FIELD OF THE INVENTION The present invention generally relates to immunogenic polypeptides associated with Staphylococci. More particularly, the invention relates to membrane-bound immunogenic polypeptides and to nucleic acids encoding said immunogenic polypeptides.

The invention also relates to a general method for isolating these immunogenic polypeptides in substantially pure form, and to uses of said immunogenic polypeptides for the production of antibodies effective against Staphylococci infections. Further, the invention relates to diagnostic probes for Staphylococci.

BACKGROUND OF THE INVENTION Staphylococci make up a medically important genera of microbes. They are known to produce invasive and toxigenic diseases. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues.

Staphylococcus aureus (hereinafter"S. aureus"), in particular, is an important human pathogen that is capable of causing a wide spectrum of infections and diseases. Infections can range from minor (e. g.,. carbuncles) to severe (e. g.,. septicemia, endocarditis, and osteomyelitis). The diversity and severity of S. aureus infections can be attributed to this microorganism's ability to produce numerous cell surface-localized proteins that bind host tissues (e. g. , fibronectin binding protein and collagen binding protein) as well as several<BR> different extracellular toxins (e. g. , toxic shock syndrome toxin 1 and alpha-hemolysin).

S. aureus is currently a major threat to the quality of health care because of its resistance to many antibiotics and its frequent association with hospitalized patients, especially those who have undergone surgical procedures. S. aureus has a propensity to invade skin and

adjacent tissues at the site of prosthetic medical devices, including intravascular catheters, cerebrospinal fluid shunts, hemodialysis shunts and vascular grafts.

The frequency of S. aureus infections has risen dramatically in the past 20 years.

Since the 1940s, S. aureus infections were successfully treated with penicillin. Antibiotic resistant strains emerged a decade later. Methicillin was developed in the 1960s; by 1968, the first cases of methicillin-resistant Staphylococci were reported. By 1997,50 percent of S. aureus infections contracted by hospital patients were antibiotic resistant, compared with just 2 percent, 25 years earlier. At the same time, according to the Centers for Disease Control and Prevention, drug-resistant infections, once largely confined to hospitals and nursing homes, started spreading to communities. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate S. aureus strains that are resistant to some or all of the standard antibiotics. The potent antibiotic, vancomycin has offered a reliable last defense against the most virulent bacteria.

However, in recent years, there has been an increasing number of reports of bacterial resistant to this drug (Gordon, 2001). It is commonly believed that bacterial and fungal resistance to antibiotics is caused by injudicious use of antimicrobial agents.

In 1975, Kohler and Milstein reported that certain mouse cell lines could be fused with mouse spleen cells to create hybridomas that would secrete pure monoclonal antibodies. With the advent of this technology, the potential existed to produce murine antibodies to any particular determinant or determinants on antigenic substances. The ability to specifically bind biologically important determinants demonstrates the potential for monoclonal antibodies use as therapeutic agents. The advantages of monoclonal antibody therapeutics-over conventional pharmaceuticals include their extremely high selectivity, multiple effector functions, and ease of molecular manipulation such as radioisotope labeling and other types of conjugation.

One therapeutic application of monoclonal antibodies is passive immunotherapy, in which the exogenously produced immunoglobulins are administered directly to the animal being treated by injection or by ingestion. Because passive immunotherapy does not rely on an immune response in the animal being treated, successful passive immunotherapy must deliver an appropriate amount of antibodies to the animal. The immunoglobulins administered must be specific for the pathogen or the undesirable target.

One advantage of passive immunotherapy is the speed with which the antibody can be contacted with the target, compared to a normal immune response. Passive immunotherapy can also be used prophylactically to prevent the onset of diseases or infections.

The seemingly unlimited number of serious diseases caused by S. aureus, not to mention its increasing resistance to available antibiotics, has re-ignited interest in vaccines and in passive immunization with human antibodies (Kelly, J. 2000). The discovery of serologically distinct capsular polysaccharides on the surface of clinical isolates has renewed the prospects for development of vaccines and passive-protective immunity against S. aureus infections. Capsular polysaccharide conjugate vaccines have now been produced and proven to be safe and immunogenic in both healthy and a significant percentage of immunocompromised patients. Antibodies generated in humans against these vaccines have been shown to mediate type-specific opsonophagocytosis, and to protect animals against lethal challenge with the appropriate S. aureus isolate (Fattom and Nasso 1996).

Nemeth and Lee (1995) studied the protective efficacy of antibodies to S. aureus capsular polysaccharide in a rat model of catheter-induced endocarditis. Capsular antibodies were induced either by active immunization with killed S. aureus or by passive immunization with hyperimmune rabbit antiserum to S. aureus. Their findings, however, indicated that antibodies to S. aureus capsular polysaccharides are not protective in the rat model of experimental S. aureus endocarditis.

Besides capsular polysaccharides as vaccine candidates, human immunoglobulins pooled from patients suffering from natural infections, and vaccines based on whole bacteria have all lead to poor antibody responses. However, recent research using animal models of several staphylococcal diseases reveals that vaccines based on recombinant staphylococcal extracellular-matrix-binding proteins are much more protective. For example, passive immunization with antibodies against collagen-binding proteins shows promise in a mouse model of sepsis (Flock et al., 1999).

Iron is an essential nutrient for the proliferation of bacteria in vivo, but is virtually unavailable (the concentration of bio-available iron is approximately 10-18 M) in avian, animal or mammalian tissues because the iron is either intracellular or extracellularlly complexed with high affinity, iron-binding proteins (Crosa 1997). To circumvent these

restrictive conditions, pathogenic bacteria and fungi have evolved high affinity iron- transport systems that are produced in iron-deficient media. Iron-transport systems consist of specific ferric iron chelators, siderophores, and iron-regulated outer membrane proteins and/or siderophore receptor proteins. Siderophore-receptor proteins are on the outer membrane of the bacterial cell (Neilands, 1983). Many of the factors associated with viability in iron-depleted environments have been linked to the virulence of these pathogens. (Crosa, 1997; Flock, 1999; Kelly 2000). However, the secretion and transport of iron-binding ligands in gram-positive organisms are poorly understood.

At least three different siderophores have been identified in Staphylococcal strains.

Staphylococcus hyicus has been shown to produce at least two siderophores, staphyloferrins A and B (Courcol et al., 1997). Both staphyloferrins A and B were also <BR> <BR> found to be produced by certain strains of S. aureus (Courcol et al. , 1997) identified a third<BR> siderophore produced by S. aureus called aureochelin (Modun et al. , 2000), described the binding of human SEIP by intact S. aureus cells and identified a 42-kDa protein that is presumably responsible for this binding. In addition, several proteins of unknown functions have been shown to be regulated by low free iron concentration in strains of S. aureus. These include proteins with apparent molecular masses of 120 kDa, 88 kDa, 57 kDa, 35 kDa, and 33 kDa and of 36 kDa and 39 kDa, all of which were repressed by high iron concentrations (Courcol et al. , 1997).

Recently, Staphylococcus ATCC 6538 was examined for production of siderophore and expression of SEIP-binding protein (SA-tbp) in normal or deferrated brain heart infusion broth (BHI). Siderophore production was earlier and greater in the deferrated BHI. The SA-tbp, detected by ligand blot assay, was expressed only in the deferrated BHI. When human SEIP was added to the deferrated BHI, siderophore production was later and lower than when SEIP was not present. Both iron-acquisition mechanisms of S. aureus, namely the production of siderophore or expression of SEIPg- binding protein, were found to be iron-repressible. Both mechanisms were responsible for utilization of human SEIP-bound iron for growth under iron-depleted condition (Modun et <BR> <BR> al. , 2000).<BR> <P>In a separate study, Lisiecki et al. , (1997), demonstrated that S. aureus clinical and environmental isolates differ by siderophore production. Systemic and local staphylococcal infections were induced in mice by inoculation of three S. aureus strains

differing by siderophore production. It was determined that the clinical S. aureus strain B 47 characterized by enhanced siderophore activity, was more virulent in both systemic and local infection models. It was also more resistant to anti-bacterial activity of neutrophils than environmental S. aureus strains B 63 and B 32 expressing weaker siderophore production. The results suggest that siderophore-dependent iron acquisition system may be crucial to S. aureus strains in their pathogenic activity.

The strategies disclosed in the art, provide methods for the isolation of iron- regulated membrane associated polypeptides, in gram-negative bacteria and a few fungal species. For example, US Patents 5, 830, 479 (Emery) and 6,027, 736 (Emery) disclose a method for isolating high quantities of immunogenically effective siderophore receptor proteins from outer membranes of a single strain or species of gram-negative bacteria such as E. coli, Salmonella and/or Pasteurella. Antisera produced using these antigens were effectively cross-reactive with some other gram-negative bacteria. However, these hyperimmune antisera were not cross-reactive with gram-positive bacteria.

For example, Shagam et al. , 1988, studied S. aureus aqueous extract and K. pneumoniae hydroxylamine vaccine by means of chemical and immunochemical analytical techniques. The preparations were found to contain, respectively, 7.0% and <BR> <BR> 53.5% of neutral monosaccharides, 6.5% and 0.7% of nucleic acids, as well as protein in approximately equal amounts (11.63%-14. 0%). In experiments of immunodiffusion, immunoelectrophoresis and rocket immunoelectrophoresis in homologous systems with hyperimmune antimicrobial sera, the preparations were characterized by serological heterogeneity. After their combination with Escherichia coli aqueous extract and Proteus hydroxylamine preparation, their serological characteristics remained unchanged. The study of cross reactions of all components of the combined preparations with hyperimmune rabbit sera to the corresponding microorganisms revealed that only Klebsiella component of the combined vaccine reacted with all hyperimmune sera. The preparation of Proteus showed the lowest activity. It reacted only with hyperimmune sera to K. pneumoniae. No reaction of S. aureus component with sera to E. coli nor reaction of the preparation of E. coli with anti-staphylococcal serum were observed.

The continued incidence and severity of infections, the continual emergence of antibiotic resistant bacterial strains, and the inherent toxicity of some antibiotics are just some of the reasons that alternative prophylactic and therapeutic approaches need to be

explored. The present invention discloses such an alternative by demonstrating surface- exposed immunogenic polypeptides in Staphylococci species for development of vaccines and passive immunization with human antibodies.

SUMMARY OF THE INVENTION The present invention generally discloses strategies for the recovery of Staphylococci surface-exposed immunogenic polypeptides (SEIPs) in a sufficient quantity and immunogenic quality for the production of therapeutic anti-staphylococcal antibodies.

Therefore, it is an object of the invention to provide surface-exposed immunogenic polypeptides of Staplzylococci species which contain receptors for siderophores or iron- binding ligands.

It is a further object of the invention to provide an isolated, purified surface- exposed immunogenic polypeptide of Staplzylococcus aureus strains wherein said polypeptide contains receptors for siderophores or iron-binding ligands.

It is still a further object of the invention to provide polypeptides having an amino acid sequence selected from the group consisting of amino acid sequences corresponding to SEQ ID NO : 1, SEQ ED NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, and SEQ ID NO : 10, or conservatively substituted variants thereof.

It is a further object of the invention to provide an isolated DNA molecule comprising a nucleotide sequence encoding surface-exposed immunogenic polypeptides of Staphylococci species, or in particular Staphylococcus aureus strains, which contain receptors for siderophores or iron-binding ligands.

It is still a further object of the invention to provide an isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequences set forth in SEQ ID NO : 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, and SEQ ID NO: 10 or variants having at least 40% homology to said amino acid sequences.

It is a further object of the invention to provide a method for the isolation and purification of surface-exposed immunogenic polypeptides of Staphylococci species comprising the propagation of Staphylococci species in iron-depleted medium ; the

recovery of membrane-associated polypeptides whereby, the siderophores or iron-binding ligands are still complexed with the said polypeptide; the separation of said polypeptides from other components of the cell wall and cell membrane of Staphylococci species; the separation of siderophores or iron-binding ligands from the said polypeptides; and the recovery of the said polypeptides in a range from 30 kDa to 120 kDa.

It is a further object of the invention to provide a method to particularly identify individual surface-exposed immunogenic polypeptides of Staphylococci species, containing siderophores or iron-binding ligands, comprising the immunization of a vertebrate host with the polypeptides of claim 5 to generate anti-polypeptide antisera, wherein the amount of the surface exposed immunogenic polypeptides in a physiologically acceptable carrier is about 25-5000 Fg/ml ; the generation of Staphylococci species expression library using genomic DNA of said organism in an appropriate expression vector; the probing of said expression library with said antisera to identify organisms expressing the surface-exposed immunogenic polypeptides; the isolation and characterization of DNA sequences coding for the individual surface-exposed immunogenic polypeptides; and the identification of amino acid sequences corresponding to the individual surface-exposed immunogenic polypeptides.

It is an object of the invention to provide a composition comprising surface- exposed immnunogenic polypeptides of Staphylococci species, or any immunogenic fragments thereof, pharmaceutically acceptable carrier and adjuvants for immunization against Staphylococcal diseases and infections.

It is a further object of the invention to provide a composition comprising polypeptides having an amino acid sequence selected from the group comprising amino acid sequences corresponding to SEQ ID NO : 1, SEQ ID NO : 2, SEQ-ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, and SEQ ID NO: 10 or conservatively substituted variants thereof or immunogenic fragments thereof, pharmaceutically acceptable carrier and adjuvants for immunization against Stapl1ylococcal diseases and infections.

It is a further object of the invention to provide a composition comprising purified anti-staphylococcal polyclonal antibodies, isolated from a suitable host in response to the surface-exposed immunogenic polypeptides of Staphylococci species or any immunogenic

fragments thereof, and pharmaceutically acceptable carrier for immunization against Staplzylococcal diseases and infections.

It is a further object of the invention to provide a composition comprising purified anti-staphylococcal polyclonal antibodies, isolated from a suitable host in response to amino acid sequences selected from the group comprising amino acid sequences corresponding to any one of SEQ ID NOS: 1 through 12, or conservatively substituted variants thereof or immunogenic fragments thereof, and pharmaceutically acceptable carrier for immunization against Staphylococcal diseases and infections.

It is a further object of the invention to provide a composition comprising purified anti-staphylococcal monoclonal antibodies, isolated from a suitable host or host cell culture in response to any of the surface-exposed immunogenic polypeptides of Staphylococci species, or any immunogenic fragments thereof, and pharmaceutically acceptable carrier for immunization against Stapltylococcal diseases and infections.

It is a further object of the invention to provide a composition comprising purified anti-staphylococcal monoclonal antibodies isolated from a suitable host or host cell culture in response to amino acid sequences selected from the group comprising amino acid sequences corresponding to SEQ ID NO : 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, or SEQ ID 10 or conservatively substituted variants thereof or immunogenic fragments thereof, and pharmaceutically acceptable carrier for immunization against Staphylococcal diseases and infections.

It is an object of the invention to provide a method of producing anti- staphylococcal single-chain Fv (scFv) monoclonal antibodies, wherein immunoglobulin gene from anti-staphylococcal monoclonal cell lines are cloned into an expression vector to produce and isolate said single-chain antibodies.

It is a further object of the invention to provide an immunodiagnostic kit comprising Staphylococci-specific polyclonal or monoclonal antibodies for use in diagnosing stapl7ylococcal diseases or infections.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses and claims strategies for the recovery of Staphylococci surface-exposed immunogenic polypeptides (SEIPs) in a sufficient quantity and immunogenic quality for the production of therapeutic anti-staphylococcal antibodies.

The SEIPs can be used individually, or in combination with each other, to produce anti- Staphylococci antibodies useful in passive or active immunization strategies to prevent disease or contain infection caused by Staphylococcal species. This invention also claims a strategy for the localization of amino acid sequences within SEIPs that are required for their physiologic function, i. e. the reception of iron-binding ligands. These physiologically active amino acid sequences in SEIPs can be used to develop in vitro diagnostic assays for Staphylococcal infections.

The present invention provides a method for the isolation and purification of membrane-associated polypeptides of Staphylococcal species that are endotoxin-free, ligand-free, and preserves the immunogenic potential of the polypeptides. Further, the invention provides a method for the isolation and purification of DNA molecules coding for the functional amino acid sequences in SEIPs that are responsible for binding to siderophores and other iron-binding ligands. The invention also provides peptides corresponding to portions of SEIPs The DNA sequences, recombinant proteins and peptides are useful for diagnosis, immunization and the generation of diagnostic reagents and immunotherapeutic agents.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e. g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and m, Second Edition-(1989), Perbal, B., A Practical Guide to Molecular Cloning (1984); the series,'Methods In Enzymology (S.

Colowick and N. Kaplan eds. , Academic Press, Inc. ) ; and Handbook of Experimental<BR> Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds. , 1986, Blackwell Scientific Publications).

The following amino acid abbreviations are used throughout the text: Alanine: Ala (A) Arginine: Arg (R) Asparagine : Asn (N) Aspartic acid: Asp (D)

Cysteine: Cys (C) Glutamin : Gln (Q) Glutamic acid: Glu (E) Glycine: Gly (G) Histidine : His (H) Isoleucine: Ile (I) Leucine: Leu (L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr (Y) Valine: Val (V) DEFINITIONS In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appended claims, the singular forms"a", "an"and"the"include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to"an Staphylococci SEIP's includes a mixture of two or more such proteins, and the like.

The terms"surface exposed immunogenic polypeptides", or a nucleotide sequence encoding the same, intends a protein or a nucleotide sequence, respectively, which is derived from an Staphylococci SEIP gene. Henceforth, surface exposed immunogenic polypeptides (SEIP's) includes any polypeptide found on the surface of a bacteria, fungi, or protozoan. These polypeptides include but are not limited to adhesins, hearnein binding proteins, siderophore receptors, and SEIP receptors.

Furthermore, the derived protein or nucleotide sequences need not be physically derived from the gene described above, but may be generated in any manner, including for example, chemical synthesis, isolation (e. g. , from Staphylococci) or by recombinant production, based on the information provided herein. Additionally, the term intends proteins having amino acid sequences substantially homologous (as defined below) to contiguous amino acid sequences encoded by the genes, which display immunological activity.

Thus, the terms intend full-length, as well as immunogenic, truncated and partial sequences, and active analogs and precursor forms of the proteins. Also included in the term are nucleotide fragments of the gene that include at least about 8 contiguous base

pairs, more preferably at least about 10-20 contiguous base pairs, and most preferably at least about 25 to 50, or more, contiguous base pairs of the gene. Such fragments are useful as probes and in diagnostic methods, discussed more fully below.

The terms also include those forms possessing, as well as lacking, the signal sequence, as well as the nucleic acid sequences coding therefor. Additionally, the term intends forms of the surface exposed immunogenic polypeptides which lack the membrane anchor region, and nucleic acid sequences encoding proteins with such deletions. Such deletions may be desirable in systems that do not provide for secretion of the protein.

Furthermore, the ligand-binding domains of the proteins, may or may not be present. Thus, for example, if the surface exposed immunogenic polypeptide will be used to purify an iron-binding molecule, the ligand-binding domain will generally be retained. If the protein is to be used in vaccine compositions, immunogenic epitopes which may or may not include the SEIP-binding domain, will be present.

The terms also include proteins in neutral form or in the form of basic or acid addition salts depending on the mode of preparation. Such acid addition salts may involve free amino groups and basic salts may be formed with free carboxyls. Pharmaceutically acceptable basic and acid addition salts are discussed further below. In addition, the proteins may be modified by combination with other biological materials such as lipids (both those occurring naturally with the molecule or other lipids that do not destroy immunological activity) and saccharides, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl side chains, oxidation of sulfhydryl groups, glycosylation of amino acid residues, as well as other modifications of the encoded primary sequence.

The proteins-of the present invention are normally found in association with lipid moieties. It is likely that the fatty acid moiety present is a palmitic acid derivative. The antigens of the present invention, even though carrying epitopes derived from lipoproteins, do not require the presence of the lipid moiety. Furthermore, if the lipid is present, it need not be a lipid commonly associated with the lipoprotein, so long as the appropriate immunologic response is elicited. In any event, suitable fatty acids, such as but not limited to, palmitic acid or palmitic acid analogs, can be conveniently added to the desired amino acid sequence during synthesis, using standard techniques. For example, palmitoyl bound to S-glyceryl-L-Cys (Pam. sub. 3-Cys) commercially available (e. g. through Boehringer

Mannheim, Dorval, Quebec) and can easily be incorporated into an amino acid sequence during synthesis. See, e. g. Deres et al. (1989) Nature 342: 561. This is a particularly convenient method for production when relatively short amino acid sequences are used.

Similarly, recombinant systems can be used which will process the expressed proteins by adding suitable fatty acids. Representative systems for recombinant production are discussed further below.

The term therefore intends deletion, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.

In this regard, particularly preferred substitutions will generally be conservative in nature, <BR> <BR> i. e. , those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic--aspartate and glutamate; (2) basic--lysine, arginine, histidine; (3) non-polar--alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar--glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule, but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein, are therefore within the definition of the reference polypeptide.

For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, so long as the desired function of the molecule remains intact. In this regard, substitutions occurring in the transmembrane binding domain and the signal sequence normally will not affect immunogenicity. One of skill in the art may readily determine other regions of the molecule of interest that can tolerate change by reference to the Hopp/Woods and Kyte-Doolittle plots.

An"isolated"nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature;

or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.

By"subunit vaccine composition"is meant a composition containing at least one immunogenic polypeptide, but not all antigens, derived from or homologous to an antigen from a pathogen of interest. Such a composition is substantially free of intact pathogen cells or particles, or the lysate of such cells or particles. Thus, a"subunit vaccine composition"is prepared from at least partially purified (preferably substantially purified) immunogenic polypeptides from the pathogen, or recombinant analogs thereof. A subunit vaccine composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or polypeptides from the pathogen.

The term"epitope"refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with"antigenic <BR> <BR> determinant"or"antigenic determinant site. "Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

An"immunological response"to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an"immunological response"includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or. gamma.. delta. T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.

Preferably, the host will display either a therapeutic or protective immunological response such that resistance of the mammary gland to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host and/or a quicker recovery time.

The terms"immunogenic"protein or polypeptide refer to an amino acid sequence which elicits an immunological response as described above. An"immunogenic"protein or polypeptide, as used herein, includes the full-length sequence of the surface exposed immunogenic polypeptide in question, with or without the signal sequence, membrane anchor domain and/or SEIP-binding domain, analogs thereof, or immunogenic fragments thereof. By"immunogenic fragment"is meant a fragment of a surface exposed

immunogenic polypeptide which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any <BR> <BR> number of epitope mapping techniques, well known in the art. See, e. g. , Epitope Mapping<BR> Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed. , 1996) Humana<BR> Press, Totowa, N. J. For example, linear epitopes may be determined by e. g. , concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e. g. , U. S. Pat. No. 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 incorporated herein by reference in their entireties. Similarly, conformational epitopes are readily <BR> <BR> identified by determining spatial conformation of amino acids such as by, e. g. , x-ray<BR> crystallography and 2-dimensional nuclear magnetic resonance. See, e. g. , Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using <BR> <BR> standard antigenicity and hydropatly plots, such as those calculated using, e. g. , the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer <BR> <BR> program employs the Hopp/Woods method, Hopp et al. , Proc. Natl. Acad. Sci USA (1981) 78: 3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al. , J. Mol. Biol. (1982) 157: 105-132 for hydropathy plots.

Immunogenic fragments, for purposes of the present invention, will usually include at least about 3 amino acids, preferably at least about 5 amino acids, more preferably at least about 10-15 amino acids, and most preferably 25 or more amino acids, of the parent surface exposed immunogenic polypeptide molecule. There is no critical upper limit to the length of the fragment, which may comprise nearly the full-length of-the protein sequence, or even a fusion protein comprising two or more epitopes of a SEIP.

"Native"proteins or polypeptides refer to proteins or polypeptides isolated from <BR> <BR> the source in which the proteins naturally occur. "Recombinant"polypeptides refer to<BR> polypeptides produced by recombinant DNA techniques; i. e. , produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.

"Synthetic"polypeptides are those prepared by chemical synthesis.

A"vector"is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A DNA"coding sequence"or a"nucleotide sequence encoding"a particular protein, is a DNA sequence which is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory elements. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e. g. , mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3'to the coding sequence.

DNA"control elements"refers collectively to promoters, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Not all of these control sequences need always be present in a recombinant vector so long as the desired gene is capable of being transcribed and translated.

"Operably linked"refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof, Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered"operably linked"to the coding sequence.

A control element, such as a promoter, "directs the transcription"of a coding sequence in a cell when RNA polymerase will bind the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

A"host cell"is a-cell which has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule.

A cell has been"transformed"by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.

"Homology"refers to the percent identity between two polynucleotide or two polypeptide moieties. Two DNA, or two polypeptide sequences are"substantially homologous"to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.

In general, "identity"refers to an exact nucleotide-to-nucleotide or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.

Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure <BR> <BR> M. O. Dayhoff ed. , 5 Suppl. 3: 353-358, National biomedical Research Foundation,<BR> Washington, D. C. , which adapts the local homology-algorithm-of Smith and Waterman (1981) Advances in Appl. Math. 2: 482-489 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis. ) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference

sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif. ). From this suite of packages the Smith- Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six).

From the data generated the"Match"value reflects"sequence identity. "Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none ; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62 ; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: http ://www. ncbi. nlm. gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease (s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e. g., Sambrook et al. , supra; DNA Cloning, supra ; Nucleic Acid Hybridization, supra.

By the term"degenerate variant"is intended a polynucleotide containing changes in the nucleic acid sequence thereof, that encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the polynucleotide from which the degenerate variant is derived.

The term"functionally equivalent"intends that the amino acid sequence of a surface exposed immunogenic polypeptide is one that will elicit a substantially equivalent or enhanced immunological response, as defined above, as compared to the response

elicited by a surface exposed immunogenic polypeptide having identity with the reference surface exposed immunogenic polypeptide, or an immunogenic portion thereof.

A"heterologous"region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a bacterial gene, the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacteria. Another example of the heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e. g. , synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.

The term"treatment"as used herein refers to either (i) the prevention of infection or reinfection (prophylaxis), or (ii) the reduction or elimination of symptoms of the disease of interest (therapy).

As used herein, a"biological sample"refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e. g. , recombinant cells, and cell components.

As used herein, the terms"label"'and"detectable label"refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e. g. , biotin or haptens) and the like.

The term"fluorescer"refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Particular examples of labels which may be used under the invention include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH and. alpha. -. beta.-galactosidase.

GENERAL METHODS

The present invention describes the methods for the identification and recovery of Staphylococci SEIP's. The identification of amino acid sequences that are useful, as immunogens for the protection of a host from pathogenic microorganisms requires information on the structural organization of surface exposed immunogenic polypeptides (SEIP's). Surface exposed immunogenic polypeptides (SEIP's) includes any polypeptide found on the surface of a bacteria, fungi, or protozoan. These polypeptides include but are not limited to adhesins, heamein binding proteins, siderophore receptors, and SEIP receptors.

A number of studies have confirmed the close relationship between the availability of iron and pathogen virulence. The ability of a microbial invader to acquire iron from its vertebrate host has been recognized as an important virulence mechanism in some pathogenic bacteria. A number of reports have detailed the identification of outer membrane polypeptides that are up-regulated in environments that are low in iron.

However, the majority of these strategies have focused on the identification of receptors of iron-binding polypeptides of gram-negative bacteria. The uptake of iron complexes into the gram-negative bacterial cell requires highly specific outer membrane receptors and specific ATP-dependent (ATP-Binding-Cassette (ABC) transport systems located in the inner membrane. The latter type of import system is characterized by a periplasmic binding protein (BPT), integral membrane proteins, and membrane-associated ATP- hydrolyzing proteins. In gram-positive bacteria lacking the periplasmic space, the binding proteins are lipoproteins tethered to the cytoplasmic membrane. Periplasmic binding proteins (PBT) are involved in the uptake of siderophores, haem and vitamin B12, and define a subclass of polytopic integral membrane proteins. The topology of these 'siderophore family'proteins differs from that of the equivalent components of other PBT systems in that each polypeptide consists of 10 membrane-spanning regions, with the N- and C-termini located in the cytoplasm. The conserved region at a distance of about 90 amino acids from the C-terminus, typical of all hydrophobic PBT proteins, is also oriented to the cytoplasm. However, in the'siderophore family'proteins this putative ATPase interaction loop is followed by four instead of two transmembrane spans. (Groeger and Koster 1999).

The cell envelope of gram positive bacteria consists of a cytoplasmic membrane, a wall, and in some species a proteinaceous surface layer. Additional surface structures

such as capsules, slimes, fimbrae, and flagella may also be present. Unlike gram-negative bacteria, gram-positve bacteria do not have outer membranes or membrane enclosed periplasms. Thus the signature four loops of the siderophore family periplasmic binding proteins tethered to the cytoplasmic membrane are surfaced exposed.

In the present invention these surface exposed polypeptides are targeted as immunogens for development of vaccines in staphylococci and taxonomic related cocci.

Previously, Scott et al. (PCT/US02/11110) identified a span of amino acid sequence in the siderophore family of periplasmic binding proteins that were conserved using Reverse Position Specific BLAST and its disclosure, herein incorporated by reference in its entirety. The consensus sequence was defined by multiple sequence alignment of periplasmic binding proteins using cobbler software programs. The unique consensus sequence identified by COBBLER was lysine rich (SEQ ID No. l). Using this sequence to query available databases using BLAST it was realized that the sequence was localized to gram-negative bacteria, gram-positive bacteria and mycobacteria. The consensus sequence could be used as a probe to evaluate sequences in microorgansim in which the compete genome sequence is available. Alternatively, the consensus sequence could be used to generate antibodies that are subsequently used to probe genomic or cDNA expression libraries to identify iron-binding ligand receptors.

Central to the present invention is the discovery of genes encoding Staphylococci surface exposed immunogenic polypeptides that are involved in the acquisition of iron. In particular, the genes for Staphylococci surface exposed immunogenic polypeptide that share homology with the periplasmic binding protein amino acid sequence see SEQ ID No. 1. The complete DNA sequences and the protein sequences for these SEIP's are shown in SEQ ID NO : 2 through SEQ ID NO : 10.

The Staphylococci surface exposed immunogenic polypeptides, immunogenic fragments thereof or chimeric proteins including one or more epitopes of Staphylococci SEIP's can be provided, either alone or in combination, in subunit vaccine compositions to treat or prevent bacterial infections caused by Staphylococci and some other taxonomically closely related gram-positive bacteria.

In addition to use in vaccine compositions, the proteins and fragments thereof, antibodies thereto, and genes coding therefor, can be used as diagnostic reagents to detect the presence of infection in a mammalian subject. Similarly, the genes encoding the

proteins can be cloned and used to design probes to detect and isolate homologous genes in other bacterial strains. For example, fragments comprising at least about 15-20 nucleotides, more preferably at least about 20-50 nucleotides, and most preferably about 60-100 or more nucleotides, will find use in these embodiments.

Staphylococci SEIP's can be used in vaccine compositions either alone or in combination with other bacterial, fungal, viral or protozoal antigens. These antigens can be provided separately or even as fusion proteins comprising one or more epitopes of the surface exposed immunogenic polypeptides fused together and/or to one or more of the above antigens.

The strategies used for the production of Staphylococci SEIP's are also encompassed by the invention. The above described surface'exposed immunogenic polypeptides and active fragments, analogs and chimeric proteins derived from the same, can be produced by a variety of methods. Specifically, surface exposed immunogenic polypeptides can be isolated directly from bacteria which express the same. The proteins can be isolated directly from Staphylococci from outer membrane preparations, as described by Scott et al (PCT/US02/11110). The desired proteins can then be further purified i. e. by column chromatography, HPLC, immunoadsorbent techniques or other conventional methods well known in the art.

Alternatively, the proteins can be recombinantly produced as described herein. As explained above, these recombinant products can take the form of partial protein sequences, full-length sequences, precursor forms that include signal sequences, mature forms without signals, or even fusion proteins (e. g. , with an appropriate leader for the recombinant host, or with another subunit antigen sequence for Staphylococci or another pathogen).

The Staphylococci SEIP genes of the present invention can be imolated based on the ability of antibodies generated using a consensus sequence in periplasmic binding protein to bind SEIP's in an ELISA assay as described below. Thus, gene libraries can be constructed and the resulting clones used to transform an appropriate host cell. Colonies can be pooled and screened for clones containing the consensus sequence.

Alternatively, once the amino acid sequences are determined, oligonucleotide probes which contain the codons for a portion of the determined amino acid sequences can

be prepared and used to screen genomic or cDNA libraries for genes encoding the subject proteins. The basic strategies for preparing oligonucleotide probes and DNA libraries, as well-as their screening by nucleic acid hybridization, are well known to those of ordinary <BR> <BR> skill in the art. See, e. g. , DNA Cloning: Vol. I, supra; Nucleic Acid Hybridization, supra;<BR> Oligonucleotide Synthesis, supra; Sambrook et al. , supra. Once a clone from the screened library has been identified by positive hybridization, it can be confirmed by restriction enzyme analysis and DNA sequencing that the particular library insert contains a surface exposed immunogenic polypeptide gene or a homolog thereof. The genes can then be further isolated using standard techniques and, if desired, PCR approaches or restriction enzymes employed to delete portions of the full-length sequence.

Similarly, genes can be isolated directly from bacteria using known techniques, such as phenol extraction and the sequence further manipulated to produce any desired <BR> <BR> alterations. See, e. g. , Sambrook et al. , supra, for a description of techniques used to obtain and isolate DNA.

Alternatively, DNA sequences encoding the proteins of interest can be prepared synthetically rather than cloned. The DNA sequences can be designed with the appropriate codons for the particular amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e. g., Edge (1981) Nature 292: 756; Nambair et al. (1984) Science 223: 1299; Jay et al. (1984) J. Biol. Chem. 259: 6311.

Once coding sequences for the desired proteins have been prepared or isolated, they can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage. lambda. (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pU61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine <BR> <BR> papilloma virus (mammalian cells). See, Sambrook et al. , supra; DNA Cloning, supra; B.

Perbal, supra.

The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control"elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.

The coding sequence may or may not contain a signal peptide or leader sequence. If a signal sequence is included, it can either be the native, homologous sequence, or a heterologous sequence. For example, the signal sequence for the particular Staphylococci surface exposed immunogenic polypeptide, can be used for secretion thereof, as can a number of other signal sequences, well known in the art. Leader sequences can be <BR> removed by the host in post-translational processing. See, e. g. , U. S. Pat. Nos. 4,431, 739; 4,425, 437; 4, 338, 397.

Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.

Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

In some cases it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i. e. , to maintain the proper reading frame. It may also be desirable to produce mutants or analogs of the surface exposed immunogenic polypeptide. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e. g., Sambrook et al. , supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese

hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e. g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp. , will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Boimbyx mori, Drosophila melanogaster, gpodoptera frugiperda, and Trichoplusla ni.

Depending on the expression system and host selected, the proteins of the present invention are produced by culturing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into the growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

The proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are <BR> <BR> known to those skilled in the art. See, e. g. , J. M. Stewart and J. D. Young, Solid Phase<BR> Peptide Synthesis, 2nd Ed. , Pierce Chemical Co. , Rockford, Ill. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J.

Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques, and M. godangky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds. , The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis. Chemical synthesis of peptides may be preferable if a small fragment of the antigen in question is capable of raising an immunological response in the subject of interest.

The surface exposed immunogenic polypeptides of the present invention, or their fragments, can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e. g., mouse, rabbit, goat, horse,

etc. ) is immunized with an antigen of the present invention, or its fragment, or a mutated antigen. Serum from the immunized animal is collected and treated according to known <BR> <BR> procedures. See, e. g. , Jurgens et al. (1985) J. Chrom. 348: 363-370. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.

Monoclonal antibodies to the surface exposed immunogenic polypeptides and to the fragments thereof, can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by using hybridoma technology is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or <BR> <BR> transfection with Epstein-Barr virus. See, e. g. , M. Schreier et al. , Hybridoma Techniques (1980) ; Hammering et al., Monoclonal Antibodies and T-cell Hybridomas (1981) ; Kennett et al., Monoclonal Antibodies (1980); see also U. S. Pat. Nos. 4,341, 761; 4,399, 121; 4,427, 783; 4,444, 887 ; 4, 4S2, 570; 4,466, 917; 4,472, 500,4, 491,632 ; and 4,493, 890. Panels of monoclonal antibodies produced against the surface exposed immunogenic polypeptides, or fragments thereof, can be screened for various properties; i. e. , for isotype, epitope, affinity, etc. Monoclonal antibodies are useful-in purification, using immunoaffinity techniques, of the individual antigens which they are directed against. Both polyclonal and monoclonal antibodies can also be used for passive immunization or can be combined with subunit vaccine preparations to enhance the immune response. Polyclonal and monoclonal antibodies are also useful for diagnostic purposes.

The surface exposed immunogenic polypeptides of the present invention can be formulated into vaccine compositions, either alone, in combination and/or with other antigens, for use in immunizing subjects as described below. Methods of preparing such formulations are described in, e. g. , Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18 Edition, 1990. Typically, the vaccines of the present invention are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in or suspension in liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. The active immunogenic ingredient is generally mixed with a compatible pharmaceutical vehicle, such as, for example, water, saline, dextrose, glycerol,

ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents.

Adjuvants which enhance the effectiveness of the vaccine may also be added to the formulation. Adjuvants may include for example, muramyl dipeptide, avridine, aluminum hydroxide, dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, and other substances known in the art.

The surface exposed immunogenic polypeptides may be linked to a carrier in order to increase the immunogenicity thereof. Suitable carriers include large, slowly metabolized macromolecules such as proteins, including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.

The surface exposed immunogenic polypeptides may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cys-thiolactone. A sulfhydryl group may also be incorporated into the carrier (or antigen) by, for example, reaction of amino functions with 2- iminothiolane or the N-hydroxysuccinimide ester of 3- (4-dithiopyridyl propionate.

Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides.

Other suitable carriers for the surface exposed immunogenic polypeptides of the present invention include VP6 polypeptides of rotaviruses, or functional fragments thereof, as disclosed in U. S. Pat. No. 5,071, 651, incorporated herein by reference, Also useful is a fusion product of a viral protein and the subject immunogens made by methods disclosed in U. S. Pat. No. 4,722, 840. Still other suitable carriers include cells, such as lymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, the proteins of the present invention may be coupled to erythrocytes, preferably the subject's own erythrocytes. Methods of coupling peptides to proteins or cells are known to those of skill in the art.

Furthermore, the surface exposed immunogenic polypeptides (or complexes thereof) may be formulated into vaccine compositions in either neutral or salt forms.

Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Vaccine formulations will contain a"therapeutically effective amount"of the active ingredient, that is, an amount capable of eliciting an immune response in a subject to which the composition is administered. In the treatment and prevention of Staphylococci infection, for example, a"therapeutically effective amount"would preferably be an amount that enhances resistance of the mammal in question to new infection and/or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host and/or a quicker recovery time.

The exact amount is readily determined by one skilled in the art using standard tests. The surface exposed immunogenic polypeptide concentration will typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate. With the present vaccine formulations, 5 to 500, mg of active ingredient per ml of injected solution, preferably 10 to 100 jig of active ingredient per ml, should be adequate to raise an immunological response when a dose of 1 to 3 ml per animal is administered.

To immunize a subject, the vaccine is generally administered parenterally, usually by intramuscular injection. Other modes of administration, however, such as subcutaneous, intraperitoneal and intravenous injection, are also acceptable. The quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. The subject is immunized by administration of the

vaccine in at least one dose, and preferably two doses. Moreover, the animal may be administered as many doses as is required to maintain a state of immunity to infection.

Additional vaccine formulations which are suitable for other modes of administration include suppositories and, in some cases, aerosol, intranasal, oral formulations, and sustained release formulations. For suppositories, the vehicle composition will include traditional binders and carriers, such as, polyalkaline glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.

Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluenta such as water, aqueous saline or other known substances can be employed with the subject invention.

The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

Controlled or sustained release formulations are made by incorporating the protein into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel@ copolymers, swellable polymers such as -hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. The surface exposed immunogenic polypeptides can also be delivered using implanted mini-pumps, well known in the art.

The surface exposed immunogenic polypeptides of the instant invention can also be administered via a carrier virus which expresses the same. Carrier viruses which will find use with a the instant invention include but are not limited to the vaccinia and other pox viruses, adenovirus, and herpes virus. By way of example, vaccinia virus recombinants expressing the novel proteins can be constructed as follows. The DNA encoding the particular protein is first inserted into an appropriate vector so that it is

adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the instant protein into the viral genome. The resulting TK recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

An alternative route of administration involves gene therapy or nucleic acid immunization. Thus, nucleotide sequences (and accompanying regulatory elements) encoding the subject surface exposed immunogenic polypeptides can be administered directly to a subject for in vivo translation thereof. Alternatively, gene transfer can be accomplished by transfecting the subject's cells or tissues ex vivo and reintroducing the transformed material into the host. DMA can be directly introduced into the host organism, i. e. , by injection (see U. S. Pat. Nos. 5, 580, 859 and 5, 589, 466; International Publication No. WO/90/11092; and Wolff et al. (1990) Science 247: 1465-1468).

Liposome-mediated gene transfer can also be accomplished using known methods. See, e. g. , U. S. Pat. No. 5,703, 055; Hazinski et al. (1991) Am. J. Respir. Cell Mol. Biol. 4: 206- 209; Brigham et al. (1989) Am. J. Med. Sci. 298: 278-281 ; Canonico et al. (1991) Clin.

Res. 39: 219A; and Nabel et al. (1990) Science 249: 1285-1288. Targeting agents, such as antibodies directed against surface antigens expressed on specific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to specific tissues and cells susceptible to infection.

The use of animal to evalute the immunotherapeutic SEIP's are also encompased by the invention.

Active Immunization SEIP antigen cocktails or peptides will be injected intramuscularly in vertebrate host, followed by three boosts of antigen at weekly intervals. One week after the last immunization the vertebrate host will be challenged with a predefined dose of desired pathogen. In addition, each group receiving a different antigen preparation will be housed separately in order to avoid contamination of the vaccinated, coccidia free layers. Two-day fecal samples will be collected five days after infection and oocyst output will be determined. One day later, blood samples are collected via cardiac puncture under

methoxyflurane anesthesia and plated on the appropriate selection agar plates. The number of colony forming units (cfu) per/ml, of blood is quantified after 24 hr. The statistical significance of differences observed in the levels of disease causing organism relative to controls is analyzed by the Student's t-test.

Passive Immunization Vertebrate hosts, such as chicken, mice, rats etc. will be immunized with immunoglobulins (1 mg/ml) obtained from hyperimmune serum. Pre-immune sera are used as negative controls. One day after immunization, the animal is inoculated with a specified pathogen at a predetermined dosage. One day later, blood samples are collected via cardiac puncture under methoxyflurane anesthesia and plated on the appropriate selection agar plates. The number of colony forming units (cfu) per ml, of blood is quantified after 24 hr. The statistical significance of differences observed in the levels of disease causing organism relative to controls is analyzed by the Student's t-test.

The use of the surface exposed immunogenic gene and/or the polypeptides that they encode as diagnostics tools are encompassed by the invention. The use of the surface exposed immunogenic polypeptides of the present invention may also be used as diagnostics to detect the presence of reactive antibodies of Staphylococciin a biological sample in order to determine the presence of Staphylococci infection. For example, the presence of antibodies reactive with surface exposed immunogenic polypeptides can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competitions direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs ; biotinfavidin type assays; radioimmunoassays ; immunoelectrophoresis ; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound antibody in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.

Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g. , in membrane or microtiter well form); polyvinylchloride (e. g. , sheets

or microtiter wells); polystyrene latex (e. g. , beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

Typically, a solid support is first reacted with a solid phase component (e. g. , one or more surface exposed immunogenic polypeptides) under suitable binding conditions such that the component is sufficiently immobilized to the support. Sometimes, immobilization of the antigen to the support can be enhanced by first coupling the antigen to a protein with better binding properties. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that can be used to bind the antigens to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules to the antigens, are well known to those of ordinary skill in the art. See, e. g. , Brinkley, M. A. Bioconjugate Chem. (1992) 3: 2-13; Hashida et al, J.

Appl. Biochem. (1984) 6: 56-63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30: 117-124.

After reacting the solid support with the solid phase component, any non- immobilized solid-phase components are removed from the support by washing, and the support-bound component is then contacted with a biological sample suspected of containing ligand moieties (e. g. , antibodies toward the immobilized antigens) under suitable binding conditions. After washing to remove any non-bound ligand, a secondary binder moiety is added under suitable binding conditions, wherein the secondary binder is capable of associating selectively with the bound-ligand. The presence of the secondary binder can then be detected using techniques well known in the art.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a S surface exposed immunogenic polypeptide. A biological sample containing or suspected of containing anti-surface exposed immunogenic polypeptide immunoglobulin molecules is then added to the coated wells. After a period of incubation sufficient to allow antibody binding to the immobilized antigen, the plate (s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added.

The secondary binding molecule is allowed to react with any captured sample antibodies,

the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Thus, in one particular embodiment, the presence of bound anti-SEIP-binding antigen ligands from a biological sample can be readily detected using a secondary binder comprising an antibody directed against the antibody ligands. A number of anti-bovine immunoglobulin (Ig) molecules are known in the art which can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, alkaline phosphatase or urease, using methods known to those of skill in the art. An appropriate enzyme substrate is then used to generate a detectable signal. In other related embodiments, competitive-type ELISA techniques can be practiced using methods known to those skilled in the art.

Assays can also be conducted in solution, such that the surface exposed immunogenic polypeptides and antibodies specific for those proteins form complexes under precipitating conditions. In one particular embodiment, surface exposed <BR> immunogenic polypeptides can be attached to a solid phase particle (e. g. , an agarose bead or the like) using coupling techniques known in the art, such as by direct chemical or indirect coupling. The antigen-coated particle is then contacted under suitable binding conditions with a biological sample suspected of containing antibodies for the surface exposed immunogenic polypeptides. Cross-linking between bound antibodies causes the formation of particle-antigen-antibody complex aggregates which can be precipitated and separated from the sample using washing and/or centrifugation. The reaction mixture can be analyzed to determine the presence or absence of antibody-antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above.

In yet a further embodiment, an immunoaffinity matrix can be provided, wherein a polyclonal population of antibodies from a biological sample suspected of containing anti- SEIP-binding molecules is immobilized to a substrate. In this regard, an initial affinity purification of the sample can be carried out using immobilized antigens. The resultant sample preparation will thus only contain anti-Staphylococci moieties, avoiding potential nonspecific binding properties in the affinity support. A number of methods of immobilizing immunoglobulins (either intact or in specific fragments) at high yield and good retention of antigen binding activity are known in the art. Not being limited by any

particular method, immobilized protein A or protein G can be used to immobilize immunoglobulins.

Accordingly, once the immunoglobulin molecules have been immobilized to provide an immunoaffinity matrix, labeled surface exposed immunogenic polypeptides are contacted with the bound antibodies under suitable binding conditions. After any non- specifically bound antigen has been washed from the immunoaffinity support, the presence of bound antigen can be determined by assaying for label using methods known in the art.

Additionally, antibodies raised to the surface exposed immunogenic polypeptides, rather than the surface exposed immunogenic polypeptides themselves, can be used in the above-described assays in order to detect the presence of antibodies to the proteins in a given sample. These assays are performed essentially as described above and are well known to those of skill in the art.

The above-described assay reagents, including the surface exposed immunogenic polypeptides, or antibodies thereto, can be provided in a kit, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above. The kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i. e. wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.

EXAMPLES It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis, and treatment of disease and-infections caused pathogenic microorganisms. A further non- limiting discussion of such uses is further presented below. These examples are not meant to limit the scope of the invention that has been set forth in the foregoing description.

Variation within the concepts of the invention is apparent to those skilled in the art. The disclosures of the cited references throughout the application are incorporated by reference herein.

Example 1 Recovery of staphylococci outer membrane associated polypeptides

In a preferred embodiment, An overnight culture of Staphylococcus aureus, designated D2-DLS03, was used to inoculate 500 ml of freshly prepared Minimal medium (Sigma Chemical Company, St. Loius, MO) supplemented with glucose (lOg/ ml) (Sigma Chemical Company); proline (40 Fg/ml) (Sigma Chemical Company); 1 M MgS04 (0.1%) (Sigma Chemical Company); and the iron-chelator, 2'2'dipyridyl (100 uM) (Sigma Chemical Company). The culture was incubated in gyration water bath 37 C until the growth density of the culture read an optical density (OD) 0.6 at A600. The S. aureus cells were concentrated by centrifuging the culture in 250 ml tubes for 10 min at 5,000 x g in a model J2-21 Beckman centrifuge. The supernatant was stored at-20 C and the bacteria cells were gently resuspended in 250 ml of ice cold HE (10 mM Hepes [ph 7.4]) (Sigma Chemical Company) and 1 mM EDTA buffer (Sigma Chemical Company) and the centrifugation step was repeated. The cell culture supernatant was analyzed for the presence of siderophores (henceforth iron reactive material) using the Chrome Azural S (CAS) the presence lipopolysaccharides was determined using Limulus amoebocyte lysate assay (Sigma Chemical Company). The D2-DLS03 supernatant was positive for iron-reactivity and LPS.

The bacterial concentrate was re-suspended in 17 ml of HE buffer in a 50 ml sterile tube, then frozen in liquid nitrogen and thawed at room temperature. This step was repeated until the solution became viscous. Next the S. aureus lysate was processed to separate and purify the surface exposed immunogenic polypeptides from other cell wall including lipolysaccharide (endotoxins). Ten milliliters (10 ml) of the viscous lysate was layered on a 0.5 ml 60% sucrose shelf and centrifuge for 1 h at 38,000-x g in a model L8- 70M ultracentrifuge (Beckman Instruments, Inc.) in a SW 41 swinging bucket rotor <BR> <BR> (Beckman Instruments, Inc. ) to differentiate the cytoplasmic and membrane fractions. The cytoplasmic and membrane fractions were analyzed for iron-reactive material and the presence of LPS as described previously. The iron reactivity and the LPS contamination were localized to the membrane fraction.

The membrane fraction was resuspended in 50 ml of solubilization buffer (10 mM Hepes [ph 7. 4] ; 1 mM EDTA; 1.2 M NaCI ; 2% Triton X-100) and incubated 1 h at 4 C.

The solubilized membranes were mixed with 10% Polyethyleneimine (PEI) (Sigma Chemical Company) by stirring until the concentration of PEI was 1% of the solubilized membrane solution. The membrane-PEI solution was stirred for 1 h in a cold room at 4 C

and then centrifuged for 15 min at 10,000-x g in a model J2-21 centrifuge (Beckman Instruments, Inc. ). The PEI supernatant and pellet (resuspended in HE buffer) were analyzed for iron-reactive material and the presence of LPS as described previously. The iron-reactivity was identified in the PEI supernatant while the LPS contamination molecules were localized to the PEI pellet.

The iron-reactive PEI supernatant (50 ml) was mixed by slow stirring with 18. 05 g of ammonium sulfate and incubated with continuous stirring at 4 C for 1 h. The ammonium sulfate precipitate was collected by centrifugation for 15 min at 10,000-x g in a model J2-21 centrifuge (Beckman Instruments, Inc. ). The ammonium sulfate supernatant and pellet (resuspended in HEU buffer) were analyzed for iron-reactivity and LPS contamination. The iron-reactive fraction was recovered in the Ammonium sulfate pellet no LPS was detectable The ammonium sulfate precipitate was resuspended in 25 ml of HE buffer (10 mM Hepes [ph 7.4] ; 1 mM EDTA) and size fractionated by tangential-flow ultra centrifugation against a membrane with an apparent cut-off of 30 Kda (Centricon 30, Amicon). The filtrate and retainate were analyzed for iron-reactivity using the CAS assay and presence of LPS. Usually, the iron-reactive material is low in molecular weight (500-1000 daltons).

Thus the iron-reactivity was expected to be found in the filtrate. In contrast the iron- reactivity was found in the retainate, no LPS were detected in either fraction.

The iron-reactivity was transferred to the filtrate by the addition of solid urea to a final concentration of 6 M. The retainate, hence for D2-DLS03 antigen cocktail, was modified with. 02% sodium azide and stored at-20 C.

Example 2. Production-of Anti-SEIP's Polyclonal Antibodies To identify antigenic determinants for protection against a specified pathogen, SEIP's from a target organism are used to immunize vertebrate host such as chickens, goats and rabbits that are known in the art for quantitative production of antibodies (Emery, . In a preferential embodiment, SEIP's were used to generate antisera in laying hens using standard techniques. Briefly, laying hens were immunized 3 times subcutaneously, at intervals of two weeks, using complete Freund's adjuvant for the first injection and incomplete Freund's adjuvant for subsequent injections.

Example 3. Evaluation of the Affinity Anti-SEIP's Immunoglobulins for Staphylococci Cells and Purified SEIP's The affinity of anti-SEIP's antisera for the surface of a specified pathogen was evaluated using a whole-cell enzyme linked immunoabsorbent assay (ELISA) method.

After determining the titer of the staphylococci anti-SEIP's antibodies, they were covalently attached to a microtiter plate by incubation in diluted 1: 10 in carbonate buffer at 37 C for 2 h. A 5 % milk-fat protein-TBS solution was as a blocking agent to eliminate non-specific binding of staphylococci cells. Next, 100 u, l aliquots of serial dilutions (100- fold) S. aureus cells were added to each well coated anti-SEIP's antisera and incubated for 1 h. After thouroughly washing away any unbound S. aureus with TTBS (TBS supplemented with 0.2% Tween 20) 100 J. l of 1: 100 dilution of anti-SEIP's was added for 1 h. After washing again with TTBS antibody binding was determine using an anti-IgY alkaline phosphatase conjugate (Sigma Chemcial Company) (diluted 1: 1000). Antibody binding was determined by adding the colorimetric substrate para-nitrophenyl phosphate (PNP) and allowing the reaction to proceed for 1 h. At the completion of the lh incubation the reaction was terminated by the addition of 100 jjl of 1 N NaOH. The color development was observed (as determined spectrophotometrically at 405 nm using a microtiter plate reader) in every well with noticeable decline as the dilution factor increased.

Next, increasing concentrations of SEIP's from S. aureus D2-DLS03 were evaluated by polyacrylamide gel electrophoresis, followed by transfer step to a nitrocellulose filter as described by Maniatis et al. The nitrocellulose filter was blocked with 5% milk fat proteins for 1 h. After blocking the filter was probed with a 1: 1000 dilution of D2-DLS01 antisera for 1 h. After thoroughly washing with TTBS (60 mM Tris-HCl pH 7.4 ; 150 mM NaCI ; 0.5% Tween 20) the filter was probed with anti-IgY alkaline phosphatase conjugate (Sigma-Aldrich, St. Louis, MO) for 1 h. The filter was washed with TTBS. After the wash step the colorimetric substrate BCIP/NBT (Sigma Chemcal Company) was added and the colorimetric assay was allowed to proceed for 30 min at room temperature. The reaction was terminated by the rinsing of the filter with sterile water. The anti-D2-DLS03 anti-SEIP's antisra recognized S. aureus SEIP's ranging in size from 30 kda-100 kda as determine using a broad range protein marker (BioRad).

Example 4. In vitro growth Inhibition Studies Desired antibodies are those that are capable of inhibiting the proliferation of pathogenic microorganisms in a complement independent manner. Thus, increasing dosage of purified immunoglobulins (monoclonal or polyclonal pools) were included in iron-deplete (100 uM 2'2'-dipyridyl) and iron-replete (50 uMFeCI3) minimal mediums and the growth of a specific pathogen was monitored by counting colony forming units (CFU) for solid medium or monitoring absorbance spectrophotometrically at 600 nm for liquid cultures.

In a B-D Falcon 48 well tissue culture plate (Fisher Scientific, Suwanee, GA) 1 ml of M9-minimal medium supplemented with maltose (lOg/ml) ; methionine (40 llg/ml) ; 1 M MgS04 (0.1%) and 120, uM 2'2'dipyridyl was added to individual wells. The minimal medium also included purified immunoglobulins at a concentration of 500 ßg/ml. An overnight culture of S. aureus was serially diluted in increments of 100-fold. One- microliter aliquots of the serial diluted bacteria were inoculated into the M9 medium and incubated at 37 C for 18 h-24 h. Simultaneously, 100 gel portion of the serial diluted bacteria were streaked on Maltose agar plates and incubated at 37 C for 18 h-24 h.

The growth rate of S. aurues in the presence of the immunoglobulins was evaluated by monitoring the absorbance spectrophotometrically at 600 nm. The concentrations of immunoglobulins did reduce the growth rate of S. aureus until we reached the 10-g dilution factor. Evaluation of the colony forming units present at this dilution factor it on the solid M9-maltose medium demonstrated that 500 jig of purified immunoglobulins are effective in neutralizing 100 or less bacteria.

Example 5. Isolation and Purification of Genes Encoding the Individual SEIP's A 100 ml culture of S. aurueus grown in M9-maltose medium supplemented with 120 uM 2, 2' dipyridyl was harvested at an optical density (OD) 0.6 at A600. The S. aureus cells were concentrated by centrifuging the culture in 50 ml tubes for 10 min at 5,000 x g in a model J2-21 Beckman centrifuge. The concentrated bacteria were resuspended in 5.0 ml DNA X-tract solution 1 (D-squared BioTechnologies Incorporated, Atlanta, GA).

The resuspended bacteria is mixed with an equal volume of DNA X-tract solution 2 (D-

squared BioTechnologies Incorporated) in a 50 ml polypropylene tube. Next 10 ml of molecular grade chloroform is added to the lysate and the mixture is made homogenous by inversion and then centrifuged at 10,000 x g for 10 min. The aqueous phase (top phase) is transferred to a new tube and the chloroform extraction step and centrifugation is repeated.

The aqueous phase is transferred to a new tube and mixed with 10.0 ml of DNA X-tract precipitation solution (D-squared BioTechnologies Incorporated) and incubated on ice for 30 min or longer. DNA is precipitated by centrifuging at 10,000 x g for 15 min in a microcentrifuge. DNA pellet is washed with 1 ml 70 % ethanol, air dried and resuspended in TE or water.

A 100 Zg aliquot of S. aureus chromosomal DNA in TE was mechanically sheared in a 1 ml syringe with a 25-gauge needle. The sheared DNA was made blunt-ended by adding water to a final volume of 405 1, 45 pL of 10x S1 nuclease buffer (2M NaCl, 50 mM NaOAc, pH 4.5, 10 mM ZnSO¢, 5% glycerol), and 1.7 pi of S1 nuclease at 100 U/gl and incubating at 37 C. for 15 min. The sample was extracted once with phenol/chloroform and once with chloroform and 1 ml of ethanol was added to precipitate the DNA. The sample was incubated on ice for 10 min or at-20 C overnight and the DNA was harvested by centrifugation in a microcentrifuge for 30 min. The DNA was washed with 70% ethanol and dried. The EcoRI sites in the DNA sequence were methylated using standard procedures. To this methylated DNA was added 5 u. l of 100 mM MgCl2, 8 ul of dNTP mix (2.5 mM each of DATP, dCTP, dGTP, and dTTP), and 4 Vtl of 5 U/, ul Klenow.

The mixture was incubated at 12 C. for 30 min. 450 ul of STE (0. 1M NaCl, 10 mM Tris- HC1, 1 mM EDTA, pH 8.0) was added, and the mixture extracted once with phenol/chloroform, and once with chloroform, before adding 1 ml of ethanol to precipitate the DNA. The sample was incubated on ice for 10 min or at-20 C. overnight. The DNA was harvested by centrifugation in a microcentrifuge for 30 min., washed with 70% ethanol and dried.

The DNA was resuspended in 7 gel of TE and to the solution was added 14 ill of phosphorylated Eco RI linkers (200 ng/lll), 3 u. l of 10X ligation buffer, 3 1ll of 10 mM ATP, and 3 1 of T4 DNA ligase (4 U/u. 1). The sample was incubated at 4 C overnight, then incubated at 68 C for 10 min. to inactivate the ligase. To the mixture was added 218 1 of H2 O, 45 gel of lOx Universal buffer, and 7 u. l of Eco RI at 30 U/u. 1. After incubation

at 37. C. for 1.5 h, 1. 5 gel of 0.5M EDTA was added, and the mixture placed on ice.

The DNA was size fractionated on a sucrose gradient, pooling fractions containing DNA of 6-10 kb. The pooled DNA was ethanol precipitated and resuspended in 5 ! ll of TE buffer. The 20 ng of insert DNA was ligated for 2-3 days at 4 C with lgg of ZAP II vector in a final volume of 5 p1. The ligation mixture was then packaged using GIGAPACK II GOLD (Stratagene, CA) and plated on E. coli SURE (Stratagene) cells on NZY plates. The library was titrated, amplified, and stored at 4 C under 0.3% chloroform.

The S. aureus lambda. ZAP library was plated on E. coli SURE cells and plaques were transferred onto nitrocellulose membranes, which had been pre-soaked in 10 mM IPTG to induce expression from the pBluescript lacZ promoter. Filters were blocked using 0.5% skim milk in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, prior to being probed with the anti- D2DLS03 polyclonal antibodies for the identification of recombinant SEIP's. The plaques of interest from the agar plate were cored and transfered to a sterile microcentrifuge tube containing 500 1 of SM buffer and 20 tel of cloroform. The microcentrifuge tube was vortexed to release the phage particles into the SM buffer. The microcentrifuge tube was then incubated for 1-2 hours at room temperature or overnight at 4 C. Next, separate overnight cultures ofXLl-Blue MRF'and SOLR cells in LB broth were grown, supplemented with 0.2% (w/v) maltose and 10 mM MgS04, at 30 C. The next day, the XLl-Blue MRF'and SOLR cells were gently spun down at 1000 x g and resuspended the XL1-Blue MRF and SOLR cells at an OD600 of 1.0 in 10 mM MgS04. In a Falcon 2059 polypropylene tube 200 Ill of XLl-Blue MRF'cells at an OD500 of 1.0 ; 250 ßl of phage stock (> 1 x 105 phage particles); 1 1 ofExAssist helper phage (> 1 x 106 pfu/Rl) were combined to release the phage particles; then the tube was incubated at 37 C for 15 min.

Next, 3 ml LB broth was added and again the tube was incubated for 2.5-3. 0 h at 37 C with shaking. At the completion of the incubation step, the tube was heated at 65 C for 20 min and then spun down at 100 x g for 15 min. The supernatant was decanted into a fresh tube. The excised pagemenids were plated by adding 200 fil of freshly grown SOLR cells (OD600 1. 0) to two 1.5 ml microcentrifue tubes. 100 Ill of the phage supernatant was added to 1 tube and 10 , l was added to the other then the tubes were incubated at 37 C for 15 min. At the completion of the incubation step the 200 jjl of the cell mixture was plated on LB-amplicillin agar plates (50 g/ml) and incubated overnight at 37 C. The next day

individual colonies were picked and grown in 10 ml of LB-amplicillin medium. The cells were concentrated by centrifuging the culture in 50 ml tubes for 10 min at 5,000 x g in a model J2-21 Beckman centrifuge. The concentrated bacteria were resuspended in 2.5 T. E. buffer ph 8.0 then mixed with an equal volume of DNA X-tract solution 1 in a 50 ml polypropylene tube. The mixture is made homogenous by inversion and then centrifuged at 10,000 x g for 10 min. The supernatant was transferred to a new tube containing an equal volume of molecular grade chloroform extraction. The mixture was again made homogenous by inversion and then centrifuged at 10,000 x g for 10 min. The aqueous phase (top phase) was transferred to a new tube and mixed with 10.0 ml of DNA X-tract precipitation solution and incubated on ice for 30 min or longer. The DNA is precipitated by centrifuging at 10,000 x g for 15 min in a microcentrifuge. The DNA pellet is washed with 1 ml 70 % ethanol, air dried and resuspended in TE or water. The purified phagemid DNA was sequenced and amino acid sequences deduced (SEQ ID Nos.

2-10).

The first step in the localization of target amino acids for the development of anti- microbial immunoglobulins is to identify polypeptides in the target pathogen that are essential to the proliferation and establishment of infection. The strategy described in this application for the recovery of immunogenic polypeptides provides invaluable information in reference to what epitopes, on the surface of a pathogenic microorganism, are accessible by the immune system of the said host. A desired target sequence must be 1) surfaced exposed; 2) immunogenic ; 3) the target sequence should be essential to the ligand contacting its membrane associated receptor; and 4) at least conserved at the species level.

Candidate amino acid sequences in target proteins should be continuous, i. e. , the determinant is an uninterrupted fragment of the primary structure of-the protein on which the determinant occurs. Furthermore, consideration should be given to the fact that in many instances, strains of the target microorganism will occur naturally. Therefore, various strains may be antigenically variable, i. e. differ from one another in the amino acid sequences of one or more of their antigenic determinants. Thus, a vaccine based on a single strain may not provide immunity in a vaccinated individual against other strains of the same microorganism, as antibodies induced by the single strain may not be reactive with antigenic determinants on other strains. This problem of antigenic variability has in fact been encountered with currently available anti-rabies vaccines (Luo et al. 1998).

Candidate target amino acid sequences must be conserved among all strains of the target microorganism. Thus, a vaccine with a synthetic peptide with such a sequence will not be limited by antigenic variability and will be effective to provide protection against all strains of the target microorganism against which the vaccine is intended to provide protection. A key advantage to the approaches of the present invention is that the target of the immune system is known. Therefore the sequence can be monitored by methods common to the art such as PCR and ELISA, to ensure that the peptide is still a useful immunogen. Thus, if mutations should arise in the target sequence a new peptide can be synthesized.

The first step in our scheme was to analyze the amino acid sequences for the presence of conserved and/or functional domains. To evaluate the structural organization of recombinant SEIP's we evaluated their sequences using a Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997). BLAST is a set of search programs designed to explore all of the available sequence databases using a heuristic algorithm which seeks local as opposed to global alignments to detect relationships among sequences which share only isolated regions of similarity (Altschul et al. , 1990). The BLAST programs have been designed for speed, with a minimal sacrifice of sensitivity to distant sequence relationships. The scores assigned in a BLAST search have a well-defined statistical interpretation, making real matches easier to distinguish from random background hits.

The Gapped BLAST algorithm allows gaps (deletions and insertions) to be introduced into the alignments that are returned. Allowing gaps means that similar regions are not broken into several segments. The scoring of these gapped alignments tends to reflect biological relationships more closely. Position-Specific Iterated BLAST (PSI-BLAST) provides an automated, easy-to-use version of a"profile"search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. PSI-BLAST may be iterated until no new significant alignments are found.

The results of the query of the amino acid SEQ ID Nos. 2-9 using BLAST suggested that the polypeptides shared sequence homology (at least 40% sequence homology) with the siderophore family of periplasmic binding proteins. More specifically

the consensus sequence of periplasmic binding proteins were conserved among gram- negative, gram-positive bacteria, and mycobacteria.

Hydrophilicity/hydrophobicity plots of amino acid SEQ ID Nos. 2-9 are particularly useful in identifying immunogens that protect a specified host from diseases caused by Staplxylococcus aureus. Furthermore cetain regions of these amino acids are also useful in broad host protection which includes gram negative bacteria, gram positive bacteria and mycobacteria in both a complement dependent and complement independent fashions. The preferred method for determining target sequences is to compare the results of Kyte and Doolittle (1982) and the Hopp and Woods algorithm (1981). The production of antibodies to these region may physical impair the iron bound ligand from contacting its receptor.

The discovery of conserved sequences within the siderophore receptor of a range of bacterial pathogens allows the selection of a minimal number of antigens having particular amino acid sequences (including in the form of synthetic peptides) to immunize against the disease caused by pathogens that have such receptors. Such conserved amino acid sequences among many bacterial pathogens permits the generation of siderophore receptor specific antibodies, including monoclonal antibodies that recognize most if not all siderophore receptors. Such antisera are useful for the detection and neutralization of most if not all bacteria that produce siderophore receptors and are also useful for both active and passive immunization against the diseases caused by such pathogens. Diagnostic assays and kits using such conserved amino acid sequences are useful to detect many if not all bacteria that produce siderophore receptor.

Example 6. Strategies for the identification of genus and/or species-specific epitopes in SEIP's As an alternative to the strategy provided above we will also employ a subtraction strategy for the localization of the immunogenic amino acid sequences in SEIP's. In a preferred embodiment, SEIP's are prepared from a target pathogen (see example 1) and used to generate anti-SEIP's antisera in vertebrate host. The anti-SEIP's antisera is pre- absorbed with SEIP's from other pathogens that you are not interested in neutralization.

Next, the pre-absorbed anti-SEIP's antisera are attached to a microtiter plate as described in example 3. The anti-SEIP's antisera is probed with peptide libraries as described in the

art. The recovered peptides are absorbed with pre-immune antisera obtained from the vertebrate host prior to immunization. The pre-immune antisera are separated from the peptides by immunoprecipitation or affinity column purification using protein A as described in the art. The resulting clones are sequenced and used as immunogens to generate genus or species-specific antisera. A key advantage of this strategy is that the recovered peptide sequences represent surface available sequences or mimic unique surface structures.

Example 7. Immunotherapeutic SEIP's The Staphylococci are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues. The use of human immunoglobulins for the treatment of staphylococcus diseases appears promising. However, commercial products are not yet readily available due to certain inherent limitations that have prevented their widespread use in the treatment of life-threatening bacterial disease.

One such limitation associated with immunoglobulin compositions is that they are assembled from large pools of plasma samples that have been pre-selected for the presence of a limited number of particular antibodies. Typically, these pools consist of samples from a thousand donors who may have low titers to some pathogenic bacteria. Thus, at best, there is only a modest increase in the resultant titer of desired antibodies.

Yet another such limitation inherent in immunoglobulin compositions is that their use results in coincident administration of large quantities of extraneous proteinaceous substances (e. g. , viruses) having the potential to cause adverse biologic effects. The combination of low titers of desired antibodies and high content of extraneous substances often limits, to sub-optimal levels, the amount of specific and thus beneficial immune globulin (s) administrable to the patient.

Another limitation is that the pre-selection process itself requires very expensive, continuous screening of the donor population to assure product consistency. Despite considerable effort, product lots can still vary between batches and geographic regions.

In addition, all approaches to recover anti-staphylococcal antibodies from humans are hampered because the immune response cannot be manipulated experimentally. The development of a transgenic mouse strain reconstituted with human immunoglobulin loci

has made it possible to generate fully human antibodies of desired therapeutic quality. For example, these transgenic mice reconstituted with human immunoglobulin loci can be immunized with the appropriate staphylococci SEIP or SEIP cocktails to produce anti- staphylococci immunoglobulins.

To reduce cost associated with large scale production of monoclonal antibodies advantage can be taken of phage display techniques to provide libraries containing a repertoire of antibodies with high affinities for the desired antigen. For production of such repertoires, it is necessary to immortalize the B cells from an immunized mouse expressing human immuoglobulins; the resulting B cells are then used as a source of <BR> <BR> DNA. The mixture of cDNAs obtained from B cells, e. g. , derived from spleens, are used to prepare an expression library, for example, a phage display library transfected into E. coli. The resulting cells are tested for immunoreactivity to the desired antigen. Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths et al. (1994). Ultimately, clones from the library are identified which produce binding affinities of a desired magnitude for the antigen, and the DNA encoding the product responsible for such binding is recovered and manipulated for standard recombinant expression. Phage display libraries may also be constructed using previously manipulated nucleotide sequences and screened in similar fashion. In general, the cDNAs encoding heavy and light chain are independently supplied or are linked to form Fv analogs for production in the phage library. The phage library is then screened for the antibodies with highest affinity for the antigen and the genetic material recovered from the appropriate clone. Further rounds of screening can increase the affinity of the original antibody isolated. The manipulations described above for recombinant production of the antibody or modification to form a desired analog can then be employed.

Where applicable the treatment of staphyloccoci infections with antibodies derived from laying hens immunized with the appropriate staphylococci SEIP or SEIP cocktails offers an attractive alternative as opposed the expensive production of human immunoglobulins. Chicken antibodies offer many advantages over mammalian antibodies. Laying hens are highly cost-effective as producers of antibodies compared with the mammals traditionally used for production. Furthermore, yolk antibodies show great acid and heat resistance. Extraction of yolk antibodies can be performed even on a large scale without costly investment, plus concentrating them from egg yolk is a

relatively straightforward process. The antibodies are not harmed by pasteurization. More importantly, the FDA regards egg antibodies as a food rather than a drug and has granted GRAS (generally accepted as safe) status. Finally, chicken antibodies have biochemical advantages over mammalian antibodies due to the phylogenetical differences between avian and mammalian species, resulting in increased sensitivity as well as a decreased background in immunological assays. In contrast to mammalian antibodies, chicken antibodies do not activate the human complement system nor will they react with rheumatoid factors, human anti-mouse IgG antibodies, or bacterial and human Fc (fragment crystallizable)-receptors (Carlander et al 2000).

Recently, it was discovered that the systemic effects of IgY relate to the absorption or translocation of fragments of orally administered antibody from the intestine into circulation. The IgY molecule is disassembled by naturally occurring enzymes in the intestine into binding fragments, which comprise peptides of the highly variable portion of the terminal domain of the antibody. The peptides of the highly variable portion of the antibody, the Fab chain, are taken into circulation. The constant, or Fc, portion of IgY is left in the intestine. Once in the circulation, these fragments randomly search out a pathogen with the matching lectan and neutralize it by binding to that site. These Fab moieties, unlike the Fc portion, do not elicit an allergic reaction, presumably because they are either too small or are unrecognized as foreign for some other reason. These Fab moieties can be added to the terminal end of the host's circulating globulin, wherein they are hidden from destruction but available for neutralization.

For therapeutic applications, the anti-staphylococci antibodies may be administered in a pharmaceutically acceptable dosage form. They may be administered by any means that enables the active agent to reach the desired site of action, for example, intravenously as by bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes.

The antibodies may be administered as a single dose or a series of treatments.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

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