HK1171653A - Immunogenic compositions of staphylococcus aureus antigens - Google Patents

Description

Immunogenic compositions of staphylococcus aureus antigens

Cross Reference to Related Applications

This application claims priority from U.S. provisional patent application No. 61/219,134, filed on 22/6/2009, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to immunogenic compositions comprising a polypeptide isolated from Staphylococcus aureus (Staphylococcus aureus) and a capsular polysaccharide. Furthermore, the present invention relates to methods of inducing an immune response against staphylococcus aureus in a subject using immunogenic compositions of staphylococcus aureus polypeptides and capsular polysaccharides. The resulting antibodies may also be used to treat or prevent staphylococcus aureus infections by passive immunotherapy.

Background

Humans are natural storage hosts for staphylococcus aureus (s. Healthy individuals can colonize the skin, nostrils and throat with staphylococcus aureus, continuously (10-35%), intermittently (20-75%), or in a non-carrying state (5-70%), without associated disease. See Vandenbergh et al, j.clin.micro.37: 3133-3140(1999). Disease subsequently occurs when an individual becomes immunocompromised due to disruption of the immune barrier, such as during surgery, placement of an indwelling catheter or other device, trauma, or wound. The resulting staphylococcus aureus infection can cause a wide variety of diseases ranging from mild skin infections to intima, osteomyelitis, bacteremia, sepsis and other forms of disease with high mortality. The vast human storage host increases the opportunity for the evolution and dissemination of adapted pathogenic clonal types.

Invasive staphylococcal infections from the gram-positive cocci staphylococcus aureus and staphylococcus epidermidis (s. epidermidis) are of particular concern because they are an increasing global public health problem. In particular, staphylococcus aureus is responsible for most hospital-acquired (nosocomial) infections, and its prevalence increases in community-onset infections. For example, the incidence of invasive methicillin-resistant Staphylococcus aureus (MRSA) in the United states was estimated to be 31.8/100,000 in 2005, including 18,650 deaths. See Klevens r.m. et al, JAMA, 298: 1763-71(2007).

Staphylococcal disease has increased dramatically over the past 20 years, with this increase being accompanied by concomitant intravascular devices and invasive procedures. This increase in disease incidence is more disconcerting due to the concomitant increase in antibiotic resistance, and therefore there is an urgent need for immunogenic compositions for use in vaccines or for eliciting polyclonal or monoclonal antibodies to confer passive immunity as a means of preventing or treating staphylococcal infections and related diseases.

Summary of The Invention

The present invention relates to a multi-antigen or multi-component immunogenic composition comprising at least 3 antigens isolated from staphylococci. Antigens that are polypeptides and polysaccharides may be obtained directly from bacteria using isolation methods known to those skilled in the art, or they may be prepared using synthetic protocols, or they may be prepared recombinantly using genetic engineering methods also known to those skilled in the art, or by a combination of any of the foregoing methods. In certain embodiments, the immunogenic compositions of the invention comprise 3 or more antigens selected from the group consisting of an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide (CP5) conjugated to a carrier protein, an isolated staphylococcus aureus type 8 capsular polysaccharide (CP8) conjugated to a carrier protein, and an isolated staphylococcus aureus MntC protein. In addition, the present invention provides methods of inducing an immune response against staphylococci, methods of preventing, reducing the severity of, or delaying the onset of a disease caused by staphylococci, and methods of preventing, reducing the severity of, or delaying the onset of at least one symptom of a disease caused by infection with staphylococci.

Accordingly, in one embodiment, the present invention provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein (CP5), and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein (CP 8).

In one embodiment, the present invention provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus clump factor b (clfb), an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein (CP5), and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein (CP 8).

In one embodiment, the present invention provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide (CP5) conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide (CP8) conjugated to a carrier protein.

In one embodiment, the present invention provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide (CP5) conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide (CP8) conjugated to a carrier protein.

In one embodiment, the present invention provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein (CP5), and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein (CP 8).

In one embodiment, the present invention provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide (CP5) conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide (CP8) conjugated to a carrier protein.

In one embodiment, the present invention provides an immunogenic composition comprising: an isolated staphylococcus aureus aggregation factor a (clfa) polypeptide, an isolated staphylococcus aureus aggregation factor b (clfb) polypeptide, and an isolated staphylococcus aureus MntC protein.

In one embodiment, the present invention provides an immunogenic composition comprising: an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide (CP5) conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide (CP8) conjugated to a carrier protein.

In one embodiment, the immunogenic composition comprises an isolated ClfA polypeptide fragment, wherein the ClfA polypeptide fragment comprises the fibrinogen binding domain of ClfA. In one embodiment, the ClfA polypeptide fragment comprises a fibrinogen binding domain comprising the N1, N2, and N3 domains of ClfA. In one embodiment, the ClfA polypeptide fragment comprises a fibrinogen binding domain comprising the N2 and N3 domains of ClfA. In one embodiment, a composition comprising the ClfA fibrinogen binding domain exhibits reduced binding to fibrinogen. In one embodiment, the fibrinogen binding domain of ClfA binds to fibrinogen at a reduced level compared to the observed binding to fibrinogen by the native fibrinogen binding domain of ClfA. In one embodiment, a composition comprising the ClfA fibrinogen binding domain exhibits reduced binding to fibrinogen and has one or more amino acid substitutions of Tyr 338, Tyr256, Pro 336, Lys 389, Ala 254, and Ile 387 in a full-length protein comprising a signal sequence. In one embodiment, a composition comprising the ClfA fibrinogen binding domain exhibits an amino acid substitution at one or more of Tyr 338, Tyr256, Pro 336, Lys 389, Ala 254, and Ile 387, wherein the amino acid substitution at any one or more of these positions is Ala or Ser. In one embodiment, the composition comprises a ClfA fibrinogen binding domain, wherein the Tyr at position 338 is substituted to Ala.

In one embodiment, the immunogenic composition comprises an isolated ClfB polypeptide fragment, wherein the ClfB polypeptide fragment comprises the fibrinogen binding domain of ClfB. In one embodiment, the ClfB polypeptide fragment comprises a fibrinogen binding domain comprising the N1, N2, and N3 domains of ClfB. In one embodiment, the ClfB polypeptide fragment comprises a fibrinogen binding domain comprising the N2 and N3 domains of ClfB. In one embodiment, a composition comprising the ClfB fibrinogen binding domain exhibits reduced binding to fibrinogen. In one embodiment, the fibrinogen binding domain of ClfB binds to fibrinogen at a reduced level compared to the observed binding to fibrinogen by the native fibrinogen binding domain of ClfB.

In one embodiment, the immunogenic composition comprises a staphylococcus aureus type 5 capsular polysaccharide (CP5), which is a high molecular weight polysaccharide of 20-1000 kDa. In one embodiment, the type 5 high molecular weight polysaccharide has a molecular weight of 50-300 kDa. In one embodiment, the type 5 high molecular weight polysaccharide has a molecular weight of 70-150 kDa.

In one embodiment, the immunogenic composition comprises 10% to 100% O-acetylated staphylococcus aureus type 5 capsular polysaccharide. In one embodiment, the immunogenic composition comprises 50% to 100% O-acetylated staphylococcus aureus type 5 capsular polysaccharide. In one embodiment, the immunogenic composition comprises 75% to 100% O-acetylated staphylococcus aureus type 5 capsular polysaccharide.

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

In one embodiment, the immunogenic composition comprises 10% to 100% O-acetylated staphylococcus aureus type 8 capsular polysaccharide. In one embodiment, the immunogenic composition comprises 50% to 100% O-acetylated staphylococcus aureus type 8 capsular polysaccharide. In one embodiment, the immunogenic composition comprises 75% to 100% O-acetylated staphylococcus aureus type 8 capsular polysaccharide.

In one embodiment, capsular polysaccharides 5 and/or 8 present in the immunogenic composition are conjugated to a carrier protein. In one embodiment, the carrier protein is a Corynebacterium diphtheriae (c. diphtheria) toxoid CRM₁₉₇。

In one embodiment, the immunogenic composition comprises staphylococcus aureus MntC, which is a lipidated protein. In one embodiment, the immunogenic composition comprises staphylococcus aureus MntC, which is not a lipidated protein.

In one embodiment, the invention provides an immunogenic composition as described herein, further comprising at least one protein from the Serine-aspartate repeat (Sdr) protein family selected from SdrC, SdrD and SdrE.

In one embodiment, the invention provides an immunogenic composition as described herein, further comprising an Iron surface determinant B (IsdB) protein.

In each of the embodiments described herein where the immunogenic composition comprises 3 or more of the recited antigens, the composition may further comprise other immunogenic and/or non-immunogenic substances. As detailed herein, optionally, in certain embodiments, each immunogenic composition can "consist essentially of" or "consist of" 3 or more of the recited antigens, and further comprise one or more non-immunogenic substances.

In one embodiment, the invention provides an immunogenic composition as described herein, further comprising any one of the following antigens: opp3a, DltD, HtsA, Ltas, IsdA, IsdC, SdrF, SdrG, SdrH, SrtA, SpA, Sbi FmtB, alpha-hemolysin (hla), beta-hemolysin, fibronectin binding protein A (fnbA), fibronectin binding protein B (fnbB), coagulase, FIG, map, Panton-Valentine leukocidin (pvl), alpha-toxin and variants thereof, gamma-toxin (hlg) and variants thereof, ica, immunodominant ABC transporter, Mg2+ transporter, Ni ABC transporter, RAP, autolysin, laminin receptor, IsaA/PisA, IsaB/PisB, SPOIE, SsaA, Ebps, SasA, CnssF, SssH, EFB (FIB), SBI, Npase, SaaP, bone acid binding protein II, sialoglobin II, protease (AUcA-597) and fragments thereof such as protein A, Sqa-25, Sasbc 4625, Sasbc, Sasabin, Sasab, Sasbc, Sa, Sasbc, Sa, poly-N-acetylglucosamine (PNAG/dPNAG) exopolysaccharide, GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin binding protein, HarA, EsxA, EsxB, enterotoxin A, enterotoxin B, enterotoxin C1, and neoautolysin. In certain embodiments of the invention, when the immunogenic composition comprises certain forms of CP5 and/or CP8, it may not further comprise PNAG.

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

In one embodiment, the immunogenic composition is used to formulate a vaccine. In one embodiment, the vaccine is for inducing an immune response against staphylococcus aureus in a subject. In one embodiment, the immunogenic composition is used to generate an antibody preparation to confer passive immunity to a subject.

In one embodiment, the invention provides a method of inducing an immune response against staphylococcus aureus comprising administering to a subject an immunogenic amount of any of the immunogenic compositions described herein, and a pharmaceutically acceptable carrier.

In one embodiment, the present invention provides a method of preventing or reducing infection with staphylococcus aureus, or preventing or reducing the severity of at least one symptom associated with infection caused by staphylococcus aureus, comprising administering to a subject an immunogenic amount of any of the immunogenic compositions described herein, and a pharmaceutically acceptable carrier.

In one embodiment, the method of inducing resistance to staphylococcus aureus comprises delivering the immunogenic composition with an adjuvant. In one embodiment, the method of inducing resistance to staphylococcus aureus provides for delivery of the immunogenic composition with a pharmaceutically acceptable carrier.

In one embodiment, the immunogenic composition described herein induces an immune response that prevents or reduces a disease or disorder associated with a staphylococcal organism in the subject, or prevents or reduces one or more symptoms associated with the staphylococcal organism in the subject. In one embodiment, the disease is selected from invasive staphylococcus aureus disease, sepsis and carriage (carriage).

In one embodiment, the immune response induced comprises the production of antibodies with opsonophagocytic activity (OPA) against staphylococcus aureus. In one embodiment, the immune response induced comprises the production of higher titers of staphylococcus aureus-specific opsonophagocytic antibodies than observed in non-immunized subjects. In one embodiment, the opsonophagocytic titer is at least 1: 20.

In one embodiment, the staphylococcus aureus against which the immune response is induced is MRSA. In one embodiment, the staphylococcus aureus against which the immune response is induced is MSSA. In one embodiment, the staphylococcus aureus against which the immune response is induced is VRSA. In one embodiment, the staphylococcus aureus against which the immune response is induced is VISA.

In one embodiment, the present invention provides a method of preventing staphylococcal infection in a subject undergoing surgery, the method comprising administering to the subject prior to the surgery an immunologically effective amount of any of the immunogenic compositions described herein. The surgical procedure may be an elective surgical procedure or a non-elective surgical procedure. In one embodiment, the surgical procedure is cardiothoracic surgery (cardiac-thoracic surgery). In one embodiment, the subject is a human, a veterinary animal or a livestock animal.

In one embodiment, the present invention provides a method of conferring passive immunity to a subject, the method comprising the steps of: (1) producing an antibody preparation using the immunogenic composition of the invention; and (2) administering the antibody preparation to the subject to confer passive immunity.

Drawings

Fig. 1 shows various forms of recombinant ClfA and discloses in order of appearance SEQ ID NO: 125 and 127 and 129.

FIG. 2 shows the cloning steps used to construct pLP1179 expressing ClfA.

FIG. 3 shows T7ClfA_(N123)Y338A expression vector pLP 1179.

Fig. 4 shows the repeating structures of CP5 and CP8 polysaccharides.

FIGS. 5A and 5B show molecular weight spectra of CP5(A) and CP8(B) produced at different broth (broth) pH.

Fig. 6A and 6B show molecular weight spectra of CP5(a) and CP8(B) produced at different temperatures.

Figure 7 demonstrates the correlation of molecular weight of purified CP5 and CP8 with treatment time for mild acid hydrolysis.

FIGS. 8A-8E show an alignment of ClfA between various strains of Staphylococcus aureus (SEQ ID NOs: 62, 64, 68, 84, 70, 104, 66, 78, 86, 88, 90, 72, 74, 76, 80, 94, 82, 92, 96, 98, 100, 102, 106, and 108, respectively, in order of appearance).

Fig. 9 shows a CffA system tree.

FIGS. 10A-10E show an alignment of ClfB between various strains of Staphylococcus aureus (SEQ ID NOs: 26, 28, 32, 18, 54, 34, 36, 30, 16, 20, 22, 24, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, and 60, respectively, in order of appearance).

Fig. 11 shows a ClfB system tree.

FIG. 12 shows an alignment of MntC between various strains of Staphylococcus aureus (SEQ ID NOS: 2, 8, 10, 4,6, 14, and 12, respectively, in order of appearance).

FIG. 13 demonstrates that polyclonal rabbit anti-ClfA antibodies reduce Staphylococcus aureus 659-018 colony counts in a mouse sepsis model.

Figure 14 demonstrates that active immunization with ClfA reduces colonization of the heart by staphylococcus aureus PFESA0003 in a rabbit infective endocarditis model.

Fig. 15A and 15B demonstrate that immunization with MntC reduces staphylococcus aureus in the blood. A: staphylococcus aureus PFESA0237 strain; b: staphylococcus aureus PFESA0266 strain.

FIG. 16 demonstrates Staphylococcus aureus CP5-CRM₁₉₇The conjugate immunogenic formulations consistently showed protection in the mouse pyelonephritis model.

FIG. 17 evidence utility CP8-CRM₁₉₇Immunization with the conjugate immunogenic formulation reduced death in a sepsis model.

FIG. 18 shows Colony Forming Units (CFU) recovered in the kidney following challenge with Staphylococcus aureus PFESA0266 in mice immunized with High Molecular Weight (HMW) CP5-CRM, Low Molecular Weight (LMW) CP5-CRM, or PP5-CRM controls.

Figure 19 shows a comparison of OPA titers (geometric means) of sera obtained from mice immunized with different formulations of polysaccharide conjugates (high molecular weight (HMW) CP5-CRM, Low Molecular Weight (LMW) CP 5-CRM). Groups consisted of 5-9 mice.

Figure 20 demonstrates OPA titers in non-human primate sera before (wk0, open symbols) and after 2 weeks (wk2, closed symbols) of immunization with different combinations of staphylococcus aureus antigens. The 3-antigen (3Ag) vaccine consists of 3 antigens, while the 4-antigen (4Ag) vaccine consists of 4 antigens. Each formulation had 2 CP conjugates and 1 or 2 peptides.

Detailed Description

Before the present methods and treatment methods are described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

The terms used herein have meanings that are recognized and known by those skilled in the art, however, for convenience and completeness, specific terms and their meanings are set forth below.

As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein, and/or one or more methods and/or steps that will become apparent to those skilled in the art upon reading this disclosure, and so forth.

The term "about" or "approximately" means within a statistically significant range of values. Such a range may be within an order of magnitude, typically within 20%, more typically still within 10%, and even more typically within 5% of a given value or range. The allowable deviation covered by the terms "about" or "approximately" depends on the particular system under study and can be readily determined by one skilled in the art. Whenever a range is mentioned within this application, every integer within the range is also contemplated as an embodiment of the invention.

An "antibody" is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless the context indicates otherwise, the term includes not only intact polyclonal or monoclonal antibodies, but also engineered antibodies (e.g., chimeric, humanized and/or derivatized to alter effector function, stability and other biological activity) and fragments thereof (such as Fab, Fab ', F (ab') 2, Fv), single chain (ScFv) and domain antibodies, including shark and camelid antibodies), as well as fusion proteins comprising an antibody portion, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and antibody fragments as described herein, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site. Antibodies include any class of antibody, such as IgG, IgA, or IgM (or subclasses thereof), and the antibodies need not be of any particular class. Depending on the antibody amino acid sequence of its heavy chain constant domain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 in humans. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

An "antibody fragment" comprises only a portion of an intact antibody, wherein said portion preferably retains at least one, preferably most or all, of the functions normally associated with the fragment when present in an intact antibody.

The term "antigen" generally refers to a biomolecule, typically a protein, peptide, polysaccharide, lipid or conjugate containing at least one epitope to which a cognate antibody can selectively bind; or in some cases immunogenic substances that stimulate the production of antibodies or T-cell responses or both in an animal, including compositions that are injected or absorbed into an animal. An immune response may be generated to the entire molecule, or to one or more different portions of the molecule (e.g., an epitope or hapten). The term may be used to refer to a single molecule, or to a homogeneous or heterogeneous population of antigenic molecules. Antigens are recognized by antibodies, T-cell receptors, or other elements of specific humoral and/or cellular immunity. The term "antigen" includes all relevant epitopes. Epitopes of a given antigen can be identified using any number of epitope mapping techniques well known in the art. See, for example, epipope Mapping Protocols in Methods in molecular biology, vol.66(Glenn E.Morris, Ed., 1996) Humana Press, Totowa, N.J.. For example, linear epitopes can be determined by, for example, simultaneously synthesizing a plurality of peptides on a solid support, the peptides corresponding to portions of a protein molecule, and reacting the peptides with an antibody while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al (1984) Proc.Natl.Acad.Sci.USA 81: 3998-4002; geysen et al (1986) Molec.Immunol.23: 709-715, all of which are incorporated herein by reference in their entirety. Similarly, conformational epitopes can be identified by determining the spatial conformation of amino acids by, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Furthermore, for the purposes of the present invention, "antigen" may also be used to refer to proteins that include modifications to the native sequence, such as deletions, additions, and substitutions (generally conserved in nature, but they may not), so long as the protein retains the ability to elicit an immune response. These modifications may be deliberate, as by site-directed mutagenesis, or by specific synthetic methods, or by genetic engineering methods, or may be accidental, as by mutation of the host, which produces the antigen. Furthermore, the antigen may be derived, obtained or isolated from a microorganism, such as a bacterium, or may be a whole organism. Similarly, oligonucleotides or polynucleotides that express antigens as in nucleic acid immunization applications are also included in the definition. Also included are synthetic antigens, e.g., polyepitopes, flanking epitopes, as well as other antigens of recombinant or synthetic origin (Bergmann et al (1993) Eur. J. Immunol.23: 27772781; Bergmann et al (1996) J. Immunol.157: 32423249; Suhrbier, A. (1997) Immunol.and CellBiol.75: 402408; Gardner et al (1998)12th World AIDS Conterence, Geneva, Switzerland, Jun.28-Jul.3, 1998).

The term "adjuvant" refers to a compound or mixture that enhances an immune response to an antigen as further described or exemplified herein.

"bacteremia" is the transient presence of bacteria in the blood. Bacteremia can progress to sepsis or sepsis, which can be considered as an infection and the persistent presence of bacteria in the blood with associated clinical signs/symptoms. Not all bacteria are able to survive in the blood. Those bacteria have specific genetic traits that provide this ability. But also host factors play an important role.

"Capsular polysaccharide" or "Capsular polysaccharide" refers to the polysaccharide capsule outside the cell wall of most isolates (isolates) of Staphylococci (staphylococi). For example, staphylococcus aureus includes a cell wall component composed of a peptidoglycan complex that enables the organism to survive adverse osmotic conditions, and also includes a unique teichoic acid attached to the peptidoglycan. Most isolates of staphylococcus aureus are coated with a thin polysaccharide capsule outside the cell wall. Such serotype-distinct capsules may be used for various isolates of serotyped staphylococcus aureus. Many clinically significant isolates have been shown to include two capsule types: serotype 5(CP5) and serotype 8(CP 8). The structures of CP5 and CP8 are shown in FIG. 4.

As used herein, a "conjugate" comprises a capsular polysaccharide, typically having a desired range of molecular weights, and a carrier protein, wherein the capsular polysaccharide is conjugated to the carrier protein. The conjugate may or may not contain an amount of free capsular polysaccharide. As used herein, "free capsular polysaccharide" refers to capsular polysaccharide that is non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or embedded in) conjugated capsular polysaccharide-carrier protein. The terms "free capsular polysaccharide", "free polysaccharide" and "free saccharide" are used interchangeably and are intended to convey the same meaning. Regardless of the nature of the carrier molecule, it may be conjugated to the capsular polysaccharide, either directly or through a linker. As used herein, "to be conjugated," "conjugated," and "conjugating" refer to the process of covalently linking a bacterial capsular polysaccharide to the carrier molecule. Conjugation enhances the immunogenicity of the bacterial capsular polysaccharide. Conjugation can be performed according to the methods described below or by methods known in the art.

As described above, the present invention relates to conjugates comprising staphylococcus aureus serotype 5 capsular polysaccharide (CP5) conjugated to a carrier protein and to conjugates comprising staphylococcus aureus serotype 8 capsular polysaccharide (CP8) conjugated to a carrier protein. An embodiment of the invention provides a conjugate comprising a staphylococcus aureus serotype 5 capsular polysaccharide conjugated to a carrier protein and a staphylococcus aureus serotype 8 capsular polysaccharide conjugated to a carrier protein, wherein: the type 5 capsular polysaccharide has a molecular weight of 50kDa to 800 kDa; the type 8 capsular polysaccharide has 50-700 kDa; the immunogenic conjugate has a molecular weight of about 1000kDa to about 5000 kDa; and the conjugate comprises less than about 30% free polysaccharide relative to total polysaccharide. In one embodiment, the conjugate comprises less than about 25%, about 20%, about 15%, about 10%, or about 5% free polysaccharide relative to total polysaccharide. In one embodiment, the type 5 or type 8 polysaccharide has a molecular weight of 20kDa to 1000 kDa.

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

Accordingly, in one embodiment, the carrier protein within the immunogenic conjugate of the invention is CRM₁₉₇And said CRM₁₉₇Covalently attached to the capsular polysaccharide through a carbamate linkage, an amide linkage, or both. The number of lysine residues conjugated to the capsular polysaccharide in the carrier protein can be characterized as a range of conjugated lysines. For example, in a given immunogenic composition, the CRM₁₉₇May comprise 5-15 of the 39 lysines, the 5-15 lysines being covalently linked to the capsular polysaccharide. Another way to express this parameter is 12% -40% CRM₁₉₇Lysine is covalently linked to the capsular polysaccharide. In some implementationsIn the scheme, covalently bound to CRM₁₉₇CRM of polysaccharides of (A)₁₉₇The moiety comprises 5-22 lysines covalently linked to the polysaccharide. In some embodiments, covalently bound to CRM₁₉₇CRM of polysaccharides of (A)₁₉₇The moiety comprises 5-23 lysines covalently linked to the polysaccharide. In some embodiments, CRM of polysaccharides covalently bound to a carrier protein₁₉₇The moiety comprises 8-15 lysines covalently linked to the polysaccharide. In some embodiments, CRM of polysaccharides covalently bound to a carrier protein₁₉₇The moiety comprises 8-12 lysines covalently linked to the polysaccharide. For example, in a given immunogenic composition, the CRM₁₉₇May comprise 18-22 of the 39 lysines, the 18-22 lysines being covalently linked to the capsular polysaccharide. Another way to express this parameter is 40% -60% CRM₁₉₇Lysine is covalently linked to the capsular polysaccharide. In some embodiments, the CRM₁₉₇Contains 5-15 of 39 lysines, and the 5-15 lysines are covalently linked to CP 8. Another way to express this parameter is 12% -40% CRM₁₉₇Lysine is covalently attached to CP 8. In some embodiments, the CRM₁₉₇Contains 18-22 lysines of 39, the 18-22 lysines are covalently linked to CP 5. Another way to express this parameter is 40% -60% CRM₁₉₇Lysine is covalently attached to CP 5.

As discussed above, the number of lysine residues conjugated to the capsular polysaccharide in the carrier protein may be characterized as a range of conjugated lysines, which may be expressed as a molar ratio. For example, the conjugated lysine to CRM ratio in the CP8 immunogenic conjugate₁₉₇May be in a molar ratio of from about 18: 1 to about 22: 1. In one embodiment, the conjugated lysine to CRM of the CP8 immunogenic conjugate₁₉₇May range from about 15: 1 to about 25: 1. In some embodiments, the conjugated lysine to CRM in the CP8 immunogenic conjugate₁₉₇May range from about 14: 1 to about 20: 1; from about 12: 1 to about 18: 1; from about 10: 1 to about 16: 1; from about 8: 1 to about 14: 1; from about 6: 1 to about 12: 1; about 4: 1 to about 10: 11; from about 20: 1 to about 26: 1; from about 22: 1 to about 28: 1; from about 24: 1 to about 30: 1; from about 26: 1 to about 32: 1; from about 28: 1 to about 34: 1; from about 30: 1 to about 36: 1; from about 5: 1 to about 10: 1; from about 5: 1 to about 20: 1; from about 10: 1 to about 20: 1; alternatively from about 10: 1 to about 30: 1. Moreover, the molar ratio of conjugated lysine to CRM197 in the CP5 immunogenic conjugate can be about 3: 1 to 25: 1. In one embodiment, the molar ratio of conjugated lysine to CRM197 in the CP5 immunogenic conjugate can range from about 5: 1 to about 20: 1. In one embodiment, the molar ratio of conjugated lysine to CRM197 in the CP5 immunogenic conjugate can range from about 4: 1 to about 20: 1; from about 6: 1 to about 20: 1; from about 7: 1 to about 20: 1; from about 8: 1 to about 20: 1; from about 10: 1 to about 20: 1; from about 11: 1 to about 20: 1; from about 12: 1 to about 20: 1; from about 13: 1 to about 20: 1; from about 14: 1 to about 20: 1; from about 15: 1 to about 20: 1; from about 16: 1 to about 20: 1; from about 17: 1 to about 20: 1; from about 18: 1 to about 20: 1; from about 5: 1 to about 18: 1; from about 7: 1 to about 16: 1; alternatively from about 9: 1 to about 14: 1.

Another way of expressing the number of lysine residues in the carrier protein conjugated to the capsular polysaccharide may be a range of conjugated lysines. For example, in a given CP8 immunogenic conjugate, the CRM₁₉₇May comprise 5-15 of the 39 lysines, the 5-15 lysines being covalently linked to the capsular polysaccharide. Alternatively, this parameter may be expressed as a percentage. For example, in a given CP8 immunogenic conjugate, the percentage of conjugated lysine may be 10% -50%. In some embodiments, 20% to 50% of the lysine may be covalently attached to CP 8. Alternatively, 30% -50% CRM₁₉₇Lysine may be covalently linked to CP 8; 10% -40% CRM₁₉₇Lysine; 10% -30% CRM₁₉₇Lysine; 20% -40% CRM₁₉₇Lysine; 25% -40% CRM₁₉₇Lysine; 30% -40% CRM₁₉₇Lysine; 10% -30% CRM₁₉₇Lysine; 15% -30% CRM₁₉₇Lysine; 20% -30% CRM₁₉₇Lysine; 25% -30% CRM₁₉₇Lysine; 10% -15% CRM₁₉₇Lysine; or 10% -12% CRM₁₉₇Lysine is covalently attached to CP 8. Moreover, in a given CP5 immunogenic conjugate, the CRM₁₉₇May comprise 18-22 of the 39 lysines, the 18-22 lysines being covalently linked to the capsular polysaccharide. Alternatively, this parameter may be expressed as a percentage. For example, in a given CP5 immunogenic conjugate, the percentage of conjugated lysine may be 40% to 60%. In some embodiments, 40% to 60% of the lysine may be covalently attached to CP 5. Alternatively, 30% -50% CRM₁₉₇Lysine may be covalently linked to CP 5; 20% -40% CRM₁₉₇Lysine; 10% -30% CRM₁₉₇Lysine; 50% -70% CRM₁₉₇Lysine; 35% -65% CRM₁₉₇Lysine; 30% -60% CRM₁₉₇Lysine; 25% -55% CRM₁₉₇Lysine; 20% -50% CRM₁₉₇Lysine; 15% -45% CRM₁₉₇Lysine; 10% -40% CRM₁₉₇Lysine; 40% -70% CRM₁₉₇Lysine; or 45% -75% CRM₁₉₇Lysine is covalently attached to CP 5.

The frequency of lysine attachment of the capsular polysaccharide chain to the carrier molecule is another parameter characterizing the capsular polysaccharide conjugate. For example, in one embodiment, CRM is performed for at least every 5-10 saccharide repeat units of the capsular polysaccharide₁₉₇And a polysaccharide, at least one covalent bond being present. In another embodiment, for every 5-10 saccharide repeat units of the capsular polysaccharide; every 2-7 saccharide repeat units, every 3-8 saccharide repeat units; every 4-9 saccharide repeat units; every 6-11 saccharide repeat units; every 7-12 saccharide repeat units; every 8-13 saccharide repeat units; every 9-14 saccharide repeat units; every 10-15 saccharide repeat units; every 2-6 saccharide repeat units, every 3-7 saccharide repeat units; every 4-8 saccharide repeat units; every 6-10 saccharide repeat units; every 7-11 saccharide repeat units; every 8-12 saccharide repeat units; every 9-13 saccharide repeat units; every 10-14 saccharide repeat units; every 10-20 saccharide repeat units; every 5-10 saccharide repeat units, CRM₁₉₇And the capsular polysaccharide is present with at least one covalent bond. In another embodiment, for said podEvery 2, 3, 4, 5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 saccharide repeat units of the membrane polysaccharide, CRM₁₉₇And the capsular polysaccharide has at least one bond present between them.

Chemical activation of the polysaccharide and subsequent conjugation to the carrier protein can be achieved by conventional methods. See, for example, U.S. Pat. nos. 4,673,574 and 4,902,506. Other methods of activation and conjugation may alternatively be used.

As used herein, "carrier protein" or "protein carrier" refers to any protein molecule that can be conjugated to an antigen (e.g., the capsular polysaccharide) against which an immune response is desired. Conjugation of an antigen, such as a polysaccharide, to a carrier protein can render the antigen immunogenic. The carrier protein is preferably a protein that is non-toxic and non-reactogenic and that can be obtained in sufficient quantity and purity. Examples of carrier proteins are toxins, toxoids or any mutated cross-reactive material (CRM) of toxins from tetanus, diphtheria, pertussis, Pseudomonas (Pseudomonas) species, escherichia coli (e.coli), Staphylococcus (Staphylococcus) species and Streptococcus (Streptococcus) species₁₉₇). The carrier protein should be amenable to standard conjugation procedures. In a particular embodiment of the invention, CRM₁₉₇Used as a carrier protein.

CRM₁₉₇(Wyeth/Pfizer, Sanford, NC) is isolated from Corynebacterium diphtheriae (Corynebacterium diphtheria) strain C7 (. beta.) grown in casamino acids and yeast extract-based medium₁₉₇) A non-toxic variant of diphtheria toxin (i.e., toxoid) of the culture of (a). Purification of CRM by ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography₁₉₇. Generating CRM₁₉₇Strain C7 (C7) of corynebacterium diphtheriae₁₉₇) Has been stored in the American type culture Collection, Rockville, Maryland, and has been assigned accession number ATCC 53281. Other diphtheria toxoids are also suitable as carrier proteins.

Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (e.g. as described in international patent application WO 2004/083251), escherichia coli LT, escherichia coli ST, and exotoxin a from Pseudomonas aeruginosa (Pseudomonas aeruginosa). Bacterial outer membrane proteins such as outer membrane protein complex c (ompc), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein a (pspa), pneumococcal adhesin protein (PsaA), clostridium difficile (c.difficile) enterotoxin (toxin a) and cytotoxin (toxin B) or Haemophilus influenzae (Haemophilus influenzae) protein D may also be used. Other proteins may also be used as carrier proteins, such as ovalbumin, Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), or tuberculin Pure Protein Derivative (PPD).

After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugate (enriched in polysaccharide-protein conjugate) is purified by various techniques. These include, for example, concentration/diafiltration operations, precipitation/elution, column chromatography and depth filtration. See examples below.

After purification of the individual conjugates, they may be combined to formulate immunogenic compositions of the invention, which may be used, for example, in vaccines. The formulation of the immunogenic compositions of the invention can be accomplished using methods well known in the art.

Note that in the present disclosure, terms such as "comprising", "including", "containing", and the like may have meanings in conformity with the united states patent law; for example, they may represent "include", "included", "including", etc. Such terms are intended to encompass a particular ingredient or group of ingredients without excluding any other ingredients. Terms such as "consisting essentially of … (of) and" consisting essentially of … (of) "have the meaning consistent with united states patent law, e.g., they allow the inclusion of additional ingredients or steps that do not depart from the novel or essential features of the present invention, i.e., they exclude additional ingredients or steps that do not depart from the novel or essential features of the present invention, and they exclude prior art ingredients or steps, e.g., documents cited or incorporated by reference herein, particularly the purpose of this document is to define an embodiment that is patentable, as new, non-obvious, inventive for the prior art, e.g., documents incorporated by reference herein. Also, the terms "consisting of … (consistency of)" and "consisting of … (consistency of)" have meanings consistent with U.S. patent laws; i.e., the terms are closed. Thus, these terms refer to the inclusion of a particular ingredient or group of ingredients and the exclusion of all other ingredients.

"conservative amino acid substitutions" refer to the substitution of one or more amino acid residues of a protein with other amino acid residues having similar physical and/or chemical properties. Substitutions of amino acids within a sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The amino acids containing aromatic ring structure are phenylalanine, tryptophan and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such changes are not expected to affect the apparent molecular weight or isoelectric point as determined by polyacrylamide gel electrophoresis. Particularly preferred substitutions are: lys replaces Arg and vice versa, so that a positive charge can be retained; glu is substituted for Asp and vice versa, so that negative charges can be retained; ser for Thr, so that free-OH can be retained; and Gln substituted for Ash, whereby free NH may be retained₂。

"fragment" refers to a protein that includes only a particular domain of a larger protein. For example, if signal sequences are included, the ClfA and ClfB proteins each contain up to 8 domains. Polypeptides corresponding to the N1N2N3, N2N3, N1N2, N1, N2, or N3 domains are each considered fragments of ClfA or ClfB. "fragment" also refers to a protein or polypeptide comprising an amino acid sequence of at least 4 amino acid residues (preferably at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues) of the amino acid sequence of a parent protein or polypeptide; or a nucleic acid comprising a nucleotide sequence of at least 10 base pairs (preferably at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 100 base pairs, at least 200 base pairs) of the nucleotide sequence of a parent nucleic acid.

As used herein, the "functional activity" of an antibody or "functional antibody" refers to an antibody that can at least specifically bind to an antigen. Additional functions are known in the art and may include additional components of the immune system that affect the clearance or killing of pathogens, for example, by opsonization, ADCC, or complement-mediated cytotoxicity. Following antigen binding, any subsequent antibody function may be mediated through the Fc region of the antibody. Antibody opsonophagocytic assay (OPA) is an in vitro assay designed to measure in vitro Ig complement-assisted bacterial killing with effector cells (leukocytes), thereby mimicking biological processes. Antibody binding can also directly inhibit the biological function of the antigen to which it binds, e.g., an antibody that binds ClfA can neutralize its enzymatic function. In some embodiments, a "functional antibody" refers to an antibody that is functional as measured by an opsonophagocytic killing assay that kills bacteria in an animal efficacy model or by confirming that the antibody kills bacteria.

The molecular weight of the staphylococcus aureus capsular polysaccharide is a consideration for use in immunogenic compositions. For example, high molecular weight capsular polysaccharides are capable of inducing certain antibody immune responses due to the presence of more expensive epitopes on the surface of the antigen. Isolation of "high molecular weight capsular polysaccharides" is contemplated for use in the compositions and methods of the invention. For example, in one embodiment of the present invention, it is contemplated to isolate type 5 high molecular weight polysaccharides having molecular weights ranging from about 50 to about 800 kDa. In one embodiment of the present invention, it is contemplated to isolate type 5 high molecular weight polysaccharides having molecular weights of from about 20 to about 1000 kDa. In one embodiment of the invention, it is contemplated to isolate and purify a type 5 high molecular weight capsular polysaccharide having a molecular weight of from about 50 to about 300 kDa. In one embodiment, it is contemplated to isolate and purify a type 5 high molecular weight capsular polysaccharide having a molecular weight in the range of 70kDa to 300 kDa. In one embodiment, it is contemplated to isolate and purify a type 5 high molecular weight capsular polysaccharide having a molecular weight in the range of 90kDa to 250 kDa. In one embodiment, it is contemplated to isolate and purify a type 5 high molecular weight capsular polysaccharide having a molecular weight in the range of 90kDa to 150 kDa. In one embodiment, it is contemplated to isolate and purify a type 5 high molecular weight capsular polysaccharide having a molecular weight in the range of 90kDa to 140 kDa. In one embodiment, it is contemplated to isolate and purify a type 5 high molecular weight capsular polysaccharide having a molecular weight in the range of 80kDa to 120 kDa. Other ranges for high molecular weight serotype 5 capsular polysaccharides that may be isolated and purified by the methods of the present invention include the molecular weight size range of about 70kDa to about 100 kDa; a molecular weight range of 70kDa to 110 kDa; a molecular weight range of 70kDa to 120 kDa; a molecular weight range of 70kDa to 130 kDa; a molecular weight range of 70kDa to 140 kDa; a molecular weight range of 70kDa to 150 kDa; a molecular weight range of 70kDa to 160 kDa; molecular weight range of 80kDa-110 kDa; molecular weight range of 80kDa-120 kDa; molecular weight range of 80kDa-130 kDa; molecular weight range of 80kDa-140 kDa; molecular weight range of 80kDa-150 kDa; molecular weight range of 80kDa-160 kDa; a molecular weight range of 90kDa to 110 kDa; molecular weight range of 90kDa-120 kDa; a molecular weight range of 90kDa to 130 kDa; a molecular weight range of 90kDa to 140 kDa; a molecular weight range of 90kDa to 150 kDa; a molecular weight range of 90kDa to 160 kDa; a molecular weight range of 100kDa to 120 kDa; a molecular weight range of 100kDa to 130 kDa; a molecular weight range of 100kDa to 140 kDa; a molecular weight range of 100kDa to 150 kDa; a molecular weight range of 100kDa to 160 kDa; and similar desired molecular weight ranges.

As discussed above, the molecular weight of the staphylococcus aureus capsular polysaccharide is a consideration for use in immunogenic compositions. For example, high molecular weight capsular polysaccharides are capable of inducing certain antibody immune responses due to the presence of more expensive epitopes on the surface of the antigen. In one embodiment of the invention, it is contemplated to isolate and purify a type 8 high molecular weight capsular polysaccharide having a molecular weight in the range of about 20kDa to about 1000 kDa. In one embodiment of the invention, it is contemplated to isolate and purify a type 8 high molecular weight capsular polysaccharide having a molecular weight in the range of about 50kDa to about 700 kDa. In one embodiment of the invention, it is contemplated to isolate and purify a type 8 high molecular weight capsular polysaccharide having a molecular weight in the range of 50kDa to 300 kDa. In one embodiment, it is contemplated to isolate and purify a type 8 high molecular weight capsular polysaccharide having a molecular weight in the range of 70kDa to 300 kDa. In one embodiment, it is contemplated to isolate and purify a type 8 high molecular weight capsular polysaccharide having a molecular weight in the range of 90kDa to 250 kDa. In one embodiment, it is contemplated to isolate and purify a type 8 high molecular weight capsular polysaccharide having a molecular weight in the range of 90kDa to 150 kDa. In one embodiment, it is contemplated to isolate and purify a type 8 high molecular weight capsular polysaccharide having a molecular weight in the range of 90kDa to 120 kDa. In one embodiment, it is contemplated to isolate and purify a type 8 high molecular weight capsular polysaccharide having a molecular weight in the range of 80kDa to 120 kDa. Other ranges for high molecular weight serotype 8 capsular polysaccharides that may be isolated and purified by the methods of the present invention include the molecular weight size range of about 70kDa to about 100 kDa; a molecular weight range of 70kDa to 110 kDa; a molecular weight range of 70kDa to 120 kDa; a molecular weight range of 70kDa to 130 kDa; a molecular weight range of 70kDa to 140 kDa; a molecular weight range of 70kDa to 150 kDa; a molecular weight range of 70kDa to 160 kDa; molecular weight range of 80kDa-110 kDa; molecular weight range of 80kDa-120 kDa; molecular weight range of 80kDa-130 kDa; molecular weight range of 80kDa-140 kDa; molecular weight range of 80kDa-150 kDa; molecular weight range of 80kDa-160 kDa; a molecular weight range of 90kDa to 110 kDa; molecular weight range of 90kDa-120 kDa; a molecular weight range of 90kDa to 130 kDa; a molecular weight range of 90kDa to 140 kDa; a molecular weight range of 90kDa to 150 kDa; a molecular weight range of 90kDa to 160 kDa; a molecular weight range of 100kDa to 120 kDa; a molecular weight range of 100kDa to 130 kDa; a molecular weight range of 100kDa to 140 kDa; a molecular weight range of 100kDa to 150 kDa; a molecular weight range of 100kDa to 160 kDa; and similar desired molecular weight ranges.

An "immune response" to an immunogenic composition is the development of a humoral and/or cell-mediated immune response in a subject to a molecule (e.g., an antigen, such as a protein or polysaccharide) present in the composition of interest. For the purposes of the present invention, a "humoral immune response" is an antibody-mediated immune response and includes the production of antibodies with affinity for the antigen present in the immunogenic composition of the invention, while a "cell-mediated immune response" is a T-lymphocyte and/or other leukocyte-mediated immune response. A "cell-mediated immune response" is elicited by the presentation of an epitope associated with a class I or class II molecule of the Major Histocompatibility Complex (MHC). This activates antigen-specific CD4+ T helper cells or CD8+ cytotoxic T lymphocytes ("CTLs"). CTLs are specific for peptide or lipid antigens that are presented in association with proteins encoded by the Major Histocompatibility Complex (MHC) or CD1 and expressed on the cell surface. CTLs help induce and promote intracellular destruction of intracellular microorganisms, or lysis of cells infected with such microorganisms. Another aspect of cellular immunity involves antigen-specific responses of helper T-cells. Helper T-cells assist the stimulatory function and focus on the activity of non-specific effector cells against cells displaying peptide antigens in association with classical or non-classical MHC molecules on the cell surface. "cell-mediated immune response" also refers to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other leukocytes, including molecules derived from CD4+ and CD8+ T-cells. The ability of a particular antigen or composition to stimulate a cell-mediated immune response can be determined by a number of assays, for example by lymphocyte proliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, by assaying for T-lymphocytes specific for the antigen in a sensitized subject, or by measuring cytokine production by T-cells in response to re-stimulation by the antigen. Such assays are well known in the art. See, e.g., Erickson et al, j.immunol. (1993) 151: 4189 + 4199; doeet al, eur.j.immunol. (1994) 24: 2369-2376.

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

An "immunogenic amount," or an "immunologically effective amount" or "dose," each used interchangeably herein, generally refers to an amount of an antigen or immunogenic composition sufficient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, as measured by standard assays known to those skilled in the art.

The amount of a particular conjugate in a composition is generally calculated based on the total polysaccharide conjugated and unconjugated to the conjugate. For example, a CP5 conjugate containing 20% free polysaccharide would contain about 80 meg conjugated CP5 polysaccharide and about 20 meg unconjugated CP5 polysaccharide in a 100 meg CP5 polysaccharide dose. The contribution of the protein to the conjugate is generally not taken into account when calculating the dose of the conjugate. The amount of conjugate may vary depending on the staphylococcal serotype. Typically, each dose will contain 0.01-100mcg of polysaccharide, particularly 0.1-10mcg, and more particularly 1-10 mcg. The "immunogenic amount" of the different polysaccharide components in the immunogenic composition can be different, and each can comprise any particular polysaccharide antigen of 0.01mcg, 0.1mcg, 0.25mcg, 0.5mcg, 1mcg, 2mcg, 3mcg, 4mcg, 5mcg, 6mcg, 7mcg, 8mcg, 9mcg, 10mcg, 15mcg, 20mcg, 30mcg, 40mcg, 50mcg, 60mcg, 70mcg, 80mcg, 90mcg, or about 100 mcg.

In another embodiment, the "immunogenic amount" of a protein component in an immunogenic composition can be from about 10 meg to about 300 meg of each protein antigen. In particular embodiments, the "immunogenic amount" of a protein component in an immunogenic composition can be from about 20 meg to about 200 meg of each protein antigen. The "immunogenic amount" of the different protein components in the immunogenic composition can vary and each comprises 10mcg, 20mcg, 30mcg, 40mcg, 50mcg, 60mcg, 70mcg, 80mcg, 90mcg, 100mcg, 125mcg, 150mcg, 175mcg, or about 200mcg of any particular protein antigen.

The effectiveness of an antigen as an immunogen can be measured by measuring the level of B cell activity measured by measuring the level of circulating antibodies specific for the antigen using immunoassays, immunoprecipitation assays, functional antibody assays such as in vitro opsonization assays, and many other assays known in the art. Another measure of the effectiveness of an antigen as a T-cell immunogen can be measured by proliferation assays, by cytolytic assays, such as chromium release assays to measure the ability of a T cell to lyse its specific target cell. Furthermore, in the present invention, an "immunogenic amount" may also be defined by measuring the serum level of antigen-specific antibodies induced following administration of the antigen, or as described above by measuring the ability of such induced antibodies to enhance the opsonophagocytic capacity of specific leukocytes. The level of protection of the immune response can be measured by challenging the immunized host with the injected antigen. For example, if the antigen for which an immune response is desired is a bacterium, the level of protection induced by an "immunogenic amount" of the antigen can be measured by measuring the percent survival or percent death following challenge of the animal with the bacterial cells. In one embodiment, the protective amount can be measured by measuring at least one symptom associated with the bacterial infection, e.g., fever associated with the infection. The amount of each antigen in a multi-antigen or multi-component vaccine or immunogenic composition will vary with respect to each other component and can be determined by methods known to the skilled person. Such methods include, for example, methods of measuring immunogenicity and/or in vivo efficacy.

The term "immunogenic composition" relates to any pharmaceutical composition containing an antigen, such as a microorganism, or a component thereof, which can be used to elicit an immune response in a subject. The immunogenic compositions of the invention may be used to treat a human susceptible to infection with staphylococcus aureus by administering the immunogenic composition by a systemic transdermal or mucosal route. These administrations may include injection by intramuscular (i.m.), intraperitoneal (i.p.), intradermal (i.d.), or subcutaneous routes; administration via a patch or other transdermal delivery device; or through the mucosa to the oral/digestive, respiratory or genitourinary tract. In one embodiment, intranasal administration is used to treat or prevent nasopharyngeal carriage of Staphylococcus aureus, thereby attenuating the infection during the initial stages of the infection. In one embodiment, the immunogenic composition may be used to prepare a vaccine or to elicit polyclonal or monoclonal antibodies that may be used to passively protect or treat an animal.

The optimal amounts of the components of a particular immunogenic composition can be determined by standard studies, including observing the appropriate immune response in a subject. Following the initial immunization, the subject may receive one or more boosters at sufficient intervals.

In one embodiment of the invention, the staphylococcus aureus immunogenic composition comprises a recombinant staphylococcus aureus aggregating factor a (clfa) fragment (N1N2N3, or a combination thereof), isolated conjugated to CRM₁₉₇And isolated conjugation to CRM₁₉₇Type 8 capsular polysaccharide of (1). In another embodiment, the staphylococcus aureus immunogenic composition is a recombinant staphylococcus aureus aggregation factor (ClfA) fragment (N1N2N3, or a combination thereof), a recombinant staphylococcus aureus aggregation factor b (clfb) fragment (N1N2N3, or a combination thereof), an isolated conjugate to CRM₁₉₇And isolated conjugation to CRM₁₉₇Sterile preparations (liquid, lyophilized, DNA vaccine, intradermal preparation) of type 8 capsular polysaccharide. In one embodiment of the invention, the staphylococcus aureus immunogenic composition comprises a recombinant staphylococcus aureus clumping factor a (clfa) fragment (N1N2N3, or a combination thereof), staphylococcus aureus iron binding protein MntC, isolated conjugated to CRM₁₉₇And isolated conjugation to CRM₁₉₇Type 8 capsular polysaccharide of (1). In one embodiment, the staphylococcus aureus immunogenic composition is a recombinant staphylococcus aureus aggregating factor (ClfA) fragment (N1N2N3, or a combination thereof), recombinant staphylococcus aureusAggregation factor B (ClfB) fragment (N1N2N3, or a combination thereof), Staphylococcus aureus iron binding protein MntC, isolated conjugated to CRM₁₉₇And isolated conjugation to CRM₁₉₇Sterile preparations (liquid, lyophilized, DNA vaccine, intradermal preparation) of type 8 capsular polysaccharide. In one embodiment of the invention, the staphylococcus aureus immunogenic composition comprises a recombinant staphylococcus aureus aggregating factor b (clfb) fragment (N1N2N3, or a combination thereof), isolated conjugated to CRM₁₉₇And isolated conjugation to CRM₁₉₇Type 8 capsular polysaccharide of (1). In one embodiment of the invention, the staphylococcus aureus immunogenic composition comprises a recombinant staphylococcus aureus clumping factor b (clfb) fragment (N1N2N3, or a combination thereof), staphylococcus aureus iron binding protein MntC, isolated conjugated to CRM₁₉₇And isolated conjugation to CRM₁₉₇Type 8 capsular polysaccharide of (1). In one embodiment of the invention, the staphylococcus aureus immunogenic composition comprises the staphylococcus aureus iron binding protein MntC, isolated conjugated to CRM₁₉₇And isolated conjugation to CRM₁₉₇Type 8 capsular polysaccharide of (1).

The immunogenic compositions of the invention may further comprise one or more additional "immunomodulatory agents", which are substances that disrupt or alter the immune system, such that up-or down-regulation of humoral and/or cell-mediated immunity is observed. In a particular embodiment, it is preferred that the humoral and/or cell-mediated up-regulation of the immune system of the moiety (arm). Examples of certain immunomodulatory agents include, for example, adjuvants or cytokines as described in U.S. patent No. 5,254,339, et al, or ISCOMATRIX (CSL Limited, Parkville, Australia). Non-limiting examples of adjuvants that can be used in the vaccines of the present invention include RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions, e.g., Freund's complete and incomplete adjuvants, block copolymers (CytRx, Atlanta Ga.), QS-21(Cambridge Biotech Inc., Cambridge Mass.), SAF-M (SAF-M;)Chiron，Emeryville Calif.)，Adjuvants, saponin, Quil a or other saponin components, monophosphoryl lipid a and an avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions useful in the vaccines of the present invention include modified formulations of SEAM62 and SEAM 1/2. The modified SEAM62 was a reagent containing 5% (v/v) squalene (Sigma), 1% (v/v)85 detergent (ICI surfactant), 0.7% (v/v) polysorbateAn oil-in-water emulsion of detergent (ICI surfactant), 2.5% (v/v) ethanol, 200. mu.g/ml Quil A, 100. mu.g/ml cholesterol and 0.5% (v/v) lecithin. The improved SEAM 1/2 is a composition comprising 5% (v/v) squalene, 1% (v/v)85 detergent, 0.7% (v/v) polysorbate 80 detergent, 2.5% (v/v) ethanol, 100 μ g/ml Quil A and 50 μ g/ml cholesterol in oil-in-water emulsion. Other "immunomodulatory agents" that may be included in the vaccine include, for example, one or more interleukins, interferons, or other known cytokines or chemokines. In one embodiment, the adjuvant may be a cyclodextrin derivative or a polyanionic polymer, as described in U.S. Pat. nos. 6,165,995 and 6,610,310, respectively. It will be appreciated that the immunomodulatory agent and/or adjuvant to be used will depend on the subject to which the vaccine or immunogenic composition is administered, the route of injection and the number of injections to be administered.

Staphylococcus aureus "invasive disease" is the isolation of bacteria from a normally sterile site with associated clinical signs/symptoms of the disease. Normally sterile body sites include blood, CSF, pleural fluid, pericardial fluid, peritoneal fluid, joint/synovial fluid, bone, internal body sites (lymph nodes, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, ovary), or other normally sterile sites. Clinical conditions characterized by invasive disease include bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis, septic shock, and the like.

The term "isolated" means that the substance is removed from its original environment (e.g., from its natural environment if it is naturally occurring, or from its host organism if it is a recombinant entity, or from one environment to a different environment). For example, an "isolated" capsular polysaccharide, protein or peptide is substantially free of cellular material or other contaminating proteins from a cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized or otherwise present in the mixture as part of a chemical reaction. In the present invention, the protein or polysaccharide may be isolated from bacterial cells or cell fragments, thereby providing them in a manner conducive to the preparation of immunogenic compositions. The term "isolated" or "isolating" may include purification (purifying) or purification (purifying), including, for example, processes for purifying a protein or capsular polysaccharide as described herein. The language "substantially free of cellular material" includes preparations of polypeptides/proteins wherein the polypeptides/proteins are isolated from cellular components of cells from which the polypeptides/proteins are isolated or recombinantly produced. Thus, a capsular polysaccharide, protein or peptide that is substantially free of cellular material includes preparations of capsular polysaccharide, protein or peptide that contain less than about 30%, 20%, 10%, 5%, 2.5% or 1% (by dry weight) of contaminating protein or polysaccharide or other cellular material. When the polypeptide/protein is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide/protein or polysaccharide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is isolated from the chemical precursors or other chemicals involved in the synthesis of the protein or polysaccharide. Accordingly, such polypeptide/protein or polysaccharide preparations contain less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide/protein or polysaccharide fragment of interest.

"non-conservative amino acid substitution" refers to the substitution of one or more amino acid residues of a protein with other amino acid residues having different physical and/or chemical properties, which use the features defined above.

The term "pharmaceutically acceptable carrier" means a carrier that is approved by a regulatory agency of the federal, a state government or other regulatory agency, or a carrier listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans and non-human mammals. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions, and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting agents, bulking agents, emulsifying agents or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, sustained release formulations and the like. Examples of suitable pharmaceutical carriers are described in e.w. martin "Remington's pharmaceutical Sciences". The formulation should be adapted to the mode of administration.

The terms "protein", "polypeptide" and "peptide" refer to a polymer of amino acid residues and are not limited to the minimum length of the product. Accordingly, peptides, oligopeptides, dimers, multimers, and the like are included within this definition. This definition encompasses full-length proteins and fragments thereof. The term also includes modifications to the native sequence, such as deletions, additions and substitutions (generally conserved in nature, but which may be non-conserved), preferably such that the protein retains the ability to elicit an immune response in the animal to which it is administered. Also included are post-expression modifications such as glycosylation, acetylation, lipidation (phosphorylation), phosphorylation, and the like.

A "protective" immune response refers to the ability of an immunogenic composition to elicit a humoral or cell-mediated immune response that helps protect a subject from infection. The protection provided need not be absolute, i.e., the infection need not be completely prevented or eradicated if there is a statistically significant improvement compared to a control population of subjects (e.g., infected animals not administered the vaccine or immunogenic composition). Protection may be limited to alleviating the severity or rate of onset of symptoms of the infection. In general, a "protective immune response" includes inducing an increase in the level of specific antibodies to a particular antigen in at least 50% of subjects, including some level of measurable functional antibodies in response to each antigen. In particular instances, a "protective immune response" may include inducing a 2-fold increase or a 4-fold increase in the level of specific antibodies to a particular antigen in at least 50% of subjects, including some level of measurable functional antibodies in response to each antigen. In certain embodiments, the opsonic antibody is associated with a protective immune response. Thus, a protective immune response can be determined by measuring the percentage reduction in bacterial count in an opsonophagocytosis assay, such as described below. Preferably, there is at least a 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more reduction in bacterial count.

As used herein, the term "recombinant" simply refers to any protein, polypeptide, or cell that expresses a gene of interest produced by genetic engineering methods. The term "recombinant" when used with respect to a protein or polypeptide refers to a polypeptide produced by expression of a recombinant polynucleotide. The proteins used in the immunogenic compositions of the invention can be isolated from natural sources or produced by genetic engineering methods, such as recombinant ClfA, recombinant ClfB or recombinant MntC. As used herein, "recombinant" further describes a nucleic acid molecule that, due to its origin or manipulation, is unrelated to all or part of its naturally associated polynucleotide. The term "recombinant" when used with respect to a host cell denotes a host cell into which a recombinant polynucleotide has been introduced.

As used herein, recombinant ClfA (rclfa) and recombinant ClfB (rclfb) refer to the forms of ClfA or ClfB used in the immunogenic compositions of the invention. In one embodiment, rClfA is a fragment of ClfA that comprises one or more N domains, such as N1N2N3, N2N3, N2, or N3, and is referred to herein as "recombinant ClfA" or "rClfA. In one embodiment, rClfB is a fragment of ClfB comprising one or more N domains of ClfB, e.g., N1N2N3, N2N3, N2, or N3, and referred to herein as "recombinant ClfB" or "rClfB.

The term "subject" refers to a mammal, bird, fish, reptile, or any other animal. The term "subject" also includes humans. The term "subject" also includes household pets. Non-limiting examples of household pets include: dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, birds, snakes, lizards, fish, turtles, and frogs. The term "subject" also includes livestock. Non-limiting examples of livestock include: alpaca, bison, camel, cattle, deer, pig, horse, llama, mule, donkey, sheep, goat, rabbit, reindeer, yak, chicken, goose and turkey.

As used herein, "treatment" (including variants thereof, e.g., "treatment" or "treated") refers to any one or more of the following: (i) preventing infection or reinfection as in traditional vaccines, (ii) reducing the severity of symptoms, or eliminating symptoms, and (iii) substantially or completely eliminating the pathogen or disorder in question. Thus, treatment can be effected prophylactically (before infection) or therapeutically (after infection). In the present invention, either prophylactic or therapeutic treatment may be used. According to particular embodiments of the present invention, compositions and methods are provided that treat a host animal against antimicrobial infections (e.g., bacteria, such as staphylococcal species), including prophylactic and/or therapeutic immunity. The methods of the invention are useful for conferring prophylactic and/or therapeutic immunity to a subject. The methods of the invention may also be administered to subjects for biomedical research applications.

The terms "vaccine" or "vaccine composition" are used interchangeably to refer to a pharmaceutical composition comprising at least one immunogenic composition that induces an immune response in an animal.

SUMMARY

The present invention relates to immunogenic compositions comprising at least 3 antigens from a staphylococcal organism, such as from staphylococcus aureus. The antigens may be isolated from the organism using biochemical isolation methods, or they may be synthesized or produced by recombinant methods. The antigen may be a polypeptide or a polysaccharide or a combination thereof. These immunogenic compositions can be used to prepare vaccines to immunize subjects against infection by staphylococcal organisms. Suitable components for use in these compositions are described in more detail below.

Staphylococcal immunogenic compositions

Staphylococcus aureus is the causative agent of a variety of human diseases ranging from superficial skin infections to life-threatening conditions such as pneumonia, sepsis, and endocarditis. See Lowy n.eng.j.med.339: 580-532(1998). In the case of invasive diseases, staphylococcus aureus can be isolated from normally sterile body sites including blood, cerebrospinal fluid CSF, pleural fluid, pericardial fluid, peritoneal fluid, joint/synovial fluid, bone, internal body sites (lymph nodes, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, ovaries) or other normally sterile sites. This can lead to life-threatening clinical conditions such as bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis, and septic shock. Adult, elderly and pediatric patients are most at risk for infection with staphylococcus aureus.

Description of embodiments of the invention selected antigens in immunogenic compositions include isolated staphylococcus aureus clump factor a (clfa) polypeptide, isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein, isolated staphylococcus aureus clump factor b (clfb), and recombinant staphylococcus aureus MntC protein. The antigens in the immunogenic composition are then characterized by a series of combinations to demonstrate that the particular combination provides an immune response that may be superior to that produced using the individual components of the immunogenic composition. Accordingly, one combination provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein. The second combination provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus clump factor b (clfb), an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein. A third combination provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein. A fourth combination provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein. A fifth combination provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein. A sixth combination provides an immunogenic composition comprising: an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein. A seventh combination provides an immunogenic composition comprising: an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein. An eighth combination provides an immunogenic composition comprising: an isolated staphylococcus aureus aggregation factor a (clfa) polypeptide, an isolated staphylococcus aureus aggregation factor b (clfb) polypeptide, and an isolated staphylococcus aureus MntC protein. In some embodiments, the above combinations further comprise at least one of the following antigens: ekes, DsqA, KesK, KrkN2, Rkas, RrkN, Knka, SdrC, SdrD, SdrE, Opp3a, DltD, HtsA, Ltas, IsdA, IsdB, IsdC, SdrF, SdrG, SdrH, SrtA, SpA, Sbi α -hemolysin (hla), β -hemolysin, fibronectin binding protein A (fnbA), fibronectin binding protein B (fnbB), coagulase, FIG, Fig, Panton-Valentin (pvl), α -toxin and variants thereof, γ -toxin (hlg) and variants thereof, ica, immunodominant ABC transporter, Mg2+ transporter, Ni ABC transporter, CnRAP, autolysin, laminin receptor, IsaIIA/Pisbb, SaaPisbsA, IsaS, ESPA/S, ESPA, FA, ESA, ESBA, ESA, ESBA, ESB, ES, TSST-1, mecA, poly-N-acetylglucosamine (PNAG/dPNAG) exopolysaccharide, GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin binding protein, HarA, EsxA, EsxB, enterotoxin A, enterotoxin B, enterotoxin C1, and neoautolysin.

Epidemiological studies of staphylococcus aureus outbreaks have shown that the evolution of staphylococcus aureus is clonal in nature, with a single clone that achieves a successful genotype spreading rapidly and causing many infections. Therefore, evolution is considered to be clonal. The bacterial genome is composed of a larger, more stable, species core genome and a more diverse set of helper genes. See Feil et al, Nature Reviews: microbiology 2: 483-495(2004). The core gene is ubiquitous in all clones of a species, while the helper genes need not be present in any given clone. Considering staphylococcus aureus, one study using a DNA microarray representing more than 90% of the staphylococcus aureus genome found 78% of the genes in the genome to be common to all staphylococcus aureus and thus represent the "species core", while the remaining 22% are "helper genes". Helper genes comprise nonessential genetic material, a number of genes encoding virulence factors, proteins mediating antibiotic resistance, and proteins that specifically interact with a particular host environment. See Fitzgerald et al, PNAS 98: 8821-8826 (2001); feil et al, Nature Reviews: microbiology 2: 483-495(2004). Overall, the core gene evolves more slowly, while the helper genes are polymorphic. See Kuhn et al, j.bact.188: 169-178(2006). Thus, an appropriate selection of the core gene provides a better target antigen for use in immunogenic compositions to prevent infection.

Surface-expressed antigens from disease-causing isolates or clonotypes of staphylococcus aureus provide a source of antigens capable of inducing immune and functional antibodies. At the macromolecular level (amino acid or polysaccharide sequences), conserved forms of antigens expressed by different disease isolates can be selected to allow for broad cross-reactivity of antibodies to those strains that may have antigenic variation of the vaccine target.

An important consideration in including antigens in the multiple antigen immunogenic compositions described herein is whether the antigens have demonstrated efficacy by providing protection in one or more animal models of bacterial infection when administered as an immunogenic composition. There are many animal models of various staphylococcus aureus diseases. Each of these models has advantages and disadvantages.

Human clearance of bacterial infections can be through opsonic killing mediated after phagocytic uptake. There are many convincing examples of this from studies using gram-positive polysaccharide antigens such as the capsular polysaccharide of Streptococcus pneumoniae (Streptococcus pneumoniae) and the capsular polysaccharide of staphylococcus aureus. See Lee et al, crit. 333-349(2003). There is less evidence for opsonic activity induced by gram-positive protein antigens. Phagocytic uptake has been observed, but direct killing is more difficult to confirm. Monoclonal antibodies to the protein were shown to confer protection against s.aureus challenge in animal models of infection; and mechanisms other than opsonophagocytic killing may be responsible for the observed protection.

Induction of antibodies with measurable functional activity such as opsonophagocytic activity (OPA) is an indication of whether a particular antigen is beneficial for inclusion in the immunogenic compositions of the invention. Other indicators include, but are not limited to, antigen expression on the cell surface during in vivo expression as measured using antigen-specific antibodies, or the ability of the antibody to inhibit/neutralize antigen function.

Species/strains

Any particular hospital or type of disease strain is useful for determining origin, clonal association, and monitoring epidemiology of outbreaks. A number of methods are available for typing Staphylococcus aureus strains. The classical practical definition of a bacterial species is a group of strains characterized by more than 70% genomic hybridization (DNA-DNA genomic hybridization of DDH) and by more than 97% sequence identity of 16S ribosomal RNA genes. See Vandamme et al, microbiol. rev.60: 407-438(1996). Phage typing (BT) is a method of typing staphylococcus aureus strains based on their susceptibility to cleavage by certain phage types. See Blair et al, bull.w.h.o.24: 771-784(1961). This older method lacks repeatability between laboratories and has 15-20% of the isolates failing to type.

Single antigen to multiple antigen immunogenic compositions

The question arises as to whether an optimal immunogenic composition against infection with a major s.aureus strain should consist of a single component or of multiple components. Many studies have shown that immunogenic compositions based on a single protein or carbohydrate component can provide some protection against attack by a strain of staphylococcus aureus expressing the component in certain animal models. Importantly, it has also been demonstrated that protection against a single antigen may depend on the strain chosen.

Surface proteins such as adhesins have been investigated as single component vaccines. For example, mice immunized with staphylococcus aureus ClfA developed less severe arthritis than mice with control proteins. See Josefsson et al, j.infect.dis.184: 1572-1580(2001). Fragments of collagen-binding adhesin (cna) provide protection in a mouse sepsis model. See Nilsson, et al, j.clin.invest, 101: 2640-2649(1998). Immunization of mice with the a domain of ClfB can reduce nasal colonization in a mouse model. See schafer et al, infect.immun.74: 2145-2153(2006).

One of 14 staphylococcus aureus iron chelatin proteins, known as IsdB, was studied in a monovalent immunogenic formulation for protection against infection with staphylococcus aureus. This protein has shown good protective effects in mice and good immunogenicity in non-human primates. See Kuklin et al, infect.immun.74: 2215-2223(2006).

Because of the great potential for staphylococcus aureus to evolve or replace different proteins to perform the same or similar functions, the best immunogenic formulation for protecting most humans from most staphylococcus aureus diseases is a multi-antigen formulation comprising 2 or more (e.g., 3, 4, 5, etc.) antigens properly selected and present in the immunogenic formulation. In certain embodiments, the immunogenic compositions of the invention comprise 3 or more antigens selected from the group consisting of an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide (CP5) conjugated to a carrier protein, an isolated staphylococcus aureus type 8 capsular polysaccharide (CP8) conjugated to a carrier protein, and an isolated staphylococcus aureus MntC protein. In certain embodiments, the immunogenic compositions of the invention comprise 4 or more antigens selected from the group consisting of an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide (CP5) conjugated to a carrier protein, an isolated staphylococcus aureus type 8 capsular polysaccharide (CP8) conjugated to a carrier protein, and an isolated staphylococcus aureus MntC protein. In certain embodiments, the immunogenic compositions of the invention comprise as antigens an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide (CP5) conjugated to a carrier protein, an isolated staphylococcus aureus type 8 capsular polysaccharide (CP8) conjugated to a carrier protein, and an isolated staphylococcus aureus MntC protein.

Adjuvant

In certain embodiments, an immunogenic composition as described herein further comprises one or more adjuvants. Adjuvants are substances that enhance the immune response when administered with an immunogen or antigen. Many cytokines or lymphokines have been shown to have immunomodulatory activity and are therefore useful as adjuvants, including, but not limited to, interleukins 1-alpha, 1-beta, 2, 4, 5, 6,7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127), 13, 14, 15, 16, 17, and 18 (and mutated forms thereof); interferon- α, β and γ; granulocyte macrophage colony stimulating factor (GM-CSF) (see, e.g., U.S. patent No. 5,078,996 and ATCC accession No. 39900); macrophage colony stimulating factor (M-CSF); granulocyte colony stimulating factor (G-CSF); and tumor necrosis factors alpha and beta. Other adjuvants useful in the immunogenic compositions described herein include chemokines, including but not limited to MCP-1, MIP-1 α, MIP-1 β, and RANTES; adhesion molecules, e.g., selectins, such as L-selectin, P-selectin and E-selectin; mucin-like molecules, e.g., CD34, GlyCAM-1, and MadCAM-1; integrin family members such as LFA-1, VLA-1, Mac-1 and p 150.95; immunoglobulin superfamily members, e.g., PECAM, ICAMs, such as ICAM-1, ICAM-2, and ICAM-3, CD2, and LFA-3; co-stimulatory molecules such as B7-1, B7-2, CD40 and CD 40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1 and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR 6; and Caspase (ICE).

Suitable adjuvants for enhancing an immune response further include, but are not limited to, MPL described in U.S. Pat. No.4,912,094^TM(3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT). Synthetic lipid a analogs or aminoalkyl phosphoglucamine compounds (AGP), or derivatives or analogs thereof, are also suitable for use as adjuvants, and are available from Corixa (Hamilton, MT) and described in U.S. patent No. 6,113,918. One such AGP is 2- [ (R) -3-tetradecanoyloxy-tetradecanoyl-amino]Ethyl 2-deoxy-4-O-phosphono-3-O4 (R) -3-tetra-decanoyloxy-tetradecanoyl]-2- [ (R) -3-tetradecanoyloxy-tetradecanoyl-amino]-b-D-glucopyranoside, which is also known as 529 (formerly RC 529). This 529 adjuvant is formulated either in Aqueous Form (AF) or as a Stable Emulsion (SE).

Other adjuvants include muramyl peptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2- (1 '-2' dipalmitoyl-sn-glycero-3-hydroxy-phosphoryl-oxy) -ethylamine (MTP-PE); oil-in-water emulsions such as MF59 (U.S. patent No. 6,299,884) (containing 5% squalene, 0.5% polysorbate 80 and 0.5% span 85 (optionally containing various amounts of MTP-PE), formulated as submicron using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, MA)) and SAF (containing 10% squalene, 0.4% polysorbate 80, 5% pluronic-block polymer L121 and thr-MDP, the microfluidizer being a submicron emulsion or vortexed to produce a larger particle size emulsion); incomplete Freund's Adjuvant (IFA); aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate; aifoil gold; alfvudine; l121/squalene; d-lactide-polylactide/glucoside; pluronic polyols; killed Bordetella (Bordetella); saponins, e.g. American patentStimulon No. 5,057,540^TMQS-21 (antibiotics, Framingham, MA.), ISCOMATRIX (CSL Limited, Parkville, Australia) and ISCOMATRIX (ISCOMATRIX) as described in U.S. Pat. No. 5,254,339; mycobacterium tuberculosis (Mycobacterium tuberculosis); bacterial lipopolysaccharides; synthetic polynucleotides, such as oligonucleotides containing CpG motifs (e.g., U.S. Pat. No. 6,207,646); IC-31(Intercell AG, Vienna, Austria), as described in European patent Nos. 1,296,713 and 1,326,634; pertussis Toxin (PT) or a variant thereof, cholera toxin or a variant thereof (e.g., U.S. patent nos. 7,285,281, 7,332,174, 7,361,355, and 7,384,640); or E.coli heat-Labile Toxin (LT) or variants thereof, particularly LT-K63, LT-R72 (e.g., U.S. Pat. Nos. 6,149,919, 7,115,730 and 7,291,588).

Candidate antigens:

ClfA: organization of structural domains

Aggregation factor a (clfa) is a staphylococcus aureus surface protein that is associated with binding to host matrix proteins through fibrinogen binding sites. ClfA is a member of a family of proteins that contain a carboxy-terminal LPXTG (SEQ ID NO: 125) motif that enables the protein to covalently bind to the cell surface. ClfA also belongs to another family of proteins (microbial surface components recognize adhesion matrix molecules or MSCRAMMs) that are associated with binding to host proteins such as fibrinogen (bound by ClfA), fibronectin binding proteins (FnbA and FnbB), collagen binding proteins (Cna), and the like. These proteins all share an amino-terminal signal sequence that mediates transport to the cell surface. MSCRAMMs also include an a-domain, which is a functional region that contains the active site for ligand binding (e.g., fibrinogen, fibronectin, elastin, keratin). The a-domain is followed by a region consisting of a serine aspartate repeat (SD repeat), which is believed to span the peptidoglycan layer. The SD repeat is followed by a transmembrane region, which includes the LPXTG (SEQ ID NO: 125) motif, for covalent attachment of proteins to peptidoglycans. ClfA is described in us patent No. 6,008,341.

Comprising an A domainThe ligand binding region of ClfA of N1N2N3 (FIG. 1) spans amino acids 40-559. The N domain of ClfA has been assigned as follows: n1 includes residues 45-220; n2 includes residues 229-; and N3 includes residues 370-559. See, Deivanayagam et al embo j.21: 6660-6672(2002). For ease of reference, the N1N2N3 domain may be referred to as N123, and likewise N2N3 may be referred to as N23. In the preparation of recombinant N1N2N3, it was found that the N1 domain was protease sensitive and was easily cleaved or hydrolyzed away from N2N3 as a stable ligand-binding recombinant fragment. See, Deivanayagam et al embo j.21: 6660-6672(2002). The crystal structure of the fibrinogen-binding N2N3 fragment of the ClfA domain showed that N2 and N3 were under control of antiparallel beta strands. In addition to antiparallel beta strands, the N2 domain contains a single folded alpha helix and two 3₁₀Helices, and the N3 domain contains three 3₁₀A helix. See, Deivanayagam et al embo j.21: 6660-6672(2002). Sequence alignment of N2 and N3 showed only 13% sequence identity and 36% sequence similarity over their length. See, Deivanayagam et al embo j.21: 6660-6672(2002). The topology of the N2 and N3 domains is similar to that of a classical IgG fold and has been proposed as a new variant of an IgG fold. See, Deivanayagam et al embo j.21: 6660-6672(2002).

Sequence of ClfA

The gene for aggregation factor protein A, designated ClfA, has been cloned, sequenced and analyzed at a molecular level (McDevett et al, mol. Microbiol. 11: 237-. The sequence identifiers of the amino acid sequences of ClfA from 111 disease-causing isolates of staphylococcus aureus are shown in table 10. The amino acid sequence of the full-length (including signal sequence) wild-type ClfA from staphylococcus aureus strain PFESA0237 is set forth in SEQ ID NO: 130. This sequence shows tyrosine at position 338, which is changed to alanine in the mutant form of ClfA. The full-length gene encoding wild-type ClfA from staphylococcus aureus strain PFESA0237 is set forth in SEQ ID NO: 131, comprising an N123 region, a repeat region, and an anchor region. The amino acid sequence of the Y338A mutant form of ClfA is set forth in SEQ ID NO: 123 are shown. However, it should be noted that the amino acid sequence of SEQ ID NO: 130 and designated Y338A is shown in the mutant form of ClfA, in the mutant form of SEQ ID NO: 123 at location 310. Furthermore, SEQ ID NO: 123 is the mature form of ClfA without the signal sequence, and thus is the mature form of ClfA shown in the amino acid sequence of SEQ ID NO: 130 and SEQ ID NO: 123 is different from each other in the position of this mutation.

ClfB: organization of structural domains

ClfB is staphylococcus aureus with fibrinogen binding activity and triggers the formation of clumps of staphylococcus aureus in the presence of plasma. ClfB is an MSCRAMM protein and exhibits a characteristic MSCRAMM domain organization including an a-domain, which is a functional region containing the active site for ligand binding (e.g., fibrinogen, fibronectin, elastin, keratin). The a-domain is followed by a region consisting of a serine aspartate repeat (SD repeat), which is believed to span the peptidoglycan layer. The SD repeat is followed by a transmembrane region, which includes the LPXTG (SEQ ID NO: 125) motif, for covalent attachment of proteins to peptidoglycans. ClfB as described in WO 99/27109 and us patent 6,680,195.

The internal organization of the ClfB N-terminal a domain is very similar to that found in ClfA. The a domain consists of three subdomains N1, N2, and N3. The ligand binding region of ClfB of N1N2N3 (fig. 1) comprising the a domain spans amino acids 44-585. For ease of reference, the N1N2N3 domain may be referred to as N123, and likewise N2N3 may be referred to as N23. The N domain of ClfB has been assigned as follows: n1 includes residue 44-₁₉₇(ii) a N2 includes residues 198-375; and N3 includes residues 375-585. In ClfA, the crystal structure of the a domain is found to have a unique form of immunoglobulin folding, and by analogy, ClfB can be presumed to be the same. See, Deivanayagam et al, emboj.21: 6660-6672(2002). Although the organization of the a domains of ClfB and ClfA is similar, the sequence identity is only 26%. See Ni Eidhin et al, mol. microbiol.30: 245-257(2002).

ClfB sequence

The gene encoding ClfB was classified as a core adhesion gene. The ClfB sequences from 92 strains of staphylococcus aureus associated with various disease states are summarized in table 11. Other sequences were obtained from GenBank.

Other MSCRAMSs

Other MSCRAMMS are contemplated for use in the immunogenic compositions of the invention. For example, the serine-aspartate repeat (Sdr) proteins SdrC, SdrD and SdrE are related to ClfA and ClfB in primary sequence and structural organization and are localized at the cell surface. SdrC, SdrD and SdrE proteins are cell wall-associated proteins with an N-terminal signal sequence and an LPXTG (SEQ ID NO: 125) motif, a C-terminal hydrophobic domain and positively charged residues. Each also has an SD repeat containing an R region of sufficient length to allow, together with the B motif, efficient expression of the ligand binding domain region a on the cell surface. Having the A regions of SdrC, SdrD and SdrE located on the cell surface, proteins can interact with proteins in plasma, extracellular matrices or molecules on the surface of host cells. Sdr proteins share some limited amino acid sequence similarity with ClfA and ClfB. Like ClfA and ClfB, SdrC, SdrD and SdrE also exhibit cation-dependent ligand binding to extracellular matrix proteins.

sdr genes are closely linked and arranged in tandem. The Sdr proteins (SdrC, SdrD, SdrE, ClfA and ClfB) characteristically contain an A region, in which there is a highly conserved amino acid sequence that can be used to obtain a consensus TYTFTDYVD (SEQ ID NO: 126) motif. This motif shows slight variation between different proteins. The consensus sequence of this variation with this motif is described in U.S. Pat. No. 6,680,195. This motif is highly conserved in the Clf-Sdr protein. The motifs can be used in immunogenic compositions to confer broad-spectrum immunity to bacterial infections and can also be used as antigens in the preparation of monoclonal or polyclonal antibodies. Such antibodies can be used to confer broad spectrum passive immunity.

The Sdr protein differs from ClfA and ClfB in containing2-5 additional repeats of 110-113 residues (B-motif) located between the A and R regions. Each B-motif contains a common Ca commonly found in eukaryotic proteins²⁺-binding EF-bracelet. Structural integrity of recombinant proteins comprising 5 SdrD B-repeats was Ca as shown by bisANS fluorescence analysis²⁺Dependent, indicating that the EF-hand is functional. When Ca is removed²⁺When this structure collapses into an unfolded conformation. By adding Ca²⁺And recovering the original structure. The C-terminal R-domain of the Sdr protein contains 132-170 SD residues. These are followed by conserved wall anchoring regions characteristic of many gram-positive bacterial surface proteins.

In the Sdr and Clf proteins, this B motif is highly conserved, while degenerate forms appear in fibronectin-binding MSCRAMMS and in the collagen-binding protein Can. The B motif associated with the R region is necessary for the display of the ligand binding domain at a distance from the cell surface. The repeat B motif is a common feature of a subset of SD repeat proteins described herein. These motifs were found in different numbers in the 3 Sdr proteins from strain PFESA 0237. There was a clear difference between each B motif. The most conserved units are those adjacent to the R region (SdrC B2, SdrD B5, and SdrE B3). They differ from the rest in several positions, in particular in the C-terminal half. A notable structural detail is that adjacent B repeats are often separated by a proline present in the C-terminal region, but proline never occurs between the last B repeat and the R region. In contrast, this linker is characterized by a short acidic segment (stretch). These differences are evidence that the terminal units have different structural or functional roles compared to other B motifs. The SdrD and SdrEN terminal B motifs are distant from other B motifs and have many amino acid changes, including small insertions and deletions, while the remaining internal B motifs are more highly conserved. Note that 3 Sdr proteins each have at least one of a variety of B motifs.

The C-terminal R-domain of the Sdr protein contains 132-170 SD residues. These are followed by conserved wall anchoring regions characteristic of many gram-positive bacterial surface proteins.

Other candidate SdrD molecules for use in the immunogenic compositions of the invention may be derived from organisms of various species, some of which include the following SdrD, from staphylococcus aureus: strain USA300FPR3757 (protein accession number SAUSA 3000547); strain NCTC8325 (protein accession number SAOUHSC 00545); strain MW2 (protein accession number MW 0517); strain MSSA476 (protein accession No. SAS 0520; and strain Mu50 (protein accession No. SAV 0562).

Other MSCRAMMS that may be considered for use in the immunogenic compositions of the invention include EkeS, DsqA, KesK, KrkN2, RkaS, RrkN and KnkA. These MSCRAMMS are described in WO 02/102829, which is incorporated herein by reference. Other MSCRAMMS identified by GenBank accession numbers include NP _373261.1, NP _373371.1, NP _374246.1, NP _374248.1, NP _374841.1, NP _374866.1, NP _375140.1, NP _375614.1, NP _375615.1, NP _375707.1, NP _375765.1, and NP _ 375773.1.

Type 5 and 8 capsular polysaccharides

Staphylococcal microorganisms that can cause invasive disease are also generally capable of producing Capsular Polysaccharides (CP) that encapsulate the bacteria and enhance their resistance to clearance by the host's innate immune system. The CP masks the bacterial cells in a protective capsule, rendering the bacteria resistant to phagocyte lysis and intracellular killing. Bacteria lacking a capsule are more susceptible to phagocyte lysis. Capsular polysaccharides are often important virulence factors for many bacterial pathogens, including haemophilus influenzae, streptococcus pneumoniae (streptococcus pneumoniae) and Group B streptococcus (Group B streptococcus).

Capsular polysaccharides may be used to serotype a particular species of bacteria. Typing is usually accomplished by reaction with specific antisera or monoclonal antibodies raised against specific structural or unique epitopes characteristic of capsular polysaccharides. Bacteria enclosed in a capsule tend to grow into smooth colonies, while colonies of bacteria that have lost their capsule appear rough. Colonies that produce a slime appearance are referred to as heavily enveloped. Staphylococcus aureus types 1 and 2 are heavily enveloped and rarely associated with disease.

Most clinical isolates of staphylococcus aureus are encapsulated by serotype 5 or 8. Type 5(CP5) and type 8(CP8) capsular polysaccharides contain similar trisaccharide repeat units consisting of N-acetamidomannic acid, N-acetyl L-fucosamine, and N-acetyl D-fucosamine. See Fournier, j.m.et al, infection.immun.45: 97-93(1984) and Moreau, M., et al, Carbohydrate Res.201: 285-297(1990). The two CPs contain the same sugar, but the sugar linkage and O acetylation sites are different to create serologically distinct immunoreactivity patterns.

In some embodiments, the serotype 5 and/or 8 capsular polysaccharides of the present invention are O-acetylated. In some embodiments, the degree of O-acetylation of the type 5 capsular polysaccharide or oligosaccharide is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In some embodiments, the degree of O-acetylation of the type 8 capsular polysaccharide or oligosaccharide is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In some embodiments, the degree of O-acetylation of the type 5 and type 8 capsular polysaccharides or oligosaccharides is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%.

The degree of O-acetylation of the polysaccharide or oligosaccharide can be determined by any method known in the art, for example, by proton NMR (Lemercinier and Jones 1996, Carbohydrate Research 296; 83-96, Jones and Lemercinier 2002, J Pharmaceutical and BiomedicalAnalysis 30; 1233-. Another commonly used method is e.g. Hestrin (1949) j.biol.chem.180; 249-261.

In some embodiments, serotype 5 and/or 8 capsular polysaccharides of the invention are used to produce antibodies that are functional as measured by an opsonophagocytic killing assay that kills bacteria in an animal efficacy model or by demonstrating that the antibodies kill bacteria. Such functionality may not be observed with assays that monitor antibody production alone, which does not show the importance of O-acetylation in potency.

Epidemiology of capsule

By monitoring the clinical isolates, associations between particular capsular serotypes and disease are possible. Of the 8 different serotypes of S.aureus that have been identified (Karakawa and Vann (1982) only serotypes 1 and 2 are heavily enveloped and these are rarely isolated. see capsularPolysaccharoids of Staphylococcus aureus, p.285-293, In J.B.Robbins, J.C.Hill and J.C.Sadoff (ed.), Seminirs In infection disease, vol.4, Bacillus vaccines, Thieme Stretton, Inc.New York). Investigations have shown that about 85-90% of Staphylococcus aureus clinical isolates express CP5 or CP8(Arbeit RD, et al, Diagn. Microbiol. Infect. Dis. (1984) Apr; 2 (2): 85-91; Karakawa WW, et al, J.Clin. Microbiol. (1985) Sep; 22 (3): 445-7; Essawi T, et al, trop. Med. int. Health. (1998) Jul; 3 (7): -83; Na' was T, et al., J.Clin. Microbiol. (1998)36 (2): 414-20. most of the strains of CP5 and CP8 inseparable type are generally of type 5 or 8 (Cocchiro) containing mutations in the cap5/8 locus, which were rapidly recovered after several passages of the encapsulated phosphate in vitro (Op. 3. 75. Op. 59. Microbiol.) strains which were not expressed in the cap5/8 locus, and were also reported to be recovered after several times of the in vitro encapsulated phosphate-encapsulated in the strain produced by Fembellizing microorganisms (Op. 3. 76, Op. 76. 7. Microbiol. multidrug) and was also reported to be recovered in the encapsulated strain of the encapsulated in the encapsulated strain of the cap5 gene, j.p.et al, j.med.microbiol.19: 275-278(1985). Some non-typeable strains become capsule positive under appropriate growth conditions.

CP5 and CP8 structures

The repeating units of CP5 and CP8 are each composed of 2-acetamido-2-deoxy-D-mannuronic acid, 2-acetamido-2-deoxy-L-fucose and 2-acetamido-2-deoxy-D-fucose. See c.jones et al, carbohydr.res.340: 1097-1106(2005). Although CP5 and CP8 have the same sugar composition, they have been shown to be immunologically distinct. They differ in glycosidic linkages and sites of uronic acid O-acetylation. Strain dependent incomplete N-acetylation of one of the FucNAc residues was observed. See Tzianabos et al, PNAS V98: 9365(2001).

Staphylococcus aureus capsular polysaccharides in immunogenic compositions

The molecular weight of the staphylococcus aureus capsular polysaccharide is an important consideration for use in immunogenic compositions. High molecular weight capsular polysaccharides are able to induce certain antibody immune responses due to the more expensive epitopes present on the surface of the antigen. The processes described herein provide for the isolation and purification of type 5 and 8 capsular polysaccharides of higher molecular weight than previously available.

MntC/SitC/saliva binding protein

The MntC/SitC/saliva binding protein is an ABC transporter and has homologues in Staphylococcus epidermidis and Staphylococcus aureus. It is referred to as MntC in the present invention. This protein is a 32kDa lipoprotein and is located in the cell wall of bacteria. See Sellman et al, and Cockayne et al, infection.immun.66: 3767(1998). In staphylococcus epidermidis, it is a component of the iron regulatory operon. It shows comparable homology to adhesins, including FimA of streptococcus paracasei (s.paraanguis), and to lipoproteins of the ABC transporter family with a confirmed or putative metalloiron transport function. (Staphylococcus aureus strains and sequences see Table 12.)

MntC protein of staphylococcus aureus

The staphylococcus aureus homolog of MntC, known as salivary binding protein, is disclosed in U.S. patent No. 5,801,234 and may be included in the immunogenic compositions of the invention. The protein sequence of the S.aureus homologue of the MntC/SitC/saliva binding protein is found in GenBank accession NP-371155 of strain Mu 50. (also known as SAV 0631.) the sequence identifier is SEQ ID NO: 119. the accession number for the nucleotide sequence of the complete genome of strain Mu50 is NC-002758.2 (coordinates 704988 and 705917).

Staphylococcus epidermidis SitC protein

The Staphylococcus epidermidis homolog of MntC/SitC/saliva binding protein is called SitC and is disclosed by Sellman et al (Sellman et al, infection. Immun.2005 October; 73 (10): 6591-. The protein sequence of the Staphylococcus epidermidis homolog of the MntC/SitC/saliva binding protein is found in GenBank accession number YP-187886.1. (also known as SERP 0290.). The sequence identifier is SEQ ID NO: 121.

the accession number for the nucleotide sequence of the complete genome of the strain RP62A is NC-002976 (coordinates 293030-293959). Other candidate SitC molecules for use in the immunogenic compositions of the invention may be derived from various species of organisms, some of which are listed in table 1 below.

TABLE 1

Protein	Species (II)	Example strains	Protein accession number
				SitC	Staphylococcus haemolyticus (S.haemolyticus)	JCSC1435	BAE03450.1
SitC	Staphylococcus epidermidis	ATCC 12228	AAO04002.1
				SitC	Staphylococcus saprophyticus (S. saprophyticus)	ATCC 15305	BAE19233.1
SitC	Staphylococcus xylosus (S.xylosus)	DSM20267	ABR57162.1
				SitC	Staphylococcus aureus (S.camosus)	TM300	CAL27186.1

Staphylococcus aureus iron binding proteins

Another potential candidate antigen contemplated for use in the immunogenic compositions of the invention includes the staphylococcus aureus surface protein iron surface determinant b (isdb). Such MSCRAMMs are described by Mazmanian et al (Mazmanian, SK et al, Proc. Natl. Acad. Sci., USA 99: 2293-. This IsdB molecule is present in various strains of staphylococcus aureus, including strain MRSA252 (protein accession number CAG 40104.1); strain Newman (protein accession BAF 67312.1); strain MSSA476 (protein accession number CAG 42837.1); strain Mu3 (protein accession number BAF 78003.1); strain RF122 (protein accession number CAI 80681.1).

Candidate antigens:

the immunogenic compositions of the invention may also include one or more of the following antigens: opp3a, DltD, HtsA, Ltas, IsdA, IsdC, SdrF, SdrG, SdrH, SrtA, SpA, Sbi α -hemolysin (hla), β -hemolysin, fibronectin binding protein A (fnbA), fibronectin binding protein B (fnbB), coagulase, FIG, map, Panton-Valentine blasticidin (pvl), α -toxin and variants thereof, γ -toxin (hlg) and variants, ica, immunodominant ABC transporter, Mg2+ transporter, Ni ABC transporter, RAP, autolysin, laminin receptor, IsaA/PisA, IsaB/PisB, SPOIE, SepaA, EbpS, Sas A, SasF, Cnsh, EFB (FIB), SBI, Npase, EBP, osteocalcin II, sialoprotein binding protein, protein precursor (AUAU), PNAS-S, PNAS-25, PNAS, GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin binding protein, HarA, EsxA, EsxB, enterotoxin A, enterotoxin B, enterotoxin C1, and neoautolysin. In certain embodiments of the invention, when the immunogenic composition comprises certain forms of CP5 and/or CP8, it may not further comprise PNAG.

Immunogenic composition formulations

In one embodiment, the immunogenic composition of the invention further comprises at least one of an adjuvant, a buffer, a cryoprotectant, a salt, a divalent cation, a non-ionic detergent, a free radical oxidation inhibitor, a diluent, or a carrier.

In addition to the various staphylococcal protein antigens and the capsular polysaccharide-protein conjugate, the immunogenic compositions of the invention may further comprise one or more preservatives. The FDA requires that biologicals in multi-dose (multi-dose) vials contain preservatives with only a few exceptions. Preservative containing vaccine products include vaccines containing benzethonium chloride (anthrax), 2-phenoxyethanol (DTaP, HepA, Lyme, polio (parenteral)), phenol (Pneumo, typhoid (parenteral), vaccinia) and thimerosal (DTaP, DT, Td, HepB, Hib, influenza, JE, Mening, Pneumo, rabies). Preservatives approved for use in injectable pharmaceuticals include, for example, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, and phenylmercuric nitrate.

The formulation of the present invention may further comprise one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, and an antioxidant such as a free radical scavenger or a chelating agent, or any combination thereof. The choice of any one component (e.g., chelating agent) can determine whether another component (e.g., scavenger) is desirable. The final composition formulated for administration should be sterile and/or pyrogen-free. The skilled artisan can empirically determine which combination of these and other components is optimal for inclusion in the preservative containing immunogenic compositions of the invention, depending on various factors, such as the particular storage and administration conditions desired.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more physiologically acceptable buffers selected from, but not limited to, tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine, and succinate. In certain embodiments, the formulation is buffered to a pH in the range of about 6.0 to about 9.0, preferably about 6.4 to about 7.4.

In certain embodiments, it may be desirable to adjust the pH of the immunogenic compositions or formulations of the invention. The pH of the formulations of the present invention can be adjusted using standard techniques in the art. The pH of the formulation may be adjusted to 3.0-8.0. In certain embodiments, the pH of the formulation may be, or may be adjusted to, 3.0-6.0, 4.0-6.0, or 5.0-8.0. In other embodiments, the pH of the formulation may be, or may be adjusted to, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 5.8, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In certain embodiments, the pH may be, or may be adjusted to range from 4.5 to 7.5, or from 4.5 to 6.5, 5.0 to 5.4, 5.4 to 5.5, 5.5 to 5.6, 5.6 to 5.7, 5.7 to 5.8, 5.8 to 5.9, 5.9 to 6.0, 6.0 to 6.1, 6.1 to 6.2, 6.2 to 6.3, 6.3 to 6.5, 6.5 to 7.0, 7.0 to 7.5, or 7.5 to 8.0. In a particular embodiment, the pH of the formulation is about 5.8.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more divalent cations, including but not limited to MgCl₂、CaCl₂And MnCl₂The concentration ranges from about 0.1mM to about 10mM, preferably up to about 5 mM.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more salts, including but not limited to sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate, present at a physiologically acceptable ionic strength for the subject upon parenteral administration, and included at a final concentration that produces a selected ionic strength or osmolarity in the final formulation. The final ionic strength or osmolality of the formulation is determined by various components (e.g., ions from buffering compounds and other non-buffering salts). The preferred salt NaCl is present in a range of up to about 250mM, with salt concentrations selected to complement other components (e.g., sugars), such that the final total osmolality of the formulation is compatible with parenteral administration (e.g., intramuscular or subcutaneous injection) and will improve the long term stability of the immunogenic components of the immunogenic composition formulation in various temperature ranges. A salt-free formulation will tolerate an increased range of one or more selected cryoprotectants to maintain the desired final osmolarity level.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more cryoprotectants selected from, but not limited to, disaccharides (e.g., lactose, maltose, sucrose, or trehalose) and polyhydroxy hydrocarbons (e.g., dulcitol, glycerol, mannitol, and sorbitol).

In certain embodiments, the formulation has an osmolality in the range of about 200mOs/L to about 800mOs/L, preferably in the range of about 250mOs/L to about 500mOs/L or in the range of about 300mOs/L to about 400 mOs/L. The salt-free formulation may contain, for example, from about 5% to about 25% sucrose, and preferably from about 7% to about 15% or from about 10% to about 12% sucrose. Alternatively, the salt-free formulation may contain, for example, about 3% to about 12% sorbitol, and preferably about 4% to 7% or about 5% to about 6% sorbitol. If a salt such as sodium chloride is added, then the effective range of sucrose or sorbitol is relatively reduced. These and other such osmolality and osmolality considerations are well within the skill of the art.

In certain embodiments, formulations of the present invention that are compatible with parenteral administration comprise one or more free radical oxidation inhibitors and/or chelating agents. Various free radical scavengers and chelators are known in the art and apply to the formulations and methods of use described herein. Examples include, but are not limited to, ethanol, EDTA/ethanol combinations, triethanolamine, mannitol, histidine, glycerol, sodium citrate, phytate, tripolyphosphate, ascorbic acid/ascorbate, succinic acid/succinate, malic acid/maleate, destens, EDDHA, and DTPA, and various combinations of two or more thereof. In certain embodiments, at least one non-reducing free radical scavenger may be added at a concentration effective to increase the long-term stability of the formulation. One or more free radical oxidation inhibitors/chelators, such as scavengers and divalent cations, may also be added in various combinations. The choice of chelating agent will determine whether or not a scavenger needs to be added.

In certain embodiments, formulations of the invention that are compatible for parenteral administration comprise one or more nonionic surfactants including, but not limited to, polyoxyethylene sorbitol fatty acid esters, polysorbate-80 (tween 80), polysorbate-60 (tween 60), polysorbate-40 (tween 40), and polysorbate-20 (tween 20), polyoxyethylene alkyl ethers including, but not limited to, Brij 58, Brij 35, and others such as triton X-100; the pluronic series of triton X-114, NP40, span 85 and nonionic surfactant (e.g., pluronic 121), preferably with the components being polysorbate-80 at a concentration of about 0.001% to about 2% (preferably up to about 0.25%) or polysorbate-40 at a concentration of about 0.001% to 1% (preferably up to about 0.5%).

In certain embodiments, the formulations of the present invention comprise one or more additional stabilizing agents suitable for parenteral administration, for example, a reducing agent comprising at least one sulfhydryl (-SH) (e.g., cysteine, N-acetylcysteine, reduced glutathione, sodium thioglycolate, thiosulfate, thioglycerol, or mixtures thereof). Alternatively or optionally, the preservative containing immunogenic composition formulations of the invention may be further stabilized by removing oxygen from the storage container, protecting the formulation from light (e.g., by using an amber glass container).

The preservative-containing immunogenic composition formulations of the present invention may comprise one or more pharmaceutically acceptable carriers or excipients, including any excipient that does not itself induce an immune response. Suitable excipients include, but are not limited to, macromolecules such as proteins, sugars, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al, 2001, Vaccine, 19: 2118), trehalose, lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such vectors are well known to the skilled person. Pharmaceutically acceptable excipients such as Gennaro, 2000, Remington: the Science and Practice of pharmacy, 20^thedition, ISBN: 0683306472, respectively.

The compositions of the invention may be in lyophilized or aqueous form, i.e. solutions or suspensions. Liquid formulations can advantageously be administered directly from their packaged form and are therefore ideal for injection without the need for reconstitution in an aqueous medium as otherwise required by the lyophilized compositions of the invention.

Delivery of the immunogenic composition of the invention directly to a subject may be accomplished by parenteral administration (intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous or to the interstitial space of a tissue); or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, otic, pulmonary or other mucosal administration. In a preferred embodiment, parenteral administration is by intramuscular injection, for example to the thigh or upper arm of a subject. The injection may be through a needle (e.g., a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is 0.5 mL. The compositions of the present invention may be prepared in various forms, for example, as liquid solutions or suspensions for injection. In certain embodiments, the compositions may be prepared as a powder or spray for pulmonary administration, e.g., in an inhaler. In other embodiments, the composition may be prepared as a suppository or pessary, or for nasal, otic, or ocular administration, e.g., as a spray, drop, gel, or powder.

The optimal amounts of the components of a particular immunogenic composition can be determined by standard studies, including observing the appropriate immune response in a subject. After the initial immunization, the subject may receive one or several booster immunizations at sufficient intervals.

Packaging and dosage form

The immunogenic compositions of the invention may be packaged in unit-dose or multi-dose form (e.g., 2 doses, 4 doses or more). For multi-dose forms, vials are typically, but not necessarily, preferred over prefilled syringes. Suitable multi-dose forms include, but are not limited to: 2-10 doses per container, 0.1-2mL per dose. In certain embodiments, the dose is a 0.5mL dose. See, for example, International patent application WO2007/127668, which is incorporated herein by reference.

The composition may be present in a vial or other suitable storage container, or may be present in a pre-filled delivery device, such as a single or multi-component syringe, which may be supplied with or without a needle. Although multi-dose, pre-filled syringes are also contemplated, the syringe will typically, but need not necessarily, contain a single dose of the preservative-containing immunogenic composition of the present invention. Likewise, a vial may comprise a single dose, but may alternatively comprise multiple doses.

Effective dose volumes can be routinely established, but a typical dose of injectable composition has a volume of 0.5 mL. In certain embodiments, the dosage is formulated for administration to a human subject. In certain embodiments, the dose is formulated for administration to an adult, teenager, adolescent, toddler, or infant (i.e., no more than one year of age) human subject, and in preferred embodiments may be administered by injection.

The liquid immunogenic compositions of the invention are also suitable for reconstitution of other immunogenic compositions in lyophilized form. When immunogenic compositions are used for such immediate reconstitution, the invention provides kits having two or more vials, two or more filled syringes, or one or more of each, the contents of the syringes being used to reconstitute the contents of the vials prior to injection, or vice versa.

Alternatively, the immunogenic compositions of the invention may be lyophilized and reconstituted, for example, using one of a variety of methods well known in the art for lyophilization to form dry, regularly shaped (e.g., spherical) particles, such as microparticles or microspheres, having particle characteristics such as average diameter size which may be selected and controlled by varying the exact method used to prepare them. The immunogenic composition may further comprise an adjuvant, which may optionally be prepared with or contained in separate dry, regularly shaped (e.g. spherical) particles (e.g. microparticles or microspheres). In such embodiments, the present invention further provides an immunogenic composition kit comprising a first component comprising the stable dry immunogenic composition, optionally further comprising one or more preservatives of the present invention, and a second component comprising a sterile aqueous solution for reconstituting the first component. In certain embodiments, the aqueous solution comprises one or more preservatives, and may optionally comprise at least one adjuvant (see, e.g., WO2009/109550 (incorporated herein by reference).

In another embodiment, the container in multi-dose form is selected from one or more of the group consisting of, but not limited to, common laboratory glassware, bottles, beakers, graduated cylinders, fermenters, bioreactors, tubes (tubing), tubes (pipe), bags, jars, vials, vial stoppers (e.g., rubber stoppers, screw caps), ampoules, syringes, dual or multi-chamber syringes, syringe stoppers, syringe plungers, rubber stoppers, plastic bottle caps, glass bottle stoppers, cartridges, disposable pens, and the like. The container of the present invention is not limited by the material of manufacture and includes materials such as glass, metals (e.g., steel, stainless steel, aluminum, etc.), and polymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers). In a particular embodiment, the form of the container is a 5mL Schott type 1 glass vial with butyl stopper. It will be appreciated by the skilled person that the forms shown above are not exhaustive lists but merely serve as guidance to the skilled person regarding the various forms available for the invention. Other forms contemplated for use in the present invention may be found in published product catalogs from laboratory equipment suppliers and manufacturers such as United States Plastic Corp. (Lima, OH), VWR.

Evaluation of immunogenic compositions

In one embodiment, the present invention provides an immunogenic composition comprising at least 3 from staphylococcus aureus organisms.

Various in vitro tests are used to assess the immunogenicity of the immunogenic compositions of the invention. For example, in vitro opsonization assays are performed by incubating staphylococcal cells, heat inactivated serum containing antibodies specific for the antigen in question and a mixture of exogenous complement sources. Opsonophagocytosis occurs during incubation of freshly isolated polymorphonuclear cells (PMN's) or differentiated effector cells such as HL60 with antibody/complement/staphylococcal cell mixtures. Antibody and complement coated bacterial cells are killed upon opsonophagocytosis. Colony forming units (cfu) of viable bacteria recovered from opsonophagocytosis were determined by plating assay mixtures. Titer is reported as the reciprocal of the highest dilution that gave 50% bacterial kill as determined by comparison to assay controls.

Whole cell ELISA assays are also used to evaluate the immunogenicity and surface exposure of the antigens in vitro, wherein a bacterial strain of interest (staphylococcus aureus) is coated on a plate, such as a 96-well plate, and test serum from the immunized animal is reacted with the bacterial cells. If any antibody that is tested for antigen specificity reacts with a surface-exposed epitope of the antigen, it can be detected by standard methods known to those skilled in the art.

Any antigen demonstrating the desired in vitro activity is then tested in an in vivo animal challenge model. In certain embodiments, the immunogenic compositions are used to immunize an animal (e.g., a mouse) by immunization methods and routes known to those of skill in the art (e.g., intranasal, parenteral, oral, rectal, vaginal, transdermal, intraperitoneal, intravenous, subcutaneous, etc.). After immunization of an animal with an immunogenic composition of a particular Staphylococcus species (Staphylococcus sp.), the animal is challenged with Staphylococcus and its resistance to Staphylococcus infection is determined.

In one embodiment, pathogen-free mice are immunized and challenged with staphylococcus aureus. For example, a mouse is immunized with one or more doses of the desired antigen in the immunogenic composition. Subsequently, mice were challenged with staphylococcus aureus and survival was monitored over time after challenge.

Immunization method

Methods of immunizing a host to prevent staphylococcal infection are also provided. In a preferred embodiment, the host is a human. Thus, an immunogenic amount of the immunogenic composition is administered to a host or subject as described herein. The immunogenic amount of an immunogenic composition can be determined by performing a dose response study in which subjects are immunized with increasing amounts of the immunogenic composition and the immune response is analyzed to determine the optimal dose. The starting point of the study can be inferred from the immunization data in the animal model. The dosage may vary depending on the individual's particular conditions. The amount can be determined in routine tests by methods known to the person skilled in the art. In some embodiments, the method of immunizing a host to prevent a staphylococcal infection, disease or condition comprises human, veterinary, animal or agricultural treatment. Another embodiment provides a method of immunizing a host to prevent a staphylococcal infection, disease or condition associated with a staphylococcal bacterium in a subject, the method comprising producing a polyclonal or monoclonal antibody preparation from an immunogenic composition described herein and using the antibody preparation to confer passive immunity to the subject.

An immunologically effective amount of the immunogenic composition is administered to a subject in an appropriate number of doses to elicit an immune response. The treated individuals should not exhibit clinical manifestations of more severe staphylococcal infection. The dosage may vary according to the particular conditions of the individual, such as age and weight. This amount can be determined in routine tests by methods known to the person skilled in the art.

In one embodiment, patients administered an immunogenic composition of the invention exhibit a reduction in the carriage of staphylococcus aureus. Such reduction in carryover or extended time intervals spent as non-carriers after administration of the immunogenic composition is significant from a medical need point of view. For example, the reduction in carriage of all staphylococcus aureus in the vector can be assessed after one dose of staphylococcus aureus multi-antigen vaccine. For example, a panel of adult screening carriers of 18-50 years old may be carried 1 day prior to administration of the immunogenic composition, and their carriage status determined by nasal and throat swabs and then cultured. The group is then administered the immunogenic composition of the invention, while one group receives a control. Nasal and throat swabs were performed weekly for a period of 12 weeks, then monthly until 6 months after administration of the immunogenic composition, and compared to placebo. One primary endpoint was to compare the carrier rate in patients after administration of the immunogenic composition to placebo at 3 month intervals post immunization.

Animal model of staphylococcal infection

Several animal models for evaluating the efficacy of any of the immunogenic compositions described herein are described below.

Mouse sepsis model (Passive or active)

Passive immunization model

Mice were passively immunized intraperitoneally (i.p.) with immune IgG or monoclonal antibodies. Mice were then challenged with a lethal dose of staphylococcus aureus 24 hours later. Bacterial challenge administered either intravenously (i.v.) or i.p, ensuring any survival can be attributed to specific in vivo interactions of antibodies with bacteria. The bacterial challenge dose was determined as the dose required to achieve lethal sepsis in approximately 20% of the non-immunized control mice. Statistical evaluation of survival studies can be performed by Kaplan-Meier analysis.

Active immunization model

In this model, mice (e.g., Swiss Webster mice) are actively immunized with the target antigen either intraperitoneally (i.p.) or subcutaneously (s.c.) at weeks 0,3, and 6 (or other similar suitably spaced immunization schedules), and then challenged with staphylococcus aureus by the intravenous route at week 8. The bacterial challenge dose was scaled to achieve about 20% survival in the control over a 10-14 day period. Statistical evaluation of survival studies can be performed by Kaplan-Meier analysis.

Infectious endocarditis model (passive or active)

A passive immunization model of Infectious Endocarditis (IE) caused by staphylococcus aureus has been previously used to show that ClfA can induce protective immunity. See Vernachio et al, antmicro. Agents & Chemo.50: 511-518(2006). In this IE model, rabbits or rats were used to simulate clinical infections, including central venous catheters, bacteremia, and hematogenous transfusion (hematogenous seeding) to the remote organs. Monoclonal or polyclonal antibodies specific for a target antigen are administered to a catheterized rabbit or rat with a sterile aortic valve neoplasm by single or multiple intravenous injections. Subsequently, animals were i.v. challenged with staphylococcus aureus or staphylococcus epidermidis strains. Then, after challenge, the heart, cardiac neoplasms and other tissues including the kidney, as well as blood were harvested and cultured. The frequency of staphylococcal infection in heart tissue, kidney and blood was then measured. In one study, polyclonal antibody preparations or monoclonal antibodies using ClfA showed a significant reduction in infection rates when animals were challenged with mrsec atcc 35984 or MRSA 67-0. See Vernachio et al, antmicro. Agents & Chemo.50: 511-518(2006).

Infectious endocarditis is also suitable for active immunization studies in rabbits and rats. Rabbits or rats were immunized intramuscularly or subcutaneously with the target antigen and challenged with staphylococcus aureus by the intravenous route 2 weeks later.

Pyelonephritis model

In the pyelonephritis model, mice are immunized with the target antigen at weeks 0,3, and 6 (or other suitably spaced immunization schedules). Subsequently, the animals were challenged with staphylococcus aureus pfesa0266i.p. or i.v. bacteria. After 48 hours, kidneys were harvested and bacterial CFUs were counted.

Antibodies and antibody compositions

The invention further provides antibodies and antibody compositions that specifically and selectively bind to one or more antigens of the immunogenic compositions of the invention. In some embodiments, the antibodies are produced upon administration of the immunogenic compositions of the invention to a subject. In some embodiments, the invention provides purified or isolated antibodies against one or more antigens of the immunogenic compositions of the invention. In some embodiments, the antibodies of the invention are functional as measured by killing bacteria in an animal efficacy model or opsonophagocytic killing assay. In some embodiments, the antibodies of the invention confer passive immunity to a subject. The invention further provides polynucleotide molecules encoding the antibodies or antibody fragments of the invention, as well as cells or cell lines (e.g., hybridoma cells or other engineered cell lines for recombinant production of antibodies) and transgenic animals that produce the antibodies or antibody compositions of the invention using techniques well known to those skilled in the art.

The antibodies or antibody compositions of the invention may be used to treat or prevent a staphylococcal infection, disease or condition associated with staphylococci in a subject, the method comprising producing a polyclonal or monoclonal antibody preparation and using the antibodies or antibody compositions to confer passive immunity to the subject. The antibodies of the invention may also be used in diagnostic methods, for example, to detect the presence of or quantify the level of one or more antigens of the immunogenic compositions of the invention.

Examples

The following examples demonstrate some embodiments of the invention. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed as being fully determinative of the conditions and scope of the present invention. It is to be understood that when typical reaction conditions (e.g., temperatures, reaction times, etc.) are given, it is generally less convenient, but that conditions above and below the specified ranges can also be used. All parts and percentages referred to herein are on a weight basis and all temperatures are expressed in degrees celsius unless otherwise indicated.

Furthermore, unless otherwise specified, the following examples are carried out using standard techniques that are well known and conventional to those skilled in the art. As noted above, the following examples appear for illustrative purposes and should not be construed as limiting the scope of the invention in any way.

Example 1: production of antigens ClfA and ClfB

Aggregation factors a (ClfA) and b (ClfB) are staphylococcus aureus surface proteins responsible for binding to host proteins including fibrinogen (ClfA, ClfB) and cytokeratin 10 (ClfB). ClfA and ClfB are members of a family of proteins that contain a carboxy-terminal LPXTG (SEQ ID NO: 125) motif that enables covalent attachment of the protein to the cell surface. Both ClfA and ClfB belong to a family of proteins (microbial surface components recognize adhesion matrix molecules or MSCRAMMs) that recognize and bind host extracellular matrix proteins such as fibrinogen (ClfA and ClfB), fibronectin (FnbA and FnbB), collagen (Cna), and the like. These proteins all share an amino-terminal signal sequence that mediates transport to the cell surface. MSCRAMMs also include an a-domain, which is a functional region containing the ligand binding sites for fibrinogen, fibronectin, elastin, and keratin. The a-domain may be followed by a region consisting of a serine-aspartate repeat (SD repeat), which is believed to span the peptidoglycan layer. The SD repeat is followed by a transmembrane region comprising the LPXTG (SEQ ID NO: 125) motif that covalently links the protein to the peptidoglycan.

The ligand binding regions of ClfA and ClfB of N1N2N3 comprising the a domain span amino acids 40-559. The N domains of ClfA/ClfB have been assigned as follows: n1 includes residues 45-220; n2 includes residues 229-; and N3 includes residues 370-559. See, Deivanayagam et al emboj.21: 6660-6672(2002). In the preparation of recombinant ClfA N1N2N3, the N1 domain was found to be protease sensitive and to be readily cleaved or hydrolyzed off N2N3 as a stable ligand-binding recombinant fragment. See, Deivanayagam et al embo j.21: 6660-6672(2002). Similarly, the N1 domain of ClfB was also shown to be protease sensitive and could be readily cleaved by s.aureus metalloproteases (mcaleesee, f.m.et al.j.biol.chem.2001, 276, pp.29969-29978). The crystal structure of the fibrinogen-binding N23 fragment of the ClfA a domain showed that N2 and N3 are under the control of antiparallel beta chains. In addition to antiparallel beta strands, the N2 domain contains a single folded alpha helix and two 3₁₀Helices, and the N3 domain contains three 3₁₀A helix. See, Deivanayagam et al embo j.21: 6660-6672(2002)。

Sequence alignment of N2 and N3 showed only 13% sequence identity and 36% sequence similarity over their length. See, Deivanayagam et al embo j.21: 6660-6672(2002). The topology of the N2 and N3 domains is similar to that of a classical IgG fold and has been proposed as a new variant of an IgG fold. See, Deivanayagam et al embo j.21: 6660-6672(2002).

Recombinant forms of ClfA for use in the immunogenic compositions described herein are ClfA fragments comprising one or more N domains, e.g., N1N2N3, N2N3, and are referred to herein as "recombinant ClfA" or "rClfA". Furthermore, any rClfA should be such that it retains the native structure of the single N domain and key epitopes, but does not interfere with the normal processing of the immunized individual after administration (i.e., does not bind fibrinogen). Mutation studies have shown that mutation Y338A (N2) completely abolished binding of the N23 fragment to fibrinogen. (this Y338A position refers to the change from tyrosine to alanine at position 338 in the immature form of the polypeptide sequence to which the leader sequence is still attached. this change can be seen at position 310 in the mature form of the mutant ClfA polypeptide of SEQ ID NO: 123 which demonstrates lack of binding to fibrinogen.) see Deivanayagam et al EMBO J.21: 6660-6672(2002). Therefore, all fragments of ClfA in the following studies have adopted the Y338A mutation.

Similarly, a recombinant form of ClfB for use in the immunogenic compositions described herein is a ClfB fragment comprising one or more N domains, e.g., N1N2N3, N2N3, and is referred to herein as "recombinant ClfB" or "rClfB. Furthermore, any rClfB should be such that it retains the native structure of the single N domain and key epitopes, but does not interfere with the normal processing of the immunized individual after administration (i.e., does not bind fibrinogen). (see, e.g., Walsh, E.J.et al. microbiology (2008), 154, 550-.

ClfA and ClfB: overview of cloning methods

Different formats for generating preclinical efficacy dataThe rClfA protein includes HisClfA_(N123)；T7ClfA_(N123)；T7ClfA_(N123)；Y338A；ClfA_(N23)And ClfA_(N23)Y338A. See fig. 1. The ClfA gene contains the a region coding sequence from staphylococcus aureus PFESA0237 corresponding to residues 40-559. The reading frame cloned from S.aureus was fused to the N-terminus of the vector with HisTag and linker sequence (MRGSHHHHHHGS SEQ ID NO: 127), with the introduction of 3 additional coding sequences (KLN) at the C-terminus. (see below for detailed procedure.) the protein expressed from this vector, which is called HisClfA, was used in all experiments_(N123)。

The different forms of rClfA were derived from the A region (residues 40-559 (top row) of ClfA expressed by Staphylococcus aureus PFESA 0237. HisClfA was expressed using the T5 promoter contained in pQE30_(N123)And all other forms were expressed using the T7-based pET expression system.

Two forms of ClfB (T7ClfB N1N2N3 and ClfB N23) were used in preclinical animal studies.

ClfA cloning method

The ClfA coding sequence from staphylococcus aureus strain PFESA0237 corresponding to amino acid residues 40-559 was cloned and the mutation Y338A was introduced to eliminate fibrinogen binding. The mutated ClfA gene was introduced into T7RNA polymerase expression vector pET9a (Novagen) to provide plasmid pLP 1179. The DNA sequence of the region of pLP1179 containing the T7 promoter and the coding region is seq id: 124. the expression vector was transformed into E.coli BLR (DE3) (Novagen) for the production of recombinant ClfA.

T7ClfA_(N123)Construction of Y338A included several steps. A summary of the cloning steps used to construct the final expression plasmid pLP1179 is shown in FIG. 2.

The clfA DNA sequence present in pQEClf40 corresponds to amino acid residues 40-559 of clfA originally cloned into the BamHI/HindIII cloning site of pQE 30. This resulted in a HisTag fusion at the N-terminus of ClfA and the addition of 3 residues at the C-terminus. Will be present in (AmpR) pQEThe ClfA coding region in Clf40 was subcloned into the KanR pET27b vector (Novagen) to generate pLP 1137. In addition, the clfA DNA sequence corresponding to amino acid residue 221-. The N-terminal HisTag of ClfA was replaced with N-terminal T7 by subcloning the BamHI-BlpI DNA fragment from pQEClf40 into pET9a (Novagen) to generate pLP 1153. T7ClfA present in pLP1153_(N123)The coding sequence of (a) contained 11N-terminal amino acid residues from the T7 tag of pET9a, followed by 3 amino acid residues from the linker sequence plus the 3C-terminal linker derived residues originally present in pQE30Clf 40. The Y338A mutation was first introduced into ClfA of pLP1134_(N23)Coding sequence to generate pLP 1168. Then ClfA from pLP1168_(N23)Replacement of the PstI-SnaBI DNA fragment containing the Y338A mutation with T7ClfA of pLP1153_(Ni23)PstI-SnaBI of the coding sequence to generate pLP 1171. Alteration of T7ClfA present in pLP1171 by silent mutations at positions 339 and 342 in the DNA of the T7rClfA Y338A ORF_(N123)The internal ribosome binding site in the coding sequence of Y338A changed from G to T and G to a, respectively. The resulting plasmid pLP1176 was then used to remove the 3 foreign residues between the T7 tag originally derived from pQE30Clf40 and the beginning of the ClfA coding region. The 3 linker-derived C-terminal residues were also removed at this time.

The resulting rClfA expressed by plasmid pLP1179 (FIGS. 2 and 3) contained only fusion to ClfA_(N123)11N-terminal amino acids from residues 40-559 of Y338A. T7rClfA comprising T7 promoter and pLP1179_(N123)The DNA sequence of a region of the coding sequence of Y338A is SEQ id no 124.

Bacterial strains and plasmids

Plasmid backbone pET9a (obtained from Novagen) was used to construct expression of T7ClfA from the T7 promoter_(N123)pLP1179 of Y338A. This plasmid contains the kanamycin resistance gene (KanR) for positive selection. BL21(DE3) E.coli host strain [ F-ompT hsdS_B(r_B ^-m_B ^-)gal dcm(DE3)](Novagen) originally used to obtain T7ClfA_(N123)Expression of Y338A. DE3 nomenclatureA Lambda lysogen containing a T7RNA polymerase gene under the control of a lacUV5(IPTG inducible) promoter for inducible expression of T7RNA polymerase, followed by proximity to ClfA present in pLP1179_(N123)Transcription from the T7 promoter of the Y338A coding sequence. When receiving information that BL21(DE3) lysogenic host strain was able to induce lytic phages upon large-scale fermentation, the host strain was changed to recA BLR (DE3) host strain [ F-ompT hsdS ]_B(r_B ^-m_B ^-)gal dcmΔ(sri-recA)306：：Tn10(TcR)(DE3)](Novagen)

ClfA production and purification

To produce ClfA, escherichia coli BLR (DE3)/pLP1179 was grown in a bioreactor in fed-batch mode with glucose in defined medium. When the culture reached an Optical Density (OD) of between 30 and 50₆₀₀) When the expression of ClfA was induced by the addition of IPTG. Cultures were harvested between 3-16 hours post-induction.

The cells were disrupted and the clear soluble fraction was collected. After addition of ammonium sulfate, the material was applied to a column containing phenyl-agarose resin and eluted. The ClfA-containing fractions were identified, dialyzed and loaded onto an anion exchange column (Q-Sepharose). After elution with a salt gradient, the ClfA-containing fractions were identified, concentrated by ultrafiltration and packed on a size exclusion column (Superdex-75). The ClfA containing fractions were identified and pooled. The purity of ClfA was about 98% at this point as measured by SDS-PAGE.

Cloning and purification of ClfB N1N2N3

The ClfB coding sequence corresponding to amino acid residues 44-542 was cloned into the T7RNA polymerase expression vector pET28a (Novagen) to provide plasmid px 1189. The expression vector was transformed into E.coli BLR (DE3) (Novagen) for the production of recombinant ClfB. (see Walsh, et al, Microbiology 154: 550-

For the production of ClfB, E.coli BLR (DE3)/pLP1179 was treated with dextrose in defined mediumGlucose fed-batch mode was grown in the bioreactor. When the culture reached an Optical Density (OD) of between 30 and 50₆₀₀) When the expression of ClfB was induced by the addition of IPTG. Cultures were harvested between 3-16 hours post-induction.

The cells were disrupted and the clear soluble fraction was collected. The pH of the soluble fraction was adjusted to about pH 3.2 and the precipitated impurities were removed. The pH of the soluble fraction containing ClfB was readjusted to about pH 8.0 and dialyzed to remove the salts. After addition of ammonium sulfate, the material was applied to a column containing phenyl-agarose resin and eluted. The ClfB-containing fractions were identified, dialyzed and loaded onto an anion exchange column (Q-sepharose). After elution with a salt gradient, the ClfB-containing fractions were identified, concentrated by ultrafiltration and packed on a size exclusion column (Superdex-75). The ClfB-containing fractions were identified and pooled. The purity of ClfB was about 94% at this point as measured by SDS-PAGE.

Example 2: production of antigen: staphylococcus Aureus (Staph Aureus) MntC

Lipidated MntC of cloned Staphylococcus aureus

Recombinant MntC was originally cloned from staphylococcus aureus strain Mu 50. The rMntC coding sequence was amplified by PCR from Staphylococcus aureus Mu50 genomic DNA. Two pairs of nested primers were used for the amplification (table 2). The first pair of primers, 5 'SA 926-MntC ups and 3' SA926-MntC down, were aligned to the upstream and downstream sequences of the open reading frame of rMntC. The second set of primers was aligned to the coding sequence of rMntC, allowing amplification of the sequence corresponding to amino acid residues 19-309. Restriction enzyme sites were added to the 5' ends of these primers to facilitate directional cloning. PCR was performed in a Peltier thermal cycler (MJ Research Inc, Walthan, MA) using TaKaRa PrimeSTAR HS DNA polymerase premix (Takara Bio USA, Madison, Wis.). The PCR product was purified by QIAEX II (Qiagen, Valencia, CA), cleaved with the appropriate restriction endonuclease (New England BioLabs, Ipswich, MA), and subcloned into the araBAD promoter driven expression vector pBAD18 Cm. The carrier also contains Haemophilus influenzaeSignal peptide of lipoprotein P4 of bacterium (h. influenza). The mntccpcr product was subcloned in-frame downstream of the P4 signal peptide to generate pLP 1194. The DNA sequence of the MntC coding region of pLP1194 is set forth in SEQ ID NO: shown at 120. MntC expressed from pLP1194 is a lipoprotein. Passing the recombinant plasmid DNA through ABI PRISM bigDye^TMTerminator V.3.1(Applied Biosystems, Foster City, Calif.) and recombinant proteins were expressed in E.coli BLR (NOVAGEN) for the production of lipidated rMntC.

Production and purification of lipidated MntC

To produce lipidated MntC, e.coli BLR/pLP1194 was grown in a bioreactor in a glucose fed-batch mode in defined medium. When the culture reached an Optical Density (OD) of about 60₆₀₀) When the feed was switched to a mixture of glucose and arabinose, the expression of rMntC was induced. Cultures were harvested approximately 24 hours after induction.

The cells were disrupted and the insoluble fraction was collected. Lipidated MntC was found to be associated with cell membranes due to lipid modification. MntC was extracted from the membrane fraction with a detergent (Zwittergent ZW-312). After removal of insoluble debris, lipidated MntC was found in the soluble fraction. The soluble fraction was applied to a column containing mixed mode resin and eluted with a linear salt and pH gradient. The MntC containing fractions were identified and pooled. Ammonium sulfate was added to the cell and the material was applied to a column containing butyl-agarose and eluted. The MntC-containing fractions were identified, desalted and loaded onto a cation exchange column (SP-sepharose). After elution with a salt gradient, the fractions containing rMntC were identified and pooled.

Clonal Staphylococcus aureus non-lipidated MntC

The DNA sequence for expression of non-lipidated rMntC was isolated by PCR amplification from plasmid pLP 1194. The resulting sequence corresponds to amino acid residues 19-309 and does not contain a signal sequence that directs secretion and lipidation. The DNA sequence of the rMntC coding region of pLP1215 is shown in DNA SEQ ID NO: 120 are found in.

To generate pLP1215, MntC was amplified by PCR from pLP 1194. The MntC DNA sequence present in pLP1215 corresponds to amino acid residues 19-309 and the first codon of this construct is introduced into the forward primer used to amplify the gene. The primers used for PCR also contained restriction enzyme sites at the 5' end to facilitate directional cloning (Table 2). PCR and purification of the amplified gene were performed as described above. The purified PCR product was cleaved with the appropriate restriction endonuclease (New England BioLabs, Ipswich, MA) and subcloned into the T7 promoter driven expression vector pET28a (Novagen, Madison, WI). Recombinant plasmid DNA pLP1215 was purified by ABI PRISMBigdye^TMTerminator V.3.1(Applied Biosystems, Foster City, Calif.) and recombinant protein was expressed in E.coli BLR (DE 3). Plasmid DNA of pLP1215 was purified and used to transform E.coli HMS174(DE3) to assess protein expression.

Table 2: MntC primer.

Synthetic oligonucleotides were used to generate the rMntC construct. Restriction endonuclease sites are underlined. The bold nucleotides show the first codon of the non-lipidated rMntC construct.

Production and purification of non-lipidated rMntC

To produce non-lipidated rMntC, e.coli HMS174(DE 3)/pLP1215 were grown in a bioreactor in a glucose fed-batch mode in defined medium. When the culture reached an Optical Density (OD) of about 60-80₆₀₀) When the expression of rMntC is induced by adding IPTG. Cultures were harvested approximately 24 hours after induction. The cells were disrupted and the clear soluble fraction was collected. The lysate was applied to a column containing cation exchange resin (SP-sepharose) and eluted with a linear salt gradient. The MntC containing fraction was identified. After addition of ammonium sulfate, the material was applied to a column containing phenyl-agarose resin and eluted. After elution, the fractions containing rMntC were identified, pooled and desalted. The purity of rMntC at this point was > 95% as measured by SDS-PAGE.

Example 3: production of capsular polysaccharides CP5 and CP8

In this example, the production of various sizes of staphylococcus aureus capsular polysaccharide types 5 and 8 is described. The structures of CP5 and CP8 polysaccharides are shown in fig. 4. The methods described herein are effective in producing CP5 and CP8 having molecular weights of about 50kDa-800 kDa. Based on growth characteristics and the quality of the capsules produced, strain PFESA0266 was selected for the production of CP5, while strain PFESA0005 or PFESA0286 was used for the production of CP 8. The capsules isolated from strains PFESA0005 and PFESA0286 were confirmed to be identical.

For the production of capsular polysaccharides, the strains are grown in a complex medium consisting essentially of a carbon source (lactose or sucrose), hydrolysed soya flour as nitrogen source and trace metals. The strains were grown in a bioreactor for 2-5 days.

Purification of CP5 and CP8 for preparation of conjugates was performed by two different methods that relied on elevated temperature and low pH to affect capsule release from the cells and reduce the molecular weight of the polysaccharide. The resulting molecular weight depends on the time, temperature and pH of the hydrolysis step.

The characterization of CP5 and CP8 was performed using the techniques specified in table 3. Capsular polysaccharides produced by this process result in pure polysaccharides with low levels of protein, NA, peptidoglycan, and TA contaminants. See tables 4 and 5.

Table 3: characterization assays for purified Staphylococcus aureus CP5 and CP8

In the first method, after the capsule is released from the cells and the molecular weight is reduced, the capsule preparation is treated with a mixture of enzymes (ribonuclease, deoxyribonuclease, lysozyme and protease) to digest impurities. After incubation, residual impurities were precipitated by adding ethanol (final concentration about 25%). After removal of residual ethanol, the pod-containing solution was loaded onto an anion exchange column (Q-sepharose) and eluted with a linear salt gradient. The fraction containing the pods was pooled and treated with sodium meta-periodate. This treatment resulted in oxidative hydrolysis of residual teichoic acid contaminants, but did not affect CP5 or CP 8. The reaction was quenched by the addition of ethylene glycol. This material was concentrated and diafiltered against dH20 to remove any residual reagents and by-products.

The second method is used to produce the capsule and does not involve the use of enzymes to digest various cell-derived impurities. In this process, after the capsules are released from the cells and the molecular weight is reduced, the hydrolytic fermentation broth is clarified by microfiltration followed by ultrafiltration and diafiltration. The solution was treated with activated carbon to remove impurities. After the charcoal treatment, the material was treated with sodium meta-periodate to oxidize residual teichoic acid and then quenched with propylene glycol. Concentrating the material and concentrating to dH₂O diafiltration to remove any residual reagents and by-products.

The capsules produced using either method result in pure polysaccharides with low levels of protein, nucleic acid, and teichoic acid contaminants. The process can be used to produce a specific range of desired high molecular weight polysaccharides simply by varying the hydrolysis conditions. A particularly advantageous range of high molecular weight polysaccharides of 70-150kDa is used to prepare immunogenic compositions by conjugating the polysaccharide to a carrier protein.

Examples of high molecular weight capsular polysaccharides obtainable by the processes described herein are shown in table 4 below. The purified batch of higher MW CP5 also had high purity as shown by the absence of TA, peptidoglycan and low residual protein. See table 4. The molecular weight range in these examples spanned 132.7kDa-800kDa, and the purified polysaccharide was highly O-acetylated, ranging from 90-100%, and 100% N-acetylated. See table 4.

Examples of lower molecular weight capsular polysaccharides obtainable by the processes described herein are shown in table 5 below. The purified batches of lower MW CP8 were of high purity as shown by the absence of Teichoic Acid (TA), peptidoglycan, and lower residual protein. See table 5. The lower molecular weight range spans 20.4kDa-65.1kDa, and the purified polysaccharide is highly O-acetylated, ranging from 75-96%. The level of nucleic acid contamination was low, ranging from 0.5-% -2.45%. See table 5.

Table 4: characterization of CP5 articles

Table 5: characterization of CP8 articles

Molecular weight selection of capsular polysaccharides

This kinetic analysis demonstrates that capsular polysaccharides of a wide range of molecular weights can be produced by the methods described herein. Initially, larger polysaccharides are produced by bacterial cells, and subsequently, the desired molecular weight range can be selected and then purified by manipulating the pH and heating conditions of the heating and hydrolysis steps.

The heat treatment of the S.aureus fermentation broth is a processing step between fermentation and CP recovery. This processing step treats the pH-adjusted culture broth with heat for a prescribed period of time. The purpose of heat treatment at low pH is to kill the cells, inactivate enterotoxins, release cell-bound polysaccharides and reduce the molecular weight to the desired size. Among these objectives, lowering the molecular weight is the slowest in terms of the processing time required for this step. Therefore, other objectives must be achieved within the considered processing time.

Heat treatment of

The temperature and pH conditions for selection of capsular polysaccharides of various molecular weight ranges were determined. The pH of the culture broth was adjusted with concentrated sulfuric acid. Then, the temperature of the culture broth was increased to the set value. The heat treatment time begins as soon as the temperature reaches the set point. When the desired treatment time was reached, the broth was cooled to room temperature. Samples were collected during the process to determine polysaccharide concentration and molecular weight by HPLC and SEC-MALLS systems, respectively. MW data were used for kinetic analysis. The MW spectrum of CP5 at pH 4.0, 4.5 and 5.0 and the MW spectrum of CP8 at pH 3.5, 4.0 and 5.0 were determined over time. See fig. 5A and 5B.

Kinetics of mild acid hydrolysis of polysaccharides using purified CP-5 and CP-8 obtained from the method. The purified polysaccharide solution was adjusted to the experimentally desired pH with sulfuric acid. The samples were placed in an oil bath equipped with a precision temperature control system. Each sample was removed at predetermined time intervals and snap cooled in an ice bucket. At the end of the experiment, an aliquot of 1M Tris buffer (pH 7.5) was added to the sample to bring the pH back to about 7. The samples were analyzed by SEC-MALLS system. MW data were used for kinetic analysis. The effect of temperature on the MW spectra of CP5 at pH4.5 and CP8 at pH 3.5 was determined over time. See fig. 6A and 6B. This acid hydrolysis process can be accomplished using fermentor cultures, or at an intermediate stage of purification, or as shown herein using purified polysaccharides. Other molecular weight reduction steps such as sonication or shearing (sheet) may be similarly accomplished.

Results

The effect of pH on MW reduction in heat treatment for CP-5 and CP-8 is shown in FIGS. 5A and 5B, respectively. It can be seen that lower pH is more effective in reducing the size of the polysaccharide. The data also show that CP-5 is more difficult to hydrolyze than CP-8 at the same pH. In view of the CP8 spectrum, a molecular weight range of 300kDa to 600kDa can be generated using pH 5 at 95 ℃ for 15 minutes to 120 minutes. Likewise, selection of pH4 at 95 ℃ for 15 minutes to 120 minutes can produce CP8 polysaccharide molecular weight ranges of 250kDa to 450 kDa. In addition, selection of pH 3.5 at 95 ℃ for 15 minutes to 120 minutes can produce CP8 polysaccharide molecular weight range of 120kDa to 450 kDa.

The effect of temperature on MW reduction is performed using purified polysaccharide recovered from the recovery process. The results are shown in FIGS. 6A and 6B. As shown, the higher the temperature, the faster the hydrolysis rate, and the broader the molecular weight range of the polysaccharide produced over time. The use of a lower temperature of 55 ℃ versus 95 ℃ at the same pH results in a narrower range of polysaccharide molecular weights.

In addition, fig. 7 demonstrates the correlation between molecular weight of purified CP5 and CP8 and mild acid hydrolysis treatment time. The purified polysaccharide is the final product obtained from the recovery process detailed previously. As shown in fig. 7, increasing the time of heat treatment of staphylococcus aureus PFESA0266 strain at pH4.5 resulted in the production of lower molecular weight CP5 polysaccharide, while shorter heat treatment time at pH4.5 resulted in the production of higher molecular weight CP5 polysaccharide. The size of the CP5 polysaccharide ranges from about 90kDa to about 220kDa, depending on the length of heat treatment at low pH (4.5). Likewise, increasing the time to heat-treat the staphylococcus aureus PFESA0005 strain at pH 3.5 resulted in the production of a smaller molecular weight CP8 polysaccharide, while shorter heat-treatment times at pH 3.5 resulted in the production of a higher molecular weight CP8 polysaccharide. The size of the CP8 polysaccharide ranges from about 80kDa to about 220kDa, depending on the length of heat treatment at low pH (3.5). As shown in this study, the correlation between the time of heat treatment at low pH and the size of the purified CP5 and CP8 polysaccharides allowed the estimation of the treatment time required to produce purified polysaccharides with molecular weights in the specified range.

It is important to note that as demonstrated above, capsular polysaccharides of full range molecular weights of CP5 and CP8 of 20kDa to over 800kDa can be produced, released and purified. The process can be used to produce a specific range of desired high molecular weight capsular polysaccharides. Particularly advantageous ranges of high molecular weight capsular polysaccharides types 5 and 8 can be produced from these processes, having molecular weights in the range of 70-150 kDa. See table 6. Capsular polysaccharides of this molecular weight range are used to prepare immunogenic compositions by conjugating the polysaccharide to a carrier protein. Alternatively, the high molecular weight capsular polysaccharides of this advantageous range are CP5 and CP8 of 80-140 kDa. See table 6. Another advantageous range of high molecular weight capsular polysaccharides CP5 and CP8 is between 90-130kDa, or between 90-120kDa CP5 and CP 8. See table 6. The conditions used to produce the CP5 capsular polysaccharide having a molecular weight range of about 100-140kDa were as follows: at 95 ℃ and at pH4.5 for 135 minutes. The conditions used to produce CP8 capsular polysaccharides having a molecular weight range of about 80-120kDa were as follows: the pH was 3.5 at 95 ℃ for 300 minutes.

Table 6: generation of specific ranges of high molecular weight CP5 and CP8

Operation of	CP8MW(kDa)	CP5MW(kDa)
			1	98	142
2	89	108
			3	108	142
4	108	108
			5	89	ND
6	100	ND
			7	99	63
8	113	72
			9	105	74
10	100	63
			11	87	ND

Not performing ND-

Example 4: conjugation of capsular polysaccharides CP5 and CP8 to CRM₁₉₇

This example describes the production of Staphylococcus aureus CP5-CRM₁₉₇And CP8-CRM₁₉₇Methods and characterization assays for conjugates. To conjugate staphylococcus aureus capsular polysaccharides CP5 and CP8 to carrier proteins, several conjugation chemistries were evaluated. Conjugation with PDPH (3-2-pyridyldithio) -propionohydrazide) resulted in covalent thioether linkages, whereas conjugation with CDI/CDT (1, 1-carbonyldiimidazole/1, 1-carbonyl-di-1, 2, 4-triazole) resulted in a 1-carbon or 0-carbon linker between the CP and the carrier protein.

Conjugation of CP to CRM by PDPH conjugation chemistry ₁₉₇

PDPH conjugation chemistry is a multi-step process that involves activation of polysaccharides, removal of thiol protecting groups, purification of activated polysaccharide intermediates, activation and purification of CRM₁₉₇The protein and the conjugated activated fraction are then purified. Introduction of thiol-containing linker into polysaccharide and haloacetyl into CRM₁₉₇Protein carrier following, staphylococcus aureus CP5 and CP8 polysaccharides were linked to the protein carrier via thioether bonds. Bromoacetyl groups are introduced to CRM by reacting amine groups with N-hydroxysuccinimide esters of bromoacetic acid₁₉₇A protein. To generate thiolated CP, the carbodiimide-activated carboxylate group of N-acetylmannosaminidonic acid (N-acetylmannosaminouronic acid) in CP is coupled to the hydrazide group of the thiol-reactive hydrazide heterobifunctional linker 3- (2-pyridyldithio) -propionhydrazide (PDPH). The sulfhydryl group of PDPH-thiolated CP, produced by reduction with DTT and purified by SEC on a Sephadex G25 column, is reacted with the bromoacetyl group of the activated protein, resulting in covalent thioether bonds formed by bromine displacement between CP and the protein. Using non-reacted bromoacetyl groupsCysteamine hydrochloride (2-mercaptoethylamine hydrochloride) "capped". The reaction mixture was then concentrated and diafiltered. The remaining unconjugated bromoacetyl groups were capped with cysteamine hydrochloride to ensure that no reacted bromoacetyl groups remained after conjugation. This forms a covalent bond between the thiol end of the cysteamine and the acetyl group on the lysine residue after bromine substitution.

Thiolation of Staphylococcus aureus capsular polysaccharide with PDPH

The polysaccharide was first activated by thiolation with PDPH. Polysaccharides were mixed with freshly prepared PDPH stock (250 mg/mL in DMSO), EDAC stock (diH)₂O90 mg/mL) and MES buffer stock (0.5M, pH 4.85) to prepare final solutions 0.1M MES, and 2 and 4mg CP/mL while maintaining CP5 at a CP: PDPH: EDAC weight ratio of 1: 5: 3 and CP8 at a CP: PDPH: EDAC weight ratio of 1: 0.6: 1.25. This mixture was incubated at room temperature for 1 hour, then distilled H was applied at a volume of 1000X using a 3500MWCO dialysis unit at 4-8 deg.C₂O dialysis 4 times to remove unreacted PDPH. PDPH linked polysaccharide was brought to 0.2M DTT and incubated at room temperature for 3 hours or at 4-8 ℃ overnight. Excess DTT as well as by-products of the reaction were separated from the activated sugars by SEC using Sephadex G25 resin and distilled water as mobile phase. Fractions were assayed by DTDP assay of thiol groups and the eluted thiol positive fractions close to the void volume of the column were pooled. The pools of fractions were assayed by PAHBAH and O-acetyl assays to determine the degree of activation expressed as the mole percent of thiol-containing repeat units (molar concentration of thiol groups/molar concentration of repeat units). The activated polysaccharide was lyophilized and stored at-25 ℃ until conjugation was required.

Activation of carrier protein

Separately, the carrier protein is activated by bromoacetylation. CRM₁₉₇Diluted to 5mg/mL with 10mM phosphate buffered 0.9% NaCl pH 7(PBS) and then made 0.1M NaHCO using a 1M stock solution₃The pH was 7.0. BAANS stock solution with 20mg/mL DMSO in CRM₁₉₇BAANS ratio 1: 0.25 (w: w) N-hydroxysuccinimide ester of bromoacetic acid (BAANS) is added. This reaction mixture was incubated at 4-8 ℃ for 1 hour and then purified on Sephadex G-25 using SEC. Purified activated CRM as determined by Lowry assay₁₉₇To determine the protein concentration, it was then diluted to 5mg/mL with PBS. Sucrose was added to 5% wt/vol as a cryoprotectant and the activated protein was frozen and stored at-25 ℃ until conjugation was required.

Coupling reaction

Once the activated capsular polysaccharide and activated carrier protein are prepared, the two are mixed in a conjugation reaction. Lyophilized and thiolated polysaccharide was dissolved in 0.16M borate pH8.95 with thawed bromoacetylated CRM₁₉₇And distilled water to prepare a final solution 0.1M Borate, CRM in a 1: 1wt/wt ratio₁₉₇CP with 1mg/mL polysaccharide (CP8) and 2mg/mL polysaccharide (CP 5). This mixture was incubated at room temperature for 16-24 hours. CRM at 1: 2(wt/wt) by using a 135mg/mL stock of cysteamine dissolved in 0.1M borate, pH8.95₁₉₇Cysteamine hydrochloride was added in a ratio to cap unreacted bromoacetyl groups on the protein and incubated at room temperature for 4 hours. Capsular polysaccharide-CRM was purified by 50-fold diafiltration against 0.9% NaCl using a 100K polyethersulfone ultrafilter₁₉₇Conjugates (conjugates).

The results from the reproducibility of the thiolation studies with CP5 and CP8 with PDPH demonstrated that the degree of activation of CP5 was in the range of 11-19%, which corresponds to approximately 1 linker molecule linked per 10 CP repeat units to 1 linker molecule linked per 5 repeat units. CP8 activation was in the range of 12-16%, which is very similar to the activation of CP 5.

CRM₁₉₇The bromoacetylation of lysine residues in (a) is very consistent, resulting in the activation of 19-25 of the 39 lysines available. This reaction produces high yields of activated protein.

Conjugation of CP to CRM by CDI/CDT conjugation chemistry ₁₉₇

CDI and CDT provide a one-step conjugation process in which polysaccharides are activated in an anhydrous environment (DMSO) to form an imidazole or triazole carbamate moiety with available hydroxyl groups and an acylimidazole or acyltriazole moiety with carboxylic acids. The addition of a protein carrier (in DMSO) results in nucleophilic displacement of imidazole or triazole by lysine, and formation of carbamate bonds (activated hydroxyl groups) and amide bonds (activated carboxylic acids). The reaction solution was diluted 10-fold into an aqueous solution to remove the activated group as unreacted, and then purified by diafiltration.

Both conjugation chemistries produced CP covalently linked to the carrier protein, as shown by the presence of sugars and proteins in the fractions from size exclusion chromatography and by amino acid analysis of either glycolaldehyde-capped or cysteamine hydrochloride-capped conjugates.

A summary of the results from the preparation of several batches of conjugates of two capsular serotypes with a polysaccharide size of 20-40kDa prepared by PDPH and CDI/CDT is shown in table 7 below. There was no significant difference in the ratio of free capsular polysaccharide to CP: protein and the yield of conjugate produced by these two conjugation methods. Conjugation did not alter the antigenicity of the conjugated CP, as shown by the same precipitin line between the conjugate and the native CP.

Table 7: SA CP5-CRM prepared by two conjugation chemistries ₁₉₇ And CP8-CRM ₁₉₇ Is characterized by

As indicated above, the processes described herein can be used to produce a specific range of desired high molecular weight capsular polysaccharides. The aim of this study was to remove the impurities from the solution which may be filtration and purificationA preselected range of high molecular weights of CP of (a) to prepare the conjugate for use in an immunogenic composition. In this example, 8 batches were selected in which the CP5 capsular polysaccharide had a molecular weight ranging from about 90kDa to about 120kDa, and conjugation was performed using triazole (CDT) activation. See table 8. The resulting conjugate had a molecular weight range of 1533kDa to 2656 kDa. Per CRM₁₉₇The number of conjugated lysines ranged from as high as 22 to as low as 15. Free capsular polysaccharides range from as high as 18% to as low as 11%. See table 8.

Table 8 conjugates of CP5 with preselected MW ranges

Table 9 summarizes the analysis of CP8 conjugates, where the CP8 capsular polysaccharide has a molecular weight ranging from about 87kDa to 113kDa and utilizes imidazole conjugation chemistry. The resulting conjugate has a molecular weight range of 595kDa to 943 kDa. Per CRM₁₉₇The number of conjugated lysines ranged from as high as 9 to as low as 3. Free capsular polysaccharides range from as high as 6% to as low as 2%. See table 9.

Table 9 conjugates of CP8 with preselected MW ranges

Both conjugation chemistries produce CPs that are covalently linked to a carrier protein. There was no significant difference in the ratio of free capsular polysaccharide to CP: protein and the yield of conjugate produced by these two methods.

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

Protein sequence heterogeneity of ClfA polypeptide fragments N1, N2, and N3 from disease-causing isolates obtained from various sources was evaluated in this example. The ClfA gene was sequenced from strains of staphylococcus aureus associated with various disease states. Sequence information from additional strains was obtained from GenBank to generate sequences from related strains. Table 10 lists the different ClfA sequences.

Sequence alignments of the ClfA protein from different disease-causing strains of staphylococcus aureus are shown in figures 8A-8E. The protein sequences were aligned using MUSCLE. See Edgar, r.c. nucleic acids research 32 (5): 1792-1797(2004). The alignment is shown using SHOWALIGN. See Rice, p.et al, "EMBOSS: the European Molecular Biology Open software "Trends in Genetics, 16 (6): 276-277(2000). Many sequences repeat many times without variation. For clarity, each unique sequence is placed in the alignment only once. See fig. 8A-8E. Only unique sequences are included in the sequence listing. For example, the protein sequence of ClfA _001 was obtained from a number of different strains without any variation. See fig. 8A-8E. Sequence listing numbers for any sequence can also be obtained from table 10: a ClfA strain and a sequence listing. An example strain containing this same ClfA _001 protein sequence is listed in table 10. This sequence is shown in the first row of the alignment in FIGS. 8A-8E. This alignment of the unique sequences of the ClfA antigen showed that the polymorphisms were distributed throughout the a region of ClfA (N1-N2-N3). In some cases, for any given unique protein sequence of ClfA, more than one nucleotide sequence was found to encode the same protein. Only the most frequently occurring DNA sequences are included in the sequence table and table 10. For ClfA, the following sequences are disclosed herein and not found in GenBank: ClfA _003, ClfA _005, ClfA _008, ClfA _009, ClfA _013, ClfA _014, ClfA _015, ClfA _016, ClfA _017, ClfA _018, ClfA _019, ClfA _020, ClfA _021, ClfA _022, ClfA _023, and ClfA _ 024.

Table 10: ClfA strain and sequence listing

Phylogeny of ClfA protein sequences was detected and phylogenetic trees were constructed. The sequences were aligned using ClustalW. See Chenna R, Sugawara H, Koike T, et al.nucleic Acids research.31 (13): 3497-3500(2003). The adjoining tree was bootstrapped (bootstrap)1000 times and displayed with MEGA 4.0. See Tamura K, et al, Molecular Biology & evolution.24 (8): 1596-1599(2007). The bootstrap values shown on the branches are the number of times the branches were repeatedly generated in 1,000 trials. Values less than 500 (50% reproducibility) are considered to be low-supported.

The ClfA sequence forms a tree with 2 major branches. See fig. 9. The separation of these two groups is well supported in the phylogeny. One branch (top) includes 9 sequences that are fairly closely related to each other (96-99% identity) but are more distantly related to the candidate sequence clfA _011, which are identical to clfA _ 01191-92%. The second group, which includes clfA _011, is more diverse and the phylogenies in this group are not well supported. These protein sequences are 93-99% identical to each other.

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

In this example, the protein sequence heterogeneity of ClfB N1, N2, and N3 polypeptide fragments from 92 disease-causing isolates isolated from various sources was evaluated. The ClfB gene was sequenced from strains of staphylococcus aureus associated with various disease states. See table 11. Information from additional strains was obtained from GenBank to generate additional sequences.

An alignment of the sequences of the ClfB protein from different disease-causing strains of staphylococcus aureus is shown in fig. 10A-10E. The protein sequences were aligned using MUSCLE. See Edgar, r.c. nucleic acids Research 32 (5): 1792-1797(2004). The alignment is shown using SHOWALIGN. See Rice, p.et al, "EMBOSS: the European Molecular Biology open software Suite "Trends in Genetics, 16 (6): 276-277(2000). See FIGS. 10A-10EClfB alignment. As with ClfA, many sequences repeat many times without variation. For clarity, each unique sequence is placed in the alignment only once. See fig. 10A-10E. Only unique ClfB sequences are included in the sequence listing. For example, the sequence of ClfB _006 was obtained from a number of different strains without any variation. This sequence is shown in the first row of the alignment in FIGS. 10A-10E. Sequence listing numbers for any sequence can also be obtained from table 11. This alignment of representative unique sequences of ClfB antigen showed that the polymorphisms were distributed throughout the a region of ClfB (N1-N2-N3). Similar to ClfA, for any given unique protein sequence of ClfB, more than one nucleotide sequence was found to encode the same protein. Only the most frequently occurring DNA sequences are included in the sequence table and table 11. For ClfB, the following sequences are disclosed herein and not found in GenBank: ClfB _001, ClfB _004, ClfB _005, ClfB _010, ClfB _011, ClfB _013, ClfB _014, ClfB _015, ClfB _016, ClfB _017, ClfB _018, ClfB _019, ClfB _020, ClfB _021, ClfB _022, ClfB _023, and ClfB _ 024. The system tree is shown in fig. 11.

Table 11: ClfB strain and sequence listing

Example 7: sequence diversity of MntC in disease-causing clones of Staphylococcus aureus

In this example, the protein sequence heterogeneity of the MntC gene from 104 disease-causing isolates obtained from various sources was evaluated. MntC sequences were sequenced from strains of staphylococcus aureus associated with various disease states. See table 12. Information from additional strains was obtained from GenBank to generate strain sequences.

An alignment of the sequences of the MntC protein from different disease causing strains of staphylococcus aureus is shown in fig. 12A-12B. The protein sequences were aligned using MUSCLE. See Edgar, r.c. nucleic acids Research 32 (5): 1792-1797(2004). The alignment is shown using SHOWALIGN. See Rice, p.et al, "EMBOSS: the European Molecular Biology open software Suite "Trends in Genetics, 16 (6): 276-277(2000). See fig. 12. As with ClfA, many sequences repeat many times without variation. For clarity, each unique sequence is placed in the alignment only once. See fig. 12. Only unique MntC sequences are included in the sequence listing. For example, the sequence of MntC _001 was obtained from different strains without any variation. See fig. 12. This sequence is shown in the first row of the alignment in FIG. 12. Sequence listing variations of any of the sequences can also be obtained from table 12. Only the most frequently corresponding DNA sequences are included in the sequence listing. For MntC, the following sequences are disclosed herein and not found in GenBank: MntC _002, MntC _006, MntC _007, MntC _008, and MntC _ 009.

Table 12: MntC strain and sequence listing

Example 8: surface expression of ClfA, CP5, CP8 and MntC in vivo during infection

Staphylococcus aureus is responsible for causing a variety of human infections. Bacteria must therefore adapt to different environmental niches by differentially expressing virulence factors required for infection. The expression of target antigens was studied in 3-individual rodent assays to evaluate their expression during infection: wound models that measure antigen expression at the primary site of infection, bacteremia models that monitor antigen expression in blood, and indwelling chamber models that monitor antigen expression under nutrient/oxygen limited conditions. For all these models, rodents were challenged with bacteria at the site of interest. Following infection, bacteria were harvested at various time points and antigen expression was assessed using immunofluorescence microscopy (wound and bacteremia) or flow cytometry (chamber) (ClfA, CP5, CP8, MntC).

Materials and methods

Expression in wound models

Wound infection experiments consisted of 5 animals/group and up to 5 groups, up to 25 animals/experiment. C57BL/6 male mice, 6-8 weeks (wk) old, were subjected to surgery to embed suture packages into the thigh muscle incisions. This provides a foreign body substrate for bacterial attachment and significantly reduces the minimum infectious dose required to produce staphylococcal wound infections. mu.L of Staphylococcus aureus or sterile saline was introduced into the 4-0 silk deep tissue sutured incision. The skin is closed with a 4-0Prolene suture or a surgical adhesive (e.g., cyanoacrylate). Animals were euthanized at time points between 30min and 10 days post infection, thigh muscles were excised, homogenized, and bacteria were counted. When infection was seen, the bacteria were analyzed for antigen expression by Immunofluorescence (IF) confocal microscopy.

Expression in bacteremia models

Groups of 10 4-week-old CD-1 or Balb/C mice were immunized with 1 μ g of protein or CP conjugate by subcutaneous injection at weeks 0,3, and 6. Animals were bled at weeks 0 and 8 after intraperitoneal challenge with s.aureus grown to late log phase in TSB. Animals were euthanized 3 hours after challenge and blood was collected for IF confocal microscopy.

Expression in indwelling dialysis bag model

S. aureus isolates were grown overnight at 37 ℃ on TSA plates. Bacteria were scraped from the plate, resuspended in sterile PBS, and OD was performed₆₀₀Adjusted to 1, about 10⁹Colony forming units (cfu)/mL. Bacteria were diluted to 10³cfu/mL and inoculated into dialysis bags. Aliquots of this suspension were plated to determine the actual number of cfu. Dialysis bags with 3.5kda mwco were prepared for implantation by bulk rinsing in sterile water and then sterile saline after 30 minutes of sterilization in 70% ethanol. A 2mL aliquot of the bacterial suspension was transferred to a dialysis bag, which was closed with a knot and then rinsed extensively with sterile saline. Male Sprague Dawley rats (6 weeks old) were anesthetized and a 2-3cm incision was made along the dorsal midline. Pockets are created at the site of the incision by gently separating the skin from the subcutaneous tissue. The bag is implanted in the pocket and the skin is closed using surgical staples. After 24h, the rats were euthanized, the bag removed, and the bacteria recovered for flow cytometry analysis.

Immunofluorescence microscopy (IF)

Blood from 5 mice was pooled into ice-cold sodium citrate, pH 7.0 (final concentration, 0.4%). Eukaryotic cells were lysed with 1% NP-40(Pierce Biotechnology). The bacteria were washed with PBS, incubated overnight at 4 ℃ with rabbit immune or preimmune serum (1: 100), and detected with ALEXA 488-conjugated goat- α -rabbit antibodies (1: 250, Invitrogen). The labeled bacteria were dried on microscope slides and the coverslips mounted with Vectashield HardSet media (Vector Laboratories, Inc.). Images were obtained with a Leica TCS SL spectroscopic confocal microscope (Leica Microsystems).

Flow cytometry analysis

Staphylococcus aureus isolates were grown as described in the rat dialysis bag model method. Will be about 10⁷The bacterial cells were blocked in staining buffer (Hank's balanced salt solution containing 10% goat serum) for 1 hour on ice water. The bacterial cells were centrifuged at 10,000rpm for 5 minutes, the supernatant was removed, and the cells were incubated with mouse antibody or isotype control antibody on ice for 30 minutes. The cells were then washed and stained with FITC-conjugated goat anti-mouse igg (jackson immunoresearch) for 30 minutes on ice. Cells were washed with staining buffer, fixed with 2% paraformaldehyde, and data were obtained and analyzed using a FACSCaliber flow cytometer and Cell quest software (Becton, Dickinson and Co.). A total of 30,000 events were collected per sample.

Table 13 a: antigen expression profile in staphylococcus aureus CP5 isolates.

The bacteremia experiment was carried out for 6 hours! Death of animals during the experiment NT not detected

Table 13 b: antigen expression in staphylococcus aureus CP8 isolates.

The bacteremia experiment was carried out for 6 hours! Death of animals during the experiment NT not detected

Table 13c. expression of staphylococcus aureus antigens in the indwelling dialysis bag.

Frequency of Positive cells (% of the total)

Results

Combinations of 19 s.aureus isolates were tested for ClfA, CP5, CP8 or MntC expression on the surface of s.aureus cells during infection (tables 13a, 13b and 13 c). These isolates included the most recently clinically relevant strains and were all different as monitored by MLST. Antigen expression depends on the strain, time point and infection model. Variations in antigen expression between isolates in different in vivo environments (blood flow versus wound) support the use of multi-antigen immunogenic compositions to induce staphylococcal isolates that cover a wide range of different infections. The antigen is expressed internally within the first 24 hours of infection and is therefore an effective component of an anti-staphylococcal immunogenic composition. Protein antigens ClfA and MntC can stain in the presence of capsular expression, indicating that the presence of the capsule does not mask antibodies to the proteins.

Most of the tested type 8 isolates did not express CP in blood until a later time point (> 4 hours) after challenge (see tables 13 a-c). These results demonstrate that CP is differentially regulated in staphylococcus aureus, depending on the microenvironment in vivo, i.e. the site of infection. These results may explain the inconsistent efficacy results reported for CP8 conjugates in animal models.

In vivo expression results indicate that no single antigen immunogenic formulation will provide broad coverage of most S.aureus infections. There is too much diversity of expression phenotypes for individual strains within the in vivo microenvironment. Thus, there is a need for immunogenic compositions consisting of more than one antigen for the prevention of staphylococcus aureus disease.

Example 9: contains ClfA, CP 5-and CP8-CRM₁₉₇Immunogenicity of multi-antigen formulations of conjugates

In this example, we evaluated ClfA, CP5-CRM₁₉₇And CP8-CRM₁₉₇The immunogenicity of the combination of (a).

A.Double antigen (CP 5-CRM) ₁₉₇ /CP8-CRM ₁₉₇ ) Immunogenic composition formulation-anti-capsular to rabbit Dose effect of antibody response

In this example, combined CP5-CRM was evaluated in rabbits₁₉₇And CP8-CRM₁₉₇Dose effect of immunogenic formulations on immunogenicity. Add 125 μ g AlPO with bivalent conjugate administered by subcutaneous injection at weeks 0,3 and 6₄Rabbits were immunized. The dose evaluated in this study was CP5-CRM₁₉₇And CP8-CRM₁₉₇0.1. mu.g, 1. mu.g or 10. mu.g each (final combined CP-CRM)₁₉₇The doses were 0.2. mu.g, 2. mu.g and 20. mu.g). The dosage of the conjugate reflects the total polysaccharide component of the proteoglycan conjugate. Rabbits were bled at weeks 0,3, 6, and 8. ELISA was performed on pooled and individual sera. The end-point antibody titer was determined to be at 0.1OD₄₀₅The reciprocal dilution of (c). The titer of the individual at week 8 was statistically analyzed. The results demonstrate that the highest CP5 and CP8 specific antibody titers were induced by immunizing rabbits with a bivalent immunogenic formulation at a CP dose of 1 μ g per component, where CP5 is 5X10⁵CP8 is 1X10⁶(data not shown).

B.Three antigen formulations (CP 5-CRM) ₁₉₇ +CP8-CRM ₁₉₇ + rClfA) -Weekly dose in rabbits (1) μ g) of each conjugate

The effect of rClfA in combination with CP5 and CP8 conjugates on the immune response of each component was examined. With bivalent Staphylococcus aureus CP5-CRM₁₉₇+CP8-CRM₁₉₇(1. mu.g dose of each conjugate) 3 different doses of T7-ClfA (N1N2N3) were combined in 3 immunization groups. The control group was immunized with unconjugated CP5 and CP8 (50 μ g each) in combination with 100 μ g T7-ClfA (N1N2N 3). Each immunogenic composition was mixed with 500. mu.g adjuvant AlPO₄And (4) preparing the components together. The immunogenic composition is administered by subcutaneous injection in the neck. Rabbits were bled at weeks 0, 6 and 8. ELISA was performed on pooled and individual sera and the end-point antibody titer was determined at 0.1OD₄₀₅The reciprocal dilution of (c).

The results show that increased amounts of rClfA do not affect the capsular antibody response when combined with a bivalent conjugate. Antibody levels for both capsular serotypes were in the same range as in rabbits immunized with the bivalent conjugate alone (data not shown). Antibody levels of CP5 and CP8 were 2.5-fold lower at 10(103K) and 100 μ g (106K) doses of rClfA compared to 1 μ g doses of ClfA (273K). There was a booster effect after the second and third injections. Unconjugated bivalent polysaccharide immunogenic formulations (CP5+ CP8, 50 μ g each) combined with 100 μ g rClfA did not induce CP-specific antibodies. The rClfA-specific antibody response was also not greatly affected by the dose, with titers at 1X10 after 3 of 1, 10 and 100 μ g doses⁵And 1X10⁶In between (data not shown). And similar levels of anti-ClfA response were achieved when administered with conjugated or unconjugated CP5 and CP8 polysaccharides.

Example 10: three antigen preparations-immunogenicity in rabbits with high pre-immune CP5, CP8, and ClfA Ab titers.

The staphylococcal immunogenic composition is targeted to an adult population of antibodies having pre-existing surface components of s. To investigate the effect of antibodies of pre-existing immunogenic formulation components on the response to the immunogenic formulation, we selected rabbits with high titers of naturally obtained anti-CP 5, anti-CP 8, and anti-ClfA antibody titers. Three antigen immunogenic formulations (CP 5-CRM) at weeks 0,3, and 6₁₉₇(1μg) And CP8-CRM₁₉₇Group 2 rabbits (N-6/7) were immunized (1 μ g) with T7-ClfA (N1N2N3) Y338A (10 μ g)). One group was used with 500. mu.g of AlPO as adjuvant₄The formulated immunogenic compositions were immunized, while the second group was immunized with the non-adjuvanted immunogenic composition. The immunogenic composition is administered by subcutaneous injection. Rabbits were bled at weeks 0,3, 6 and 8. Antibody titers of CP5, CP8, and rClfA were determined by ELISA as the endpoint antibody titer for pooled and individual sera (determined at 0.1OD₄₀₅Reciprocal dilution of (d).

The results show that rabbits with pre-existing antibody titers induced by natural infection respond to trivalent immunogenic preparations with elevated levels of antibodies to all immunogenic preparation components CP5, CP8 and rClfA. Even with 1x10⁶Shows a 5-10 fold increase in Ab levels of the respective antigens in the animals of the antibody titer of (a). The presence of adjuvant in the immunogenic formulation resulted in higher antibody titers compared to the group immunized without adjuvant (data not shown).

Example 11: effect of adjuvants on the response to the capsular polysaccharide component

A.Two different doses of AlPO ₄ For bivalent CP5-CRM in rabbits ₁₉₇ /CP8-CRM ₁₉₇ Conjugation Effect of the response of the immunogenic composition of matter

Study of AlPO adjuvant in rabbits₄Dose effects against CP5 and CP8 responses. Bivalent Staphylococcus aureus CP5-CRM at weeks 0,3, and 6₁₉₇+CP8-CRM₁₉₇Rabbits were immunized (1 μ g dose of each conjugate). One group (n-5/group) was used with 125 μ g AlPO as adjuvant₄The immunogenic compositions formulated together were immunized, while the second group was with 500. mu.g AlPO₄As an adjuvant. The immunogenic composition is administered by subcutaneous injection in the neck. Rabbits were bled at weeks 0, 6 and 8 and anti-capsular antibody was determined by ELISA as the endpoint antibody titer determined at 0.1OD₄₀₅The reciprocal dilution of (c). The results are shown in 125. mu.g or 500. mu.g AlPO₄In the immunized rabbit, the immune response of the rabbit is improved,there was no difference in CP 8-specific antibody responses. Formulations containing 125 μ g adjuvant gave higher CP5 antibody responses. Furthermore, all rabbits in the 125 μ g group had a higher CP5 antibody response, whereas in the 500 μ g adjuvant group, 2 rabbits had a low response to the formulation.

B.AlPO ₄ Effect on immunogenicity of Tri-antigen formulations

CP5-CRM used at weeks 0,3, and 6₁₉₇(1. mu.g) and CP8-CRM₁₉₇Rabbits were immunized with a three-antigen preparation consisting of (1 μ g) T7-ClfA (N1N2N3) Y338A (10 μ g) (NZW, N6/7 rabbits per group). A group of rabbits contains 500. mu.g of AlPO₄A second group formulated without adjuvant and a third group at week 0 with a vaccine containing 500 μ g of AlPO₄And at weeks 3 and 6 with the non-adjuvanted immunogenic formulation. The immunogenic formulation was administered by subcutaneous injection, rabbits were bled at weeks 0,3, 6 and 8, and sera were evaluated by antigen-specific ELISA. The results showed that the presence of adjuvant in the immunogenic formulation had no effect on anti-CP 5 or anti-CP 8 responses in rabbits (data not shown). The GMT titers of abs of both capsules were comparable. However, there was an adjuvant effect on ClfA specific antibody responses in the groups immunized with the adjuvant present in all 3 immunizations. AlPO-free in rabbits initially with an immunogenic formulation containing an adjuvant₄The second and third boosts of the immunogenic formulation of (a) give a higher ClfA response compared to the group without adjuvant.

Examples 12 to 29: staphylococcus aureus ClfA, MntC, CP5-CRM₁₉₇And CP8-CRM₁₉₇Preclinical evaluation of (a):

the results of preclinical evaluation of CP5 and CP8 conjugates, ClfA, and MntC are described in examples 12-29 below. The examples demonstrate the efficacy of these antigens in preclinical animal models. The examples also demonstrate that antibodies produced by CP conjugates, ClfA and MntC are functionally active in vitro assays.

Two different chemistries were used to conjugate CPTo CRM₁₉₇However, no difference in efficacy was observed for the conjugates prepared by the different methods. It was demonstrated that O-acetylation of capsular polysaccharides affects priming function antibodies. Including CP5-CRM₁₉₇、CP8-CRM₁₉₇And ClfA showed no interference with specific antibody (Ab) levels of each immunogenic formulation component.

Materials and methods

ELISA

Maxisorp microtiter ELISA plates (Nalge Nunc International, Rochester, N.Y.) were coated with 1. mu.g/mL ClfA antigen in PBS pH 7.5 for 18 hours at 4 ℃ or 90min at 37 ℃. Plates were washed 5 times in PBST (1X PBS, 0.1% polysorbate 20) and blocked with 1% (w/v) skim milk in PBS containing 0.05% polysorbate 20 for 1h at room temperature. Plates were washed with PBST, serial dilutions (3-fold) and individual week 0,3, 6 and 8 rabbit antisera were added to the plates and incubated overnight at 4 ℃ or 2h at 37 ℃. Plates were washed and bound primary antibody was detected with horseradish peroxidase conjugated goat anti-rabbit IgG in PBST (1: 1000 dilution). Plates were incubated at 37 ℃ for 1h, then washed, and developed with ABTS-peroxidase substrate solution (KPL, inc., Gaithersburg, MD) at room temperature for about 20 minutes. The reaction was stopped by adding 1% (v/v) SDS solution. Absorbance was measured at 405nm in an automatic microplate reader (Molecular Devices Corporation, Sunnyvale, Calif.). Antibody titers were expressed as the reciprocal of the highest serum dilution with an absorbance value of 0.1. The student's t-test using JMP software (SAS Institute, Cary, NC) was used to determine the differences in antibody titers between the different groups. A probability of less than 0.05 is considered to indicate a statistically significant difference.

Mouse sepsis model

The mouse sepsis model simulates a blood-borne disease. For passive immunization, a group of 15 Swiss-Webster mice were intraperitoneally (i.p.) treated with IgG. After 24 hours, mice were challenged with a single intravenous (i.v.) injection (0.1ml) of S.aureus 659-018 via the tail vein. All animals were closely attended for 14-15 days, at which point all remaining mice were sacrificed.

For active immunization, mice were immunized with antigen at weeks 0, 2 and 4 and challenged with staphylococcus aureus at week 6 via the intravenous route.

Active immune rabbit endocarditis model

Adult New Zealand white rabbits were immunized 4 times intramuscularly with 25. mu.g of antigen. One day after surgery, animals were i.v. challenged with a bolus of staphylococcus aureus and the number of colony forming units (cfu) in heart tissue was determined 24 hours after challenge.

Bacteremia in mice

The 3hr bacteremia model was used to determine the effect of early immunization during infection on bacterial numbers. Mice were immunized with antigen at weeks 0,3 and 6, and then challenged with s.aureus i.p. at week 8. After 3 hours, animals were bled and serially diluted blood plated to count bacteria.

Mouse model of pyelonephritis

The mouse pyelonephritis model mimics the spread of staphylococcus aureus from bacteremia. Groups of 10 female CD-1 mice, 4 weeks old, were immunized with antigen at weeks 0,3 and 6. Mice were challenged by i.p. injection of staphylococcus aureus. 48 hours after challenge, mice were sacrificed and bacteria in the kidney and blood were counted.

Rat endocarditis model

The rat endocarditis model mimics human endocarditis, in which colonization occurring after a blood-borne infection results in the colonization of damaged cardiac tissue. 5 male Sprague-Dawley rats (Charles River, Kingston, NY) of 5 weeks of age were dosed at weeks 0, 2 and 4 with 100. mu.g AlPO₄1 μ g formulated together CP5-CRM₁₉₇The conjugates are immunized. Animals were bled at the end of weeks 0 and 5 prior to immunization. After 72 hours, a catheter (PE-10 tube) was surgically placed into the left ventricle of the heart through the carotid artery. The placement of the catheter results in the formation of sterile neoplasms to which the staphylococci can attach at the time of infection. To prevent infection from surgery, animals were treated with the antibiotic Baytril (5mg/kg) at and 8hr post-surgery. 48 hours after surgery, with PFESA0266 (about 4X 10)⁸cfu) or SA315 (about 1X 10)⁹cfu) rats were challenged by intraperitoneal injection. 48 hours after challenge, rats were euthanized, hearts and kidneys were removed and placed in 3mL Phosphate Buffered Saline (PBS). The organ was then homogenized with a tissue homogenizer (Kinematica AG, Luzernerstrasse, Germany) and supplemented to 10mL with PBS. The homogenate was then serially diluted and plated for bacterial enumeration.

Monitoring functional antibodies using opsonophagocytic killing assays

Differentiated effector cells from cell lines (e.g., HL60) or polymorphonuclear cells (PMNs) usingPoly solution (Cedarlane laboratories limited, Ontario, Canada) was isolated from donor human blood according to the manufacturer's protocol. The effector cells were treated at about 2X10⁷The concentration of individual cells/ml was resuspended in assay buffer (modified eagle's medium containing 1% bovine serum albumin) and placed in a 37 ℃ incubator until ready for use. The staphylococcus aureus strain PFESA0266 was grown overnight on tryptic soy agar plates. Bacterial cells were scraped, washed 2 times and resuspended to OD in assay buffer containing 5% glycerol₆₀₀This is equal to about 5X10 ═ 1⁸concentration of cfu/ml. 1ml aliquots of the bacterial suspension were frozen and stored at-40 ℃ until ready for use. The frozen bacterial suspension was thawed and adjusted to 10 in assay buffer⁶cfu/ml and placed on ice. The assay was performed using sterile 96 deep well 1ml polypropylene platesAnd (4) determining. A2-fold serial dilution of the antibody sample (50. mu.l) was prepared, and then 300. mu.l of assay buffer was added to the antibody mixture. Cells were added (50. mu.l) to the plate and placed on a rotary shaker at 4 ℃ for 30 minutes. After the conditioning step 50 μ l of human complement (1% final concentration) was added. Finally, 50. mu.l of effector cells (10)⁷Concentration of individual cells/ml) was added to the plate and the suspension was mixed well by repeated pipetting. 50 μ l aliquots of the suspension were serially diluted 10-fold in sterile 1% saponin solution, vortexed to minimize bacterial aggregation, and plated in duplicate on tryptic soy agar. The assay plates were incubated at 37 ℃ for 1 hour and mixed continuously using an electric rotisserie style shaker. At the end of the incubation, 50 μ Ι aliquots of the suspension were serially diluted 10-fold in sterile 1% saponin solution, mixed by vortexing to minimize bacterial aggregation, and plated in duplicate on tryptic soy agar. Percent kill was calculated by determining the ratio of the number of cfu surviving at 60 minutes in wells containing bacteria, antibody, complement and effector cells to the number of cfu surviving in tubes lacking antibody but containing bacteria, complement and effector cells. Controls containing bacteria, complement and serum were included to adjust for any reduction in cfu due to aggregation.

Complement adsorption

Sera from human donors adsorbed against staphylococcus aureus strains PFESA0266, PFESA0286, and PFESA0270 can be used as a source of complement in the assay. Staphylococcus aureus strains were grown overnight at 37 ℃ on TSA plates. Cells were scraped from the plate and resuspended in sterile PBS. The bacterial cells were centrifuged at 10,000rpm for 10 minutes at 4 ℃, and the cell pellet was resuspended in human serum for adsorption. The serum was incubated with the bacteria at 4 ℃ for 30 minutes on an octotor. The cells were centrifuged, the serum was transferred to another tube containing the bacteria, and the adsorption step was repeated again for 30 minutes. Finally, the cells were centrifuged and the serum was passed through a 0.2 micron filter, and then 0.5ml aliquots were frozen in liquid nitrogen.

Method II-OPA Using HL-60 cells

HL-60 cells were differentiated according to S.Romero-Steiner, et al, Clin Diagn Lab Immunol 4(4) (1997), pp.415-422. Harvesting HL-60 cells at about 10⁸Individual cells/ml were resuspended in assay buffer (modified eagle's medium containing 1% bovine serum albumin) and placed in a 37 ℃ incubator until ready for use. Staphylococcus aureus was grown overnight on tryptic soy agar plates. Bacterial cells were scraped, washed twice and resuspended to OD in assay buffer containing 5% glycerol₆₀₀This is equal to about 5X10 ═ 1⁸cfu/ml. 1ml aliquots of the bacterial suspension were frozen and stored at-40 ℃ until ready for use. The frozen bacterial suspension was thawed and adjusted to 10 in assay buffer⁶cfu/ml and placed on ice. The assay was performed using sterile 96 deep well 1ml polypropylene plates. Monoclonal antibody samples (25. mu.l) were prepared at 2-fold serial dilutions, and then 150. mu.l of assay buffer was added to the antibody suspension. Bacteria were added (25 μ l) to the plate and placed on a rotary shaker at 4 ℃ for 30 minutes, followed by 25 μ l of human complement (1% final concentration). Finally, 25. mu.l of HL-60 cells (10)⁷cells/ml) were added to the plate and the suspension was mixed well by repeated pipetting. Aliquots of 25 μ l of the suspension were 10-fold serially diluted in sterile 1% saponin solution, mixed by vortexing to minimize bacterial aggregation, and plated in duplicate on tryptic soy agar. The assay plates were incubated at 37 ℃ for 1 hour and mixed continuously using an electric rotisserie style shaker. At the end of the incubation, 25 μ Ι aliquots of the suspension were serially diluted 10-fold in sterile 1% saponin solution, mixed by vortexing, and plated in duplicate on tryptic soy agar. Percent kill was calculated by determining the ratio of the number of cfu surviving at 60 minutes in wells containing bacteria, antibody, complement and HL-60 cells to the number of cfu surviving in tubes lacking antibody but containing bacteria, complement and HL-60 cells. Controls containing bacteria, complement and mAb were included to adjust for any reduction in cfu due to aggregation.

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

To evaluate whether polyclonal rabbit antibodies raised against ClfA could reduce staphylococcus aureus colony counts in the mouse sepsis model, purified rabbit polyclonal anti-ClfA IgG was used in two doses (0.8mg and 1.6mg) in a passive immunization study (fig. 13). The S.aureus challenge strain was the most recent clinical isolate 659-018. Both antibody doses resulted in a significant reduction in bacterial colony counts in the mouse sepsis model (1.8mg dose p ═ 0.0134, and 0.8mg dose p ═ 0.0013). This experiment has been repeated with other S.aureus isolates with similar results (data not shown).

Example 13: heart colonization with active ClfA immunization for reduction of staphylococcus aureus

Active immunization of rabbits with ClfA resulted in protection in a rabbit endocarditis model. We found animals immunized with ClfA with negative controls (PBS or AlPO)₄) The immunized animals had 3-4 orders of magnitude less staphylococcus aureus cfu recovered from cardiac neoplasms compared to the animals immunized (fig. 14).

Example 14: protective effects of MntC in vivo animal models

Active immunization with MntC has been shown to consistently protect mice from early time points after s. Bacterial counts in the blood of mice receiving i.p. s.aureus challenge were significantly reduced compared to controls immunized with PBS (fig. 15A and 15B). 4 of 6 individual studies showed a significant reduction in cfu/ml blood in the immunized animals. The protection mediated by MntC immunity was confirmed using 2 different s.aureus challenge strains PFESA0237 (fig. 15A) and PFESA0266 (fig. 15B).

Example 15: CP5 conjugate protection in a mouse model of pyelonephritis

The ability of CP5 conjugate to protect mice in an active immunization pyelonephritis model was evaluated. Figure 16 shows results from several studies. Bacterial counts in the blood of mice receiving i.p. s.aureus challenge were significantly reduced compared to controls immunized with pbs (fig. 16). 6 of the 6 individual studies showed a significant reduction in cfu/ml kidney in the immunized animals. The data show a consistent reduction in kidney colonization following active immunization with CP5 conjugate.

Example 16: CP5 conjugates prepared by different conjugation chemistries to protect mice against experimental infection

Active immunization studies in the mouse pyelonephritis model were performed using CP5 conjugates prepared by PDPH or CDT chemistry. Conjugation of CP5 or CP8 to CRM₁₉₇The method of (a) is as described above. The results show that both conjugates significantly reduced colonization in mice compared to sham-immunized animals (table 14).

Table 14: effect of PDPH on CDT conjugation in models of pyelonephritis

Example 17: CP5 conjugate protection in rat endocarditis model

With CP5-CRM₁₉₇PDPH conjugates and unrelated conjugates (PP 5-CRM)₁₉₇) 4 studies were performed at a dose of 1 μ g. In 2 out of 3 experiments in which the challenge type 5 challenge strain was PFESA0266, the CP5 conjugate significantly reduced colonization in the heart and kidney (table 15). In the third study, the Geometric Mean Titer (GMT) anti-CP 5 titer was the lowest of these 3 experiments, but it was only slightly lower than in the previous experiments (51,000 vs 67,000).

Table 15: CP5-CRM ₁₉₇ Cfu in immunodepression rat endocarditis model

Example 18: CP5-CRM in pyelonephritis model₁₉₇Conjugates

A preliminary study was conducted to investigate the efficacy of the conjugate with 25kDa MW CP 5. Improvements in the fermentation process resulted in the production of high MW polysaccharides, which were conjugated to protein carriers and tested in parallel with 25kDa CP5 conjugates. Conjugates containing CP with MW of 25kDa (low MW) and 300kDa (high MW) were prepared using CDT conjugation chemistry and evaluated in a mouse pyelonephritis model. HMW conjugates at 3 doses (0.01, 0.1 and 1. mu.g) were tested and compared to a control LMWCP5-CRM at a 1. mu.g dose₁₉₇And unrelated conjugates (PP 5-CRM)₁₉₇) And (6) comparing. The results show a significant reduction in CFU of staphylococcus aureus PFESA0266 recovered from the kidney at a1 μ g dose. There was no statistical difference between the protection from conjugates prepared with different sizes of CP5 at the 1 μ g dose (table 16). Lower doses (0.01 μ g and 0.1 μ g) of the conjugate did not elicit an immune response sufficient to significantly reduce infection. The experiment was repeated using the same immunization and challenge protocol. In repeated experiments, only 1. mu.g dose of LMWCP5-CRM₁₉₇Resulting in a significant reduction in colonization (p ═ 0.01). HMW CP5-CRM at 1. mu.g dose₁₉₇Cfu in the kidney was reduced, but the reduction was not statistically significant (p ═ 0.056).

TABLE 16 CP5 conjugate protection in mouse pyelonephritis model

Example 19: polysaccharide O-acetylation is important for inducing a protective antibody response to CP5 conjugate immunogenic formulations

To evaluate the importance of O-acetylation of CP5, native CP5 was de-O-acetylated (dOAc), andand to CRM using PDPH conjugation chemistry₁₉₇(dOAc-CRM₁₉₇). dOAcCP-CRM in mouse pyelonephritis model₁₉₇Potency of conjugate with CP5-CRM₁₉₇And (4) performing parallel comparison. The results show that the O-acetyl deficient conjugates (dOAc CP5-CRM) as demonstrated by no significant change in bacterial colonization in the kidney₁₉₇) Are not valid in this model. These data (table 17) show that O-acetylation is important for eliciting functional antibodies against CP 5.

Table 17: by using de-O-acetylated CP5-CRM ₁₉₇ Immunization does not protect mice from kidney colonization

Example 20: immunity reduction of death in sepsis model with CP 8-conjugates

Evaluation of CP8-CRM in a mouse sepsis model following challenge with Staphylococcus aureus PFESA0268 (type 8)₁₉₇Potency of the conjugate. Swiss Webster mice (n-30) were incubated with 100. mu.g of AlPO₄1 μ g CP8-CRM formulated together₁₉₇And saline for active immunization by subcutaneous injection. This study showed that AlPO alone was used₄Sepsis was significantly reduced compared to immunized mice (p ═ 0.0308). See fig. 17.

Example 21: evaluation of conjugated native and base-treated CP8 in a mouse bacteremia model

For the CP8 conjugate, the importance of the O-acetyl group present on native CP8 prior to conjugation to induce a functional antibody response was evaluated. O-acetylation of CP8 polysaccharide under mild alkaline conditions, and NMR and dissociationThe sub-chromatography (IC) proves that the removal of O-Ac-CRM from CP8₁₉₇In the absence of O-acetylation.

Mouse bacteremia model to evaluate conjugation to CRM₁₉₇The natural potency of alkali-treated CP 8. Groups of female BALB/c mice (15/group) were de-O-Ac-CRM at weeks 0,3 and 6 with 1 μ g CP8₁₉₇Or 1. mu.g of CP8O-Ac-CRM₁₉₇And (4) immunization. Immunogenic formulations with 22 μ g AlPO₄And (4) preparing the components together. Animals were challenged with s.aureus PFESA 0003. Mice were sacrificed 3 hours after challenge and bacteria in the blood were counted. The data show a statistically significant (p 0.0362) reduction in bacterial cfu recovered from blood of animals immunized with untreated native CP8 conjugate as determined by the student's t-test (table 18). In animals immunized with the alkali treated CP8 conjugate, the bacterial cfu recovered from the blood was similar to the saline control group.

Table 18: CP8-CRM ₁₉₇ Conjugate reduces bacteremia staphylococcus aureus in mice PFESA0003

Example 22: the importance of O-acetylation as a functional epitope for CP5 was demonstrated by OPA using MAbs with known specificity

OP killing activity of the CP5 monoclonal antibody with specificity for CP5OAc + (CP5-7-1), CP5OAc +/- (CP5-5-1) and CP5OAc- (CP5-6-1) against type 5 strain PFESA0266 was evaluated (Table 19). A CP8OAc + specific MAb CP8-3-1 was used as a negative control. The results show that CP5-7-1mAb (CP5OAc + specific) mediates killing of the tested type 5 strains. Also mAb CP5-5-1, recognizing an epitope shared by CP5OAc + and CP5OAc-, mediated killing of the PFESA0266 strain. MAb specific for an epitope present on CP5 OAc-polysaccharide did not mediate killing of the PFESA0266 strain. These results show that the O-acetyl epitope on CP5 is involved in the functional activity of CP5 specific antibodies.

TABLE 19O-acetylated (+) CP5 and O-and De-O-acetylated (+/-) CP5 specific mAbs are Opsonized against staphylococcus aureus PFESA0266 (type 5).

The data was reported as percent kill and was calculated by determining the ratio of the number of cfu surviving at 60 minutes in wells containing bacteria, antibody, complement and HL-60 cells to the number of cfu surviving in wells lacking antibody but containing bacteria, complement and HL-60 cells.

Example 23: induced opsonic Activity of mouse antibodies to high and Low MW CP5 conjugates

Opsonizing activity of 1 μ g of high molecular weight and low molecular weight group serum from example 18 from mice (n ═ 5) with high CP5ELISA titers was compared using staphylococcus aureus PFESA 0266. OPA results show that both conjugates elicited opsonic antibodies in mice (table 20). There is a trend observed that high MW conjugates elicit higher titers of opsonizing antibodies. Data are shown as mean% kill of serum from 5 individual mice ± SEM. Antibodies need to be functional as measured by killing bacteria in an animal efficacy model or by an opsonophagocytic killing assay that demonstrates that antibodies kill bacteria. Functional killing may not be demonstrated using an assay that only monitors the production of antibodies alone, which is not an indication of the importance of high molecular weight conjugates in efficacy.

Table 20: both LMW and HMW CP5 elicit opsonic antibodies

Example 24: opsonic Activity of sera from mice immunized with Natural and chemically modified CP8 conjugates

Mouse sera from the study in example 21 with high CP8 titers (n-5) were selected and their opsonic activity compared using PFESA0005 strain. OPA results (table 21) show that opsonized antibodies in mice were elicited by conjugates prepared only by conjugation with native CP 8. It is noteworthy that the deoac CP8 conjugate was immunogenic in mice, but the elicited antibody was not opsonized in this assay. OPA titers are reported as the reciprocal of the dilution at which 40% killing was observed. Antibodies need to be functional as measured by killing bacteria in an animal efficacy model or by an opsonophagocytic killing assay that demonstrates that antibodies kill bacteria. Functional killing may not be demonstrated using an assay that only monitors antibody production alone, which is not an indication of the importance of O-acetylation in efficacy.

TABLE 21 Natural CP8 vs. de-O-Ac CP8-CRM ₁₉₇ Of (2) a conditioning activity

Example 25: addition of natural CP8 to inhibit CP8 conjugate non-human primate antiserum kills type 8 strains

To demonstrate the specificity of killing activity in the serum of non-human primates immunized with CP 8-conjugate, assays were performed in the presence of native CP 8. OP method II was used with the following modifications. Antibody samples (25. mu.l) were prepared in 2-fold serial dilutions, and then 150. mu.l (Pn14 competitor) or 125. mu.l (CP8 competitor) of assay buffer was added to the antibody suspension. The competitor was purified CP8 polysaccharide (CP8 mer), and the unrelated pneumococcal polysaccharide (Pn14 mer) was used as a control. The polysaccharide was added (50 μ g) to the antibody suspension and the plate incubated at 4 ℃ for 30 minutes with mixing reversed. After incubation with polysaccharide, bacteria were added (25 μ l) to the plate and placed on a rotary shaker at 4 ℃ for 30 minutes, followed by addition of 25 μ l of human complement (1% final concentration). The results (table 22) show that the presence of native CP8 in the reaction mixture inhibited opsonophagocytic killing of staphylococcus aureus type 8. These results demonstrate that opsonophagocytic killing of immune sera is mediated by capsule-specific abs.

TABLE 22 addition of CP8 polysaccharide inhibits opsonophagocytosis of immune serum to kill Staphylococcus aureus.

Example 26: naturally-obtained antibody-mediated opsonophagocytosis of ClfA to kill staphylococcus aureus

Humans in the population are naturally exposed to staphylococcus aureus and therefore contain pre-existing antibodies to this bacterium in their blood circulation. We affinity purified an anti-ClfA antibody from human serum and evaluated whether the antibody could mediate opsonic killing. Antibodies to ClfA were confirmed to be opsonic for staphylococcus aureus capsular polysaccharide (data not shown). The strain PFESA0266 was grown overnight in Columbia broth containing 2% NaCl. Bacteria were opsonized with ClfA affinity purified human IgG or irrelevant antigen affinity purified human IgG (negative control, streptococcal SCP protein) and opsonizing activity was detected. Differentiated HL-60 cells were used in an opsonophagocytosis assay at an effector cell/target ratio of 100: 1. As an additional control, CP5mAb was included in this experiment to confirm that CP5 was present on the surface. Results are the average of two independent experiments. Antibodies specific for ClfA and CP5 did mediate opsonic killing, whereas SCP-specific (negative control) antibodies were not active in this assay.

Example 27: CP5-CRM₁₉₇Conjugate elicited opsonic antibodies in non-human primates (NHPs)

For higher molecular weight versus lower molecular weight CP5-CRM₁₉₇Functionality of the conjugate in NHP, groups of 5 monkeys were dosed with 2 and 20 μ g with or without AlPO₄Conjugate immunization with adjuvant. Monkeys received a first and second immunization on day 0 and day 28, respectively. The OP activity of blood from days 0, 14, 28 and 42 was measured. The results are summarized in Table 23. The 20 μ g HMW conjugate with the highest OP titer was compared to the other groups. Moreover, OP positive monkeys were more frequent in both doses of the high MW group than in the corresponding low MW group. These results demonstrate a trend towards HMW CP5-CRM in NHP₁₉₇The conjugate elicited a better OP response than the LMW CP5 conjugate.

Table 23 OPA of NHP sera after immunization with CP5 conjugate.

Example 28: capsular polysaccharide conjugates comprising high molecular weight polysaccharides exhibit enhanced immunogenicity as compared to conjugates comprising low molecular weight polysaccharides

Non-human primate (NHP) studies were performed to evaluate the differencesImmunogenicity of capsular conjugate formulations. Two formulations were tested at two different dose levels (2 and 20 μ g). The first formulation consists of conjugation to CRM₁₉₇Of High Molecular Weight (HMW) polysaccharide (about 130 kDa). The second formulation contained a conjugate to CRM₁₉₇Low Molecular Weight (LMW) polysaccharide (about 25 kDa). Groups of 5 primates were immunized with a single dose of either vaccine and the immune titers were monitored prior to immunization and 2 weeks post-immunization. OPA titer is defined as the dilution of serum required to kill 40% of the s.aureus strain PFESA0266 in the OPA assay. Antibody titers were also monitored by ELISA. Enhanced activity of HMW vaccine compared to LMW formulation was observed (table 24), as evidenced by a 10-fold increase in antibody titer of HMW vaccine compared to LMW vaccine. The OPA effector rate of NHP receiving HMW vaccine was also higher (80% vs 40%).

TABLE 24 increase in HMW polysaccharide conjugate vaccine compared to LMW polysaccharide conjugate vaccine was observed Strong immunogenicity.

Example 29: double antigens in non-human primates (CP 5-CRM)₁₉₇And ClfA) preparation-antibody response

To evaluate the immunogenicity of a single dose of two antigen immunogenic compositions (CP 5-CRM) in NHP₁₉₇And ClfA), groups of 5 monkeys were given different doses of no AlPO₄The two antigens of (1) are immunized. Opsonophagocytosis (OP) activity was tested for blood from days 0, 14 and 28, and ELISA titers and results are summarized in table 24. The results show that OP activity was consistently observed in animals immunized with CP5 compared to the CP5 sham group. Overall, the 100 μ g group had the highest ELISA and OP titers compared to the other groups. No OP killing activity was observed in sera from the ClfA group alone. No interference was observed in the group administered increasing doses of ClfA or CP 5. See table 25.

Table 25: to comeOPA results from bivalent immunization studies in NHP

Animal models demonstrate the potential of the staphylococcus aureus CP5 and CP8 capsular polysaccharide antigens

CP5-CRM₁₉₇And CP8-CRM₁₉₇The conjugates all induced capsular serotype specific antibody responses in mice, rats, rabbits and non-human primates (NHPs). The conjugate-induced antibodies were functional in an in vitro functional opsonophagocytic killing assay. Data were generated to demonstrate that O-acetylation is important for eliciting protective antibodies to both CP5 and CP8, and that O-acetyl is OPA against CP5⁺Part of the epitope recognized by the mAb. Mabs recognizing O-acetylated native CP5 function in OPA and mediate killing of bacteria. The CP8 conjugate induces functional antibodies in mice and rabbits that mediate killing of type 8 strains in OPA. Specificity of polyclonal or monoclonal antibody killing was demonstrated by abolishing killing after addition of the homologous native polysaccharide to the assay. Various active immunization models were used to show CP 5-and CP8-CRM₁₉₇Preclinical efficacy of the conjugate. The CP5 conjugate showed consistent efficacy in the mouse pyelonephritis model and the rat endocarditis model. The importance of O-acetylation of CP5 was demonstrated in a mouse pyelonephritis model, in which conjugation to CRM was performed₁₉₇The de-O-acetylated CP5 failed to protect the animals against experimental infections.

The combination of conjugates in the dual antigen formulation induced antibodies to capsular CP5 and CP8 and did not interfere with the levels of specific antibodies induced as compared to single antigen immunization. The combination of the conjugate in the triantigen preparation with ClfA induced high CP5, CP8, and ClfA levels, and did not interfere with the induced antibody responses against any of the antigens present in the combination. The tri-antigen immunogenic composition induces an antibody (Ab) response in rabbits with high pre-immune titers, wherein the antibody (Ab) response to all 3 components is capable of boosting.

These results indicate conjugation to CRM₁₉₇CP5 and CP8 should be included as immunogenic formulation components of a protective staphylococcus aureus immunogenic composition.

Example 30: different antigens are required to prevent a wide range of possible S.aureus diseases

Staphylococcus aureus causes a wide range of infections, ranging from relatively mild skin infections to more severe and invasive infections such as endocarditis, necrotizing fasciitis, osteomyelitis, septic arthritis and pneumonia. Each of these in vivo sites is unique and bacteria may respond to differences in environmental stimuli by changing their antigen expression profile to that which is best suited for individual strains to colonize, grow and ultimately cause disease. As shown in example 12, staphylococcus aureus strains exhibited a diversity of antigen expression in vivo. Multicomponent immunogenic compositions consisting of different antigens are more likely to protect against various disease manifestations caused by staphylococcus aureus.

ClfA was demonstrated to protect in rodent endocarditis and sepsis models. ClfB has been reported to be important in nasal colonization of staphylococcus aureus. MntC comprises mice in a mouse bacteremia model. CP5 conjugate was protected in pyelonephritis and endocarditis, and CP8 conjugate was protected in rodent models of pyelonephritis and sepsis. These results demonstrate that multicomponent vaccines containing these antigens will protect against multiple types of S.aureus disease.

In vivo animal models approximate the course of actual infection and help in exploring which antigens may be beneficial in protecting against a particular disease. Table 26 summarizes the results from a number of experiments performed in various in vivo models. The results are reported in each block as 4 numbers separated by a slash, e.g. ClfA in the sepsis model has the number 27/1/3/31. The first number represents the number of experiments in which ClfA immunization produced statistically significant positive protection results. The second number represents the number of experiments in which ClfA immunization produced positive protection results that tended to be significant, but not statistically significant. The third number represents the number of experiments in which ClfA immunization produced negative results, but the negative results were not statistically significant. The fourth number is the total number of experiments performed. The first three numbers should add up to equal the fourth number.

TABLE 26 summary of Staphylococcus aureus antigen protection in animal models

	CIfA	CP5	CP8	MntC20
					Bacteremia	1/4/0/5	3/0/3/6	1/1/1/3	6/2/5/13
Sepsis	27/1/3/31	1/0/0/1	NT	NT
					Pyelonephritis	0/4/2/6	13/1/0/14	NT	1/0/4/525
Endocarditis of heart	3/6/1/10	3/2/2/7	NT	NT

NT: not detected

Example 31: various multi-antigen immunogenic compositions are tested in vitro and in vivo.

The immunogenicity and efficacy of various multi-antigen staphylococcal immunogenic formulations comprising 3, 4 or 5 antigens selected from the following polypeptides and/or polysaccharides were tested in various in vivo models: ClfA, ClfB, MntC, CP 5-and CP 8. The immunogenic composition is as follows:

(1) an immunogenic composition comprising: isolated Staphylococcus aureus aggregation factor A (ClfA) polypeptide, isolated conjugated to CRM₁₉₇S. aureus type 5 capsular polysaccharide and isolated conjugation to CRM₁₉₇S. aureus type 8 capsular polysaccharide;

(2) the second combination provides aggregation factor A (ClfA), aggregation factor B (ClfB), isolated MntC, isolated conjugation to CRM₁₉₇And an isolated conjugate to CRM₁₉₇Staphylococcus capsular polysaccharide CP 8;

(3) a third combination provides an immunogenic composition comprising: isolated staphylococcus aureus aggregation factor a (clfa) polypeptide, isolated staphylococcus aureus aggregation factor b (clfb) polypeptide, or isolated staphylococcus aureus MntC protein, isolated conjugated to CRM₁₉₇S. aureus type 5 capsular polysaccharide, and isolated conjugation to CRM₁₉₇S. aureus type 8 capsular polysaccharide; separated from each other

(4) A fourth combination provides an immunogenic composition comprising: isolated Staphylococcus aureus aggregation factor B (ClfB) polypeptide, isolated conjugated to CRM₁₉₇S. aureus type 5 capsular polysaccharide and isolated conjugation to CRM₁₉₇S. aureus type 8 capsular polysaccharide;

(5) a fifth combination provides an immunogenic composition comprising: isolated staphylococcus aureus clumping factor b (clfb) polypeptide, isolated staphylococcus aureus MntC protein, isolated conjugated to CRM₁₉₇S. aureus type 5 capsular polysaccharide and isolated conjugation to CRM₁₉₇S. aureus type 8 capsular polysaccharide; and

(6) a sixth combination provides an immunogenic composition comprising: an isolated staphylococcus aureus aggregation factor a (clfa) polypeptide, an isolated staphylococcus aureus aggregation factor b (clfb) polypeptide, and an isolated staphylococcus aureus MntC protein.

rClfA and rClfB were prepared and purified as described in example 1. MntC was prepared and purified as described in example 2. Isolated CP5 and CP8 were prepared and purified as described in example 3, and conjugated to CRM as described in example 4₁₉₇。

More particularly, the methods described in the previous examples above were used to measure immunogenicity and potency. A study was conducted to determine whether each of the 3, 4 or 5 components induced an immune response when delivered separately or together. These same studies were used to determine whether the presence of any of the 4 or 5 components interfered with the ability of any of the other 3 or 4 components to induce an immune response. In addition, studies were conducted to determine whether the 4 or 5 components, when tested alone or together, would confer protection in any one or more of the animal models described above. As described in the previous examples above, the 4 or 5 components are administered to an animal, e.g., a mouse, rat, rabbit or non-human primate, as a single dose or as multiple doses. The animals were bled, sera collected, and tested for the presence of antibodies for each of the 4 or 5 fractions. The presence of antigen-specific antibodies is measured by any immunoassay known to those skilled in the art, e.g., ELISA (see examples 11-29) or western blot (see example 1) is used to assess the presence or absence of antigen-specific antibodies. In addition, opsonophagocytosis assays are used to determine whether antigen-specific antibodies are effective in mediating phagocytic cell killing of staphylococcal organisms (see examples 11-29).

In vivo efficacy is also assessed using any one or more of the animal studies described above, such as, but not limited to, an indwelling catheter model; a mouse bacteremia model; a wound infection model; mouse pyelonephritis model; rat endocarditis models and mouse sepsis models (see examples 11-30).

Example 32: the combination of staphylococcus aureus antigens produces antibodies in non-human primates that promote killing of staphylococcus aureus strain Pfe 5-1.

Enhanced efficacy was observed with the combination of antigens as measured with the OPA assay. A non-human primate study was performed in which groups of 3-10 monkeys were immunized with the multicomponent vaccine. Animals received a single dose of vaccine and OPA titers were monitored on day 0 and 2 weeks post-immunization. The OPA titer is defined as the dilution of serum required to kill 50% of staphylococcus aureus strain Pfe5-1 in the OPA assay. Enhanced activity was observed for the combination of 4 antigens compared to the 3-antigen vaccine formulation (p ═ 0.0272; fig. 18).

Claims (90)

1. An immunogenic composition comprising at least 3 components selected from the group consisting of: an isolated staphylococcus aureus (s.aureus) aggrekine a (clfa) polypeptide, an isolated staphylococcus aureus aggrekine b (clfb) polypeptide, an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein.

2. An immunogenic composition comprising: an isolated staphylococcus aureus clump factor a (clfa) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein.

3. The immunogenic composition of claim 2, further comprising an isolated Staphylococcus aureus aggregating factor B (ClfB) polypeptide.

4. The immunogenic composition of claim 2 or 3, further comprising an isolated Staphylococcus aureus MntC protein.

5. The immunogenic composition of any one of claims 1-4, wherein the ClfA or ClfB polypeptide is a polypeptide fragment comprising the fibrinogen binding domain of ClfA or ClfB.

6. The immunogenic composition of claim 5, wherein the ClfA or ClfB polypeptide fragment is a fibrinogen binding domain comprising the N1, N2, and N3 domains of ClfA or ClfB.

7. The immunogenic composition of claim 5, wherein the ClfA or ClfB polypeptide fragment is a fibrinogen binding domain comprising the N2 and N3 domains of ClfA or ClfB.

8. The immunogenic composition of any one of claims 5, 6, or 7, wherein the fibrinogen binding domain of ClfA binds to fibrinogen at a reduced level compared to the observed binding to fibrinogen by the native fibrinogen binding domain of ClfA.

9. The immunogenic composition of claim 8, wherein the fibrinogen binding domain of ClfA binds to fibrinogen at a reduced level compared to the observed binding to fibrinogen by the native fibrinogen binding domain of ClfA by having an amino acid substitution at one or more of Tyr 338, Tyr256, Pro 336, Lys 389, Ala 254, and Ile 387.

10. The immunogenic composition of claim 9, wherein the amino acid substitution at one or more of Tyr 338, Tyr256, Pro 336, Lys 389, Ala 254, and Ile 387 is to Ala or Ser.

11. The immunogenic composition of claim 10, wherein said Tyr 338 is substituted with Ala.

12. The immunogenic composition of any one of claims 1-11, wherein said ClfA, said ClfB, or MntC are recombinantly produced.

13. The immunogenic composition of any one of claims 1-12 wherein the type 5 capsular polysaccharide is a high molecular weight capsular polysaccharide of 20-1000 kDa.

14. The immunogenic composition according to claim 13, wherein the high molecular weight type 5 capsular polysaccharide has a molecular weight of 70-300 kDa.

15. The immunogenic composition of any one of claims 1-14, wherein the type 5 capsular polysaccharide is 10% -100% O-acetylated.

16. The immunogenic composition of any one of claims 1-14, wherein the type 5 capsular polysaccharide is 50-100% O-acetylated.

17. The immunogenic composition of any one of claims 1-14, wherein the type 5 capsular polysaccharide is 75% -100% O-acetylated.

18. The immunogenic composition of any one of claims 1-17 wherein the type 8 capsular polysaccharide is a high molecular weight capsular polysaccharide of 20-1000 kDa.

19. The immunogenic composition of claim 18 wherein the high molecular weight type 8 capsular polysaccharide has a molecular weight of 70-300 kDa.

20. The immunogenic composition of any one of claims 1-19, wherein the type 8 capsular polysaccharide is 10% -100% O-acetylated.

21. The immunogenic composition of any one of claims 1-19, wherein the type 8 capsular polysaccharide is 50-100% O-acetylated.

22. The immunogenic composition of any one of claims 1-19, wherein the type 8 capsular polysaccharide is 75% -100% O-acetylated.

23. The immunogenic composition of any one of claims 1-22, wherein the carrier protein is a corynebacterium diphtheriae (c.diphtheria) toxoid CRM 197.

24. The immunogenic composition of any one of claims 1 or 4-23, wherein the MntC protein of Staphylococcus aureus is a lipidated protein.

25. The immunogenic composition of any one of claims 1 or 4-23, wherein the MntC protein of Staphylococcus aureus is not a lipidated protein.

26. An immunogenic composition comprising: an isolated staphylococcus aureus clump factor b (clfb) polypeptide, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein.

27. The immunogenic composition of claim 23, further comprising an isolated staphylococcus aureus MntC protein.

28. The immunogenic composition of claim 26 or 27, wherein the ClfB polypeptide is a polypeptide fragment comprising the fibrinogen binding domain of ClfB.

29. The immunogenic composition of claim 28, wherein the ClfB polypeptide fragment is a fibrinogen binding domain comprising the N1, N2, and N3 domains of ClfB.

30. The immunogenic composition of claim 28, wherein the ClfB polypeptide fragment is a fibrinogen binding domain comprising N2 and N3 domains of ClfB.

31. The immunogenic composition of any one of claims 28, 29, and 30, wherein the fibrinogen binding domain of ClfB binds to fibrinogen at a reduced level as compared to the observed binding to fibrinogen by the native fibrinogen binding domain of ClfB.

32. The composition of any one of claims 26-31, wherein said ClfB or said MntC protein is recombinantly produced.

33. The immunogenic composition of any one of claims 26-32 wherein the type 5 capsular polysaccharide is a high molecular weight capsular polysaccharide of 20-1000 kDa.

34. The immunogenic composition of any one of claims 26-32 wherein the high molecular weight type 5 capsular polysaccharide has a molecular weight of 70-300 kDa.

35. The immunogenic composition of any one of claims 26-34, wherein the type 5 capsular polysaccharide is 10% -100% O-acetylated.

36. The immunogenic composition of any one of claims 26-34, wherein the type 5 capsular polysaccharide is 50-100% O-acetylated.

37. The immunogenic composition of any one of claims 26-34, wherein the type 5 capsular polysaccharide is 75% -100% O-acetylated.

38. The immunogenic composition of any one of claims 26-37 wherein the type 8 capsular polysaccharide has a molecular weight of 20-1000 kDa.

39. The immunogenic composition of any one of claims 26-37 wherein the type 8 capsular polysaccharide has a molecular weight of 70-300 kDa.

40. The immunogenic composition of any one of claims 26-39, wherein the type 8 capsular polysaccharide is 10% -100% O-acetylated.

41. The immunogenic composition of any one of claims 26-39, wherein the type 8 capsular polysaccharide is 50-100% O-acetylated.

42. The immunogenic composition of any one of claims 26-39, wherein the type 8 capsular polysaccharide is 75% -100% O-acetylated.

43. The immunogenic composition of any one of claims 27-42, wherein the Staphylococcus aureus MntC is a lipidated protein.

44. The immunogenic composition of any one of claims 27-42, wherein the Staphylococcus aureus MntC is not a lipidated protein.

45. The immunogenic composition of any one of claims 26-44, wherein the carrier protein is Corynebacterium diphtheriae CRM 197.

46. An immunogenic composition comprising: an isolated staphylococcus aureus aggregation factor a (clfa) polypeptide, an isolated staphylococcus aureus aggregation factor b (clfb) polypeptide, and an isolated staphylococcus aureus MntC protein.

47. The immunogenic composition of claim 46, wherein the ClfA polypeptide is a polypeptide fragment comprising the fibrinogen binding domain of ClfA.

48. The immunogenic composition of claim 47, wherein the ClfA polypeptide fragment is a fibrinogen binding domain comprising the N1, N2, and N3 domains of ClfA.

49. The immunogenic composition of claim 47, wherein the ClfA polypeptide fragment is a fibrinogen binding domain comprising the N2 and N3 domains of ClfA.

50. The immunogenic composition of any one of claims 47, 48, and 49, wherein the fibrinogen binding domain of ClfA binds to fibrinogen at a reduced level compared to the observed binding to fibrinogen by the native fibrinogen binding domain of ClfA.

51. The immunogenic composition of claim 50, wherein the fibrinogen binding domain exhibits reduced binding to fibrinogen by having an amino acid substitution at one or more of Tyr 338, Tyr256, Pro 336, Lys 389, Ala 254, and Ile 387.

52. The immunogenic composition of claim 51, wherein the amino acid substitution at one or more of Tyr 338, Tyr256, Pro 336, Lys 389, Ala 254, and Ile 387 is a substitution to Ala or Ser.

53. The immunogenic composition according to claim 52, wherein said Tyr 338 is substituted with Ala.

54. The immunogenic composition of any one of claims 46-53 wherein the Staphylococcus aureus ClfB polypeptide is a polypeptide fragment comprising the fibrinogen binding domain of ClfB.

55. The immunogenic composition of claim 54, wherein the Staphylococcus aureus ClfB polypeptide fragment is a fibrinogen binding domain comprising the N1, N2, and N3 domains of ClfB.

56. The immunogenic composition of claim 54, wherein the Staphylococcus aureus ClfB polypeptide fragment is a fibrinogen binding domain comprising the N2 and N3 domains of ClfB.

57. The immunogenic composition of any one of claims 46-56, wherein the Staphylococcus aureus MntC is a lipidated protein.

58. The immunogenic composition of any one of claims 46-56, wherein the Staphylococcus aureus MntC is not a lipidated protein.

59. An immunogenic composition comprising: an isolated staphylococcus aureus MntC protein, an isolated staphylococcus aureus type 5 capsular polysaccharide conjugated to a carrier protein, and an isolated staphylococcus aureus type 8 capsular polysaccharide conjugated to a carrier protein.

60. The composition of claim 59, wherein the MntC protein is recombinantly produced.

61. The immunogenic composition of claim 59 or 60 wherein the type 5 capsular polysaccharide is a high molecular weight capsular polysaccharide of 20-1000 kDa.

62. The immunogenic composition of any one of claims 59-61 wherein the high molecular weight type 5 capsular polysaccharide has a molecular weight of 70-300 kDa.

63. The immunogenic composition of any one of claims 59-62, wherein the type 5 capsular polysaccharide is 10% -100% O-acetylated.

64. The immunogenic composition of any one of claims 59-62, wherein the type 5 capsular polysaccharide is 50-100% O-acetylated.

65. The immunogenic composition of any one of claims 59-62, wherein the type 5 capsular polysaccharide is 75% -100% O-acetylated

66. The immunogenic composition of any one of claims 59-65, wherein the type 8 capsular polysaccharide has a molecular weight of 20-1000 kDa.

67. The immunogenic composition of any one of claims 59-65, wherein the type 8 capsular polysaccharide has a molecular weight of 70-300 kDa.

68. The immunogenic composition of any one of claims 59-67, wherein the type 8 capsular polysaccharide is 10% -100% O-acetylated.

69. The immunogenic composition of any one of claims 59-67, wherein the type 8 capsular polysaccharide is 50% -100% O-acetylated.

70. The immunogenic composition of any one of claims 59-67, wherein the type 8 capsular polysaccharide is 75% -100% O-acetylated.

71. The immunogenic composition of any one of claims 1-70, further comprising at least one protein from the serine-aspartate repeat (Sdr) protein family selected from the group consisting of SdrC, SdrD, and SdrE.

72. The immunogenic composition of any one of claims 1-71, further comprising an iron surface determinant B (IsdB) protein.

73. The immunogenic composition of any one of claims 1-72, further comprising an adjuvant.

74. The immunogenic composition of any one of claims 1-73, further comprising a pharmaceutically acceptable carrier.

75. The immunogenic composition of any one of claims 1-74, further comprising any one of the following antigens: opp3a, DltD, HtsA, Ltas, IsdA, IsdC, SdrF, SdrG, SdrH, SrtA, SpA, Sbi FmtB, alpha-hemolysin (hla), beta-hemolysin, fibronectin binding protein A (fnbA), fibronectin binding protein B (fnbB), coagulase, FIG, map, Panton-Valentine leukocidin (pvl), alpha-toxin and variants thereof, gamma toxin (hlg) and variants thereof, ica, immunodominant ABC transporter, Mg2+ transporter, Ni ABC transporter, RAP, autolysin, laminin receptor, IsaA/PisA, IsaB/PisB, SPOIE, SsaA, EbpS, SasA, CnsF, CsH, SBB (EFB), SBI, Npase, EBP, SatSUI, SfSATSII binding protein, AUTSII binding protein, PGA-S-P, PNAS-P46 55, PNA, PNAS, GehD, EbhA, EbhB, SSP-1, SSP-2, HBP, vitronectin binding protein, HarA, EsxA, EsxB, enterotoxin A, enterotoxin B, enterotoxin C1, and neoautolysin.

76. A method of inducing an immune response against Staphylococcus aureus comprising administering to a subject an immunologically effective amount of the immunogenic composition of any one of claims 1-75.

77. The method of claim 76, wherein the immune response prevents or reduces a disease or disorder associated with a staphylococcal organism in the subject or prevents or reduces one or more symptoms associated with the staphylococcal organism in the subject.

78. The method of claim 77, wherein the disease is selected from the group consisting of invasive Staphylococcus aureus, sepsis, and carryover.

79. The method of claim 76, wherein the subject is undergoing a surgical procedure.

80. The method of claim 79, wherein the surgery is elective surgery or non-elective surgery.

81. The method of claim 79, wherein the surgery is cardiothoracic surgery.

82. The method of claim 76, wherein the induced immune response comprises production of antibodies with opsonophagocytic activity (OPA) against Staphylococcus aureus.

83. The method of claim 76, wherein the induced immune response comprises production of a significantly higher titer of Staphylococcus aureus-specific opsonophagocytic antibodies than observed in an unimmunized subject.

84. The method of claim 83, wherein the opsonophagocytic titer is at least 1: 20.

85. The method of any one of claims 76-84, wherein the Staphylococcus aureus is MSSA.

86. The method of any one of claims 76-84, wherein the Staphylococcus aureus is VRSA.

87. The method of any one of claims 76-84, wherein said Staphylococcus aureus is VISA.

88. The method of any one of claims 76-84, wherein the Staphylococcus aureus is MRSA.

89. The method of any one of claims 76-88, wherein the subject is a human, a domestic pet, or a livestock.

90. A method of conferring passive immunity to a subject, the method comprising the steps of: (1) producing an antibody preparation using the immunogenic composition of any one of claims 1-75; and (2) administering the antibody preparation to the subject to confer passive immunity.