HK1175704A

HK1175704A - Immunogenic composition for use in vaccination against staphylococcei

Info

Publication number: HK1175704A
Application number: HK13102994.7A
Authority: HK
Inventors: Castado Cindy; Pierre Femand Lecrenier Nicolas; Anne Neyt Cecile; Poolman Jan
Original assignee: Glaxosmithkline Biologicals S.A.
Priority date: 2004-09-22
Filing date: 2013-03-11
Publication date: 2013-07-12

Description

Immunogenic compositions for vaccination against staphylococci

The present application is a divisional application of the chinese patent application 200580039690.0 entitled "immunogenic composition for vaccination against staphylococcus", filed on 9/20/2005.

Technical Field

The present invention relates to staphylococcal immunogenic compositions and vaccines, their manufacture and use of these compositions in medicine. More particularly, it relates to vaccine compositions comprising a combination of antigens for the treatment or prevention of staphylococcal infection. The use of said vaccines in medicine and methods for their preparation are also provided.

Background

In recent years, as the use of intravascular devices has increased, the number of infections obtained in the community and in hospitals has increased. Hospital acquired (nosocomial) infections are a major cause of morbidity and mortality, and more particularly in the united states, it affects over 200 million patients per year. According to various studies, approximately 6% of us patients will acquire an infection during their stay in the hospital. The economic burden in the United states was estimated to exceed $ 45 billion in 1992 (Emori and Gaynes, 1993, Clin. Microbiol. Rev.6; 428). The majority of frequent infections are urinary tract infections (33% of UTI-infections), followed by pneumonia (15.5%), surgical site infections (14.8%) and primary bloodstream infections (13%) (Emori and Gaynes, 1993, Clin. Microbiol. Rev.6; 428).

Staphylococcus aureus, coagulase-negative staphylococci (mostly Staphylococcus epidermidis), enterococcus species, Escherichia coli and Pseudomonas aeruginosa are the major iatrogenic pathogens. Although those pathogens cause almost the same number of infections, the severity of the disorders they can cause, plus the frequency of antibiotic-resistant isolates, balances this ranking with Staphylococcus aureus and Staphylococcus epidermidis, which are among the most important nosocomial pathogens.

Staphylococcus aureus is the most common cause of iatrogenic infections with significant morbidity and mortality (Romero-Vivas et al 1995, infection. Dis.21; 1417). It is responsible for some cases of osteomyelitis, endocarditis, septic arthritis, pneumonia, abscesses, and toxic shock syndrome.

Staphylococcus epidermidis is a normal skin commensal organism and is also a significant opportunistic pathogen causing infection of implanted medical devices and infections at surgical sites. Medical devices infected with staphylococcus aureus include cardiac pacemakers, cerebrospinal fluid shunts, continuous flow peritoneal dialysis catheters, orthopedic devices, and prosthetic heart valves.

Antibiotics were used to treat staphylococcus aureus and staphylococcus epidermidis infections, penicillin was the drug of choice, and vancomycin was used for methicillin-resistant isolates. Since the 1980 s, the percentage of staphylococcal strains that exhibited broad-spectrum resistance to antibiotics has become increasingly prevalent (Panlilo et al 1992, feed. control. Hosp. epidemic.13; 582), posing a threat to effective antimicrobial therapy. In addition, the recent emergence of vancomycin resistant staphylococcus aureus has raised fear that methicillin resistant staphylococcus aureus strains will develop and spread for which no effective treatment is available.

An alternative approach to the use of antibodies against staphylococcal antigens in passive immunotherapy has been investigated. Therapies involving the administration of polyclonal antisera are under development (WO 00/15238, WO 00/12132), and treatment with monoclonal antibodies against lipoteichoic acid (WO 98/57994).

An alternative approach would be to use active vaccination to generate an immune response to staphylococci. Several candidates have been identified for inclusion as vaccine components. These candidates include fibronectin binding protein (US5840846), MHC II analogues (US5648240), fibrinogen binding protein (US6008341), GehD (US2002/0169288), collagen binding protein (US6288214), SdrF, SdrG and SdrH (WO 00/12689), mutant SEA and SEB exotoxin (WO 00/02523) and 52kDa vitronectin binding protein (WO 01/60852).

The S.aureus genome has been sequenced and a number of coding sequences have been identified (EP786519, WO 02/094868). The same is true for Staphylococcus epidermidis (WO 01/34809). As an improvement of this approach, others have identified proteins recognized by hyperimmune serum from patients with staphylococcal infections (WO 01/98499, WO 02/059148).

The first generation of vaccines targeting staphylococcus aureus or the exoproteins it produces has met with limited success (Lee 1996 Trends microbiol.4; 162). There remains a need to develop effective vaccines against staphylococcal infections.

Accordingly, the present invention provides an immunogenic composition comprising at least two different proteins or immunogenic fragments thereof selected from at least two groups of proteins or immunogenic fragments:

group a) at least one staphylococcal extracellular component binding protein or immunogenic fragment thereof 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, esterase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, vitr-2, HBP, SSP connexin binding protein, fibrinogen binding protein, coagulase, Fig and MAP;

group b) at least one staphylococcal transporter protein or immunogenic fragment thereof selected from the group consisting of immunodominant ABC transporter, IsdA, IsdB, Mg²⁺Transporters, SitC and Ni ABC transporters;

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

Drawings

FIG. 1-polypeptide sequence of preferred proteins. Table 1 provides information about which protein each SEQ ID represents.

FIG. 2-nucleotide sequence encoding a preferred protein. Table 1 provides information about which protein is encoded by each SEQ ID.

Figure 3-purification of alpha toxin under native conditions. Panel A shows a Coomassie Brilliant blue stained SDS-PAGE of samples prepared during alpha toxin purification. Lane 1-molecular weight marker, lane 2-containing soluble fraction overexpressing alpha toxin, lane 3-flow through Ni-NTA column, lane 4-fraction eluted with 10% buffer B, lane 5-fraction eluted with 20% buffer B, lane 6-fraction eluted with 30% buffer B, lane 7-fraction eluted with 50% buffer B, lane 8-fraction eluted with 75% buffer B, lane 9and lane 10-fractions eluted with 100% buffer B, lane 11-inducing bacteria before T ═ 0, lane 12-inducing bacteria after T ═ 4, lane 13-cell lysate, lane 14-soluble fraction, lane 15-insoluble fraction.

Panel B shows Coomassie blue stained SDS-PAGE of 10, 5,2 and 1 μ l purified alpha toxin.

FIG. 4-purification of SdrC under denaturing conditions. Panel A shows a Coomassie Brilliant blue stained SDS-PAGE of samples prepared during alpha toxin purification. Lane M-molecular weight marker, lane Start-supernatant formed from insoluble fraction containing overexpressed SdrC, lane FT 1-flow through Ni-NTA column, lane C-fraction eluted with rinse buffer C, lane D-fraction eluted with buffer D, lane E-fraction eluted with buffer E.

Panel B shows Coomassie Brilliant blue stained SDS-PAGE of 1,2, 5 and 10. mu.l of purified SdrC.

FIG. 5-ELISA results for anti-staphylococcal protein antisera on plates coated with purified protein.

Pooled mice pre-results using pooled sera extracted from pre-vaccinated mice. Pooled mouse Post III-results using pooled mouse sera extracted after immunization. Pooled rabbit pre-results using pooled sera extracted from pre-vaccinated rabbits. Pooled rabbit Post III-results using pooled rabbit sera extracted after immunization. Blc-negative control.

FIG. 6-ELISA results of mouse antisera raised against staphylococcal proteins on plates coated with killed staphylococci.

Panel A uses plates coated with intact cells killed by Staphylococcus aureus serotype 5. Panel B uses plates coated with intact cells killed by s.aureus serotype 8. Panel C uses plates coated with intact cells killed by Staphylococcus epidermidis.

The line marked with a square symbol shows the results of ELISA using antisera from mice immunized three times with the indicated staphylococcal proteins. The line marked with diamond symbols shows the ELISA results of the mouse sera before immunization.

FIG. 7-ELISA results of rabbit antisera raised against staphylococcal proteins on plates coated with killed staphylococci.

The line marked with a square symbol shows the results of ELISA using antisera from rabbits, which were immunized three times with the indicated staphylococcal proteins. The line marked with diamond symbols shows the ELISA results of pre-immune rabbit sera.

Detailed Description

Specific combinations of staphylococcal antigens are disclosed which, when combined, result in the production of an immunogenic composition effective in the treatment or prevention of staphylococcal infection. The immunogenic compositions of the invention suitably incorporate antigens involved in infection with different staphylococci. The immunogenic compositions target immune responses against different aspects of staphylococcal function and are therefore capable of inducing a particularly preferred immune response.

Staphylococcal infections progress through several different stages. For example, the life cycle of staphylococci includes commensal proliferation, initiation of infection by contact with adjacent tissues or blood stream, anaerobic proliferation in the blood, interaction between virulence determinants of staphylococcus aureus and host defense mechanisms, and induction of complications including endocarditis, metastatic abscess formation and sepsis syndrome. Different molecules on the surface of the bacteria will participate in different steps of the infectious cycle. By targeting an immune response against a combination of effective amounts of specific antigens involved in different steps of a staphylococcal infection, a staphylococcal immunogenic composition or vaccine with enhanced efficacy can be produced.

In particular, a combination of certain antigens from different classes, some of which are involved in adhesion to host cells, some of which are involved in iron capture or other transporter functions, some of which are toxins or virulence modulators and immunodominant antigens, are capable of eliciting an immune response that prevents multi-stage infections.

The effectiveness of the immune response can be determined by animal model assays as described in the examples and/or using opsonophagocytosis assays as described in the examples.

An additional advantage of the present invention is that combinations of antigens of the invention from different protein families in immunogenic compositions will be able to produce protection against a wider range of strains.

The present invention relates to immunogenic compositions comprising a plurality of proteins selected from at least two different classes of proteins having different functions of staphylococci. Examples of such protein classes are extracellular binding proteins, transport proteins such as Fe capture proteins, toxins or virulence modulators and other immunodominant proteins. The vaccine combination of the invention is effective against homologous staphylococcal strains (strains giving antigens), and preferably also against heterologous staphylococcal strains.

The immunogenic composition of the invention comprises equal to or more than 2,3, 4,5 or 6 proteins selected from the group of 2 or 3 proteins:

For example, the first protein is selected from group a), b) or c), the second protein is selected from a group selected from groups a), b) and c), which does not comprise the second protein.

In a preferred embodiment, the immunogenic composition of the invention comprises at least one protein selected from group a) and additional proteins selected from group b) and/or group c).

In another preferred embodiment, the immunogenic composition of the invention comprises at least one antigen selected from group b) and an additional protein selected from group c) and/or group a).

In another preferred embodiment, the immunogenic composition of the invention comprises at least one antigen selected from group c) and an additional protein selected from group a) and/or group b).

The immunogenic compositions of the invention suitably comprise proteins from staphylococcus aureus and/or staphylococcus epidermidis.

Protein

The immunogenic composition of the invention comprises an isolated protein comprising an amino acid sequence having at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% or complete identity to any of the sequences of figure 1.

Where reference is made explicitly to a protein herein, it preferably refers to the native or recombinant, full-length protein or mature protein optionally from which any signal sequence has been removed. The protein may be isolated directly from the staphylococcal strain or produced by recombinant DNA technology. Immunogenic fragments of the proteins may be added to the immunogenic compositions of the invention. These fragments are fragments comprising at least 10 amino acids, preferably 20 amino acids, more preferably 30 amino acids, more preferably 40 amino acids or 50 amino acids, most preferably 100 amino acids consecutively selected from the amino acid sequence of the protein. In addition, such immunogenic fragments are immunoreactive with antibodies raised against staphylococcal proteins or antibodies raised by infection with staphylococci. Immunogenic fragments also include fragments that, when administered in an effective amount, (either alone or as a hapten conjugated to a carrier), elicit a protective immune response against staphylococcal infection, more preferably protection against s. Such immunogenic fragments may include, for example, proteins lacking an N-terminal leader sequence, and/or a transmembrane domain and/or a C-terminal anchoring domain. In a preferred aspect, the immunogenic fragment according to the invention comprises substantially all of the extracellular domain of a protein having at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% identity to a sequence selected from figure 1 over the entire length of the fragment sequence.

The immunogenic compositions of the invention may also contain a fusion protein of a staphylococcal protein or an immunogenic fragment of a staphylococcal protein. Such fusion proteins may be recombinantly produced and may comprise a portion of at least 2,3, 4,5, or 6 staphylococcal proteins. Alternatively, the fusion protein may comprise portions of at least 2,3, 4, or 5 staphylococcal proteins. These fusion proteins may combine different staphylococcal proteins or fragments thereof into the same protein. Alternatively, the invention also includes fusion proteins of staphylococcal proteins or fragments thereof alone, as fusion proteins with heterologous sequences such as T cell epitopes or providers of purification markers, for example: beta-galactosidase, glutathione-S-transferase, Green Fluorescent Protein (GFP), epitope tags such as FLAG, myc tag, polyhistidine, or viral surface proteins such as influenza virus hemagglutinin, or bacterial proteins such as tetanus toxoid, diphtheria toxoid, CRM 197.

TABLE 1

The following table lists the SEQ ID numbers of the protein and DNA sequences found in fig. 1 and 2, respectively. SA denotes a sequence derived from Staphylococcus aureus, and SE denotes a sequence derived from Staphylococcus epidermidis.

Extracellular component binding proteins

An extracellular component binding protein is a protein that binds to an extracellular component of a host. The term includes, but is not limited to, adhesins.

Examples of extracellular component-binding proteins include laminin receptor (Naidu et al J.Med.Microbiol. 1992, 36; 177), SitC/MntC/saliva-binding protein (US5801234, Wiltshire and Foster Infect.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.Crein.Microbiol.33; 2347), SBI (Sazhang et al FEMS. Med.Microbiol.2000, 28; Im211), autolysin (Runal 2001, J.Infect.Dis.183; Clefa.1038), SamseMS. FEMS. Med.Med.2000, Sdhool.32; Sdhool.32, Sdhool.32; Sdhool.103; Sdhool.19, Sdhool.51, Sdhool.11; Sdhool.51, Sdhool, Sdhool.32; Sdhool, S, collagen binding protein Cna (Visai et al 2000, J.biol. chem.275; 39837), ClfB (WO 99/27109), FbpA (Phonimdaeng et al 1988 J.GenMicrobiol.134; 75), Npase (Flock 2001 J.Bacteriol.183; 3999), IsaA/PisA (Lonenz et al FEMS Immuno.Med.Microbiol.2000, 29; 145), SsaA (Lang et al FEMS Immuno.Med.Microbiol.2000, 29; 213), EPB (Hussain and Hermann, Denmark 14-17)^thSeminar on staphylococci, 2000), SSP-1(Veenstra et al 1996,bacteriol.178; 537) SSP-2(Veenstra et al 1996, J.Bacteriol.178; 537) the 17kDa heparin binding protein HBP (Fallgren et al 2001, j.med.microbiol.50; 547) vitronectin binding protein (Li et al 2001, curr. microbiol.42; 361) fibrinogen binding protein, coagulase, FIG (WO 97/48727) and MAP (US 5648240).

SitC/MntC/saliva binding protein

This is an ABC transporter, which is a homologue of the staphylococcal adhesin PsaA. It is a highly immunogenic 32kDa lipoprotein distributed throughout the bacterial cell wall (Cockayneet al infection. Immun.199866; 3767). It is expressed as a 32kDa lipoprotein in Staphylococcus aureus and Staphylococcus epidermidis, and a 40kDa homologue is present in Staphylococcus hominis. In Staphylococcus epidermidis, it is a component of the iron regulatory operon. It shows considerable homology to adhesins including FimA of streptococcus paracasei, and to lipoproteins of the ABC transporter family with a confirmed or putative metal ion transport function. SitC is thus included as extracellular binding proteins and metal ion transporters.

The saliva binding proteins disclosed in US5,801,234 are also in the form of sitcs and may be included in the immunogenic compositions of the invention.

ClfA and ClfB

Both proteins have fibrinogen binding activity, which causes aggregation of Staphylococcus aureus in the presence of plasma. They contain the LPXTG motif common to cell wall-associated proteins.

ClfA is described in US6008341 and ClfB in WO 99/27109.

Coagulase (FbpA)

This is a fibrinogen-binding protein that causes staphylococcus aureus to form aggregates in the presence of plasma. It is described in references relating to coagulase: phnimdaeng et al (J.Gen.Microbio.1988, 134: 75-83), Phnimdaeng et al (MolMicrobio 1990; 4: 393-404), Cheung et al (infection Immun 1995; 63: 1914-1920) and Shopsin et al (J.CLin.Microbio.2000; 38: 3453-3456).

Preferred fragments for inclusion in the immunogenic compositions of the invention include mature proteins from which the signal peptide (the C-terminal 27 amino acids) has been removed.

Coagulase has three distinct domains. Amino acids 59-297 are coiled coil regions, amino acids 326-505 are proline and glycine rich regions, and the C-terminal domain from amino acids 506 to 645 has a beta-sheet conformation. Each of these domains is a preferred fragment of the invention.

SdrG-Fbe-EfB/FIG

Fbe is a fibrinogen-binding protein found in many isolates of Staphylococcus epidermidis and has a deduced molecular weight of 119kDa (Nilsson et al 1998. infection. Immun.66; 2666). Its sequence correlates with that of the aggregation factor of Staphylococcus aureus (ClfA). anti-Fbe antibodies can block the binding of Staphylococcus epidermidis to fibrinogen-coated plates and catheters (Pei and Flock 2001, J.Infect.Dis.184; 52). This protein is also described as SdrG in WO 00/12689. SdrG is found in coagulase-negative staphylococci and is a cell wall-associated protein containing LPXTG sequences.

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

Fbe has a putative signal sequence with a cleavage site between amino acids 51 and 52. Thus, a preferred fragment of Fbe contains the mature form of Fbe, extending from amino acid 52 to the C-terminus (amino acid 1092).

The domain from amino acid 52 to amino acid 825 of Fbe is responsible for fibrinogen binding. Thus a preferred fragment of Fbe comprises or consists of amino acids 52-825.

The region between amino acids 373 and 516 of Fbe shows the greatest conservation between Fbe and ClfA. Thus, preferably the fragment will contain amino acids 373-516 of Fbe.

Amino acid 825-1041 of Fbe contains a highly repetitive region consisting of tandem repeats of aspartic acid and serine residues.

Preferred fragments of SdrG include polypeptides from which the signal peptide and/or SD repeat sequence and anchoring domain have been removed. These polypeptides include or consist of SEQ ID NO: 70, amino acid 50-825, amino acid 50-633, amino acid 50-597 (SEQ ID NO 2 of WO 03/76470), amino acid 273-597 (SEQ ID NO 4 of WO 03/76470), amino acid 273-577 (SEQ ID NO 6 of WO 03/76470), amino acid 1-549, amino acid 219-549, amino acid 225-549, amino acid 219-528, and amino acid 225-528.

Preferably, the peptide will be identical to SEQ ID NO: 70. 20 or 21 has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% homology to the immunogenic composition of the invention.

The compositions of the invention optionally comprise fragments of the SdrG polypeptides described above.

Preferably the fragment has the signal peptide and/or the SD repeat and/or the anchor domain removed. For example, a sequence corresponding to amino acids 1-713, 1-549, 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 SEQ ID 76 or a sequence having 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID 70 or 20 or 21.

Preferred fragments with the signal peptide removed have a methionine residue at the N-terminus of the fragment to ensure correct translation.

More preferred fragments have the following sequence:

MEENSVQDVKDSNTDDELSDSNDQSSDEEKNDVINNNQSINTDDNNQIIKKEETNNYDGIEKRSEDRTESTTNVDENEATFLQKTPQDNTHLTEEEVKESSSVESSNSSIDTAQQPSHTTINREESVQTSDNVEDSHVSDFANSKIKESNTESGKEENTIEQPNKVKEDSTTSQPSGYTNIDEKISNQDE

LLNLPINEYENKARPLSTTSAQPSIKRVTVNQLAAEQGSNVNHLIKVTDQSITEGYDDSEGVIKAHDAENLIYDVTFEVDDKVKSGDTMTVDIDKNTVPSDLTDSFTIPKIKDNSGEIIATGTYDNKNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQRPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGST

IIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYDNTIAFSTSSGQGQGDLPPEKTYKIGDYVWEDVDKDGIQNTNDNEKPLSNVLVTLTYPDGTSKSVRTDEDGKYQFDGLKNGLTYKITFETPEGYTPTLKHSGTNPALDSEGNSVWVTINGQDDMTIDSGFYQTPKYSLGNY

VWYDTNKDGIQGDDEKGISGVKVTLKDENGNIISTTTTDENGKYQFDNLNSGNYIVHFDKPSGMTQTTTDSGDDDEQDADGEEVHVTITDHDDFSIDNGYYDDE

EbhA and EbhB

EbhA and EbhB are proteins expressed in both S.aureus and S.epidermidis (Clarke and Foster infection. Immun.2002, 70; 6680, Williams et al infection. Immun.2002, 20; 6805) that bind to fibronectin. Since fibronectin is an important component of the extracellular matrix, EbhA and EbhB have important functions in adhering staphylococci to the extracellular matrix.

The Ebh protein is large and has a molecular weight of 1.1 megaDalton. Due to the ease of production and formulation, it is advantageous to use fragments of the Ebh protein rather than the complete sequence. The central region of the protein contains an incomplete repeat sequence that contains a fibronectin binding site. The following fragments containing one or more repeat domains are preferred fragments for incorporation into the immunogenic compositions of the invention.

The Ebh protein contains an incomplete repeat unit of 127 amino acids in length, characterized by containing the consensus sequence:

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

preferably

.{19}L.G.{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.......

Wherein ". times" represents any amino acid, "{ 10 }" represents any 10 amino acids, and I/V represents an alternative to an amino acid.

Reference Kuroda et al (2001) Lancet 357; 1225-1240 and Table 2, the repetitive sequences in the Ebh protein are readily deduced.

Preferred fragments for inclusion in the immunogenic compositions of the invention include proteins comprising one, two, three, four, five, six, seven, eight, nine, ten or more than 10 repeating units of 127 amino acids. Such fragments may consist of 1,2, 3,4, 5,6, 7, 8, 9, 10 or more repeats of a 127 amino acid repeat region, or may consist of 1,2, 3,4, 5,6, 7, 8, 9, 10 or more repeats with additional amino acid residues at either or both termini of the fragment. Another preferred fragment is an approximately 44kDa H2 polypeptide spanning the three repeats (amino acids 3202-3595) as described in Clarke et al Infection and Immunity 70, 6680-6687, 2002. Such fragments will preferably be capable of binding fibronectin and/or eliciting antibodies active against the entire Ebh protein.

The Ebh protein is capable of binding fibronectin. Preferred fragments of these polypeptide sequences retain the ability to bind to fibronectin. Binding to fibronectin can be assessed by ELISA as described by Clarke et al (Infection and identity 70; 6680-.

Yet another preferred fragment is those comprising B cell or T helper cell epitopes, such as those fragments/peptides described in tables 3 and 4.

Table 2. repeat sequences in the full length Ebh sequence.

The full-length sequence of Ebh is disclosed in Kuroda et al (2001) Lancet 357; 1225-1240. The following table shows the amino acid residues at the beginning and end positions of a 127 amino acid repeat in the full-length sequence.

Table 3B cell epitopes predicted for a 127 amino acid repeat sequence:

full length sequences are disclosed in Kuroda et al (2001) Lancet 357; 1225-1240. One of these repeats was selected for epitope prediction as the repeat encoded by amino acids 3204 and 3331 of the full-length sequence:

MDVNTVNQKAASVKSTKDALDGQQNLQRAKTEATNAITHASDL

NQAQKNALTQQVNSAQNVHAVNDIKQTTQSLNTAMTGLKRGV

ANHNQVVQSDNYVNADTNKKNDYNNAYNHANDIINGNAQHPVI

start of	End up	Epitope sequences	Initiation of	Terminate
					5	10	TVNQKA	3208	3213
14	19	KSTKDA	3217	3222
					21	33	DGQQNLQRAKTEA	3224	3236
42	51	DLNQAQKNAL	3245	3254
					66	74	DIKQTTQSL	3269	3277
100	112	ADTNKKNDYNNAY	3303	3315
					117	123	DIINGNA	3320	3326

The columns "start" and "end" represent the positions of the predicted B-cell epitopes in a 127 amino acid repeat

The columns "start" and "stop" represent the positions of predicted B-cell epitopes in the full-length Ebh sequence

Table 4 predicted T helper epitopes in Ebh：

Full-length sequences are disclosed in the TrEMBL database, sequence index Q8NWQ 6. One of these repeats was selected for epitope prediction as encoded by amino acids 3204 and 3331 of the full-length sequence:

MDVNTVNQKAASVKSTKDALDGQQNLQRAKTEATNAITHASDL

NQAQKNALTQQVNSAQNVHAVNDIKQTTQSLNTAMTGLKRGV

ANHNQVVQSDNYVNADTNKKNDYNNAYNHANDIINGNAQHPVI

position in the repetitive sequence	Epitope sequences	Position in sequence
			1	MDVNTVNQK	3204
3	VNTVNQKAA	3206
			6	VNQKAASVK	3209
26	LQRAKTEAT	3229
			37	ITHASDLNQ	3240
43	LNQAQKNAL	3246
			51	LTQQVNSAQ	3254
55	VNSAQNVHA	3258
			61	VHAVNDIKQ	3264
64	VNDIKQTTQ	3267
			67	IKQTTQSLN	3270
74	LNTAMTGLK	3277
			78	MTGLKRGVA	3281
81	LKRGVANHN	3284
			85	VANHNQVVQ	3288
91	VVQSDNYVN	3294
			92	VQSDNYVNA	3295
97	YVNADTNKK	3301
			98	VNADTNKKN	3302
108	YNNAYNHAN	3311
			112	YNHANDIIN	3315
118	IINGNAQHP	3321
			119	INGNAQHPV	3322

The column "position in the repetitive sequence" indicates the position of the predicted T-cell epitope in the repetitive sequence

The column "position in sequence" indicates the position of the predicted T-cell epitope in the full-length Ebh sequence

Fragments of the polypeptides of the invention may be used to produce the corresponding full-length polypeptides by peptide synthesis; thus, these fragments may be used as intermediates in the production of full-length polypeptides of the invention.

Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted or added in any combination.

Elastin binding protein (EbpS)

EbpS is a 83kDa protein of 486 amino acids. It is associated with the cytoplasmic membrane of Staphylococcus aureus, with three hydrophobic regions that retain proteins in the membrane (Down et al 2002, J.biol. chem.277; 243; Park et al 1996, J.biol. chem.271; 15803).

The two regions between amino acids 1-205 and 343-486 were surface exposed on the outer surface of the cytoplasmic membrane. The ligand binding domain of EbpS is located between residues 14-34 at the N-terminus (Parket al 1999, J.biol.chem.274; 2845).

Preferred fragments for incorporation into the immunogenic compositions of the invention are surface exposed fragments (amino acids 1-205) containing the elastin binding region. Some preferred fragments do not contain an intact exposed loop, but should contain an elastin binding region (amino acids 14-34). An alternative fragment that can be used consists of the amino acids forming the second surface exposed loop (amino acids 343-486). Alternative fragments containing as few as 1,2, 5, 10, 20, 50 amino acids at one or both ends are also possible.

Laminin receptors

The laminin receptor of staphylococcus aureus plays an important role in pathogenicity. The infection is typically characterized by a blood stream invasion that allows the formation of extensive metastatic abscesses. Blood invasion requires the ability to flow through the vascular basement membrane. This is achieved by the binding of laminin receptors to laminin (Lopes et al Science 1985, 229; 275).

Laminin is surface exposed and is present in many strains of staphylococcus, including staphylococcus aureus and staphylococcus epidermidis.

SBI

Sbi is a second IgG-binding protein, in addition to protein A, which is expressed in most S.aureus strains (Zhang et al 1998, Microbiology 144; 985).

The N-terminus of the Sbi sequence has a typical signal sequence with a cleavage site after amino acid 29. Preferred fragments of Sbi for incorporation into the immunogenic composition of the invention therefore start at amino acid residues 30, 31, 32 or 33 and continue to the C-terminus of Sbi, e.g. as shown in seq id NO: 26.

the IgG-binding domain of Sbi has been identified as the region from amino acids 41-92 toward the N-terminus of the protein. This domain is homologous to the IgG binding domain of protein a.

The minimal IgG binding domain of Sbi contains the following sequence:

-indicates amino acids that are similar between IgG binding domains

Preferred fragments of Sbi included in the immunogenic compositions of the invention contain an IgG binding domain. This fragment contains the consensus sequence of the IgG binding domain, indicated by x as indicated in the sequence above. Preferably, the fragment contains or consists of all of the sequences shown 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, 33-170, 33-180, 33-190, 33-200, 33-205 or 33-210 of Sbi, such as the amino acid sequence of SEQ ID NO: 26.

preferred fragments may contain 1,2, 3,4, 5,6, 7, 8, 9, 10 amino acid substitutions from the indicated sequence.

Preferred fragments may contain multiple repeats (2, 3,4, 5,6, 7, 8, 9 or 10) of the IgG binding domain.

EFB-FIB

Fib is a 19kDa fibrinogen binding protein which is secreted by Staphylococcus aureus into the extracellular matrix. It was produced by all S.aureus isolates tested (Wastfeland Flock 1995, J.Clin.Microbiol.33; 2347).

Staphylococcus aureus aggregates and binds to fibrinogen-coated surfaces in the presence of fibrinogen. This ability aids in the proliferation of staphylococcal colonies of both catheters and endothelial cells.

Fib contains a signal sequence at the N-terminus of the protein with a putative cleavage site at about amino acid 30. Thus preferred fragments for incorporation into the immunogenic compositions of the invention will contain the sequence of the mature protein (from about amino acid 30 to the C-terminus of the protein).

IsaA/PisA

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

The first 29 amino acids of the IsaA sequence are considered to be the signal sequence. Thus the IsaA preferred sequence included in the immunogenic composition of the invention will contain from amino acid residue 30 to the end of the coding sequence.

Fibronectin binding proteins

Fibronectin binding protein A (FnbA) is described in US5320951 and Schenings et al (1993, Mircob. Patholog.15; 207). Fibronectin-binding protein a contains several domains involved in binding fibronectin (WO 94/18327). These domains are referred to as D1, D2, D3 and D4. Preferred fragments of fibronectin binding protein A or B comprise 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 proteins contain a 36 amino acid signal sequence. For example:

VKNNLRYGIRKHKLGAASVFLGTMIVVGMGQDKEAA

optionally, a mature protein lacking the signal sequence is included in the immunogenic composition of the invention.

Transporter proteins

The cell wall of gram-positive bacteria acts as a barrier to prevent free diffusion of metabolites into the bacteria. A series of proteins cooperate to form pathways for essential nutrients into bacteria and are therefore essential for bacterial survival. The term transporter includes proteins involved in the initial step of binding to a metabolite such as iron as well as those involved in the actual transport of the metabolite into the bacteria.

Molecular iron is an essential cofactor for bacterial growth. The siderophore, which binds free iron, is secreted and then captured by the iron-delivering bacterial surface receptors for transport across the cytoplasmic membrane. Iron detection is critical for determining human infection so that the development of an immune response against such proteins can lead to loss of staphylococcal viability.

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

Immunodominant ABC transporters

The immunodominant ABC transporter is a well-conserved protein that may be able to generate cross-protective immune responses against different Staphylococcus strains (Mei et al 1997, mol. Microbiol.26; 399). Antibodies against this protein have been found in patients with sepsis (Burnie et al 2000, infection. Immun.68; 3200).

Preferred fragments of immunodominant ABC transporters will include the peptides DRHFLN, GNYD, RRYPF, KTTLLK, gvtsls, VDWLR, RGFL, more preferably KIKVYVGNYDFWYQS, TVIVVSHDRHFLYNNV and/or TETFLRGFLGRMLFS, as these sequences contain epitopes recognized by the human immune system.

IsdA-IsdB

The isd gene (iron regulated surface determinant) of staphylococcus aureus encodes a protein responsible for hemoglobin binding and transport of heme iron to the cytoplasm, where it serves as an essential nutrient. IsdA and IsdB are located in the cell wall of staphylococci. IsdA appears to be exposed on the bacterial surface as it is sensitive to proteinase K digestion. IsdB was partially digested, indicating that it was partially exposed on the bacterial surface (Mazmanian et al 2003 Science 299; 906).

Both IsdA and IsdB are heme-binding 29kDa proteins. Their expression is regulated by the availability of iron via the Fur repressor. Their expression will be higher during infection of the host where the iron concentration will be lower.

They are also known as FrpA and FrpB (Morrissey et al 2002, feed. Immun.70; 2399). FrpA and FrpB are major surface proteins with high charge. They have been shown to provide the primary function of adhesion to plastics.

In one embodiment, the immunogenic composition of the invention comprises an IsdA and/or IsaB fragment, which is described in WO 01/98499 or WO 03/11899.

Toxins and virulence modulators

Members of this protein family include toxins such as alpha toxin, hemolysin, enterotoxin B and TSST-1, and proteins that modulate toxin production such as RAP.

Alpha toxin (Hla)

Alpha toxin is an important virulence determinant produced by most staphylococcus aureus strains. It is a pore-forming toxin with hemolytic activity. Antibodies against alpha toxin have been shown to neutralize the deleterious and lethal effects of alpha toxin in animal models (Adlam et al 1977 infection. Immun.17; 250). Human platelets, endothelial cells and monocytes are sensitive to the effects of alpha toxin.

The high toxicity of alpha toxin requires that it should be detoxified before use as an immunogen. This can be achieved by chemical treatment, for example by treatment with formaldehyde, glutaraldehyde or other cross-linking reagents or by chemically conjugating it to the bacterial polysaccharide as described below.

A further way to remove toxicity is to introduce point mutations that remove toxicity while maintaining the antigenicity of the toxin. Introduction of a point mutation at amino acid 35 of the alpha toxin, which mutation is a substitution of a leucine residue for a histidine residue, results in the removal of toxicity while maintaining immunogenicity (Menzies and Kernode 1996; infection. Immun.64; 1839). Histidine 35 appears to be critical for the correct oligomerization required for pore formation, and mutation of this residue results in loss of toxicity.

When added to the immunogenic composition of the invention, the alpha toxin is preferably detoxified by His35 mutation, most preferably by substituting His35 with Leu or Arg. In an alternative embodiment, the alpha toxin is detoxified by conjugation to other components of the immunogenic composition, preferably a capsular polysaccharide, most preferably to a staphylococcus aureus type V polysaccharide and/or a staphylococcus aureus type VIII polysaccharide and/or PNAG.

RNA III activating protein (RAP)

RAP itself is not a toxin. It is a regulator of virulence factor expression. RAP is produced and secreted by staphylococci. It activates the agr regulatory system of other staphylococci and activates expression and subsequent release of virulence factors such as hemolysin, enterotoxin B and TSST-1.

The immune response generated against RAP will not kill the bacteria, but will interfere with their pathogenicity. This has the advantage of providing less selective pressure for the development of new resistant strains.

It would have the second advantage that the resulting immune response would be helpful in reducing the incidence of infection.

It is particularly advantageous to combine RAP with other antigens in the vaccine, particularly where the additional antigen will provide an immune response capable of killing the bacteria.

Other proteins

Accumulation-binding protein (Aap)

Aap is a 140kDa protein essential for the accumulation of Staphylococcus epidermidis strains on the surface (Hussain et al infection. Immun.1997, 65; 519). Strains expressing this protein produce significantly greater amounts of biofilm, Aap appears to be involved in biofilm formation. anti-Aap antibodies are capable of inhibiting biofilm formation and inhibiting the accumulation of staphylococcus epidermidis.

Preferred fragments of Aap are conserved domains comprising or consisting of amino acids 550-1069.

Staphylococcal secreted antigen SsaA

SsaA is a strong immunogenic protein of 30kDa found in both Staphylococcus aureus and Staphylococcus epidermidis (Lang et al 2000 FEMS Immunol. Med. Microbiol. 29; 213). Its expression during endocarditis suggests a virulence effect specific for the onset of infectious diseases.

SsaA contains an N-terminal leader sequence and a signal peptidase cleavage site. The leader peptide is followed by a hydrophilic region of approximately 100 amino acids from residue 30 to residue 130.

Preferred SsaA fragments for addition to the immunogenic compositions of the invention consist of the mature protein (amino acid 27 to the C-terminus or amino acid 30 to the C-terminus).

Another preferred fragment contains the hydrophobic region of SsaA from amino acid 30 to amino acid 130.

Preferred combinations

Preferred combinations of proteins in the immunogenic composition of the invention comprise a laminin receptor and 1,2, 3,4 or 5 additional antigens selected from the group consisting of immunodominant ABC transporter, IsdA, IsdB, Mg²⁺Transporter, SitC, Ni ABC transporter, alpha toxin H35L or H35R mutant, RAP, Aap and SsaA.

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

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

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

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

Another preferred combination of proteins in the immunogenic composition of the invention comprises efb (fib) and 1,2, 3,4 or 5 further antigens selected from the group consisting of immunodominant ABC transporter, IsdA, IsdB, Mg²⁺Transporter, SitC, Ni ABC transporter, alpha toxin H35L or H35R mutant, RAP, Aap and SsaA.

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

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

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

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

Another preferred combination of proteins in the immunogenic composition of the invention comprises SdrG and 1,2, 3,4 or 5 further antigens selected from immunodominantABC transporter, IsdA, IsdB, Mg²⁺Transporter, SitC, Ni ABC transporter, alpha toxin H35L or H35R mutant and RAP.

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

Another preferred combination of proteins in the immunogenic composition of the invention comprises the esterase GehD and 1,2, 3,4 or 5 further antigens selected from the group consisting of immunodominant ABC transporter, IsdA, IsdB, Mg²⁺Transporter, SitC, Ni ABC transporter, alpha toxin H35L or H35R mutant, RAP, Aap and SsaA.

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

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

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

Another preferred protein in the immunogenic compositions of the inventionThe combination comprises Cna and 1,2, 3,4 or 5 further antigens selected from the group consisting of immunodominant ABC transporter, IsdA, IsdB, Mg²⁺Transporter, SitC, Ni ABC transporter, alpha toxin H35L or H35R mutant, RAP, Aap and SsaA.

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

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

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

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

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

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

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

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

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

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

Another preferred combination of proteins in the immunogenic composition of the invention comprises a fibrinogen binding protein and 1,2, 3,4 or 5 further antigens selected from the group consisting of immunodominant ABC transporter, IsdA, IsdB, Mg²⁺Transporter, SitC, Ni ABC transporter, alpha toxin H35L or H35R mutant, RAP, Aap and SsaA.

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

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

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

Another preferred combination of proteins in the immunogenic composition of the invention comprises an immunodominant ABC transporter and 1,2, 3,4 or 5 additional 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, esterase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, fiag, MAP, alpha toxin H35L or H35R mutant, RAP, p and SsaA.

Another preferred combination of proteins in the immunogenic composition of the invention comprises IsdA and 1,2, 3,4 or 5 further 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, esterase 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 H35L or H35R mutant, RAP, p and SsaA.

Another preferred combination of proteins in the immunogenic composition of the invention comprises IsdB and 1,2, 3,4 or 5 further antigens selected from the group consisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), efb (fib), SBI, autolysin, cifa, SdrC, SdrG, SdrH, esterase GehD, SasA, FnbA, FnbB, Cna, cifb, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinogen binding protein, coagulase, Fig, MAP, alpha toxin H35L or H35R mutant, RAP, p and SsaA.

Another preferred combination of proteins in the immunogenic composition of the invention comprises SitC and 1,2, 3,4 or 5 further 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, esterase 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 H35L or H35R mutant, RAP, p and SsaA.

Another preferred combination of proteins in the immunogenic composition of the invention comprises an alpha toxin and 1,2, 3,4 or 5 further 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, esterase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, vitronectin binding protein, fibrinAlbumin binding protein, coagulase, FIG, MAP, immunodominant ABC transporter, IsdA, IsdB, Mg²⁺Transporters, SitC, Ni ABC transporters, Aap and SsaA.

Another preferred combination of proteins in the immunogenic composition of the invention comprises the alpha toxin H35L or H35R mutant and 1,2, 3,4 or 5 further 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, esterase 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⁺Transporters, SitC, Ni ABC transporters, Aap and SsaA.

Another preferred combination of proteins in the immunogenic composition of the invention comprises RAP and 1,2, 3,4 or 5 further 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, esterase 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⁺Transporters, SitC, Ni ABC transporters, Aap and SsaA.

Another preferred combination of proteins in the immunogenic composition of the invention comprises Aap and 1,2, 3,4 or 5 further 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, esterase 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, immunodominantABC transporter, IsdA, IsdB, Mg of (1)²⁺Transporter, SitC, Ni ABC transporter, RAP, alpha toxin and H35L OR H35R alpha toxin.

Another preferred combination of proteins in the immunogenic composition of the invention comprises SsaA and 1,2, 3,4 or 5 further 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, esterase 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 certain antigens generate particularly effective immune responses within the context of antigen mixtures. Thus, one embodiment of the invention is an immunogenic composition comprising IsaA and a staphylococcal transporter protein or IsaA and a staphylococcal regulator or toxin of virulence, or comprising Sbi and a staphylococcal transporter protein or Sbi and a staphylococcal regulator or toxin of virulence, or comprising SdrC and a staphylococcal transporter protein or SdrC and a staphylococcal regulator or toxin of virulence, or IsaA and Sbi, or IsaA and SdrC, or IsaA and an autolysin, or IsaA and Ebh, or Sbi and SdrC, or Sbi and an autolysin, or Sbi and Ebh, or SdrC and an autolysin, or SdrC and Ebh, or autolysin-glucanase and Ebh. For each of these combinations, the protein may be full-length or a fragment having a sequence at least 85%, 90%, 95%, 98%, or 100% identical to the sequence of fig. 1.

In the above and below combinations, the particular protein may optionally be present in the immunogenic composition of the invention as a fragment or fusion protein as described above.

Preferred immunogenic compositions of the invention do not include the protein sequences disclosed in WO 02/094868.

Combination of three proteins

Preferred immunogenic compositions of the invention comprise three protein components of an alpha-toxin, an extracellular component binding protein (preferably an adhesin) and a transporter protein (preferably an iron binding protein) in combination.

In such combinations, the alpha toxin may be chemically detoxified or genetically detoxified by introducing a point mutation, preferably a His35Leu point mutation. The a toxin is present as a free protein or alternatively is conjugated to a polysaccharide or LTA component of the immunogenic composition.

Preferred combinations include:

an immunogenic composition comprising an alpha toxin, IsdA and an extracellular component binding protein 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, esterase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, vitr-2, HBP, SSP connexin binding protein, fibrinogen binding protein, coagulase, Fig and MAP.

An immunogenic composition comprising an alpha toxin, IsdB and an extracellular component binding protein 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, esterase 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.

An immunogenic composition comprising an alpha toxin, IsdA and adhesin 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.

An immunogenic composition comprising an alpha toxin, IsdB and adhesin 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.

An immunogenic composition comprising an alpha toxin, IsdA and a laminin receptor.

An immunogenic composition comprising an alpha toxin, IsdA and EbhA.

An immunogenic composition comprising an alpha toxin, IsdA and EbhB.

An immunogenic composition comprising an alpha toxin, IsdA and EbpS.

An immunogenic composition comprising an alpha toxin, IsdA and efb (fib).

An immunogenic composition comprising an alpha toxin, IsdA and SdrG.

An immunogenic composition comprising an alpha toxin, IsdA and ClfA.

An immunogenic composition comprising an alpha toxin, IsdA and ClfB.

An immunogenic composition comprising an alpha toxin, IsdA and FnbA.

An immunogenic composition comprising an alpha toxin, IsdA and a coagulase.

An immunogenic composition comprising an alpha toxin, IsdA and Fig.

An immunogenic composition comprising an alpha toxin, IsdA and SdrH.

An immunogenic composition comprising an alpha toxin, IsdA and SdrC.

An immunogenic composition comprising an alpha toxin, IsdA and MAP.

An immunogenic composition comprising IsaA and Sbi.

An immunogenic composition comprising IsaA and IsdB.

An immunogenic composition comprising IsaA and IsdA.

An immunogenic composition comprising IsaA and SdrC.

An immunogenic composition comprising IsaA and Ebh or fragments thereof as described above.

An immunogenic composition comprising Sbi and SdrC.

An immunogenic composition comprising Sbi and Ebh or fragments thereof as described above.

An immunogenic composition comprising IsaA, Sbi or SdrC.

Selection of antigens expressed in different clonal lines

Analysis of the presence of virulence factors associated with the staphylococcus aureus population structure indicates that variable virulence genes are present in natural populations of staphylococcus aureus.

Of the clinical isolates of Staphylococcus aureus, at least five clonal lines have been shown to be very common (Booth et al, 2001 infection Immun.69 (1): 345-52). A-hemolysin (hla), fibronectin binding protein A (fnbA) and aggregation factor A (clfA) were shown to be present in most isolates, indicating an important role for these proteins in the survival of Staphylococcus aureus, regardless of strain identity (Booth et al, 2001 infection Immun.69 (1): 345-52). Furthermore, according to Peacock et al 2002, fnbA, clfA, coagulase, spa, map, pvl (Panton-Valentine leukocidin), hlg (gamma-toxin), alpha-toxin and ica appear to be independent of potential cloning structures, suggesting considerable horizontal transfer of these genes.

In contrast, other virulence genes such as fibronectin binding protein b (fnbb), beta-hemolysin (hlb), collagen binding protein (can), TSST-1(tst) and methicillin resistance gene (mecA) are strongly associated with specific strains (Booth et al, 2001 infectimmun.69 (1): 345-52). Likewise, Peacock et al 2002 (infection Immun.70 (9): 4987-96) showed that the distribution of enterotoxin, tst, exitein (eta and etb), β -and δ -toxins, sdr genes (sdrD, sdrE and bbp), cna, ebpS and efb in the population was quite significantly correlated with MLST-derived clonal complexes.

MLST data provide no evidence that the iatrogenic strains represent a distinct subpopulation from those causing community acquired disease or recovered from healthy carriers (Feil et al, 2003J bacteriol.185 (11): 3307-16).

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

In one embodiment, this is achieved by comprising 1,2, 3,4, preferably at least 1 protein expressed in most staphylococcal isolates. Examples of such proteins include alpha-hemolysin (hla), fibronectin binding protein A (fnbA) and aggregation factor A (clfA), coagulase, spa, map, pvl (Panton-Valentine leukocidin), hlg (gamma-toxin) and ica. We have also identified immunodominant ABC transporters, RAP, autolysins (Rupp et al 2001, J. infection. Dis. 183; 1038), laminin receptors, SitC, IsaA/PisA, SPOIIIE (), SsaA, Ebps, SasF (Roche et al 2003, Microbiology 149; 643), EFB (FIB), SBI, ClfB, IsdA, IsdB, FnbB, Npase, EBP, bone sialic acid binding protein II, IsaB/PisB (Lorenz et al FEmmMSituno. Med. Microb.2000, 29; 145), SasH (Roche et al 2003, Biology 149; 643), MRPI, SasD (Roche al 2003, 149; 643), Microbiol (Roche al 2003, 643), autolysin precursors (Rupp et 149)/Sep (Sabiol 1), and autolysins precursors.

In an alternative embodiment, 2 or more proteins expressed in different clonal strain groups are included in an immunogenic composition of the present invention. Preferably, the antigen combination will enable the generation of an effective immune response against a plurality of clonal strains, most preferably against all clonal strains. Preferred combinations include FnbB and β -hemolysin, FnbB and Cna, FnbB and TSST-1, FnbB and mecA, FnbB and SdrD, FnbB and SdrF, FnbB and EbpS, FnbB and Efb, β -hemolysin and Cna, β -hemolysin and TSST-1, β -hemolysin and mecA, β -hemolysin and SdrD, β -hemolysin and SdrF, β -hemolysin and EbpS, β -hemolysin and Efb, Cna and TSST-1, Cna and mecbpb, Cna and SdrD, TSa and SdrF, Cna and EbpS, Cna and Efb, TSST-1 and Mefb, TSST-1 and Sdrb, SdrS-1 and SdrB, SdrS and Edbs, MerF and SdrB, SdrF and SdrF, Sdrb and ErdF.

The above preferred combinations may be combined with the above additional ingredients.

Selection of antigens expressed at different stages of growth

Staphylococci undergo an exponential growth phase in which a specific set of proteins is expressed. This includes many extracellular component binding proteins and transporters. After the exponential growth phase, staphylococci return to the post-exponential phase where they grow slower and regulate protein expression. Many proteins expressed during the exponential growth phase are down-regulated, while other proteins, such as enzymes and most toxins, including alpha toxin, are expressed at higher levels.

Preferred immunogenic compositions of the invention comprise proteins expressed at higher levels during the exponential growth phase and proteins expressed at higher levels during the post-exponential phase.

By "higher level" is meant that the expression level is higher in one stage relative to the other.

In a preferred embodiment, the immunogenic composition of the invention comprises an alpha toxin and an extracellular component binding protein (preferably FnbA, FnbB, ClfA and ClfB) or transporter.

More preferably, it comprises an alpha toxin or Cna or esterase GehD and a protein selected from the group consisting of: layer adhesionProtein receptors, SitC/MntC/saliva binding proteins, elastin binding proteins (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 proteins, fibrinogen binding proteins, coagulase, FIG, MAP, immunodominant ABC transporter, IsdA, IsdB, Mg²⁺Transporters, SitC, Ni ABC transporters, Aap and SsaA.

In the combinations described above, the alpha toxin may be genetically or chemically detoxified as described above, and may or may not be conjugated to a polysaccharide as described below.

Polysaccharides

The immunogenic compositions of the invention preferably further comprise capsular polysaccharides, including one or more type V and/or type VIII capsular polysaccharides of PIA (also known as PNAG) and/or staphylococcus aureus and/or type I and/or type II and/or type III capsular polysaccharides of staphylococcus epidermidis.

PIA(PNAG)

It is now clear that the various forms of staphylococcal surface polysaccharides identified as PS/A, PIA and SAA are the same chemical entity-PNAG (Maira-Litran et al Vaccine 22; 872-879 (2004)). The term PIA or PNAG thus includes all such polysaccharides or oligosaccharides derived therefrom.

PIA is a polysaccharide intercellular adhesin consisting of a polymer of- (16) -linked glucosamine substituted with N-acetyl and O-succinyl components. The polysaccharide is present in both Staphylococcus aureus and Staphylococcus epidermidis and can be isolated from either source (Joyce et al 2003, Carbohydrate Research 338; 903; Maira-Litran et al 2002, Infect. Imun.70; 4433). For example, PNAG may be isolated from staphylococcus aureus strain MN8m (WO 04/43407).

PIA isolated from staphylococcus epidermidis is a constituent of biofilms. It is responsible for mediating cell-cell adhesion and perhaps also acts to prevent growing colonies from the host's immune response.

This polysaccharide has previously been referred to as poly-N-succinyl-beta-, (6) Glucosamine (PNSG), which recently showed no expected structure, since it was determined that N-succinylation was incorrect (Maira-Litran et al 2002, feed. imun.70; 4433). Thus, polysaccharides formally known as PNSG and now found to be PNAG are also encompassed by the term PIA.

The PIA (or PNAG) may be oligosaccharides of varying sizes consisting of up to 30 repeating units (a- (16) -linked glucosamine polymer substituted with N-acetyl and O-succinyl moieties) varying from over 400kDa, to between 75 and 400kDa, to between 10 and 75 kDa. Any size of PIA polysaccharide or oligosaccharide may be used in the immunogenic composition of the invention, however sizes above 40kDa are preferred. Size fractionation can be obtained by any method known in the art, for example by microfluidization, ultrasound irradiation or by chemical lysis (WO03/53462, EP497524, EP 497525).

Preferred sizes of PIA (PNAG) are 40-400kDa, 50-350kDa, 40-300kDa, 60-300kDa, 50-250kDa and 60-200 kDa.

PIA (pnag) PIA may have varying degrees of acetylation due to the substitution of the amino group by acetate. The PIA produced in vitro is almost completely substituted on the amino group (95-100%). Alternatively, deacetylated pia (pnag) with less than 60%, preferably less than 50%, 40%, 30%, 20%, 10% acetylation may be used. The use of deacetylated Pia (PNAG) is preferred because the non-acetylated epitope of PNAG is effective in mediating opsonin killing of gram positive bacteria, preferably staphylococcus aureus and/or staphylococcus epidermidis. More preferably, the pia (pnag) has a size between 40kDa and 300kDa and is deacetylated such that less than 60%, 50%, 40%, 30% or 20% of the amino groups are acetylated.

The term deacetylating PNAG (dPNAG) refers to PNAG polysaccharides or oligosaccharides with less than 60%, 50%, 40%, 30%, 20% or 10% of the amino groups acetylated.

In one embodiment, PNAG is deacetylated by chemical treatment of the natural polysaccharide to form dPNAG. For example, native PNAG is treated with an alkaline solution to raise the pH above 10. For example with 0.1-5M, 0.2-4M, 0.3-3M, 0.5-2M, 0.75-1.5M or 1M NaOH, KOH or NH₄OH treatment of PNAG. The treatment is carried out for at least 10 or 30 minutes, alternatively 1,2, 3,4, 5, 10, 15 or 20 hours, at a temperature of 20-100, 25-80, 30-60 or 30-50 or 35-45 ℃. The dPNAG may be prepared as described in WO 04/43405.

The polysaccharides included in the immunogenic compositions of the invention are preferably conjugated to a carrier protein, as described below, or alternatively are not conjugated.

Type 5 and 8 polysaccharides from Staphylococcus aureus

Most staphylococcus aureus strains that cause infections in humans contain type 5 or type 8 polysaccharides. About 60% of the human strains are of type 8 and about 30% are of type 5. The structures of type 5 and type 8 capsular polysaccharide antigens are described in Moreau et al Carbohydrate res.201; 285(1990) and Fournier et al Infect.Immun.45; 87 (1984). Both have FucNAcp in their repeat units and ManNAcA, useful for introducing sulfhydryl groups. The reported structures are:

type 5

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

8 type

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

Recently (Jones Carbohydrate Research 340, 1097-:

type 5

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

8 type

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

The polysaccharides can be extracted from suitable staphylococcus aureus strains using methods well known to those skilled in the art, for example as described in US 6294177. For example, ATCC 12902 is a type 5 strain of Staphylococcus aureus and ATCC 12605 is a type 8 strain of Staphylococcus aureus.

The polysaccharide is of natural size or alternatively may be size fractionated by, for example, microfluidization, ultrasonic irradiation or by chemical treatment. The present invention also includes oligosaccharides derived from staphylococcus aureus type 5 and type 8 polysaccharides.

The type 5 and type 8 polysaccharides included in the immunogenic compositions of the invention are preferably conjugated to a carrier protein as described below, or alternatively are not conjugated.

Alternatively, the immunogenic composition of the invention comprises a type 5 or type 8 polysaccharide.

Staphylococcus aureus 336 antigen

In one embodiment, the immunogenic composition of the invention comprises the staphylococcus aureus 336 antigen described in US 6294177.

The 336 antigen comprises a β -linked hexosamine, free of O-acetyl groups, that specifically binds to antibodies directed against the type 336 staphylococcus aureus deposited as ATCC 55804.

In one embodiment, the 336 antigens are of native size or alternatively may be size fractionated by, for example, microfluidization, ultrasound irradiation or by chemical treatment. The present invention also includes oligosaccharides derived from the 336 antigen.

The 336 antigen included in the immunogenic composition of the invention is preferably conjugated to a carrier protein as described below, or alternatively is not conjugated.

I, II and type III polysaccharides from Staphylococcus epidermidis

Staphylococcus epidermidis strains ATCC-31432, SE-360 and SE-10 are characterized by three different capsules I, II and type III, respectively (Ichiman and Yoshida 1981, J.Appl.Bacteriol.51; 229). Capsular polysaccharides extracted from each of the staphylococcus epidermidis serotypes constitute the I, II and type III polysaccharides. Polysaccharides can be extracted by several methods, including the method described in US4197290 or according to Ichiman et al 1991, j.appl.bacteriol.71; 176.

In one embodiment of the invention, the immunogenic composition comprises a type I and/or II and/or III polysaccharide or oligosaccharide from staphylococcus epidermidis.

The polysaccharide is of natural size or alternatively may be size fractionated by, for example, microfluidization, ultrasonic irradiation or by chemical lysis. The invention also includes oligosaccharides extracted from a staphylococcus epidermidis strain.

These polysaccharides are not conjugated or preferably conjugated as described below.

Conjugation of polysaccharides

Among the problems associated with the use of polysaccharides in vaccination is the fact that polysaccharides are themselves weak immunogens. Strategies that have been designed to overcome this lack of immunogenicity include the attachment of polysaccharides to larger protein carriers, which provide bystander T cell help. Preferably, the polysaccharides utilized in the present invention are linked to a protein carrier that provides bystander T cell help. Examples of such carriers that may be conjugated to the polysaccharide immunogen include diphtheria and tetanus toxoids (DT, DT Crm197 and TT respectively), Keyhole Limpet Haemocyanin (KLH), and purified protein derivatives of tuberculin (PPD), pseudomonas aeruginosa exoprotein a (repa), protein D from haemophilus influenzae, pneumolysin or fragments of any of the foregoing. Fragments suitable for use include fragments comprising a T helper epitope. In particular, a protein D fragment will preferably comprise the N-terminus 1/3 of the protein. Protein D is an IgD binding protein from haemophilus influenzae (EP 0594610B 1) and is a potential immunogen.

In addition, staphylococcal proteins can be used as carrier proteins in the polysaccharide conjugates of the invention. Staphylococcal proteins described below can be used as carrier proteins; for example, laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB, elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, esterase 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⁺Transporters, SitC and Ni ABC transporters, alpha toxin (Hla), alpha toxin H35R mutant, RNA III activating protein (RAP), or fragments thereof.

A novel carrier protein particularly advantageous for use in staphylococcal vaccines is staphylococcal alpha toxoid. The native form can be conjugated to the polysaccharide because the conjugation step reduces toxicity. Genetically detoxified alpha toxins such as His35Leu or His35Arg variants are preferably used as vectors because of lower residual toxicity. Alternatively, the alpha toxin is chemically detoxified by treatment with a cross-linking reagent, formaldehyde or glutaraldehyde. The genetically detoxified alpha toxin is optionally chemically detoxified, preferably by treatment with a cross-linking reagent, formaldehyde or glutaraldehyde to further reduce toxicity.

The polysaccharide can be linked to the carrier protein by any known method (e.g., by Likhite, U.S. Pat. No.4,372,945, Armor, U.S. Pat. No.4,474,757, and Jennings et al, U.S. Pat. No.4,356,170). Preferably, CDAP conjugation chemistry is performed (see WO 95/08348).

In CDAP, the cyanating reagent 1-cyano-dimethylaminopyridine tetrafluoroborate (CDAP) is preferably used for the synthesis of polysaccharide-protein conjugates. The cyanation reaction can be carried out under relatively mild conditions, which avoids hydrolysis of the alkali-sensitive polysaccharide. This synthesis allows direct coupling to the carrier protein.

The polysaccharide may be dissolved in water or a salt solution. CDAP can be dissolved in acetonitrile and added immediately to the polysaccharide solution. The CDAP reacts with the hydroxyl groups of the polysaccharide to form cyanate esters. After the reaction step, a carrier protein is added. The amino group of lysine reacts with the activated polysaccharide to form an isourea covalent linkage. After the coupling reaction, a large excess of glycine is then added to quench the remaining activated functional groups. The product is then passed through a gel permeation column to remove unreacted carrier protein and residual reagents.

Conjugation preferably involves creating a direct bond between the carrier protein and the polysaccharide. Optionally, a spacer, such as adipic Anhydride (ADH), may be introduced between the carrier and the polysaccharide.

Protective effect against staphylococcus aureus and staphylococcus epidermidis

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

One use of the immunogenic compositions of the invention is to prevent iatrogenic infections by vaccination prior to hospital treatment. At this stage, it is difficult to predict with precision which staphylococcal strains a patient will be exposed to. It is therefore advantageous to vaccinate with a vaccine that is capable of generating an effective immune response against a variety of staphylococcal strains.

An effective immune response is defined as producing significant protection in a mouse challenge model or opsonophagocytosis assay as described in the examples. Significant protection in a mouse challenge model such as example 5 is defined as at least a 10%, 20%, 50%, 100% or 200% increase in LD50 compared to a vector-inoculated mouse. Significant protection in a cotton rat challenge model such as example 8 is defined as at least a 10%, 20%, 50%, 70% or 90% reduction in the average log CFU/nose observed. The presence of opsonic antibodies is known to correlate with protection, and thus a reduction in bacterial count of at least 10%, 20%, 50%, 70% or 90% in an opsonophagocytosis assay, such as that of example 7, indicates significant protection.

Several proteins including immunodominant ABC transporter, RNA III activator, laminin receptor, SitC, IsaA/PisA, SsaA, EbhA/EbhB, EbpS and Aap are fairly conserved between staphylococcus aureus and staphylococcus epidermidis, example 8 shows that IsaA, ClfA, IsdB, SdrG, HarA, FnbpA and Sbi are capable of generating cross-reactive immune responses (e.g., cross-reactivity between at least one staphylococcus aureus and at least one staphylococcus epidermidis strain). PIA is also well conserved between staphylococcus aureus and staphylococcus epidermidis and is capable of inducing cross-immune responses.

Thus in a preferred embodiment, the immunogenic composition of the invention will comprise two, three or four of the above proteins, preferably further comprising pia (pnag).

Polynucleotide vaccine

In another aspect, the invention relates to the use of the polynucleotide of FIG. 2 in the treatment, prevention or diagnosis of a staphylococcal infection. Such polynucleotides include isolated polynucleotides comprising a polypeptide that encodes at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity to the amino acid sequence of figure 1 over the entire length of the sequence. In this regard, polypeptides having at least 97% identity are highly preferred, while polypeptides having at least 98-99% identity are more highly preferred, with polypeptides having at least 99% identity being most highly preferred.

Other polynucleotides useful in the present invention include isolated polynucleotides comprising a nucleotide sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, more preferably at least 95% identical, to the nucleotide sequence encoding the protein of the present invention throughout the coding region. In this regard, highly preferred are polynucleotides having at least 97% identity, while polynucleotides having at least 98-99% identity are more highly preferred, with polynucleotides having at least 99% identity being most highly preferred.

Other polynucleotides include isolated polynucleotides comprising a nucleotide sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, more preferably at least 95% identical to the sequence of figure 1. In this regard, highly preferred are polynucleotides having at least 97% identity, while polynucleotides having at least 98-99% identity are more highly preferred, with polynucleotides having at least 99% identity being most highly preferred. The polynucleotide may be inserted into a suitable plasmid or recombinant microbial vector and used for immunization (see, e.g., Wolff et al., Science 247: 1465-.

The invention also provides nucleic acids encoding the aforementioned proteins of the invention and their use in medicine. In a preferred embodiment, the isolated polynucleotide of the invention may be single-stranded (coding or antisense) or double-stranded, and may be a DNA (genomic, cDNA or synthetic) or RNA molecule. Other coding or non-coding sequences may, but need not, be present in the polynucleotides of the invention. In other related embodiments, the present invention provides polynucleotide variants having significant identity to the sequences disclosed in fig. 2; polynucleotides having at least 75% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a polynucleotide sequence of the invention using the methods described herein (e.g., BLAST analysis using standard parameters). In related embodiments, an isolated polynucleotide of the invention will comprise a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95% and more identity to the amino acid sequence of figure 1 over the entire length of the sequence of figure 1; or a nucleotide sequence complementary to the isolated polynucleotide.

The present invention also relates to the use of polynucleotides complementary to all of the above polynucleotides.

The invention also provides the use of a fragment of a polynucleotide of the invention, which fragment has the same immunogenic characteristics as the polynucleotide of figure 1, when administered to a subject.

The invention also provides the use of a polynucleotide encoding an immunogenic fragment of the protein of figure 1 as defined herein. Also contemplated is the use of fragments having a level of immunogenic activity that is at least about 50%, preferably at least about 70%, more preferably at least about 90% of the level of immunogenic activity of the polypeptide sequence encoded by the polynucleotide shown in figure 2.

Polypeptide fragments for use in the present invention preferably comprise at least about 5, 10, 15, 20, 25, 50 or 100 or more contiguous amino acids, including all intermediate lengths of the polypeptide components described herein, such as those listed above.

Polynucleotides for use in the invention can be obtained from cDNA libraries of mRNA from cells derived from human pre-tumor or tumor tissue (e.g., lung) using standard Cloning and screening techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2)^ndEd., Cold Spring harbor Laboratory Press, Cold Spring harbor, N.Y. (1989)). Polynucleotides of the invention may also be obtained from natural sources such as genomic DNA libraries, or may be synthesized using well-known and commercially available techniques.

There are several methods available and well known to those skilled in the art to obtain full-length cDNAs or extended short cDNAs, such as those based on the Rapid Amplification of CDNA Ends (RACE) (see, e.g., Frohman et al, PNAS USA 85, 8998-. Recent improvements to this technique, e.g. Marathon^TMThe technology (clontech laboratories Inc.) significantly simplifies the search for longer cDNAs. In Marathon^TMIn the technique, cDNAs are prepared from mRNA extracted from a selected tissue and "adaptor" sequences are ligated to each end. Nucleic acid amplification (PCR) is then performed to amplify the "missing" 5' ends of the cDNA with a combination of gene-specific and adaptor-specific oligonucleotide primers. The PCR reaction is then repeated with "nested" primers, i.e. primers designed to anneal within the amplified product (typically adaptor-specific primers that anneal more 3 'in the adaptor sequence and gene-specific primers that anneal more 5' of the known gene sequence). The products of this reaction can then be analyzed by DNA sequencing, by constructing full-length cDNAs by ligating the products directly to the existing cDNAs to obtain the complete sequence, or by performing a separate full-length PCR with new sequence information for the design of the 5' primers.

Vectors comprising said DNA, hosts transformed with said vectors and their truncated or hybrid proteins expressed as follows all form part of the invention.

The expression vector may also be a recombinant live microorganism, such as a virus or bacterium. The gene of interest may be inserted into the genome of a live recombinant virus or bacterium. Vaccination and in vivo infection with the live vector will result in vivo expression of the antigen and induction of an immune response.

Thus, in certain embodiments, polynucleotides encoding immunogenic polypeptides for use in the present invention are introduced into suitable mammalian host cells for expression using any of a variety of known virus-based systems. In an illustrative embodiment, retroviruses provide a convenient and efficient platform for gene delivery systems. Selected nucleotide sequences encoding polypeptides for use in the present invention may be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. No.5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-.

In addition, a number of illustrative adenovirus-based systems have been described. Unlike retroviruses which integrate into the host genome, adenoviruses remain extrachromosomal, thus minimizing the risk associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J.Virol.57: 267-274; Bett et al (1993) J.Virol.67: 5911-5921; Mitterer et al (1994) Human 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 Therapy 4: 461-476).

Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, for example, U.S. Pat. nos. 5,173,414 and 5,139,941; international publication nos. WO 92/01070 and WO 93/03769; lebkowski et al (1988) molecular. 3988-; vincent et al (1990) Vaccines 90(Cold Spring harbor laboratory Press); carter, B.J, (1992) Current Opinion in biotechnology 3: 533-; muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158: 97 to 129; kotin, R.M. (1994) HumanGene Therapy 5: 793-801; shelling and Smith (1994) Gene Therapy 1: 165-169; and Zhou et al (1994) j.exp.med.179: 1867-1875.

Other viral vectors useful for delivering nucleic acid molecules encoding polypeptides of the invention by gene transfer include those derived from the poxvirus family, such as vaccinia virus and avipoxvirus. For example, vaccinia virus recombinants expressing the molecule of interest can be constructed as follows. The DNA encoding the polypeptide is first inserted into a suitable vector so that it is proximal to the vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding Thymidine Kinase (TK). This vector was then used to transfect cells that were also infected with vaccinia. Homologous recombination is used to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The TK.sup. (-) recombinants produced can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and selecting viral plaques resistant thereto.

Vaccinia-based infection/transfection systems can be conveniently used to provide inducible, transient expression or co-expression of one or more of the polypeptides described herein in a host cell of an organism. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant encoding bacteriophage T7RNA polymerase. This polymerase showed precise specificity, transcribing only the template with the T7 promoter. Following infection, cells are transfected with a polynucleotide driven by the T7 promoter or a polynucleotide of interest. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into a polypeptide by the host's translation machinery. The method provides for the production of high levels, transient, cytoplasmic quantities of RNA and its translation products. See, e.g., ElroyStein and Moss, proc.natl.acad.sci.usa (1990) 87: 6743-6747; fuerst et al proc natl acad sci usa (1986) 83: 8122-8126.

Alternatively, avian poxviruses such as fowlpox (fowlpox) and fowlpox (canarypox) viruses may also be used to deliver the coding sequence of interest. Recombinant fowlpox viruses expressing immunogens from mammalian pathogens are known to confer protective immunity when administered to non-avian species. The administration of Avipox vectors in humans and other mammalian species is particularly desirable because members of the Avipox genus replicate productively only in susceptible avian species and are therefore not infectious in mammalian cells. Methods for producing recombinant fowlpox viruses are known in the art and employ genetic recombination, as described above in relation to the production of vaccinia virus. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used to deliver the polynucleotide compositions of the present invention, such as those described in U.S. patent nos. 5,843,723; 6,015,686, respectively; 6,008,035 and 6,015,694. Certain Venetian Equine Encephalitis (VEE) based vectors may also be used, illustrative examples of which may be found in U.S. patent nos. 5,505,947 and 5,643,576.

In addition, molecules may also be conjugated to carriers, such as Michael et al.j.biol.chem. (1993) 268: 6866-: the adenovirus chimeric vectors described in 6099-6103 are used for gene delivery according to the present invention.

Additional illustrative information on these and other well-known viral-based delivery systems can be found, for example, in Fisher-Hoch et al, proc.natl.acad.sci.usa 86: 317 and 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 nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. patent No.4,777,127; GB 2,200,651; EP 0,345,242; 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-; Kass-Eisleret al, proc.natl.acad.sci.usa 90: 11498. 11502, 1993; guzman et al, Circulation 88: 2838 2848, 1993; and Guzman et al, cir.res.73: 1202-1207, 1993.

The recombinant live microorganisms described above may be virulent or attenuated in various ways to obtain live vaccines. Such live vaccines also form part of the present invention.

In certain embodiments, the polynucleotide may be integrated into the genome of the target cell. The integration can occur at specific locations and orientations by homologous recombination (gene replacement) and can be at random, non-specific locations (gene enhancement). In a further embodiment, the polynucleotide may be stably maintained in the cell as a separate, additional fragment of DNA. The polynucleotide fragment or "episome" encodes a sequence sufficient to be maintained and replicated independently of, or in synchrony with, the host cell cycle. The manner in which the expression construct is delivered to the cell and the polynucleotide is maintained in the cell is independent of the expression of the construct employed.

In another embodiment of the invention, the polynucleotide of the invention is administered/delivered as "naked" DNA, for example as described in Ulmer et al, Science 259: 1745. cndot. 1749, 1993 and Cohen, Science 259: 1691 1692, 1993. Uptake of naked DNA can be increased by coating the DNA in biodegradable beads that are efficiently transported into the cell.

In another embodiment, the compositions of the present invention may be delivered by particle bombardment methods, many of which have been described. In one illustrative example, gas-driven particle acceleration can be achieved using devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject vaccines Inc. (Madison, Wis.), some examples of which are described in U.S. Pat. Nos. 5,846,796; 6,010,478, respectively; 5,865,796, respectively; 5,584,807; and EP Patent No. 0500799. This method provides a needle-free delivery method in which a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high velocity within a helium gas jet produced by a hand-held device, propelling the particles into the target tissue of interest.

Other devices and methods useful for gas-driven, needle-free injection of compositions of the invention in related embodiments include those provided by Bioject, inc. (Portland, OR), some examples of which are described in U.S. patent nos.4,790,824; 5,064,413, respectively; 5,312,335, respectively; 5,383,851, respectively; 5,399,163; 5,520,639 and 5,993,412.

Vaccine

In a preferred embodiment, the immunogenic composition of the invention is mixed with a pharmaceutically acceptable excipient, more preferably an adjuvant, to form a vaccine.

The vaccine of the invention is preferably adjuvanted. Suitable adjuvants include aluminium salts such as aluminium hydroxide gel (alum) or aluminium phosphate, but may also be calcium, magnesium, iron or zinc salts, or may be an insoluble suspension of acylated tyrosine or acylated sugars, cationically or anionically derivatised polysaccharides or polyphosphazenes.

Preferably the adjuvant selected is a preferential inducer of a TH1 or TH2 type response. High levels of Th 1-type cytokines tend to aid in the induction of cell-mediated immune responses to particular antigens, while high levels of Th 2-type cytokines tend to aid in the induction of humoral immune responses to antigens.

It is important to remember that the distinction between Th1 and Th2 type immune responses is not absolute. Virtually everyone will maintain an immune response described as either a Th1 preponderance or a Th2 preponderance. However, it is often convenient to consider the cytokine family in terms of the different patterns of lymphokine secretion described by Mosmann and Coffman in the murine CD4+ ve T cell clone (Mosmann, T.R. and Coffman, R.L. (1989) TH1 and TH2 cells: different functional properties (TH1 and TH2 cells: differential patterns of lymphokine differentiation lead to differential functions). Annual Review of Immunology, 7, p 145-173). Traditionally, Th1 type responses have been associated with INF-gamma and IL-2 cytokines produced by T-lymphocytes. Other cytokines that are often directly associated with the induction of Th 1-type immune responses are not produced by T cells, such as IL-12. In contrast, Th2 type responses are associated with secretion of IL-4, IL-5, IL-6, IL-10. Suitable adjuvant systems that promote a preferential response to Th1 include: monophosphoryl lipid a or a derivative thereof, in particular 3-deoxy-acylated monophosphoryl lipid a (3D-MPL) (for its preparation see GB 2220211A); combinations of monophosphoryl lipid a, preferably 3-deoxy-acylated monophosphoryl lipid a, plus aluminium salts (e.g. aluminium phosphate or aluminium hydroxide) or oil-in-water emulsions. In such a combination, the antigen and 3D-MPL are contained within the same particulate structure so as to enable more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further enhance the immunogenicity of alum-adsorbed antigens [ Thoelen et al vaccine (1998) 16: 708-14; EP 689454-B1 ].

The enhancement system involves the combination of monophosphoryl lipid a with a saponin derivative, in particular QS21 and 3D-MPL as disclosed in WO94/00153, or a less reactive composition in which QS21 is quenched with cholesterol as disclosed in WO 96/33739. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO95/17210, which is a preferred formulation. Preferably, the vaccine additionally comprises a saponin, more preferably QS 21. The formulation may also comprise an oil-in-water emulsion and tocopherol (WO 95/17210). The invention also provides a method of producing a vaccine formulation comprising mixing together a protein of the invention and a pharmaceutically acceptable excipient such as 3D-MPL. Unmethylated CpG-containing oligonucleotides (WO 96/02555) are also preferred inducers of TH1 responses and are suitable for use in the present invention.

Preferred compositions of the invention are those that form liposome structures. Compositions wherein the sterol/immunologically active saponin fraction forms an ISCOM structure also form an aspect of the invention.

The ratio of QS21 to sterol will typically be about 1: 100 to 1: 1 by weight. Preferably, an excess of sterol is present, the ratio of QS 21: sterol being at least 1: 2 w/w. For human administration, the QS21 and sterol will typically be present in the vaccine in the range of about 1 μ g to about 100 μ g, preferably about 10 μ g to about 50 μ g per dose.

The liposomes preferably contain natural lipids, such as lecithin, preferably non-crystalline at room temperature, such as egg yolk lecithin, dioleoyl lecithin or dilauryl group lecithin. Liposomes may also contain charged lipids that increase the structural stability of the liposome-QS 21, since liposomes are composed of saturated lipids. In these cases, the amount of charged lipid is preferably 1-20% w/w, most preferably 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), most preferably 20-25%.

Preferably, the compositions of the present invention contain MPL (3-deacylated monophosphoryl lipid A, also known as 3D-MPL). From GB 2220211 (Ribi), 3D-MPL is known as a mixture of 3-type deoxyacylated monophosphoryl lipid A with 4,5 or 6 acylated chains, produced by Ribi Immunochem, Montana. The preferred form is disclosed in international patent application 92/116556.

Suitable compositions of the invention are those in which the liposomes are initially prepared without MPL and then MPL is added, preferably to 100nm particles. Thus MPL is not contained inside the vesicle membrane (called MPL outside). Compositions in which MPL is contained within the membrane of the vesicle (referred to as MPL internally) also form an aspect of the invention. The antigen may be contained within the vesicle membrane or may be contained outside the vesicle membrane. Preferably, the soluble antigen is external and the hydrophobic or lipidated antigen is contained within or outside the membrane.

The vaccine formulations of the present invention are useful for protecting or treating mammals from susceptibility to infection by administering the vaccine via systemic or mucosal routes. Such administration may include injection by intramuscular, intraperitoneal, intradermal or subcutaneous routes; or transmucosal administration to the oral/esophageal, respiratory, genitourinary tracts. Intranasal administration of a vaccine for the treatment of pneumonia or otitis media is preferred (as nasopharyngeal delivery of pneumococci can be more effectively prevented, thereby reducing infection at its earliest stage). Although the vaccine of the invention may be administered as a single dose, the components may also be co-administered simultaneously or at different times (e.g. the pneumococcal polysaccharides may be administered separately, either simultaneously with administration of any bacterial protein component of the vaccine or 1-2 weeks later, for optimal coordination of the immune responses with each other). For co-administration, the Th1 adjuvant may optionally be present in any or all of the different administrations, however it is preferred that it is present in association with the bacterial protein component of the vaccine. In addition to a single route of administration, 2 different routes of administration may also be used. For example, the polysaccharide may be administered IM (or ID) and the bacterial protein may be administered IN (or ID). IN addition, the vaccine of the present invention can be administered IM for a sensitizer and IN for a booster.

The amount of conjugated antigen in each dose of vaccine is selected to induce an immunoprotective response in a typical vaccine without significant adverse side effects. Such amounts will vary depending on which particular immunogen is employed and how it is presented. Generally, it is desirable that each dose will contain 0.1-100. mu.g of polysaccharide, preferably 0.1-50. mu.g of polysaccharide conjugate, preferably 0.1-10. mu.g, more preferably 1-10. mu.g, with 1 to 5. mu.g being a more preferred range.

The amount of protein antigen in the vaccine will typically be in the range 1-100. mu.g, preferably 5-50. mu.g, most typically 5-25. mu.g. After the initial vaccination, the subject may receive one or several booster immunizations at appropriate intervals.

Vaccine preparation is generally described in Vaccine Design ("subunit and adjuvant approach") (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York). Encapsulation in liposomes is described in U.S. Pat. No.4,235,877 to Fullerton.

The vaccines of the present invention may be stored in solution or lyophilized. Preferably, the solution is lyophilized in the presence of a sugar such as sucrose, trehalose or lactose. It is still further preferred that they are lyophilized and reconstituted in situ prior to use. Lyophilization may result in a more stable composition (vaccine), possibly resulting in higher antibody titers in the presence of 3D-MPL and in the absence of aluminum-based adjuvant.

Antibodies and passive immunization

Another aspect of the invention is a method for preparing an immunoglobulin for use in the prevention or treatment of staphylococcal infection comprising the steps of immunizing a recipient with a vaccine of the invention and isolating the immunoglobulin from the recipient. Immunoglobulins prepared by this method are a further aspect of the invention. Pharmaceutical compositions comprising the immunoglobulins of the present invention and a pharmaceutically acceptable carrier are further aspects of the invention, which may be used in the manufacture of a medicament for the treatment or prevention of staphylococcal diseases. A further aspect of the invention is a method for treating or preventing a staphylococcal infection comprising the step of administering to a patient an effective amount of a pharmaceutical formulation of the invention.

Inocula for polyclonal antibody production are typically prepared by dispersing the antigenic composition in a physiologically compatible diluent such as saline or other suitable adjuvant for human use to form a water-soluble composition. An immunostimulatory amount of the inoculum is administered to the mammal, and the inoculated mammal is then maintained for a time sufficient for the antigenic composition to induce protective antibodies.

Antibodies can be isolated to the extent desired by well-known techniques such as affinity chromatography (Harlow and Lane Antibodies; a laboratory manual 1988).

The antibody may include antisera from various commonly used animals, such as goats, primates, donkeys, pigs, horses, guinea pigs, rats or humans. Blood was collected from the animals and serum was recovered.

Immunoglobulins produced according to the present invention may include whole antibodies, antibody fragments or subfragments. The antibody may be any type of whole immunoglobulin, such as IgG, IgM, IgA, IgD, or IgE, a chimeric antibody or a hybrid antibody having bispecific properties against two or more antigens of the invention. They may also be fragments, such as F (ab ') 2, Fab', Fab, Fv and the like, including hybrid fragments. Immunoglobulins also include natural, synthetic or genetically engineered proteins that act like antibodies by binding to a specific antigen to form a complex.

The vaccines of the present invention may be administered to a recipient who then acts as a source of immunoglobulin in response to challenge from a particular vaccine. Thus, the treated subject will provide plasma from which the hyperimmune globulin will be obtained via conventional plasma fractionation methods. The hyperimmune globulin is to be administered to another subject in order to confer resistance against or treat a staphylococcal infection. The hyperimmune globulin of the invention is particularly useful for treating or preventing staphylococcal disease in infants, immunocompromised individuals, or for raising antibodies to the individual in response to vaccination without time when treatment is required.

A further aspect of the invention is a pharmaceutical composition comprising two or more monoclonal antibodies (or fragments thereof; preferably human antibodies or humanized antibodies) reactive against at least two components of the immunogenic composition of the invention, useful for the treatment or prevention of infection by gram-positive bacteria, preferably staphylococci, more preferably Staphylococcus aureus or Staphylococcus epidermidis.

Such pharmaceutical compositions comprise monoclonal antibodies which may be whole immunoglobulins of any type, for example IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid antibodies having specificity for two or more antigens of the invention. They may also be fragments, such as F (ab ') 2, Fab', Fab, Fv and the like, including hybrid fragments.

Methods for making monoclonal Antibodies are well known in the art and may include the fusion of spleen cells with myeloma cells (Kohler and Milstein 1975Nature 256; 495; Antibodies-a laboratory Manual Harlow and Lane 1988). Alternatively, monoclonal Fv fragments can be obtained by screening suitable phage display libraries (Vaughan TJ et al 1998Nature Biotechnology 16; 535). Monoclonal antibodies can be humanized or partially humanized by known methods.

Method of producing a composite material

The invention also includes methods of making the immunogenic compositions and vaccines of the invention.

A preferred method of the invention is a method of preparing a vaccine comprising the steps of mixing antigens to prepare an immunogenic composition of the invention and adding a pharmaceutically acceptable excipient.

Method of treatment

The invention also includes methods of treating or preventing staphylococcal infections, particularly nosocomial infections.

The immunogenic compositions or vaccines of the invention are particularly advantageous for use in elective surgery cases. Such patients will know the surgical date in advance and can be vaccinated in advance. Since it is not known whether a patient will be exposed to a staphylococcus aureus or staphylococcus epidermidis infection, it is preferred to vaccinate with the vaccine of the present invention which protects both as described above. Preferably, adults over 16 years of age awaiting elective surgery are treated with the immunogenic compositions and vaccines of the present invention.

It is also advantageous to vaccinate medical personnel with the vaccine of the present invention.

The vaccine formulations of the present invention are useful for protecting or treating mammals from susceptibility to infection by administering the vaccine via systemic or mucosal routes. Such administration may include injection by intramuscular, intraperitoneal, intradermal or subcutaneous routes; or transmucosal administration to the oral/esophageal, respiratory, genitourinary tracts.

The amount of conjugated antigen in each dose of vaccine is selected to induce an immunoprotective response in a typical vaccine without significant adverse side effects. Such amounts will vary depending on which particular immunogen is employed and how it is presented. The protein content of the vaccine will typically be in the range 1-100. mu.g, preferably 5-50. mu.g, most typically 10-25. mu.g. Generally, it is desirable that each dose will contain 0.1-100. mu.g of the polysaccharide presented, preferably 0.1-50. mu.g, preferably 0.1-10. mu.g, more preferably 1-10. mu.g, with 1 to 5. mu.g being the most preferred range. The optimal amount for a particular vaccine can be determined by standard studies, including observation of an appropriate immune response in a subject. After the initial vaccination, the subject may receive one or several booster immunizations at appropriate intervals.

Although the vaccine of the present invention may be administered by any route, administration of the vaccine into the skin (ID) constitutes one embodiment of the present invention. Human skin comprises an outer "horny" stratum corneum, called stratum corneum, which covers the epidermis. Beneath this epidermis is a layer called the dermis, which in turn covers the subcutaneous tissue. Researchers have demonstrated that injection of vaccines into the skin, particularly the dermis, stimulates an immune response, which may also be associated with a number of additional advantages. Intradermal vaccination with the vaccines described herein constitutes a preferred feature of the present invention.

The conventional technique of intradermal injection, the "mantoux method", involves the steps of cleansing the skin, then stretching with one hand, and inserting a narrow gauge needle (26-31 gauge) at an angle of 10-15 ° towards the bevel of the upper portion of the needle. Once the bevel of the needle is inserted, the needle cannula is lowered, advanced further, while providing a depression to lift it under the skin. The liquid is then injected very slowly to form a bubble or bulge on the skin surface, followed by slow withdrawal of the needle.

More recently, devices specifically designed to administer liquid agents into or through the skin have been described, such as the devices described in WO 99/34850 and EP 1092444, and rapid injection devices described in, for example, WO 01/13977; US5,480,381, US5,599,302, US5,334,144, US5,993,412, US5,649,912, US5,569,189, US5,704,911, US5,383,851, US5,893,397, US5,466,220, US5,339,163, US5,312,335, US5,503,627, US5,064,413, US5,520,639, US4,596,556, US4,790,824, US4,941,880, US4,940,460, WO 97/37705 and WO 97/13537. Alternative methods of intradermal administration of vaccine formulations may include conventional syringes and needles, or devices designed for shock delivery of solid vaccines (WO 99/27961), or patches (WO 97/48440; WO 98/28037); or applied to the skin surface (transdermal or percutaneous delivery WO 98/20734; WO 98/28037).

When the vaccine of the present invention is administered to the skin, or more particularly to the dermis, the vaccine is a lower liquid volume, particularly a volume between about 0.05ml and 0.2 ml.

The content of antigen in the dermal or transdermal vaccine of the invention may be similar to conventional doses present in intramuscular vaccines (see above). However, a feature of dermal or transdermal vaccines is that the formulation may be a "low dose". Thus the protein antigen in the "low dose" vaccine is preferably present at only 0.1 to 10 μ g per dose, preferably 0.1 to 5 μ g per dose; the polysaccharide (preferably conjugated) antigen may be present in the range of 0.01-1 μ g per dose, preferably between 0.01 and 0.5 μ g of polysaccharide per dose.

As used herein, the term "transdermal delivery" means the delivery of a vaccine to a dermal region in the skin. However, the vaccine will not necessarily be located entirely in the dermis. The dermis is a layer of skin that is located between about 1.0 and about 2.0mm from the surface of the human skin, but there is a certain amount of variation between individuals and in different parts of the body. Typically, it is expected that the dermis is reached by 1.5mm below the skin surface. The surface of the dermis is the stratum corneum and epidermis, and the subcutaneous layer is beneath. Depending on the mode of delivery, the vaccine may end up entirely or primarily in the dermis, or it may end up distributed in the epidermis and dermis.

A preferred embodiment of the invention is a method of preventing or treating staphylococcal infection or disease comprising the step of administering an immunogenic composition or vaccine of the invention to a patient in need thereof.

In a preferred embodiment, the patient is awaiting elective surgery.

Another preferred embodiment of the invention is the use of an immunogenic composition of the invention in the manufacture of a vaccine for the treatment or prevention of staphylococcal infection or disease, preferably post-surgical staphylococcal infection.

The term "staphylococcal infection" includes infections caused by staphylococcus aureus and/or staphylococcus epidermidis as well as other strains of staphylococci capable of causing infection in a mammalian, preferably human, host.

In each case, the inventors believe that the terms "comprising," including, "and" comprising, "as used herein, are optionally substituted by" consisting.

All references or patent applications cited in this patent specification are hereby incorporated by reference.

In order that the invention may be better understood, the following examples are provided. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

Examples

EXAMPLE 1 construction of plasmid for recombinant protein expression

A: and (4) cloning.

Design of specific to staphylococcal gene oligonucleotides in the appropriate restriction sites allow the PCR product directional cloning into E.coli expression plasmid pET24d or pQE-30, so that the protein can be expressed in N-or C-terminal containing (His)6 affinity chromatography marker fusion protein.

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’GGAACTGCAGTTATTTCCAGAATGATAATAAATTAC-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’-GCATCCATGGCACCATCACCATCACCACGAAGTAAAC

GTTGATCAAGC-3' and

5’-AGCACTCGAGTTAGAATCCCCAAGCACCTAAACC-3’

HarA-5'-GCACCCATGGCAGAAAATACAAATACTTC-3' and

5’TTTTCTCGAGCATTTTAGATTGACTAAGTTG-3’

autolysin gluceramide enzyme-5' -CAAGTCCCATGGCTGAGACGACACAAGA

TCAAC-3 ' and 5'-CAGTCTCGAGTTTTACAGCTGTTTTTGGTTG-3'

Autolysin amidase-5'-AGCTCATATGGCTTATACTGTTACTAAACC-3'

And 5 'GCGCCTCGAGTTTATATTGTGGGATGTCG-3'

IsdA-5’-CAAGTCCCATGGCAACAGAAGCTACGAACGCAAC-3’

And 5 'ACCAGTCTCGAGTAATTCTTTAGCTTTAGAGCTTG-3'

IsdB-5'-TATTCTCGAGGCTTTGAGTGTGTCCATCATTTG-3' and

5’GAAGCCATGGCAGCAGCTGAAGAAACAGGTGG-3’

MRPII-5'-GATTACACCATGGTTAAACCTCAAGCGAAA-3' and

5’AGGTGTCTCGAGTGCGATTGTAGCTTCATT-3’

the PCR product was first introduced into the pGEM-T cloning vector (Novagen) using Top10 bacterial cells according to the manufacturer's instructions. This intermediate construct was prepared to facilitate further cloning into an expression vector. Transformants containing the DNA insert were selected by restriction enzyme analysis. After digestion, approximately 20. mu.l of the reaction sample was analyzed by gel electrophoresis (0.8% agarose in Tris-acetate-EDTA (TAE) buffer). The DNA fragments were visualized by UV irradiation after gel electrophoresis and ethidium bromide staining. DNA molecule size standards (1Kb gradients, Life technologies) were electrophoresed in parallel with the test samples for estimating the size of the DNA fragments. The plasmid purified from the selected transformants of each clone was then digested to completion sequentially with the appropriate restriction enzymes as recommended by the manufacturer (Life Technologies). The digested DNA fragment was then purified using a silica gel-based spin column, followed by ligation with pET24d or pQE-30 plasmids. Cloning of Ebh (H2 fragment), AaA, IsdA, IsdB, HarA, Atl-amidase, Atl-glucosamine, MRP, IsaA was performed using pET24d plasmid, and cloning of ClfA, SdrC, SdrE, Fnbpa, SdrG/Fbe, alpha toxin and Sbi was performed using pQE-30 plasmid.

B: and (3) producing an expression vector.

To prepare the expression plasmids pET24d or pQE-30 for ligation, digestion with the appropriate restriction enzymes was likewise complete. Ligation was performed using approximately 5-fold molar excess of the digestion fragments to the prepared vector. Standard ligation reactions of about 20. mu.l (about 16 ℃ C., about 16 hours) were performed using T4DNA ligase (about 2.0 units/reaction, Life Technologies) using methods well known in the art. M15(pREP4) or BT21:: DE3 electrocompetent cells were transformed with ligation samples (about 5. mu.l) according to methods known in the art. After a growth period of about 2-3 hours at 37 ℃ in about 1.0ml of LB broth, the transformed cells were placed on LB agar plates containing ampicillin (100. mu.g/ml) and/or kanamycin (30. mu.g/ml). Antibiotics were included in the selection. The plates were incubated at 37 ℃ for about 16 hours. Individual ApR/KanR clones were picked with sterile toothpicks and used to "prime" inoculate fresh LB ApR/KanR plates and approximately 1.0ml LB Ap/Kan broth. Both the supplemented plate and broth media were incubated overnight at 37 ℃ in a standard incubator (plate) or a shaking water bath. Whole cell based PCR analysis was used to demonstrate that the transformants contained DNA inserts. Here, about 1.0ml of overnight LB Ap/Kan broth was transferred to a 1.5ml polypropylene tube and cells were collected by centrifugation in a Beckmann microfuge (about 3 minutes, room temperature, about 12,000 Xg). The cell pellet was suspended in about 200. mu.l of sterile water and about 50. mu.l of a final volume of PCR reaction containing the forward and reverse amplification primers was performed using about 10. mu.l of the sample. The initial 95 ℃ denaturation step was increased to 3 minutes to ensure thermal destruction of the bacterial cells and release of plasmid DNA. Using an ABI Model9700 thermal cycler and a 32 cycle three-step thermal amplification program at 95 ℃ for 45 seconds; BASB203 fragments were amplified from lysed transformant samples at 55-58 deg.C, 45 seconds, 72 deg.C, and 1 minute. After thermal amplification, approximately 20. mu.l of the reaction sample was analyzed by gel electrophoresis (0.8% agarose in Tris-acetate-EDTA (TAE) buffer). The DNA fragments were visualized by UV irradiation after gel electrophoresis and ethidium bromide staining. DNA molecule size standards (1Kb gradients, Life technologies) were electrophoresed in parallel with the test samples for estimating the size of the PCR product. Transformants producing PCR products of the expected size were identified as strains containing the protein expression construct. The strains containing the expression plasmids were then analyzed for inducible expression of the recombinant protein.

C: expression analysis of PCR-positive transformants.

An overnight seed culture aliquot (about 1.0ml) was inoculated into a 125ml Erlenmeyer flask containing about 25ml of LBAp/Kan broth and cultured with shaking (about 250rpm) at 37 ℃ until the culture turbidity reached about 0.5 O.D.600, i.e., mid-log phase (typically about 1.5-2.0 hours). At this point, approximately half of the culture (approximately 12.5ml) was transferred to a second 125ml flask and expression of the recombinant protein was induced by adding IPTG (1.0M stock prepared in sterile water, Sigma) to a final concentration of 1.0 mM. IPTG induced and uninduced cultures were continued for an additional about 4 hours at 37 ℃ with shaking. Samples (about 1.0ml) of induced and non-induced cultures were removed after the induction period and cells were harvested by centrifugation in a microcentrifuge for about 3 minutes at room temperature. The individual cell pellets were suspended in about 50. mu.l of sterile water, then mixed with an equal volume of 2X Laemelli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placed in a boiling water bath for about 3 minutes to denature the protein. Equal volumes (about 15. mu.l) of crude IPTG-induced and non-induced cell lysates were loaded on duplicate 12% Tris/glycine polyacrylamide gels (Mini gel 1mm thick, Novex). The lysate samples induced and not induced were electrophoresed with a pre-stained molecular weight marker (SeeBlue, Novex) under conventional conditions using standard SDS/Tris/glycine running buffer (BioRad). After electrophoresis, an aliquot of the gel was stained with Coomassie Brilliant blue R250(BioRad) and then destained to visualize new IPTG inducible proteins. A second gel was electroblotted on PVDF membrane (0.45 micron pore size, Novex) for about 2 hours at 4 ℃ using a BioRad Mini-ProteanII blotting apparatus and Towbin's methanol (20%) transfer buffer. Membranes were blocked and antibody incubations were performed according to methods well known in the art. The expression and recognition of recombinant proteins was first confirmed using a monoclonal anti-rgs (his)3 antibody followed by a rabbit anti-mouse secondary antibody conjugated to hrp (qiagen). The observation of the anti-His antibody response pattern was achieved using ABT insoluble substrates or using Hyperfilm with Amersham ECL chemiluminescence system.

Example 2: production of recombinant proteins

The cell mass for recombinant protein purification was produced using E.coli M15(pREP4) containing plasmid (pQE30) or BL21 containing plasmid pET24d, DE3 encoding staphylococcal protein.

Culture medium

The fermentation medium for recombinant protein production consists of 2X YT broth (Difco) containing 100. mu.g/ml Ap and/or 30. mu.g/ml Km. Antifoam was added at 0.25ml/L (antifoam 204, Sigma) to the medium of the fermentor. To induce expression of the recombinant protein, IPTG (isopropyl. beta. -D-thiogalactoside) was added to the fermentor (1mM final concentration).

Production of recombinant proteins

Under natural conditions

IPTG was added at a final concentration of 1mM and the cultures were grown for an additional 4 hours. The culture was then centrifuged at 6,000rpm for 10 minutes and the pellet was resuspended in phosphate buffer (50mM K) containing a mixture of protease inhibitors₂HPO₄，KH₂PO₄pH 7). The sample was subjected to French pressure lysis (2 times) using a pressure of 1500 bar. After centrifugation at 15,000rpm for 30 minutes, the supernatant was retained for further purification and NaCl was added to 0.5M. The sample was then loaded to 50mM K₂HPO₄，KH₂PO₄Ni-NTA resin in pH7 environment (XK 16 column Pharmacia, Ni-NTA resin Qiagen). After loading, buffer A (0.2M NaH) was used₂PO₄pH7, 0.3M NaCl, 10% glycerol). To elute bound proteins, a step gradient was used, in which buffer B (0.2M NaH) was added to buffer A in varying proportions₂PO₄pH7, 0.3M NaCl, 10% glycerol and 200mM imidazole). The proportion of buffer B was gradually increased from 10% to 100%. After purification, the fractions containing the protein were pooled and concentrated to 0.002M KH₂PO₄/K₂HPO₄pH7, 0.15M NaCl dialysis.

The method was used to purify ClfA, SdrG, IsdA, IsaB, HarA, Atl-glucosamine, and alpha toxin.

Under denaturing conditions

IPTG was added at a final concentration of 1mM and the cultures were grown for an additional 4 hours. The culture was then centrifuged at 6,000rpm for 10 minutes and the pellet resuspended in a suspension including proteinPhosphate buffer (50mM K) of enzyme inhibitor cocktail₂HPO₄，KH₂PO₄pH 7). The sample was subjected to French pressure lysis (2 times) using a pressure of 1500 bar. After centrifuging the sample at 15,000rpm for 30 minutes, the pellet was rinsed with phosphate buffer including 1M urea. After centrifugation at 15,000rpm for 30 minutes, 8M urea, 0.1M NaH₂PO₄The pellet was resuspended in 0.5M NaCl, 0.01M Tris-HCl pH8 and kept overnight at room temperature. The sample was centrifuged at 15000rpm for 20 minutes and the supernatant collected for further purification. The sample was then loaded into 8M urea, 0.1M NaH₂PO₄0.5M NaCl, 0.01M Tris-HCl pH8 environment of Ni-NTA resin (XK 16 column Pharmacia, Ni-NTA resin Qiagen). After flowing through the channel, buffer A (8M urea, 0.1M NaH) was used₂PO₄0,5M NaCl, 0.01M Tris, pH 8.0), buffer C (8M Urea, 0.1M NaH)₂PO₄0.5M NaCl, 0.01M Tris, pH 6.3), buffer D (8M Urea, 0.1M NaH)₂PO₄0.5M Na Cl, 0.01M Tris, pH 5.9) and buffer E (8M Urea, 0.1M NaH)₂PO₄0.5M NaCl, 0.01M Tris, pH 4.5) continuous rinsing of the column. The recombinant protein was eluted from the column with buffers D and E during the rinsing. The denatured recombinant protein can be dissolved in a solution lacking urea. For this purpose, the denatured protein contained in 8M urea was reacted with 4M urea, 0.1M Na₂PO₄0.01M Tris-HCl, pH7.1, 2M urea, 0.1M NaH₂PO₄0.01M Tris-HCl, pH7.1, 0.5M arginine and 0.002M KH₂PO₄/K₂HPO₄Continuous dialysis was performed at pH7.1, 0.15M NaCl, 0.5M arginine.

This method was used to purify Ebh (fragment H2), AaA, SdrC, FnbpA, Sbi, Atl-amidase and IsaA.

The purified protein was analyzed by SDS-PAGE. The results for one protein purified under native conditions (alpha toxin) and one protein purified under denaturing conditions (SdrC) are shown in figures 3 and 4.

EXAMPLE 3 preparation of polysaccharide

Such as Joyce et al 2003, Carbohydrate Research 338; 903-922 for PIA (PNAG).

Such as Infection and Immunity (1990)58 (7); 2367 type 5 and 8 polysaccharides are extracted from Staphylococcus aureus.

Activation and coupling chemistry:

the natural polysaccharide was dissolved in NaCl 2M or water. The optimal polysaccharide concentration was estimated for all serotypes, between 2mg/ml and 5 mg/ml.

CDAP (CDAP/PS ratio: 0.75mg/mg PS) was added to the polysaccharide solution from a stock solution of 100mg/ml in acetonitrile. After 1.5 minutes, 0.2M triethylamine was added to obtain a specific activation pH (pH 8.5-10.0). Polysaccharide activation was carried out at 25 ℃ during 2 minutes at this pH. The carrier protein was added to the activated polysaccharide in an amount sufficient to produce a molar ratio of 1/1, and the coupling reaction was carried out at the specified pH for 1 hour.

The reaction was then quenched with glycine at 25 ℃ for 30 minutes and at 4 ℃ overnight.

The conjugate was purified by gel filtration using a Sephacryl 500HR gel filtration column equilibrated with 0.2M NaCl.

The sugar and protein content of the eluted fractions was determined. The conjugates were pooled and sterile filtered on a 0.22 μm sterile membrane. The PS/protein ratio in the conjugate preparation was determined.

And (3) characterization:

the protein and polysaccharide content of each conjugate was characterized.

Polysaccharide content was determined by resorcinol test and protein content was determined by Lowry test. The final PS/PD ratio (w/w) was determined by the concentration ratio.

Residual DNAP content (ng/. mu.g PS):

activation of the polysaccharide with CDAP introduces cyanate groups into the polysaccharide and releases DMAP (4-dimethylamino-pyrimidine). Residual DMAP content was determined by specific assays developed and validated at GSK.

Free polysaccharide content (%):

the free polysaccharide content of the conjugates, placed at 4 ℃ or stored at 37 ℃ for 7 days, was determined from the supernatant obtained after incubation with alpha-carrier antibody and saturated ammonium sulphate followed by centrifugation.

The free polysaccharide in the supernatant was quantified using an alpha-PS/alpha-PS ELISA. The absence of conjugate can also be controlled by an alpha-carrier/alpha-PS ELISA.

Example 4 formulations

Adjuvant compositions

The proteins from the above examples may be formulated alone or together in combination with staphylococcal polysaccharides and as adjuvants the formulations may comprise a mixture of 3-deoxy-acylated-monophosphoryl ester a (3D-MPL) and aluminium hydroxide, or a mixture of 3-deoxy-acylated-monophosphoryl ester a (3D-MPL) and aluminium phosphate, or a mixture of 3D-MPL and/or QS21, optionally in an oil/water emulsion, optionally formulated alone with cholesterol or an aluminium salt, preferably aluminium phosphate.

3D-MPL: is a chemically detoxified form of Lipopolysaccharide (LPS) of the gram-negative bacterium salmonella minnesota.

Experiments performed in GSK Biologicals have shown that 3D-MPL in combination with various carriers strongly enhances humoral and TH1 cellular immunity.

QS 21: is a saponin purified from crude extract of bark of Quillaja Saponaria Molina tree, which has strong adjuvant activity: it activates antigen-specific lymphocyte proliferation as well as CTLs against several antigens.

Vaccines with the antigens of the invention comprising 3D-MPL and alum may be prepared in a similar manner as described in WO93/19780 or 92/16231.

Experiments conducted in GSK Biologicals have demonstrated a clear synergistic effect of the combination of 3D-MPL and QS21 in inducing both humoral and TH1 cellular immune responses. Vaccines containing such antigens are described in US 5750110.

The oil/water emulsion consisted of 2 oils (tocopherol and squalene) and PBS containing tween 80 as emulsifier. The emulsion comprised 5% squalene, 5% tocopherol, 0.4% tween 80, having an average particle size of 180nm, was designated SB62 (see WO 95/17210).

Experiments conducted in GSK Biologicals have demonstrated that the addition of MPL/QS21 to the O/W emulsions further increases their immunostimulatory properties.

Preparation of emulsion SB62 (2-fold concentration)

Tween 80 was dissolved in Phosphate Buffered Saline (PBS) to produce a 2% PBS solution. To provide 100ml of double concentration emulsion, 5g of DL alpha tocopherol and 5ml of squalene were vortexed to mix thoroughly. 90ml of PBS/Tween solution were added and mixed thoroughly. The resulting solution was then passed through a syringe and finally microfluidised by using a microfluidizer. The resulting oil droplets had a size of about 180 nm.

Example 5

And (5) animal experiments.

Female CD-1 mice, 8 to 10 weeks old, were obtained from Charles River Laboratories, Kingston, Mass. For lethality studies, five groups of 9 to 11 CD-1 mice were challenged intraperitoneally (i.p.) with serial dilutions of Staphylococcus aureus cultured on CSA plates. The inoculum size is about 10¹⁰To 10⁸CFU/mouse range. Mortality was assessed on a daily basis for 3 days. Estimation of 50% Lethal Dose (LD) by using a probabilistic Unit model of dose-response relationship₅₀). Testing of generic LDs by likelihood ratio testing₅₀The null hypothesis of (a). By using about 2x 10⁶CFU/mouse intravenous (i.v.) route or with approximately 2x 10⁷CFU/mouse groups of 8 to 20 mice were challenged by the i.p. route to cause sub-lethal bacteremia. After inoculation, separate groups of animals were bled from the tail at specific times and bacteremia levels were assessed by repeating quantitative plate counts twice on tryptic soy agar plates with 5% sheep blood (Becton dickinson microbiology Systems). Statistical significance was determined using Welch's correction of unpaired student's t-test.

Example 6

General method for determining antibody responses in different mammals

Sera were tested for IgG antibodies to staphylococcal polysaccharides by ELISA. Briefly, purified capsular polysaccharides from ATCC (Rockville, Md, 20852) were coated in Phosphate Buffered Saline (PBS) at a concentration of 25 μ g/ml on high binding microtiter plates (NuncMaxisorp) overnight at 4 ℃. The plates were blocked with 10% Fetal Calf Serum (FCS) for 1 hour at 37 ℃. Serum samples were preincubated with 20 μ g/ml cell wall polysaccharide (Statens Serum Institute, Copenhagen) and 10% FCS at room temperature for 30 min to neutralize antibodies against the antigen. The samples were then diluted on a microplate in 10% FCS twice in PBS and equilibrated for 1 hour at room temperature with stirring. After rinsing, the microplate was equilibrated with peroxidase-labeled anti-human IgG Fc monoclonal antibody (HP6043-HRP, Stratechscientific Ltd) diluted 1: 4000 in 10% FCS in PBS for 1 hour at room temperature with stirring. Rat IgG was assayed by ELISA using a 1: 5000 Jackson Immunolaboratories Inc. peroxidase-conjugated AffiniPure goat anti-rat IgG (H + L) (code 112. sup. 035. sup. 003). Titration curve identification was performed for standard sera of each serotype using logistic log comparison of SoftMax Pro. The concentration of polysaccharide used to coat the ELISA plates was 10-20. mu.g/ml. 4mg OPD (Sigma) plus 14. mu.l H per 10ml 0.1M citrate buffer pH 4.5₂O₂The color was developed at room temperature in the dark for 15 minutes. With 50. mu.l HClThe reaction was stopped and the optical density was read at 490nm relative to 650 nm. IgG concentrations were determined by reference to the titration points of the calibration curve model using a 4-parameter logistic logarithmic equation calculated by SoftMax Pro software.

The ELISA for determination of mouse and rat IgG against staphylococcal polysaccharides was similar, with the following exceptions. Bound IgG was detected using Jackson immunology laboratories inc.

HP6043-HRP and rhesus purified IgG and the same reaction, so the reagent was used for rhesus antiserum.

Protein ELISA was performed similarly to polysaccharide ELISA with the following modifications. The protein was coated overnight in PBS at 2.0. mu.g/ml. Serum samples were diluted in PBS containing 10% fetal bovine serum and 0.1% polyvinyl alcohol. Bound human antibodies were detected using a Sigma peroxidase conjugated goat affinity purified antibody (index a-2290) against human IgG Fc.

Example 7

Opsonophagocytosis assay.

According to Xu et al 1992 feed.immun.60; 1358 the in vitro opsonophagocytic killing of Staphylococcus aureus by human polymorphonuclear leukocytes (PMNs) is described. Human PMNs were prepared from heparinized blood by precipitation in 3% dextran T-250. Opsonin reaction mixture (1ml) contained about 10% in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum⁶PMN, about 10⁸CFU of staphylococcus aureus, and 0.1ml of test serum or IgG preparations. Hyperimmune rabbit serum was used as a positive control and 0.1ml of nonimmune rabbit serum was used as a complete source of IgG samples. The reaction mixture was incubated at 37 ℃, the bacterial samples were transferred to water at 0, 60 and 120 minutes, subsequently diluted, spread on tryptic soy agar plates, and incubated at 37 ℃Incubations were performed overnight for bacterial counts.

Example 8

Immunogenicity of staphylococcal proteins in mice and rabbits

Animals were immunized with purified staphylococcal protein to produce hyperimmune serum. Mice were immunized three times with 10 μ g of each protein adjuvanted with Specol (days 0, 14 and 28). Rabbits were immunized three times with 20 μ g of each protein with Specol as adjuvant (days 0, 21 and 42). Immune sera were collected and evaluated in ELISA against protein and against killed whole cells.

Anti-protein ELISA:

1 μ g/ml of purified protein in Phosphate Buffered Saline (PBS) was coated onto a high binding microtiter plate (Nunc Maxisorp) overnight at 4 ℃. The blocking plate was stirred with PBS-BSA 1% for 30 min at room temperature. The test sample 1/1000 was then diluted and incubated at room temperature for 1 hour with stirring. After rinsing, bound murine or rabbit antibodies were detected using Jackson Immunolaboratories Inc. peroxidase conjugated affiniPure goat anti-mouse IgG (H + L) (index: 115-035-003) or Affinipure goat anti-rabbit IgG (H + L) (index: 11-035-003) diluted 1: 5000 in PBS-Tween 0.05%. The detection antibody was incubated at room temperature for 30 minutes with stirring. 4mg OPD (Sigma) + 5. mu. l H in 0.1M citrate buffer pH 4.5 per 10ml were used₂O₂The color was developed at room temperature in the dark for 15 minutes. The reaction was stopped with 50. mu.l HCl and the optical density was read at 490nm versus 650 nm.

The o.d. of 1/1000 dilution of Post III was compared to that obtained with preimmunized serum.

The results produced with mouse and rabbit sera are in fig. 5A. Good seroconversion was observed for each antigen. Evaluation of sera directed against SBI was reduced due to the Ig-binding activity of the protein.

ELISA against killed whole cells:

killed whole cells (heat-or formaldehyde-inactivated) from staphylococcus aureus type 5 and 8 or staphylococcus epidermidis strain Hay at 20 μ g/ml in Phosphate Buffered Saline (PBS) were coated on high binding microtiter plates (Nunc Maxisorp) and evaporated overnight at 4 ℃. The blocking plate was stirred with PBS-BSA 1% for 30 min at room temperature. Protein A was neutralized by adding 10. mu.g/ml affinity purified chicken anti-protein A (ICL index: CPA-65A-2) diluted in PBS-Tween 0.05% followed by incubation for 1 hour at room temperature. The test samples were then incubated on a microplate with stirring at room temperature for 1 hour with two-fold dilution from the initial dilution of 1/10 to PBS-0.05%. After rinsing, bound murine or rabbit antibodies were detected using Jackson Immunolaboratories Inc. peroxidase conjugated affiniPure goat anti-mouse IgG (H + L) (index: 115-035-003) or Affinipure goat anti-rabbit IgG (H + L) (index: 11-035-003) diluted 1: 5000 in PBS-Tween 0.05%. The detection antibody was incubated for 30 minutes. 4mg OPD (Sigma) + 5. mu. l H in 0.1M citrate buffer pH 4.5 per 10ml were used₂O₂The color was developed at room temperature in the dark for 15 minutes. The reaction was stopped with 50. mu.l HCl and the optical density was read at 490nm versus 650 nm.

It should be noted that the expression level of a protein in staphylococci will vary depending on the culture conditions. Negative results may therefore reflect incorrect selection of culture conditions rather than lack of immunogenicity.

The results using mouse serum are shown in table 5 and some graphs are shown in fig. 6. Weak recognition of Staphylococcus aureus strain 5 was observed with sera to SdrC, FnbpA, Ebh, Sbi and IsaA. Only with serum directed against Sbi was the recognition of s.aureus strain 8 observed. Weak recognition of Staphylococcus epidermidis Hay was observed with sera against Atl amidase, MRP, IsdA, IsaA, Ebh, Aaa and Sbi.

The results of selectivity using rabbit serum are shown in fig. 7 and summarized in table 6. Very good identification of the three strains was observed with IsaA and IsdB. Weak recognition of the three strains was observed with HarA, even though animals received only one injection instead of three injections for other proteins.

TABLE 5

Name of protein	Reaction to SA5	Reaction to SA8	Reaction to SE Hay
				IsaA	(+)	(+)	(+)
ClfA	-	(+)	(+)
				Atl amidase	-	-	++
SdrG	-	-	-
				Glucosamidase	-	-	-
IsdA	-	-	++
				Alpha toxin	-	-	-
SrdC	++	(+)	-
				Ebh	+	-	+
AaA	-	-	++
				MRP	-	-	++
Sbi	++	++	+++
				FnbpA	+	+	(+)

TABLE 6

Name of protein	Reaction to SA5	Reaction to SA8	Reaction to SE Hay
				IsaA	+++	+++	+++
ClfA	+	++	++
				Atl amidase	-	++	+
IsdB	+++	+++	+++
				SdrG	+	+	+
Glucosamidase	-	-	-
				HarA (1 time injection)	+	+	+
IsdA	-	-	-
				Alpha toxin	-	-	+
SrdC	-	-	-
				Ebh	-	+	-
AaA	-	-	-
				MRP	-	-	++
Sbi	-	+++	-
				FnbpA	-	++	++

Example 8

Efficacy of staphylococcal protein combinations in a nasal colony proliferation model.

Fifteen groups of three cotton rats were inoculated with a combination of eight staphylococcal antigens and five cotton rats as controls were not treated with antigen. These sixteen groups are as follows:

group 1-Atl-glucosamine, 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-amidases, IsdA, MRP, AAA, IsaA, SdrG, Ebh, Fnbpa

Group 8-control

Groups 9-Atl-glucosamine, IsdA, MRP, alpha toxin, IsaA, SdrC, Sbi, Fnbpa

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

Groups 11-Atl-amidase, MRP, IsdB, alpha toxin, ClfA, IsaA, Ebh, Sbi

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

Group 13-Atl-amidases, HarA, IsdB, AAA, IsaA, SdrC, Sbi, Fnbpa

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

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

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

Each antigen mixture contained 3 μ g of each antigen mixed with an adjuvant prepared from liposomes containing MPL and QS 21. Cotton rats were inoculated three times on days 1, 14 and 28 of the experiment. Two weeks after inoculation, as described by Kokai-Kun et al (2003) antimicrob. Agents. Chemother.47; 1589 nasal colony proliferation assay described in 1597 to assess immune efficacy.

Classical multiple linear regression analysis was performed on the data using "Design Expert 6" software. The presence of antigen is encoded as 1 and the absence of antigen is-1. Using the formulation of the model, it was possible to determine which antigens were key antigens to produce a large reduction in the number of colonies per nose.

Results

The results of the nasal colony proliferation assay are shown in table 7. The control group had an average log CFU/nose of 3.51335, and a reduction in nasal colony proliferation was seen in all cotton rat groups inoculated with staphylococcal protein. Groups 4, 9and 13 showed the greatest reduction in nasal colony proliferation with over 2 log CFU/nose reduction. Good results were also obtained for groups 12 and 16, showing a reduction of about 2 log CFU/nose.

TABLE 7

Group of	Average log CFU/nose observed	Predicted logarithm CFU/nose
			1	1.77527	2.03560
2	2.90435	2.52684
			3	1.96556	2.23033
4	1.27748	1.21872
			5	1.67304	1.93128
6	2.79745	2.98193
			7	2.21481	2.30705
8	3.51355	3.47317
			9	1.22480	1.44080
10	2.03085	1.93204
			11	2.02522	1.81581
12	1.53402	1.70996
			13	1.36063	1.49100
14	2.31201	1.73909
			15	2.22979	1.98223
16	1.58109	1.44004

Multiple linear regression analysis of nasal colony proliferation data was used to calculate the contribution of a particular antigen in an antigen mixture. The final model contained the seven best antigens. The results for these antigens are shown in table 8. Inclusion of HarA produced the greatest reduction in proliferation of nasal colonies in the protein mixture, followed by IsaA, Sbi, SdrC, autolysin-glucosamine, MRP and Ebh.

Influence of the differences in the logarithmic CFU/nasal and CFU/nasal ratios of the seven best antigens in the model of Table 8 And the corresponding p-value.

Antigens	prob＞F	Estimating impact	Reduction of the ratio	Cumulative effect	Cumulative ratio
						HarA	0.033	-0.596	3.9	-0.596	3.9
IsaA	0.046	-0.558	3.6	-1.154	14.3
						Sbi	0.077	-0.491	3.1	-1.645	44.2
SdrC	0.22	-0.337	2.2	-1.982	96.0
						Atl-glucosamine	0.238	-0.324	2.1	-2.306	202.2
MRP	0.239	-0.323	2.1	-2.629	425.3
						Ebh	0.297	-0.286	1.9	-2.914	821.0

Claims

1. An immunogenic composition comprising at least two different proteins or immunogenic fragments thereof selected from at least two groups of proteins or immunogenic fragments:

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

2. The immunogenic composition of claim 1, wherein at least one protein or immunogenic fragment is selected from group a).

3. The immunogenic composition of claim 1 or 2, wherein at least one protein or immunogenic fragment is selected from group b).

4. The immunogenic composition of claims 1-3, wherein at least one protein or immunogenic fragment is selected from group c).

5. The immunogenic composition of claims 1-4, wherein at least one protein or immunogenic fragment is selected from group a), group b) and group c).

6. The immunogenic composition of claims 1-5, comprising at least one protein or immunogenic fragment from Staphylococcus aureus.

7. The immunogenic composition of claims 1-6, comprising at least one protein or immunogenic fragment from Staphylococcus epidermidis.

8. The immunogenic composition of claims 1-7, further comprising a PIA polysaccharide or oligosaccharide.

9. The immunogenic composition of claims 1-8, further comprising a type V and/or type VIII capsular polysaccharide or oligosaccharide from Staphylococcus aureus.

10. The immunogenic composition of claims 1-9, further comprising a type I and/or type II and/or type III capsular polysaccharide or oligosaccharide from staphylococcus epidermidis.

11. The immunogenic composition of claims 1-10, further comprising lipoteichoic acid from a gram positive bacterium.

12. The immunogenic composition of claim 11, wherein the lipoteichoic acid is extracted from staphylococci.

13. The immunogenic composition of claims 8-12 wherein the staphylococcal capsular polysaccharide is conjugated to a protein carrier.

14. The immunogenic composition of claims 11-13 wherein the lipoteichoic acid is conjugated to a protein carrier.

15. The immunogenic composition of claims 13-14 wherein the protein carrier is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM197, haemophilus influenzae protein D, pneumolysin and alpha toxoid.

16. The immunogenic composition of claims 1-15, which is capable of generating an effective immune response against both staphylococcus aureus and staphylococcus epidermidis.

17. A vaccine comprising the immunogenic composition of claims 1-16 and a pharmaceutically acceptable excipient.

18. A method of making a vaccine comprising the steps of mixing antigens to make the immunogenic composition of claims 1-16 and adding a pharmaceutically acceptable excipient.

19. A method of preventing or treating staphylococcal infection comprising the step of administering the vaccine of claim 17 to a patient in need thereof.

20. Use of the immunogenic composition of claims 1-16 in the manufacture of a vaccine for the treatment or prevention of staphylococcal infection.

21. A method of preparing immunoglobulins for use in the prevention or treatment of staphylococcal infection comprising the steps of immunizing a recipient with the vaccine of claim 17 and isolating immunoglobulins from the recipient.

22. A pharmaceutical composition comprising two or more monoclonal antibodies or fragments thereof reactive with at least two components of the immunogenic composition of any one of claims 1-16, wherein the at least two components are selected from at least two of groups a), b) and c).

23. A pharmaceutical composition comprising the immunoglobulin of claim 22 and a pharmaceutically acceptable excipient.

24. A method of treating or preventing staphylococcal infection comprising the step of administering to a patient an effective amount of the pharmaceutical composition of claim 22 or 23.

25. Use of a pharmaceutical composition according to claim 22 or 23 in the manufacture of a medicament for the treatment or prevention of staphylococcal infection.