Immunogenic composition applicable for staphylococcus vaccination

FIELD: medicine, pharmaceutics.

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

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

23 cl, 8 tbl, 7 dwg, 8 ex

 

The scope of the invention

The present invention relates to the field of staphylococcal immunogenic compositions and vaccines, their production and use of such compositions in medicine. More specifically, it relates to vaccine compositions containing a combination of antigens for the treatment or prevention of staphylococcal infection. Also suggested ways in which such vaccines in medicine and methods for their preparation.

Prior art

The number of both community-acquired and hospital-acquired infections has increased in recent years with the increased use of intravascular devices. Hospital-acquired (nosocomial) infections are a major cause of morbidity and mortality, more specifically in the USA, where they affect more than 2 million patients annually. According to various studies, about 6 percent of American patients acquire an infection during their hospital stay. Estimated that the economic burden in the United States in 1992 gpiiia more than 4.5 billion dollars (Emori and Cup, 1993, Clin. Environ. Rev. 6; 428). The most frequent infections are urinary tract infections (UTI-33% of infections), then pneumonia (15,5%), surgical wound infection (14.8%), primary infection of the blood (13%) (Emori and Cup, 1993, Clin. Environ. Rev. 6; 428)).

Staphylococcus aureus, coagulase-negative staphylococci (most everything about Staphylococcus epidermidis), Enterococcus spp, Esherichia coli and Pseudomonas aeruginosa are major nosocomial pathogens. Although these pathogens are unlikely to cause the same number of infections, the severity of the disorders that they can cause, along with the frequency of antibiotic-resistant isolates, shift this equilibrium towards S. aureus and S. epidermidis as the most important nosocomial pathogens.

Staphylococcus aureus is the most frequent cause of nosocomial infections with significant morbidity and mortality (Romero-Vivas et al., 1995, Infect. Dis. 21; 1417). He is the cause of some cases of osteomyelitis, endocarditis, septic arthritis, pneumonia, abscesses and toxic shock syndrome.

S. epidermidis is a normal skin commensal, which is also an important opportunistic pathogen responsible for infections of implanted medical devices and infection of surgical wounds. Medical devices infected with S. epidermidis, include pacemakers, cerebrospinal shunts, catheters for continuous peritoneal dialysis outpatient, orthopedic devices and artificial heart valves.

Infection of S. aureus and S. epidermidis treated with antibiotics and penicillin is the drug of choice, while vancomycin is used for methicillin-resistant isolates. The percentage of strains of the article is filatochev, exhibiting a wide range of resistance to antibiotics has become increasingly grow 1980s(Panlilo et al., 1992, Infect. Control. Hosp. Epidemiol. 13; 582), posing a threat to effective antimicrobial therapy. In addition, the recent emergence of vancomycin-resistant strain of S. aureus has caused concern that will appear and spread of methicillin-resistant strains of S. aureus, for which there is no effective therapy.

Explored an alternative approach using antibodies against staphylococcal antigens in passive immunotherapy. Therapy, including the introduction of polyclonal antisera, is under development (WO 00/15238, WO 00/12132), and treatment with monoclonal antibody against lipoteichoic acid (WO 98/57994).

An alternative approach could be the use of active immunization for the induction of an immune response against Staphylococcus. Identified several candidates for inclusion as a vaccine component. These include fibronectin-binding protein (US5840846), similar to MHC II (US5648240), fibrinogen-binding protein (US6008341), GehD (US 2002/0169288), collagen-binding protein (US6288214), SdrF, SdrG and SdrH (WO 00/12689), mutant SEA and SEB exotoxins (WO 00/02523) and vitronectin-binding protein of 52 kDa (WO 01/60852).

Genome sequenced S. aureus, and many coding sequences identified (EP 786519, WO 02/094868). The same in the RNO and S. epidermidis (WO 01/34809). Improving this approach, others have identified proteins that are recognized by hyperimmune sera from patients who suffered a staph infection (WO 01/98499, WO 02/059148).

The first generation of vaccines against S. aureus or against absoprtion, which he produces, did not give great results (Lee 1996 Trends Environ. 4; 162). There is still a need for effective vaccines against staphylococcal infections.

Accordingly, the present invention proposed immunogenic composition comprising at least two different protein or immunogenic fragment is selected from at least two groups of proteins or immunogenic fragments, selected from the following groups:

Group (a): at least one staphylococcal binding protein extracellular component, or immunogenic fragment selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase (neutral phosphatase), IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, and MAP.

Group (b): at least one staphylococcal transport protein or immunogenic fragment selected from the group consisting of IMM is codominance ABC Transporter, IsdA, IsdB, Mg2+ Transporter, Site and Ni ABC Transporter.

Group (C): at least one staphylococcal regulator of virulence, toxin or immunogenic fragment selected from the group consisting of alpha-toxin (HIa), mutant alpha toxin H35R, RNA Ill-activating protein (RAP).

Description of graphic materials

Figure 1. Polypeptide sequence is preferred proteins. Table 1 shows the information which protein presents each of SEQ ID.

Figure 2. The nucleotide sequence encoding the preferred proteins. Table 1 provides information, which protein is encoded by each of SEQ ID.

Figure 3. Purification of alpha-toxin in native conditions. Panel a shows SDS page-ordinator (polyacrylamide gel with dodecyl sodium sulfate), colored Kumasi, samples obtained during purification of alpha-toxin. Lane 1 - molecular weight markers, lane 2, soluble fraction, containing sverhagressivny alpha-toxin, lane 3 - penetrations from the Ni-NTA column, lane 4 - fractions, erwerbende 10%buffer B, track 5 - fractions, erwerbende 20%buffer B, lane 6 - fractions, erwerbende 30%buffer B, track 7 - fractions, erwerbende 50%buffer B, lane 8 - faction, erwerbende 75%buffer B, lane 9 and 10 fractions, erwerbende 100%buffer B, track 11 - bacteria at T=0 before induction, d is the horn 12 - bacteria at T=4 hours after induction, lane 13 cell lysate, lane 14 - soluble fraction, lane 15 - insoluble fraction.

On the panel shown In colored Kumasi PAG-ordinator 10, 5, 2 and 1 µl of the purified alpha-toxin.

Figure 4. Cleaning SdrC under denaturing conditions. Panel a shows colored Kumasi PAG-ordinator of samples obtained during purification of alpha-toxin. Lane M - molecular weight markers, lane Start supernatant obtained from the insoluble fraction containing sverhagressivny SdrC, track FT1 - penetrations from the Ni-NTA column, the track - faction, erwerbende wash buffer C track D - fraction, erwerbende buffer D track E - faction, erwerbende buffer that is On the panel shown In colored Kumasi PAG-LTO 1, 2, 5 and 10 ál of purified SdrC.

Figure 5. The results of ELISA for antisera against staphylococcal proteins in the tablets coated with purified proteins.

Pool mice pre - result using the United sera taken from mice before immunization. Pool mice Post III is the result of a pooled mouse sera taken after immunization. Pool rabbit pre - result using the United sera obtained from rabbits before immunization. Pool rabbit Post III is the result of a pooled rabbit sera taken after immunization. BIc - negative control

6 - results of ELISA for murine antisera raised against staphylococcal proteins, tablets, coated killed staphylococci.

For the panel And use the tablets covered with killed whole cells of S. aureus serotype 5. For the panel To use the tablets covered with killed whole cells of S. aureus serotype 8. For the panel To use the tablets covered with killed whole cells of S. epidermidis.

The line marked with squares shows the result of ELISA using antisera from mice immunized three times specified staphylococcal protein. The line marked with diamonds shows the result of ELISA for preimmune mouse sera.

Fig.7 - ELISA results for rabbit antisera raised against staphylococcal proteins, tablets, coated killed staphylococci.

For the panel And use the tablets covered with killed whole cells of S. aureus serotype 5. For the panel To use the tablets covered with killed whole cells of S. aureus serotype 8. For the panel To use the tablets covered with killed whole cells of S. epidermidis.

The line marked with squares shows the result of ELISA using antisera from rabbits immunized three times specified staphylococcal protein (except HarA, where he made only one immunization). The line marked with diamonds, showing the characteristic result of ELISA for preimmune rabbit sera.

Detailed description

In the present invention have been disclosed for a particular combination of staphylococcal antigens, which when combined result in an immunogenic composition, which is effective in the treatment or prevention of staphylococcal infection. Immunogenic compositions according to the invention respectively include antigens that are involved in different functions of staphylococci. Such immunogenic compositions are targeting the immune response to different aspects of the function of staphylococci and therefore able to induce particularly effective immune response.

Staphylococcal infections go through several different stages. For example, the life cycle of staphylococci includes commensal colonization, the initiation of infection by providing access to adjacent tissues or the bloodstream, anaerobe reproduction in the blood, the interaction between epitopes virulence of S. aureus and protective mechanisms of the host, and the induction of complications, including endocarditis, education metastatic abscess and sepsis syndrome. Different molecules on the surface of the bacteria will be involved in the different stages of the infectious cycle. Targeting the immune response against an effective amount of the combination of the specific antigens involved in different processes staphylococcal infection, you can get staph immunogenic to the position or the vaccine with increased efficiency.

In particular, a combination of some antigens from different classes, some of which are involved in adhesion to host cells, some of which are involved in the absorption of iron or other transport functions, some of which are toxins or regulators of virulence, and immunodominant antigens can induce an immune response that protects against many stages of infection.

The effectiveness of the immune response can be measured in tests on animal models, as described in the examples, and/or using opsonophagocytosis analysis, as described in the examples.

An additional advantage of the invention is that the combination of antigens according to the invention from different families of proteins in the immunogenic composition is capable of protecting against a wider range of strains.

The invention relates to immunogenic compositions containing a lot of proteins, selected from at least two different types of protein have different functions in staphylococci. Examples of such categories of proteins are extracellular binding proteins, transport proteins, such as proteins, engaged in the capture of Fe; toxins or regulators of virulence and other immunodominant proteins. Vaccine combinations according to the invention are effective against homologous strains of staphylococci (strains, the C which occur these antigens) and preferably also against heterologous strains of staphylococci.

Immunogenic composition according to the invention contains some proteins, the number of which is equal to or greater than 2, 3, 4, 5 or 6, selected from 2 or 3 of the following groups:

group (a) at least one staphylococcal binding protein extracellular component, or immunogenic fragment selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, and MAP;

group (b) at least one staphylococcal transport protein or immunogenic fragment selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC and Ni ABC Transporter;

group (b) at least one staphylococcal regulator of virulence, toxin or immunogenic fragment selected from the group consisting of alpha-toxin (HIa), mutant alpha toxin H35R, RNA III activating protein (RAP).

For example, the first protein selected from the group (a), (b) or (C), and the second protein selected from the group selected from groups (a), (b) and (C), which does not include the second protein.

In the preferred embodiment of the immunogenic composition according to the invention contains at measures is one protein, selected from the group (a), and additional protein selected from the group (b) and/or group (s).

In another embodiment of the immunogenic composition according to the invention contains at least one antigen selected from the group (b), and additional protein selected from the group (b) and/or group (s).

In another embodiment of the immunogenic composition according to the invention contains at least one antigen selected from the group (b), and additional protein selected from the group (a) and/or group (b).

Immunogenic composition according to the invention accordingly contains proteins of S. aureus and/or S. epidermidis.

Proteins

Immunogenic compositions of the invention include isolated protein containing amino acid sequence that has at least 85%identity, preferably at least 90%identity, more preferably at least 95%identity, most preferably at least 97-99%or strict identity with any sequence of figure 1.

When the protein is specifically mentioned in this description, the preferred reference to native or recombinant, full-length protein or perhaps Mature protein was removed signal sequence. This protein can be isolated directly from staphylococcal strain or obtained by the methods recombinant the x DNA. Immunogenic fragments of the protein can be included in the immunogenic composition according to the invention. They represent fragments containing at least 10 amino acids, preferably 20 amino acids, more preferably 30 amino acids, more preferably 40 amino acids, or about 50 amino acids, most preferably 100 amino acids, taken continuously from the amino acid sequence of the protein. In addition, such immunogenic fragments react immunologically with antibodies raised against staphylococcal proteins, or antibodies induced by infection of a mammalian host by Staphylococcus. Immunogenic fragments also include fragments that with the introduction of effective dose (either alone or in the form of the hapten associated with the media), is called a protective immune response against staphylococcal infections, more preferably it is protective against infection with S. aureus and/or S. epidermidis. Such immunogenic fragment may include, for example, a protein containing N-terminal leader sequence and/or transmembrane domain, and/or C-terminal anchor domain. In a preferred aspect the immunogenic fragment according to the invention contains essentially the entire extracellular domain of a protein that has at least 85%identity, preferably at least 90-percent identity, more preferably at least 95%identity, most preferably at least 97-99%identity with a sequence that is selected from 1, compared to the full length sequence of the fragment.

In immunogenic compositions according to the invention also includes fused protein consisting of staphylococcal proteins, or immunogenic fragments of staphylococcal proteins. Such fused proteins can be obtained recombinante and can contain one section of at least 2, 3, 4, 5 or 6 staphylococcal proteins. Alternatively, the protein may contain many sections of at least 2, 3, 4 or 5 staphylococcal proteins. They can combine different staphylococcal proteins or immunogenic fragments of the same protein. Alternatively, the invention also includes a separate fused proteins staphylococcal proteins or immunogenic fragments in the form of a fused protein with heterologous sequences, such as the source of T-cell epitope or a tag for purification, for example: β-galactosidase, glutathione-8-transferase, green fluorescent proteins (GFP), epitope tags such as FLAG, myc-tag, polyhistidine, or viral surface proteins, such as hemagglutinin of influenza virus, or bacterial proteins, such as tetanus toxoid, diphtheria toxoid, CRM197.

Tab the Itza 1

The following table shows the SEQ ID numbers preferred protein sequences and DNA sequences that are located on figure 1 and figure 2 respectively. SA indicates the sequence of the S. aureus and SE indicates the sequence of S. epidermidis.

NameProtein sequenceThe DNA sequence
Immunodominant ABC Transporter
SASEQ ID 1SEQ ID 34
SESEQ ID 2SEQ ID 35
The laminin receptor
SASEQ ID3SEQ ID 36
SESEQ ID 4SEQ ID 37
Secretory antigen SsaA A
SA1SEQ ID 5SEQ ID 38
SA2SEQ ID 6SEQ ID 39
5THSEQ ID 7SEQ ID 40
SitC
SASEQ ID 8SEQ ID 41
SESEQ ID 9SEQ ID 42
IsaA/PisA (IssA)
SASEQID10SEQ ID 43
SESEQID11SEQ ID 44
EbhA/B
SA EbhASEQID12SEQ ID 45
SA EbhBSEQID 13SEQ ID 46
SE EbhASEQ ID 14SEQ ID 47
SE EbhBSEQID 15SEQ ID 48
Protein associated with the accumulation of AAR
SASEQ ID 16SEQ ID 49
SESEQ ID 17SEQ ID 50
RNA III activating protein RAP
SASEQ ID 18SEQ ID 51
SESEQ ID 19SEQ ID 52
FIG/SdrG
SASEQ ID 20SEQ ID 53
SESEQ ID 21SEQ ID 54
Elastin-binding white is EbpS
SASEQ ID 22SEQ ID 55
SESEQ ID 23SEQ ID 56
Extracellular protein EFB SASEQ ID 24SEQ ID 57
alpha-toxin SASEQ ID 25SEQ ID 58
SBI SASEQ ID 26SEQ ID 59
IsdA SASEQ ID 27SEQ ID 60
IsdB SASEQ ID 28SEQ ID 61
SdrC SASEQ ID 29SEQ ID 62
ClfA SASEQ ID 30SEQ ID 63
FnbA SASEQ ID 31SEQ ID 64
ClfB SA SEQ ID 32SEQ ID 65
Coagulase SASEQ ID 33SEQ ID 66
FnbB SASEQ ID 67SEQ ID 71
MAP SASEQ ID 68SEQ ID 72
SdrC SASEQ ID 69SEQ ID 73
SdrG SASEQ ID 70SEQ ID 74

Proteins that bind extracellular components

Proteins that bind extracellular components are proteins that bind to the extracellular components of the host. The term includes, but is not limited to adesanmi.

Examples of proteins that bind extracellular components include laminin receptor (Naidu et al. J. Med. Environ. 1992, 36; 177), SitC/MntC/saliva binding protein (US5801234, Wiltshire and Foster Infec. Immun. 2001, 69; 5198), EbhA (Williams et al. Infect. Immun. 2002, 70; 6805), EbhB, elastin binding protein (EbpS) (Park et al., 1999, J. Biol. Chem. 274; 2845), EFB (FIB) (Wastfelt and Flock 1995, J. Clin. Environ. 33; 2347), SBI (Zhang et al. FEMS Immun. Med. Environ. 2000, 28; 211), autolysin (Rupp et al., 2001, J. Infect. Dis. 183; 1038), ClfA (US6008341, McDevitt et al. Mol. Environ. 1994, 11; 237), SdrC, SdrG (McCrea et al. Microbiology 2000, 146; 155), SdrH (McCrea et al. Microbiology 2000, 146; 1535), lipase GehD (US2002/0169288), SasA, FnbA (Flock et al. Environ Mol. 1994, 12; 599, US6054572), FnbB (WO 97/14799, Booth et al., 2001 Infec. Immun. 69; 345), collagen-binding protein Cna (Visai et al., 2000, J. Biol. Chem. 275; 39837), ClfB (WO 99/27109), FbpA (Phonimdaeng et al., 1988 J. Gen Environ. 134; 75), Npase (2001 Flock J. Bacteriol. 183; 3999), IsaA/PisA (Lonenz et al. FEMS Measurement. Med. Environ. 2000, 29; 145), SsaA (Lang et al. FEMS Immunol. Med. Environ. 2000, 29; 213), EPB (Hussain and Hermann symposium on Staph Denmark 14-17th 2000), SSP-1 (Veenstra et al., 1996, J. Bacteriol. 178; 537), SSP-2 (Veenstra et al., 1996, J. Bacteriol. 178; 537), heparin-binding protein HBP 17 kDa (Fallgren et al., 2001, J. Med. Environ. 50; 547), vitronectin binding protein (Li et al., 2001, Curr. Environ. 42; 361), fibrinogen binding protein, coagulase, Fig (WO 97/48727) and MAP (US 5648240) SitC/MntC/saliva binding protein

He is an ABC-transport protein which is a homologue of adhesin PsaA in S. pneumoniae. He is a highly immunogenic lipoprotein 32 "Yes, which is distributed in the bacterial cell wall (Cockayne et al. Infect. Immun. 1998 66; 3767). It is expressed in S. aureus and S. epidermidis in the form of lipoprotein 32 "Yes, and the homologue of 40 kDa is present in S. hominis. In S. epidermidis, it is a component of the iron-regulated operon. It shows significant homology to both adhesins, including FimA Streptococcus parasanguis, and lipoproteins family of ABC-transporters with proven or putative transport functions of the metal iron. So SitC include as extracellular binding be the spacecraft and as a conveyor of metal ions.

Binding protein saliva, disclosed in US 5801234, also represents a form SitC and can be included in the immunogenic composition according to the invention.

ClfA and ClfB

Both of these protein fibrinogen-binding activity and stimulates S. aureus to form colonies in the presence of plasma. They contain an LPXTG-motif shared by proteins associated with the wall.

ClfA is described in US6008341, and ClfB described in WO 99/27109. Coagulase (FppA)

It is a fibrinogen-binding protein that induces S. aureus to form colonies in the presence of plasma. It is described in the references related to coagulase: Phonimdaeng et al. (J. Gen. Microbio. 1988, 134:75-83), Phonimdaeng et al. (Environ Mol 1990; 4:393-404), Cheung et al. (Infect Immun 1995; 63:1914-1920) and Shopsin et al. (J. Clin. Environ. 2000; 38:3453-3456).

Preferred fragments for inclusion in immunogenic composition according to the invention include the Mature protein, which was deleted signal peptide (amino acids 27 to C-end).

Coagulase has three different domain. Amino acids 59-297, which are bipedally plot, amino acids 326-505, which is a Proline - and glycine-rich segment, and the C-terminal domain from amino acids 506-645, which has beta-layer conformation. Each of these domains is the preferred fragment according to the invention.

SdrG - Fbe - EfB/FIG

Fbe is a fibrinogen-binding Bel is to, which are found in many isolates of S. epidermidis and has a deduced molecular weight of 119 kDa (Nilsson et al., 1998. Infect, Immun. 66; 2666). Its sequence related sequence of factor agglutination of S. aureus (ClfA). Antibodies against Fbe can block the binding of S. epidermidis with fibrinogen-coated tablets and catheters (Pei and Flock 2001, J. Infect. Dis. 184; 52). This protein is also described as SdrG in WO 00/12689. SdrG found in coagulase-negative staphylococci and is associated with a cell wall protein containing the LPXTG sequence.

SdrG contains a signal peptide (amino acids 1-51), the area containing the fibrinogen-binding sites and the collagen-binding site (amino acids 51-825), two CnaB domain (amino acids 627-698 and 738-809), repetitive plot SD (amino acids 825-1000) and an anchoring domain (amino acids 1009-1056).

Fbe has estimated signal sequence with a cleavage site between amino acids 51 and 52. Therefore, the preferred fragment Fbe contains Mature form Fbe, continued from amino acids 52 to C-end amino acid 1092).

Domain Fbe from amino acids 52 to 825 amino acids responsible for binding to fibrinogen. Therefore, the preferred fragment Fbe consists of amino acids 52-825 or contains them.

The area between amino acids 373 and 516 Fbe demonstrates the greatest conservatism between Fbe and ClfA. P is this the preferred fragment will contain amino acids 373-516 Fbe.

Amino acids 825-1041 Fbe contain highly repetitive plot, consisting of tandemly repeated residues aspartic acid and serine.

Preferred fragments of SdrG include polypeptides in which the signal peptide and/or repetitions SD and anchor domain have been removed. They include polypeptides containing or consisting of amino acids 50-825, amino acids 50-633, amino acids 50-597 (SEQ ID NO 2 from WO 03/76470), amino acids 273-597 (SEQ ID NO 4 of WO 03/76470), amino acids 273-577 (SEQ ID NO 6 of WO 03/76470) amino acids 1-549, amino acids 219-549, amino acids 225-549, amino acids 219-528, amino acids 225-528 of SEQ ID ON:70.

Preferably, the polypeptide SdrG having a sequence at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% homologous to the sequence of SEQ ID 70, 20 or 21, included in the immunogenic composition according to the invention.

The composition of the invention may contain a fragment of the polypeptide SdrG described above.

Preferred fragments are deleteriously signal peptide and/or repetitive domain SD, and/or anchor domain. For example, the sequence corresponding to amino acids 1-713, 1-549, 225-549, 225-529, 24-717, 1-707, 1-690, 1-680, 1-670, 1-660, 1-650, 1-640, 1-630, 1-620, 1-610, 1-600, 34-707, 44-697, 36-689 of SEQID 76 or sequences with 85%-, 90%-, 92%-, 95%-, 97%-, 98%-, 99%- or 100%identity with SEQ ID 70, or 20, or 21.

A preferred fragment with deletionism signal pept the house has meinenemy residue at the N-end of the fragment to ensure correct translation.

More preferred fragment has the following sequence:

MEENSVQDVKDSNTDDELSDSNDQSSDEEKNDVINNNQSINTDDNNQIIKKEETNNYDGIEKRSEDRTESTINVDENEATFLQKTPQDNTHLTEEEVKESSSVESSNSSIDTAQQPSHTTINREESVQTSDNVEDSHVSDFANSKIKESNTESGKEENTIEQPNKVKEDSTTSQPSQYTNIDEKISNQDE

LLNLPINEYENKARPLSTTSAQPSIKRVTVNQLAAEQGSNVNHLIKVTDQSITEGYDDSEGVIKAHDAENLIYDVTFEVDDKVKSGDTMTVDIDKNTVPSDLYDSFTIPKIKDNSDEIIATGTYDNKNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQRPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEDST

IIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQYVTNQTTINEYTGEFRTASYDNTIAFSTSSGQGQGDLPPEKTYKIGDYVWEDVDKDGIQNTNDNEQPLSNVLVTLTYPDGTSKSVRTDEDGKYQFDGLKNGLTYKITFETPEGYTPTLKHSGTMPALDSEGNSVWVTINGQDDNTIDSGFYQTPKYSLGNY

VWYDTNKDGIQGDDEKDISGVKVTLKDENGNIISTTTTDENGKYQFDNLNSGNYIVHFDKPSGMTQTTTDSGDDDEQDADGEEVHVTITDHDDFSIDNGYYDDE

EbhA and EbhB

EbhA and EbhB represent proteins that are expressed in S. aureus and S. epidermidis (Clarke and Foster Infect. Immun. 2002, 70; 6680, Williams et al. Infect. Immun. 2002, 20; 6805), and which are associated with fibronectin. Since fibronectin is an important component of the extracellular matrix, EbhA and EbhB perform an important function in the attachment of staphylococci to the extracellular matrix of the host.

Protein Ebh are great, having a molecular weight of 1.1 megadalton. It is preferable to use a fragment of the protein Ebh, not the full sequence due to the ease of obtaining and cooking in the form of the drug. The Central part of the protein contains a partial repeats, which contain fibronectin-binding sites. Fragments containing one or more repeating domains, described below, are preferred which considerable fragments for inclusion in immunogenic composition according to the invention.

Protein Ebh contain incomplete repeating unit length of 127 amino acids, which are characterized by the content of the consensus sequence:

L.G.{10}A.{13}q{26}L...M..L.{33}A

Preferably,

.{19}LG{10}A.{13}q {26}L...M..L.{33}A.{12}

More preferably,

.....I/V..A...I/V..AK.ALN/DG..NL..AK..A.{6}L..LN.AQK..L..QI/V..A..V..V{6}A..LN/D.AM..L...I/V.D/E...TK.S.NY/F.N/DAD..K..AY/F..AV..A..I/V.N/D.......

Where '.' means any amino acid, and '.{10}' means any 10 amino acids, and the I/V indicates the alternate choice of amino acids.

By reference to the sequence disclosed in Kuroda et al., (2001) Lancet 357; 1225-1240, and Table 2, easily remove duplicate sequences in proteins Ebh.

Preferred fragments that can be included in the immunogenic composition according to the invention include polypeptides containing one, two, three, four, five, six, seven, eight, nine, ten, or more than 10 127-amino acid repeating units. Such fragments may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats 127-amino acid repeating section or may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repetitions with additional amino acid residues that are present either on one or on both ends of this fragment. Another preferred fragment is a polypeptide H2 of about 44 kDa, covering three repeats (amino acids 3202-3595), as described in Clrke et al. Infection and Immunity 70, 6680-6687, 2002. Such fragments preferably are capable of binding fibronectin and/or activate antibodies that are reactive against a protein Ebh.

The Ebh polypeptide is able to connect with fibronectin. Preferred polypeptide fragments of these sequences retain the ability to bind to fibronectin. The binding of fibronectin can be assessed using ELISA, as described by Clarke et al.(Infection and Immunity 70; 6680-6687 2002).

Another preferred fragments are fragments that contain b-cell or T-helper epitope, for example those fragments/peptides, which are described in Tables 3 and 4.

TABLE 2. Repeated sequences in the full-size sequence Ebh.

Full sequence Ebh disclosed in Kuroda et al. (2001) Lancet 357; 1225-1240. The following table shows amino acid residues, in which 127-amino acid repeats start and end within the full sequence.

7097
StartEnd
132043330
233313457
334573583
435833709
537093835
638353961
739614087
842004326
943264452
1044524578
1145784704
1247044830
1348304956
1449565082
1550825208
1652085334
1753345460
1854605586
1955855711
2057115837
2158375963
2259636089
2360896215
2462156341
2563416467
2664676593
2765936719
2867196845
2968456971
3069717097
317223
3272237349
3373497475
3474757601
3576017727
3677277853
3778527978
3879788104
3981048230
4082308356
4183568482
4284828608
4386048730
4488588984

Table 3. Prediction of b-cell epitope for a 27-amino acid repeat:

Full sequence disclosed in Kuroda et al. (2001) Lancet 357; 1225-1240. One of these repeats encoded by amino acids 3204-3331 full sequence was chosen for implementation of predicting epitopes:

MDVNTVNQKAASVKSTKDALDGQQNLQRAKTEATNAITHASDLNQAQKNALTQQVNSAQNVHAVNDIKQTTQSLNTAMTGLKRGVANHNQVVQSDNYVNADTNKKNDYNNAYNHANDIINGNAQHPVI

StartEndThe sequence of the epitopeStartStop
510TVNQKA32083213
1419KSTKDA32173222
2133DGQQNLQRAKTEA32243236
4251DLNQAQKNAL32453254
6674DIKQTTQSL32693277
100112ADTNKKNDYNNAY33033315
117123DIINGNA33203326

- Columns “Start” and “End” represent the position of the predicted b-cell epitopes in a 127-amino acid repeat

- Columns “Start” and “Stop” represent the position of the predicted b-cell epitopes in the full sequence Ebh

Table 4. Prediction of T-helper cell epitope in Ebh: Full sequence disclosed in the TrEMBL database, the reference sequence Q8NWQ6. One of these repeats encoded by amino acids 3204-3331 full sequence was chosen for implementation of predicting epitopes:

MDVNTVNQKAASVKSTKDALDGQQNLQRAKTEATNAITHASDLNQAQKNALTQQVNSAQNVHAVNDIKQTTQSLNTAMTGLKRGVANHNQVVQSDNYVNADTNKKNDYNNAYNHANDIINGNAQHPVI

Position repeatThe sequence of the epitopePosition sequence
1MDVNTVNQK3204
3VNTVNQKAA 3206
6VNQKAASVK3209
26LQRAKTEAT3229
37ITHASDLNQ3240
43LNQAQKNAL3246
51LTQQVNSAQ3254
55VNSAQNVHA3258
61VHAVNDIKQ3264
64VNDIKQTTQ3267
67IKQTTQSLN3270
74LNTAMTGLK3277
78MTGLKRGVA3281
81LKRGVANHN3284
85VANHNQWQ3288
913294
92VQSDNYVNA3295
97YVNADTNKK3301
98VNADTNKKN3302
108YNNAYNHAN3311
112YNHANDIIN3315
118IINGNAQHP3321
119INGNAQHPV3322

- Column “Position repeat” represents the position of the predicted T-cell epitopes in the redo

- Column “Position sequence” represents the position of the predicted T-cell epitopes in the full sequence Ebh

Fragments of the polypeptides according to the invention can be used to obtain the corresponding full-length polypeptide by peptide synthesis; therefore, these fragments can be used as an intermediate to obtain the full-length polypeptides according to the invention.

Especially preferred are the variants, in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid(a) substituted, deleterow or added in any combination.

The elastin binding protein (EbpS)

EbpS is a protein containing 486 amino acids with a molecular mass of 83 kDa. It is associated with the cytoplasmic membrane of S. aureus and has three hydrophobic stretch that hold the protein in the membrane (Downer et al., 2002, J. Biol. Chem. 277; 243; Park et al., 1996, J. Biol. Chem. 271; 15803).

Two sites between amino acids 1-205 and 343-486 surface exposed on the outer side of the cytoplasmic membrane. The ligand-binding domain EbpS is located between residues 14-34 at N-end (Park et al., 1999, J. Biol. Chem. 274; 2845).

A preferred fragment that can be included in the immunogenic composition according to the invention can be a surface-exposed fragment containing the elastin-binding site (amino acids 1-205). Some preferred fragments do not contain the full exposed loop, but must contain elastin-binding site (amino acids 14-34). Alternative fragment that could be used, consists of amino acids forming the second surface-exposed loop (amino acids 343-486). Possible alternative fragments containing up to 1, 2, 5, 10, 20, 50 and less of the amino acids at one or both ends.

The laminin receptors

The laminin receptor from S. aureus plays an important role in pathogenicity. The hallmark of infection is invasion into the bloodstream, which makes it possible systemic spread of metastatic abscess. For invasion into the bloodstream need to be able to penetrate through the basal membrane of blood vessels. This is achieved through binding to laminin via the laminin receptor (Lopes et al. Science 1985, 229; 275).

The laminin receptors exposed on the surface and are present in many strains of staphylococci, including S. aureus and S. epidermidis.

SBI

Sbi is a protein with lgG-binding site and apolipoprotein N-binding domain, and it is expressed in most strains of S. aureus (Zhang et al., 1998, Microbiology 144; 985).

N-end of the sequence Sbi has a typical signal sequence with a cleavage site after amino acid 29. Therefore, a preferred fragment of the Sbi, which can be included in the immunogenic composition according to the invention begins at amino acid residue 30, 31, 32 or 33 and continues to the end of Sbi, for example of SEQ ID NO: 26.

lgG-binding domain Sbi was identified as parcel near N-Terminus of the protein from amino acids 41-92. This domain is homologous lgG-binding domains of protein A.

Minimum lgG-binding domain Sbi contains the following sequence:

QTTQNNYVTDQQKAFYQVLHLKGITEEQRNQYIKTLREHPERAQEVFSESLK

∗∗ ∗∗∗ ∗ ∗∗∗ ∗ ∗ ∗ ∗ ∗

∗ - indicates amino acids that are similar between lgG-binding domains

A preferred fragment of Sbi to be included in the immunogenic composition according to the invention contains lgG-binding domain. This fragment contains a consensus sequence for lgG-binding domain, denoted by ∗, as shown in the above sequence. Preferably, the fragment contains or consists of a full sequence above. More preferably, the fragment contains or consists of amino acids 30-92, 33-92, 30-94, 33-94, 30-146, 33-146, 30-150, 33-150, 30-160, 33-160, 33-170, 33-180, 33-190, 33-200, 33-205 or 33-210 Sbi, for example of SEQ ID NO:26.

A preferred fragment may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions of these two sequences.

Preferred fragments can contain multiple repetitions(2, 3, 4, 5, 6, 7, 8, 9 or 10) lgG-binding domain.

EFB-FIB

Fib is a fibrinogen-binding protein of 19 kDa, which is secreted in the extracellular environment of S. aureus. It is produced by all the tested isolates of S. aureus (Wastfelt and Flock 1995, J. Clin. Environ. 33; 2347).

S. aureus aggregates in the presence of fibrinogen and binds to fibrinogen-coated surfaces. This ability facilitates staphylococcal colonization of catheters and endothelial cells.

Fib sod is RIT signal sequence at the N-end of the protein with the putative cleavage site about amino acid 30. Therefore, the preferred fragment, which is subject to the introduction in the immunogenic composition according to the invention will contain the sequence of the Mature protein (about amino acid 30 to C-end of the protein).

IsaA/PisA

IsaA is a protein of 29 kDa, also known as PisA, which was shown to be an immunodominant staphylococcal protein during sepsis in hospital patients (Lorenz et al., 2000, FEMS Immunol. Med. Microb. 29; 145).

Suppose that the first 29 amino acids of the sequence IsaA represent a signal sequence. Therefore, a preferred fragment of the IsaA, be included in the immunogenic composition according to the invention will contain amino acid residues 30 to the end of the encoded sequence.

Fibronectin-binding protein

Fibronectin-binding protein A (FnbA) is described in US 5320951 and Schennings et al.(1993, Microb. Pathog. 15; 227). Fibronectin-binding protein And contains several domains, which are involved in binding to fibronectin (WO 94/18327). They are called D1, D2, D3 and D4. Preferred fragments of fibronectin-binding protein a, or contain or consist of D1, D2, D3, D4, D1-D2, D2-D3, D3-D4, D1-D3, D2-D4 or D1-D4 (as described in WO 94/18327).

Fibronectin-binding protein contains a signal sequence of 36 amino acids. For example:

VKNNLRYGIRKHKLGAASVFLGTMIWGMGQDKEAA

Perhaps the Mature protein, except for the receiving of this signal sequence, include in the immunogenic composition according to the invention.

Transport proteins

The cell wall of gram-negative bacteria acts as a barrier that prevents the free diffusion of metabolites in bacteria. A family of proteins controls the movement of essential nutrients in bacteria and therefore it is necessary for the viability of the bacteria. The term “transport protein” includes proteins involved in the initial stage of binding to metabolites, such as iron, as well as proteins involved essentially in the transportation of metabolite in bacteria.

Molecular iron is an essential cofactor for bacterial growth. Are secreted of siderophore that bind free iron and then captured bacterial surface receptors that deliver iron for transport across the cytoplasmic membrane. Absorption of iron is critical for the establishment of human infections, so developing an immune response against this class of proteins leads to loss of viability of Staphylococcus.

Examples of transport proteins include immunodominant ABC Transporter (Burnie et al., 2000 Infect. Imun. 68; 3200), IsdA (Mazmanian et al., 2002 PNAS 99; 2293), IsdB (Mazmanian et al., 2002 PNAS 99; 2293), Mg2+ Transporter, SitC (Wiltshire and Foster 2001 Infect. Immun. 69; 5198) and Ni ABC Transporter.

Immunodominant ABC-transport is Ter

Immunodominant ABC Transporter is a very conservative protein that can induce an immune response that cross-protects against different strains of staphylococci (Mei et al., 1997, Mol. Environ. 26; 399). Antibodies against this protein were detected in patients with septicemia (Burnie et al., 2000, Infect. Immun. 68; 3200).

Preferred fragments of immunodominant ABC Transporter shall include peptides DRHFLN, GNYD, RRYPF, KTTLLK, GVTTSLS, VDWLR, RGFL, more preferably KIKVYVGNYDFWYQS, TVIWSHDRHFLYNNV and/or TETFLRGFLGRMLFS, because these sequences contain epitopes that are recognized by the human immune system.

IsdA-IsdB

Genes isd (iron-regulated surface epitope) of S. aureus encode proteins responsible for binding to hemoglobin and the transition of iron heme into the cytoplasm, where it acts as a necessary nutrient elements. IsdA and IsdB are located in the cell wall of staphylococci. IsdA, apparently exposed on the surface of bacteria, because it is sensitive to digestion by proteinase K. IsdB was partially digested, suggesting that it is partially exposed on the surface of bacteria (Mazmanian et al., 2003 Science 299; 906).

IsdA and IsdB both represent protein 29 kDa that bind heme. Their expression is regulated by the availability of iron through the Fur repressor. Their expression is high during infection in x is Saine, where the iron concentration is low.

They are also known as FrpA and FrpB (Morrissey et al., 2002, Infect. Immun. 70; 2399). FrpA and FrpB represent a major surface proteins with a large charge. It has been shown that they provide the main contribution to the adhesion to the plastic.

In one embodiment of the immunogenic composition according to the invention contains a fragment of IsdA and/or IsdB, which is described in WO 01/98499 or WO 03/11899.

Toxins and regulators of virulence

Members of this protein family include a toxin, such as alpha-toxin, hemolysin, enterotoxin b and TSST-1, as well as proteins that regulate the production of toxins, such as RAP.

Alpha-toxin (Hla)

Alpha-toxin is an important epitope virulence produced by most strains of S. aureus. He is a pore-forming toxin with hemolytic activity. It was shown that antibodies against the alpha-toxin neutralizing harmful and lethal effects of alpha-toxin in animal models (Adlam et al., 1977 Infect. Immun. 17; 250). Human platelets, endothelial cells and mononuclear cells sensitive to the effects of alpha-toxin.

High toxicity of alpha-toxin require detoxification before using as immunogen. This can be achieved by chemical treatment, for example by treatment with formaldehyde, glutaraldehyde or other cross-linking Rea the customers, or by chemical conjugation with bacterial polysaccharides, as described below.

Another way to remove toxicity consists in the introduction of point mutations that remove toxicity, cochranae while the antigenicity of the toxin. The introduction of point mutations at amino acid 35 alpha-toxin, where his-tag remnant replace latinoam residue, removes toxicity, whereas the immunogenicity remains (Menzies and Kernodle 1996; Infect. Immun. 64; 1839). Histidine 35, apparently, is critical for proper oligomerization required for steam formation, and mutation of this residue leads to the loss of toxicity.

For inclusion in immunogenic compositions according to the invention the alpha-toxin preferably detoxify through His mutation 35, most preferably by substitution 35 on His Leu or Arg. In an alternative embodiment the alpha-toxin detoxify by conjugation with other components of the immunogenic composition, preferably capsular polysaccharides, most preferably a polysaccharide of type V from S. aureus and/or polysaccharide type VIII and/or PNAG from S. aureus.

RNA III activating protein (RAP)

The RAP itself is not a toxin, but is a regulator of the expression of virulence factors. RAP is produced and secreted by Staphylococcus. He activates the agr regulatoryauthority other staphylococci and activates expression and subsequent release of virulence factors, such as hemolysin, enterotoxin b and TSST-1.

The immune response induced against RAP, will not kill the bacteria but will prevent their pathogenicity. This has the advantage of providing less selective pressure for the emergence of new resistant strains.

This will be the second advantage in obtaining immune response, which will act as a tool in reducing the incidence of infection.

Especially, it is preferable to combine RAP with other antigens in the vaccine, in particular where additional antigen will provide an immune response that is capable of killing bacteria.

Other proteins

Protein associated with the accumulation (AAR)

AAR is a protein of 140 kDa, which is necessary for the accumulation of strains of S. epidermidis surface (Hussain et al. Infect. Immun. 1997, 65; 519). Strains expressing this protein, produced significantly higher amounts of biofilm, and AAR, apparently involved in the formation of biofilms. Antibodies against AAR able to inhibit biofilm formation and to inhibit the accumulation of S. epidermidis.

A preferred fragment of the AAR is conservative domain containing or consisting of amino acids 550-1069.

Staphylococcal secretory antigen SsaA

SsaA is a highly immunogenic protein of 30 kDa, identified as S. aureus and S. pidermidis (Lang et al., 2000 FEMS Immunol. Med. Environ. 29; 213). His expression when endocarditis led me to think about the role of virulence characteristic of the pathogenesis of infectious diseases.

SsaA contains an N-terminal leader sequence and the cleavage site of the signal peptidases. For the leader peptide should hydrophilic area of approximately 100 amino acids from residue 30 to residue 130.

A preferred fragment of the SsaA, which shall be included in the immunogenic composition according to the invention consists of the Mature protein (amino acids 27 to C-end or amino acids 30 to C-end).

Other preferred fragments contain hydrophilic region SsaA from amino acid 30 to amino acids 130.

Preferred combinations

The preferred combination of proteins in the immunogenic composition according to the invention includes a receptor for laminin and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes SitC and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes EbhA and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes EbhB and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes EbpS and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes EFB(FIB), and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in immunore the Noah compositions according to the invention includes SBI and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes autolysin and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes ClfA and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes SdrC and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes SdrG and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of IMM is codominance ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R and RAP.

Another preferred combination of proteins in the immunogenic composition according to the invention includes SdrH and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes lipase GehD, and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes SasA and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes FnbA and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant al the a-toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes FnbB and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes Cna and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes ClfB and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes FbpA and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in immunogen the second composition according to the invention includes Npase and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes IsaA/PisA, and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes SsaA and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes EUAs and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes an SSP-1 and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immuno is dominantnogo ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes SSP-2, and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes NRW and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes a vitronectin-binding protein 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes a fibrinogen-binding protein 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ TRANS is ortera, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes coagulase and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes Fig 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of proteins in the immunogenic composition according to the invention includes MAP and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of protein in the immunogenic composition according to the invention includes the immunodominant ABC Transporter and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, alpha-toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of protein in the immunogenic composition according to the invention includes IsdA and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, alpha-toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of protein in the immunogenic composition according to the invention includes IsdB and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, alpha-toxin mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of protein in the immunogenic composition according to the invention includes SitC and 1, 2, 3, 4 or 5 others who of tiganov, selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, alpha-toxin the mutant alpha toxin H35L or H35R, RAP, AAP and SsaA.

Another preferred combination of protein in the immunogenic compositions of the invention include alpha-toxin and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, AAP and SsaA.

Another preferred combination of protein in the immunogenic composition according to the invention includes a variant of the alpha toxin H35L or H35R and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen-binding of the corresponding protein, coagulase, Fig, MAP, immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, AAP and SsaA.

Another preferred combination of protein in the immunogenic composition according to the invention includes RAP and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, AAP and SsaA.

Another preferred combination of protein in the immunogenic composition according to the invention includes AAR and 1, 2, 3, 4 or 5 other antigens selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, RAP, alpha-toxin and H35L or H35R alpha-toxin.

Another preferred combination of protein in the immunogenic composition according to the invention includes SsaA and 1, 2, 3, 4 or 5 other antig the new, selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, RAP, alpha-toxin and H35L or H35R alpha-toxin.

The inventors have demonstrated that some antigens produce particularly effective immune response in the context of a mixture of antigens. Accordingly, one embodiment according to the invention is an immunogenic composition comprising IsaA and staphylococcal transport protein, or IsaA and staphylococcal regulator of virulence or toxin, or containing Sbi and staphylococcal transport protein, or Sbi, and staphylococcal regulator of virulence or toxin, or containing SdrC and staphylococcal transport protein, or SdrC and staphylococcal regulator of virulence or toxin, or IsaA and Sbi, or IsaA and SdrC, or IsaA and autolysin, or IsaA and Ebh, or Sbi, and SdrC, or Sbi, and autolysin, or Sbi, and Ebh, or SdrC and autolysin or SdrC and Ebh, or autolysin-glucosaminidase and Ebh. For each of these combinations of proteins can be full-length or fragments having a sequence at least 85%, 90%, 95%, 98% or 100%identical to the sequences in figure 1.

In the above and the below combinations of these proteins may be present in the immunogenic composition according to the invention in the form of a fragment or fused protein, as described above.

Preferred immunogenic compositions according to the invention do not include the protein sequence disclosed in WO 02/094868.

The combination of three proteins

Preferred immunogenic composition of the invention includes three protein component in combination alpha-toxin binding protein extracellular component (preferably adhesin), and transport protein (preferably iron-binding protein).

In this combination of alpha-toxin can be chemically detoxified or genetically detoxified by introducing point(s) of mutation(s), preferably a point mutation His35Leu. Alpha-toxin is present in the form of a free protein or, alternatively, anywhereman with polysaccharide or LTA-component of the immunogenic composition.

Preferred combinations include:

Immunogenic composition comprising alpha toxin, IsdA and binding protein extracellular component selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin-tie the living protein, fibrinogen binding protein, coagulase, Fig, and MAP.

Immunogenic composition comprising alpha toxin, IsdB and binding protein extracellular component selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin-binding protein, fibrinogen binding protein, coagulase, Fig, and MAP.

Immunogenic composition comprising alpha toxin, IsdA and adhesion, selected from the group consisting of laminin receptor, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), ClfA, SdrC, SdrG, SdrH, autolysin, FnbA, FnbB, Cna, ClfB, FbpA, Npase, SSP-1, SSP-2, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, and MAP.

Immunogenic composition comprising alpha toxin, IsdB and adhesion, selected from the group consisting of laminin receptor, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), autolysin, ClfA, SdrC, SdrG, SdrH, FnbA, FnbB, Cna, ClfB, FbpA, Npase, SSP-1, SSP-2, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, and MAP.

Immunogenic composition comprising alpha toxin, IsdA and the laminin receptor.

Immunogenic composition comprising alpha toxin, IsdA and EbhA.

Immunogenic composition comprising alpha toxin, IsdA and EbhB.

Immunogenic composition comprising alpha toxin, sdA and EbpS.

Immunogenic composition comprising alpha toxin, IsdA and EFB (FIB).

Immunogenic composition comprising alpha toxin, IsdA and SdrG.

Immunogenic composition comprising alpha toxin, IsdA and ClfA.

Immunogenic composition comprising alpha toxin, IsdA and ClfB.

Immunogenic composition comprising alpha toxin, IsdA and FnbA.

Immunogenic composition comprising alpha toxin, IsdA and coagulase.

Immunogenic composition comprising alpha toxin, IsdA and Fig.

Immunogenic composition comprising alpha toxin, IsdA and SdrH.

Immunogenic composition comprising alpha toxin, IsdA and SdrC.

Immunogenic composition comprising alpha toxin, IsdA and MAP.

Immunogenic composition comprising IsaA and Sbi.

Immunogenic composition comprising IsaA and IsdB.

Immunogenic composition comprising IsaA and IsdA.

Immunogenic composition comprising IsaA and SdrC.

Immunogenic composition comprising IsaA and Ebh or its fragment as described above.

Immunogenic composition comprising Sbi and SdrC.

Immunogenic composition comprising Sbi and Ebh or its fragment as described above.

Immunogenic composition according to the invention, containing IsaA, Sbi or SdrC.

The selection of antigens expressed in different clonal lines

Analysis of the occurrence of virulence factors in accordance with the population structure of Staphylococcus aureus showed different is ricotta virulence genes in natural populations of S. aureus.

It is shown that among clinical isolates of Staphylococcus aureus of at least five clonal lines are common (Booth et al., 2001 Infect Immun. 69(1):345-52). It is shown that alpha-hemolysin (l/a), fibronectin-binding protein A (fnbA) and factor agglutination A (clfA) are present in most isolates, regardless of the type of line that speaks about the important role of these proteins in the survival of S. aureus (Booth et al., 2001 Infect Immun. 69(1):345-52). Moreover, according to Peacock et al.(2002), distribution of fnbA, clfA, coagulase, spa, map, pvl (leukocidin Panton-Valentine, eng. Panton-Valentine leukocidin), hlg (gamma-toxin), alpha-toxin and ica, apparently not connected with the main clonal structure, which involves considerable horizontal transfer of these genes.

In contrast, other virulence genes such as fibronectin-binding protein B (fnbB), beta-hemolysin (hlb), collagen binding protein (cna), TSST-1 (tst) and the gene of resistance to methicillin (TEC), is strongly associated with specific lines (Booth et al., 2001 Infect Immun. 69(1):345-52). Similarly, Peacock et al. in 2002 (Infect Immun. 70(9):4987-96) showed that the distribution of enterotoxins, tst, exfolation (eta and etb), beta -, and Delta-toxins, sdr genes (sdrD, sdrE and bbp), cna, ebpS and efb in the population is significantly associated with MLST-derived clonal complexes.

Data MLST (Multilocus sequence typing, Multiloci typing sequences give no evidence, the strains responsible for nosocomial disease, represent a special subpopulation of strains causing community-acquired disease, or strains extracted from asymptomatic carriers (Feil et al., 2003 J Bacteriol. 185(11):3307-16).

Preferred immunogenic compositions according to the invention are effective against staphylococci from different clonal lines.

In one embodiment this is achieved by incorporating 1, 2, 3, 4, preferably at least 1 protein, which is expressed in most isolates of staphylococci. Examples of such proteins include alpha-hemolysin (hla), fibronectin-binding protein A (fnhA) and factor agglutination A (clfA), coagulase, spa, map, pvl (leukocidin Panton-Valentine, eng. Panton-Valentine leukocidin), hlg (gamma-toxin) and ica. The inventors have also identified immunodominant ABC Transporter, RAP, autolysin (Rupp et al., 2001, J. Infect. Dis. 183; 1038), laminin receptor, SitC, IsaA/PisA, SPOIIIE (), SsaA, EbpS, SasF (Roche et al., 2003, Microbiology 149; 643), EFB(FIB), SBI, ClfB, IsdA, IsdB, FnbB, Npase, EBP, sialo-binding protein II of the bone, IsaB/PisB (Lorenz et al. FEMS Measurement. Med. Microb. 2000, 29; 145), SasH (Roche et al., 2003, Microbiology 149; 643), MRPI, SACD playback (Roche et al., 2003, Microbiology 149; 643), SasH (Roche et al., 2003, Microbiology 149; 643), the predecessor of aureolin (AUR)/Sepp1 and new autolysin.

In alternative embodiment 2 or more proteins that are expressed in different groups clonal strains are immunogenic composition according to izopet the tion. Preferably, the combination of antigens will induce an immune response that is effective against many clonal strains, most preferably, against all clonal strains. Preferred combinations include FnbB and beta-hemolysin, FnbB and Cna, FnbB and TSST-1, FnbB and mecA, FnbB and SdrD, FnbB and SdrF, FnbB and EbpS, FnbB and Efb, beta-hemolysin and Cna, beta-hemolysin and TSST-1, beta-hemolysin and mecA, beta-hemolysin and SdrD, beta-hemolysin and SdrF, beta-hemolysin and EbpS, beta-hemolysin and Efb, Cna and TSST-1, Cna and mecA, Cna and SdrD, Cna and SdrF, Cna and EbpS, Cna and Efb, TSST-1 and mecA, TSST-1 and SdrD, TSST-1 and SdrF, TSST-1 and EbpS, TssT-1 and Efb, MecA and SdrD, MecA and SdrF, MecA and EbpS, MecA and Efb, SdrD and SdrF, SdrD and EbpS, SdeD and Efb, SdrF and EbpS, SdrF and Efb, and EbpS and Efb.

Preferred combinations described above can be combined with additional components described below.

The selection of antigens expressed during different phases of growth

Staphylococcus pass through exponential growth phase, during which will be expressed particular set of proteins. They include many proteins that bind extracellular components, and transport proteins. After a period of exponential growth of Staphylococcus return to postexperimental phase, during which growth slows, and protein expression changes. Many proteins expressed during exponential phase growth, suppressed, whereas other proteins which, such as enzymes and most of the toxins, including alpha-toxin is expressed at higher levels.

Preferred immunogenic compositions according to the invention contain protein, expressed at higher levels during the exponential phase of growth, and the protein is expressed at higher levels during postexposure phase.

The term “higher levels” refers to an expression level that is higher in one phase compared to other phases.

In the preferred embodiment of the immunogenic composition according to the invention contains alpha-toxin and protein binding extracellular component (preferably FnbA, FnbB, ClfA and ClfB), or transport protein.

More preferably, it contains alpha-toxin, or Cna, or a lipase GehD and protein selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, SasA, FnbA, FnbB, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, AAP and SsaA.

In the combinations described above, alpha-toxin can be genetically or chemically detoxified, as described above, and may not be anywhereman or anywhereman with polishuri the Ohm, as is described below.

Polysaccharides

Immunogenic compositions according to the invention preferably additionally contain capsular polysaccharides comprising one or more than one PIA (also known as PNAG), and/or capsular polysaccharide of type V or type VIII S. aureus, and/or capsular polysaccharide of type I and/or type II and/or type III S. epidermidis.

PIA (PNAG)

At the present time, it is obvious that various forms of staphylococcal surface polysaccharides, identifizierung as PS/A, PIA and SAA have the same chemical structural unit - PNAG (Maira-Litran et al. Vaccine 22; 872-879 (2004)). Therefore, the term PIA or PNAG cover all of these polysaccharides or oligosaccharides derived from them.

PIA is a polysaccharide intercellular adhesion and consists of a polymer of β-(1→6)-linked glucosamine, substituted N-acetyl and O-coccinellini components. This polysaccharide is present in S. aureus and S. epidermidis and can be isolated from both the source (Joyce et al., 2003, Carbohydrate Research 338; 903; Maira-Litran et al., 2002, Infect. Imun. 70; 4433). For example, PNAG can be isolated from a strain of S. aureus MN8m (WO 04/43407).

PIA isolated from S. epidermidis, is an integral component of biofilms. He is responsible for mediating cell-cell adhesion and may also function to protect a growing colony of the host immune response.

Recently, it was shown that the polysaccharide, RAS is e known as poly-N-succinyl-β-(1→6)-glucosamine (PNSG), no proposed structure, since the identification of N-succinylcholine was wrong (Maira-Litran et al., 2002, Infect. Imun. 70; 4433). Therefore, the polysaccharide, formally known as PNSG and, as now found, representing PNAG, also covered by the term PIA.

PIA (or PNAG) can have different sizes, ranging from more than 400 "Yes to 75-400 kDa up to 10-75 kDa for oligosaccharides, consisting of up to 30 repeating units (β-(1→6)-linked glucosamine, substituted N-acetyl and O-coccinellini components). Any size PIA polysaccharide or oligosaccharide may be used in immunogenic compositions according to the invention, however, it is preferable larger than 40 kDa. The sizes can be determined by any method known in this field, for example by microfluidizer, ultrasonic treatment or by chemical cleavage (WO 03/53462, EP 497524, EP 497525).

The preferred size ranges PIA (PNAG) is 40-400 kDa, 40-300 kDa, 50-350 kDa, 60-300 kDa, 50-250 60-200 kDa and kDa.

PIA (PNAG) may have a different degree of acetylation in the replacement of the amino acetate. PIA, in vitro, is almost completely substituted on the amine groups (95-100%). Alternatively, it may be used deacetylating PIA (PNAG), with less than 60%, preferably less than 50%, 40%, 30%, 20%, 10% acetylation. Application deacetylating PIA (PNAG) which is preferred, because deatsetilirovanie epitopes PNAG effective in mediating an opsonic lysis of gram-positive bacteria, preferably S. aureus and/or S. epidermidis. Most preferably, PIA (PNAG) has a size of from 40 kDa to 300 kDa and deacetylation so that less 60%, 50%, 40%, 30% or 20% of the amino groups azetilirovanny.

The term deacetylating PNAG (dPNAG) refers to PNAG the polysaccharide or oligosaccharide in which less 60%, 50%, 40%, 30%, 20% or 10% of the amino groups azetilirovanny.

In one embodiment of PNAG deacetylase education dPNAG by chemical treatment of the native polysaccharide. For example, native PNAG is treated with an alkaline solution so that the pH is above 10. For example, PNAG is treated with 0.1 to 5 M, 0.2-4 M, 0.3 To 3 M, 0.5-2 M, 0.75 To 1.5 M or 1 M NaOH, KOH or NH4OH. Processing is carried out for at least 10 or 30 min, or 1, 2, 3, 4, 5,10, 15 or 20 hours at a temperature of 20-100, 25-80, or 30-60 30-50 or 35-45°C. dPNAG may be obtained as described in WO 04/43405.

The polysaccharide(s)included in the immunogenic composition according to the invention, preferably conjugated to a protein carrier, as described below or, alternatively, not conjugated.

Polysaccharides type 5 and type 8 S. aureus

Most strains of S. aureus that cause infection in humans, contain polysaccharides either type 5 or type 8. Approximately 60% of human strains are type 8 and bring the flax 30% are type 5. The structure of the capsular polysaccharide antigen of type 5 and type 8 are described in Moreau et al. Carbohydrate Res. 201; 285 (1990) and Fournier et al. Infect. Immun. 45; 87 (1984). Both have FucNAcp in its repeating unit, and ManNAcA, which can be used to introduce sulfhydryl groups. The structure known as:

Type 5

→4)-β-D-ManNAcA(3OAc)-(1→4)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→

Type 8

→3)-(3-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→

Recently (Jones Carbohydrate Research 340, 1097-1106 (2005)NMR spectroscopy explained before:

Type 5

→4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-β-D-FucNAc-(1→

Type 8

→3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-α-D-FucNAc(1→

Polysaccharides can be isolated from a suitable strain of S. aureus using the method well known to the specialist, for example as described in US 6294177. For example, ATSC 12902 is a strain of S. aureus type 5, and ATSS 12605 is a strain of S. aureus type 8.

Polysaccharides have a native size or, alternatively, may be brought to the desired size, for example, by microfluidizer, ultrasonic treatment or by chemical treatment. The invention also covers oligosaccharides originating from a polysaccharide types 5 and 8 of S. aureus.

Polysaccharides type 5 and 8, included in the immunogenic composition according to the invention, preferably conjugated to a protein carrier, as described below, or, alternatively, are conjugial is low.

Immunogenic compositions of the invention contain alternative polysaccharide type 5 or type 8.

The 336 antigen of S. aureus

In one embodiment of the immunogenic composition according to the invention contains the 336 antigen from S. aureus, as described in US6294177.

The 336 antigen contains β-linked hexosamine, contains no O-acetyl groups and specifically binds with antibodies to S. aureus type 336 deposited under the number of ATSS 55804.

In one embodiment of the 336 antigen is a polysaccharide, which has a native size or, alternatively, may be brought to the desired size, for example, by microfluidizer, ultrasonic treatment or by chemical treatment. The invention also covers oligosaccharides originating from the 336 antigen.

The 336 antigen included in the immunogenic composition according to the invention, preferably anywhereman with protein carrier, as described below, or, alternatively, is unconjugated.

Polysaccharides of type I, II and III of S. epidermidis

The strains of ATSS-31432, SE-360 SE-10 S. epidermidis are characterized by three different capsular types I, II and III respectively (Ichiman and Yoshida 1981, J. Appl. Bacteriol. 51; 229). Capsular polysaccharides isolated from each serotype S. epidermidis, are polysaccharides of type I, II and III. Polysaccharides can be allocated in several ways, including the method described in US4197290, which is described in Ichiman et al., 1991, J. Appl. Bacteriol. 71; 176.

In one embodiment of the invention, the immunogenic composition contains polysaccharides or oligosaccharides of type I and/or II and/or III of S. epidermidis.

Polysaccharides have a native size or, alternatively, may be brought to the desired size, for example, by microfluidizer, ultrasonic treatment or chemical cleavage. The invention also covers oligosaccharides isolated from strains of S. epidermidis.

These polysaccharides are unconjugated or preferably conjugated, as described below.

Conjugation of polysaccharides

One of the problems associated with the use of polysaccharides by vaccination, is the fact that polysaccharides are themselves weak immunogenum. Strategies that have been developed to overcome this lack of immunogenicity include the linking of the polysaccharide with large protein-carriers, which provide assistance to non-specific T-cells. Preferably, the polysaccharides used in the invention, have been associated with protein carrier, which provides assistance to non-specific T-cells. Examples of such carriers which can be conjugated to polysaccharide immunogenum include diphtheria and tetanus toxoids (DT, DT, crm197 and TT, respectively), hemocyanin lymph snails (KLH) and cleaned up the e protein derivative of tuberculin (PPD), absoprtion A Pseudomonas aeruginosa (rEPA), protein D from Haemophilus influenzae, pneumolysin or fragments of any of the above. Fragments suitable for use include fragments covering the T-helper epitopes. In particular, a fragment of the protein D will preferably contain N-terminal 1/3 of this protein. Protein D is a lgD-binding protein from Haemophilus influenzae (EP 0594610 B1) and is a potential immunogen.

In addition, staphylococcal proteins can be used as a protein carrier in the polysaccharide conjugates according to the invention. Staphylococcal proteins, described below, can be used as a protein carrier; for example, the laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, ClfA, SdrC, SdrG, SdrH, lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC, Ni ABC Transporter, alpha-toxin (Hla), the mutant alpha toxin H35R, RNA III activating protein (RAP), or fragments thereof.

New a carrier protein, which would be especially preferred for use in the case of staphylococcal vaccines, is a staphylococcal alpha toxoid. The native form can be conjugated to the polysaccharide, as the process con is ugorowanie reduces toxicity. Preferably, genetically detoxified enduring alpha toxin, such as options His35Leu or His Arg 35, are used as carriers, as residual toxicity is less. Alternatively, the alpha-toxin chemically detoxify by processing the cross-linking reagent, formaldehyde or glutaraldehyde. Genetically detoxified enduring alpha-toxin may chemically detoxify, preferably by treatment with a crosslinking reagent, formaldehyde or glutaraldehyde, to further reduce toxicity.

The polysaccharides may be associated with the protein(s)carrier(s) by any known method (for example, Likhite, U.S. patent No. 4372945, Armor et al., U.S. patent No. 4474757, and Jennings et al., U.S. patent No. 4356170). Preferably, carry out the chemistry of conjugation with CDAP (see WO 95/08348).

In CDAP, cunilingusi reagent 1-cyano-dimethylaminopyridine tetrafluoroborate (CDAP) is preferably used for the synthesis of polysaccharide-protein conjugates. The reaction cinelibre can be carried out under relatively mild conditions, which avoid hydrolysis of the alkali sensitive polysaccharides. This synthesis makes possible direct binding to the protein carrier.

Polysaccharide solubilizer in water or physiological solution. CDAP is dissolved in acetonitrile and added directly to the polysaccharide solution. CDAP with hydroxyl groups of the polysaccharide with the formation of ester cyanic acid. After stage activation add a carrier protein. Amino group of lysine reacts with the activated polysaccharide with the formation of ismokemenhol covalent bond. After the reaction, the combination of a large excess of glycine then add to extinguish the remaining activated functional groups. The product is then passed through the column for gel permeation chromatography to remove unreacted protein carrier and the remaining reagents.

Conjugation preferably includes obtaining a direct link between protein carrier and a polysaccharide. Between a protein carrier and polysaccharide perhaps you can enter a spacer (such as adipic digital (ADH)).

Protection against S. aureus and S. epidermidis

In the preferred embodiment of the invention the immunogenic composition provides an effective immune response against more than one strain of Staphylococcus, preferably against strains of both S. aureus and S. epidermidis. More preferably, a protective immune response is induced against S. aureus serotypes 5 and 8. More preferably, a protective immune response is induced against numerous strains of S. epidermidis, for example, strains of at least two serotypes I, II and III of S. epidermidis.

One of the applications of immunogenic compositions according to the invention consists in the prevention of nosocomial infections through vaccination PE the ed treatment in the hospital. At this stage it is difficult to accurately predict what strains of staphylococci will be exposed to the patient. Therefore, it is preferable to inoculate the vaccine is able to induce an effective immune response against various strains of Staphylococcus.

An effective immune response is defined as an immune response, which gives significant protection in the mouse model of infection or in the analysis of opsonophagocytosis, as described in the examples. Significant protection in the mouse model of infection, such as the model of example 5, is defined as the increase in LD50 compared to mice vaccinated with the media, at least 10%, 20%, 50%, 100% or 200%. Significant protection in models of infection of cotton hamster, for example the model of example 8, is defined as the reduction in the average measured LogKOE/nose at least 10%, 20%, 50%, 70% or 90%. The presence opsoniziruyuschih antibodies known to correlate with protection, so significant protection indicates a decrease in the number of bacteria by at least 10%, 20%, 50%, 70% or 90% in the analysis of opsonophagocytosis, for example, in the analysis of example 7.

Some proteins, including the immunodominant ABC Transporter, I'll RNA-activating protein, laminin receptor, SitC, IsaA/PisA, SsaA, EbhA/EbhB, EbpS and AAR are very conservative between S. aureus and S. epidermidis, and example 8 shows that IsaA, ClfA, IsdB, SdrG, HarA, FnbpA and Sbi can cause Perek the local immune response (for example, cross-reaction between at least one strain of S. aureus and at least one strain of S. epidermidis). PIA is also a very conservative between S. aureus and S. epidermidis and is able to induce cross-protective immune response.

Therefore, in the preferred embodiment of the immunogenic composition according to the invention will contain two, three or four of the above-mentioned protein, preferably additionally contains PIA (PNAG).

Polynucleotide vaccines

In another aspect, the present invention relates to the use of polynucleotides from figure 2 in the treatment, prevention or diagnosis of staphylococcal infection. Such polynucleotide include a dedicated polynucleotide containing the nucleotide sequence encoding a polypeptide that has at least 70%identity, preferably at least 80%identity, more preferably at least 90%identity, even more preferably at least 95%identity with the amino acid sequence of figure 1 relative to the full sequence. In this regard, polypeptides which have at least 97%identity are highly preferred, whereas polypeptides with at least 98-99%identity are more preferred, and polypeptides with at IU the f 99%identity are most preferred.

Other polynucleotide, which are used in the present invention include a dedicated polynucleotide containing a nucleotide sequence that has at least 70%identity, preferably at least 80%identity, more preferably at least 90%identity, even more preferably at least 95%identity with a nucleotide sequence that encodes a protein according to the invention, relative to the full coding of the site. In this respect, polynucleotide, which have at least 97%identity are highly preferred, while polynucleotide with at least 98-99%identity are more preferred, and polynucleotide with at least 99%identity are most preferred.

Other polynucleotide include a dedicated polynucleotide containing a nucleotide sequence that has at least 70%identity, preferably at least 80%identity, more preferably at least 90%identity, even more preferably at least 95%identity with the sequences of figure 1. In this respect, polynucleotide, which have at least 97%identity are highly preferred and, while polynucleotide with at least 98-99%identity are more preferred, and polynucleotide with at least 99%identity are most preferred. The specified polynucleotide can be embedded into a suitable plasmid or recombinant mikroorganismen vector and used for immunization (see, for example, Wolff et. al., Science 247:1465-1468 (1990); Corr et. al., J. Exp. Med. 184:1555-1560 (1996); Doe et. al., Proc. Natl. Acad. Sci. 93:8578-8583 (1996)).

The present invention also proposed a nucleic acid encoding the above proteins of the present invention, and their use in medicine. In preferred embodiments the selected polynucleotide according to the invention can be single-stranded (coding or antimuslim) or double-stranded and may represent a molecule DNA (genomic, cDNA or synthetic) or RNA. Additional coding or non-coding sequences can be represented, but not necessarily, within polynucleotide of the present invention. In other related embodiments of the present invention proposed polynucleotide variants having substantial identity to the sequences disclosed in this specification, figure 2; polynucleotide variants containing at least 70%sequence identity, preferably n is at least 75%-, 80%-, 85%-, 90%-, 95%-, 96%-, 97%-, 98%- or 99%or higher sequence identity compared to a polynucleotide sequence of this invention using the methods described in this description (e.g., BLAST analysis using standard parameters). In a related embodiment, the selected polynucleotide according to the invention will contain a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95%or higher identity with the amino acid sequence in figure 1 relative to the full-size sequence in figure 1, or a nucleotide sequence complementary to the specified dedicated polynucleotide.

The invention also suggested the use of polynucleotides, which are complementary to all of the above polynucleotides.

The invention also suggested the use of a fragment of polynucleotide according to the invention that when administered to a subject has the same immunogenic properties as polynucleotide in figure 1.

The invention also suggested the use of polynucleotide encoding immunological protein fragment in figure 1, as defined in the description above. It also assumes the use of such fragments, which have a level of immunogenic activity of at least about 50%, being the equipment at least about 70% and more preferably at least about 90% of the level of immunogenic activity of the polypeptide sequence, encoded by a polynucleotide sequence shown in figure 2.

Polypeptide fragments for use according to the invention preferably contain at least about 5, 10, 15, 20, 25, 50 or 100 contiguous amino acids or more, including all intermediate lengths, of a polypeptide compositions set forth herein, such as those listed above.

Polynucleotide for use in the invention can be obtained using methods standard cloning and screening of libraries of DNA derived from mRNA in cells of human preneoplastic or tumor tissue (e.g. lung) (for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotide according to the invention can also be obtained from natural sources such as genomic DNA library, or can be synthesized using well known and commercially available methods.

There are several methods available and well known to specialists in this field to obtain a full DNA or for lengthening short cDNA, for example methods based on rapid amplification of cDNA ends (RACE) (see, for example, Frohman et al., PNAS USA 85, 8998-9002,1988). Modern modifications of this method is given as an example in technology Marathon™ (Clontech Laboratories Inc.), for example, they who have significantly simplified the search for longer cDNA. In technology Marathon™, cDNA was obtained from mRNA extracted from the selected tissue, and adaptarea” sequence, legirovannoi on each end. Amplification of nucleic acids (PCR) is then carried out to amplify the “missing” 5'-end of the cDNA using a combination of gene-specific and adaptor-specific oligonucleotide primers. The PCR reaction is then repeated, using "nested" primers, i.e., the primers designed for hybridization within the amplified product (typically an adaptor-specific primer, which hybridizes additionally with 3' adaptorname sequence, and a gene-specific primer, which hybridizes additional 5' in known gene sequences). The products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA, constructed either by joining the product directly to the existing cDNA with obtaining a complete sequence, or by implementing a separate full-sized PCR using information about new sequence to construct a 5'-primer.

Vectors containing such DNA, hosts transformed with them, in and of themselves shortened or hybrid proteins expressed, as described in this invention below, are all h is STU invention.

Expressing the system can also be a recombinant a living microorganism such as a virus or bacterium. Of interest, the gene can be integrated into the genome of a live recombinant virus or bacteria. Immunization and infection in vivo this live vector will lead to the expression of the antigen in vivo and induction of immune responses.

Therefore, in some embodiments polynucleotide encoding immunogenic polypeptides for use according to the present invention, is introduced into a suitable host mammalian cells for expression using any of many known viral systems. In one illustrative embodiment of retroviruses provide a convenient and effective basis for systems of gene delivery. A selected nucleotide sequence encoding a polypeptide for use in the present invention may be incorporated into a vector and packaged in retroviral particles using techniques known in the field. The recombinant virus can then be selected and delivered to the subject. Described a number of illustrative retroviral systems (e.g., U.S. patent No. 5219740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop.3:102-109.

In addition, was also described a number of Illustra the active adenoviral systems. In contrast to retroviruses, which integrate into the host genome, adenoviruses persist extrachromosomal, thus minimizing the risks associated with the insertion mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476).

To deliver polynucleotides were also developed various adeno-associated viral (AAV) vector system. AAV-vectors can be easily constructed using methods well known in the field. See, for example, U.S. patent No. 5173414 and 5139941; international publication nos WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B.J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Environ, and Immunol. 158:97-129; Kotin, R. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165 to 169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

Additional viral vectors useful for delivery of nucleic acid molecules encoding polypeptides for use in the present invention, by gene transfer include molecules originating from the family of poxviruses, such as vaccinia virus and avian poxvirus. As an example, recombinants on the basis of vaccinia virus expressing interest molecules, can be MSE is strayaway as follows. DNA encoding the polypeptide is first inserted into an appropriate vector so that it is bordered by the promoter of vaccinia virus and flanking DNA sequences from vaccinia virus, such as a sequence encoding timedancing (TC). This vector is then used for transfection of cells, which simultaneously infect the vaccinia virus. Homologous recombination is used to insert a promoter of vaccinia virus plus gene encoding the desired polypeptide, in the viral genome. Received TK.sup.(-) recombinant can be selected by culturing cells in the presence of 5-bromosuccinimide and selection of viral plaques, steady with him.

System infection/transfection-based vaccinia virus can be conveniently used to obtain inducible, and temporal expression or co-expression of one or more of the polypeptides described in this invention, the master cells of the body. In this particular system cells initially infect in vitro by recombinant-based vaccinia virus that encodes the RNA polymerase of bacteriophage T7. This polymerase shows strong specificity in that it Transcriber only matrix carrying the T7 promoters. After infection cells transferout interesting(and) polynucleotides or polynucleotide administered by promote the om T7. Polymerase expressed in the cytoplasm of recombinant-based vaccinia virus, Transcriber transtitional DNA into RNA which is then translated into the polypeptide translational apparatus of the owner. The method provides a high-level temporary cytoplasmic production of large quantities of RNA and products of its transmission. See, for example, Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

Alternatively, avipoxvirus, such as viruses smallpox-diphtheria birds and smallpox Canaries, can also be used to deliver interested in coding sequences. Recombinant avipoxvirus expressing immunogen of pathogens of mammals is known to provide protective immunity in the introduction of species not related to birds. The use of vector-based avipoxviruses particularly preferably in humans and other mammalian species, as members of the genus Avipox can only effectively be replicated in sensitive bird species and are therefore non-infectious in mammalian cells. Methods preparation of recombinant avipoxviruses known in this area and the use of genetic recombination, as described above in relation to the production of viruses ospowiki. See, for example, WO 91/12882, WO 89/03429 and WO 92/03545.

Any of many alphavirus vectors t is the train can be used to deliver the polynucleotide compositions for use in the present invention, like vectors, are described in U.S. patent No. 5843723; 6015686; 6008035 and 6015694. Some vectors based on virus Venezuelan encephalomyelitis of horses (VEE), can also be used, illustrative examples can be found in U.S. patent No. 5505947 and 5643576.

Moreover, the molecular conjugate vectors, such as adenoviral chimeric vectors, described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery according to the invention.

Additional illustrative information about these and other known delivery systems based viruses can be found, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. patent No. 4603112, 4769330 and 5017487; WO 89/01973; U.S. patent No. 4777127; GB 2200651; EP 0345242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91-215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; Guzman et al., Cir. Res. 73:1202-1207, 1993.

Recombinant live microorganisms described above, can be virulent or attenuated in various ways to produce live vaccines. Such live vaccines are also part of the invention.

In some embodiments polynucleotide can be integrated into the genome of target cells. This integration can occur the specific position and orientation by means of homologous recombination (gene replacement), or it can be embedded in a random, non-specific position (gene amplification). In other embodiments polynucleotide can stably maintained in the cell as a separate episunago segment of DNA. Such polynucleotide segments or “episome” encode sequence, sufficient to provide for the maintenance and replication, independent or synchronized with the cell cycle of the host. The way in which expressing the design is delivered into the cell, and in which polynucleotide remains in the cell depends on the type of expressing design.

In another embodiment of the invention polynucleotide introduced/delivered as “naked” DNA, for example, as described in Ulmer et al., Science 259:1745-1749, 1993, and reviewed by Cohen, Science 259:1691-1692,1993. The capture of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

In yet another embodiment the composition according to the present invention can be delivered using the approach in the bombardment of particles, many of which have already been described. In one illustrative example, the acceleration of particles in the gas stream can be achieved using devices such as devices manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, Wl), some examples of which are described in U.S. patent is No. 5846796, 6010478, 5865796, 5584807 and the patent EP 0500799. This approach offers a needleless delivery method, where a dry powder preparation of microscopic particles, such as a polynucleotide or polypeptide of the particle is accelerated to a high velocity gas stream of helium produced by the portable device, push the particles of interest to the target tissue.

In a related embodiment, other devices and methods that may be useful for needleless injection of the compositions of the present invention in the gas stream include those proposed by the company Bioject, Inc. (Portland, OR), some examples of which are described in U.S. patents№№4790824, 5064413, 5312335, 5383851, 5399163, 5520639 and 5993412.

Vaccines

In the preferred embodiment of the immunogenic composition according to the invention is mixed with pharmaceutically acceptable excipients, more preferably with an adjuvant, with the formation of the vaccine.

Vaccines of the present invention preferably contain adjuvant. Suitable adjuvants include an aluminium salt, such as a gel of aluminum hydroxide (alum) or aluminum phosphate, but may also be a salt of calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationic or anionic derivatives of polysaccharides or polyphosphazene.

Pre is respectfully, to be selected adjuvant, which is the preferred inductor or TN-or TN-type response. High levels of cytokines Th1-type are usually contribute to the induction of cell-mediated immune responses to the antigen, whereas high levels of cytokines of the Th2-type usually contribute to the induction of humoral immune responses to this antigen.

It is important to remember that the difference in the immune response of Th1 - and Th2-type is not absolute. In fact, the individual will support an immune response that has been described as being primarily or predominantly Th1 Th2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 +ve T cell clones Musmanno and Coffman (Mosmann, T.R. and Coffman, R.L. (1989) TH1 and T-cells: different patterns of secretion of lymphokines lead to different functional properties. Annual Review of Immunology, 7, p145-173). Traditionally, answers TM-type is associated with the production of cytokines INF-γ and IL-2 by T lymphocytes. Other cytokines, often directly associated with the induction of immune responses of the Th1-type, is not produced by T-cells, such as IL-12. In contrast, the responses of the Th2-type is associated with the secretion of IL-4, IL-5, IL-6, IL-10. Suitable adjuvant systems that promote a predominantly Th1 response include: monophosphorylated And or its derivative, particularly 3-de-O-acylated monof strolled A (3D-MPL) (relative to its preparation, see GB 2220211 A); and a combination of monophosphorylated A, preferably 3-de-O-acylated monophosphorylated And, together, or with a salt of aluminum (such as aluminum phosphate or aluminum hydroxide)or an emulsion of oil in water. In combinations of antigen and 3D-MPL are contained in the same dispersive media, providing more efficient delivery of antigen and immunostimulatory signals. Studies have shown that 3D-MPL is able to additionally increase the immunogenicity of the antigen adsorbed on alum [Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1].

The improved system includes a combination of monophosphorylated and derived saponin, particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 weaken cholesterol as disclosed in WO 96/33739. Particularly strong adjuvant drug, involving QS21, 3D-MPL and tocopherol in an oil emulsion in water, is described in WO 95/17210, and is the preferred drug. Preferably, the vaccine further comprises a saponin, more preferably QS21. The preparation may also contain emulsion water-in-oil and tocopherol (WO 95/17210). The present invention also proposed a method of obtaining a vaccine preparation comprising mixing the protein of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL. Demetriou the major CpG-containing oligonucleotides (WO 96/02555) are also preferred inducers IN response and suitable for use in the present invention.

Preferred compositions according to the invention are compositions that form the liposomal structure. Compositions in which the Sterol/immunological active sapojnikova fraction forms the ISCOM structure, are also aspect of the invention.

The ratio of QS21:Sterol will typically be in the range of from 1:100 to 1:1 wt./wt.

Preferably, when there is excess Sterol, the ratio of QS21:Sterol is at least 1:2 wt./wt. Typically, with regard to the introduction of man, QS21 and Sterol will be present in a vaccine in the range from about 1 μg to about 100 μg, preferably from about 10 μg to about 50 μg per dose.

The liposomes preferably contain a neutral lipid, for example phosphatidylcholine, which at room temperature is preferably non-crystalline, for example phosphatidylcholine from egg yolk, dioleoylphosphatidylcholine or dilaurylracglycerol. Liposomes may also contain a charged lipid, which increases the stability of the structure of liposome-QS21 for liposomes composed of saturated lipids. In these cases, the number of charged lipid is preferably 1-20% wt./wt., most preferably 5-10%. The ratio of Sterol to phospholipid is 1-50% (mol./mol.), most preferably 20-25%.

Preferably, the composition according to the invention will emerge MPL (3-describeany monophosphorylated And, also known as 3D-MPL). 3D-MPL is known from GB 2220211 (Ribi) as a mixture of 3 de-O-acylated monophosphorylated And with 4, 5 or 6 acylated chains and produced by firm Ribi Immunochem, Montana. The preferred form is disclosed in international patent application 92/116556.

Suitable compositions according to the invention are compositions in which the liposomes are first obtained without MPL and MPL is then added, preferably in the form of particles 100 nm. Therefore, the MPL is not contained in the vesicular membrane (known as MPL “outside”). Compositions in which the MPL is contained in the vesicular membrane (known as MPL “inside”)are also an aspect of the invention. The antigen can be contained within the vesicle membrane or contained with the outer side of the vesicular membrane. Preferably, soluble antigens are on the outside and hydrophobic or limitirovanie antigens are either inside or outside the membrane.

The vaccine preparations of the present invention can be used to protect or treat a mammal susceptible to infection, through the introduction of specified vaccines via the system path or through mucous membranes. This introduction may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; or introduction through the mucous membranes in the oral/digestive track is, respiratory, urinary ways. Intranasal administration of vaccines for the treatment of pneumonia or inflammation of the middle ear is preferred (because it is possible to more effectively prevent nasopharyngeal carriage of pneumococci, weakening thus the infection is at its very early stage). Although the vaccine according to the invention can be introduced in the form of a single dose, its components can also be administered simultaneously or at different times (e.g., pneumococcal polysaccharides can be entered separately, simultaneously or within 1-2 weeks after the introduction of any bacterial protein component of the vaccine for optimal coordination of immune responses relative to each other). As for the joint introduction, the optimal TM-adjuvant may be present in any or all of the various introductions, however, preferably, if it is present in combination with bacterial protein component of the vaccine. In addition to one way of introduction you can use 2 different way of introduction. For example, polysaccharides can be entered in a/m (or intradermal), and bacterial proteins can be introduced intranasally (or intradermal). In addition, the vaccine according to the invention can be put into/m for the primary dose and route for repeated doses.

The number of conjugated antigen in each vaccine dose is selected as the number is creation, which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending on which specific immunogen will use and in what quantity. Usually expected that each dose will contain 0.1 to 100 µg of polysaccharide, preferably 0.1 to 50 μg polysaccharide conjugates, preferably 0.1 to 10 μg, more preferably 1-10 μg, of which 1-5 µg exceed the preferred range.

The content of protein antigens in the vaccine will typically be in the range of 1-100 μg, preferably 5-50 μg, most typically in the range 5-25 μg. After the initial vaccination, subjects may receive one or several booster immunizations, appropriately spaced in time.

Vaccine formulation in General is described in Vaccine Design (“the Subunit and adjuvant approach (eds M.F. Powell & Newman M.J.) (1995) Plenum Press New York). Encapsulation in liposomes described Fullerton, U.S. patent No. 4235877.

Vaccines of the present invention can be stored in solution or liofilizirovanny. Preferably, the solution lyophilizer in the presence of a sugar such as sucrose, trehalose or lactose. Even more preferably, that their lyophilizer and dissolved immediately before use. Lyophilization can give a more stable component is iciu (vaccine) and may, may result in higher antibody titers in the presence of 3D-MPL and in the absence of adjuvant aluminium-based.

Antibodies and passive immunization

Another aspect of the invention is a method of producing immunoglobulin for use in the prevention or treatment of staphylococcal infections, including stage immunization of the recipient of the vaccine according to the invention and selection of immunoglobulin from the recipient. Immunoglobulin obtained in this way is another aspect of the invention. A pharmaceutical composition comprising the immunoglobulin according to the invention and a pharmaceutically acceptable carrier, is another aspect of the invention, which can be used in the manufacture of a medicinal product for the treatment or prevention of staphylococcal disease. A method of treating or preventing staphylococcal infections, including the stage of the introduction to the patient an effective amount of a pharmaceutical preparation according to the invention is another aspect of the invention.

The inoculum to obtain polyclonal antibodies are typically prepared by dispersing the antigenic composition in a physiologically acceptable solvent, such as saline or other adjuvants suitable for use in humans, with formation water compositions. Significant quantities of the inoculum is administered to a mammal, and vaccinated mammal is then maintained for a time sufficient to antigenic composition induced protective antibodies.

Antibodies can be selected in the desired degree of well known methods, such as affinity chromatography (Harlow and Lane, Antibodies; A laboratory manual, 1988).

Antibodies may include preparations of antisera from many traditionally used animals, such as goats, primates, donkeys, pigs, horses, Guinea pigs, rats or humans. Animals take blood and get the serum.

Immunoglobulin obtained according to the present invention, may include whole antibodies, antibody fragments or subfragments. The antibody may be a whole immunoglobulins of any class such as IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid antibodies with dual specificity to two or more antigens according to the invention. They can also represent fragments such as F(ab')2, Fab', Fab, Fv and the like, including hybrid fragments. Immunoglobulin also includes natural, synthetic or genetically engineered proteins that act like an antibody by binding with specific antigens with the formation of the complex.

The vaccine of the present invention can be administered to the recipient, which then acts in Kacha is TBE source of immunoglobulin, produced in response to the stimulus of specific vaccines. The subject, which is treated in this way, will serve as the donor plasma, from which is obtained hyperimmunoglobulin using standard methodology fractionation of plasma. Hyperimmunoglobulin will introduce another subject to make it more stable against staphylococcal infection or its treatment. Hyperimmunoglobulin according to the invention are particularly useful for treating or preventing staphylococcal disease in infants, individuals with immune disorders or where treatment is required, and the individual has no time to produce antibodies in response to vaccination.

An additional aspect of the invention relates to pharmaceutical compositions containing two or more monoclonal antibodies or their fragments; preferably human or humanized)reacting at least two components of the immunogenic compositions according to the invention, which can be used for treating or preventing infections caused by gram-positive bacteria, preferably Staphylococcus, more preferably S. aureus or S. epidermidis.

Such pharmaceutical compositions include monoclonal antibodies, which can be a whole immunoglobulins of any class such as IgG, IgM, IgA, IgD or IgE, himem the e antibodies or hybrid antibodies with specificity to two or more antigens according to the invention. They can also represent fragments such as F(ab')2, Fab', Fab, Fv and the like, including hybrid fragments.

Methods for obtaining monoclonal antibodies are well known in this field and may include the fusion of splenocytes with myeloma cells (Kohler and Milstein 1975 Nature 256; 495; Antibodies - a laboratory manual, Harlow and Lane 1988). Alternatively, monoclonal Fv-fragments can be obtained by screening appropriate ragovoy display library (Vaughan TJ, et al., 1998, Nature Biotechnology 16; 535). Monoclonal antibodies can be humanitarian or partially humanitarian by known methods.

Ways

The invention also encompasses a method of making immunogenic compositions and vaccines according to the invention.

A preferred method according to the invention is a method of making a vaccine comprising a stage of mixing the antigen with obtaining immunogenic composition according to the invention and adding a pharmaceutically acceptable excipient.

Methods of treatment

The invention also encompasses a method of treating staphylococcal infections, especially nosocomial infections acquired in the hospital.

This immunogenic composition or vaccine according to the invention is particularly preferred for use in cases of elective surgery. Such patients will know the date of the transaction in advance and may be vaccinated in advance. the as is not known, will the patient be exposed to infection with S. aureus or S. epidermidis, it is preferable to inoculate the vaccine according to the invention, which protects against both, as described above. Preferably, adult after 16 years of waiting for elective surgery, treat immunogenic compositions and vaccines according to the invention.

Also, it is preferable to vaccinate healthcare workers vaccine according to the invention.

The vaccine preparations of the present invention can be used to protect or treat a mammal susceptible to infection, through the introduction of specified vaccines via the system path or through mucous membranes. This introduction may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; or introduction through the mucous membranes in the oral/alimentary tract, respiratory, urinary path.

The amount of antigen in each vaccine dose is chosen as the amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending on which specific immunogen will use and in what quantity. The protein in the vaccine will typically be in the range of 1-100 μg, preferably 5-50 μg, most typically in the range of 10-25 µg. Typically expect each the dose will contain 0.1 to 100 μg of the polysaccharide, if present, preferably 0.1 to 50 μg, preferably 0.1 to 10 μg, of which 1-5 µg are the most preferred range. The optimum amount for a particular vaccine is defined by standard studies involving observation of appropriate immune responses in subjects. After the initial vaccination, subjects may receive one or several booster immunizations, appropriately spaced in time.

Although the vaccine of the present invention can type in any way, the introduction describes the vaccine into the skin (intradermal) forms one of the embodiments of the present invention. Human skin contains the outer “dead” cuticle called the Horny layer, which covers the epidermis. Under the epidermis is a layer called the dermis, which, in turn, covers the subcutaneous tissue. Researchers have shown that injection of the vaccine into the skin and, in particular, in the dermis stimulates the immune response, which can also be associated with a number of additional advantages. Intradermal vaccination vaccines described in this description is a preferred characteristic of the present invention.

The traditional method of intradermal injection, “Mantoux test”, includes the stage of purification of the skin and then her stretching one hand, and bevel narrow needle(gauge 26-31) facing up insert the needle at an angle from 10°to 15°. After inserting the end section of the needle cylinder, the needles are lowered and additionally promoting, providing a slight pressure to promote it under the skin. The liquid is then injected very slowly, forming a bubble or bump on the skin surface, followed by slow removal of the needle.

Recently been described devices, which are specially designed for the introduction of liquid agents into or through the skin, for example, the device described in WO 99/34850 and EP 1092444 and device for ink-jet injection, for example described in WO 01/13977; US 5480381, US 5599302, US 5334144, US 5993412, US 5649912, US 5569189, US 5704911, US 5383851, US 5893397, US 5466220, US 5339163, US 5312335, US 5503627, US 5064413, US 5520639, US 4596556, US 4790824, US 4941880, US 4940460, WO 97/37705 and WO 97/13537. Alternative ways intradermal vaccine preparations may include traditional syringes and needles or devices designed for ballistic delivery of particulate vaccines (WO 99/27961), or transdermal patches (WO 97/48440; WO 98/28037); or attached to the surface of the skin (transdermal or transcutaneous delivery WO 98/20734; WO 98/28037).

When the vaccine of the present invention are intended for administration to the skin or, more specifically, in the dermis, then the vaccine is in a small volume of liquid, in particular the amount is from about 0.05 ml to 0.2 ml.

The content of the antigens in the skin or intradermal vaccines is about the present invention can be similar to traditional dose as found in intramuscular vaccines (see above). However, the feature of the skin or intradermal vaccine is that drugs can be “low doses”. Accordingly, the protein antigens in the “low dose” vaccines are preferably present in an amount of from 0.1 to 10 μg, preferably 0.1 to 5 μg per dose; and polysaccharide (preferably conjugated) antigens may be present in the range of 0.01-1 μg, and preferably from 0.01 to 0.5 μg of polysaccharide per dose.

As used herein, the term “intradermal delivery” means delivery of the vaccine in the area of the dermis in the skin. However, the vaccine does not need to be placed exclusively in the dermis. The dermis is a layer of skin located from about 1.0 to about 2.0 mm from the surface of human skin, but there is some variation between individuals and in different parts of the body. In most cases you can expect to achieve the dermis with the passage of 1.5 mm below the skin surface. The dermis is located between the stratum corneum and epidermis on the surface and the subcutaneous layer below. Depending on the shipping method, the vaccine may be ultimately placed solely or mainly in the dermis, or it can be distributed eventually in the epidermis and the dermis.

The preferred embodiment of the invention, the stand is made by a method of preventing or treating staphylococcal infection or disease, including the stage of introduction of the immunogenic composition or vaccine according to the invention to a patient in need of it.

In the preferred embodiment, the patient waits for elective surgery.

Another preferred embodiment of the invention is a use of the immunogenic compositions according to the invention in the manufacture of a vaccine for treatment or prevention of staphylococcal infection or disease, preferably postoperative staphylococcal infections.

The term “staphylococcal infection” encompasses the infection caused by S. aureus and/or S. epidermidis and other strains of staphylococci, are able to cause infection in a mammal, preferably a human host.

The terms “include”, “contain” and “contains” in this description is intended by the inventors for possible replacement in each case, respectively, the terms “comprising”, "comprise" and “comprises”.

All references or patent applications cited in the description of this application, are included in this description by reference.

For a better understanding of the present invention are the following examples. These examples are presented only for illustration purposes and are not intended to limit the scope of the invention in any way.

Examples

Example 1. Construction of plasmids for expression of recombinant proteins

Who: cloning.

Suitable restriction sites designed oligonucleotides specific for staphylococcal gene, allowing directional cloning of PCR product in expressing plasmid E. coli pET24d or pQE-30 so that the protein can be expressed in the form of a fused protein containing (His)6 tag for affinity chromatography on N - or C-end.

The primers used were:

Alpha-toxin - 5'-CGCGGATCCGCAGATTCTGATATTAATATTAAAAC-3, and 5'CCCAAGCTTTTAATTTGTCATTTCTTCTTTTTC-3'

EbpS - 5'-CGCGGATCCGCTGGGTCTAATAATTTTAAAGATG-3' and 5'CCCAAGCTTTTATGGAATAACGATTTGTTG-3'

ClfA - 5'-CGCGGATCCAGTGAAAATAGTGTTACGCAATC-3' and 5'CCCAAGCTTTTACTCTGGAATTGGTTCAATTTC-3'

FnbpA - 5'-CGCGGATCCACACAAACAACTGCAACTAACG-3' and 5'CCCAAGCTTTTATGCTTTGTGATTCTTTTTCAAAC3'

Sbi - 5'-CGCGGATCCAACACGCAACAAACTTC-3' and 5'GGAACTGCAGTTATTTCCAGMTGATMTAMTTAC-3'

SdrC - 5'-CGCGGATCCGCAGAACATACGAATGGAG-3' and 5'CCCAAGCTTTTATGTTTCTTCTTCGTAGTAGC-3'

SdrG - 5'-CGCGGATCCGAGGAGAATTCAGTACAAG-3' and 5'CCCAAGCTTTTATTCGTCATCATAGTATCCG-3'

Ebh - 5'-AAAAGTACTCACCACCACCACCACC-3' and 5'AAAAGTACTCACTTGATTCATCGCTTCAG-3,

Aaa - 5'-GCGCGCCATGGCACAAGCTTCTACACAACATAC-3' and 5'GCGCGCTCGAGATGGATGAATGCATAGCTAGA-3'

IsaA - 5'-GCATCCATGGCACCATCACCATCACCACGAAGTAAACGTTGATCAAGC-3' and 5'-AGCACTCGAGTTAGAATCCCCAAGCACCTAAACC-3'

HarA - 5'-GCACCCATGGCAGAAAATACAAATACTTC-3' and 5TTTTCTCGAGCATTTTAGATTGACTAAGTTG-3'

Authorizing.dominate - 5'-CAAGTCCCATGGCTGAGACGACACAAGATCAAC-3' and 5'-CAGTCTCGAGTTTTACAGCTGTTTTTGGTTG-3'

Outlinedata - 5'-AGCTCATATGGCTTATACTGTTACTAAACC-3' and 5'GCGCCTCGAGTTTATATTGTGGGATGTCG-3'

IsdA - 5'-CAAGTCCCATGGCAACAGAAGCTACGAACGCAAC-3' and 5'ACCAGTCTCGAGTAATTCTTTAGCTTTAGAGCTTG-3'

IsdB - 5'-TATTCTCGAGGCTTTGAGTGTGTCCATCATTTG-3, and 5'GAAGCCATGGCAGCAGCTGAAGAAACAGGTGG-31

MRPII - S'-GATTACACCATGGTTAAACCTCAAGCGAAA-S' and 5'AGGTGTCTCGAGTGCGATTGTAGCTTCATT-3'

PCR products were first introduced in the clone vecto the pGEM-T (Novagen), using bacterial Top10 cells according to the manufacturer's instructions. This intermediate construct was done to facilitate further cloning in expressing vector. Transformants containing the insert DNA were selected using restricting analysis. After digestion an aliquot of the reaction ~20 µl were analyzed by gel electrophoresis in agarose (0.8% of agarose in Tris-acetate-EDTA (TAE) buffer). The DNA fragments were visualized by UV light after gel electrophoresis and staining bromide by ethidium. Standard molecular weight DNA (1 TPN ladder, Life Technologies) were subjected to electrophoresis in parallel with the analyzed samples and used to estimate the size of DNA fragments. Plasmid purified from selected transformants for each clone then digested to completion with appropriate restriction enzymes as recommended by the manufacturer (Life Technologies). Digested DNA fragment was then purified using a centrifuge (spin) column with silica gel before legirovaniem with plasmid pET24d or pQE-30. Cloning Ebh (H2 fragment), AAA, IsdA, IsdB, HarA, Atl amidase, Atl-glucosamine, MRP, IsaA was carried out using plasmid pET24d and cloning ClfA, SdrC, SdrE, FnbpA, SdrG/Fbe, alpha-toxin and Sbi was carried out using plasmid pQE-30.

In: Obtaining expressing vector.

In order to get expr serwisow plasmid pET24d or pQE-30 for ligating, it similarly digested to completion with appropriate restriction enzymes. Approximately 5-fold molar excess of digested fragments relative to the received vector used for programming the ligation reaction. Standard ligation reaction in a volume of ~20 ál (~16°C, ~16 h)using methods well-known in this field was performed using DNA T4 ligase (~2.0 units per reaction, Life Technologies). An aliquot of ligation (~5 µl) was used for transformation M15(pREP4) or electrocompetent cells BT21::DE3 according to methods well known in the field. After ~2-3 h period of growth at 37°C in ~1.0 ml of LB broth, the transformed cells were sown on plates with LB agar containing ampicillin (100 μg/ml) and/or kanamycin (30 µg/ml). The antibiotics included in the selection. The cups were incubated over night at 37°C for ~16 hours of Individual colonies ApR/KanR was picked with sterile toothpicks and used for local (“patch”) contamination of fresh LB ApR/KanR cups, and ~1.0 ml LB Ap/Kan broth culture. As a locally infected (patch) cups and broth culture incubated overnight at 37°C either in a standard incubator (Cup)or in a water bath-shaker. PCR-based analysis of whole cells was used to confirm that the transformants contained the insert DNA. At this stage ~1.0 ml night is th LB Ap/Kan broth culture was transferred into a 1.5 ml polypropylene tube, and the cells were collected by centrifugation in microcentrifuge Beckmann (~3 min, room temperature, ~12000 X g). Cellular precipitate suspended in ~200 µl of sterile water and an aliquot of 10 μl was used for programming the PCR reactions with a final volume of 50 µl, containing both direct and reverse primers for the amplification. The initial stage of denaturation at 95°C was increased to 3 min to ensure thermal destruction of bacterial cells and release of plasmid DNA. For amplification WE W-fragment of the samples lysed transformants used thermal cycler ABI Model 9700 and 32-circular profile three-stage thermal amplification, i.e. 95°C, 45 s; 55 to 58°C 45 s, 72°C, 1 min After thermal amplification, an aliquot of the reaction ~20 µl were analyzed by gel electrophoresis in agarose (0.8% of agarose in Tris-acetate-EDTA (TAE) buffer). The DNA fragments were visualized by UV light after gel electrophoresis and staining bromide by ethidium. Standard molecular weight DNA (1 TPN ladder, Life Technologies) were subjected to electrophoresis in parallel with the analyzed samples and used to estimate the size of PCR products. Transformants which have produced PCR product of the expected size were identified as strains containing protein expressing design. Strains containing expressing plasmid, the ATEM analyzed in relation to inducible expression of recombinant protein.

With: the Analysis of gene PCR-positive transformants.

An aliquot night seed culture (~1.0 ml) was inoculable in a 125 ml Erlenmeyer flask containing ~25 ml of LB broth Ap/Kan and grown at 37°C With shaking (~250 rpm) up until the turbidity of the culture reached about 0.5 at OD600, i.e. mid-logarithmic growth (usually about 1.5-2.0 hours). At this stage, approximately half of the culture (~12.5 ml) was transferred to a second 125 ml flask, and the expression of recombinant protein was induced by adding IPTG (1.0 M source solution prepared in sterile water, Sigma) to a final concentration of 1.0 mm. Incubation as IPTG-induced and reinducing cultures continued for another ~4 h at 37°C With shaking. Samples (~1.0 ml) as induced and reinducing cultures were removed after a period of induction, the cells were collected by centrifugation in microcentrifuge at room temperature for ~3 min to Separate the cell sediments suspended in ~50 ál of sterile water, then mixed with an equal volume of 2X Laemelli PAG-ordinator of sample buffer containing 2-mercaptoethanol, and placed in a boiling water bath for ~3 min to denature the protein. Equal volumes (about 15 µl) of both untreated IPTG-induced and reinducing cell lysates were applied to duplicate 12%Tris/glycine polyacrylamide g is l (Miniheli a thickness of 1 mm, Novex). Samples of induced and reinducing lysate was subjected to electrophoresis along with pre-stained molecular weight markers (SeeBlue, Novex) under standard conditions using standard LTO/Tris/glycine buffer for separation (BioRad). After electrophoresis, one gel was stained with Kumasi brilliant blue R250 (BioRad) and then decolorized to visualize new(s) IPTG-inducible(s) protein(s). The second gel was subjected to electroblotting on a PVDF membrane (pore size of 0.45 microns, Novex) for ~2 h at 4°C, using the device for blotting BioRad Mini-Protean II and methanol-containing buffer to wrap Tubino (Towbin). Blocking of the membrane and incubation with antibodies was carried out according to methods well known in the field. Monoclonal anti-RGS (His)3 antibody, then the second rabbit anti-mouse antibody conjugated with HRP (QiaGen)was used to confirm expression and identification of the recombinant protein. A visual image of the specific anti-His antibody was achieved using either insoluble substrate WAVEGUIDE, or Hyperfilm with chemiluminescent system Amersham ECL.

Example 2: preparation of recombinant protein

Bacterial strain

Recombinant expressing the E. coli strain M15(pREP4)containing plasmid (pQE30), or BL21::DE3 containing plasmid pET24d encoding staphylococcal protein is the objects of study were to produce cell mass with the purpose of purification of the recombinant protein.

Environment

Fermentation medium used for the production of recombinant protein consisted of 2X YT broth (Difco)containing 100 μg/ml AP and/or 30 μg/ml Km. Antifoam was added to the medium to the fermenter at a concentration of 0.25 ml/l Antifoam 204, Sigma). For induction of expression of recombinant protein in the fermenter was added IPTG (isopropyl-β-D-togetheranother) (1 mm final).

The preparation of recombinant proteins

In native conditions

IPTG was added at a final concentration of 1 mm and the culture was grown for another 4 h of the Culture is then centrifuged at 6000 rpm for 10 min and the precipitate resuspendable in phosphate buffer (50 mm K2HPO4KH2PO4, pH 7), including cocktail protease inhibitors. This sample was subjected to lysis under pressure (French), using a pressure of 1500 bar (150 MPa) (2 passes). After centrifugation for 30 min at 15,000 rpm, the supernatant was stored for further purification and was added NaCl to 0.5 M of the Sample is then applied to Ni-NTA resin (column HC 16 Pharmacia, Ni-NTA resin Qiagen), equilibrated in 50 mm K2NRA4KN2RHO4pH 7. After application of the sample the column was washed with buffer A (0.2 M NaH2PO4pH 7, 0.3 M NaCl, 10% glycerol). For elution of the bound protein was using a stepped gradient, where different proportions of buffer (0.2 M NaH2PO4at pH 7, 0.3 M NaCl, 10% glaze the ins and 200 mm imidazole) was added to the buffer A. The proportion of the buffer gradually increased from 10% to 100%. After cleaning elyuirovaniya fractions containing the protein were collected, concentrated and were dialyzed against 0,002 M KN2RHO4/K2NRA4at pH 7, 0.15 M NaCl.

This method is used for cleaning ClfA, SdrG, IsdA, IsaB, HarA, Atl-glucosamine and alpha-toxin.

Under denaturing conditions

IPTG was added at a final concentration of 1 mm and the culture was grown for another 4 h of the Culture is then centrifuged at 6000 rpm for 10 minutes and the precipitate resuspendable in phosphate buffer (50 mm K2HPO4KH2PO4, pH 7), including cocktail protease inhibitors. This sample was subjected to lysis under pressure (French), using a pressure of 1500 bar (150 MPa) (2 passes). After centrifugation for 30 min at 15000 rpm and the precipitate washed with phosphate buffer containing 1 M urea. The sample was centrifuged for 30 min at 15,000 rpm, and the precipitate resuspendable in 8 M urea, 0.1 M NaH2PO4, 0.5 M NaCl, 0.01 M Tris-HCl, pH 8, and stored overnight at room temperature. The sample was centrifuged for 20 min at 15,000 rpm, and the supernatant was collected for further purification. The sample is then applied to Ni-NTA resin (column HC 16 Pharmacia, Ni-NTA resin Qiagen), equilibrated in 8 M urea, 0.1 M NaH2PO4, 0.5 M NaCl, 0.01 M Tris-HCl, pH 8. After passing flow column was washed sequentially with buffer A (8 M urea, 0.1 M NaH2PO4, 0.5 M NaCl, 0.01 M Tris, pH 8.0)buffer (8 M urea, 0.1 M NaH2PO4, 0.5 M NaCl, 0.01 M Tris, pH 6.3), buffer D (8 M urea, 0.1 M NaH2PO4, 0.5 M NaCl, 0.01 M Tris, pH 5,9) and buffer E (8 M urea, 0.1 M NaH2PO4, 0.5 M NaCl, 0.01 M Tris, pH 4.5). Recombinant protein was suirable from the column during washing buffer D and E. Denatured recombinant protein can be solubilisate in solution without urea. For this purpose, a denatured protein in 8 M urea, gradually were dialyzed against 4 M urea, 0.1 M Na2PO4, 0.01 M Tris-HCl, pH 7,1, 2 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 7,1, 0.5 M arginine and 0,002 M KH2PO4/K2HPO4pH of 7.1, 0.15 M NaCl, 0.5 M arginine.

This method is used for cleaning Ebh (H2 fragment), AAA, SdrC, FnbpA, Sbi, Atl amidase and IsaA.

Purified proteins were analyzed using SDS page-ordinator. The results for the same protein, purified in native conditions (alpha-toxin), and one protein, purified under denaturing conditions (SdrC), shown in figure 3 and 4.

Example 3. Obtaining polysaccharides

PIA (PNAG) receive, as described in Joyce et al., 2003, Carbohydrate Research 338; 903-922.

Polysaccharides type 5 and type 8 are extracted from S. aureus, as described in Infection and Immunity 58(7); 2367.

Chemistry activation and combinations:

Native polysaccharide is dissolved in 2 M NaCl or in the water. The optimal concentration at which ishared appreciate for all serotypes and 2 mg/ml to 5 mg/ml

Of 100 mg/ml initial solution in acetonitrile CDAP (CDAP/PS ratio:0.75 mg/mg PS) is added to the polysaccharide solution. After 1.5 min of 0.2 M triethylamine is added to obtain specific activation of pH (pH 8.5 to 10.0). Activation of the polysaccharide is carried out at this pH for 2 min at 25°C. the carrier Protein is added to an activated polysaccharide in an amount sufficient to obtain a molar ratio of 1/1, and the reaction mix is carried out at a specific pH within 1 hour

Then the reaction quenched with glycine for 30 min at 25°C and over night at 4°C.

Conjugates purified by gel-filtration using a gel-filtration column Sephacryl 500HR, equilibrated with 0.2 M NaCl.

Determine the content of carbohydrates and protein in buervenich fractions. Conjugates pooled and sterile filtered on a sterilizing membrane 0.22 μm. Determine the ratio PS/protein drug conjugates.

Feature:

Each conjugate is characterized by the content of protein and polysaccharides.

The content of polysaccharides was measured using resorcinol test, and the protein content by the method of Lowry. The final ratio of PS/PD (wt./wt.) determined by the concentration ratio.

The residual content of DMAP (ng/μg PS):

Activation of the polysaccharide using CDAP introduces tiantou group in the polysaccharide and releases DMAP (4-dimethylaminopyridine). Ostatok the second content DMAP define specific analysis, developed and tested at GSK.

The content of free polysaccharide (%):

The content of free polysaccharide in the conjugate stored at 4°C or stored 7 days at 37°C, determined in the supernatant obtained after incubation with α-carrier antibodies and saturated ammonium sulfate, followed by centrifugation.

α-PS/α-PS ELISA kit is used for quantitative determination of free polysaccharide in the supernatant. The absence of conjugate is also monitored by the α-media/α-PS ELISA.

Example 4. Drug

Adjuvant composition

Protein, either separately or together, of the above examples can be prepared in the form of the drug with a combination of staphylococcal polysaccharide in the form of the adjuvant, the preparation may contain a mixture of 3 de-O-acylated monophosphorylated A (3D-MPL) and aluminum hydroxide, or 3-de-O-acylated monophosphorylated A (3D-MPL) and aluminum phosphate, or 3D-MPL and/or QS21, perhaps, emulsion oil in water, and possibly made in the medication with cholesterol, or only aluminium salt preferably the aluminum phosphate.

3D-MPL is a chemically detoxificating form of lipopolysaccharide (LPS) of gram-negative bacteria Salmonella minnesota.

Experiments performed at GSK Biologicals, showed that 3D-MPL, combined with various media dramatically increases to the to humoral, and TN type of cellular immunity.

QS21: represents one saponin purified from the crude extract, soap bark Typically Saponaria Molina, who has strong adjuvant activity: as antigen-specific lymphoproliferation and CTL (cytotoxic T lymphocytes) to multiple antigens.

The vaccine containing the antigen according to the invention, containing 3D-MPL and alum can be obtained similarly as described in WO 93/19780 or 92/16231. Experiments performed at GSK Biologicals, showed a clear synergistic effect of combinations of 3D-MPL and QS21 in the induction of both humoral and cellular immune responses TN-type. Vaccine containing the antigen, such antigens are described in US 5750110.

Emulsion oil-in-water may consist of 2 oil (tocopherol and squalene), and PBS containing Tween 80 as emulsifier. The emulsion contained 5% squalene, 5% tocopherol, and 0.4% Tween 80, and had an average particle size of 180 nm and is known as SB62 (see WO 95/17210).

Experiments performed at GSK Biologicals, proved that adding this O/W emulsion to MPL/QS21 additionally increases their immunostimulatory properties.

The SB62 emulsion preparation (2-fold concentrate)

Tween 80 dissolved in phosphate-buffered saline (PBS) with a 2%solution in PBS. To obtain 100 ml of double concentrated emulsion, 5 g of DL-alpha-tocopherol and 5 ml squall is on shaken for thorough mixing. Add 90 ml PBS/Tween and thoroughly mix. The resulting emulsion is then passed through a syringe and end up putting microfluidizer using the apparatus for microfluidizer M110S. The obtained oil droplets have a size of approximately 180 nm.

Example 5

Experiments on animals.

Female mice of the CD-1 at the age of 8-10 weeks obtained from Charles River Laboratories, Kingston, Mass. For mortality studies, five groups of 9-11 mice CD-1 infected intraperitoneally (b) serial dilutions of S. aureus grown on tablets CSA. The amount of inoculum ranged from ~1010up to 108CFU/mouse. Mortality assessed daily for 3 days. 50%lethal dose (LD50) estimated using a probit model based dose-response. The null hypothesis General LD50 tested using the criterion of odds ratios. Sub-lethal bacteremia initiated by infecting groups of 8-20 mice intravenous (IV) the introduction of ~2×106CFU/mouse or/b introduction ~2×107CFU/mouse. After infection in separate groups of animals take blood from the tail at the specified time points, and the levels of bacteremia assessed by determining the number of bacteria plating method, implemented in a dual replications on cups with trypticase soy agar with 5% sheep blood (Becton Dickinson Microbiology Systems). The statistical significance of the definition is given using unpaired f-test, student's t modification Welch (Welch).

Example 6

General methodology of determining or antibody-based test answers from different mammals

Serum was tested for the presence of IgG antibodies to staphylococcal polysaccharide using ELISA. Briefly, purified capsular polysaccharides from ATS (Rockville, Md, 20852) applied at a concentration of 25 μg/ml in phosphate-buffered saline (PBS) on microtiter tablets with high binding (Nunc Maxisorp) overnight at 4°C. the Tablets are blocking with 10% fetal calf serum (FCS), 1 h at 37°Sobrance serum pre-incubated with 20 μg/ml of polysaccharide cell wall (Statens Serum Institute, Copenhagen) and 10% FCS at room temperature for 30 min for neutralizing antibodies to this antigen. The samples are then diluted two times on the microplate in 10% FCS in PBS and balance at room temperature for 1 h with stirring. After washing the microplate balance with the Fc-fragment of monoclonal antibodies to human IgG labeled with peroxidase (NR-HRP, Stratech Scientific Ltd.), diluted 1:4000 in 10% FCS in PBS for 1 h at room temperature with stirring. ELISA carried out to measure IgG rat using conjugated with peroxidase, affinity purified antibodies goats against IgG (H+L) rat from Jackson ImmunoLaboratories Inc. (code 112-035-003) at 1:5000. Titration curves for referenced standard serum for each serotype is using logistic log comparison using SoftMax Pro. The concentration of polysaccharides used for coating tablets for ELISA, are 10 to 20 µg/ml of the Color developing when using 4 mg OPD (ortho-phenylenediamine) (Sigma) in 10 ml of 0.1 M citrate buffer, pH 4.5, with 14 μl of N2About2within 15 min in the dark at room temperature. The reaction is stopped with 50 μl of HCl, and the optical density recorded at 490 nm relative to 650 nm. The concentration of IgG determined by reference to the point of titration against a calibration curve is modeled using a 4-parameter logistic log equations defined using the software SoftMax Pro.

ELISA for the measurement of mouse and rat IgG to staphylococcal polysaccharide similar with the following exceptions. Conjugated with peroxidase, affinity purified antibody (H+L) goat against mouse IgG and affinity purified antibody (H+L) goat against rat IgG from Jackson ImmunoLaboratories Inc. used to identify features associated IgG.

HP6043-HRP reacts equally with human and rhesus purified IgG, and therefore, this reagent is used for RH antisera.

Protein ELISA carried out similarly polysaccharide ELISA with the following modifications. Protein is applied over night at 2.0 μg/ml in PBS. Serum samples were diluted in PBS containing 10% fetal calf serum and 0.1% polyvinyl alcohol. Wired the e human antibody is determined using conjugated to peroxidase, affinity purified antibody to goat IgG Fc man from Sigma (reference A-2290).

Example 7

Analysis of opsonophagocytosis.

Opsonophagocytosis killing in vitro S. aureus by human polymorphonuclear-leukocyte (PMN) is carried out, as described in Xu et al., 1992 Infect. Immun. 60; 1358. Human PMN obtained from heparinized blood by sedimentation in 3%dextran T-250. The reaction mixture for opsonization (1 ml) contains ~106PMN in medium RPMI 1640, supplemented with 10% V / V heat inactivated fetal calf serum, ~108SOME S. aureus and 0.1 ml of the analyzed serum or drug IgG. Hyperimmunizing rabbit serum used as positive controls, and 0.1 ml of non-immune rabbit serum was used as a comprehensive source of IgG samples. The reaction mixture is incubated at 37°C, and bacterial samples are transferred at 0, 60 and 120 min in water and then diluted, causing the Cup with trypticase soy agar and incubated at 37°C for counting the number of bacteria after an overnight incubation.

Example 8

Immunogenicity of staphylococcal protein in mice and rabbits

Animals were immunized purified staphylococcal protein to obtain hyperimmune sera. Mice were immunized three times (day 0, 14 and 28) 10 μg of each protein in Freund Specol. Rabbits were immunized three times (day 0, 21 and 42) 20 mcg every what about the protein in Freund Specol. Immune serum was collected and evaluated in ELISA against the protein and cons of killed whole cells.

ELISA against protein:

Purified protein was applied at a concentration of 1 μg/ml in phosphate-buffered saline (PBS) on microtiter tablets with high binding (Nunc Maxisorp) overnight at 4°C. the plates were blocked with 1% BSA in PBS for 30 min at room temperature with stirring. The analyzed samples were then diluted 1/1000 and incubated at room temperature for 1 h with stirring. After washing, the bound mouse or rabbit antibody was determined using conjugated with peroxidase, affinity purified antibodies goats against IgG (H+L) mouse from Jackson ImmunoLaboratories Inc. (reference: 115-035-003) or affinity purified antibodies goats against IgG (H+L) rabbit (link: 11-035-003), diluted 1:5000 in PBS with 0.05% of tween. Antibodies for detection incubated for 30 min at room temperature with stirring. Color was developed using 4 mg OPD (Sigma)+5 μl of N2About210 ml of 0.1 M citrate buffer, pH 4.5, for 15 min in the dark at room temperature. The reaction was stopped using 50 μl of HCL, and the absorbance was recorded at 490 nm relative to 650 nm. OD for 1/1000 dilution Post III was compared with the OD obtained with the same breeding preimmune sera.

The results obtained with mouse and rabbit what aforetime, presented in figure 5. Watched a good seroconversion against each antigen. Evaluation of sera directed against SBI, was distorted due to lg-binding activity of this protein.

ELISA against the killed whole cells:

Killed whole cells (inactivated by heating or formaldehyde) from S. aureus type 5 and 8 or S. epidermidis strain Hay was applied at a concentration of 20 μg/ml in phosphate-buffered saline (PBS) on microtiter tablets with high binding (Nunc Maxisorp) overnight at 4°C with evaporation. The plates were blocked with 1% BSA in PBS for 30 min at room temperature with stirring. Protein And neutralized by adding 10 μg/ml affinity purified chicken antibodies against protein A (ICL link: CPA-65A-2), diluted in PBS with 0,05% tween followed by incubation for 1 h at room temperature. The analyzed samples were then diluted two times on the microplate in PBS-0.05 per cent of the original 1/10 dilution and incubated for 1 h at room temperature with stirring. After washing, the bound mouse or rabbit antibody was determined using conjugated with peroxidase, affinity purified antibodies goats against IgG (H+L) mouse from Jackson ImmunoLaboratories Inc. (reference: 115-035-003) or affinity purified antibodies goats against IgG (H+L) rabbit (link: 11-035-003), diluted 1:5000 in PBS with 0.05% of tween. These antibodies for detection incubated in the course the e 30 min at room temperature with stirring. Color was developed using 4 mg OPD (Sigma)+5 ál of H2O210 ml of 0.1 M citrate buffer, pH 4.5, for 15 min in the dark at room temperature. The reaction was stopped using 50 μl of HCl, and the absorbance was recorded at 490 nm relative to 650 nm.

It should be noted that the levels of protein expression in Staphylococcus will vary depending on the cultivation conditions. Therefore, a negative result may indicate poor conditions of cultivation, and not about the lack of immunogenicity.

Results using mouse sera are shown in Table 5, and some graphs shown in Fig.6. Weak recognition of S. aureus strain 5 observed with sera directed against SdrC, FnbpA, Ebh, Sbi and IsaA. Detection of S. aureus strain 8 see only with serum directed against Sbi. Weak recognition of S. epidermidis Hay observed with sera directed against Atl amidase, MRP, IsdA, IsaA, Ebh, AAA and Sbi.

The results obtained using rabbit sera shown figure 7 and summarized in Table 6. Very good recognition of the three strains was observed with IsaA and IsdB. Weak recognition of the three strains was observed with HarA, although animals received only one injection instead of three injections used for other proteins.

Table 5
The name proteinReaction to SA5Reaction to SA8Response to SE Hay
IsaA(+)(+)(+)
ClfA(+)(+)

Atl-amidase--++
SdrG--
Glucosidase---
IsdA--++
Alpha-toxin---
SrdC++(+)
Ebh+- +
AaA-++
MRP--++
Sbi+++++++
FnbpA++(+)

td align="center"> +
Table 6
The name proteinReaction to SA5Reaction to SA8Response to SE Hay
IsaA+++++++++
ClfA+++++
Atl-amidase-+++
IsdB+++++++++
SdrG++
Glucosidase-
HarA (1 injection)+++
IsdA-
Alpha-toxin--+
SrdC--
Ebh-+-
AaA---
MRP-++
Sbi-+++-
FnbpA-++++

Example 8

the power of combinations of staphylococcal proteins in models of nasal colonization.

Fifteen groups of cotton hamsters were inoculated with combinations of the eight staphylococcal antigens, and five cotton hamsters, which served as controls, were treated without antigens. These sixteen groups are the following:

Group 1 - Atl-glucosamine, Atl-amidase, AAA, alpha-toxin, SdrC, SdrG, Ebh, Sbi

Group 2 - Atl-glucosamine, Atl-amidase, IsdA, IsdB, ClfA, SdrC, Ebh, FnbpA

Group 3 - Atl-glucosamine, Atl-amidase, HarA, IsdA, MRP, IsdB, AAA, alpha-toxin

Group 4 - Atl-glucosamine, HarA, IsdA, AAA, ClfA, IsaA, Ebh, Sbi

Group 5 - HarA, MRP, AAA, alpha-toxin, ClfA, SdrC, Ebh, FnbpA

Group 6 - IsdA, IsdB, AAA, alpha-toxin, ClfA, SdrG, Sbi, FnbpA

Group 7 - Atl-linedata, IsdA, MRP, AAA, IsaA, SdrG, Ebh, FnbpA

Group 8 Control

Group 9 - Atl-glucosamine, IsdA, MRP, alpha-toxin, IsaA, SdrC, Sbi, FnbpA

Group 10 - Atl-glucosamine, MRP, IsdB, AAA, ClfA, IsaA, SdrC, SdrG

Group 11 - Atl-linedata, MRP, IsdB, alpha-toxin, ClfA, IsaA, Ebh, Sbi

Group 12 - Atl-glucosamine, HarA, IsdB, alpha-toxin, IsaA, SdrG, Ebh, FnbpA

Group 13 - Atl-amidase, HarA, IsdB, AAA, IsaA, SdrC, Sbi, FnbpA

Group 14 - Atl-glucosamine, Atl-amidase, HarA, MRP, ClfA, SdrG, Sbi, FnbpA

Group 15 - Atl-amidase, HarA, IsdA, alpha-toxin, ClfA, IsaA, SdfC, SdrG

Group 16 - HarA, IsdA, MRP, IsdB, SdrC, SdrG, Ebh, Sbi

Each mixture of antigens containing 3 μg of each antigen was mixed with adjuvant consisting of liposomes containing MPL and QS21. Cotton hamsters were inoculated three times at 1, 14 and 28 days of experiment. Two weeks after instill and evaluated the effectiveness of immunization, using analysis of nasal colonization, as described in Kokai-Kun et al., (2003) Antimicrob.Agents.Chemother. 47; 1589-1597.

Classical multiple regression analysis was performed on data using the software Design Expert 6”. The presence of the antigen coded as +1, and the absence of antigen as-1. Using the equation of this model, you can determine which antigens were key antigens, which gave a significant reduction in the number of colonies in the nose.

Results

The results of the analysis of nasal colonization is shown in Table 7. The control group had an average logKOE/nose 3,51335, and reduced nasal colonization could be observed for all groups of cotton hamsters inoculated staphylococcal proteins. Groups 4, 9 and 13 showed the highest reduction of nasal colonization with a decrease of more than 2 logarithms in CFU/nose. Group 12 and 16 also gave good results, showing a decrease of about 2 logarithms in CFU/nose.

Table 7
GroupThe average observed LogKOE/nosePredicted LogKOE/nose
11,775272,03560
2 2,904352,52684
31,965562,23033
41,277481,21872
51,673041,93128
62,797452,98193
72,214812,30705
83,513553,47317
91,224801,44080
102,030851,93204
112,025221,81581
121,534021,70996
131,360631,49100
142,312011,73909
152,229791,98223
161,581091,44004

The contribution of specific antigens in a mixture of antigens was determined using multiple regression analysis data of nasal colonization. The final model contains seven best antigens. The results for these antigens are shown in Table 8. In the context of the mixture of proteins enable HarA gave the greatest reduction in nasal colonization, followed by IsaA, Sbi, SdrC, autolysin-glucosamine, MRP and Ebh.

Table 8
Effects difference logKOE/nose and the ratio of CFU/nose for the seven best antigens in the model and the corresponding p-values.
antigenprob>FAssessment of effectThe reduction ratioThe cumulative effectThe cumulative ratio
HarA0,033-0,596a 3.9-0,596a 3.9
IsaA0,046-0,5583,6 -1,15414,3
Sbi0,077-0,4913,1-1,64544,2
SdrC0,22-0,3372,2-1,98296,0
Atl-glucose0,238-0,3242,1-2,306202,2
MRP0,239-0,3232,1-2,629425,3
Ebh0,297-0,2861,9-2,914821,0

prob - probability

1. Immunogenic composition for immunization against staphylococcal infections, containing an effective amount of at least two different proteins or immunogenic fragments, selected from at least two groups of proteins or immunogenic fragments, selected from the following groups:
Group (a): at the ore one of staphylococcal protein linking extracellular component, or immunogenic fragment selected from the group consisting of SdrG, laminin receptor, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrH, lipase GehD, FnbA, FnbB, Cna, ClfB, FbpA, Npase (neutral phosphatase), IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin-binding protein, coagulase, and MAP;
Group (b): at least one staphylococcal transport protein or immunogenic fragment selected from the group consisting of immunodominant ABC Transporter, IsdA, IsdB, Mg2+ Transporter, SitC and Ni ABC Transporter;
Group (C): at least one staphylococcal regulator of virulence, toxin or immunogenic fragment selected from the group consisting of alpha-toxin (Hla), mutant alpha toxin H35L, mutant alpha toxin H35R and RNA III activating protein (RAP);
provided that the composition does not contain at least one protein or immunogenic fragment selected from the group (a), together with at least one protein or immunogenic fragment selected from the group (b).

2. Immunogenic composition according to claim 1, where at least one protein or immunogenic fragment selected from the group (a) and at least one protein or immunogenic fragment selected from the group (b).

3. Immunogenic composition according to claim 1, where at least one protein or immunogenic fragment selected from the group (b) and IU is greater least one protein or immunogenic fragment selected from the group (b).

4. Immunogenic composition according to claim 1, containing at least one protein or immunogenic fragment of S. aureus.

5. Immunogenic composition according to claim 1, containing at least one protein or immunogenic fragment from S. epidermidis.

6. Immunogenic composition according to claim 1, additionally containing PIA polysaccharide or oligosaccharide.

7. Immunogenic composition according to claim 1, additionally containing capsular polysaccharide or oligosaccharide of type V or type VIII of S. aureus.

8. Immunogenic composition according to claim 1, additionally containing capsular polysaccharide or oligosaccharide of type I and/or type II and/or type III from S. epidermidis.

9. Immunogenic composition according to claim 6, where staphylococcal capsular polysaccharide anywhereman with protein carrier.

10. Immunogenic composition according to claim 9, where the carrier protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM197, protein D of Haemophilus influenzae, pneumolysin and alpha toxoid.

11. Immunogenic composition according to claim 1 capable of inducing an effective immune response against S. aureus, and against S. epidermidis.

12. Immunogenic composition according to claim 1, where at least one protein or immunogenic fragment selected from the group (a)represents the ClfA or immunogenic fragment.

13. Immunogenic composition according to claim 1, where at least one protein or immunogenic fragment, select the config from the group (b), is an alpha-toxin (Hla), the mutant alpha toxin H35L, mutant alpha toxin H35R or immunogenic fragment.

14. Immunogenic composition according to claim 1, containing ClfA or immunogenic fragment and alpha-toxin (Hla), the mutant alpha toxin H35L, mutant alpha toxin H35R or immunogenic fragment.

15. Vaccine for immunization against staphylococcal infections, containing an effective amount of the immunogenic composition according to any one of claims 1 to 14 and a pharmaceutically acceptable excipient.

16. A method of producing a vaccine, comprising a stage of mixing the antigen with obtaining immunogenic composition according to any one of claims 1 to 14 and add pharmaceutically acceptable excipient.

17. A method of preventing or treating staphylococcal infections, including stage vaccine at 15 to a patient in need of it.

18. The use of the immunogenic composition according to any one of claims 1 to 14 in the manufacture of a vaccine for treatment or prevention of staphylococcal infection.

19. The method of producing immunoglobulin for use in the prevention or treatment of staphylococcal infections, including stage immunization vaccine recipient indicated in paragraph 15 and excretion of immunoglobulin from the recipient.

20. Pharmaceutical composition for preventing or treating staphylococcal infections, containing an effective quantity:
(1) monoclone inogo antibody or its fragment, which react with staphylococcal protein that binds the extracellular component of group (a)as defined in claim 1, or its immunogenic fragment;
(2) the monoclonal antibody or its fragment, which react with staphylococcal transport protein group (b)as defined in claim 1, or its immunogenic fragment; and
(3) the monoclonal antibody or its fragment, which react with staphylococcal regulator of virulence or toxin group (b)as defined in claim 1, or its immunogenic fragment;
provided that the composition does not contain simultaneously (1) and (2).

21. Pharmaceutical composition for preventing or treating staphylococcal infections, containing an effective amount of an antibody obtained by the method according to p. 19, and a pharmaceutically acceptable excipient.

22. A method of treating or preventing staphylococcal infections, including the stage of the introduction to the patient an effective amount of the pharmaceutical composition according to claim 20 or 21.

23. The use of the pharmaceutical composition according to claim 20 or 21 for the treatment or prevention of staphylococcal infection.



 

Same patents:

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SUBSTANCE: invention discloses composition for production of antibodies SNAP-25, capable of binding with epitope, which contains C-end (carboxylic) on residue P1 of cleaved bond of cleavage site BoNT/A of SNAP-25 cleavage product, which contains carrier, flexible linker, containing at least three amino acids, and SNAP-25 antigen, for which amino acid sequence is given. Described are versions of antibodies α-SNAP-25 with said capability and their application in method of detecting BoNT/A activity with application of cells from stable cell line, sensitive to BoNT/A intoxication, and method of determining immunoresistance to BoNT/A in mammal with application of tested animal sample and cells of stable cell lime, whose cells are sensitive to BoNT/A intoxication.

EFFECT: application of invention ensures reliability, simplicity and makes it possible to exclude necessity of animal tests from botulinum toxin analyses.

15 cl, 11 dwg, 14 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. There are presented versions (aCt1 and aCt2) of one-domain antibodies specifically binding the Chlamydia trachomatis antigen. There are described versions of the method of inhibiting an infection caused by Chlamydia wherein the method involves the preparation of elementary bodies C.trachomatis by a therapeutically effective amount of the nanoantibody aCt1 or aCt2 before being attached to infected target cells.

EFFECT: use of the invention provides the antibodies to detect and block the infections Chlamydia trachomatis that can find application in medicine.

6 cl, 4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: there are presented antibodies specifically binding the lipid-associated antigen of M.hominis with the characterised amino acid and nucleotide sequences, as well as a method of treating a Mycoplasma M.hominis infection involving administering to said mammal a therapeutically effective amount of the nanoantibodies.

EFFECT: invention can find further application in treating the mycoplasma infection.

4 cl, 2 tbl, 6 ex, 5 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a method for preparing an anthrax diagnostic serum by hyperimmunisation of ox producers with an antigen of the strain Bacillus anthracis M-71. Hyperimmunisation is conducted in increasing doses: first subcutaneous introduction of the antigen 100-120 mln microbial cells together with saponin 2.5-3 mcg; 12-14 days later, the antigen is introduced intracutaneously in a dose of 2.5-3.0 bln microbial cells together with saponin 2.5-3 mcg; 6-7 days later, every 3-4 days, the antigen is introduced intravenously 12-14 times in increasing doses 10.0 to 210 bln. microbial cells. It is followed by blood sampling, keeping at temperature 37-38°C for 2-3 hours, placing in a fridge at 2-8°C for 3-5 days, separating serum. The prepared serum is preserved in 5-7% carbolic acid in isotonic solution in ratio (9-10):1 respectively. The ready serum is sterile and has an AR titre min. 1:1000, a CFT titre min. 1:20. A diagnostic set for anthrax diagnosis comprises the antigen of the strain Bacillus anthracis M-71, the anthrax diagnostic serum and healthy bovine's native serum.

EFFECT: invention provides higher specific activity of the serum and diagnostic effectiveness for an anthrax agent.

5 cl, 1 tbl, 3 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: invention discloses a purified and/recombinant antigen polypeptide possessing toxin activity, recovered from Clostridium perfringens with specified amino acid sequence. The invention discloses the recovered or recombinant polynucleotide coding such polypeptide, an expression vector and a host cell expressing the polypeptide. The invention discloses a method for preparing the polypeptide, an antibody specifically bound with the polypeptide, immunogenic compositions and vaccines containing the given polypeptide or a polynucleotide thereby providing a specifically immune response to the polypeptide. There are disclosed a method for inducing the immune response, a method of determining the fact whether an individual has been exposed to a pathogen (versions), a method of screening, an agonist or an antagonist modulating activity of the polypeptide, a method of animal vaccination, e.g. hens for inducing active immunity, as well as passive immunity in hen off-springs which becomes less sensitive to clostridial diseases. What is disclosed is a transgenic plant containing the exogenous polynucleotide coding the polypeptide under the invention, applicable for animal feeding.

EFFECT: polypeptide is used as an ingredient of a forage or a beverage for preventing a disease caused by bacteria expressing the polypeptide under the invention.

39 cl, 8 dwg, 6 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: hybrid cultured cell strain of the animals Mus museums 13F8 is produced by immunisation of Balb/c mice. The mice are immunised by four introductions of the recombinant antigen preparation F1 100 mcg/mouse. The third post-immunisation day is followed by splenocyte hybridisation of the immunised mice (1x108 cells) and mice myeloma cells P3-X63 Ag/8-653 (1×107cells). Polyethylene glycol (Sigma, the USA) is used as a fusion agent. The hybridisation is followed by hybridoma selection, screening, cloning and cryopreservation. Hybridoma is deposited in the State Collection of Pathogens and Cell Cultures of GKPM-Obolensk, No. N-18.

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7 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: polypeptide (versions) immunogenic with respect to meningococcal infections contains: an amino acid sequence at least 90 % identical to a sequence presented in the description (SEQ ID NO: 32), or said amino acid sequence, or a fragment of 80 sequenced amino acids of said sequence. What is described is an antibody which contacts with the polypeptide under the invention and which may be used as a drug. What is described is nucleic acid of the preset structure which codes the polypeptide or its versions and which may be used for treating or preventing a disease and/or an infection caused by Neisseria meningitides. The invention provides additional polypeptides applicable in advanced vaccines for preventing and/or treating meningococcal meningitis. The peptides can also find application in diagnosing of the disease and as targets of antibiotics.

EFFECT: higher clinical effectiveness for meningococcal meningitis.

19 cl, 2 tbl

FIELD: medicine, pharmaceutics.

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EFFECT: use of the invention enables enhanced immune response at height of the disease, ensures effective rehabilitation and prevents recurrences of the disease.

14 cl, 4 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention describes novel polynucleotide and amino acid sequences of Brachyspira hyodysenteriae, which can be used to diagnose diseases in animals, caused by B. hyodysenteriae, to treat or prevent diseases associated with infection with B. hyodysenteriae. The invention describes a cell containing a plasmid containing a polynucleotide, for treating and preventing a disease associated with infection of an animal with B. Hyodysenteriae. The invention describes immunogenic and vaccine compositions for generating immune response in an animal, which contain a polypeptide, a polynucleotide, a cell or a plasmid for treating or preventing infection of animals by B. hyodysenteriae, as well as sets of instruments for diagnosis which contain a monoclonal antibody, capable of biding the disclosed polypeptide or a polypeptide or polynucleotide. The invention enables to successfully diagnose diseases caused by B. hyodysenteriae, prevent or treat animals infected with B. hyodysenteriae. The sequences described herein can be used for diagnosis and therapeutic and/or preventive treatment of animals from diseases caused by other types of Brachyspira, including B. intermedia, B. suantatina, B. alvinipulli, B. aalborgi, B. innocens, B. murdochii and B. pilosicoli.

EFFECT: high efficiency of using the composition.

39 cl, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical formulations and is applicable for providing bactericidal efficacy. A pharmaceutical formulation contains two various antibiotics as active ingredients in the form of a synergetic combination of a fixed dose in a parenteral dosage form. The first antibiotic represents carbapenem or its pharmaceutically acceptable salts, while the second antibiotic represents aminoglycoside which represents etimycin or its pharmaceutically acceptable salts. The above first antibiotic and the above second antibiotic are found in weight ratio of 6:1 to 13:1. Besides, the pharmaceutical formulation contains one or more additives specified from a group of synthetic/natural amino acids/vitamins/stabilisers/polymers/antioxidants/micronutrient elements.

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8 cl, 3 tbl, 6 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: composition includes a bactericidal substance - catapol - in amount of 2.1-2.5 wt %, zosterin in amount of 1.1-5.0 wt % and distilled water.

EFFECT: providing a composition which stimulates a reparative process in external protective tissue, having anti-inflammatory and radioprotective action.

1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry and medicine and deals with novel 4-(pyrrolidine-1-yl)quinoline compounds, a method of their obtaining and application for treatment of bacterial or fungal infection.

EFFECT: invention provides extension of arsenal of means for fighting "latent" bacteria.

17 cl, 3 dwg, 1 tbl, 47 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of pharmaceutics, namely represents compositions for treating nail and nail bed diseases and methods of treating nail diseases.

EFFECT: claimed compositions do not form a film when applied on the nail surface and contain a carrier, in which suspended, dispersed or emulsified are all components of the composition, a non-volatile solvent, a moistening preparation and a pharmaceutically active ingredient, soluble in the non-volatile solvent and/or in a mixture of the carrier and the non-volatile solvent, with the composition being efficient in treatment of nail or nail bed diseases.

30 cl, 2 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: in a complex preparation containing a carrier representing an enterosorbent; the enterosorbent is modified by immobilising high-disperse silver - nanosilver in a concentration of 0.01 - 1.0 wt % on its surface. The enterosorbent represents activated carbon, kaolin, bentonit, or enterodesum, or monocrystalline cellulose. A modifying silver-containing solution - a nanosilver source - is silver clusters in an aqueous solution.

EFFECT: higher specific antimicrobial activity.

2 cl, 3 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and concerns a local topical composition characterised by the fact that it contains synthetic lipoic acid; 95% ethyl alcohol; 1,2-propylene glycol, polyacrylic acid (any other hydrophilic gelling agent), triethanolamine, purified water; a method for preparing the local topical composition characterised by the fact that synthetic lipoic acid is dissolved while stirring in mixed ethyl alcohol and propylene glycol; at the same time polyacrylic acid is dispersed in a specified amount of water sufficient to reduce the ratio of the ingredients up to 100 wt %, after 3-hour swelling, the prepared solution is added to synthetic lipoic acid dissolved in mixed ethyl alcohol and propylene glycol and mixed up; the prepared composition is neutralised by a triethanolamine solution to pH 5.5-6.5 and mixed to form a light-yellow transparent gel-like mass.

EFFECT: group of inventions provides high antirheumatic, anti-inflammatory and analgesic activity, as well as effective antibacterial and anti-burn action.

4 cl, 4 ex, 1 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to obstetrics and gynaecology, and may be used for preconception preparation in early recurrent miscarriage caused by chronic genital inflammation or inflammatory hormonal ovarian insufficiency. That is ensured by 12 intravaginal introductions of disposable injectors with Dead sea mud gel as a therapeutic agent daily for 30 minutes within one therapeutic course upon completion of the anti-inflammatory treatment following the early miscarriage.

EFFECT: invention provides 50% higher safety and effectiveness of preconception preparation in the given group of patients.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to a composition containing encapsulated triterpenic acid: betulinic acid, ursolic acid or derivatives thereof in the form of salts and esters, or triterpene alcohol - betulin, which may be used in medicine for treating and preventing viral infections caused by DNA and RNA-containing viruses, such as influenza viruses, oncogenic viruses, herpes virus, herpes zoster virus, as well as infections caused by gram-positive and gram-negative bacteria: Staphylococcus spp., Streptococcus spp., Enterococcus spp., Shigella spp., Escherichia spp., Salmonella spp., Proteus spp., Acinetobacter spp., Citrobacter spp., Pseudomonas spp., Serratia spp., Klebsiella spp., Antracoides spp., Cryptococcus spp., pathogenic fungi of the genus Microsporum, Trichophyton, Nocardia, Aspergillus, yeast-like fungi of the genus Candida, including multiresistant strains, as well as Actinomycetes and some pathogenic protozoa: Entamoeba histolytica, Trichomonas vaginalis. The invention presents the composition containing an active ingredient presented by 0.5 wt % of betulin or 0.5 wt % of encapsulated triterpenic acid: betulinic acid, ursolic acid or derivatives thereof in the form of salts and esters and others, and carriers presented by: β-cyclodextrins, fullerene, lecithins and polymers binding to the ingredients to form ingredient-carrier complexes, and excipients.

EFFECT: higher efficacy of using the composition.

3 cl

FIELD: chemistry.

SUBSTANCE: invention relates to field of food industry, biotechnology and deals with antibacterial composition and strain of bacteriophage Escherichia coli, used for obtaining said composition. Characterised composition includes filtrate of Escherichia coli phage lysate, obtained with application of strain of bacteriophage Escherichia coli, deposited in collection of museum of microorganisms of Federal Budget Institution of Science "State Research Centre for Applied Microbiology and Biotechnology" of the Federal Service on Customers' Rights Protection and Human Well-being Surveillance (FBIS SRC AMB of Rospotrebnadzor) under number Ph 64, filtrate of Escherichia coli phage lysate, containing coli bacteriophage, filtrate of staphylococcus phage lysate, filtrate of salmonella phage lysate, filtrate of Listeria monocyctogenes phage lysate and target additives in amount 1.0÷95.0 wt % of composition weight.

EFFECT: claimed composition has required spectrum of specific activity due to inclusion of polyvalent bacteriophage and can be used in production of biologically active additives and food additives.

11 cl, 1 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to pharmacology and medicine and concerns using a dipeptide of a general formula Tyr-Pro-X, where X - OH, NH2, OCH3, OC2H5, as an anti-inflammatory, antibacterial, wound-healing, regenerative, analgesic and burntreating agent for external application, as well as a dosage form for external application containing this dipeptide.

EFFECT: group of inventions provides preparing the effective agent for external applications with no side effects.

3 cl, 1 tbl, 22 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary medicine, and concerns an associated salmonellosis, escherichiosis and rabbit viral haemorrhagic disease vaccine. The presented vaccine contains antigens in the form of pure-growth suspended cells of salmonellosis Sal.typhimurium, escherichiosis E.coli and rabbit viral haemorrhagic disease prepared from target affected organ of dead rabbits in a local epizootic centre; the vaccine also contains glucose, formalin and aluminium hydroxide in the specified amounts.

EFFECT: presented invention enables providing higher specificity and efficacy of the prepared vaccine, as well as eliminating side effects in the animals and may be used for biopharmaceutical engineering.

1 tbl, 5 ex

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