to the 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, e.g the measures WO2009/109550 (included here by reference).

In one another embodiment of the container-parenteral multi-dose format selected from one or more than one group, consisting of, but not limited to, standard laboratory glassware, bottles, beakers, measuring cylinders, fermentors, bioreactors, hoses, tubes, bags, cans, containers, lids of vessels (for example, rubber caps, caps with thread), vials, syringes, two - or multi-chamber syringes, syringe caps, pistons of syringes, rubber caps, plastic lids, glass lids, cartridges and disposable pens and the like. The container of the present invention is not limited to the material from which it is made, and includes such materials as glass, metals (e.g. steel, stainless steel, aluminum and so on) and polymers (e.g., thermoplastics, elastomers, thermoplastic elastomers). In a particular embodiment of the container of this format represents a 5 ml glass vessel of SCHOTT type 1 (Schott Type 1) with a butyl stopper. A qualified expert will understand that the formats outlined above are by no means an exhaustive list, but simply serve as a guide to the specialist relatively the variety of formats available for the present invention. Additional formats considered for use in the present from which reenie, can be found in the published directories from suppliers and manufacturers of laboratory equipment, such as United States Plastic Corp. (Lima, OH), VWR.

Evaluation of immunogenic compositions

In one embodiment according to the present invention proposed immunogenic compositions containing at least three antigen from an organism S. aureus.

Used different in vitro tests to assess the immunogenicity of immunogenic compositions according to the invention. For example, opsonic the in vitro assays carried out through co-incubation of a mixture of staphylococcal cells, serum inactivated by heating, containing specific antibodies to the antigens, and an exogenous source of complement. Opsonophagocytic goes for incubation of freshly isolated polymorphically cells (PMN) or differentiated effector cells such as HL60, and a mixture of antibody/complement/staphylococcal cells. Bacterial cells that are coated with antibody and complement, wordplays when opsonophagocytosis. Colony-forming units (CFU) surviving bacteria that are recovered after opsonophagocytosis, determined by seeding the Cup of the analyzed mixture. Titles lead as the return value from the highest dilution that gives 50% killing of bacteria, as determined by comparison with controls analysis.

DL the assessment of immunogenicity in vitro and finding the antigen on the surface also use the ELISA (enzyme-linked immunosorbent assay) of whole cells, when interested in a bacterial strain (S. aureus) cover the tablet, such as a 96-well plate, and conducting the reaction of the tested serum from the immunized animal and bacterial cells. If any antibody that is specific for the test antigen reacts with the surface epitope of the antigen, it can be detected by standard methods known to the expert in this field.

Any antigen, demonstrating the desired activity in vitro, and then tested in animal models of infection in vivo. In certain embodiments in immunization of the animal (e.g. mouse) use the immunogenic compositions ways, immunization, well-known specialists in this field (e.g., intranasal, parenteral, oral, rectal, vaginal, transdermal, intraperitoneal, intravenous, subcutaneous, and so on). After immunization of an animal of a particular immunogenic composition based on Staphylococcus sp., animal infecting Staphylococcus sp.and analyzed for resistance to staphylococcal infection.

In one embodiment of mice that do not contain the pathogen, are subjected to immunization and infect S. aureus. For example, mice are subjected to immunization of one or more than one dose of the desired antigen in the immunogenic composition. Then mice infect S. aureus and monitor survival over a period of time postaraisya.

Methods immunization

Proposed ways immunization of the host to prevent staphylococcal infections. In the preferred embodiment of the owner is the person. Thus, the owner or the subject is administered the immunogenic amount of the immunogenic composition as herein described. Immunogenic amount of the immunogenic composition can be determined by the implementation of the study, the dose-response, in which subjects are subjected to immunization gradually increasing amounts of immunogenic compositions, and analyze the immune response to determine the optimal dosage. A starting point for research can be derived from data on immunization in animal models. The amount of dosages can vary depending on the specific conditions of the individual. The number can be defined in the traditional testing methods known to experts in this field. In some embodiments, the method of immunization of the host to prevent staphylococcal infection, disease or condition includes treatment of human and veterinary treatment or treatment of farm animals. According to another embodiment, a method of immunization of the host to prevent staphylococcal infection, disease or condition associated with Staphylococcus sp., the subject, including the preparation of polyclo the social or monoclonal antibodies described here immunogenic compositions and the use of this drug antibodies to make the subject of the passive immunity.

Immunologically effective amount of the immunogenic composition is administered to a subject in a suitable number of doses to invoke an immune response. Processed the individual must not show more severe clinical manifestations of staphylococcal infection. The amount of dosages can vary depending on the specific conditions of the individual, such as age and body weight. This size can be defined in the traditional testing methods known to experts in this field.

In one embodiment of the patients who entered the immunogenic composition according to the invention demonstrate reduced carriage of S. aureus. Such a reduction in the carriage or a long period of time after administration of the immunogenic composition, which is held in the status of nanocytes is important from the point of view of medical necessity. For example, the decrease in the total carriage of S. aureus carriers can be evaluated after a single dose multiantigenic vaccine against S. aureus. For example, up to 1 day before the introduction of the immunogenic composition of the group of adults aged 18-50 years of age can be screened for carriage by strokes of the nose and throat, followed by cultivation to determine their condition of carriage. For the eat group, you can enter the immunogenic composition according to the invention, having the group receiving the control treatment. Swabs from the nose and throat taken weekly during the 12-week period, and monthly until 6 months after the introduction of immunogenic compositions, in comparison with placebo. One of the primary stage is a comparison of the carriage in patients after administration of immunogenic compositions compared to placebo with 3-month intervals after immunization.

Animal models of staphylococcal infection

Here are a few animal models for use in assessing the effectiveness of any of these immunogenic compositions.

Murine model of sepsis (passive or active)

Model passive immunization

Mice passively were immunized intraperitoneally (i.p.) immune IgG or monoclonal antibody. Subsequently, after 24 hours, the mice were infected with a lethal dose of S. aureus. Bacterial infection was injected intravenously (i.v.) or i.p., ensuring that any survival could be attributed to specific interactions in vivo antibodies to the bacterium. The dose of bacterial contamination is defined as the dose required to achieve lethal sepsis in approximately 20% of the unimmunized control mice. Statistical evaluation studies survival analysis can be carried out by Kaplan-Meier.

The model of active immunization

In this model, we is it (for example, mice Swiss Webster) actively subjected to immunization administered intraperitoneally (i.p.) or subcutaneously (s.c.) target antigen at 0, 3 and 6 weeks (or according to another similar scheme vaccination with appropriate intervals in time) and then infect S. aureus at week 8 intravenously. The dose of bacterial contamination are calibrated to achieve approximately 20% survival in the control group over a 10-14 day period. Statistical evaluation studies survival analysis can be carried out by Kaplan-Meier.

The model of infective endocarditis (passive or active)

The model of passive immunization for infectious endocarditis (IE) caused by S. aureus, used previously to show that ClfA can induce protective immunity. See Vernachio et al., Antmicro. Agents & Chemo. 50:511-518 (2006). In this model, IE rabbits or rats are used to simulate clinical infections, which include Central venous catheter bacteremia and hematogenous seeding in distal organs. Catheterized rabbits or rats with sterile enlargement of the aortic valve impose one or more intravenous injection of monoclonal or polyclonal antibodies that are specific against the target antigen. Then animals i.v. infect a strain of S. aureus or S. epidermidis. Then after infection, heart, heart expansion and additional cloth is, including the kidneys and the blood is collected and cultivated. Then measure the frequency of staphylococcal infections in cardiac tissue, kidneys and blood. In one study, when infected animals or MRSE was ATSS 35984, or MRSA 67-0 have shown significant reduction in the level of infection when using any preparty polyclonal antibodies or monoclonal antibodies to ClfA. See Vernachio et al., Antmicro. Agents & Chemo. 50:511-518 (2006).

The model of infective endocarditis is also adapted for research active immunization as in rabbits and rats. Rabbits or rats were immunized intramuscularly or subcutaneously target antigen and were infected with S. aureus in two weeks by intravenous route.

The model of pyelonephritis

In the model of pyelonephritis mice subjected to immunization at weeks 0, 3 and 6 (or according to another scheme immunization with appropriate intervals in time) antigens on target. Then infect animals i.p.or i.v. S. aureus PFESA0266. After 48 h select the kidneys, and the bacterial count CFU (colony forming unit).

The antibodies and compositions of antibodies

According to the invention additionally proposed antibodies and compositions of antibodies that are specific and selectively associated with one or more than one antigen in the immunogenic compositions of the present invention. In some embodiments the antibodies are produced by introduction of the subject of them is ungenau compositions of the present invention. In some embodiments according to the invention proposed peeled or dedicated antibodies directed against one or more than one antigen in the immunogenic compositions of the present invention. In some embodiments the antibodies of the present invention are functional when measuring for killing bacteria or in an animal model of efficiency, either through analysis of opsonophagocytosis killing. In some embodiments the antibodies according to the invention give the subject of passive immunity. According to the present invention is additionally proposed molecules polynucleotide encoding the antibody or antibody fragment according to the invention, the cell or cell line (such as hybridoma cells or other engineered cell lines for recombinant production of antibodies), and transgenic animal that produces an antibody or antibody composition according to the invention using techniques well known to specialists in this field.

Antibodies or compositions of the antibodies according to the invention can be used in a method of treatment or prevention of staphylococcal infection, disease or condition associated with Staphylococcus sp., for a subject, comprising obtaining a preparation of polyclonal or monoclonal antibodies, and the use of the indicated antibodies or compositions of the anti-Christ. ate to make the subject of passive immunity. Antibodies according to the invention may also be useful in diagnostic methods, for example, determine the presence or quantitatively measuring levels of one or more than one antigen in the immunogenic compositions of the present invention.

EXAMPLES

The following examples illustrate some embodiments of the present invention. However, it should be understood that these examples are intended only for illustration and are not intended to fully define the terms and scope of this invention. It should be understood that when given a typical reaction conditions (e.g., temperature, reaction time and so on) can also be used in conditions both above and below certain intervals, although usually with less convenience. All parts and percentages referred to here are based on weight, and all temperatures are in degrees Celsius unless otherwise indicated.

In addition, the following examples were carried out using standard techniques that are well known and customary for specialists in this area, except in those cases where described otherwise. As noted above, the following examples are presented for illustrative purposes and in no way should be vtolkovivau as limiting the scope of the invention.

Example 1: retrieving antigens ClfA and ClfB

Factors SL is company A (ClfA) and b (ClfB) are the surface proteins of S. aureus, responsible for binding to host proteins, including fibrinogen (ClfA, ClfB) and cytokeratin 10 (ClfB). ClfA and ClfB are members of a family of proteins containing carboxykinase motif LPXTG (SEQ ID NO:125), which enables covalent binding of the protein to the cell surface. And ClfA, and ClfB belong to the family of proteins (the adhesive matrix molecule that recognize microbial components of the surface or MSCRAMM) that recognize and bind to proteins of the extracellular matrix of the host, such as fibrinogen (ClfA and ClfB), fibronectin (FnbA and FnbB), collagen (Cna) and others. All these proteins have aminobenzene signal sequence that mediates the transport to the cell surface. MSCRAMM also include domain a, which represents a functional area containing legendbase.ui site for fibrinogen, fibronectin, elastin and keratin. After the a-domain can be followed by a region consisting of serine-aspartate repeats (SD-repeat), which, according crosses peptidoglycan layer. After SD retry should cross the membrane area, which includes the motif LPXTG (SEQ ID NO:125) for covalent binding of protein to the peptidoglycan.

Landscapemode region of ClfA and ClfB, containing N1N2N3 AND domain comprise the amino acids 40-559. N-domains of ClfA/ClfB were defined as follows: N1 covers the remains of 45-220; N2 cover which indicates the remains 229-369; and N3 covers the remains of 370-559. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). In preparations of recombinant N1N2N3 ClfA found that the domain N1 is protectchristian and easily cleaved or hydrolyzed, leaving N23 in the form of stable landscapebased recombinant fragment. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). Similarly, it was demonstrated that the domain N1 ClfB is also protectchristian and can easily split metalloproteases S. aureus (McAleese, F. M. et al. J. Biol. Chem. 2001, 276, pp.29969-29978). Crystal structure fibrinogenesis fragment N23 And domain ClfA has revealed that as in N2, and N3 are dominated by antiparallel beta-strands. In addition antiparallel beta-strands of the domain N2 contains one turn of an alpha helix and two 310spiral, and N3 domain contains three 310the spiral. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002).

The alignment of the sequence N2 and N3 reveals only 13% sequence identity and 36% similarity to the sequence according to their length. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). The topology of the domain N2 and N3 is similar to the classical styling IgG, and it was suggested that it represents new ways of laying IgG. See Deivanayagam et al. EMBO J. 21:6660-6672 (2002).

A recombinant form of ClfA used in described here immunogenic compositions, represent fragments of ClfA containing one Il is more than one domain N, for example, N1N2N3, N2N3, and are referred to here as "recombinant ClfA or rClfA". In addition, any rClfA should be a rClfA, which preserves the native structure of individual domains N and critical epitopes, but does not interfere with the normal processes of the immunized individual after administration (i.e., does not bind fibrinogen). Mutational studies have shown that matirovanie Y338A (N2) eliminated binding fragment N23 with fibrinogen. (This provision Y338A refers to the substitution of tyrosine to alanine at position 338 in the immature form of the polypeptide sequence from the still attached leader sequence. This replacement can be seen in the position 310 in the Mature mutated form of the ClfA polypeptide with SEQ ID NO:

123, which demonstrates the absence of binding to fibrinogen). See Deivanayagam et al. EMBO J. 21:6660-6672 (2002). Therefore, mutation Y338A took all fragments ClfA in the following studies.

Similarly, a recombinant form of ClfB used in described here immunogenic compositions, represent fragments of ClfB, containing one or more than one domain N, for example, N1N2N3, N2N3, and are referred to here as "recombinant ClfB or rClfB". In addition, any rClfB should be a rClfB, which preserves the native structure of individual domains N and critical epitopes, but does not interfere with the norms of the global processes in immunized individual after administration (i.e., does not bind fibrinogen). (See, for example, Walsh, E. J. et al. Microbiology(2008), 154, 550-558).

ClfA and ClfB: overview of the cloning strategy

Different forms of protein rClfA used to obtain preclinical data on efficiency include HisClfA(N123); T7ClfA(N123); T7ClfA(N123); Y338A; ClfA(N23)and ClfA(N23)Y338A. See The Fig.1. ClfA gene contains a region And the coding sequence of PFESA0237 S. aureus corresponding to residues 40-559. - Reading frames cloned from S. aureus, was merged with N-terminal His-tag (HisTag) and linker sequences of the vector (MRGSHHHHHHGS SEQ ID NO:127), along with three additional coding sequences (KLN), introduced at the C-end. (See below for the detailed methodology). The protein expressed from this vector was used for all experiments, where it is called HisClfA(N123).

Different forms rClfA originated in region (residues 40-559 ClfA expressed PFESA0237 S. aureus (top row). HisClfA(N123)is expressed using the T5 promoter contained in pQE30, and all other forms expressed using the expression of RET based on the T7.

Two forms of ClfB (T7ClfB N1N2N3 and ClfB N23) used for pre-clinical animal studies.

Methods cloning ClfA

Cloned the coding sequence of ClfA corresponding to amino acid residues 40-59 from strain S. PFESA0237 aureus, and introduced the mutation Y338A to eliminate binding of fibrinogen. The mutated gene ClfA was introduced in T7 RNA polymerase expression vector, RETA (Novagen) to obtain plasmid pLP1179. The sequence of the DNA region containing the T7 promoter and coding region in pLP1179 represents SEQ ID NO:124. The expression vector was transformed into BLR(DE3) E. coli (Novagen) for production of recombinant ClfA.

Design T7ClfA(N123)Y338A included several stages. Generalization stages of cloning used to design the final expression plasmid, pLP1179 shown in Fig.2.

The DNA sequence clfA present in pQECIf40 corresponds to amino acid residues 40-559 ClfA, which were initially cloned into the cloning site BamHI/HindIII pQE30. This creates a fusion with a His-tag at N-end ClfA and the addition of three residues at the C-end. The coding region of ClfA present in (AmpR) pQECIf40, was subcloned into the vector KanR pET 27b (Novagen) to create pLP1137. In addition, the DNA sequence clfA, corresponding to amino acid residues 221-559, cloned into the cloning site NdeI-HindIII pET27b to create pLP1134. N-terminal His-tag ClfA was replaced by N-terminal T7 by sublimirovanny DNA fragment BamHI-BlpI of pQECIf40 in RETA (Novagen) to create pLP1153. The coding sequence of the T7ClfA(N123)present in pLP1153, contains 11 N-terminal amino acid is the action of T7 tag rate, with the subsequent three amino acid residues of the linker sequences plus three C-terminal residue originating from the linker, originally present in pQE30Clf40. Mutation Y338A was first introduced in the coding sequence of ClfA(N23)pLP1134 creating pLP1168. Later, the DNA fragment > PST -SnaBI containing the mutation Y338A of ClfA(N23)pLP1168, was moved in the coding posledovatelnosti > PST -SnaBI T7ClfA(N123)pLP1153 creating pLP1171. Internal binding site of ribosomes present in the coding sequence T7ClfA(N123)Y338A pLP1171, changed silent mutations at positions 339 and 342 DNA ORF (open reading frame) T7 rClfA Y338A, replacing G to T and G to A, respectively. The resulting plasmid, pLP1176, then used to remove the three extraneous residues, initially originating from pQE30Clf40 between T7-tag and the beginning of the coding region of ClfA. At this time we also removed three C-terminal residue originating from the linker.

rClfA expressed resulting plasmid, pLP1179 (Fig.2 and 3), contains only the 11 N-terminal amino acids, fused with the remnants of 40-559 ClfA(N123)Y338A. The sequence of the DNA region containing the T7 promoter and the coding sequence T7 rClfA(N123)Y338A pLP1179 represents SEQ ID NO 124.

Bacterial strains and plasmids

Plasmid based rate (obtained from Novagen) is designed and used for the Finance pLP1179, which expresses T7ClfA(N123)Y338A from the T7 promoter. This plasmid contains the gene for resistance to kanamycin (KanR) for positive selection. The original used the host strain is E. coli BL21(DE3) [F - ompT hsdSB(rB-mB-) gal dcm (DE3)] (Novagen) for expression T7ClfA(N123)Y338A. Designation DE3 means lambda lysogen, containing the gene for RNA polymerase T7 under control of the lacUV5 promoter (induced by IPTG (isopropylthioxanthone)), which is used for induced expression of the RNA polymerase T7 and subsequent transcription from the T7 promoter, proximal with respect to the coding sequence ClfA(N123)Y338A present in pLP1179. When receiving information about what lysogeny strain-host BL21(DE3) is able to induce lytic phage when fermentation on a large scale, strain-owner replaced the strain-host recA BLR(DE3) [F - ompT hsdSB(rB-mB-) gal dcm Δ(sri-recA)306::Tn10(TcR) (DE3)] (Novagen).

Production and purification of ClfA

For the production of ClfA E. coli BLR(DE3)/pLP1179 were grown in defined medium in bioreactors under periodic way with water glucose (glucode fed-batch mode). When the culture optical density (OD600from 30 to 50 the expression of ClfA was induced by adding IPTG. The culture was collected through 3-16 hours after induction.

Cells were destroyed, and collected clarified R is storemay faction. After adding ammonium sulfate material was applied to a column containing phenyl-separato resin, and suirable. The fractions containing ClfA identified, deliberately and inflicted on anion-exchange column (Q-Sepharose). After elution salt gradient fractions containing ClfA identified, was concentrated by ultrafiltration and applied to a column for gel filtration (Superdex-75). The fractions containing ClfA identified and combined. Purity ClfA at this point was approximately 98% when the dimension SDS-PAGE (polyacrylamide gel electrophoresis with sodium dodecyl sulfate).

Cloning and purification of N1N2N3 ClfB

The coding sequence of ClfB, corresponding to amino acid residues 44-542, cloned in the expression vector RNA polymerase T7, RETA (Novagen), to obtain plasmid RRH. The expression vector was transformed into E. coli BLR(DE3) (Novagen) for production of recombinant ClfB. (See Walsh, et al., Microbiology 154:550-558 (2008).)

For the production of ClfB E. coli BLR(DE3)/pLP1179 were grown in defined medium in bioreactors under periodic way with water glucose. When the culture optical density (OD600from 30 to 50 the expression of ClfB was induced by adding IPTG. The culture was collected through 3-16 hours after induction.

Cells were destroyed, and collecting the clarified soluble fraction. the pH of the soluble fraction was brought is to a pH of approximately 3.2 and removed fallen to precipitate impurities. the pH of the soluble fraction containing ClfB, brought to a pH of about 8.0 and deliberately to remove salts. After adding ammonium sulfate material was applied to a column containing phenyl-separato resin, and suirable. The fractions containing ClfB, identified, deliberately and inflicted on anion-exchange column (Q-Sepharose). After elution salt gradient fractions containing ClfB, identified, was concentrated by ultrafiltration and applied to a column for gel filtration (Superdex-75). The fractions containing ClfB, identified and combined. Purity ClfB at this point was approximately 94% when the dimension SDS-PAGE.

Example 2: production of antigens: MntC Staph aureus

Cloning limitirovannoe S. aureus MntC

Recombinant MntC originally cloned from a strain of S. aureus Mu50. The coding sequence rMntC amplified PCR (polymerase chain reaction) from genomic DNA of S. aureus Mu50. Were used for amplification two pairs nested (nested) primers (table 2). The first pair of primers, 5'SA926-MntC ups and 3'SA926-MntC down, aligned with the sequence above and below relative to the open reading frame rMntC. The second set of primers is aligned with the coding sequence rMntC, allowing you to amplify a sequence that corresponds to amino acid residues 19-309. Sites of restriction enzymes were included at the 5'-ends of the data is x primers to facilitate directional cloning. PCR was performed in a Peltier Thermal Cycler (MJ Research Inc, Walthan, MA) with TaKaRa PrimeSTAR HS DNA Polymerase Premix (Takara Bio USA, Madison, WI). The PCR product was purified QIAEX II (Qiagen, Valencia, CA), were digested with appropriate restriction endonucleases (New England BioLabs, Ipswich, MA) and was subcloned into the expression vector pBAD18Cm, managed by the araBAD promoter. This vector also contains a signal peptide lipoprotein P4 of N. influenzae. The PCR product MntC was subcloned into the reading frame downstream from the signal peptide P4 with the creation of pLP1194. The DNA sequence coding region MntC pLP1194 shown in SEQ ID NO:120. MntC expressed from pLP1194, is a lipoprotein. Recombinant plasmid DNA sequenced by the ABI PRISM BigDye™ Terminator V. 3.1 (Applied Biosystems, Foster City, CA), and recombinant protein expressed in E. coli BLR (NOVAGEN) for products limitirovannoe rMntC.

Production and purification limitirovannoe MntC

For products limitirovannoe MntC E. coli BLR/pLP1194 were grown in defined medium in bioreactors under periodic way with water glucose. When the culture optical density (OD600) about 60 expression rMntC induced switching of feeding on a mixture of glucose and arabinose. The culture was collected approximately 24 hours after induction.

Cells were destroyed and collected insoluble fraction. Libidinously MntC found associated with cellular membranes due to the modification of lipids MntC was extracted from the membrane fraction of the detergent (Zwittergent ZW-312). After removal of insoluble debris, libidinously MntC found in the soluble fraction. The soluble fraction was applied to a column containing a resin mixed principle, and suirable linear gradient of salt and pH. The fractions containing MntC identified and combined. To the combined fractions were added ammonium sulfate, and the substance was applied to a column containing boutiliers, and suirable. The fractions containing MntC identified, absoluely and loaded on a cation exchange column (SP-Sepharose). After elution salt gradient fractions containing MntC identified and combined.

Cloning nelimitirovanogo S. aureus MntC

The DNA sequence used for expression nelimitirovanogo rMntC, was isolated PCR amplification from a plasmid pLP1194. The resulting sequence corresponds to amino acid residues 19-309 and does not contain a signal sequence, which controls the secretion and limitirovanie. The DNA sequence coding region rMntC pLP1215 is in the DNA of SEQ ID NO:120.

To create pLP1215 MntC PCR amplified from pLP1194. The DNA sequence MntC present in pLP1215 corresponds to amino acid residues 19-309, and the first codon for this design was introduced in direct primer used in the amplification of the gene. The primers used for PCR, also contain what the AITA restriction enzymes at the 5'ends to facilitate directional cloning (table 2). PCR and purification of the amplified gene was performed as described above. Purified PCR product was digested with appropriate restriction endonucleases (New England BioLabs, Ipswich, MA) and was subcloned into the expression vector rate driven by the T7 promoter (Novagen, Madison, WI). DNA recombinant plasmids pLP1215 sequenced by the ABI PRISM BigDye™ Terminator V. 3.1 (Applied Biosystems, Foster City, CA), and recombinant protein expressed in E. coli BLR(DE3). Plasmid DNA for pLP1215 was purified and used to transform E. coli HMS174(DE3) to assess the expression of the protein.

Table 2
Primers MntC
Expression designName of primerSequence (5'-3')
Libidinously MntC (pLP1194)5'SA926-MntCupsCAC AAA ATT TAC GAA AAA TAG GAA ACG AG (SEQ ID NO:109)
3'SA926-MntCdownAAA ATA TTG GAG ATA CCA ATA TTT TAG GTT G (SEQ ID NO:110)
5'BamHISA926_MntCTTT CTTGGA TCCGGT ACT GGT GGT AAA CAA AGC AGT G (SEQ ID NO:111)
3'SphlSA926_MntCTTT CTT TTA TTT CAT GCT TCC GTG TAC AGT TTC (SEQ ID NO:112)
Neopalimovsky MntC (pLP1215)5'NcoIMntCTTT CTTCCA TGGGTA CTG GTG GTA AAC AAA GCA G (SEQ ID NO:113)
3'BlpIMntCTTT CTTGCT CAG CAT TAT TTC ATG CTT CCG TGT ACA G (SEQ ID NO:114)

To generate structures rMntC used synthetic oligonucleotides. Sites of restriction endonucleases are underlined. Nucleotides in bold indicate the first codon for construction nelimitirovanogo rMntC.

Production and purification nelimitirovanogo rMntC

For products nelimitirovanogo rMntC E. coli HMS174(DE3)/pLP1215 were grown in defined medium in bioreactors under periodic way with water glucose. When the culture optical density (OD600) from about 60 to 80 expression rMntC induced by adding IPTG. The culture was collected approximately 24 hours after induction. Cells were destroyed and collected the clarified soluble fraction. The lysate was applied to a column containing cation exchange resin (SP-Sepharose) and suirable linear salt gradient. Identify fractions containing the MntC. After adding ammonium sulfate material was applied to a column containing phenyl-separato resin, and suirable. After elution fractions, rMntC, identified, consolidated and absoluely. Purity rMntC at this point was >95% when the dimension SDS-PAGE.

Example 3: production of capsular polysaccharides SR and SR

This example describes the obtaining of capsular polysaccharide types 5 and 8 S. aureus in different sizes. The structure of the polysaccharides SR and SR shown in Fig.4. The described methods are effective for products SR and SR with molecular masses ranging from about 50 kDa to 800 kDa. On the basis of growth characteristics and quantity of biogas produced capsules was selected strain PFESA0266 for products SR, whereas strains PFESA0005 or PFESA0286 were used for the production SR. It was shown that capsules isolated from strains PFESA0005 and PFESA0286 are identical.

For the production of capsular polysaccharides strains were grown in complex medium consisting primarily 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.

Cleaning SR and SR used to obtain conjugates, conducted in two different ways, which are based on high temperature and low pH to effect the release of the capsule from the cells and reduce the molecular weight of the polysaccharide. The obtained molecular mass depends on time and, temperature and pH of the hydrolysis step.

Characterization SR and SR was performed using the methods specified in Table 3. Capsular polysaccharides obtained with this technique are pure polysaccharides with low levels of impurities protein, NA (nucleic acids), peptidoglycan and THAT tahaawee acid). See Tables 4 and 5.

Table 3
Analyses for the characterization of purified SR and SR S. aureus
SpecificityAnalysis
Residual proteinColorimetric analysis at Lowry
Residual nucleic acidsScanning at 260 nm
The residual tagaeva acidColorimetric analysis of phosphate
Residual peptidoglycanHPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection)
SizeSEC-MALLS (gel filtration, combined with detection by scattering of the hole the aqueous radiation under many angles)
CompositionHPAEC-PAD
Identity1H-NMR (1H-nuclear magnetic resonance) or reaction with a specific mAb (monoclonal antibody)
O-acetylation1H-NMR
ConcentrationMALLS-RI or HPAEC-PAD

In the first method, after the release of the capsule from the cells and reduce the molecular weight, the drug capsule is treated with a mixture of enzymes (ribonuclease, deoxyribonuclease, lysozyme and protease for cleavage of impurities. After incubation, residual impurities precipitated by adding ethanol (final concentration approximately 25%). After removal of residual ethanol, the solution containing capsule, loaded on anion-exchange column (Q-Sepharose) and suirable linear salt gradient. The fractions containing capsules were combined and treated with meta-periodate sodium. This treatment resulted in oxidative hydrolysis of residual impurities tahaawee acid but had no effect on SR or SR. The reaction was suppressed by the addition of ethylene glycol. The substance was concentrated and subjected diafiltration against distilled H2O to remove any residual reagents and by-products.

The second way, use the p to obtain capsules did not include the use of enzymes for degradation of different impurities originating from the cells. In this way, after the release of the capsule from the cells and reduce the molecular weight hydrolysate fermentation broth was osvetleni by microfiltration followed by ultrafiltration and diafiltration. To remove impurities, the solution was treated with activated carbon. After treatment with charcoal, the substance was treated with meta-periodate sodium for oxidation of the residual tahaawee acid with subsequent quenching with propylene glycol. The substance was concentrated and subjected diafiltration against distilled H2O to remove any residual reagents and by-products.

Capsules produced using any of the two methods yield the pure polysaccharides with low levels of impurities proteins, nucleic acids and tahaawi acid. The described methods can be used to obtain polysaccharides with specific desirable intervals of high molecular mass simply by varying the conditions of hydrolysis. Having special advantages in the range of polysaccharides with high molecular weight, ranging from 70 to 150 kDa, is useful for obtaining immunogenic compositions by conjugation of polysaccharide with protein carrier.

Examples of capsular polysaccharides with high molecular mass is, you can get the ways described here, is shown in Table 4 below. The party cleared SR with higher MW (molecular weight) also had a high purity, as indicated by the absence of THAT (tahaawee acid), peptidoglycan and low level of residual protein. See Table 4. The interval of molecular masses in these examples covered from 132,7 kDa to 800 kDa, and purified polysaccharides were highly O-acetylated - in the range of 90-100%, and 100% with respect to N-acetylation. See Table 4.

Examples of the capsular polysaccharide of smaller molecular weight, which can be obtained is described here by the methods shown in Table 5 below. The party cleared SR lower MW was of high purity, as indicated by the absence of tahaawee acid (TA), peptidoglycan and low level of residual protein. See Table 5. Interval smaller molecular masses covered from of 20.4 kDa to 65.1 kDa, and purified polysaccharides were highly O-acetylated in the interval 75-96%. Pollution levels nucleic acids were low, varying from 0.5% to 2.45%. See Table 5.

CF (mg/ml)
Table 4
Characterization of drugs SR
CF-5 SAMW (kDa)O-acetyl (%) NMRIdentity NMRN-acetyl (%) NMR
1800,13,164100Test passed100
2132,71,17290Test passed100
3335,40,97590Test passed100
4366,80,86590Test passedND

Table 5
Characterization of drugs SR
CF-8 SATotal purified WED mgMW (kDa) (g/mol)Protein (Lowry) % (wt./mass.)NK (SK the plan at 260 nm) % (wt./mass.) O-Acetyl NMR %
531027,01,20,94100
643829,02,42100
717920,40,370,12108
810146,9Below the threshold of detection0,594
99165,11,152,4596
1057835,52,470,6575

The choice of molecular weight capsular polysaccharides

This kinetic analysis demonstrates that through the methods described here can generate the wide range of potential molecular masses of capsular polysaccharides. The original bacterial cells were produced larger polysaccharides, and later you can select the desired value of the molecular weight and then do the cleaning, manipulating pH and heating conditions in the stages of heat and hydrolysis.

Heat treatment of fermentation broth of S. aureus was the stage of the way between fermentation and isolation CF. At this stage of the method used for heating treatment of the broth with pH adjusted within a certain period of time. The purpose of heat treatment at low pH was killing cells, inactivation of enterotoxins, the release associated with cell polysaccharide and reducing molekulyarnoi mass to the desired size. Among these goals, the decrease in molecular weight was the slowest in terms of processing required at this stage. Therefore, other goals inevitably have been achieved within the time of processing.

Thermal processing

Were defined conditions of temperature and pH for different intervals molecular weight capsular polysaccharides. the pH of the broth was adjusted with concentrated sulfuric acid. Then the temperature of the broth was increased to the target value. The time of heat treatment was started as soon as the temperature reached the set point. When was achieved we wish the e processing time, the broth was cooled to room temperature. Samples that are in the process of treatment, were selected to determine the concentration and molecular weight polysaccharides via HPLC (high performance liquid chromatography) and SEC-MALLS, respectively. Data on MW used in the kinetic analysis. Profiles MW was determined period of time at pH 4.0, 4.5, 5.0mm for SR and at pH 3,5; 4,0 and 5,0 for SR. See The Fig.5A and 5B.

The kinetics of mild acid hydrolysis of the polysaccharides was determined using purified CF-5 and CF-8 obtained in this way. The solution of purified polysaccharide brought up to the desired experiment the pH with sulfuric acid. The samples were placed in an oil bath, equipped with a system for precise temperature control. Each sample was collected with a specified time interval and extinguished (reaction) in the vessel with ice. At the end of the experiment the sample was added to an aliquot of 1 M Tris buffer (pH 7.5) and brought the pH back up to a value of about 7. The samples were analyzed system SEC-MALLS. Data on MW used in the kinetic analysis. Determined the effect of temperature on the profiles MW CP5 at pH 4.5 and SR at pH 3.5 during the period of time. See The Fig.6A and 6B. This method of acid hydrolysis can be accomplished with the use of culture in the fermenter or in an intermediate state of purification, or, as shown here, using PTS is on the polysaccharide. Similarly, you can take other steps to reduce the molecular weight, such as sonication or shear force.

Results

The effect of pH on the reduction of MW during the heat treatment shown in Fig.5A and 5B for CF-5 and CF-8, respectively. You can see that the lower pH was more effective in reducing the size of the polysaccharide. The data also suggests that the CF-5 was more difficult to hydrolyze than CF-8 at the same pH. Looking at the profiles SR, intervals molecular mass of 300 kDa to 600 kDa can be generated using pH 5 at 95°C for a period of time from 15 minutes to 120 minutes. Similarly, the choice of pH 4 at 95°C for a period of time from 15 minutes to 120 minutes can give intervals molecular weight polysaccharide SR from 250 to 450 kDa kDa. In addition, the choice of pH 3.5 at 95°C for a period of time from 15 minutes to 120 minutes can give intervals molecular weight polysaccharide SR from 120 kDa to 450 kDa.

The effect of temperature on the reduction of MW investigated with the use of purified polysaccharides isolated from the allocation process. The results are shown in Fig.6A and 6B. As shown, the higher the temperature, the faster the rate of hydrolysis and the wider the range of molecular weights of polysaccharides formed over time. The use of a lower temperature, 55°C, compared with 95 which, at the same pH gives a more narrow range of molecular weights of polysaccharides.

In addition, in Fig.7 illustrates the correlation between the molecular weight of purified CP5 and SR and processing time for mild acid hydrolysis. The purified polysaccharide is a final product, obtained as a result of the allocation process, described in detail previously. As shown in Fig.7, increasing the time of heat treatment strain PFESA0266 S. aureus at pH 4.5 leads to the generation of polysaccharides SR lower molecular weight, while more than a short time heat treatment at pH 4.5 leads to the generation of polysaccharides SR with higher molecular weight. The size of the polysaccharides SR varied from about 90 kDa to about 220 kDa, depending on the length of time of heat treatment at low pH (4,5). Similarly, the increasing time of heat treatment strain PFESA0005 S. aureus at pH 3.5 leads to the generation of polysaccharides SR lower molecular weight, while more than a short time heat treatment at pH 3.5 leads to the generation of polysaccharides SR with higher molecular weight. The size of the polysaccharides SR varied from about 80 kDa to about 220 kDa, depending on the length of time of heat treatment at low pH (3,5). The correlation between the time of heat treatment at Eskom pH and the amount of purified polysaccharides SR and SR, as shown in this study provides an estimate of the processing time required for the production of purified polysaccharide with a certain interval of molecular weight.

It is important to note that, as demonstrated above, can be produced, released and cleared the full range of potential molecular masses of capsular polysaccharides for SR and SR from 20 kDa to more than 800 kDa. The described methods can be used for specific intervals desired capsular polysaccharides with high molecular weight. On the basis of these methods can be obtained particularly useful interval capsular polysaccharides type 5 and type 8 with high molecular weight, having molecular weights ranging from 70 to 150 kDa. See Table 6. This range of molecular masses capsular polysaccharide is useful for obtaining immunogenic compositions by conjugation of polysaccharide with protein carrier. Alternatively, this useful interval capsular polysaccharides SR and SR high molecular mass ranging from 80 to 140 kDa. See Table 6. Another useful range of capsular polysaccharides SR and SR with high molecular weight is from 90 to 130 kDa, or from 90 to 120 kDa for SR and SR. See Table 6. The conditions used to generate the capsular polysaccharide SR, and housego interval of molecular weight from about 100 to 140 kDa, are as follows: 95°C, pH 4.5 within 135 minutes. The conditions used to generate the capsular polysaccharide SR with the interval of molecular weight from about 80 to 120 kDa, are as follows: 95°C, pH 3.5 for 300 minutes.

Table 6
Getting specific interval SR and SR high molecular mass
RunMW SR (kDa)MW SR (kDa)
198142
289108
3108142
4108108
589ND
6100ND
79963
811372
910574
1010063
1187ND
ND = not done

Example 4: conjugation of capsular polysaccharides SR and SR

CRM197

This example describes methods and characterizing the analyses used in obtaining conjugates of S. aureus CP5-CRM197and S. aureus CP8-CRM197. It was estimated several chemical methods for conjugation of conjugation of capsular polysaccharides SR and SR S. aureus protein carrier. Conjugation using PDPH (3-2-pyridyldithio)-propenylidene) leads to the formation of covalent thioester communication, and CDI/CDT (1,1-carbonyldiimidazole/1,1-carbol-di-1,2,4-triazole) leads to the formation of one or carbon linker between the CF and the protein carrier.

Conjugation with CF CRM197through the conjugation chemistry with PDPH

The conjugation chemistry PDPH is a multi-step method, which involves the activation of the polysaccharide, remove tylenol protective group, treatment of activated polysaccharide intermediate connection, activation and protein purification CRM197and conjugation activated the s components with subsequent cleaning. After the introduction of a linker containing Tilney group in the polysaccharide and halogenoacetyl groups in the carrier protein CRM197, polysaccharides SR and SR S. aureus have been associated with protein carrier via a thioester bond. In protein CRM197introduced bromacetyl group by reaction of amine groups with N-hydroxysuccinimide ester bromoxynil acid. To generate tarirovannogo WED, carboxylate group, N-acetylaminophenol acid on WED, activated by a carbodiimide were subjected to combination with the hydrazide group reacts with sulfhydryl hydrazide heterobifunctional linker 3-(2-pyridyldithio)propionitrile (PDPH). Thiols PDPH-tarirovannogo WED, generated by recovery with DTT (dithiothreitol) and purified SEC on a column of Sephadex G25, were subjected to reaction with bromacetyl groups activated protein, leading to covalent thioester bond formed by substitution of bromine between CF and protein. Unreacted bromacetyl group "covered" by the hydrochloride group probably facilitates (hydrochloride of 2-aminoethanethiol). The reaction mixture was then concentrated and subjected diafiltration. The remaining unconjugated bromacetyl group covered hydrochloride group probably facilitates to ensure that after conjugation does not remain reactive bromoacetyl groups. This formed valentno the relationship between tilenum end group probably facilitates and acetyl group on the lysine residue after substitution of bromine.

Milirovanie capsular polysaccharide of S. aureus PDPH

The polysaccharide is first activated by milirovanie with PDPH. The polysaccharide was mixed with a freshly prepared mother liquor PDPH (250 mg/ml in DMSO (dimethylsulfoxide)), uterine EDAC solution (90 mg/ml in distilled H20) and mother liquor MES (morpholinepropanesulfonic acid) buffer (0.5 M, pH 4,85) to obtain the final solution of 0.1 M MES, and 2 and 4 mg CU/ml, maintaining the ratio by weight CP:PDPH:EDAC 1:5:3 for SR and 1:0,6:1.25 for SR. 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 MWCO (nominal cut-off molecular weight of 3500 Da) at 4°-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 4°-8°C. the Excess of DTT, as well as side reaction products were separated from the activated saccharide SEC using resin Sephadex G25 and distilled water as the mobile phase. Fractions were analyzed by analysis with DTDP (zitieren) tirinya group, and diapositive faction, which was loirevalley close to the "free volume" column were combined. The combined fractions were analyzed RANJAN (hydrazide pair-hydro is cibenzoline acid) and tests for O-acetyl to determine the degree of activation, which is 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 it was required for conjugation.

Activation of carrier protein

Separately, the carrier protein activated by bromotetradecane. CRM197diluted to 5 mg/ml of 0.9% NaCl, buffered with 10 mm phosphate, pH 7 (PBS (phosphate saline buffer solution), and then added NaHCO3, pH 7, to a concentration of 0.1 M using a 1 M mother liquor. Was added N-hydroxysuccinimidyl ether bromoxynil acid (BAANS) at a ratio of CRM197:BAANS 1:0,25 (mass.:mass.), using the mother liquor BAANS 20 mg/ml in DMSO. This reaction mixture is incubated at 4-8°C for 1 hour, then was purified using SEC on Sephadex G-25. Purified activated CRM197analyzed by analysis on Lowry to determine protein concentration and then diluted in PBS to 5 mg/ml as a cryoprotectant was added sucrose to 5% (wt./vol.), and the activated protein was frozen and stored at -25°C until it was required for conjugation.

The reaction mix

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

Results from studies of reproducibility of etiolirovaniya SR and SR with PDPH demonstrated that the degree of activation SR ranged from 11% to 19%, which corresponds to approximately one molecule of linker attached to ten repeating units of CF, to one molecule of linker five repeating units. Activation SR ranged from 12 to 16%, which was very similar to activation SR.

Bromotetradecane lysine residues CRM197it was very consistent, leading to activation 19-25 lysine from DOS is available 39 lysine. The reaction gave high outputs activated protein.

Conjugation with CF CRM197the conjugation chemistry CDI/CDT

CDI and CDT provide one-step method of conjugation, when the polysaccharide is activated in an anhydrous environment (DMSO) with the formation of the imidazole or triazole urethane groups with the 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). The reaction solution is diluted 10 times in water solution to remove unreacted activated groups and then purified by diafiltration.

Both chemical method of conjugation gave WED, covalently linked to a protein carrier, which was indicated the presence of saccharide and protein in the fractions from gel filtration and amino acid analysis of the conjugate is "covered" by glycolaldehyde or hydrochloride group probably facilitates.

The generalization of results from two or more parties conjugates obtained as chemically with PDPH and CDI/CDT, for both capsular serotypes with the size of the polysaccharides in the range from 20 to 40 kDa, are shown in Table 7 below. Was no significant time is of ice free capsular polysaccharides, the ratio CF:protein and outputs conjugates generated by these two types of conjugation. Antigenicity conjugated CF has not been modified by conjugation, as shown by the identity of the lines precipitation between conjugates and native CF.

Table 7
Characterization of SA (S. aureus) CPS-CRM197and SR-CRM197received two chemical methods of conjugation
ConjugateChemistryThe output of SR(%)The protein yield (%)The ratio of outputsFree saccharide (%)Free protein (%)Modified LisinaSize (MW or kDa (% less than 0.3), Mar./Bel.))
SA CP5-CRM
197
CDT19-27350,5-0,810-40less than 118-22from 38/61 to 76/74
PDPH 26-5240-990,4-1,023-50NDND7,5×105-2,3×106
SA CP8-CRM
197
CDI46-6254-550,8-0,922-25less than 17-834/57-60/57
PDPH34-7061-830,6-0,915-41ND11-1674-92%

As shown above, the described methods can be used for specific intervals desired capsular polysaccharides with high molecular weight. The aim of this study was to obtain conjugates of the pre-selected interval of high molecular mass, which could be filtered and purified CF for use in immunogenic compositions. In this example we've selected eight parties, where capsular polysaccharide SR ranged in molecular weight from about 90 kDa to about 10 kDa, and was conducted by conjugation using activation triazole (CDT). See Table 8. The molecular weight of the resulting conjugates ranged from 1533 kDa to 2656 kDa. The number of conjugated lysine on CRM197varied from a high - 22 to low 15. The level of free capsular polysaccharide varied from a high of 18% to a low of 11%. See Table 8.

Table 8
Compared with the pre-selected interval MW SR
RunMW of the polymer (kDa)The output of the saccharide (%)Free saccharide (%)MW, certain SEC-MALLS (kDa)Modified Lisina
11216311213019
2927216153322
311974265615
41156318191115

Table 9 summarizes the analysis of conjugates SR where capsular polysaccharide SR ranged in molecular weight from about 87 to 113 kDa kDa, and used imidazole conjugation chemistry. The molecular weight of the resulting conjugates ranged from 595 kDa to 943 kDa. The number of conjugated lysine on CRM197varied from high 9 to low -3. The level of free capsular polysaccharide varied from a high of 6% to a low of 2%. See Table 9.

Table 9
Compared with the pre-selected interval MW SR
RunMW polysaccharide (kDa)The output of the saccharide (%)Free saccharide (%)MW, certain SEC-MALLS (kDa)Modified Lisina
199886 9434
21137358413
31057937197
41008626309
5879035956

Both chemical method of conjugation gave WED, covalently linked to a protein carrier. No significant differences in free capsular polysaccharide, the ratio CF:protein and yield conjugates generated by these two methods.

Example 5: a variety of sequence polypeptide fragments N1, N2 and N3 ClfA

In this example evaluated the heterogeneity of the protein sequence of the polypeptide fragments N1, N2 and N3 ClfA from isolates that cause disease, obtained from different sources. Genes ClfA sequenced from strains of S. aureus associated with the many painful conditions. Sequence information from additional strains were obtained from GenBank for generating sequences of relevant strains. Table 10 lists the different sequence ClfA.

The alignment of the ClfA protein sequences from different strains of S. aureus that cause the disease, as shown in Fig.8A-8E. Protein sequences were aligned using MUSCLE. See Edgar, R. C. Nucleic Acids Research 32 (5):1792-1797 (2004). The alignment showed using SHOWALIGN. See Rice, P. et al., "EMBOSS: The European Molecular Biology Open Software Suite' Trends in Genetics, 16(6):276-277 (2000). Many of the sequences were repeated many times without variation. For clarity, each unique sequence was placed in the alignment only once. See The Fig.8A-8E. Only unique sequences were included in the list of sequences (sequence listing presented in the graphics part). For example, the protein sequence ClfA_001 was obtained from many different strains without any variation. See The Fig.8A-8E. The number in the sequence listing for any sequence can also be obtained from Table 10: strains of ClfA and sequence listings. Table 10 lists one typical strain that contained the same protein sequence ClfA_001. This sequence is shown in the first row is iraniana in Fig.8A-8E. This unique alignment sequence of ClfA antigen indicates that the SNPs were distributed across the entire area A (N1-N2-N3) ClfA. In some cases, for any given unique protein sequence ClfA was opened more than one nucleotide sequence encoding the same protein. Only the most frequently occurring DNA sequences were included in the sequence listing and Table 10. For ClfA the following sequences disclosed here and not found in GenBank: ClfA_003, ClfA_005, ClfA_008, ClfA_009, ClfA_013, ClfA_014, ClfA_015, ClfA_016, ClfA_017, ClfA_018, ClfA_019, ClfA_020, ClfA_021, ClfA_022, ClfA_023 and ClfA_024.

Table 10
Strains of ClfA and the sequence listing
Example of strainDNA-ClfAHT* SEQ ID NO:Protein-ClfAAK** SEQ ID NO:% Identity to the antigen
PFESA0131clfA_001-161clfA_0016299
PFESA0074clfA_002-163 clfA_0026492
PFESA0072clfA_003-165clfA_0036699
PFESA0159clfA_004-167clfA_0046894
PFESA0154clfA_005-169clfA_0057091
PFESA0096clfA_006-171clfA_0067291
PFESA0269clfA_007-173clfA_0077491
PFESA0081clfA_008-175clfA_0087697
PFESA0005clfA_009-177 clfA_0097895
PFESA0139clfA_010-179clfA_0108099
PFESA0237clfA_011-181clfA_01182100
PFESA0157clfA_012-183clfA_0128496
PFESA0069clfA_013-185clfA_0138692
PFESA0002clfA_014-187clfA_0148898
PFESA0147clfA_015-189clfA_0159091
PFESA0094clfA_016-191 clfA_0169298
PFESA0143clfA_017-193clfA_0179497
PFESA0129clfA_018-195clfA_0189699
PFESA0128clfA_019-197clfA_0199892
PFESA0148clfA_020-199clfA_02010091

Example of strainDNA-ClfANT* SEQ ID NO:Protein-ClfAAK** SEQ ID NO:% Identity to the antigen
PFESA0140clfA_021-1101clfA_02110298
PFESA0152clfA_022-1103clfA_02210491
PFESA0141clfA_023-1105clfA_02310696
PFESA0160clfA_024-1107clfA_02410894
NT* nucleotide; AK** amino acid

Investigated the phylogeny of protein sequences ClfA and built a phylogenetic tree. Sequences were aligned using ClustalW. See Chenna R, Sugawara H, Koike engineering Germany T., et al. Nucleic Acids Research. 31(13):3497-3500 (2003). Connecting adjacent branches of the tree (neighbor-joining trees) was downloaded 1000 times and demonstrated by MEGA 4.0. See Tamura K, et al., Molecular Biology &Evolution. 24(8):1596-1599 (2007). The values of the bootstrap values indicated on the branches represent the number of times the branch was reproduced in 1000 trials. Values less than 500 (50% reproducibility) are treated as weakly confirmed.

Sequence ClfA form a tree with 2 main branches. See The Fig.9. The separation of these the two groups are very well supported in phylogeny. One branch (top) includes 9 sequences, which are quite closely related to each other (96-99% identity), but have a more distant relationship with sequence-candidate clfA_011 with which they are identical to 91-92%. The second group, which includes clfA_011 is more diverse, and phylogeny in this group is also not confirmed. These protein sequences are 93-99% identical to each other.

Example 6: the sequence diversity of polypeptide fragments of N1, N2 and N3 ClfB

In this example evaluated the heterogeneity of the protein sequences of polypeptide fragments of N1, N2 and N3 ClfB of the 92 isolates causing disease, obtained from different sources. Genes ClfB sequenced from strains of S. aureus associated with many painful conditions. See Table 11. Information from additional strains were obtained from GenBank for generating additional sequences.

Alignment of sequences of ClfB proteins from different strains of S. aureus that cause the disease, as shown in Fig.10A-10D. Protein sequences were aligned using MUSCLE. See Edgar, R. C. Nucleic Acids Research 32(5):1792-1797 (2004). Alignment was using SHOWALIGN. See Rice, P. et al., "EMBOSS: The European Molecular Biology Open Software Suite' Trends in Genetics, 16(6):276-277 (2000). See the alignment of ClfB in Fig.0A-10D. As in the case of ClfA, many of the sequences were repeated many times without variation. For clarity, each unique sequence was placed in the alignment only once. See The Fig.10A-10D. Only unique sequences ClfB were included in the list of sequences. For example, the sequence ClfB_006 was obtained from many different strains without any variation. This sequence is shown in the first row of the alignment in Fig.10A-10D. The number in the sequence listing for any sequence can also be taken from Table 11. This alignment is representative of the unique sequences of the antigen ClfB indicates that the SNPs were distributed across the entire area A (N1-N2-N3) ClfB. Similarly, ClfA, for any given unique protein sequence ClfB was opened more than one nucleotide sequence encoding the same protein. Only the most frequently occurring DNA sequences were included in the sequence listing and Table 11. For ClfB the following sequences disclosed here and not found in GenBank: ClfB_001, ClfB_004, ClfB_005, ClfB_010, ClfB_011, ClfB_013, ClfB_014, ClfB_015, ClfB_016, ClfB_017, ClfB_018, ClfB_019, ClfB_020, ClfBJ)21, ClfB_022, ClfB_023 and ClfB_024. The phylogenetic tree shown in Fig.11.

table 11
Strains of ClfB and the sequence listing
Example of strainDNA-ClfBSEQ ID NO:Protein-ClfBSEQ ID NO:% Identity to the antigen
PFESA0286clfB_001-115clfB_0011695
PFESA0159clfB_002-117clfB_0021895

Example of strainDNA-ClfBSEQ ID NO:Protein-ClfBSEQ ID NO:% Identity to the antigen
RF122clfB_003-119clfB_0032094
PFESA0271clfB_004-121clfB_00495
PFESA0081clfB_005-123clfB_0052495
PFESA0080clfB_006-125clfB_00626100
PFESA0270clfB_007-127clfB_0072899
PFESA0269clfB_008-129clfB_0083095
PFESA0145clfB_009-131clfB_0093294
PFESA0069clfB_010-133clfB_0103495
PFESA0002clfB_011-135clfB_011 3696
PFESA0128clfB_013-137clfB_0133896
PFESA0129clfB_014-139clfB_0144095
PFESA0136clfB_015-141clfB_0154299
PFESA0139clfB_016-143clfB_0164499
PFESA0140clfB_017-145clfB_0174696
PFESA0141clfB_018-147clfB_0184894
PFESA0144clfB_019-149clfB_019 5097
PFESA0150clfB_020-151clfB_0205296
PFESA0152clfB_021-153clfB_0215496
PFESA0156clfB_022-155clfB_0225696
PFESA0163clfB_023-157clfB_0235894
PFESA0211clfB_024-159clfB_0246099

Example 7: the diversity of sequences MntC in clones of S. aureus causing disease

In this example evaluated the heterogeneity of the protein sequences of genes from MntC 104 isolates causing disease, obtained from different sources. Genes MntC sequenced from strains of S. aureus associated with many painful with what conditions. See Table 12. Information from additional strains were obtained from GenBank for generating sequences of strains.

The alignment of the MntC protein sequences from different strains of S. aureus that cause the disease, as shown in Fig.12A-12B. Protein sequences were aligned using MUSCLE. See Edgar, R. C. Nucleic Acids Research 32(5):1792-1797 (2004). Alignment was using SHOWALIGN. See Rice, P. et al., "EMBOSS: The European Molecular Biology Open Software Suite' Trends in Genetics, 16(6):276-277 (2000). See The Fig.12. As in the case of ClfA, many of the sequences were repeated many times without variation. For clarity, each unique sequence was placed in the alignment only once. See The Fig.12. Only unique sequences MntC were included in the list of sequences. For example, the sequence MntC_001 was obtained from different strains without any variation. See The Fig.12. This sequence is shown in the first row of the alignment in Fig.12. The number in the sequence listing for any sequence can also be obtained from Table 12. Most frequently, the corresponding DNA sequence was included in the list of sequences. For MntC following sequence disclosed here and not found in GenBank: MntC_002, MntC_006, MntC_007, MntC_008 and MntC_009.

Table 12
Strains MntC and the sequence listing
StrainDNA-MntCSEQ ID NO:Protein-MntCSEQ ID NO:% Identity to the antigen
PFESA0129MntC_001-11MntC_001299
PFESA0142MntC_002-13MntC_002499
PFESA0139MntC_003-15MntC_003699
PFESA0286MntC_006-17MntC_006899
PFESA0136MntC_007-19MntC_0071099
PFESA0150MntC_008-111MntC_0081299
PFESA0153MntC_009-113MntC_0091499

Example 8: expression of ClfA, CP5, SR and MntC on the surface of the in vivo during infection

S. aureus is responsible for calling the variety of human infections. Accordingly, the bacteria must adapt to different ecological niches by differential expression of virulence factors required for infection. The expression of antigen targets investigated in three tests on rodents in vivo to assess their expression during infection: wound model for measuring the expression of the antigen in the primary site of infection; models of bacteremia, which monitored the expression of the antigen in the blood, and model implanted (indwelling) of the camera which tracks the expression of the antigen during conditions with limited access) nutrients/oxygen. For all these models rodents were infected with bacteria at the place of study. After infection, the bacteria were collected at different time points and the expression of the antigen (ClfA, SR, SR, MntC) was estimated using immunofluor the interest microscopy (wound and bacteremia) or flow cytometry (camera).

MATERIALS AND METHODS

Expression in wound model

Experiments on wound infections consisted of 5 animals per group and up to 5 groups with up to 25 animals per experiment. Male C57BL/6 mice aged six to eight weeks (weeks). underwent surgery for inserting loops of the seam slit hip muscles. This gives the alien a substrate for the attachment of the bacteria and reduces the minimum infectious dose required to obtain staphylococcal wound infection. In the section under deep tissue suture of 4-0 silk has introduced five µl of S. aureus or sterile saline. The skin was closed with sutures from Prolene 4-0 or surgical adhesive means (e.g., cyanoacrylate). Animals were subjected to euthanasia in time from 30 minutes to 10 days after infection, and femoral muscle was excised, homogenized, and counted bacteria. In the field of visible infection were analyzed bacteria on the expression of the antigen by immunofluorescent (IF) confocal microscopy.

The expression in the model of bacteremia

A group of 10 four-week mice CD-1 or Balb/C mice were immunized with 1 mg of the protein or conjugate (CF subcutaneous injection of weeks. 0, 3 and 6. The animals took the blood in weeks. 0 and 8, with subsequent intraperitoneal infection of S. aureus grown to late logarithmic phase in TSB (Tr is picazo-soy broth). Three hours after infection, animals were subjected to euthanasia, and the blood was collected for confocal IF microscopy.

The expression in the model implanted (indwelling) of the dialysis tube

Isolates of S. aureus were grown overnight on tablets TSA at 37°C. the Bacteria were scraped off from the plates and resuspendable in sterile PBS (phosphate-saline buffer solution) and OP600brought up to 1, approximately 109colony forming units (CFU)/ml of Bacteria were diluted to a concentration of 103CFU/ml and was inoculable in the dialysis tube. An aliquot of the suspension was dropped off at the Cup to determine the actual number of CFU. Dialysis tubing with MWCO (cutoff molecular weight of 3.5 kDa were prepared for implantation by sterilization in 70% ethanol for 30 minutes, followed by abundant rinsing in sterile water and then in sterile saline. 2 ml aliquot of bacterial suspension was transferred into a dialysis tube, the bag was closed by the host, and then abundantly washed with sterile saline solution. Male rats Sprague Dawley (aged 6 weeks) were anestesiology and did 2-3 cm incision along the midline of the back. At the site of incision was created pockets soft separation of skin from underlying tissue. A tube implanted in his pockets, and the skin was closed using surgical staples. After 24 hours, rats were subjected Autana the AI, remove the tube and was isolated bacteria for the analysis of flow cytometry.

Immunofluorescence microscopy (IF)

Blood from 5 mice were pooled in ice-cold sodium citrate, pH 7.0 (final concentration of 0.4%). Eukaryotic cells literally 1% NP-40 (Pierce Biotechnology). Bacteria were washed in PBS and incubated overnight at 4°C with rabbit immune or preimmune serum (1:100) and were detected with the antibody goat against rabbit antibody conjugated with ALEXA488 (1:250, Invitrogen). Labeled bacteria were dried on a glass slide and mounted covering the glass with medium Vectashield HardSet (Vector Laboratories, Inc.). Images were obtained spectral confocal microscope (Leica TCS SL (Leica Microsystems).

Running cytometrics analysis

Isolates of S. aureus were grown as described above for the rat model with a dialysis tube. Approximately 107bacterial cells blocked in staining buffer (balanced salt solution Hanks with 10% goat serum) for 1 hour on ice. Bacterial cells were centrifuged for 5 minutes at 10000 rpm./min, supernatant was removed and cells were incubated with mouse antibody or ezotericheskim control antibody for 30 minutes on ice. Then cells were washed and stained with the antibody goat against mouse IgG, conjugated with FITC (fluoresceinisothiocyanate) (Jackson ImmunoResearch)on ice for 30 minutes. Bacteria were washed with buffer for staining were fixed with 2% paraformaldehyde and data were acquired and analyzed using a flow cytometer FACS Caliber and program Cell quest (Becton, Dickinson and Co.). For each sample were collected 30000 events.

-
Table 13A
Profiles the expression of antigens in isolates of S. aureus with CF type 5
AntigenCFClfAMntC
Time [h]T01462472T01462472T01462472
PFESA0266bacteremia +-±+//+-±+//NTNTNTNTNTNT
wound+---±++-±+++-±±±±±
PFESA0272bacteremia++++//+ -++//----//
wound+---+!+-±±±!---±!!
PFESA0094bacteremia+-±+//+-±!//-- --//
wound+--++++--+++---±±±
PFESA0093bacteremia+-±+//+-±+//NTNTNTNTNTNT
wound+-±+++---±±---±±±
PFESA0028bacteremia+--+//+-±±//--±±//
wound+-----+--±±---±±-
PFESA0029bacteremia--±+//+--+//---+//
wound----±±+----±---±-
/ = experiments bacteremia was performed within 6 hours
! = animal died during the experiment
NT = not tested

Table 13B
Profiles the expression of antigen in isolates of S. aureus with CF type 8
AntigenCFClfAMntC
Time (h)T01462472T01462472T01462472
PFESA0003Bacteremia+± ++//+-±+//NTNTNTNTNTNT
Wound+-±++++-±++!NTNTNTNTNTNT
PFESA0286Bacteremia+--+//+-- +//NTNTNTNTNTNT
Wound+--+!!+-++!!NTNTNTNTNTNT
PFESA0005Bacteremia+-±±//++++//NTNTNTNT NTNT
WoundNTNT!!NTNTNTNTNTNTNTNTNTNTNTNT
PFESA0002Bacteremia+---//+±--//-±±+//
Wound+--+ +!+---±NT--±±±+
PFESA0269Bacteremia+---//+---//--±±//
Wound+--±+++--±NT----±±
PFESA0268Bacteremia++++//++--//-±-+//
Wound+---+++---±NT---±± -
PFESA0025Bacteremia+---//++--//NTNTNTNTNTNT
Wound+---+±+----NT----±+
PFESA0283Bacteremia+-- -//+++-//±±±±//
Wound+--+!!+--+!NT---±±±
PFESA0027Bacteremia+--±//+-±± //NTNTNTNTNTNT
Wound+-±±+++--±+NT----±+
PFESA0001Bacteremia+---//+---//NTNTNTNTNT NT
Wound+--±±++---±NT-----±
PFESA0095Bacteremia+--±//+--+//NTNTNTNTNTNT
+---+ ++---±NT----±
PFESA0271Bacteremia+---//++--//NTNTNTNTNTNT
Wound+---+++--++NT --±±±+
PFESA0271Bacteremia+-±+//+-±±//NTNTNTNTNTNT
Wound+---+++---+NT----±±
Table 13B
The expression of the antigens of S. aureus in implanted dialysis tube.
The frequency of positive cells (% of total cells)
S. aureus PFESA0266Time (h)036,09,013,018,030,0
ClfA69,813,78,58,012,58,816,4
SR28,01,91,86,17,15,29,6
MntC91,44,35,6 2,920,837,033,2
S. aureus PFESA0005Time (h)036,09,013,018,024,0
ClfA98,663,969,724,336,198,699,0
SR77,343,018,07,511,896,494,0
MntC5,97,712,52,62,9 9,39,9

Results

A combination of nineteen isolates of S. aureus were tested for the expression of ClfA, SR, SR or MntC on the cell surface of S. aureus during infection (table 13A, 13B and 13C). These isolates included new clinically relevant strains and were varied while monitoring MLST (multiloci sequencing-typing). The expression of the antigen was dependent on strain, time and models of infection. Variation in the expression of antigen between isolates in different environments in vivo (blood flow compared with wound) supports the use multiantigenic immunogenic composition to induce a wide coverage of staphylococcal isolates with many different infections. Antigens expressionlist on the surface within the first 24 hours of infection and, thus, are suitable components for immunogenic compositions against staphylococci. Protein antigens of ClfA and MntC were available for staining in the presence of capsule expression, indicating that the presence of a capsule does not mask the proteins from the antibodies directed against them.

Most of the tested isolates of type 8 is not expressed in CF blood to late time points after infection (>4 h) (See Table 13A). These results demonstrate that S. aureus CF differentially regulated, is dependent on the microenvironment in vivo, i.e., from the site of infection. These results may explain the conflicting results on the efficacy reported for conjugates SR in animal models.

Results expression in vivo suggests that none of the immunogenic composition based on a single antigen will not provide broad coverage against the majority of S. aureus infections. There is too much variety of expression phenotypes of individual strains within microenvironments in vivo. Therefore, to prevent diseases caused by S. aureus, is required immunogenic composition comprising more than one antigen.

Example 9: immunogenicity multiantigenic compositions containing ClfA and conjugates SR - and CP8-CRM197

In this example, the inventors evaluated the immunogenicity combinations ClfA, CPS-CRM197and CPS-CRM197.

A. Dhandhania (CPS-CRM197/CP8-CRM197) drug immunogenic composition is a dose effect on the responses of antibodies against the capsule in rabbits

In this example evaluated the effect of dose on immunogenicity United immunogenic composition CPS-CRM197and CPS-CRM197In rabbits. Rabbits were immunized at week 0, 3 and 6 bivalent conjugate plus 125 mcg AlPO4introduced by subcutaneous injection. Doses evaluated in this study was 0.1 μg, 1 μg or 10 μg of each of the CPS-CRM197 and CP8-CRM197(final combined doses of CP-CRM1970,2 μg, 2 μg and 20 μg). The dose of conjugate reflects the total polysaccharide component of the conjugate protein-polysaccharide. In rabbits took the blood at week 0, 3, 6, and 8. ELISA was performed on pooled and individual sera. The antibody titers in the endpoint was determined as the reciprocal dilution at OP4050,1. Statistical analysis was performed on the individual captions in week 8. The results showed that the highest titers of specific antibodies against SR and SR: 5×105for SR and 1×106for SR were induced by vaccination of rabbits bivalent immunogenic composition at a dose of 1 μg of each component CF (data not shown).

B. Trichanthera composition (CPS-CRM197plus CP8-CRM197plus rClfA). study on the dose interval rClfA with a fixed dose (1 μg) of each conjugate in rabbits

Tested the effect of combination rClfA and conjugates SR and SR on the immune response to each component. Three groups were immunized bivalent CPS-CRM197plus CP8-CRM197S. aureus (dose of each conjugate (1 μg), combined with T7-ClfA (N1N2N3) in three different doses (1, 10 and 100 µg. The control group were immunized unconjugated SR and SR (50 μg each), combined with 100 μg T7-ClfA (N1N2N3). Each immunogenic composition were prepared in the form of the drug with 500 m is g adjuvant AlPO 4. Immunogenic compositions were administered by subcutaneous injection in the neck. In rabbits took the blood at week 0, 6 and 8. ELISA was performed on pooled and individual sera and antibody titers in the endpoint was determined as the reciprocal dilution at OP4050,1.

The results showed that an increased amount rClfA when combined with a bivalent conjugate had no effect on the response of antibodies to capsular polysaccharides. Levels of antibodies to both capsular serotypes were in the same interval as that of the rabbits immunized with only bivalent conjugate (data not shown). Levels of antibodies to SR and SR were 2.5 times lower than the dose of 10 (103 K) and 100 μg (106 K) compared with the dose of 1 μg rClfA (273 K). There was the booster effect after the second and third injections. Unconjugated bivalent immunogenic polysaccharide composition (SR+SR, 50 μg each), combined with 100 μg rClfA, not induced antibodies that are specific against CF. Antibody responses specific to rClfA were not significantly affected by the dose, where titers ranged from 1×105up to 1×106after three doses for doses of 1, 10 and 100 μg (data not shown). The levels of response against ClfA, which can only be achieved with the introduction of conjugated or unconjugated polysaccharides SR and SR, were similar.

Example 10: trichanthera composition, IMM is noganet in rabbits with high Ab titers against SR, SR and ClfA to immunization

Staphylococcal immunogenic composition is directed at the adult population who have preexisting antibodies to the components of the surface of S. aureus. To study the effect of preexisting antibodies to components of the immunogenic composition in response to this immunogenic composition, the inventors have selected rabbits with high titers of naturally derived antibodies against SR against SR and against ClfA. Two groups of rabbits (n=6/7) were immunized at week 0, 3 and 6 trichanthera immunogenic composition (CP5-CRM197(1 μg) and CP8-CRM197(1 μg) and T7-ClfA (N1N2N3)Y338A (10 μg)). One group was immunized immunogenic composition prepared in the form of the drug with 500 µg AlPO4as the adjuvant, and the second group were immunized immunogenic composition that does not contain adjuvant. Immunogenic compositions were administered by subcutaneous injection. In rabbits took the blood in weeks. 0, 3, 6, and 8. Antibody titers to SR, SR and rClfA was determined by ELISA as antibody titers at the end point at the joint and individual sera (defined as the inverse of the dilution in OP4050,1).

The results showed that rabbits with preexisting titers of antibodies induced by natural infection, answered trivalent immunogenic composition increased levels of antibodies to all of the components immunodeficiency is agenoy composition SR, SR and rClfA. 5-10-fold increase in Ab levels for each antigen was shown even in animals with antibody titers of 1×106. The presence of adjuvant in an immunogenic composition resulted in higher antibody titers compared to the group immunized without adjuvant (data not shown).

Example 11: effect of adjuvant on the responses to the components of the capsular polysaccharides

A. Effect of two different doses of AlPO4in response to a bivalent immunogenic composition with conjugates CP5-CRM197/CP8-CRM197in rabbits

Investigated the effect of dose adjuvant AlPO4responses against SR and SR in rabbits. Rabbits were immunized at week 0, 3 and 6 bivalent CP5-CRM197plus CP8-CRM197(dose of each conjugate (1 μg) of S. aureus. One group (n=5/group) were immunized immunogenic composition prepared in the form of the drug with 125 µg, and the second group - with 500 µg AlPO4adjuvant. Immunogenic compositions were administered by subcutaneous injection in the neck. In rabbits took the blood at week 0, 6 and 8, and antibodies against the capsule was determined by ELISA as antibody titers at the end point, defined as the inverse of the dilution, if OP4050,1. The results showed that there were differences in the responses of specific antibodies against SR in rabbits immunized with either 125 mcg, or 500 mcg AlPO4. Composition with 125 μg adjuvant was given more than the strong responses of antibodies against SR. All rabbits in the group immunized with 125 µg answered stronger antibody responses against SR, whereas in the group immunized with 500 μg adjuvant, had two rabbit with a weak response to the composition.

B. Effect AlPO4on the immunogenicity trichanthera composition

Rabbits (NZW, n=6/7 rabbits per group) were immunized at week 0, 3 and 6 trichanthera composition containing CP5-CRM197(1 µg), and CP8-CRM197(1 µg), and T7-ClfA (N1N2N3)Y338A (10 µg). One group of rabbits were immunized immunogenic composition with 500 µg AlPO4the second group were immunized without adjuvant, the third group were immunized at week 0 immunogenic composition with 500 µg AlPO4and in weeks 3 and 6 immunogenic composition without adjuvant. Immunogenic compositions were administered by subcutaneous injection in rabbits took the blood at week 0, 3, 6 and 8 and the serum was assessed by antigen specific ELISA. The results showed that the presence of adjuvant in an immunogenic composition had no effect on responses against SR or against CF-8 in rabbits (data not shown). GMT (geometric mean titer) antibody titers in both capsules were comparable. However, had the effect of adjuvant on ClfA-specific antibody responses, shown in the groups immunized with adjuvant present in all three vaccinations. The second and third booster immunization (boost) IMM is Noynoy composition, not containing AlPO4, rabbits, premirovany immunogenic composition containing the adjuvant, gave stronger responses to ClfA in comparison with the group without adjuvant.

Examples 12-29: preclinical evaluation of ClfA, MntC, CPS-CRM197and SR-CRM197S. aureus:

Below in the Examples 12-29 described the results of a preclinical evaluation of conjugates SR and SR, ClfA and MntC. The examples demonstrate the effectiveness of these antigens in preclinical animal models. The examples also demonstrate that the antibodies generated by the conjugates WED, ClfA and MntC, have functional activity in vitro.

For conjugation with CF CRM197used two different chemical method, but did not observe differences in efficacy for conjugates obtained in different ways. It was shown that O-acetylation of the capsular polysaccharides affect the induction of functional antibodies. Evaluation of the United immunogenic compositions containing CP5-CRM197, CP8-CRM197and ClfA, showed no disturbing influence on the levels of specific antibodies (Ab) on each component of the immunogenic composition.

Materials and methods

ELISA

Tablets ELISA for micrometrology Maxisorp (Nalge Nunk International, Rochester, NY) were coated for 18 hours at 4°C or for 90 min at 37°C With 1 µg/ml of ClfA antigen in PBS, pH 7.5. The tablets were washed five times in PBST (1X PBS, 0.1% Polysorbate 20) and blokirovki% (wt./about.) skim milk in PBS with 0.05% Polysorbate 20 for 1 h at room temperature. The tablets were washed in PBST, and serially diluted (3-fold), and, at weeks 0, 3, 6, and 8 were added in tablets rabbit anticigarette and incubated either overnight at 4°C or for 2 h at 37°C. the Tablets were washed and bound peroxidase primary antibodies were detected by antibody goat against rabbit IgG, conjugated with horseradish peroxidase (dilution 1:1000 in PBST. The plates were incubated for 1 h at 37°C, then washed and showed a solution of peroxidase substrate ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) (KPL, Inc., Gaithersburg, MD) at room temperature for approximately 20 minutes. The reaction was stopped by adding 1% (vol./about.) solution of SDS (sodium dodecyl sulphate). The absorption was measured at 405 nm in an automatic tablet reader (Molecular Devices Corporation, Sunnyvale, CA). Antibody titers were expressed as the inverse of the most high serum dilution with absorption values of 0.1. To determine differences in antibody titers between different groups used the t-student test using the program JMP (SAS Institute, Cary, NC). Believed that the probability is less than 0.05 indicates a statistically significant difference.

Murine models of sepsis

Murine model of sepsis mimics the disease is transmitted through blood. For passive immunization, groups of 15 mice Swiss-Webster administered intraperitoneally (i.p. was treated with IgG. Twenty-four hours later the mice were infected with S. aureus 659-018 by a single intravenous (i.v.) injections (0.1 ml) via tail vein. All animals were observed for a period of 14-15 days, the moment in time at which all remaining mice were killed.

For active immunization of mice were immunized with antigen in weeks 0, 2 and 4 and were infected in week 6 of S. aureus injection.

The rabbit model of endocarditis active immunization

Adult rabbits New Zealand White 4 times were immunized intramuscularly 25 μg of antigen. Through one day after surgery, animals were infected i.v. a bolus of S. aureus and to determine the number of colony forming units (CFU) in cardiac tissue after 24 hours of infection.

Murine bacteremia

Model 3 h bacteremia used to determine the effect of vaccination on the number of bacteria at the early stage of infection. Mice were immunized at weeks 0, 3 and 6 antigen, with the next i.p. infection of S. aureus in week 8. Animals were bled after 3 hours and serial dilutions of blood were sown on Petri dishes for counting bacteria.

Murine model of pyelonephritis

Murine model of pyelonephritis simulates the spread of S. aureus in the bacteremia. A group of 10 four-week female mice CD-1 were immunized with antigen in weeks 0, 3 and 6. The mice were infected i.p. injection of S. aureus. Forty-eight hours the after infection, mice were killed and counted the number of bacteria in the kidney and in blood.

Rat model of endocarditis

Rat model of endocarditis simulates human endocarditis, in which colonization occurs as infection, blood-borne, leads to the colonization of damaged cardiac tissue. Five 5-week-old male rats Sprague-Dawley (Charles River, Kingston, NY) were immunized at weeks 0, 2 and 4 1 µg conjugate CPS-CRM197prepared in the form of a preparation with 100 µg AlPO4. The animals took the test prior to vaccination in weeks. 0 and at the end of the week. 5. In seventy-two hours in the left ventricle via the carotid artery was surgically installed a catheter (tube D-10). The catheter leads to the formation of sterile growth, to which the infection can be attached staphylococci. To prevent infections resulting from surgical procedures, animals were treated with the antibiotic baytril (5 mg/kg) during the surgical intervention and 8 hours after surgery. Forty-eight hours after surgery, rats were infected PFESA0266 (approximately 4×108SOME or SA315 (approximately 1×109SOME) intraperitoneally injection. Forty-eight hours after infection, rats were subjected to euthanasia and the heart and the kidneys were removed and placed in 3 ml of phosphate buffered saline (PBS). These bodies then Homo who was anosirovala tissue homogenizer (Kinematica AG, Luzernerstrasse, Germany) and brought volume up to 10 ml PBS. The homogenates are then serially diluted and were sown on Petri dishes for counting bacteria.

Monitoring of functional antibodies using analyses opsonophagocytosis killing

For this analysis, you can use differentiated effector cells of cell lines (e.g., HL60s) or polymorphically cells (PMN) isolated from human donor blood using solution LYMPHOLYTE®-poly (Cedarlane laboratories limited, Ontario, Canada) according to the manufacturer's Protocol. Effector cells resuspendable buffer for analysis (modified atmosphere Needle containing 1% bovine serum albumin) at a concentration of approximately 2 X 107cells/ml and placed in an incubator with 37°C until they were ready for use. Strain PFESA0266 S. aureus were grown overnight on plates with trypticase soy agar. Bacterial cells were scraped off, washed twice and resuspendable buffer for analysis containing 5% glycerin, and OP600=1, which is equal to the concentration of approximately 5×108CFU/ml 1 ml aliquots of the bacterial suspension was frozen and stored at -40°C until time of use. Frozen bacterial suspension was thawed and brought a concentration of up to 106CFU/ml in the buffer for analysis and placed on Le is. The analysis was performed using sterile 96-well polypropylene tablets deep 1 ml wells. Received twofold serial dilution of antibodies (50 μl) followed by addition of 300 μl of buffer for analysis to the mixture of antibodies. Bacteria were added (50 μl) in tablets and was placed on a rotary shaker for 30 minutes at 4°C. After 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 the suspension was well mixed by repeated pipetting. a 50 μl aliquot of the suspension 10-fold serially diluted in sterile 1% solution of saponin, shook to minimize aggregation of bacteria and landed on trypticase soy agar in duplicate. Analysis tablet incubated at 37°C for 1 hour with continuous stirring using Rotisserie shaker style. At the end of incubation, 50 μl aliquot of the suspension 10-fold serially diluted in sterile 1% solution of saponin were mixed by shaking to minimize aggregation of bacteria and landed on trypticase soy agar in duplicate. The percentage of killing was calculated by determining the ratio between the number of CFU surviving in 60 minutes in the wells with bacteria and, antibodies, complement and effector cells to the number of CFU surviving in test tubes containing no antibodies, but containing bacteria, complement and effector cells. Included controls containing bacteria, complement and serum, to correct any reduction in CFU caused by aggregation.

Adsorption of complement

As the source of complement in this analysis, you can use the serum from human donors undergoing adsorption against strains strains PFESA0266, PFESA0286 and PFESA0270 S. aureus. Strains of S. aureus were grown overnight on tablets (TSA trypticase soy agar) at 37°C. the Cells were scraped off with a tablet and resuspendable in sterile PBS. Bacterial cells were centrifuged at 10000 rpm./min for 10 minutes at 4°C, and the cell sediment resuspendable in human serum adsorption. Serum incubated with bacteria on the rocking chair (nutator) 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 again repeated for 30 minutes. Finally, cells were centrifuged and the serum was passed through the 0.2 micron filter before 0.5 ml aliquots were frozen in liquid nitrogen.

Method II. ORA using cells HL-60

Cells HL-60 were mutated (differentiated) according to S. Romero-Steiner, et al., Clin Diagn Lab Immunol 4 (4) (1997),pp.415-422. The collected cells HL-60 resuspendable buffer for analysis (modified atmosphere Needle containing 1% bovine serum albumin) at a concentration of approximately 108cells/ml and placed in an incubator at 37°C until time of use. S. aureus was grown overnight on plates with trypticase soy agar. Bacterial cells were scraped off, washed twice and resuspendable buffer for analysis containing 5% glycerin, and OP600=1, which is approximately 5×108CFU/ml 1 ml aliquots of the bacterial suspension was frozen and stored at -40°C until time of use. Frozen bacterial suspension was thawed and brought to a concentration of 106CFU/ml in the buffer for analysis, and was placed on ice. The analysis was performed using sterile 96-well polypropylene tablets deep 1 ml wells. Received twofold serial dilution of a monoclonal antibody (25 μl) and then added to a suspension of antibody 150 ál of buffer for analysis. Bacteria were added (25 μl) in tablets and was placed on a rotary shaker for 30 minutes at 4°C, followed by addition of 25 μl of human complement (final concentration 1%). Finally, the tablet was added 25 μl of cells HL-60 (107cells/ml), and the suspension was well mixed by repeated pipetting. 25 µl of al the quota suspension of 10-fold serially diluted in sterile 1% solution of saponin, mixed by shaking to minimize aggregation of bacteria and landed on trypticase soy agar in duplicate. Analysis tablet incubated at 37°C for 1 hour with continuous stirring using Rotisserie shaker style. At the end of incubation, 25 μl aliquot of the suspension 10-fold serially diluted in sterile 1% solution of saponin, mixed by shaking and landed on trypticase soy agar in duplicate. The percentage of killing was calculated by determining the ratio between the number of CFU surviving in 60 minutes in the wells with bacteria, antibodies, complement and cells HL-60 to the number of CFU surviving in test tubes containing no antibodies, but containing bacteria, complement and cells HL-60. Included controls containing bacteria, complement and mAb, to correct any reduction in CFU caused by aggregation.

Example 12: demonstration of the protective effect ClfA in animal models in vivo

To assess whether they were able polyclonal rabbit antibodies induced against ClfA, to reduce the number of colonies of S. aureus in a murine model of sepsis, used purified rabbit polyclonal IgG against ClfA in two doses (0.8 mg and 1.6 mg) in the study by passive immunization (Fig.13). Infecting strain of S. aureus was a new clinical isolate, 659-018. Both desire the key antibodies led to the fall was entirely decrease in the number of bacterial colonies in a murine model of sepsis (p=0,0134 for dose 1.8 mg, and p=0,0013 for a dose of 0.8 mg). This experiment was repeated with additional isolates of S. aureus with similar results (data not shown).

Example 13: active immunization ClfA reduced colonization of the heart of S. aureus

Active immunization of rabbits ClfA led to protection in a rabbit model of endocarditis. The inventors have discovered a 3-4 log reduction in CFU of S. aureus isolated from cardiac enlargement, animals immunized with ClfA, compared with animals of the negative control, PBS immunized or AlPO4(Fig.14)

Example 14: protective effect MntC in animal models in vivo

Active immunization MntC showed long-lasting protection in mice from an early time points after infection with S. aureus. The number of bacteria in the blood of mice receiving i.p. infection with S. aureus was significantly reduced compared with controls immunized with PBS (Fig.15A and 15B). Four of the six individual studies showed a significant decrease in CFU/ml of blood from immunized animals. Protection mediated immunization MntC was demonstrated using 2 different infecting strains of S. aureus, PFESA0237 (Fig.15A) and PFESA0266 (Fig.15B).

Example 15: conjugates SR protect in a murine model of pyelonephritis

Conjugates SR assessed on their ability to protect mice in a model of pyelonephritis active immunization. In Fig.16 shows the results of several is the space of a few studies. The number of bacteria in the blood of mice receiving i.p. infection with S. aureus was significantly reduced compared with controls immunized with PBS (Fig.16). Six out of six individual studies showed a significant reduction in CFU/ml of kidneys from immunized animals. The data showed a strong reduction of colonization of the kidney after active immunization with conjugate SR.

Example 16: conjugates SR obtained by different chemical methods of conjugation, protects mice against experimental infections

Research active immunization in a murine model of pyelonephritis was performed using a conjugate SR received either chemically with PDPH, or CDT. Methods of conjugating SR or SR with CRM197have been described above. The results showed that both conjugate significantly reduce colonization in mice compared to animals with imitation immunization (table 14).

Table 14
The effect of conjugation PDPH compared with kongugirovaniem CDT in the model of pyelonephritis
No. studiesAntigensStrain/Doselog CFU/KidneySignificance/td>
Study 1Saline + AlPO4PFESA0266of 5.53±1,90--
1 µg SR - CRM197(PDPH) + AlPO42×1083,01±1,83p<0,001
1 µg SR - CRM197(CDT) + AlPO41,67±0,23p<0,0001
Study 2Saline + AlPO4PFESA02666,17±1,76--
1 µg SR - CRM197(PDPH) + AlPO4of 2.7×1083,06±1,69p<0,0001
1 µg SR - CRM197(CDT) + AlPO41,87±0,69p<0,0001

Example 17: conjugate SR protects in a rat model of endocarditis

There were four studies with conjuga the om CP5-CRM 197PDPH and unrelated conjugate (PP5-CRM197) at a dose of 1 µg. Conjugates SR significantly reduced the colonization of the heart and kidney in 2 of 3 experiments, in which the infecting strain Type 5 was PFESA0266 (table 15). In the third study, the geometric mean titer (GMT) of titer antibodies against SR was the smallest of the three experiments, but it was only slightly lower than the titer in the previous experiment (51000 compared with 67000).

tr>
Table 15
Immunization CF-CRM197reduces SOME rat model of endocarditis
logKOE selectedSignificanceGMT
Immunogenic compositionInfecting strain/DoseHeartKidneyHeartKidneyTitre CF
1 µg CP5-CRM197PFESA02664,34±1,78to 3.92±1,73 103000
1 µg PP5-CRM197of 2.21×108SOME7,94±0,786,77±0,79p<0,001p<0,05
1 µg CP5-CRM197PFESA02664,43±2,303,109±2,3351000
Saline6,5×107SOME5,63±2,484,19±2,05NoNo
1 µg CP5-CRM197PFESA02664,01±2,493,90±1,9267000
Saline4,0×108SOME7,52±1,386,52±1,17p<is 0.0002p<is 0.0002
1 µg CP5-CRM197SA3158,17±1,026,92±1,20186000
Saline1×109SOME8,25±0,606,74±0,95NoNo

Example 18: conjugates CP5-CRM197in the model of pyelonephritis

Original research, which studied the efficacy of the conjugates were conducted with SR with MW 25 kDa. Improvement of the fermentation process resulted in the production of polysaccharide with high MW, which was anywhereman with protein carrier and tested simultaneously with the 25 kDa conjugate SR. Conjugates containing CF with MW 25 kDa (low MW) and 300 kDa (high MW) was obtained with the use of the conjugation chemistry with CDT and was evaluated in a murine model of pyelonephritis. Were tested three doses of 0.01, 0.1 and 1 µg) conjugates HMW (high molecular weight) and they were compared with a control LMW (low molecular weight) CP5-CRM197and unrelated conjugate (PP5-CRM197) at a dose of 1 µg. The results showed a significant reduction in CFU of S. aureus PFESA0266 isolated from kidneys, e is ze 1 µg. No statistical differences between the protection of conjugates obtained SR of different sizes, at a dose of 1 μg (table 16). Lower doses of 0.01 μg and 0.1 μg) conjugate was not able to induce an immune response sufficient to greatly reduce infection. The experiment was repeated using identical methods of immunization and infection. In the second experiment, only the dose of 1 μg LMW CP5-CRM197led to a significant reduction in colonization (p=0.01). Dose 1 µg HMW CP5-CRM197reduced CFU in the kidneys, however, the decrease was not statistically significant (p=0,056).

Table 16
Conjugates SR protect in a murine model of pyelonephritis
ResearchAntigenStrain/Doselog CFU/KidneySignificance (p value)
11 µg PP5-CRM197PFESA0266 of 1.7×1085,340,0048
1 µg 25 kDa CP5-CRM1972,94
1 µg 300 kDa CP5-CRM 2,740,0056
0.1 ág 300 kDa CP5-CRM1975,59
0,01 µg 300 kDa CP5-CRM1974,70
21 µg PP5-CRM197PFESA0266 of 1.7×1085,35
1 µg 25 kDa CP5-CRM1973,250,01
1 µg 300 kDa CP5-CRM1973,780,06
0.1 ág 300 kDa CP5-CRM197of 4.45
0,01 µg 300 kDa CP5-CRM197between 6.08

Example 19: O-polysaccharide acetylation is important for the induction of protective antibody responses to immunogenic composition conjugate SR

To assess the importance of O-acetylation SR, native SR was de-O-acetiminophen (dOAc) and anywhereman with CRM197(dOAc-CRM197) using conjugation chemistry with PDPH. The effectiveness of the conjugate dOAcCP-CRM197comparing the time with CP5-CRM 197in a murine model of pyelonephritis. The results showed that the conjugate having no O-acetyl groups (dOAc CP-CRM197), is not effective in this model, as shown by no significant changes in bacterial colonization in the kidneys. These data (table 17) indicate that O-acetylation was important for the induction of functional antibodies against SR.

Table 17
Immunization de-O-acetylated CP5-CRM197does not protect mice from the colonization of the kidney
No. studiesAntigensStrain/Doselog CFU/KidneySignificance
Study 11 µg RR-CRM197PFESA02663,89±2,24
1 µg dOAc CP5-CRM1977×1084,20±1,75
1 µg CP5-CRM1971,75±0,39 The value of p<0,008
Study 2SalinePFESA0266 of 2.4×1085,08±1,96
1 µg dOAc CP5 - CRM197of 5.89±1,29
1 µg CP5-CRM1972,93±2,11The value of p<0,02

Example 20: Immunization with conjugate SR reduces mortality in models of sepsis

The effectiveness of the conjugate CP8-CRM197was evaluated in a murine model of sepsis after infection with S. aureus PFESA0268 (Type 8). Mice Swiss Webster (n=30) were actively immunized by subcutaneous injection of 1 μg CPS-CRM197and saline solution, both of which were prepared in the form of a preparation with 100 µg AlPO4. The study showed a significant attenuation of sepsis (p=0,0308) compared with mice immunized with only AlPO4. See The Fig.17.

Example 21: assessment of conjugated native and processed by the base SR in a murine model of bacteremia

For conjugate SR was rated the importance of O-acetyl groups present on native SR up to conjugation, on the I induction responses of functional antibodies. Polysaccharide SR de-O-azetilirovanie under mild alkaline conditions, and as NMR and ion chromatography (IC) confirmed the absence of O-acetylation in de-O-AC (de-O-acetylated) CP8-CRM197.

Murine model of bacteremia used to assess the effectiveness of native SR compared with the processed base SR, conjugated to CRM197. Groups of female BALB/c mice (15/group) were immunized at weeks 0, 3 and 6 1 µg de-O-AC CP8-CRM197or 1 μg O-AC CP8-CRM197. Immunogenic compositions were prepared in the form of the drug with 22 µg AlPO4. Animals were infected with S. aureus PFESA0003. Three hours after infection, mice were killed, and counted the number of bacteria in the blood. The data showed that there were statistically significant (p=0,0362) reduction in bacterial CFU isolated from the blood of animals immunized with untreated conjugate native SR, as defined by the t-student test (table 18). In animals which were immunized with a conjugate SR treated base, bacterial CFU isolated from the blood were similar to the control group, which were immunized with saline.

Table 18
Conjugate CP8-CRM197reduces bacteremia S. aureus PFESA0003 in mice
AntigenStrain/Doselog CFU/BloodSignificance (p value)
SalinePFESA0003 to 1.14×1084,35
de-O-AC CR - CRM197of 4.45
O-AC CR - CRM1973,930,03

Example 22: the acknowledgement of the importance of O-acetylation as a functional epitope SR by ORA using MAY known specificnosti

Monoclonal antibodies against SR with specificnosti to SR SLA+ (SR-7-1), SR SLA+/- (SR-5-1) and SR SLA- (SR-6-1) was evaluated on the activity of the PR (opsonic) killing against the strain PFESA0266 type 5 (table 19). MAb SR-3-1, specific to SR SLA+, was used as negative control. The results showed that MAb SR-7-1 (specific SR SLA+) mediates the killing of both tested strains of type 5. Also mAb SR-5-1 recognizes an epitope that have SR SLA+, and SR SLA-, posredovano killing strain PFESA0266. mAb, specific epitopes present on the polysaccharide SR SLA-did not oposredovany umers is giving strain PFESA0266. These results indicate that O-acetyl epitopes on SR involved in the functional activity of antibodies specific against SR.

Table 19
mAb, specific for O-acetylated (+) SR and O - de-O-acetylated (+/-) SR are opsonic against S. aureus PFESA0266 (Type 5)
SR-5-1 (O-AC +/-) (µg)SR-6-1 (O-AC) (µg)SR-7-1 (O-AC +) (ug)SR-3-1 (Ref. control (g)
mcg201052,5201052,5201052,5201052,5
% killing283330 21-12-5-12-531464955-18-3-13-5

Data are reported as the percentage of killing and calculated by determining the ratio between the number of CFU surviving in 60 minutes in the wells with bacteria, antibodies, complement and cells HL-60 to the number of CFU surviving in the wells containing no antibodies, but containing bacteria, complement and cells HL-60.

Example 23: opsonic activity of mouse antibodies induced by conjugates SR with high and low MW

Serum from mice (n=5) with high titers ELISA SR of the groups immunized with 1 μg SR with high molecular weight and SR with low molecular weight from Example 18, were compared opsonic activity using S. aureus PFESA0266. The results of PRA showed that both conjugate induced opsonic antibodies in mice (table 20). There was a trend observed for conjugates with high Mw, to induce higher titers of opsonic antibodies. Data are shown as average % killing ± SEM for 5 individual mouse sera. Antibodies should be fun the elegance when measuring for killing bacteria or in an animal model of efficiency, either through analysis opsonophagocytosis killing, which demonstrates that the antibodies kill the bacteria. Functional killing may not be demonstrated by analysis, which simply tracks the generation of the same antibody that does not indicate the importance of conjugates with high molecular weight in efficiency.

Table 20
As conjugates LMW SR and conjugates HMW SR induce opsonic antibodies
Antigen: 1 µg SR - CRM197(25 kDa)Antigen: 1 µg SR - CRM197(300 kDa)
Title ORA weeks. 0Title ORA weeks. 8Title ORA weeks. 0Title ORA weeks. 8
<100400<1006400
<100<100<100800
Antigen: 1 µg SR - CRM197(25 kDa)Antigen: 1 µg SR - CRM197(CD)
<100400<1003200
<1003200<1003200
<100<100<1003200

Example 24: opsonic activity of serum from mice immunized with the conjugates of native and chemically modified SR

Selected mouse serum (n=5) with high titers SR from the study in Example 21 were compared opsonic activity using strain PFESA0005. The results of PRA (table 21) show that only the conjugates obtained by conjugation of native SR, induced opsonic antibodies in mice. It is noteworthy that the conjugate de OSA SR was immunogenic in mice, but induced antibodies were not opsonic in this analysis. Titles ORA reported as values, reverse breeding, which was watched by 40% killing. Antibodies should be functional when measuring for killing bacteria or in an animal model of efficiency, either through analysis of opsonophagocytosis killing, which demonstrates that the antibodies kill the bacteria. Functional amarsul the tion may not be demonstrated by analysis, which simply tracks the generation of the same antibody that does not indicate the importance of O-acetylation in efficiency.

Example 25: the killing strains of type 8 anticorodal primates, non-human, conjugate SR inhibited by adding native SR

To confirm the specificity of the killing activity in the serum of primates, non-human immunized with conjugate SR, the analysis was performed in the presence of native SR. Used method II PR with the following modifications. Received twofold serial dilution of antibody (25 μl), and then adding to the suspension of antibodies or 150 ál (competitor Pn 14) or 125 ál (competitor SR) buffer for analysis. The competitor was a purified polysaccharide SR (SR poly), and unrelated pneumococcal polysaccharide (Pn 14 poly) was used as control. Polysaccharides were added (50 μg) in a suspension of antibody, and the plate is incubated at 4°C for 30 minutes with stirring by stirring from the bottom to the lid (end over end mixing). After incubation with polysaccharides in the tablets was added bacteria (25 μl), and put the tablets on a rotary shaker for 30 min at 4°C, followed by addition of 25 μl of human complement (final concentration 1%). The results (table 22) showed that the presence in the reaction mixture of native SR inhibited opsonophagocytosis the killing of S. aureus type 8. These results confirm that opsontai the container killing immune serum was mediated Ab, specific to the capsule.

Table 21
Opsonic activity of native SR compared with de-O-AC CP8-CRM197
De-O-AC CR - CRM197SR - CRM197
OP titer serum weeks. 0PR titer serum weeks. 8PR titer serum weeks. 0PR titer serum weeks. 8
<50<5050150
<50<50<501350
<50<50<50450
<50<50<501350
<50<50<504050
Table 22
Adding polysaccharide SR inhibits opsonophagocytosis killing of S. aureus immune serum
MonkeySample serumTitle ORA
02D133Weeks. 0<50
Weeks. 84050
Weeks. 0 + 20 µg SR poly<50
Weeks. 8 + 20 µg SR poly<50
Weeks. 0 + 20 ug Pn 14 poly<50
Weeks. 8 + 20 ug Pn 14 poly4050
Weeks. 0<50
Weeks. 84050

MonkeySample serumTitle ORA
A4N122Weeks. 0 + 20 µg of the R8 poly <50
Weeks. 8 + 20 µg SR poly<50
Weeks. 0 + 20 ug Pn 14 poly<50
Weeks. 8 + 20 ug Pn 14 poly1350

Example 26: acquired by natural antibodies to ClfA mediate opsonophagocytic killing of S. aureus

People in the population naturally exposed to S. aureus and, thus, have preexisting antibodies to the bacteria in their bloodstream. The authors of this invention have conducted affinity purification of antibodies against ClfA from human serum and appreciated, could antibodies to mediate opsonic killing. It was shown that antibodies to ClfA are opsonic against the capsular polysaccharide of S. aureus (data not shown). Strain PFESA0266 were grown overnight in nutrient medium Columbia with 2% NaCl. Bacteria were optionsoracle human IgG, affinity purified on ClfA, or human IgG, affinity purified on the irrelevant antigen (negative control, streptococcal protein SCP), and tested opsonic activity. In the analysis of opsonophagocytic activity used differentiated cells HL-60 in the ratio of effector/target 100:1. As an additional control in experiment included mAb against SR to demonstrate the presence SR on the surface. The results represent the average from two independent experiments. Antibodies that are specific against ClfA and SR, in fact oposredovany opsonic killing, and antibodies that are specific against SCP (negative control) had no activity in this assay.

Example 27: CP5 conjugate-CRM197induces opsonic antibodies in primates, non-human (NHP)

For comparison functionality conjugates CP5-CRM197high molecular weight with respect to the functionality of conjugates SR-CRM197with low molecular weight from NHP, the group of five monkeys were immunized with doses of conjugates 2 and 20 ág with adjuvant AlPO4or without it. Monkeys received the first and second vaccination on day 0 and 28, respectively. Blood samples taken at day 0, 14, 28 and 42 were tested on the PR activity. The results are summarized in Table 23. 20 μg of HMW conjugate gave the highest titers of EOS compared with other groups. The frequency of the PR-positive monkeys was higher in the groups immunized with two doses of conjugate with high MW than in the respective groups immunized with conjugate with low MW. These results demonstrate that for conjugate HMW CPS-CRM197there is a tendency to induce the best answers to the PR than in the case of conjugate LMW CP5 at NHP.

Table 23
ORA NHP serum after immunization with conjugates CP5
Title ORA (40% killing)
GroupID monkeysDay 0Day 14Day 28Day 42
20 µg CP5 (HMW) + 0.5 mg/ml AlPO4A2N053450135040504050
149N<100405040504050
A4L069<100450150<100
A1N097<100405013501350
A4L014<100<100<100 <100
2 µg CP5 (HMW)) + 0.5 mg/ml AlPO402D125<100150150<100
A4L081<100150150150
A2N055150450150150
A4N084<100<100<100<100
A1N085<1001504504050
2 µg CP5 (HMW) without AlPO4A4L084150150<100<100
97N004150450450450
A4L055<100&t; 100<100<100
97N123<100<100150150
225N<100<100<100<100
20 µg CP5 (LMW)) + 0.5 mg/ml AlPO402D017<100<100<100<100
A4N100<1001501504050
257N<100<100<100<100
A4L046<100<100<100<100
A1N098<100150<100<100
2 µg CP596N02 15015045Q 150

Title ORA (40% killing)
GroupID monkeysDay 0Day 14Day 28Day 42
(LMW)) + 0.5 mg/ml AlPO402D005<10013504501350
02D113<100150150<100
A2N040150150<100<100
A4L056150150<100<100

Example 28: conjugates of capsular polysaccharides containing polysaccharides with high molecular weight, show increased IMM is noganet compared with conjugates, containing polysaccharides of low molecular mass

Conducted research on primates, non-human (MPR), to assess the immunogenicity of different compositions capsule conjugates. Tested two compositions at two different dosage levels (2 and 20 μg). The first track consisted of a polysaccharide with a high molecular weight (HMW) (approximately 130 kDa) conjugated to CRM197. The second composition contained polysaccharide of low molecular weight (LMW) (approximately 25 kDa) conjugated to CRM197. Groups of five primates were vaccinated with a single dose of one of the vaccines, and tracked immune titers prior to vaccination and two weeks after vaccination. The title of the PR was defined as the serum dilution required to killing 40% strain PFESA0266 S. aureus in the analysis of the ORA. Antibody titers were also monitored by ELISA. Increased activity was observed for vaccine HMW compared with LMW composition (table 24), which follows from a ten-fold increase in antibody titers to vaccine HMW compared with vaccine LMW. The percentage of responders ORA for the MPR, which received the vaccine HMW, also was higher (80% compared with 40%).

Table 24
For vaccines conjugate polysaccharide HMW there is increased and monogenist compared with conjugate vaccine polysaccharide LMW
The dose SR-CRM197 (ICG) on animalThe geometric mean PD1*The percentage of responders ORA (%)±
HMW (125 kDa)203280
22180
LMW (25 kDa)20340
2840
* The fold increase was calculated from the titer SR ELISA 2 weeks after vaccination compared with titers prior to vaccination.
± The percentage of responders was calculated on the basis of data obtained in monkeys, which were generated by the increase in titer of PR after one dose of vaccine within 2 weeks after vaccination.
Each group contained 5 rhesus monkeys, and vaccines were prepared in the form of the drug with AlPO4(250 μg/dose).

Example 29: DUJOTEKANA (CP5-CRM197and ClfA) composition - answers antibodies in primates, non-human

To assess immune response to one dose dhandhania immuno the military songs (CP5-CRM 197and ClfA) NHP group of five monkeys were immunized with different doses of the two antigens without adding AlPO4. Blood samples taken at day 0, 14 and 28 were tested on opsonophagocytic (ER) activity, and titers by ELISA and the results are summarized in Table 24. The results showed that the PR activity was systematically observed in animals immunized SR, compared with the group with imitation immunization SR. In General, the group immunized with 100 µg, had the highest titers of ELISA and ER compared with other groups. Mortifying the PR activity was not observed with serum from groups immunized with one ClfA. In the groups who were given increasing doses of ClfA or SR, there were no interfering influence. See Table 25.

Table 25
The results of the ORA of research bivalent immunization in NHP
Title ORA (40% killing)
no groupID numberWeek: 0Week: 2Week: 4
180 mcg ClfA + 20 µg 130 kDa SRA4R054 <100150<100
A4R056<100150150
A4N087<100450450
97N152<1004501350
A4R027<10013501350
180 mcg ClfA + 2 µg 130 kDaA4R062<100150<100
97N149<100150<100

Title ORA (40% killing)
no groupID numberWeek: 0Week: 2Week: 4
SRA4R131 <100450150
97N025<100450450
A4N064<100450450
60 µg ClfA + 20 µg 130 kDa SRA4L005<100<100<100
A4R029<10013501350
A3N015<100<100<100
98N02115040504050
A4R137<100<100<100
60 µg ClfA + 2 µg 130 kDa SRA1N040<100150150
A2N104<1001350 <100
A4L033<100150<100
96N048<100<100<100
A4R032<100<100<100
2 µg 130 kDa SRA4R135<100450150
A1N118<100150150
A4R061<100<1001350
A4R101<10040501350
97N137<10013501350
20 µg 130 kDa SRA4R135<100<100<100
A4N115 <100150150
95N038<100<100<100
A4N120<100450450
96N004<100150150
100 mcg 130 kDa SRA4N116<100450450
A3N097<100450450
A4N108<10013501350
98N034<100450150
99N034<10013504050
60 µg ClfA97N057<100<100<100
A4R112150<100150

Title ORA (40% killing)
no groupID numberWeek: 0Week: 2Week: 4
A4L022<100<100<100
97N100<100<100<100
99N041150150150

Animal models demonstrate the potential of antigens on the basis of capsular polysaccharides SR and SR S. aureus

As CP5-CRM197and CP8-CRM197conjugates induced specific serotype capsules antibody responses in mice, rats, rabbits and primates, non-human (NHP). The antibodies induced by the conjugates were functional in functional analysis opsonophagocytosis killing in vitro. Data were obtained on what I show you that O-acetylation is important for the induction of protective antibodies against both SR and SR, and that the O-acetyl groups are part of the epitope recognized ORA+mAb against SR. MAb that recognize native SR that is O-acetylated, are functional in the ER and mediates the killing of bacteria. Conjugate SR induced functional antibodies as mice and rabbits, which were oposredovany killing strain of type 8 in the OP. The specificity of killing polyclonal or monoclonal antibodies was confirmed by the elimination of killing after adding in the analysis of homologous native polysaccharide. To demonstrate preclinical efficacy as SR-and CP8-CRM197conjugates used different models of active immunization. Conjugate SR showed systematic efficacy in a murine model of pyelonephritis in the rat model of endocarditis. The importance of O-acetylation SR confirmed in a murine model of pyelonephritis, where de-O-acetylated SR conjugated with CRM197could not protect animals from experimental infection.

The combination of conjugates in dvuhantennoy composition induced antibodies to both capsules - SR and SR, and no interfering effect on the levels of induced specific antibodies compared with one immunizations and what Tigana. The combination of conjugates and ClfA in trichanthera composition induced high levels SR, SR and ClfA, and no interfering effect on the antibody responses induced against any antigen present in combination. Trichanthera immunogenic composition induced responses of antibodies (Ab), allowing the booster gain with respect to all three components in rabbits with high preimmune titles.

These results indicate that SR and SR conjugated to CRM197should be included as components of the immunogenic compositions of protective immunogenic compositions against S. aureus.

Example 30: the necessity of different antigens for protection against numerous diseases caused by S. aureus

S. aureus causes a wide spectrum of infections, ranging from relatively mild skin infections to more serious invasive infections such as endocarditis, necrotizing fasciitis, osteomyelitis, septic arthritis and pneumonia. Each of these sites in vivo is unique, and bacteria, it seems, respond to differences in environmental stimuli by changing their profiles the expression of antigens on the profiles, the most suitable for the individual strain for colonization, growth and, ultimately, call of the disease. As illustrated in Example 12, the strains of S. aureus demonstrate the diversity of expr the hurt antigens in vivo. Multicomponent immunogenic composition consisting of different antigens are more likely to protect against a variety of disease symptoms caused by S. aureus.

It was shown that ClfA protects in models of endocarditis and sepsis in rodents. Reported that ClfB is important for nasal colonization of S. aureus. MntC protected mice in a murine model of bacteremia. Conjugate SR defended with pyelonephritis and endocarditis, and conjugate SR defended in models of pyelonephritis and sepsis in rodents. These results demonstrate that a multicomponent vaccine containing these antigens will protect against many types of diseases caused by S. aureus.

Animal models in vivo approximate course real infection and help determine which antigens can be useful for protection against specific diseases. In Table 26 summarizes the results of the numerous experiments conducted on different models in vivo. The results are shown in each block in the form of four numbers separated by a slash, for example, ClfA in models of sepsis has a number 27/1/3/31. The first number represents the number of experiments, when immunization ClfA gave a statistically significant positive result protection. The second number represents the number of experiments, when immunization ClfA was positive protection is s, which showed a trend towards significance, but was not statistically significant. The third number represents the number of experiments, when immunization ClfA gave a negative result, but it was not statistically significant. The fourth number represents the total number of experiments. The first three numbers with the addition should give the fourth number.

Table 26
Generalization of protection in animal models for antigens of S. aureus
ClfASRSRMntC20
Bacteremia1/4/0/53/0/3/61/1/1/36/2/5/13
Sepsis27/1/3/311/0/0/1NTNT
Pyelonephritis0/4/2/613/1/0/14NT1/0/4/525
Endocarditis3/6/1/10 3/2/2/7NTNT
NT: not tested

Example 31: testing different multiantigenic immunogenic compositions in vitro and in vivo

Different multiantenna staphylococcal immunogenic compositions containing three, four or five antigens selected from the following polypeptides and/or polysaccharides: ClfA, ClfB, MntC, CP5 - and SR, tested for immunogenicity and efficacy in various models in vivo. Immunogenic compositions are as follows:

(1) immunogenic composition containing the selected polypeptide of clumping factor A of S. aureus (ClfA), isolated capsular polysaccharide type 5 S. aureus, conjugated to CRM197and selected capsular polysaccharide type 8 S. aureus, conjugated to CRM197;

(2) in the second combination is proposed clumping factor A (ClfA), clumping factor B (ClfB), a dedicated MntC allocated staphylococcal capsular polysaccharide SR conjugated with CRM197and selected staphylococcal capsular polysaccharide SR conjugated with CRM197;

(3) the third proposed combination immunogenic composition containing the selected polypeptide of clumping factor A of S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB) or isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 S. aureus, injuriously with CRM 197and selected capsular polysaccharide type 8 S. aureus, conjugated to CRM197;

(4) in the fourth combination of the proposed immunogenic composition containing the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated capsular polysaccharide type 5 S. aureus, conjugated to CRM197and selected capsular polysaccharide type 8 S. aureus, conjugated to CRM197;

(5) in the fifth combination proposed immunogenic composition containing the selected polypeptide of the clumping factor In S. aureus (ClfB), isolated protein of S. aureus MntC allocated capsular polysaccharide type 5 S. aureus, conjugated to CRM197and selected capsular polysaccharide type 8 S. aureus, conjugated to CRM197; and

(6) in the sixth combination proposed immunogenic composition containing the selected polypeptide of clumping factor A of S. aureus (ClfA), the selected polypeptide of the clumping factor In S. aureus (ClfB) and isolated protein of S. aureus MntC.

rClfA and rClfB receive and purified as described in Example 1. MntC will receive and purified as described in Example 2. Selected SR and SR receive and purified as described in Example 3, and kongugiruut with CRM197as described in Example 4.

More specifically, the techniques described in the previous examples above, is used to measure the immunogenicity and efficacy. Studies conducted to determine induces whether each is three, four or five components of the immune response when delivered singly or jointly. The same study is used to determine whether the presence of any one of the four or five components ability of any of the other three or four components to induce an immune response. In addition, studies to determine whether four or five components, tested, tested singly or jointly, to give protection to in any one or more than one animal model described above. Four or five components is administered to an animal, for example, mice, rats, rabbits or primates, non-human, in a single dose or as many doses as noted in the previous examples above. Animals take blood and serum are collected and tested for the presence of antibodies to each of the four or five components. The presence of antigen specific antibodies measured by any immunoassay known to specialists in this field, for example, to assess the presence or absence of antigen specific antibodies using ELISA (see Examples 11-29) or Western blotting (see Example 1). In addition, to determine whether antigen specific antibodies effective in mediating killing of staphylococcal organisms cells phagocytes, use the form analysis opsonophagocytic activity (see Examples 11-29).

Efficiency in vivo is also assessed using any one or more than one animal studies described above, such as the model of the implanted tube; a murine model of bacteremia; model wound infections; murine model of pyelonephritis; rat model of endocarditis and murine model of sepsis (see examples 11-30), but not limited to.

Example 32: a combination of antigens of S. aureus produce antibodies in primates, non-human, which increase the killing strain Pfe5-1 S. aureus

When using combinations of antigens observed increased efficiency, as measured by analysis of the ORA. Conducted study of primates, non-human, where a group of 3-10 monkeys were immunized multicomponent vaccines. Animals received a single dose of the vaccine, and the titles of ERS monitored at day 0 and two weeks after vaccination. The title of the PR was defined as the serum dilution required for killing 50% of the bacteria strain Pfe5-1 S. aureus in the analysis of the ORA. Increased activity was observed for the combination of the 4 antigens compared with 3-antigenic composition of the vaccine (p=0,0272; Fig.18).

1. The composition for inducing an immune response against Staphylococcus aureus containing
a) an effective amount of capsular polysaccharide type 5 S. aureus conjugated to protein-is the LEM CRM 197,
b) an effective amount allocated capsular polysaccharide type 8 S. aureus conjugated to a protein carrier CRM197and
at least one component selected from the group consisting of an effective amount of a selected polypeptide of clumping factor A of S. aureus (ClfA), an effective amount of a selected polypeptide of the clumping factor In S. aureus (ClfB) and effective number of selected protein of S. aureus MntC,
where capsular polysaccharide type 5 is a capsular polysaccharide of high molecular weight from 70 to 300 kDa, where the capsular polysaccharide type 8 is a capsular polysaccharide of high molecular weight from 70 to 300 kDa, from 5 to 23 lysine CRM197are conjugated and where the conjugates SR and SR have a molecular weight from about 200 kDa to about 5000 kDa.

2. The composition for inducing an immune response against Staphylococcus aureus, comprising: an effective amount of a selected polypeptide of clumping factor A of S. aureus (ClfA), effective number of allocated capsular polysaccharide type 5 S. aureus conjugated to a protein carrier CRM197and an effective amount allocated capsular polysaccharide type 8 S. aureus conjugated to a protein carrier CRM197where capsular polysaccharide type 5 is a capsular polysaccharide with high mo is collaroy weighing from 70 to 300 kDa, where capsular polysaccharide type 8 is a capsular polysaccharide of high molecular weight from 70 to 300 kDa, from 5 to 23 lysine CRM197are conjugated and where the conjugates SR and SR have a molecular weight from about 200 kDa to about 5000 kDa.

3. The composition according to p. 2, additionally containing an effective amount of the protein of S. aureus MntC.

4. The composition according to p. 2, additionally containing the selected polypeptide of the clumping factor In S. aureus (ClfB).

5. The composition for inducing an immune response against Staphylococcus aureus, comprising: an effective amount of the protein of S. aureus MntC, the effective amount of capsular polysaccharide type 5 S. aureus conjugated to a protein carrier CRM197and an effective amount allocated capsular polysaccharide type 8 S. aureus conjugated to a protein carrier CRM197where capsular polysaccharide type 5 is a capsular polysaccharide of high molecular weight from 70 to 300 kDa, where the capsular polysaccharide type 8 is a capsular polysaccharide of high molecular weight from 70 to 300 kDa, from 5 to 23 lysine CRM197are conjugated and where the conjugates SR and SR have a molecular weight from about 200 kDa to about 5000 kDa.

6. The composition according to p. 5, additionally containing selected by peptide factor of adhesion In S. aureus (ClfB).

7. Composition according to any one of paragraphs.1-4, where the ClfA polypeptide is a polypeptide fragment selected from the group consisting of fibrinogenesis domain; domains N1, N2 and N3; and N2 and N3 domains ClfA.

8. Composition according to any one of paragraphs.1, 4, or 6, where the polypeptide ClfB is a polypeptide fragment selected from the group consisting of fibrinogenesis domain; domains N1, N2 and N3; and N2 and N3 domains ClfB.

9. Composition according to any one of paragraphs.1-4, where the ClfA polypeptide binds to the fibrinogen to a lesser extent compared with the binding of fibrinogen observed for native ClfA.

10. Composition according to any one of paragraphs.1-4, where the ClfA polypeptide has an amino acid substitution of one or more than one of Tyr 338, Tyr 256, Pro 336, Lys 389, 254 Ala or Ile 387.

11. The composition according to p. 10, where the amino acid substitution is a substitution to Ala or Ser.

12. The composition according to p. 11, where Tyr 338 replaced by Ala.

13. Composition according to any one of paragraphs.1-6, where the capsular polysaccharide of type 5 is a capsular polysaccharide of high molecular weight from 70 to 150 kDa.

14. Composition according to any one of paragraphs.1-6, where the capsular polysaccharide of type 5 is O-acetylated at 10-100%, O-acetylated by 50-100% or O-acetylated at 75-100%.

15. Composition according to any one of paragraphs.1-6, where the capsular polysaccharide type 8 is a capsular polysaccharide with high mol the molecular weight from 70 to 150 kDa.

16. Composition according to any one of paragraphs.1-6, where the capsular polysaccharide type 8 is O-acetylated at 10-100%, O-acetylated by 50-100% or O-acetylated at 75-100%.

17. Composition according to any one of paragraphs.1, 3, 5, or 6, where the protein of S. aureus MntC is libidinously or Neopalimovsky protein.

18. Composition according to any one of paragraphs.1-6, optionally containing adjuvant.

19. Composition according to any one of paragraphs.1-6, optionally containing pharmaceutically acceptable carrier.

20. Composition according to any one of paragraphs.1-6, optionally containing an antigen selected from the group consisting of Opp3a, DltD, HtsA, LtaS, IsdA, IsdB, IsdC, SdrC, SdrD, SdrE, SdrF, SdrG, SdrH, SrtA, SpA, Sbi, FmtB, alpha-hemolysin (hla), beta-hemolysin, fibronectins protein A (fnbA), fibronectins protein B (fnbB), coagulase, Fig, map, leukocidin Panton-Valentine (pvl), alpha-toxin and its variants, gamma-toxin (hlg) and options, ica, immunodominant ABC Transporter, Transporter Mg2+, ABC Transporter Ni, RAP, autolysin, laminin receptor, IsaA/PisA, IsaB/PisB, SPOIIIE, SsaA, EbpS, SasA, SasF, SasH, EFB (FIB), SBI, Npase, EBP, SIAL-bonded protein bone II, the predecessor of aureolin (AUR)/Sepp1, Spa and their fragments, such as M55, TSST-1, mecA, exopolysaccharide poly-N-acetylglucosamine (PNAG/dPNAG), GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin protein, HarA, exhibiting esxa, EsxB, enterotoxin a, enterotoxin, enterotoxin C1 and a new autolysin.

21. The way and is dotirovanija immune response against Staphylococcus aureus, includes introduction to the subject immunologically effective amount of the immunogenic composition according to any one of paragraphs.1-6.

22. The method according to p. 21, where the immune response prevents or weakens the subject has a disease, condition or at least one symptom associated with staphylococcal organism.

23. The method according to p. 22, where the disease is selected from the group consisting of invasive disease caused by S. aureus sepsis and carriage.

24. The method according to p. 21, where the induced immune response includes the generation of antibodies with opsonophagocytic activity (OPA) against S. aureus.

25. The way to make passive immunity against Staphylococcus aureus subject, comprising the stages of (1) obtaining a preparation of antibodies using immunogenic compositions according to any one of paragraphs.1-6 and (2) the injection of antibodies to a subject to give passive immunity.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to oligosaccharide carrier conjugates effective for treating and preventing infectious diseases and containing an oligosaccharide conjugated with the carrier through a linker of formulas

VIIIa

or

VIIIb:

wherein n is more than 1, m is specified in 1-10, p is specified in 1-20 and R represents H or alkyl; the linker is bound to an oligosaccharide oxygen atom through a terminal CH2 group, and the linker is bound through a terminal CO group with an amino group of the carrier compound by an amide bond; the oligosaccharide is β-1-6 related glucosamine, and the carrier is peptide, protein, polysaccharide, nucleic acid, lipid or tetanus toxoid.

EFFECT: there are presented new conjugates effective to stimulate the immune response, and a method for preparing them.

21 cl, 12 ex, 23 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to microbiology and biotechnology. What is presented is the strain Enterococcus mundtii GKPM (State Collection of Industrial Microorganisms) - Obolensk V-7424 producing a peptide substance having the antilisterial activity.

EFFECT: antimicrobial peptide substance Enterococcus mundtii GKPM - Obolensk V-7424 possesses the activity about 6400 - 12800 antigen units/ml on the test strain Listeria monocytogenes 776 and can be used for the decontamination of bioproducts, as well as materials infected by bacterial pathogens.

2 dwg, 4 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry and microbiology and concerns a biologically active peptide. The characterised peptide hominin KLP-1 is produced by the strain Staphylococcus hominis KLP-1 and possesses an antibacterial activity on the representatives of the following bacterial genera: Arthrobacter, Bacillus, Corynebacterium, Enterococcus, Lactobacillus, Micrococcus, Mycobacterium, Propionibacterium, Rhodococcus, Staphylococcus, Streptococcus, it has a molecular weight of 2985 Da, is sensitive to proteases, possesses high thermal stability and has the following amino acids: 51.1% cationic and 31.7% hydrophobic amino acids, as well as a specific amino acid - lanthionine. The peptide is characterised by higher stability to heating, alkaline environment, as well as to DNAase and RNAase. The antibacterial activity of the peptide is considerably reduced or disappears in the acid environment and under the action of the enzymes pepsin, trypsin and proteinase K.

EFFECT: presented invention can find application in biotechnology, medicine, veterinary science, food industry, as well as in research studies.

1 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology and molecular genetics and pertains to a recombinant polypeptide A2, DNA coding said polypeptide, a strain which produces polypeptide A2 and methods of using such a recombinant polypeptide. The disclosed recombinant polypeptide A2 is characterised by an amino acid sequence of 346 amino acids in which the first 13 amino acids are coded by the plasmid sequence pQE 32 and are covalently bonded with the next 333 amino acids which are coded by a sequence of the HSA-binding fragment of chromosomal DNA of the strain DG 13 of streptococci of the group G-CFG.

EFFECT: group of inventions can be used in diagnosis, eg, when making a test system for determining microalbuminuria based on a laboratory criterion of the preclinical phase of diabetic nephropathy, as well as a reagent for separating human serum albumin by affinity chromatography and for freeing serum from HAS, which enables to determine other proteins present in the serum in lower concentrations.

9 cl, 11 dwg, 1 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a cleaned mutant protein of O-streptolysin with SEQ ID NO:1 sequence, where amino acids in positions P427 and W535 are replaced with another amino acid, or with SEQ ID NO:25 sequence. With that, hemolytic activity of SLO mutant protein is reduced at least by 50% in comparison to SLO of a wild type. The invention also refers to vaccine compositions for prophylaxis of an infection caused by S. pyogenes and containing the above streptolysin protein.

EFFECT: invention allows obtaining mutant shapes of O-streptolysin, which are non-toxic, but which still maintain the ability of inducing protection against S pyogenes and can be used in vaccine compositions for induction of protection against S pyogenes.

15 cl, 26 dwg, 21 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a number of polynucleotides and polypeptides β-hemolytic streptococci, in particular polypeptides and polynucleotides of Streptococcus pyogenes, and based on them immunogenic compositions, used for prevention or reduction of symptoms of colonisation or infection, caused by β-hemolytic streptococci. Immunogenic composition (versions) contains mixture of two or more polypeptides, encoded by sequence of nucleic acid (NA), which has, at least, 90% identity of sequence of NA, selected from group, consisting of peptidase C5a (SCP), open reading frame (OPC)554, OPC 1218, OPC 1358 and OPC 2459. One of versions of immunogenic composition contains polypeptide SCP, polypeptide peptidylpropylisomerase and, at least, one other polypeptide. Also disclosed are methods of protecting susceptible mammal against colonisation or infection, caused by β-hemolytic streptococcus, by immunisation of immunogenic composition by invention.

EFFECT: invention provides immunogenic compositions and methods for prevention or reduction of symptoms of infections, caused by β-hemolytic streptococci of group A, B, C and G, as well as ensures immunity to wide spectrum of bacteria BHS.

41 cl, 16 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: Enterococcus faecium LVP 1073 strain is deposited in the Collection of Microorganisms of the State Research Centre of Applied Microbiology and Biotechnology, Collection No. B-5736 - a producer of bacteriocin E1073 active against bacterial pathogens. The strain is recovered from the blind gut contents of a 42-day healthy broiler. Produced bacteriocin E1073 belongs to cation peptides (pi 8.2) and has molecular weight 3256 Da, consists of 34 amino acids. Presented is Lactobacillus plantarum 1 LVP7 strain deposited in the Collection of Microorganisms of the State Research Centre of Applied Microbiology and Biotechnology, Collection No. B-5943 - a bacteriocin E1073 synthesis inducer. Presented is a specific signal peptide SP1073, a bacteriocin E1073 synthesis regulator, recovered from a culture medium of a bacteriocin producing E.faecium LVP 1073 strain. Use of bacteriocin E 1073 for lifetime finish processing of poultry promotes production of pathogen-free and safe carcasses.

EFFECT: invention provides in vitro manifested antagonist activity against a number of gram-positive and gram-negative pathogens, including gram-positive spore-forming clostridia, gram-negative campilobacteria and salmonellas.

6 cl, 10 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention can be used in producing vaccines against Streptococcus agalactiae - a representative of streptococci group B (SGB) in diagnostics of the diseases - for creation of a detection system of immunoglobulin A level in biological fluids, in immunochemistry as accessible immunochemical reagents (affine recovered IgA fragments). Offered unique recombinant DNA are produced by polymerase chain reaction (PCR) with using chromosomal DNA of strain 219/4849 Ibc of serotype SGB and unique primers. One recombinant DNA contains three nucleotide substitutes in comparison with an initial site of chromosomal DNA. The following cloning of amplified fragments is carried out in a linear vector pGEM-T Easy, and at the final stage by the system of express ionic vectors pQE30/31/32 in E coli JM 109. The produced recombinant DNA code amino acid sequences of recombinant polypeptides exhibiting ability to connect selectively various molecular forms of IgA and designated as P6, P7, P8. Polypeptide P6 causes synthesis of long circulating high-affine anti-Rb antibodies possessing protective properties against SGB.

EFFECT: application of the invention provides production of recombinant polypeptides based N-terminal conservative part of surface Bac SGB of Ibc serotype and containing a first IgA-connecting site A with changed or native sequence MLKKIE, polypeptide exhibits immunologically relevant and protective properties, and they also high selectively connect IgA.

12 cl, 20 dwg, 4 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: invention can be used in manufacturing of vaccines for Streptococcus pyogenes - streptococci of group A (SGA) and Streptococcus agalactiae - streptococci of group B (SGB). Substance of the invention involves development of recombinant DNA pB1 derived from PCR with using chromosomal DNA of strain 090R Ia of serotype SGB, primers Pb1 and Pb2 and following cloning with using expression plasmid pQE-30 in E coli M15. Recombinant DNA pB1 codes recombinant protein PB1 expressing protective properties in relation to specified streptococci which has no enzymatic activity and causes synthesis of anti-Pb1 antibodies expressing protective properties in relation to Streptococcus pyogenes and Streptococcus agalactiae. In the invention there is developed recombinant plasmid DNA pQE-pB1 representing plasmid DNA pQE-30 that bears recombinant DNA pB1, and strain-producer E. coli M15-PB1 enabling to express recombinant protein PB1.

EFFECT: no enzymatic activity of produced recombinant protein allows application as an ingredient of the vaccine for Streptococcus pyogenes and Streptococcus agalactiae.

7 cl, 7 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: method involves immunising with 5×107 microbial cells Vibrio cholerae 5879; 10 days later, a common pool of anticholeraic Ig is recovered from enteric washing liquid. That is combined with producing the primary enterocyte culture from intact mice. Thereafter, the doubly-diluted anticholeraic Ig are applied in an amount of 3 ml on a monolayer of erythrocytes formed in wells of culture panels. The latter are added with alive choleraic vibrions of the strain Vibrio cholerae 5879, 40 m.c. per 1 erythrocyte and incubated for 3 hours. Then, the panels are washed three times, fixed with ethanol for 20 minutes and stained by Romanowsky-Giemsa. Local humoral anticholeraic immunity is assessed under the microscope by the number of vibrions stained in dark blue and attached to one erythrocyte.

EFFECT: method enables assessing specific local humoral immunity and intensifying the anti-adhesive activity of the common pool of anticholeraic immunoglobulin.

4 cl, 2 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new bacitracin compounds with antibiotic activity

,

wherein at least one of R1, R2 and R3 represents -CH=CH2 and wherein R1, R2 and R3 independently represent -H, -CH3 or -CH=CH2.

EFFECT: preparing the new bacitracin compounds.

13 cl, 8 dwg, 7 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula I and their pharmaceutically acceptable salts, method for preparing them by the fermentation of a microorganism of the species Streptomyces (PM0626271/MTCC 5447), and to their pharmaceutical compositions containing one or more compounds of formula I as an active ingredient.

EFFECT: compounds of formula I are used for treating and preventing the diseases caused by bacterial infections.

4 cl, 3 dwg, 6 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a producing method of cream containing fusidic acid, which involves a stage of application of sodium fusidate as an initial active ingredient and conversion of the above sodium fusidate in situ to fusidic acid in an oxygen-free medium by immediate addition of the acid to a cream base containing a preservative, an acid, a cosolvent, an emulsifier, a wax-like product and water.

EFFECT: obtaining cream having high stability at storage and smaller particles of an active ingredient.

9 cl, 11 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents dermatological cream, intended for local treatment of bacterial skin infections and for healing wounds associated with them, which contains framycetin sulfate and biopolymer, included into cream base, which contains at least one substance from each of the following groups: preservative, primary and secondary emulsifier, selected from the group which contains ketostearyl alcohol, ketomacrogol 1000, polysorbate-80 and Span-80; paraffin as wax-like product, cosolvent, selected from the group, including propylene glycol, hexylene glycol and polyethylene glycol-400; nitric acid or lactic acid and water, with said biopolymer preferably being chitosan.

EFFECT: invention provides higher therapeutic effect.

8 cl, 10 tbl, 2 dwg

FIELD: medicine.

SUBSTANCE: invention represents cream for skin infections, containing fusidic acid formed in situ in an oxygen-free medium with the use of sodium fusidate. The above cream contains fusidic acid prepared in situ by the conversion of sodium fusidate with adding acid slowly, with a particle size of the active substance of 2.33 mcm to 16.3 mcm, and the cream base containing at least one ingredient of each type: primary and secondary emulsifying agents specified in a group containing ketostearyl alcohol, ketomacrogol-1000, Polysorbate-80 and Span-80, paraffin as wax, a combined dissolving agent specified in a group including propylene glycol, hexylene glycol, polyethylene glycol 400, nitric acid or lactic acid and water.

EFFECT: invention provides high stability of the active ingredient throughout the whole shelf life.

10 cl, 16 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and concerns a pneumococcal vaccine containing saccharide of 4, 6B, 9V, 14, 18C, 19F and 23F serotypes, separately conjugated with CRM197, and at least one Toll-like receptor-9 (TLR-9) agonist as an adjuvant, wherein the above Toll-like receptor-9 agonist represents CpG-oligonucleotide. Also, the group of inventions concerns using the pneumococcal vaccine for preparing the therapeutic agent for preventing or treating diseases caused by S.pneumoniae; a method for individual's immunisation against the diseases caused by S.pneumoniae infection, involving administering the immunoprotective dose of the vaccine into the above individual.

EFFECT: adding B-class CpG to the conjugated pneumococcal vaccine provides the considerable increase of the proportion of highly responsive patients, good tolerance and cell memory induction in relation to pneumococcal polysaccharides.

45 cl, 9 dwg, 4 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention represents an antimicrobial agent for preventing an implant-associated infection containing gentamicin sulphate 0.96 g, dioxidine 1.0 g, medium-molecular-weight medical collidone (molecular weight 30000D) 10.0 g, distilled water up to 100.0 ml.

EFFECT: extending the range of products preventing the developing implant-associated infection and prevents preserving the antimicrobial activity of the tissues surrounding the implantation area.

2 cl, 1 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: agent possessing the anti-inflammatory, antipyretic and antimicrobial action representing a dry extract of drug hedge hyssop leaves and blossom by grinding them, extracting in 96% alcohol on a water bath to a boil, and boiling, evaporating, diluting the evaporated residue by distilled water first, adding chloroform then, cooling to a room temperature and centrifuging, separating a water fraction and drying it in the certain environment.

EFFECT: agent possesses the pronounced anti-inflammatory, antipyretic and antimicrobial action.

5 dwg, 5 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and represents a composition possessing the antioxidant and antibacterial activity and containing lithium ascorbate, differing by the fact that it additionally contains lithium benzoate in the following proportions, wt %: lithium ascorbate - 50; lithium benzoate - 50.

EFFECT: invention provides extending the range of antioxidants with antibacterial activity possessing normothymic activity.

1 ex, 2 tbl

FIELD: biotechnology.

SUBSTANCE: method provides culturing in the liquid culture medium of the virulent strain of bacteria Staphylococcus aureus No. 6 followed by separation of the culture medium from the bacteria by filtration through the filter PLASMAFILTER PLASMAFLUX PSu 2S with obtaining the filtrate. Ammonium sulphate to 80% saturation is added to the resulting filtrate to obtain the precipitate. The resulting precipitate is separated by centrifugation at 10000 g for 30 minutes and dissolved in phosphate buffer at pH 7.4 with subsequent microfiltration of the protein-containing fraction through the 0.22 mcm membrane and desalting on the column PD-10, purification and concentration by carrying out ion-exchange chromatography on the column of Q-sepharose, elution with 0.15 M NaCl and ultrafiltration on two filters of 100 and 30 kDa to obtain protein containing fraction with the molecular weight of 30-90 kDa and protein content of 0.5-1.0 mg/ml.

EFFECT: invention enables to obtain the products based on protective secreted protein-containing compound.

1 dwg, 5 tbl, 1 ex

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