MX2007004834A - Immunogenic compositions of staphylococcus epidermidis polypeptide and polynucleotide antigens - Google Patents

Abstract

The present invention relates to immunogenic compositions, comprising polypeptides isolated fromStaphylococcus epidermidis. The invention also relates to polynucleotides encodingStaphylococcus epidermidispolypeptides and their use in immunogenic compostions. In addition, the invention relates to methods of inducing an immune response in mammals againstStaphylococcus epidermidisandStaphylococcus aureususing immunogenic compostions of theStaphylococcus epidermidispolypeptides and polynucleotides. The invention also relates to methods for detectingStaphylococcus epidermidisin a biological sample.

Description

IMMUNOGENIC COMPOSITIONS OF POLYPEPTIDE ANTIGENS STAPHYLOCOCCUS EPIDERMIDIS The present invention relates to immunogenic compositions, comprising polypeptides isolated from Staphylococcus epidermidis. The invention also relates to polynucleotides encoding Staphylococcus epidermidis polypeptides and their use in immunogenic compositions. In addition, the invention relates to methods for inducing an immune response in mammals against Staphylococcus epidermidis and Staphylococcus aureus using immunogenic compositions of the polypeptides and polynucleotides Staphylococcus epidermidis. The invention also relates to methods for detecting Staphylococcus epidermidis in a biological sample.

BACKGROUND OF THE INVENTION Staphylococcus epidermidis is a major component of the normal microbial flora in humans in the skin and mucous membrane and is once considered a contaminant when grown from an infected patient. See Heilmann, C. and G. Peters, Biology and pathogenicity of Staphylococcus epidermidis, in Gram-positive pathogens, V.A. Fischetti, Editor. 2000, American Society for Microbiology: Washington, D.C. p. 442-449; von Eiff, C, et al., Lancet Infect Dis, 2 (11): p. 677-85 (2002). Now this is widely accepted as an opportunistic pathogen of great importance and the main cause of bloodstream infections in hospitals. See Am J Infect Control, 27: p. 520-32 (1999); Diekema, D.J., et al., Int J Antimícrob Agents, 20 (6): p. 412-8 (2002); Edmond, M.B., et al., Clin Infect Dis, 29 (2): p. 239-44 (1999). These infections are mainly associated with the presence of a continuous external polymer body such as a permanent catheter, prosthetic joint or prosthetic heart valve. See Heilmann, C. and G. Peters, Biology and pathogenicity of Staphylococcus epidermidis, in Gram-positive pathogens, V.A. Fischetti, Editor. 2000, American Society for Microbiology: Washington, D.C. p. 442-449; von Eiff, C, et al., Lancet Infect Dis, 2 (11): p. 677-85 (2002). The infection is thought to result from the introduction of Staphylcoccus epidermidis into the patient's skin following the insertion of the prosthetic device. Colonization and subsequent biofilm formation can lead to bacteremia with the potential for hematogenous dispersion to other sites in the body. These infections are often difficult to treat, arise from the reduced death of bacteria within a biofilm by antibiotics and also an increase in antibiotic resistance among clinical isolates. See Diekema, D.J., ef al., Int J Antimicrob Agents, 20 (6): p. 412-8 (2002); Edmond, M.B., et al., Clin Infect Dis, 29 (2): p. 239-44 (1999); Lewis, K., Antimicrob Agents Chemother, 45 (4): p. 999-1007 (2001); Raad, I. ef al., Clin Infect Dis, 26 (5): p. 1 182-7 (1998). It has been reported that Staphylococcus epidermidis with reduced susceptibility vancomycin. See Sanyal, D. and D. Greenwood, J Med Microbiol ,. 39 (3): p. 204-10 (1993); Sanyal, D., et al., Lancet, 337 (8732): p. 54 (1991). The difficult treatment of these infections necessitates the use of immunization as a means to prevent infection. Biofilm formation is a major virulence determinant for Staphylcoccus epidermidis infections. Therefore, research on surface proteins of Staphylcoccus epidermidis has focused on those proteins involved in biofilm formation. These proteins have been subdivided into groups based on their involvement in the two main stages of biofilm formation: 1) primary coupling, staphylococcal surface protein-1, autolysin (AtlE), Fbe (SdrG) and GehD and 2) cell accumulation bacterial, protein (Bhp) homologous Bap, accumulation of associated protein (AAP) and autolysin (AtlE). See von Eiff, C, et al., Lancet Infect Dis, 2002. 2 (11): p. 677-85; Vuong, C, er a /., J Infect Dis, 188 (5): p. 706-18 (2003); Veenstra, G.J., et al., J Bacteriol., 178 (2): p. 537-41 (1996); Rupp, M.E., et al., J Infect Dis, 183 (7): p. 1038-42 (2001); Hussain, M., et al., Infect Immun, 65 (2): p. 519-24 (1997); Nilsson, M., et al., Infect Immun, 66 (6): p. 2666-73 (1998); Davis, S.L., et al., J Biol Chem, 276 (30): p. 27799-805 (2001); and Bowden, M.G., ef al., J Biol Chem, 277 (45): p. 43017-43023 (2002). Comparatively, less effort has been exerted towards the identification of surface proteins expressed in the exposure to environmental factors within the host or those involved in host-parasite interactions. Staphylcoccus epidermidis must undergo a transition from commensal to pathogenic and must adapt to its microenvironment within the host. For a diner in the transition to pathogen must gain access to host tissue, mutate their senses to their environment, alter the expression of the gene so that it is able to evade the host's defenses, join and adhere to host factors, grow and divide in the presence of different nutrients and host defenses. The proteins on the bacterial surface make an initial contact with the new environment within the host. The many functions of these proteins include perception of the environment, collecting and transporting nutrients, defending against the host's immune system and binding host proteins. Proteins exposed to the surface can also serve as a point of contact or recognition by the host's immune system and can be targeted for a humoral immune response against the bacterium. Josefsson, E., et al., J Infect Dis, 184 (12): p. 1572-80 (2001); Swiatlo, E., et al., Infect Immun, 71 (12): p. 7149-53 (2003); Grifantini, R., et al., Nat Biotechnol, 20 (9): p. 914-21 (2002). Thus, there is an immediate need to identify promising candidates among the Staphylococcus epidermidis proteins for use in immunogenic compositions that induce an immune response to diseases caused by Staphylococcus epidermidis serotypes.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides an immunogenic composition comprising a polypeptide having an amino acid sequence that is chosen from one or more of SEQ ID NO: 1 to SEQ ID NO: 32, a biological equivalent thereof, or a fragment thereof. . In a particular embodiment, the polypeptide is immunoreactive with antibodies in the serum of rabbits infected with Staphylococcus epidermidis. In another embodiment, the polypeptide binds to one or more rabbit serum proteins. In certain embodiments, the immunogenic composition additionally comprises a pharmaceutically acceptable carrier. In other embodiments, the immunogenic compositions of the invention also comprise one or more adjuvants. In still another embodiment, the immunogenic composition additionally comprises a polysaccharide antigen of Staphylococcus epidermidis. In still another embodiment, the immunogenic composition additionally comprises a Staphylococcus aureus polypeptide or polysaccharide antigen. The present invention provides immunogenic compositions, comprising a polypeptide isolated from Staphylococcus epidermidis. The present invention provides an immunogenic composition comprising a Staphylococcus epidermidis polypeptide wherein the polypeptide further comprises heterologous amino acids. In a particular embodiment, the polypeptide is a fusion polypeptide. In another embodiment, the polypeptide is a recombinant polypeptide. In still another embodiment, the invention provides an immunogenic composition comprising a Staphylococcus epidermidis polypeptide wherein the polypeptide comprises a neutralizing epitope of Staphylococcus epidermidis. In a certain embodiment, the polypeptide is a lipoprotein. The present invention further provides immunogenic compositions, comprising a Staphylococcus epidermidis polypeptide, wherein the polypeptide is encoded by a polynucleotide comprising a nucleotide sequence having at least about 95% identity to a nucleotide sequence that is chosen from of SEQ ID NO: 33 to SEQ ID NO: 64 or a degenerate variant thereof, or a fragment thereof. In a particular embodiment, the polynucleotide sequence of Staphylococcus epidermidis is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 62, or a degenerate variant thereof, or a fragment of this. The present invention provides an immunogenic composition, wherein the polynucleotide is derived from Staphylococcus epidermidis. In a particular embodiment, the polynucleotide additionally comprises heterologous nucleotides. In another embodiment, the polynucleotide is in an expression vector. In yet another embodiment, the expression vector is a DNA plasmid. In a certain embodiment, the polynucleotide is a recombinant polynucleotide. In another embodiment, the polynucleotide is operably linked to one or more gene expression regulatory elements. In still another embodiment, the polynucleotide directs the expression of a neutralizing epitope of Staphylococcus epidermidis.

The invention also provides an immunogenic composition comprising a polypeptide of Staphylococcus epidermides encoded by a polynucleotide, wherein the immunogenic composition additionally comprises an agent that facilitates transfection. In a particular embodiment, said agent facilitating transfection is bupivicaine. The present invention also provides a method for inducing an immune response against Staphylococcus epidermidis which comprises administering to a mammal an immunogenic amount of a composition comprising: a polypeptide having an amino acid sequence that is chosen from one or more of SEQ ID NO: 1 to SEQ ID NO: 32 or a biological equivalent of this, or a fragment thereof, and a pharmaceutically acceptable carrier. In addition, the present invention provides a method for inducing an immune response against Staphylococcus epidermidis which comprises administering to a mammal an immunogenic amount of a composition comprising: a polynucleotide having a nucleotide sequence that is chosen from one or more of the SEQ ID NO: 33 to SEQ ID NO: 64, a degenerate variant thereof, or a fragment thereof and a pharmaceutically acceptable carrier. In one embodiment, the invention provides an immunogenic composition comprising a polynucleotide having a nucleotide sequence which is chosen from SEQ ID NO: 33 to SEQ ID NO: 64, a degenerate variant thereof, or a fragment of it is included in an expression vector. In another embodiment, the polynucleotide is derived from Staphylococcus epidermidis. In yet another embodiment the polynucleotide comprises heterologous nucleotides.

In a certain embodiment, the invention provides a method for the detection and / or identification of Staphylococcus epidermidis in a biological sample comprising: (a) contacting the sample with an oligonucleotide probe of a polynucleotide comprising the nucleotide sequence which is chosen from SEQ ID NO: 33 to SEQ ID NO: 64, or a degenerate variant thereof, or a fragment thereof, under conditions that allow hybridization; and (b) detecting the presence of hybridization complexes in the sample, wherein the hybridization complexes indicate the presence of Staphylococcus epidermidis in the sample. In other embodiments, the invention provides a method for the detection and / or identification of antibodies with Staphylococcus epidermidis in a biological sample comprising: (a) contacting the sample with a polypeptide comprising an amino acid sequence that is chosen from from SEQ ID NO: 1 to SEQ ID NO: 32 or a biological equivalent thereof, or a fragment thereof, under conditions that allow immune complex formation; and detect the presence of immune complex in the sample, where immune complex indicates the presence of Streptococcus pneumoniae in the sample. In a particular embodiment, the immunogenic composition comprises a Staphylococcus epidermidis polypeptide sequence that is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, one equivalent biological of this, or a fragment of it. In another embodiment, the immunogenic composition comprises a Staphylococcus epidermidis polynucleotide sequence that is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 62, or a degenerate variant of this, or a fragment of it. In yet another embodiment, the invention provides a method for inducing an immune response against Staphylococcus aureus which comprises administering to a mammal an immunogenic amount of a composition comprising: a Staphylococcus epidermidis polypeptide sequence that is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO : 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, a biological equivalent of this, or a fragment thereof. In a particular embodiment, the invention provides a method for inducing an immune response against Staphylococcus aureus which comprises administering to a mammal an immunogenic amount of a composition comprising: a Staphylococcus epidermidis polynucleotide sequence that is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 62, or a degenerate variant thereof, or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts protein expression profiles of cell wall fractions of S. epidermidis 0-47 growing in TSB (1A and 1 C) or 70% rabbit serum (1 B and 1 D), which are compared by 2D gel electrophoresis. Proteins are separated in IPG strips of pH 4-7 in the first dimension followed by SDS-PAGE in the second dimension, transferred to nitrocellulose and detected by fluorescent strain (1A and 1 C). The immunoreactive proteins are visualized with immune serum (1 B and 1 D) of rabbits infected with S. epidermidis 0-47. Label molecular weight markers are labeled on the left. Figure 2 depicts fluorescent staining blot (2A) and immunoblot (2B) of a cell surface fraction of S. epidermidis 0-47 growing in 70% rabbit serum separated into IPG strips of pH 4-7 in the first dimension and SDS-PAGE in the second dimension. The proteins in the spots are identified by mass spectrometric analysis. Figure 3 describes proteins, which are eluted from the surface of S. epidermidis0-47 that grows in TSB or 70% rabbit serum with increased concentrations of NaCl or 4.0 M of urea. Asterisks indicate enriched proteins eluted from the surface of S. Epi that grow in the presence of serum. The bacteria are washed 3X with TBS then sequentially with 0.5 M and 1.0 M NaCl and 4.0 M urea. Protein concentrations are determined for each of the samples and 0.75 g runs on 4-20% gradient gel. No protein is detected by the protein assay in samples eluted from the surface of bacteria growing in TSB (lines 2-5), such that 25 μ? They are loaded in the gel. Line 1, rabbit serum; Lines 2 and 6 - 0.15 M of NaCl eluate; Lines 3 and 7 - 0.5 M NaCl, Lines 4 and 8 - 1.0 M NaCl; line 5 and 9 - 4.0 M urea. Figure 4 depicts 2D cell surface transfer proteins of S. epidermidis, which is fluorescently stained for protein (4A) and probed with biotinylated whey proteins (4B) eluted from S. epidermidis that grow in 70% serum rabbit. The spots are visualized with a streptavidin-alkaline phosphatase conjugate. The proteins in the spots are identified by mass spectroscopy.

DETAILED DESCRIPTION OF THE INVENTION After exposure of the host's bloodstream, the invading bacteria find a specific indicator environment for the new environment. These indicators are detected by bacteria and adaptive signal changes in protein expression that may be detectable in purified cell wall proteins. Often, proteins and carbohydrates in bacterial cell walls are candidates for the inclusion of immunogenic compositions to treat or prevent bacterial infections. If a positively regulated protein interacts with the host or plays a role in nutrient acquisition, it is important for the bacteria and therefore plays a role in bacterial survival and pathogenesis. The growth of the bacteria in bodily fluids (ie serum, peritoneal dialysate fluids, and urine) has been used as a model system to mimic some of the bacterial signals found within the host. See Wiltshire, M.D. and S.J. Foster, Infect Immun, 69 (8): p. 5198-202 (2001); Shepard, B.D. and M.S. Gilmore, Infect Immun, 70 (8): p. 4344-52 (2002); Smith, D.G., et al., Infect Immun, 59 (2): p. 617-24 (1991); McDermid, K.P., et al., Infect Immun, 61 (5): p. 1743-9 (1993). One or more of these culture conditions is found to alter gene expression in Enterococcus faecalis, S. aureus and Staphylcoccus epidermidis and those proteins identified to be increased in expression under altered culture conditions are found to belong to different classes of proteins that have a variety of functions. See Wiltshire, M.D. and S.J. Foster, Infecí Immun, 69 (8): p. 5198-202 (2001); Shepard, B.D. and M.S. Gilmore, Infect Immun, 70 (8): p. 4344-52 (2002). The most common predisposing factor for an infection with Staphylcoccus epidermidis is the implantation of a prosthetic device. An implanted prosthetic device becomes coated with plasma and the matrix proteins include fibrinogen, vitronectin, von Willebrand factor and fibronectin. See von Eiff, C, et al.,. Eur J Clin Microbiol Infect Dis, 18 (12): p. 843-6 (1999). These proteins often act as ligands for surface proteins of Staphylococcal epidermidis, thus allowing the bacteria to attach and colonize the prosthetic device. Staphylcoccus epidermidis is known to express proteins that bind to fibrinogen, vitronectin and fibronectin. See Nilsson, M., er al., A fibrinogen-binding protein of Staphylococcus epidermidis. Infect Immun, 66 (6), p. 2666-73 (1998); Davis, S.L., et al., J Biol Chem, 276 (30), p. 27799-805 (2001); Williams, R.J., er al., Infect Immun ,. 70 (12), p. 6805-10 (2002); Heilmann, C, ef al., Mol Microbiol, 24 (5), p. 1013-24 (1997). It is reasonable to expect that Staphylcoccus epidermidis binds additionally to whey proteins in the transition from commensal to pathogenic. The invention described herein is directed to the need for immunogenic compositions for Staphylococcus epidermidis that effectively prevent or treat most or all of the disease caused by Staphylococcus epidermidis serotypes. The invention is further directed to the need for diagnostic methods of infection by infection with Staphylococcus epidermidis. The present invention has identified open reading frames for Staphylococcus epidermidis hereinafter ORF, which encode antigenic polypeptides. More particularly, the polypeptides encoding the ORF Staphylococcus epidermidis serve as potential antigenic polypeptides in immunogenic compositions. In certain embodiments, the invention comprises ORF polynucleotide Staphylococcus epidermidis that encodes the localized, exposed, secreted surface or membrane associated with polypeptide antigens. In other embodiments, the invention comprises vectors comprising ORF sequences and host cells or animals transformed, transfected or infected with these vectors. The invention also encompasses ORF transcriptional gene products Staphylococcus epidermidis, such as, for example, mRNA, antisense RNA, antisense oligonucleotides and ribosome molecules, which can be used to inhibit or control the growth of microorganisms. The invention also relates to methods for detecting these nucleic acids or polypeptides and kits for diagnosing Staphylococcus epidermidis infection. The invention also relates to immunogenic compositions for the prevention and / or treatment of bacterial infection, in particular infection caused by or exacerbated by Staphylococcus epidermidis. In particular embodiments the immunogenic composition is used for the treatment or prevention of systemic diseases, which are induced or exacerbated by Staphylococcus epidermidis. In other embodiments, the immunogenic compositions are used for the treatment or prevention of non-systemic diseases, which are induced or exacerbated by Staphylococcus epidermidis.

A. Staphylococcus epidermidis ORF polypeptides and polynucleotides Purified and isolated Staphylococcus epidermidis ORF polynucleotides are identified which are used in the production of Staphylococcus epidermidis polypeptides for inclusion in immunogenic compositions. More specifically, in certain embodiments, the ORF encodes the surface of localized, exposed Staphylococcus epidermidis, the associated membrane or secreted polypeptides, particularly antigenic polypeptides. Thus, in one aspect, the present invention identifies purified and isolated polynucleotides (ORFs) that encode the surface of localized, exposed Staphylococcus epidermidis, the associated membrane or secreted polypeptides for inclusion in immunogenic compositions. In particular embodiments, a polynucleotide of the present invention is a DNA molecule, wherein the DNA can be genomic DNA, chromosomal DNA, plasmid DNA or cDNA. In another embodiment, a polynucleotide is a recombinant polynucleotide, which encodes a Staphylococcus epidermidis polypeptide comprising an amino acid sequence having at least 95% identity with an amino acid sequence of one of SEQ ID NO: 1 to SEQ ID NO: 32 or a fragment of it. In another embodiment, an isolated and purified ORF polynucleotide comprises a nucleotide sequence having at least 95% identity with one of the ORF nucleotide sequences of SEQ ID NO: 33 to SEQ ID NO: 64, a degenerate variant of this, or a complement of this. In one embodiment, an ORF polynucleotide of one of SEQ ID NO: 33 to SEQ ID NO: 64 is comprised in a plasmid vector and expressed in a prokaryotic host cell. As used herein, the term "polynucleotide" means a nucleotide sequence connected by phosphodiester linkages. The polynucleotides are presented hereinafter in the 5 'to 3' direction. A polynucleotide of the present invention may comprise from about 10 to about several hundred moles of base pairs. In one embodiment, a polynucleotide comprises from about 10 to about 3,000 base pairs. Example lengths of particular polynucleotides are set forth below.

A polynucleotide as described herein may be a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or DNA or RNA analogs generated using nucleotide analogs. The nucleic acid molecule can be single helix or double helix, but preferably it is double helix DNA. When a polynucleotide is a DNA molecule, this molecule can be a gene, a cDNA molecule or a genomic DNA molecule. The nucleotide bases are indicated hereinafter by a single code letter: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U). "Isolated" means altered "altered by the hand of man" from the natural state. If a composition or substance occurs in nature, in order to be considered "isolated" it should have been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is used hereafter. As used herein, an "isolated" polynucleotide is free of sequences that naturally flank the nucleic acid (ie, sequences located at the 5 'and 3' ends of nucleic acid) in the genomic DNA of an organism for which the nucleic acid is derived. For example, in various embodiments, the isolated Staphylococcus epidermidis nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences that naturally flank the molecule of nucleic acid in the genomic DNA of the cell from which the nucleic acid is derived. However, the nucleic acid molecule of Staphylococcus epidermidis can be fused to another protein encoding or regulatory sequences and still be considered isolated.

The ORF polynucleotides and the polypeptides thus described herein can be obtained using standard selection and cloning techniques from a cDNA library derived from mRNA. The polynucleotides of the invention can also be obtained from natural sources such as libraries of genomic DNA (eg, a library of Staphylococcus epidermidis) or can be synthesized using commercially available and well-known techniques. Also encompassed herein are nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 33 to SEQ ID NO: 64 (and fragments thereof) due to the degeneracy of the genetic code and thus the encoding of the same polypeptide Staphylococcus epidermidis as that encoded by the nucleotide sequence shown in SEQ ID NO: 33 to SEQ ID NO: 64. Allelic and orthologous variants of the Staphylococcus epidermidis polynucleotides can be easily identified using methods well known in the art. The allelic and orthologous variants of the polynucleotides will comprise a nucleotide sequence that is typically at least about 70-75%, more typically at least about 80-85%, and more typically at least about 90-95% or more homologous to the sequence of nucleotide shown in SEQ ID NO: 33 to SEQ ID NO: 64, or a fragment of these nucleotide sequences. Such nucleic acid molecules can be easily identified by being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ ID NO: 33 to SEQ ID NO: 64, or a fragment of these nucleotide sequences. Moreover, the polynucleotides may comprise only one fragment of the coding region of a Staphylococcus epidermidis polynucleotide or gene, such as a fragment of one of SEQ ID NO: 33 to SEQ ID NO: 64. In certain embodiments, such fragments encode immunogenic fragments.

When these ORF Staphylococcus epidermidis polynucleotides of the invention are used for the recombinant production of Staphylococcus epidermidis polypeptides for inclusion in immunogenic compositions, the polynucleotide can include the coding sequence for the mature polypeptide, itself, or the coding sequence for the mature polypeptide in the reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other portions of fusion peptide. For example, a marker sequence that facilitates the purification of the fused polypeptide that can be ligated to the coding sequence (see Gentz et al., Proc. Nati, Acad. Sci. USA, 86: 821-824, 1989, incorporated as reference here in its entirety). Thus, the preparation of fusion polypeptides encoding polynucleotides that allow His-tag purification of expression products is contemplated herein. The polynucleotide may also contain 5 'and 3' non-coding sequences, such as transcribed, untranslated sequences, polyadenylation and splicing signals. Thus, a polynucleotide that encodes a polypeptide for inclusion in immunogenic compositions of the present invention, which includes homologs and orthologs of species other than Staphylococcus epidermidis, such as Staphylococcus aureus can be obtained by a process comprising the steps of selecting a library suitable under stringent hybridization conditions with a labeled probe having the sequence of one of SEQ ID NO: 33 to SEQ ID NO: 64, a fragment thereof; and isolating genomic clones and full length cDNA containing the polynucleotide sequence. Such hybridization techniques are well known to the person skilled in the art. The person skilled in the art will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in the coding region for the polypeptide that is short at the 5 'end of the cDNA. This is a consequence of the transcriptase, inversely, an enzyme inherently low in "processability" (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction), which fails to complete a DNA copy of the mRNA template during the first synthesis of cDNA helix. Thus, in certain embodiments, the polynucleotide sequence information provided herein allows the preparation of relatively short DNA oligonucleotide sequence (or RNA) having the ability to specifically hybridize to the gene sequences of the selected polynucleotides described hereinafter. The term "oligonucleotide" as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, usually more than three (3), and typically more than ten (10) and up to one hundred (100) or more (though preferably between twenty and thirty). The exact size will depend on many factors, which instead depend on the last function or use of the oligonucleotide. Thus, in particular embodiments, the nucleic acid probe of an appropriate length is prepared based on a consideration of a selected nucleotide sequence, for example, a sequence such as that shown in SEQ ID NO: 33 to SEQ ID NO: 64 The ability of such a nucleic acid probe to specifically hybridize to a polynucleotide that encodes a Staphylococcus epidermidis polypeptide lends itself particular utility in a variety of modalities. More importantly, the probes can be used in a variety of assays to detect the presence of complementary sequences in a given sample. In certain embodiments, it is advantageous to use oligonucleotide primers. These primers can be generated in any form, including chemical synthesis, DNA replication, reverse transcription, or a combination of these. The sequence of such primers is designated using a polynucleotide described herein for use in the detection, amplification or mutation of a defined segment of an ORF polynucleotide that encodes a Staphylococcus epidermidis polypeptide of prokaryotic cells using polymerase chain reaction (PCR) technology. . In certain embodiments, it is advantageous to employ a polynucleotide described herein in combination with an appropriate tag to detect hybrid formation. A wide variety of appropriate labels are known in the art, including radioactive, enzymatic or other ligands, such as avidin / biotin, which are capable of giving a detectable signal. Polynucleotides that are identical or sufficiently identical to a nucleotide sequence contained in one of SEQ ID NO: 33 to SEQ ID NO: 64, or a fragment thereof, can be used as hybridization probes for cDNA and genomic DNA or as initiators for a nucleic acid amplification reaction (PCR), to isolate full-length cDNA and genomic clones encoding polypeptides described herein and to isolate the cDNA from genomic clones of other genes (which include genes encoding homologs and orthologs of species) different from Staphylococcus epidermidis) having a high sequence similarity to the polynucleotide sequences set forth in SEQ ID NO: 33 to SEQ ID NO: 64, or a fragment thereof. Typically, these nucleotide sequences are at least about 70% identical to at least about 95% identical to that of the reference polynucleotide sequence. The probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.

There are several methods available and well known to those skilled in the art for obtaining full-length cDNA, or short extended cDNA, for example those based on the Rapid Amplification of cDNA ends (RACE) method. See Frohman et al., Proc. Nati Acad. Sci. USA 85, 8998-9002, 1988. Recent modifications of the technique, exemplified by Marathon ™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for major cDNAs. In Marathon ™ technology, the cDNA has been prepared from mRNA extracted from a chosen tissue and an "adapter" sequence linked at each end. Nucleic acid amplification (PCR) is then carried out to amplify the missing 5 '"end of the cDNA using a combination of specific adapter oligonucleotide and specific gene primers. The PCR reactions are then repeated using "nested" primers, that is, primers designated for hardening within the amplified product (typically a specific adapter initiator that further hardens 3 'in the adapter sequence and a gene-specific primer that additionally hardens 5' in the known gene sequence). The products of this reaction can then be analyzed by DNA sequencing and full-length cDNA constructed by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the initiator 5 '. To provide certain of the advantages according to the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays include probe molecules that are complementary to at least about 10 to about 70 long-leg nucleotides of a polynucleotide encoding a Staphylococcus epidermidis polypeptide, such as that shown in one of SEQ ID NO: 33 to SEQ ID NO: 64. A size of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred though, in order to increase the stability and selectivity of the hybrid, and therefore improve the quality and the degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having a complementary stretch of gene of 25 to 40 nucleotides, 55 to 70 nucleotides, or a greater where desired. Such fragments can be easily prepared, for example, by directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as PCR technology (US Pat. No. 4,683,202, incorporated herein by reference) or by excising DNA fragments. selected from recombinant plasmids containing appropriate inserts and suitable enzyme restriction sites. In another embodiment, it is contemplated that an isolated and purified polynucleotide comprises a nucleotide sequence that is identical or complementary to a segment of at least 10 continuous bases of SEQ ID NO: 33 to SEQ ID NO: 64, wherein the polynucleotide hybridizes to a polynucleotide that encodes a Staphylococcus epidermidis polypeptide. Preferably, the purified and isolated polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 to about 70 continuous bases of SEQ ID NO: 33 to SEQ ID NO: 64. For example, the polynucleotide may comprise base segments identical or complementary to 40 or 55 continuous bases of the nucleotide sequences described.

Accordingly, a polynucleotide probe molecule can be used for its ability to selectively form duplex molecules with a complementary stretch of the gene. Depending on the proposed request, one would like to employ variant hybridization conditions to achieve a degree of selectivity variation of the probe towards the target sequence (see Table 1 below). For applications that require a high degree of selectivity, one would typically want to employ relatively stringent conditions to form the hybrids. For some applications, for example, when one wishes to prepare mutants employing a mutant initiator helix hybridized to a prominent template or where one seeks to isolate a polypeptide coding sequence of Staphylococcus epidermidis homologous from other cells, functional equivalent, or the like, less Stringent hybridization conditions are typically necessary to allow heteroduplex formation (see Table 1). The cross-hybridized species can therefore be easily identified as hybridization signals positively with respect to control hybridizations. Thus, hybridization conditions are easily manipulated, and thus will generally be a method of choice depending on the desired results. For some applications, for example, when one wishes to prepare mutants employing a hybridized mutant primer helix for a prominent template or when one seeks to isolate an homologous polypeptide coding sequence from other cells, functional equivalents, or the like, fewer hybridization conditions strictures are typically necessary to allow heteroduplex formation. The cross hybridization species are therefore easily identified as positive hybridization signals with respect to control hybridizations. In any case, it is generally appreciated that the conditions may be more stringent by the addition of increased amounts of formamide, which serve to destabilize the hybrid duplex in the same way as the temperature increases. Thus, hybridization conditions are easily manipulated, and thus will generally be a method of choice depending on the desired results. Also described herein are polynucleotides capable of hybridizing under stringent stringent conditions, more preferably stringent conditions, and more preferably highly stringent conditions, for polynucleotides described hereafter. Examples of stringent conditions are shown in Table 1 below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; Strict conditions are at least as strict as, for example, G-L conditions; and stringent stringent conditions are at least as strict as, for example, M-R conditions.

Table 1 Strict Conditions (bp) 1: the hybrid length is that anticipated by the hybridized regions of the hybridization polynucleotides. When a polynucleotide is hybridized to a target polynucleotide of an unknown sequence, the length of the hybrid is assumed to be that of the hybridization polynucleotide. When the polynucleotides of the known sequences are hybridized, the length of the hybrid can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. Shock absorber ": SSPE (IxSSPE is 0.15M NaCl, 10mM NaH2P04, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffer The washes are developed for 15 minutes after the hybridization is complete TB to TR: the hybridization temperature for hybrids anticipated to be less than 50 base pairs in length must be 5-10 ° C less than the fusion temperature ( Tm) of the hybrid, where Tm is determined according to the following equations: For hybrids less than 18 base pairs in length, Tm (° C) = 2 (# of A + T bases) + 4 (# of G + C) bases) For hybrids between 18 and 49 base pairs in length, Tm (° C) = 81.5 + 16.6 (log10 [Na +]) + 0.41 (% G + C) - (600 / N), where N is the number of bases in the hybrid, and [Na *] is the concentration of sodium ions in the hybridization buffer ([Na +] for 1xSSC = 0. 165 M).

Additional examples of stringent conditions for polynucleotide hybridization are provided in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Ausubel et al., 1995, Current Protocols in Molecular Biology, eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.

B. Staphylococcus epidermidis polypeptides In particular embodiments, the present invention provides purified and purified Staphylococcus epidermidis polypeptides for use in immunogenic compositions. Preferably, a Staphylococcus epidermidis polypeptide used in an immunogenic composition of the invention is a recombinant polypeptide. In certain embodiments, a Staphylococcus epidermidis polypeptide comprises the amino acid sequence having at least 95% identity to the amino acid sequence of one of SEQ ID NO: 1 to SEQ ID NO: 32, a biological equivalent thereof, or a fragment of this. A Staphylococcus epidermidis polypeptide used in an immunogenic composition of the present invention encompasses a polypeptide comprising: 1) the amino acid sequence shown in one of SEQ ID NO: 1 to SEQ ID NO: 32; 2) variants of natural functional and non-functional occurrence or biological equivalents of Staphylococcus epidermidis polypeptides of SEQ ID NO: 1 to SEQ ID NO: 32; 3) recombinantly produced variants or biological equivalents of Staphylococcus epidermidis polypeptides of SEQ ID NO: 1 to SEQ ID NO: 32; and 4) polypeptides isolated from organisms other than Staphylococcus epidermidis (Staphylococcus epidermidis polypeptide orthologs).

A biological equivalent or variant of such a Staphylococcus epidermidis polypeptide encompasses 1) a polypeptide isolated from Staphylococcus epidermidis; and 2) a polypeptide that contains substantially homology to a Staphylococcus epidermidis polypeptide. Biological equivalents or variants of Staphylococcus epidermidis include functional and non-functional polypeptides of Staphylococcus epidermidis. Functional biological equivalents or variants are naturally occurring amino acid variants of a Staphylococcus epidermidis polypeptide that maintains the ability to elicit an immunological or antigenic response in a subject. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 1 to SEQ ID NO: 32, or substitution, deletion or insertion of non-critical residues in non-critical regions of the polypeptide (eg, not in regions containing antigenic determinants or protective epitopes). The present invention additionally provides non-Staphylococcus epidermidis orthologs of Staphylococcus epidermidis polypeptides. Staphylococcus epidermidis polypeptide orthologs are polypeptides that are isolated from non-Staphylococcus epidermidis organisms and possess antigenic capacity of Staphylococcus epidermidis polypeptide. Orthotics of a Staphylococcus epidermidis polypeptide can be easily identified since it comprises an amino acid sequence that is substantially homologous to one of SEQ ID NO: 1 to SEQ ID NO: 32. Changes and modifications can be made to the structure of a polypeptide of the present invention and still obtain a molecule having antigenicity for Staphylococcus epidermidis. For example, certain amino acids can be substituted by other amino acids in a sequence without appreciable loss of antigenicity. Due to the ability and interactive nature of a polypeptide that defines the biological functional activity of the polypeptide, certain amino acid sequence distributions can be made in a polypeptide sequence (or, of course, its DNA coding sequence highlighted) and not However, obtain a polypeptide with similar properties. In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring an interactive biological function on a polypeptide is generally understood in the art (Kyte and Doolittle, J Mol Biol, 157: p.105-132, 1982). It is known that certain amino acids can be substituted by other amino acids having a similar hydropathic index or classification and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and loading characteristics. Those indices are: isoleucine (+4.5); valina (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid residue determines the secondary and tertiary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtains a functionally equivalent polypeptide. In such changes, substitution of amino acids whose hydropathic indices are within + 1-2 is preferred, those that are within +/- 1 are particularly preferred, and those within +/- 0.5 are even more particularly preferred.

Similar amino acid substitutions can also be made on the basis of hydrophilicity, particularly when the biological functional equivalent polypeptide or polypeptide so created is intended for use in immunological modalities. U.S. Patent 4,554,101, incorporated herein by reference, states that the average higher hydrophilicity of a polypeptide, governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, ie with a biological property of the polypeptide. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); Alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tripiptofan (-3.4). It is understood that an amino acid can be substituted by another having a similar hydrophilicity value and still obtain a biological equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, substitution of amino acids whose hydrophilicity values are within ± 2 are preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. As noted above, amino acid substitutions are therefore generally based on the relative similarity of the side chain amino acid substituents, eg, their hydrophobicity, hydrophilicity, charge, size, and the like. Example substitutions which take several of the above characteristics into consideration well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and soleucine (See Table 2, below). The present invention thus contemplates immunogenic compositions comprising biological or functional equivalents of a Staphylococcus epidermidis polypeptide as set forth below. Table 2 Amino acid substitutions Biological or functional equivalents of a polypeptide can also be prepared using site-specific mutagenesis. Site-specific mutagenesis is a useful technique in the preparation of second generation polypeptides, or equivalent biologically functional peptides or polypeptides, derived from their sequences, through specific DNA-encoding mutagenesis. As noted above, such changes may be desirable where amino acid substitutions are desired. The technique additionally provides a ready-to-use ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more changes of nucleotide sequences in the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to deliver an initial sequence of sufficient size and complexity of sequence to form a stable duplex on both sides of the elimination junction that is transverse. Typically, an initiator of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

In general, the site-specific mutagenesis technique is well known in the art. As will be appreciated, the technique typically employs a phage vector that can exist in a single helix and double helix shape. Typically, the site-directed mutagenesis according to this is developed by first obtaining a double helix vector that includes within its sequence a DNA sequence encoding all or a portion of the selected Staphylococcus epidermidis polypeptide sequence. An oligonucleotide primer carrying the desired mutated sequence is prepared (eg, synthetically). This initiator is then hardened with the simple helix vector, and extended by the use of enzymes such as Klenow I fragment of E. coli polymerase, in order to complete the synthesis of the helix carrying the mutation. Thus, a heteroduplex is formed in which one strand encodes the original non-mutated sequence and the second helix carries the desired mutation. This heteroduplex vector is then used to transform appropriate cells such as E. coli cells and the clones are selected which include recombinant vectors carrying the mutation. Commercially available kits come with all the necessary reagents, except oligonucleotide primers. A Staphylococcus epidermidis polypeptide or polypeptide antigen used in an immunogenic composition of the present invention is understood to be any Staphylococcus epidermidis polypeptide that comprises substantial sequence similarity, structural similarity and / or functional similarity to a Staphylococcus epidermidis polypeptide comprising the sequence of amino acid of one of SEQ ID NO: 1 to SEQ ID NO: 32. Additionally, such a polypeptide of Staphylococcus epidermidis or polypeptide antigen is not limited to a particular source. Thus, the invention provides general detection and isolation of polypeptides from a variety of sources.

It is contemplated in the present invention, that a Staphylococcus epidermidis polypeptide can advantageously be cleaved into fragments for use in further functional or structural analysis, or in the generation of reagents such as polypeptides related to Staphylococcus epidermidis and antibodies specific for Staphylococcus epidermidis. This can be achieved by treating purified or unpurified Staphylococcus epidermidis polypeptides with a peptidase such as endoproteinase glu-C (Boehringer, Indianapolis, IN). Treatment with CNBr is another method by which peptide fragments can be produced from natural Staphylococcus epidermidis polypeptides. Recombinant techniques can also be used to produce specific fragments of a Staphylococcus epidermidis polypeptide.

Fragments of Staphylococcus epidermidis polypeptides are also included in the immunogenic compositions of the invention. A fragment is a polypeptide having an amino acid sequence that is completely the same as part, but not all, of the amino acid sequence. The fragment may comprise, for example, at least 7 or more contiguous amino acids (eg, 8, 10, 12, 14, 16, 18, 20, or more) of an amino acid sequence of one of SEQ ID NO: 1 to SEQ ID NO: 32. The fragments can be "independent" or comprised within a larger polypeptide of which they form a part or region, more preferably as a single, continuous region. In one embodiment, the fragments include at least one epitope of the mature polypeptide sequence. "Fusion protein" refers to a protein or polypeptide encoded by two, often unrelated, fused genes or fragments thereof. For example, fusion proteins or polypeptides comprising several portions of constant region immunoglobulin molecules together with another human protein or part thereof have been described. In many cases, employing an immunoglobulin Fe region as a part of a fusion protein or polypeptide is advantageous for use in therapy and diagnosis which results in, for example, improved pharmacokinetic properties (see for example, International Application EP-A 0232 2621). On the other hand, for some uses it is desirable to be able to remove the Fe part after the polypeptide or fusion protein has been expressed, detected and purified. It is contemplated that the Staphylococcus epidermidis polypeptides can be isolated from Staphylococcus epidermidis or recombinantly prepared as described herein.

C. STAPHYLOCOCCUS POLYPEPTIDE AND POLYCYCLEOTIDE VARIANT EPIDERMIDIS "Variant" as the term is used hereinafter, is a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide respectively, but retains essential properties. A typical variant of a polynucleotide differs from a nucleotide sequence to another, polynucleotide reference. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by reference polynucleotide. The nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in one amino acid sequence from another, reference polypeptide. In general, the differences are limited such that the sequences of the reference polypeptide and the variant are closely similar and, in many regions, identical. A reference polypeptide and variant may differ in the amino acid sequence by one or more substitutions, additions, deletions in any combination. An inserted or substituted amino acid residue may or may not be encoded by the genetic code. A variant of a polynucleotide or polypeptide can be of natural occurrence such as an allelic variant, or this can be a variant that is known to be of natural occurrence. Variants of unnatural occurrence of polynucleotides and polypeptides can be made by mutagenesis techniques or by direct synthesis. "Identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotides, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relationship between the polynucleotide or polypeptide sequences, as the case may be, as determined by the case between the helices of such sequences. "Identity" and "similarity" can be easily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988) Identity determination methods are designed to give the largest case among the tested sequences.The methods for determining identity and similarity are encoded in computer programs. Only available: computer program methods to determine identity and similit Those between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12 (1): 387, 1984), BLASTP, BLASTN, TBLASTN and FASTA (Altschul et al., J Molec. Biol. 215: 403-410, 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul er a /., J. Molec. Biol. 215: 403-410 , 1990.). The well-known Smith-Waterman algorithm can also be used to determine identity. By way of example, a polynucleotide sequence described herein may be identical to the reference sequence of one of SEQ ID NO: 33 to SEQ ID NO: 64, which is 100% identical, or it may include up to a certain number whole of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, which includes transition and transversion, or insertion, and wherein said alterations may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually between the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in one of SEQ ID NO: 33 to SEQ ID NO: 64 by the numerical percentage of the respective identity percentage (divided by 100) and subtracting that product from said total number of nucleotides in one of SEQ ID NO: 33 to SEQ ID NO: 64. For example, an isolated Staphylococcus epidermidis polynucleotide comprising a nucleotide sequence having at least 70% identity with the acid sequence nucleic of one of SEQ ID NO: 33 to SEQ ID NO: 64; a degenerate variant of this or a fragment thereof, wherein the polynucleotide sequence may include up to n "alterations of nucleic acid over the entire polynucleotide region of the nucleic acid sequence of one of SEQ ID NO: 33 to SEQ ID NO: 64, where nn is the maximum number of alterations and is calculated by the formula: nn < xn- (xn «y), wherein x "is the total nucleic acid number of one of SEQ ID NO: 33 to SEQ ID NO: 64 and has a value of 0.70, wherein any integer product of x" yy is rounded to the nearest whole before subtracting such product from x ". Of course, and can also have a value of 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, etc. Alterations of a polynucleotide sequence encoding one of the polypeptides of SEQ ID NO: 1 to SEQ ID NO: 32 can create read frame shift, nonsense mutation or nonsense mutation in this coding sequence and thus both alter the polypeptide encoded by the polynucleotide after such alterations. Similarly, a polypeptide sequence described herein may be identical to the reference sequence of SEQ ID NO: 1 to SEQ ID NO: 32, which is 100% identical, or it may include up to a certain number of alterations of amino acid as compared to the reference sequence such that% identity is less than 100%. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the polypeptide sequence. reference or anywhere between those terminal positions, intermixed either individually between the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a percent identity is determined by multiplying the total number of amino acids in one of SEQ ID NO: 1 to SEQ ID NO: 32 by the numerical percentage of the respective identity percentage (divided by 100) and then subtracting that product from said total number of amino acids in one of SEQ ID NO: 1 to SEQ ID NO: 32, or: where na is the number of amino acid alterations, xa is the total number of amino acids in one of SEQ ID NO: 1 to SEQ ID NO: 32, and y is, for example, 0.70 for 70%, 0.80 for 80 %, 0.85 for 85% etc., and where any non-integer product of xa and y is rounded to the nearest integer before subtracting it from xa.

D. Vectors, Host Cells and Polypeptides Staphylococcus epidermidis Recombinant In one embodiment, the present invention provides expression vectors comprising ORF polynucleotides that encode Staphylococcus epidermidis polypeptides for use in immunogenic compositions. Staphylococcus epidermidis expression vectors comprise ORF polynucleotides encoding Staphylococcus epidermidis polypeptides comprising the amino acid residue sequence of one of SEQ ID NO: 1 to SEQ ID NO: 32. Alternatively, expression vectors comprise a polynucleotide comprising the nucleotide base sequence of one of SEQ ID NO: 33 to SEQ ID NO: 64. In other embodiments, the expression vectors of the invention comprise a polynucleotide operably linked to a promoter-enhancer. In yet other embodiments, the expression vectors comprise a polynucleotide operably linked to a prokaryotic promoter. Alternatively, the expression vectors comprise a polynucleotide operably linked to an enhancer promoter that is a eukaryotic promoter. The expression vectors additionally comprise a polyadenylation signal that is positioned 3 'to the carboxy terminal amino acid and within a transcriptional unit of the encoded polypeptide. Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing inducible or constitutive promoters that direct the expression of fusion or non-fusion proteins. The fusion vectors add a number of amino acids to a protein encoded there, usually upon terminating amino of the recombinant protein. Such fusion vectors typically serve three purposes: 1) increase the expression of the recombinant protein; 2) increase the solubility of the recombinant protein; and 3) aid in the purification of the recombinant protein by acting as a ligand in the affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion portion and the recombinete protein to allow separation of the recombinant protein from the subsequent fusion portion for protein purification. fusion. Such enzymes, and their cognate recognition sequences, include factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc., Smith and Johnson, Gene 67: 31-40, 1988), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuses glutathione S-transferase (GST), maltose E binding protein or protein A, respectively, for the target recombinant protein. In one embodiment, the coding sequence of the polynucleotide Staphylococcus epidermidis is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from N-terminus to C-terminus, GST-thrombin cleavage of polypeptide site-Sfapfty / ococcus epidermidis. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant polypeptide Staphylococcus epidermidis not fused to GST that can be recovered by cleavage of the fusion protein with thrombin. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69: 301-315, 1988), pET lid (Studier et al., "Gene Expression Technology" Methods in Enzymology 185, 60- 89, 1990), pBAD and pCRT7. The expression of the target gene of the pTrc vector rests on the transcription of RNA polymerase host of a hybrid trp-lac fusion promoter. The target gene expression of the pET vector lies in the transcription of a 0-lac gn1 T7 fusion promoter mediated by a viral RNA polymerase co-expressed gnl J7. This viral polymerase is supplied by host strains BL21 (DE3) or HMS I 74 (DE3) from a resident profago that hosts a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. A strategy to maximize the expression of recombinant protein in E. coli is expressing the protein in a host bacterium with a damaged capacity to proteolytically cleave the recombinant protein. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector such that the individual codons for each amino acid are those preferentially used in E. coli. Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA mutagenesis or synthesis techniques. In another embodiment, the expression vector of the polynucleotide Staphylococcus epidiermidis is a yeast expression vector. Examples of expression vectors in yeast S. cerevisiae include pYepSec I (Baldari, et al., Embo J, 6: p.229-234.1987), pMFa (Kurjan and Herskowitz, Cell, pp. 933-943, 1982) , pJRY88 (Schultz et al., Gene, 54: p.13-123, 1987), and pYES2 (Invitrogen Corporation, San Diego, CA). Alternatively, a Staphylococcus epidermidis polynucleotide can be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol Cell Biol, 3: p 2156-2165, 1983) and the pVL series ( Lucklow and Summers, Virology, 170: pp. 31-39, 1989). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells that utilize a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature, 329: p.840, 1987) and pMT2PC (Kaufman et al., EMBO J, 6: p.187-195, 1987). When used in mammalian cells, the control functions of the expression vector are often supplied by viral regulatory elements. As used herein, a promoter is a region of a DNA molecule typically within about 100 even nucleotides in front of (upstream of) the point at which transcription starts (i.e., a transcription initiation site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes. As used herein, the term "promoter" includes what is referred to in the art as an upstream promoter region, a promoter region or a promoter of a generalized eukaryotic RNA polymerase II transcription unit. Another type of discrete transcription regulatory sequence element is an enhancer. An enhancer provides specificity of time, location and level of expression for a particular coding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. Likewise, a promoter or an enhancer can function when they are located at variable distances from the transcription start sites while a promoter is present. As used hereafter, the phrase "promoter-enhancer" means a composite unit containing enhancer elements and promoters. A promoter-enhancer is operably linked to a coding sequence that encodes at least one gene product. As used hereafter, the phrase "operatively linked" means that a promoter-enhancer is connected to a coding sequence in such a way that the transcription of this coding sequence is controlled and regulated by that promoter-enhancer. Means for operably linking a promoter-enhancer to a coding sequence is well known in the art. They are also well known in the art, the precise orientation and relative location for a coding sequence whose transcription is controlled, is dependent inter alia on the specific nature of the promoter-enhancer. Thus, a minimum TATA box promoter is typically located from about 25 to about 30 base pairs upstream of a transcription initiation site and an upstream promoter element is typically located from about 100 to about 200 base pairs upstream of a site. of transcription initiation. In contrast, an enhancer can be located downstream of the initiation site and that site can be a considerable distance away. A promoter-enhancer used in a vector construct described herein can be any promoter-enhancer that controls the expression in a cell to be transfected. By employing a promoter-enhancer with well-known properties, the level and pattern of gene product expression can be optimized.

For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other expression systems suitable for prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook er a /., "Molecular Cloning: A Laboratory Manual "2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, incorporated herein by reference. In another embodiment, the recombinant mammalian expression vector is capable of directing the expression of the nucleic acid preferentially in a particular cell type (for example, tissue-specific regulatory elements are used to express the nucleic acid). The tissue-specific regulatory elements are known in the art.

Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., Genes Dev, 1: p.268-277, 1987), specific lymphoid promoters (Caiame and Eaton, Adv Immunol, 43: P. 235-275, 1988), in particular, T cell receptor promoters (Winoto and Baltimore, EMBO J, 8: p.729-733, 1989) and immunoglobulins (Banerji et al., Cell, 33: p. 729-740, 1983), (Queen and Baltimore, Cell, 33: p.741-748, 1983), neuro-specific promoters (eg, the neurofilament promoter, Byrne and Ruddle, PNAS, 86: p.5473-5477). , 1989), pancreas-specific promoters (Edlund et al., Science, 230: p.912-916, 1985), and mammary gland-specific promoters (eg, milk whey promoter, US Patent 4,873,316 and International Application EP 264,166). . Promoters of regulated development are also encompassed, for example the murine hox promoters (Kessel and Gruss, Science, 249: p.374-379, 1990) and the α-fetoprotein promoter (Campes and Tilghman, Genes Dev, 3: p. 537-546, 1989). Also provided herein is a recombinant expression vector comprising a DNA molecule encoding a Staphylococcus epidermidis polypeptide cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a form that allows the expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to mRNA Staphylococcus epidermidis. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which directs the continuous expression of the antisense RNA molecule in a variety of cell types. For example, viral promoters and / or enhancers or regulatory sequences can be chosen that target tissue-specific or cell-specific expression, constituting antisense RNA. The antisense expression vector can be in the form of a recombinant, faegenic or attenuated plasmid in which the antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the type cell in which the vector is introduced. The recombinant expression vectors described herein can be inserted into any suitable host cell. The terms "host cell" and "recombinant host cells" are used interchangeably herein. It is understood that such terms refer not only to the target cell or particular object, but to the progeny or potential progeny of such a cell. Because certain modifications can occur in later generations due to mutation or environmental influences, such progeny can not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a Staphylococcus epidermidis polypeptide can be expressed in bacterial cells such as E. coli, insect cells (such as Sf9, Sf21), mammalian cells or starch (such as Chinese hamster ovary (CHO) cells)., VERO cell, chicken embryo fibroblasts, BHK cells or COS cells). Other suitable host cells are well known to those skilled in the art. The vector DNA is introduced into the eukaryotic prokaryotic cells by transfection, infection or conventional transformation techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of techniques recognized by the art for introducing external nucleic acid (e.g., DNA) into a host cell, including calcium phosphate coprecipitation. calcium chloride, dextran-mediated DEAE transfection, lipofection, ultrasound or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. ("Molecular Cloning: A Laboratory Manual" 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals. A host cell described herein, such as a eukaryotic or prokaryotic host cell in culture, is used to produce (ie, express) a Staphylococcus epidermidis polypeptide. Accordingly, methods for producing a Staphylococcus epidermidis polypeptide using such host cells are also described herein. In one embodiment, the method comprises culturing the host cell (in which a recombinant expression vector encoding a Staphylococcus epidermidis polypeptide has been introduced) in a suitable medium until the Staphylococcus epidermidis polypeptide is produced. In another embodiment, the method further comprises isolating the Staphylococcus epidermidis polypeptide from the medium or host cell.

A coding sequence of an expression vector is operably linked to a transcription termination region. The RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are hereinafter referred to as transcription termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA). The transcription-termination regions are well known in the art. Examples of such transcription-termination regions are SV40 polyadenylation signals and the protamine gene.

An expression vector comprises a polynucleotide that encodes a Staphylococcus epidermidis polypeptide. Such a polypeptide means that it includes a base nucleotide sequence that encodes a Staphylococcus epidermidis polypeptide sufficient in length to distinguish the segment of a polynucleotide segment encoding a non-Staphylococcus epidermidis polypeptide. Such a polypeptide may also encode biologically functional polypeptides or peptides having variant amino acid sequences, such as selected changes based on considerations such as relative hydropathic classification of the amino acids being exchanged. These variant sequences are those isolated from natural sources or induced in the sequences described hereinafter using a mutagenic method such as site-directed mutagenesis. In certain embodiments, the expression vectors described herein comprise polynucleotides encoding polypeptides comprising the amino acid residue sequence of one of SEQ ID NO: 1 to SEQ ID NO: 32. An expression vector can include a Staphylococcus epidermidis polypeptide which encodes the region itself of any of the Staphylococcus epidermidis polypeptides noted above or may contain coding regions that carry selected alterations or modifications in the basic coding region of such a Staphylococcus epidermidis polypeptide. Alternatively, such vectors or fragments may encode larger polypeptides or polypeptides which nevertheless include the basic coding region. In any event, it should be appreciated that due to codon redundancy as well as biological functional equivalence, this aspect is not limited to the particular DNA molecules corresponding to the polypeptide sequences noted above.

Example vectors include the mammalian expression vectors of the pCMV family including pCMV6b and pCMV6c (Chiron Corp., Emeryville CA.). In certain cases, and specifically in the case of these individual mammalian expression vectors, the resulting constructs may require counter-infection with a vector containing a selectable marker such as pSV2neo. The co-transfection pathway in a Chinese hamster ovary cell line of reductase-deficient dihydrofolate, such as DG44, can detect clones expressing Staphylococcus epidermidis polypeptides by virtue of DNA incorporated into such expression vectors. A DNA molecule can be incorporated into a vector by a number of techniques that are well known in the art. For example, the pUC18 vector has been shown to be of particular value in cloning and gene expression. Probably, the related vectors M13mp18 and M13mp19 can be used in certain embodiments of the invention, in particular, in the development of dideoxy sequencing. An expression vector described herein is useful as a means to prepare amounts of DNA encoding Staphylococcus epidermidis polypeptide, and as a means to prepare encoded peptides and polypeptides. It is contemplated that where Staphylococcus epidermidis polypeptides are made by recombinant means, one can use eukaryotic prokaryotic expression vectors as transport systems. In another aspect, the recombinant host cells are prokaryotic host cells. Preferably, the recombinant host cells of the invention are bacterial cells of the DH5 strain of Escherichia coli. In general, prokaryotes are preferred for the initial cloning of DNA sequence and construct the vectors useful in the invention. For example, K12 E. coli strains can be particularly useful. Other microbial strains that can be used include E. coli B, and E. co // x1976 (ATCC No. 31537). These examples are, of course, intended to be illustrative as opposed to limitative. The strains mentioned above, as well as E. coli W31 10 (ATCC No. 273325), E. coli BL21 (DE3), E. coli Top10, bacilli such as Bacillus subtilis, or other enterobacteria such as Salmonella typhimurium (or other strains) of Salmonella attenuated as described in US Pat. No. 4,837,151) or Serratia marcesans, and several species of Pseudomonas can be used. In general, plasmid vectors containing control and replicon sequences, which are derived from species compatible with host cells are used in relation to these hosts. The vector ordinarily carries a replication site, as well as marker sequences that are capable of delivering phenotypic selection in transformed cells. For example, E. coli can be transformed using pBR322, a plasmid derived from an E. coli species (Bolivar, et al., 1977). pBR322 that contains genes for resistance to ampicillin and tetracycline and thus provides easy means to identify transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters that can be used by microbial organisms for expression of their own polypeptides. Those promoters most commonly used in the construction of recombinant DNA include ß-lactamase (penicillinase) and lactose promoter systems (Chang, er a /.; Itakura., Er a /. 1977, Goeddel, et al. 1979; Goeddel, er a /. 1980) and a tri-trophoan promoter system (TRP) (EP 0036776, Siebwenlist et al., 1980). While these are the most commonly used, other microbial promoters have been discovered and used, and details related to their nucleotide sequences have been published, allowing a skilled worker to introduce functional promoters into plasmid vectors (Siebwenlist, er a /. . In addition to the prokaryotic, eukaryotic microbes such as yeast can also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in saccharomyces, plasmid YRp7, for example, is commonly used (Stinchcomb, et al., 1979; Kingsman, et al., 1979; Tschemper, er., 1980). This plasmid already contains the trpl gene which supplies a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977). In the presence of the trpl lesion as a characteristic of the yeast host cell genome then it provides an effective environment to detect transformation by growth in the absence of tryptophan. Suitable promoter sequences in yeast vectors include promoters for 3-phosphoglycerate kinase (Hitzeman., Et al., 1980) or other glycolytic enzymes (Hess, et al., 1968; Holland, et al., 1978) such as enolase, glyceraldehyde-3. -phosphate dehydrogenase, hexokinase, decarboxylase pyruvate, phosphofructokinase, isomerase glucose-6-phosphate, 3-phosphoglycerate mutase, pyruvate kinase, isomerase triosephosphate, phosphoglucose isomerase, and glucokinase. In the construction of suitable expression plasmids, the termination sequences associated with these genes are also introduced into the expression vector downstream of the sequences to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are promoter regions for dehydrogenated alcohol 2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, and glyceraldehyde-3-phosphate dehydrogenase, and enzymes mentioned above responsible for the use of maltose and galactose. Any plasmid vector containing a promoter compatible with yeast, origin or replication or termination sequences are suitable. In addition to the microorganisms, cultures of cells derived from multicellular organisms can also be used as hosts. In principle, any such cell culture is workable either from vertebrate or invertebrate culture. However, there has been great interest in vertebrate cells, and the spread of vertebrate cells in cultures (tissue cultures) has become a routine procedure in recent years. Examples of such useful host cell lines are AtT-20, VERO, HeLa, NSO, PER C6, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7, 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream of the gene to be expressed, together with any necessary ribosome binding site, RNA separation sites, polyadenylation sites, and transcriptional terminator sequences. When expression of recombinant Staphylococcus epidermidis polypeptides is desired and a eukaryotic host is contemplated, it is more desirable to employ a vector such as a plasmid that incorporates a eukaryotic origin of replication. Additionally, for the purposes of expression in eukaryotic systems, one wishes to position the coding sequence of Staphylococcus epidermidis adjacent to and under the control of an effective eukaryotic promoter such as promoters used in combination with Chinese hamster ovary cells. Carry a coding sequence under the control of a promoter, be it eukaryotic or prokaryotic, the 5 'end of the translation initiation region of the appropriate translation reading frame of the polypeptide should be positioned between about 1 and about 50 nucleotides 3' of or downstream of the selected promoter. Additionally, where eukaryotic expression is anticipated, one could typically wish to incorporate within the transcription unit a polynucleotide that encodes the Staphylococcus epidermidis polypeptide. Means of transformation or cellular transfection with exogenous polynucleotide such as DNA molecules are well known in the art and include techniques such as dextran-DEAE or calcium-phosphate-mediated transfection, protoplast fusion electroporation, liposome-mediated transfection, direct microinjection and infection by adenovirus (see for example, Sambrook, Fritsch and Maniatis, 1989). The most widely used method is transfection mediated by calcium phosphate or dextran-DEAE although the mechanism remains dark, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of cultured cells can be transfected at one time. Because this is highly efficient, transfection mediated by calcium phosphate or dextran-DEAE is the method of choice for experiments that require transient expression of external DNA in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that integrate copies of the external DNA, which are usually arranged in tandem head-to-tail assays in the host cell genome. In the protoplast fusion method, protoplasts derived from bacteria carrying high copy numbers of a plasmid of interest are mixed directly with cultured mammalian cells. After fusion of the cell membranes (usually with polyethylene glycol), the contents of the bacteria are delivered into the cytoplasm of the mammalian cells and the plasmid DNA is transported to the nucleus. Protoplast fusion is not as efficient as transfection for many of the cell lines that are commonly used for transient expression assays, but it is useful for cell lines in which DNA endocytosis occurs inefficiently. Protoplast fusion often produces multiple copies of the tandem plasmid DNA integrated into the host chromosome. The application of short high voltage electrical pulses to a variety of plant and mammalian cells leads to the formation of nanometer-sized pores in the plasma membrane. The DNA is carried directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompany the closing of the pores. Electroporation can be extremely efficient and can be used for transient expression of cloned genes and for the establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently elevates cell lines that carry one, or at least a few, integrated copies of the external DNA. The liposome transfection involves the encapsulation of DNA and RNA within liposomes, followed by the fusion of the liposomes with the cell membrane. The mechanism of how much DNA is supplied in the cell is unclear but the transfection efficiency can be as high as 90%. Direct microinjection of a DNA molecule in the nucleus has the advantage of not exposing DNA to cell compartments such as low endosomes in ph. Microinjection is therefore used primarily as a method to establish cell lines that carry integrated copies of the DNA of interest. The use of adenovirus as a vector for cellular transfection is well known in the art. Cell transfection mediated by adenovirus vector has been reported for several cells (Stratford-Perricaudet, et al. 1992).

A transfected cell can be prokaryotic or eukaryotic. Preferably, the host cells of the invention are prokaryotic host cells. Where it is of interest to produce a Staphylococcus epidermidis polypeptide, the cultured prokaryotic host cells are of particular interest. Also contemplated herein is a process or method for preparing polypeptides Staphylococcus epidermidis which comprises transforming, transfecting or infecting cells with a polynucleotide encoding a Staphylococcus epidermidis polypeptide to produce transformed host cells; and maintaining the transformed host cells under biological conditions sufficient for expression of the polypeptide. In a particular embodiment, the transformed host cells are prokaryotic cells. Alternatively, the host cells are eukaryotic cells. More particularly the prokaryotic cells are bacterial cells of the strain DH5-ct of Escherichia coli. Alternatively, the transfected polynucleotide in the transformed cells comprises the nucleic acid sequence of one of SEQ ID NO: 33 to SEQ ID NO: 64. Additionally, transfection is accomplished using an expression vector described above. A host cell used in the process is capable of expressing a functional recombinant Staphylococcus epidermidis polypeptide. After transfection, the cell is maintained under culture conditions for a period of time sufficient for expression of a Staphylococcus epidermidis polypeptide. Culture conditions are well known in the art and include ionic composition and concentration, temperature, pH and the like. Typically, the transfected cells are maintained under culture conditions in a culture medium. Suitable media for various cell types are well known in the art. In certain embodiments, the culture temperature is from about 20 ° C to about 50 ° C, more preferably from about 30 ° C to about 40 ° C and even more preferably about 37 ° C. The ph is preferably from about a value of 6.0 to a value of about 8.0, more preferably near a value of about 6.8 to a value of about 7.8, and more preferably about 7.4. The osmolality is preferably from about 200 milliosmoles per liter (MOSM / L) to about 400 MOSM / L and, more preferably from about 290 MOSM / L about 310 MOSM / L. Other biological conditions necessary for transfection and expression of an encoded protein are well known in the art. The transfected cells are maintained for a sufficient period of time for expression of a Staphylococcus epidermidis polypeptide. An adequate time depends inter alia on the cell type used and is easily determined by a person skilled in the art. Typically, the maintenance time is from about 2 to about 14 days. The recombinant polypeptide Staphylococcus epidermidis is recovered or harvested from the transfected cells or the medium in which those cells are cultured. Recovery comprises isolating and purifying the Staphylococcus epidermidis polypeptide. Isolation and purification techniques for polypeptides are well known in the art and include those methods such as precipitation, filtration, chromatography, electrophoresis and the like.

E. Immunogenic compositions The present invention provides immunogenic compositions comprising one or more Staphylococcus epidermidis polypeptides selected as described in the examples below, and physiologically acceptable carriers. In certain embodiments, the immunogenic compositions comprise one or more Staphylococcus epidermidis polypeptides comprising the amino acid residue sequence of one or more of SEQ ID NO: 1 through SEQ ID NO: 32. In other embodiments, the immunogenic compositions of The invention comprises polynucleotides that encode Staphylococcus epidermidis polypeptides, and physiologically acceptable carriers. For example, the immunogenic compositions of the present invention comprise Staphylococcus epidermidis polypeptides comprising the amino acid sequence of one or more of SEQ ID NO: 1 to SEQ ID NO: 32. Alternatively, the immunogenic compositions comprise polynucleotides comprising the sequence of nucleotide of one or more of SEQ ID NO: 33 to SEQ ID NO: 64. Several assays are used to evaluate the in vitro immunogenicity of the polypeptides of the invention. For example, an in vitro opsonic assay is conducted by incubating together a mixture of Staphylococcus epidermidis cells, the inactive human serum containing antibodies specific for the polypeptide in question, and a source of exogenous complement. Opsonophagocytosis progresses during incubation of fresh isolated human polymorphonuclear cells (PMN) and the anti-body / complement / Sfapfty / ococcus cell mixture. Bacterial cells that are coated with antibody and complement each other are killed after opsonophagocytosis. Colony forming units (cfu) of surviving bacteria that escape opsonophagocytosis are determined by plating the assay mixture. The titles are reported as the reciprocal of the highest dilution that gives >; 50% bacterial death, as determined by comparison for assay controls. Specimens showing less than 50% of deaths in the lowest serum dilution assayed (1: 8) are reported to have an OPA titre of 4. The highest dilution tested is 1: 2560. Samples with death > 50% in the highest dilution are repeated, starting with a higher initial dilution. The method described above is a modification of Gray's method (Gray, Conjugate Vaccines Supplement, pp. 694-697, 1990). A control serum assay, which contains test serum plus bacterial cells and hot inactive complement, is included for each individual serum. This control is used to assess whether the presence of antibiotics or other serum components are capable of directly killing the bacterial strains (ie in the absence of complement or PMN). A human serum with known opsonic titer is used as a positive human serum control. The opsonic antibody titer for each unknown serum is calculated as the reciprocal of the initial dilution of serum given the 50% reduction of cfu compared to the control without serum. A complete cell ELISA assay is also used to evaluate the in vitro immunogenicity and surface exposure of the polypeptide antigen, wherein the bacterial strain of interest. { Staphylococcus epidermidis) is covered on a plate, such as a 96-well plate, and the test serum of an immunized animal is reacted with the bacterial cells. If any antibody, specific for the test polypeptide antigen, is reactive with an exposed surface epitope of the polypeptide antigen, it can be detected by standard methods known to one skilled in the art. Any polypeptide that demonstrates the desired in vitro activity is then assayed in an inoculated animal model in vivo. In certain embodiments, immunogenic compositions are used in the immunization of an animal (e.g., a mouse) by methods and immunization routes known to those skilled in the art (e.g., intranasal, parenteral, oral, rectal, vaginal, transdermal, intraperitoneal, intravenous, subcutaneous, etc.). After immunization of the animal with a particular immunogenic composition of Staphylococcus epidermidis, the animal is inoculated with Staphylococcus epidermidis and tested for resistance to Staphylococcus epidermidis infection. The polynucleotides and polypeptides of Staphylococcus epidermidis are incorporated into immunogenic compositions suitable for administration to a subject, for example, a human. Such compositions typically comprise the nucleic acid molecule or the protein, together with a pharmaceutically acceptable carrier. As used in the following, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and fungal agents, isotonic and delaying absorption agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances are well known in the art. Except so far as any conventional medium or agent is incompatible with the active compound, such medium can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions. An immunogenic composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intraperitoneal), transmucosal (e.g., oral, rectal, intranasal, vaginal, respiratory) and transdermal (topical). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for tonicity adjustment such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be included in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where they are soluble in water) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremofor EL ™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid in the proportion in which there is easy application with a syringe. It must be understandable under the conditions of processing and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and the like. In many cases, isotonic agents are included, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought to include in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a Staphylococcus epidermidis polypeptide or an anti-Staphylococcus epidermidis antibody) in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required. , followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the other ingredients required from those enumerated above. In the case of the sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying which produce a powder of the active ingredient plus any additional desired ingredient from the previously sterile filtered solution thereof. Oral compositions generally include an inert diluent or an edible vehicle. They can be included in gelatin capsules or compressed into tablets. For the purposes of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid vehicle for use as a mouth rinse, wherein the compound in the fluid vehicle is applied orally and clicked and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a linker such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or esters; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as pepper, methyl salicylate, orange flavoring. For administration by inhalation, the compounds are supplied in the form of an aerosol spray from a pressurized container or a dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, the appropriate penetrants to the barrier to be perneated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for the administration of transmucosal, detergents, bile salts, and fusidic acid derivatives. The administration of transmucosal can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, ointments, gels, or creams as are generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as the controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (which include liposomes intended for cells infected with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. 4,522.81 1, which is incorporated herein by reference. It is especially advantageous to formulate oral or parenteral compositions in unit dose form for easy administration and uniformity of dosage. The unit dosage form as used hereafter refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounds such as an active compound for the treatment of individuals. The combination of immunogenic compositions is provided by including two or more of the polypeptides of the invention, as well as by combining one or more of the polypeptides of the invention with one or more of the known non-epidermal Staphylococcus polypeptides such as Staphylococcus polypeptides. aureus.

The following twelve Staphylococcus epidermidis polypeptide sequences SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 30 have polypeptide sequences with at least 90% identity with homologues from Staphylococcus aureus . Therefore, these twelve polypeptides can also be used in immunogenic compositions against Staphylococcus aureus. In addition, the following twelve Staphylococcus epidermidis polynucleotide sequences encoding polypeptides with at least 90% identity to the Staphylococcus aureus homologs can also be used in immunogenic compositions: SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 62, or a degenerate variant thereof, or a fragment thereof. In other embodiments, the combination of the immunogenic compositions is provided by combining one or more of the polypeptides of the invention with one or more of the known polysaccharides or polysaccharide-protein conjugates of S. epidermidis. See, for example, the capsular polysaccharide adhesin of Staphylococcus epidermidis and Staphylococcus aureus, PNSG, poly-N-succinyl beta-1-6 N-acetyl glucosamine (also known as PIA, PS / A, PNAG). See Mckenney, D., et al., Infecí. Immun. 66: 471 1 (1998) and Mckenney, D., et al., Science 284: 1523 (1998), the descriptions of which are hereby incorporated by reference in their entirety. In other embodiments, the combination of the immunogenic compositions is provided by combining one or more polypeptides of the invention with one or more S. aureus polysaccharides or polysaccharide-S. aureus protein conjugates. For example, of the 12 known capsular serotypes of S. aureus, serotype 5 (CP5) and serotype 8 (CP8) account for approximately 85-90% of all clinical isolates []. Most isolates of S. aureus resistant to methicillin express CP5]. Antibodies to CP5 and CP8 induce type-specific opsonophagocytic death by human polymorphonuclear neutrophils in vitro and confer protection in animals [Karakawa, W. W., Sutton, A., et al., Infect Immun 56 (5): 1090-1095 (1988); Fattom, A. I., Sarwar, J., et al., Infection & Immunity 64 (5): 1659-1665 (1996)]. Several laboratories have synthesized immunogenic conjugates consisting of CP5 and CP8 covalently bound to protein. These conjugates are highly immunogenic in mice and humans and induce antibodies that opsonize microencapsulated S. aureus for phagocytosis [Fattom, A., Schneerson, R, et al., Infect Immun 61 (3): 1023-1032 (1993); Gilbert, F.B., Poutrel, B., et al., Vaccine 12 (4): 369-374 (1994); Reynaud-Rondier, L, Voiland, A., et al., FEMS Microbiol Immunol 3 (4): 193-199 (1991)]. Monovalent immunogenic compositions containing CP5 conjugated to recombinant exotoxin A of Pseudomonas aeruginosa are immunogenic and well tolerated in healthy adults and in patients with end-stage renal disease [Welch, PG, Fattom, A., Moore, J. Jr., et al. al., J. Am. Soc. Nephrol. 7: 247-253 [Abstract] (1996)]. In a double-blind trial involving patients with end-stage renal disease who were receiving hemodialysis, a bivalent conjugate vaccine composed of CP5 and CP8 covalently linked to recombinant Pseudomonas aeruginosa exotoxin A conferred partial immunity against S. aureus bacteremia for approximately 40 weeks, after which protection decreased to the extent that antibody levels decreased [Shinefield, H., Black, S., et al., N Engl J Med 346 (7): 491-496 (2002) ] The result of this test indicates a need for an improved immunogenic composition that could contribute to a broader and more complete protection. As described above, in certain embodiments, the combination of the immunogenic compositions is delivered by combining one or more polypeptides of the invention with one or more known S. aureus polysaccharide-protein conjugates. The "protein component" of the carbohydrate-protein conjugates is known as a carrier protein. The term "carrier proteins", as a group, are preferably proteins that are non-toxic and non-reactogenic and obtainable in sufficient quantity and purity. Vehicle proteins must be treatable in standard conjugation procedures. In a particular embodiment of the present invention, CRM197 is used as the carrier protein. CRM197 (Wyeth, Sanford, NC) is a nontoxic (ie, toxoid) variant of diphtheria toxin isolated from cultures of the Corynebacterium diptheria C7 (ß197) strain grown in casamino acids and medium based on yeast extract. CRM 97 is purified by ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. Other diphtheria toxoids are also suitable for use as vehicle proteins. The immunogenic composition may further comprise an adjuvant, such as an aluminum-based adjuvant, such as aluminum phosphate, aluminum sulfate and aluminum hydroxide. Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (eg, as described in International Patent Application WO2004 / 083251 [38]), E. coli IT, E. coli ST , and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as the outer membrane c complex (OMPC), purines, transferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), or Protein D of Haemophilus influenza, can also be used. Other proteins, such as ovalbumin, lame huecodellave hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein derivative (PPD) can also be used as carrier proteins. Immunogenic compositions comprising the polynucleotides are delivered to the recipient by a variety of vectors and expression systems. Such systems include, among others, chromosomal systems, episomal and virus derivatives, for example, vectors derived from bacterial plasmids, attenuated bacteria such as Salmonella (US Pat. No. 4,837, 151), coming from bacteriophage, coming from transposons, coming from yeast episomes, coming from insertion elements, from yeast chromosomal elements, from viruses such as vaccinia and other poxviruses, adenoviruses, baculoviruses, papota viruses, such as SV40, bird poxviruses, seudorabies and retroviruses, alphaviruses such as encephalitis virus equine from Venezuela (US Pat. No. 5,643,576), sindbis virus and semilíki forest virus, non-segmented negative strand RNA viruses such as vesicular stomatitis virus (US Patent 6, 168,943), and vectors derived from combinations of these, such as those derived from the plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Expression systmust include control regions that regulate as well as engender expression, such as promoters and other regulatory elements (such as a polyadenylation signal). In general, any system or vector suitable for maintaining, propagating or expressing the polynucleotides to produce a polypeptide in a host can be used. The appropriate nucleotide sequence can be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., "Molecular Cloning: A Laboratory Manual" 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. A pharmaceutically acceptable carrier is meant to mean a compound or a combination of compounds that enter a pharmaceutical or immunogenic composition that does not cause side effects and which makes it possible, for example, to facilitate the administration of the active compound, to increase its life and / or its effectiveness in the body, to increase its solubility in solution or alternatively to improve its preservation. These pharmaceutically acceptable carriers are well known and will be adapted by persons skilled in the art according to the nature and mode of administration of the active compound chosen. As defined below, an "adjuvant" is a substance that serves to enhance the immunogenicity of an "antigen" or immunogenic compositions comprising one or more polypeptide antigens having an amino acid sequence selected from one of SEQ ID NO. : 1 to SEQ ID NO: 32. Thus, adjuvants are often given to reinforce or modulate the immune response and are well known to the skilled artisan. Examples of adjuvants contemplated in the present invention, but not limited to, aluminum salts (alum) such as aluminum phosphate and aluminum hydroxide, Mycobacterium tuberculosis, bacterial lipopolysaccharides, aminoalkyl glucosaline phosphate (AGP) compounds, or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT) and which are described in US Pat. No. 6,1 13,918; one such AGP is 2 - [(R) -3-Tetradecanoyloxytetradecanoylaminojetyl 2-Deoxy-4-0-phosphono-3-0 [(R) -3-tetradecanoioxytetradecanoyl] -2 - [(R) -3-tetradecanoioxytetradecanoylamino]] -bD-glucopyranoside, which is also known as 529 (formerly known as RC529), which is formulated as an aqueous form or as a stable emulsion, MPL (3-0-deacetylated monophosphoryl lipid A) (Corixa) described in US Pat. Number 4,912,094, synthetic polynucleotides such as oligonucleotides containing the CpG motif (U.S. Patent Number 6,207,646), polypeptides, saponins such as Quil A or STIMULON ™ QS-21 (Antigenics, Framingham, Mass.), Described in U.S. Pat. Number 5,057,540, a pertussis toxin (PT), or a heat-labile toxin of E. coli. { 11), particularly LT-K63, LT-R72, CT-S109, PT-K9 / G129; see, for example, International Patent Publications No. WO 93/13302 and WO 92/19265, the cholera toxin (in a wild type or in a mutant form, for example, wherein the glutamic acid at the amino acid position 29 is replaced by another amino acid, preferably a histidine, according to published International Patent Application No. WO 00/18434). Similar cholera toxin mutants are described in published International Patent Application No. WO 02/098368 (wherein the isoleusin at amino acid position 16 is replaced by another amino acid, either alone or in combination with serine replacement). at the position of amino acid 68 by another amino acid, and / or where valine at amino acid position 72 is replaced by another amino acid). Other mutants of cholera toxin are described in published International Patent Application No. WO 02/098369 (wherein the arginine at the amino acid position is replaced by another amino acid, and / or an amino acid is inserted at the position of the amino acid. 49, and / or two amino acids are inserted at amino acid positions 35 and 36). Several cytokines and lymphokines are suitable for use as adjuvants. One such adjuvant is the granulocyte-macrophage colony stimulating factor (GM-CSF), which has a nucleotide sequence as described in U.S. Pat. Number 5,078,996. A plasmid containing GM-CSF cDNA has been transformed into E. co // and has been deposited with the American Type Culture Collection (ATCC), 1081 University Boulevard, Manassas, VA 20110-2209, under accession number 39900. The cytokine interleukin-12 (IL-12) is another adjuvant described in the US Patent Number 5,723,127. Other cytokines or lymphokines have been shown to have immuno-modulating activity, including, but not limited to, the interleukins 1-alpha, 1-beta, 2, 4, 5, 6, 7, 8, 10, 13, 14, 15, 16, 17 and 18, interferons-alpha, beta and gamma, granulocyte colony-stimulating factor, and tumor necrosis factors alpha and beta, are suitable for use as adjuvants. A composition of the present invention is typically administered parenterally in unit dose formulations containing well known non-toxic, physiologically acceptable vehicles, adjuvants, and vehicles as desired. Injectable preparations, for example, aqueous sterile injectables and oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or a suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the vehicles and acceptable solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspension medium. For this purpose any soft fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Preferred carriers include neutral saline solutions buffered with phosphate, lactate, Tris, and the like. Of course, when viral vectors are administered, one purifies the vector sufficiently to render it essentially free of undesirable contaminants, such as defective interfering adenovirus particles or endotoxins and other pyrogens so that it does not originate any unaddressed reactions in the individual receiving the construction of the vector. A preferred means for purifying the vector involves the use of buoyant density gradients, such as gradient centrifugation of cesium chloride. A vehicle can also be a liposome. The means for using the liposomes as delivery vehicles are well known in the art (see, for example, Gabizon et al., 1990, Ferruti et al., 1986, and Ranade, 1989). The immunogenic compositions of this invention also comprise a polynucleotide sequence of this invention operatively associated with a regulatory sequence that controls the expression of the gene. The polynucleotide sequence of interest is engineered into an expression vector, such as a plasmid, under the control of regulatory elements that will promote DNA expression, that is, the promoter and / or enhancer elements. In a preferred embodiment, an immediate-early human cytomegalovirus promoter / enhancer is used (U.S. Patent 5, 168,062). The promoter can be a cell-specific and allow substantial transcription of the polynucleotide only in predetermined cells. The polynucleotides of the invention are introduced directly into the host and as "naked" DNA (US Pat. No. 5,580,859) or formulated in compositions with facilitators, such as bupivicaine and other local anesthetics (US Patent 5,593,972) and cationic polyamines (US Patent 6,127,170). ), which are incorporated herein by reference in their entirety.

In this polynucleotide immunization method, the polypeptides of the invention are expressed on a transient basis in vivo; no genetic material is inserted or integrated into the chromosomes of the host. This procedure must be distinguished from gene therapy, where the goal is to insert or integrate the genetic material of interest into the chromosome. One assay is used to confirm that polynucleotides administered by immunization do not give rise to a transformed phenotype in the host (U.S. Patent 6,168,918).

H. Uses and Methods of the Invention The polynucleotides, polypeptides and Staphylococcus epidermidis polypeptide homologs described herein are used in immunization methods. The isolated polynucleotides are used to express Staphylococcus epidermidis polypeptides (for example, via the recombinant expression vector in a host cell or in polynucleotide immunization applications).

As described in detail here in the Examples, Staphylococcus epidermidis grew in the presence of serum to stimulate the expression of proteins and carbohydrates in the bacterial cell wall which may be significant for systemic bacterial infection. As a result, thirty-two polypeptides and the corresponding polynucleotides were identified as expressed by Staphylococcus epidermidis when grown in 70% serum. In addition, twenty-four of these proteins were found to be reactive to the immune serum of rabbits infected with Staphylococcus epidermidis.

The genes that correspond to the proteins expressed when Staphylococcus epidermidis grew in serum were cloned and used to express the proteins recombinantly. The recombinant proteins were used to immunize mice and twenty-five of twenty-six proteins induced antibodies that reacted with whole cell lysates of Staphylococcus epidermidis. In addition, eighteen of these sera also reacted with Staphylococcus aureus whole cell lysates. Finally, when immunized mice were inoculated with Staphylococcus epidermidis it was found that eleven of the proteins had induced antibodies that reduced the amount of detectable bacteria found in the vessel after inoculation. The invention further provides immunogenic compositions comprising one or more polypeptides already described, which have an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 32, a biological equivalent thereof or a fragment thereof. The immunogenic composition may further comprise a pharmaceutically acceptable carrier. In certain embodiments, the immunogenic composition will comprise one or more adjuvants.

In another embodiment, the invention provides immunogenic compositions comprising a polynucleotide having a nucleotide sequence selected from one of SEQ ID NO: 33 to SEQ ID NO: 64, wherein the polynucleotide is comprised in a recombinant expression vector. Preferably the vector is a plasmid DNA. The polynucleotide may further comprise heterologous nucleotides, for example, the polynucleotide is operably linked to one or more gene expression regulatory elements, and further comprises one or more adjuvants. In a preferred embodiment, the immunogenic polynucleotide composition directs the expression of one or more neutralizing epitopes of Staphylococcus epidermidis. Methods for immunizing a host against Staphylococcus epidermidis infection are also provided. In a preferred embodiment, the host is a human. Thus, a host or subject is administered an immunizing amount of an immunogenic composition comprising a polypeptide having an amino acid sequence selected from one of SEQ ID NO: 1 to SEQ ID NO: 32, a biological equivalent of this or a fragment thereof and a pharmaceutically acceptable carrier. An immunizing amount of an immunogenic composition is determined by developing a dose response study in which subjects are immunized with gradually increasing amounts of immunogenic composition and the immune response analyzed to determine the optimal dose. The starting points of the study are inferred from the immunization data in animal models. The amount of dose may vary depending on the specific conditions of the individuals. The amount is determined in routine tests by means known to those skilled in the art. An immunologically effective amount of the immunogenic composition in an appropriate number of doses is administered to the subject to elicit an immune response. The immunologically effective amount, as used herein, means the administration of that amount to a mammalian (preferably human) host, either in a single dose or as part of a series of doses, sufficient to at least cause the immune system of the treated individual to generate a response that reduces the clinical impact of the bacterial infection. Ideally, the treated individual will not exhibit the most serious clinical manifestations of the infection with Staphylococcus epidermidis or Staphylococcus aureus. The amount of dose may vary depending on the specific conditions of the individuals, such as age and weight. This amount can be determined in routine tests by means known to those skilled in the art. All patents and publications cited herein are incorporated by reference.

EXAMPLES The following examples were carried out using standard techniques, which are well known and the routine of those skilled in the art, except where otherwise described in detail. All chemicals were obtained from Sigma (Sigma Chemical Co., St. Louis, MO) unless otherwise stated. The following examples are presented for illustrative purposes, and should not be considered in any way limiting the scope of this invention.

Example 1 Bacterial Growth in 70% Serum The following examples were developed using the clinical isolate Staphylococcus epidermidis 0-47. The non-annotated genomic sequence was available for this isolate from Incyte Corporation of Palo Alto, CA. See Heilmann, C, et al., Infect Immun, 64 (1): p. 277-82 (1996). To stimulate the expression of the proteins, which may be clinically relevant to pathogenicity, the cultures of the bacteria were grown overnight in a 100% tryptic soy broth (TSB) or in rabbit serum: TSB 70: 30 with stirring (200 rpm) at 37 ° C. Rabbit serum was obtained from Life Technologies, Rockville, MD. The bacteria were diluted from an overnight culture at an OD600 ~ 0.1 and grown for 4 h to a medium trunk phase. In the mid-stem phase, the cells were harvested by centrifugation and further processed as described in the following examples.

Example 2 Preparation of Cell Wall Fractions for 2-D Gel Electrophoresis The cell walls of Staphylococcus epidermidis 0-47 that grew as described in Example 1 were isolated and then prepared for two-dimensional gel electrophoresis. The bacterial blots were resuspended in OD600 ~ 20 and washed twice with vasculation for 15 minutes at 4 ° C using Tris buffered saline (TBS, 20mM Tris, pH 8.0, 150mM NaCl). The serum proteins bound to the surface of the bacteria were removed by washing for 15 minutes at 4 ° C with 20 mM Tris, pH 8.0 containing 1 M NaCl. The growth of the bacteria in TBS was treated in the same way as the growth of bacteria in serum. The bacteria were again palletized by centrifugation. To create the protoplasts, the bacteria were then resuspended in OD60o ~ 40 in TBS containing 30% sucrose, 100 μg / ml lysostaphin, 10 pg / ml DNase, 1 pg / ml Pefablock (Boehringer Mannheim, Indianapolis, IN ), 10 μg / ml of lysozyme and 100 units / ml of mutanolysin and incubated at 37 ° C for 1 hour. The resulting protoplasts were palletized by centrifugation at 5000 rpm for 10 minutes and the supernatant containing the cell wall material was decanted. The decanted supernatants containing the cell wall fractions were supplemented with Complete Mini protease inhibitor tablets (Roche Diagnostics, Indianapolis, IN) and dialyzed overnight against water at 4 ° C using 10,000 kD MWCO dialysis membrane (Pierce Biotechnology, Inc., Rockford, IL). After dialysis, the fractions of the cell wall were frozen at -20 ° C.

After isolation, samples of the cell wall fraction were prepared for 2-D gel electrophoresis as follows: the frozen cell wall extracts were thawed and precipitated with 70% acetone on ice for 4 hours. The protein precipitate was palletized, dried in a SpeedVac (Thermo Savant, Holbrook, NY) and solubilized with ReadyPrep (BioRad) SEQUENTIAL EXTRACTION REAGENT 3, containing 5 M urea, 2 M thiourea, 2% (w / v) of CHAPS, 2% (w / v) of SB 3-10, 40 mM of Tris and 0.2% of Bio-Lyte 3/10. Cell wall fraction samples prepared were loaded onto strips with 1 1 cm immobilized pH gradient (IPG), pH 4-7 (BioRad) by allowing each sample to re-hydrate a gel strip during an incubation throughout the night at room temperature. The sample size was 250 pg in a total volume of 200 μ ?. During the incubation all night, the strips were covered with mineral oil (BioRad) to avoid evaporation. After completing the rehydration of the strips, the excess mineral oil was stirred on drying paper that was saturated with water and the hydrated strips were then loaded into an isoelectric focusing pHaser (IEF) apparatus (Genomic Solutions Inc., Ann Arbor , MY). The strips were pre-focused with a current limit of 50 mA / strip with a gradually increasing voltage from 250 V to 5,000 V. The voltage was then kept constant at 5,000 V for a total of 50 kVh (~ 16h). The second-dimensional SDS-PAGE was carried out using 12.5% pre-cast Criterion gels (BioRad). For the mass spectrometric analysis, the gels were stained with Sypro Ruby protein gel stain (BioRad) according to the manufacturer's instructions. The profiles of the two-dimensional gel (2D) of the cell wall associated with Staphylococcus epidermidis proteins grown in TSB or 70% rabbit serum were compared. See Figure 1. Growth in 70% rabbit serum resulted in a change in the expression profile of the cell wall protein associated with Staphylococcus epidermidis proteins that was easily detectable in fluorescent dye transfers from 2D gels ( Figures 1a and 1 B). Eight proteins were detected by fluorescent dyeing to be differentially regulated between Staphylococcus epidermidis grown on TSB or in the presence of rabbit serum. See Table 3 and Figure 1. Most notable was the increase in fluorescent staining of the traces of three proteins between 25 kDa and 37 kDa in cells grown in 70% serum (Figure 1B, spots e, gyh) .

Table 3 List of Identified Stains3 15 25 aLista of spots detected on 2D blots by reactivity with immune serum or binding to serum components. b Some spots that contained more than one protein. ° Method by which the spot was detected after transfer to nitrocellulose, reactive immune serum from infected rabbits, I, or serum binding components, S. dNGID = no gene in the database.

Example 3 Binding of Immune Serum and Biotinylated Serum Proteins to Cell Wall Proteins After completion of first and second dimension electrophoresis, the protein content of the gels was transferred onto nitrocellulose for binding assays. Specifically, the protein content of the gels was electroblotted onto nitrocellulose membranes (BioRad) using a semi-dry blotter apparatus (Owl Separations Systems, Portsmouth, NH) at 12V for 1 hour. The protein containing nitrocellulose membranes (blots) was then stained with blot dyeing Sypro Ruby protein (BioRad) following the manufacturer's instructions and visualized in a FluorS Imager (BioRad). Each blot was incubated in a blocking buffer (PBS with 0.05% Tween 20 and 5% dry milk) for 10 minutes at room temperature and then incubated overnight with a 1: 2000 dilution of the immune serum (Western blot) or 40 mg / ml of biotinylated whey proteins (see below). After overnight incubation, the blots were washed 3x with wash buffer (PBS with 0.5% Tween 20) and incubated with goat anti-rabbit IgG alkaline phosphatase conjugate (Biosource International, Camarillo, CA) or phosphatase conjugate alkaline streptavidin (Biosource) for 2 hours at room temperature in blocking buffer. The blots were again washed three times with wash buffer and visualized with a BCIP / NBT membrane phosphatase substrate system (KPL, Inc., Gaithersburg, MD). The photographs were taken in FluorS. All analyzes of the 2D gels were developed using Melanie 3.0 software.

The concentration of the protein was assayed using a BioRad protein assay kit (BioRad). The changes in the protein expression profile of the proteins associated with the cell wall were more pronounced when considering the Western blots of the nitrocellulose membranes that contained the proteins transferred from the 2D gels. In Western blots, nitrocellulose membranes were incubated with pooled immune sera from rabbits repeatedly infected with Staphylococcus epidermidis 0-47. See Figures 1 C and 1 D. These positively regulated proteins were also strongly immunoreactive, suggesting that they were expressed during the infection of the rabbits. Five of other traces or immunoreactive stains from cells grown in serum were expressed at lower or undetectable levels in cells grown in TSB. See Figure 1, spots a, b, c, d and f.

Example 4 Analysis of Serum Proteins that Interact with Proteins Associated with the Cell Wall of Staphylococcus epidermidis Elusion of Whey Proteins from Staphylococcus epidermidis Staphylococcus epidermidis 0-47 grew in rabbit serum 70% at 37 ° C to OD60o ~ 0.8 and the cells were palletized. The cells were resuspended in OD600 ~ 20 and washed three times with TBS although pivoting at 4 ° C. The bound serum proteins were eluted sequentially with 20 mM Tris, pH 8.0 containing 0.5 M NaCl, 1.0 M NaCl or 4.0 M urea for 1 hour with pivoting at 4 ° C. The bacteria were then removed by centrifugation and the supernatant was collected. The supernatants contained whey proteins eluted from the surface of the bacteria.

Proteins eluted under different conditions were analyzed by SDS-PAGE using 4-20% gradient Tris-glycine gels (Cambrex Biosciences Rockland, Inc., Rockland, ME).

Biotinylation of whey proteins The eluted serum proteins were dialyzed overnight against PBS at 4 ° C. The IgG were reduced by overnight incubation with protein G sepharose (Amersham-Pharmacia, Piscataway, NJ) at 4 ° C. Assuming an average protein mass of 50 kDa in the eluted fraction, the proteins were labeled with a 15 molar excess of EZ-Link® NHS-biotin (Pierce Biotechnology) for 1.5 hours at 4 ° C. The reaction was quenched with excess glycine and dialyzed (10.0000 MWCO, Pierce) overnight against PBS.

Identification of Serum Proteins United to the Surface of Staphylococcus epidermidis The serum proteins eluted from the bacteria under these conditions were compared with SDS-PAGE to normal rabbit serum and to bacterial proteins eluted from the surface of Staphylococcus epidermidis grown on TSB. See Figure 3. Buffers containing 0.5 NaCl, 1 M NaCl and 4 M urea each eluted bound serum proteins from the bacterial cells. These eluted whey proteins represent a group of proteins eluted from the bacterial surface that is enriched for whey proteins. Some bacterial proteins are probably present in this group, however no bacterial proteins detectable by the protein assay were eluted from the growth of bacteria in TSB. Although some faded protein bands were detected by silver staining to be eluted from bacteria grown with TSB, they did not correspond to the more intensely stained proteins eluted from the surface of bacteria grown in serum. Elution with 1 M NaCl was the least denaturing condition that eluted most of the proteins and was used to elute the proteins for the following examples. In order to identify the proteins associated with the cell wall involved in the binding serum components, the biotin-labeled serum proteins were used to probe 2D transfers by incubating a solution of the proteins labeled with cell wall proteins attached to the cell wall. nitrocellulose transferred from a 2D gel. To isolate serum proteins that bind to Staphylococcus epidermidis, bacteria grown in 70% rabbit serum were washed with 1 M NaCl. The eluted whey proteins were harvested and reported in PBS. Then, the naturally occurring immune IgG that may be present in the eluted serum proteins was emptied by incubation with protein G sepharose. Removal of IgG reduces the likelihood of identifying a protein that is reactive with host antibodies. The eluted serum proteins were then labeled with biotin as described above and used to probe a 2D blot of cell surface proteins of Staphylococcus epidermidis. See Fig 4A and 4B. Thirty-four spots and regions were visualized by this method and are probably involved in the interaction of Staphylococcus epidermidis with host serum proteins. Of the 34 spots consistently found to interact with serum components all but 4 were found to react with immune serum from infected rabbits. See Fig 2 and Table 3.

The growth of Staphylococcus epidermidis in serum had serum proteins bound to the bacterial surface proteins that were eluted with 0.5M and 1 M NaCl. Under the same conditions few Staphylococcus proteins were eluted from the growth of bacteria in TSB, however it is possible that the Staphylococcus protein expressed only in serum was eluted by a treatment with high salt.

Example 5 Identification of Mass Spectroscopy of Positively Regulated Proteins of Serum Bacterial growth in 70% of serum was used in subsequent proteomic experiments and analyzes, working under the presumption that changes detected during growth in serum may more accurately reflect alterations in the expression of the gene made by means of the bacterium in response to environmental situations seen within a host. In the following mass spectroscopy studies, isolated proteins from spots on separations of 2D gel electrophoresis were first subjected to mass spectrometry flight time. If a positive, unambiguous identification was obtained, then it is not necessary to develop a spectrometric analysis of additional mass. See Table 4. In the case where ambiguity persists after flight time mass spectroscopy, such as when multiple proteins resolved the same spot on 2D gel, then electrospray mass spectroscopy was developed to resolve the ambiguity. See Table 4 Table 4 Proteins Identified by Mass Spectroscopy Sample Preparation Before developing the mass spectroscopy, the white protein spots were subjected to tryptic gel digestion. The protein spots were removed from the gel and cut into ~ 1 mm pieces. The gel pieces were washed three times with 0.2 ml of 50% (v / v) acetonitrile (Burdick &Jackson, Muskegon, MI) in 10 mM of ammonium bicarbonate (JT Baker, Phillipsburg, NJ) for 15 minutes with shaking occasional. The gel pieces were dehydrated with acetonitrile for 5 minutes, lyophilized, and stored frozen at -20 ° C. The proteins in the gel were then digested with 50 μ? of 12 ng / ml of modified trypsin-grade sequencing (Promega Corporation, Madison, Wl) overnight at 37 ° C. The trypsin solution was then removed and the gel again dehydrated in 50 μ? of acetonitrile. The acetonitrile containing peptide was then removed and the gel pieces washed in 50 μ? of 5% formic acid (Riedel-de Haén, Seelze, Germany) for 15 minutes at room temperature in a bath sonicator (Branson Cleaning Equipment Co., Shelton, CT). The supernatant containing the peptide was removed and combined with the initial acetonitrile wash. The gel was again washed in acetonitrile and the supernatant combined with two previous extraction steps and dried in a SpeedVac (Thermo Savant) to ~ 10 μ ?, then diluted to 100 μ? with 0.1% (v / v) of aqueous formic acid. The sample was then loaded onto a P10 Zip-TipCia column (Waters Corporation, Milford, MA) and eluted at 50 μ? of acetonitrile 50% / formic acid 0.1%. Samples were transferred to a Teflon-coated stainless steel plate with 96 X 2 wells (PerSeptive Biosystems, Framingham, MA) for mass fingerprint analysis on the MALDI-ToF instrument (Biosystems PerSeptive) glass nanopulverized tips ( New Objective Inc., Woburn, MA) to be sprayed into the hole of the mass spectrometer with ion trap.

Peptide Mass Fingerprint Using ToF Mass Spectrometry Each sample was applied to the plate, 96 X 2 stainless steel wells, coated with Teflon with application of a thin layer of a-cyano-4-hydroxycinnamic acid. The samples were allowed to dry at room temperature. The mass spectral data were acquired on a Voyager DE-STR MALDI-ToF mass spectrometer (PerSeptive Biosystems) equipped with delayed extraction technology, and a reflector. The mass spectrometer was equipped with a nitrogen laser at 337 nm and a laser index of 3 Hz. The acceleration voltage was adjusted to 20 kV, the operating mode (reflector), the extraction mode (delayed), polarity (positive), grid voltage (65%), mirror voltage index (1.12), extraction delay time (200 nsec), mass index (800-3500 Da), and laser shots per spectrum (200) .

Mass Spectroscopy with Static Nanopulsed Ion Trap The mass spectral data were acquired on a ThermoFinnigan LCQ DECA (ThermoFinigan, San Jose, CA) quad-ion mass spectrometer equipped with a nano-electrospray inferium. The nano-electrospray interface consisted of a silica spray needle, ~ 27 mm in length by 120/69 μ? of OD / ID, hole diameter 2 μ ?? (New Objective Inc.). The tip of the glass was mounted on a support with x, y, z axis (ThermoFinnigan) supported on a base located on the front of the mass spectrometer detector. Electrical current was applied to the standard glass tip coating to provide an electrical connection for the electrospray interface through a metal connection on the static nanopulver probe (ThermoFinnigan). The nanospray supplied a flow of 20-80 nl / min. Two to five microliters of the tryptic digest was analyzed using a nanopulverized glass tip directed towards the orifice of the mass spectrometer. Peptide analyzes were conducted on the LCQ-DECA ion trap mass spectrometer (Thermofinnigan) operating at a variable spray voltage of -1 kV, and using a heated capillary temperature of 200 ° C. The adjusted data were acquired in an automated MS / MS mode using data acquisition software supplied with the instrument. The acquisition method included a 1 MS exploration (400-1800 m / z) followed by MS / MS exploration of the three highest abundant ions in the MS scan. The dynamic exclusion function was used to increase the number of peptide ions that were analyzed (configurations: 3 amu = exclusion width, 0.5 minutes = exclusion duration). The current experiment was analyzed in groups of samples and manually. Automated analysis of mass fingerprint data was developed using MSFIT (Protein Prospector) and search engines with a MASCOT software database (Matrix Science) using an Incyte PathoSeq database (c) Staphylococcus epidermidis 0-47 . The resulting spectra were processed with baseline correction, noise removal, and peak "de-isotoping" before using the search engines. The search parameters of the database were adjusted to the following levels: MW (1000 - 150 kDa), pl (3-10), Digestion (trypsin), max number. of cleavages lost (2), pfactor of lost cleavages (0.4), static modification (cysteine modified by acrylamide), Terminal N (hydrogen), Terminal C (free acid), variable mods (methionine oxidation, N-terminal acetylation, phosphorylation of cerin, threonine, and tyrosine), mass (monoisotopic), minimum number of peptides required for matching (4) with a mass tolerance of 300 ppm, and the application of iterative calibration (Intelcast) with a mass tolerance of 15 ppm. Protein identifications were determined by a MOWSE rating and a 95% confidence rating by MS-FIT and MASCOT respectively. Automated analyzes of the MS / MS data were developed using SEQUEST incorporated in the Finnigan Bioworks data analysis package (ThermoFinnigan). See Eng, J.K., et al., J Amer Soc Mass Spec, 5 (11): p. 976-89 (1994). The following variable modifications were allowed in the software: modification of cysteine acrylamide and oxidation of methionine. The search parameters were adjusted in the following designations: mass range (400-3500 Da), MS signal of lower intensity (1 e +5), peptide tolerance (2.0 Da), min. of ion fragment (15), min. of explorations in a group (1), and maximum number of lost cleavages (2). All protein identifications were manually verified for accuracy.

Protein Identification by Mass Spectrometry.

The spots were consistently detected in both fluorescent and immunostained blots of Staphylococcus epidermidis growth in 70% serum, were calculated and labeled for identification by mass spectroscopy. See Figures 2A and 2B. A total of 40 immunoreactive spots were cut and subjected to mass spectrometric analysis for identification. See Table 4. The complete protein sequences are shown in the sequence list (SEQ ID NO: 1-32). Protein-containing gel spots were cut from a gel and identified by mass fingerprint analysis using MALDI-ToF followed by the search of an Incyte PathSeq database (c) Staphylococcus epidermidis 0-47 for the corresponding coding region . See Table 4. Stains with multiple protein hits or questionable signals were further analyzed using a static nanopulver. A total of 32 proteins were identified, with some spots containing more than one protein. See Tables 3 and 4. Twenty-four of the proteins identified were immunoreactive, and 26 bound to serum components and 20 of the proteins were both immunoreactive and bound to serum. This long overlap was expected, because most of the proteins on the surface of Staphylococcus epidermidis involved in binding to serum factors would probably elicit an immune response.

Six proteins were consistently present in the immunostaining spots, but none of the corresponding spots was visibly present on the fluorescent dye transfers. See Figure 2B, white circles and arrow. Although these proteins were probably expressed during an infection and elicited an immune response they are not expressed at levels that allow their detection by fluorescent protein staining under the conditions used in these experiments.

Example 6 Prediction of Protein Function The predicted function of the proteins was determined by comparison with the homologues of the complete genome of ATCC12228. See Zhang, Y.Q., et al., Mol Microbiol, 49 (6), p. 1577-93 (2003). The predicted functions shown in Table 5 are attributed to the respective ORFs by the previous publications that involve the specific protein or by the homology to the previously characterized proteins that occur in other organisms.

Table 5 Predicted functions of the proteins of S. epidermidis a The predicted function of the proteins was determined by comparison with the homologues of the complete genome of ATCC12228. b Method by which the stain was detected after transfer to nitrocellulose, reactive immune serum from infected rabbits, I, or serum-binding components, S.

As discussed above, the expression profile of the cell wall-associated proteins of Staphyiococcus epidermidis 0-47 grown in 70% rabbit serum was analyzed by 2D gel electrophoresis. The profile of the total expression in the serum was determined to be significantly different from that which occurs after growth in TSB. Numerous proteins that were positively regulated during growth in serum were identified by mass spectroscopy and their predicted functions by sequence comparison. See Table 5. The three proteins predicted to be involved in nutrient acquisition, 305, 1450, and 1703 were significantly increased. See Tables 4 and 5. All three proteins form veins through the gel. See Figure 4. Without wanting to be bound by theory, this may be the consequence of multiple charge isomers, or related to their predicted lipoprotein composition. Additionally, all three proteins are highly reactive with an immunosorbent from rabbits infected with Staphylococcus epidermidis 0-47 suggesting that these proteins are also expressed in the host during an infection. See Figure 2 and Table 5. In total, 24 of these proteins were identified as reactive with immune serum from infected rabbits. Not only are these proteins expressed during growth in the serum, but they also elicited an immune response in an infected animal. See Example 8. Taken together, these data suggest that these antigens are all expressed during an infection. The expression of the transcripts from these ORFs within the bloodstream of an infected mouse was confirmed by RT-PCR for all the identified proteins (data not shown).

Example 7 Cloning and Expression of Recombinant Proteins Genes were cloned using primers designed based on proteins identified by mass spectrometry of the expressed proteins and from the Staphylococcus epidermidis database 0-47. Individual genes were amplified by polymerase chain reaction (PCR) using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) and adenine overhangs were added with a Taq DNA polymerase (Roche Diagnostics). The reaction products were cloned into pCRT7 / NT-TOPO or pBAD / TOPOThio (Invitrogen, Carisbad, CA) following the manufacturer's instructions and transformed into E. coli Top10 (Invitrogen). Positive clones were detected by colony PCR using the ReddyMix PCR mastermix (ABgene, Rochester, NY) and sequenced to ensure that spurious mutations did not arise. Plasmids from pCRT7 clones were purified and transformed into E. coli BL21 (DE3) (invitrogen) for expression using T7 polymerase. Proteins were expressed by growth of the positive clones in HySoy broth (1% HySoy, Intl Cheese, Stockbridge, GA), 0.5% yeast extract, 100 mM NaCl, 50 mM Na2HP04-7H20, 40 mM NaH2P04 -H20) supplemented with 100 Mg / ml of ampicillin at 37 ° C with shaking (200 rpm) until OD60o ~ 1 0. The expression of the protein was induced with 1 mM IPTG (pCRT7) or 0.2% arabinose (pBAD) and the crops grew an additional 3 hours. The cells were then harvested by centrifugation and the expression was assessed by SDS-PAGE of whole cell lysates.

Purification of Recombinant Proteins Cell pellets were suspended in 100 ml of TBS (20 mM Tris, pH 8.0, 150 mM NaCl) and lysates by passing through a French pressure cell (SLM-Aminco, Rochester, NY). The samples were then separated into a soluble fraction or an insoluble globule by centrifugation at 10,000 x g for 10 minutes. The localization of recombinant protein was evaluated by SDS-PAGE. If a recombinant protein was in a soluble fraction, then the protein was loaded onto the iminodiacetic acid agarose resin loaded with Ni 2+. See Table 6. Then, the column was washed with 30 mM imidazole in TBS. The bound proteins were eluted with 300 mM imidazole in TBS. If an additional purification step was required, the proteins were dialysed in 20 mM Tris, pH 8.0, containing 50 mM NaCl, 1 mM EDTA and loaded onto a column packed with POROS-Q resin (Applied Biosystems, Foster City , CA). The bound proteins were eluted with a gradient of 50 mM to 500 mM NaCl in 20 mM Tris, pH 8.0, 1 mM EDTA. Fractions containing the protein of interest were determined by SDS-PAGE and frozen at -20 ° C. If a recombinant protein was found in the insoluble fraction, then the insoluble fraction was treated with 100 ml of 1% Triton X-100 in TBS for 4 hours at 4 ° C. See Table 6. The insoluble proteins were palletized by centrifugation and the supernatant was discarded. The insoluble globule was then extracted with 100 ml of 8 M urea in TBS for at least 8 hours at room temperature. The insoluble wastes were pelleted and the protein was purified as above except that all the buffers contained 2M urea. After purification Triton X-100 was added to the final concentration of 0.1%. The proteins were then dialysed in TBS containing 0.1% Triton and stored at -20 ° C.

Liquid chromatography was developed using an AKTA scanner (Amersham-Pharmacia Biotech, Piscataway, NJ). All SDS-PAGE were developed using a 4-20% gradient of Tris-glycine gels (Cambrex).

Table 6 Solubility of the Recombinant Protein X indicates that the protein was found in that fraction. NT indicates not tested Example 8 Immunogenic Compositions Using Recombinant Staphylococcus epidermidis Proteins Mouse is four week Balb / C female (Charles River Laboratories, Wilmington, MA) were immunized at 0.3 and 6 weeks with 10 g of recombinant protein formulated with 20 g of STIMULON ™ QS-21 by subcutaneous injection. The mice were bled at week 0 before the first immunization and at week 8. Two days after the final bleeding, the mice were inoculated by intraperitoneal injection of 5 x 108 cfu of Staphylococcus epidermidis 0-47 which grew overnight in a Columbia salt agar (Columbia 1x agar, 0.1% glucose, 1% yeast extract, 0.5% NaCl). Twenty-four hours after inoculation, the mice were sacrificed and the bacteria enumerated in the vessel and in the blood.

Active Immunization of mice with recombinant proteins Twenty-seven orfs coding for serum-binding proteins or immunoreactive ones were cloned from Staphylococcus epidermidis 0-47 and the recombinant proteins were expressed in E. coli with a hexahistidine tag (the His tag was used as a matter of convenience; immunogenic composition of this invention would contain proteins expressed in the His tag). See Table 7. These proteins were purified using a Ni chelate column followed by ion exchange chromatography. The remaining three cloned orf (2006, 2975 and 2907) were cloned but not expressed at levels sufficient for purification. Balb / C mice were immunized at 0.3 and 6 weeks with individual recombinant proteins. The animals were bled at weeks 0 and 8 and inoculated (i.p.) at week 8 with Staphylococcus epidermidis 0-47. Twenty-four hours after the inoculation, the animals were euthanized and the number of bacteria present was enumerated in the blood and in the vessel. This initial selection of candidates for immunogenic composition was developed in groups of 5 animals to enable selection of the numerous proteins. The resulting data are not statistically significant but they provide valuable information on the potential of the immunogenic composition of a large number of candidates. Eight of the twenty-seven recombinant proteins reduced the number of bacteria recovered from the vessel and / or blood by one log or more. See Table 7 (NT = not tested).

Table 7 Reduction in Bacterial Counts After Unproved Immunization The serum obtained from the immunized mice was evaluated for the reactivity of the antibody to the bacterial proteins. See Table 8. Twenty-three or twenty-four immune sera tested reacted with the native proteins, determined by the western blots of the whole cell lysates of Staphylococcus epidermidis which grew to the log log phase in rabbit serum. See Table 8 (NT = not tested) Table 8 Reactivity of Antibody to Staphylococcus dica protein not tested As shown in Table 8, many of the animals immunized with Staphylococcus epidermidis antigens also developed antibody responses to Staphylococcus aureus. Therefore, immunogenic compositions against Staphylococcus epidermidis antigens could be effective in the treatment or prevention of Staphylococcus aureus as well as Staphylococcus epidermidis. See Table 8. A subset of the recombinant proteins used in the above immunogenic compositions were used to immunize large groups of mice. Groups of 10 female Balb / C mice (4 weeks old) were immunized by subcutaneous injection with saline or 10 pg of antigen with 20 pg of STIMULON ™ QS-21 as adjuvant. Two weeks after the last immunization, the mice were inoculated with ~ 5 x 108 cfu of S. epidermidis 0-47 by intraperitoneal injection. Twenty-four hours after the inoculation, the bacteria were enumerated in the blood and the vessel. See Table 9. The reduction in log CFU was determined compared to a control of STIMULON ™ QS-21 in saline. The data were analyzed using the student's T test with p values of? 0.05 or "0.01.

Table 9 Proteins Used in Immunogenic Compositions 'p-value < 0.05"p-value <0.01 The Staphylococcus epidermidis proteins shown in Table 9 showed the greatest effectiveness when used in immunogenic compositions that reduced the severity of a bacterial infection after a subsequent inoculation.

Example 9 Protection of Inoculation with Staphylococcus aureus after Immunization with Staphylococcus epidermidis Proteins As suggested by the antibody binding data in Example 8, (Table 8), the immunogenic compositions against the antigens of Staphylococcus epidermidis could be effective in the treatment or prevention of Staphylococcus aureus. Therefore, an inoculation using Staphylococcus aureus was developed after immunization with immunogenic compositions of Staphylococcus epidermidis antigens. Four week old female CD-1 mice (Charles River Laboratories, Wilmington, MA) were immunized at 0.3 and 6 weeks with 10 pg of recombinant protein in 20 pg of STIMULON ™ QS-21 by subcutaneous injection. Mice were bled at week 0 before the first immunization and at week 8. Two days after final bleeding the mice were inoculated by intraperitoneal injection of 3 x 108 cfu of S. aureus Reynolds grown overnight in a Columbia salt (Columbia 1x agar, 0.1% glucose, 1% yeast extract, 0.5% NaCl). Twenty-four hours after inoculation, the mice were sacrificed and the bacteria were enumerated in the kidney. Table 10 Inoculation with Staphylococcus aureus As shown in Table 10, certain antigens of Staphylococcus epidermidis were effective in inducing antibodies that they recognized and bound to Staphylococcus aureus. In addition, the induced antibodies had the beneficial effect of reducing the level of enumerated bacteria after an inoculation with Staphylococcus aureus. The percent identity of the amino acid sequence of the Staphylococcus epidermidis polypeptide antigens of SEQ ID NO: 1 to SEQ ID NO: 32 were compared to the amino acid sequence of their homologs from Staphylococcus aureus. The results are shown in Table 1 1.

Table 11 Identity between Staphylococcus epidermidis and Staphylococcus aureus Homology was determined between the polypeptide sequences

Claims (1)

1. CLAIMS An immunogenic composition comprising a polypeptide having an amino acid sequence selected from one or more of SEQ ID NO: 1 to SEQ ID NO: 32, a biological equivalent thereof, or a fragment thereof. The immunogenic composition of claim 1, wherein the polypeptide is immunoreactive with antibodies in the serum of rabbits infected with Staphylococcus epidermidis. The immunogenic composition of claim 1 or 2, wherein the polypeptide binds to one or more of the rabbit serum proteins. The immunogenic composition according to any one or more of claims 1 to 3, further comprising a pharmaceutically acceptable carrier. The immunogenic composition according to any one or more of claims 1 to 4, further comprising one or more adjuvants. The immunogenic composition according to any one or more of claims 1 to 5, wherein the polypeptide is derived from Staphylococcus epidermidis. The immunogenic composition according to any one or more of claims 1 to 6, wherein the polypeptide further comprises heterologous amino acids. The immunogenic composition according to any one or more of claims 1 to 7, wherein the polypeptide is a fusion polypeptide. The immunogenic composition according to any one or more of claims 1 to 8, wherein the polypeptide is a recombinant polypeptide. The immunogenic composition according to any one or more of claims 1 to 9, wherein the polypeptide is isolated from epidermal Staphylococcus. The immunogenic composition according to any one or more of claims 1 to 10, wherein the polypeptide comprises a neutralizing epitope of Staphylococcus epidermidis. The immunogenic composition according to any one or more of claims 1 to 11, wherein the polypeptide is a lipoprotein. The immunogenic composition according to any one or more of claims 1 to 12, said composition additionally comprises a polysaccharide antigen of Staphylococcus epidermidis. The immunogenic composition according to any one or more of claims 1 to 13, said composition additionally comprises a polysaccharide or Staphylococcus aureus polypeptide antigen. The immunogenic composition according to any one or more of claims 1 to 14, wherein the polypeptide comprises a polypeptide sequence of Staphylococcus epidermidis selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO : 27, and SEQ ID NO: 30, a biological equivalent of this, or a fragment of it. The immunogenic composition according to any one or more of claims 1 to 15, wherein the polypeptide is encoded by a polynucleotide comprising a nucleotide sequence having at least about 95% identity with a nucleotide sequence selected from one of SEQ ID NO: 33 to SEQ ID NO: 64 or a degenerate variant thereof, or a fragment thereof. The immunogenic composition according to any one or more of claims 1 to 16, wherein the polynucleotide is derived from Staphylococcus epidermidis. The immunogenic composition according to any one or more of claims 1 to 17, wherein the Staphylococcus epidermidis polynucleotide sequence is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 62, or a degenerate variant thereof, or a fragment thereof. The immunogenic composition according to any one or more of claims 1 to 18, wherein the polynucleotide further comprises heterologous nucleotides. An immunogenic composition comprising a polynucleotide having a nucleotide sequence selected from one of SEQ ID NO: 33 to SEQ ID NO: 64, a degenerate variant of these, or a fragment thereof and is comprised in an expression vector. The immunogenic composition of claim 20, wherein the Staphylococcus epidermidis polynucleotide sequence is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 62, or one Degenerate variant of this, or a fragment of this. The immunogenic composition of claim 20 or 21, wherein the vector is a plasmid DNA. The immunogenic composition according to any one or more of claims 20 to 22, wherein the polynucleotide is a recombinant polynucleotide. The immunogenic composition according to any one or more of claims 20 to 23, wherein the polynucleotide is derived from Staphylococcus epidermidis. The immunogenic composition of claim 24, wherein the polynucleotide comprises heterologous nucleotides. The immunogenic composition according to any one or more of claims 20 to 23, wherein the polynucleotide is operably linked to one or more gene expression regulatory elements. The immunogenic composition of claim 24, wherein the polynucleotide directs the expression of a neutralizing epitope of Staphylococcus epidermidis. The immunogenic composition according to any one or more of claims 20 to 23, further comprising an agent that facilitates transfection. The immunogenic composition of claim 28, wherein said agent facilitating transfection is bupivicaine. A method for inducing an immune response against Staphylococcus epidermidis which comprises administering to a mammal an immunogenic amount of a composition comprising: a polypeptide having an amino acid sequence selected from one or more of SEQ ID NO: 1 to SEQ ID NO : 32 or biological equivalent thereof, or a fragment thereof, and a pharmaceutically acceptable carrier. The method of claim 30, wherein the polypeptide further comprises heterologous amino acids. The method of claim 31, wherein the polypeptide is a fusion polypeptide. The method of claim 30, further comprising an adjuvant. The method of claim 30 or 31, wherein the polypeptide is a recombinant polypeptide. A method for inducing an immune response against Staphylococcus epidermidis which comprises administering to a mammal an immunogenic amount of a composition comprising: a polynucleotide having a nucleotide sequence selected from one or more of SEQ ID NO: 33 to SEQ ID NO: 64, a degenerate variant thereof, a fragment thereof and a pharmaceutically acceptable carrier. The method of claim 35, further comprising a vector. The method of claim 35, wherein the vector is a plasmid DNA. The method of claim 35, wherein the polynucleotide is a recombinant polynucleotide. The method of claim 35, wherein the polynucleotide further comprises heterologous nucleotides. The method of claim 35, wherein the polynucleotide is operably linked to one or more of the gene expression regulatory elements. The method of claim 35, further comprising an adjuvant. The method of claim 35, wherein said composition additionally comprises a transfection facilitating agent. The method of claim 42, wherein said transfection facilitating agent is bupivicaine. A method for inducing an immune response against Staphylococcus aureus which comprises administering to a mammal an immunogenic amount of a composition comprising: a polypeptide sequence of Staphylococcus epidermidis selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10 , SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 30, a biological equivalent thereof, or a fragment thereof. A method for inducing an immune response against Staphylococcus aureus which comprises administering to a mammal an immunogenic amount of a composition comprising: a Staphylococcus epidermidis polynucleotide sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 62, or a degenerate variant thereof, or a fragment thereof. A method for the detection and / or identification of Staphylococcus epidermidis in a biological sample comprising: (a) contacting the sample with an oligonucleotide probe of a polynucleotide comprising the selected nucleotide sequence of one of SEQ ID NO: 33 to SEQ ID NO: 64, or a degenerate variant thereof, or a fragment thereof, under conditions that allow hybridization; and (b) detecting the presence of hybridization complexes in the sample, wherein the hybridization complexes indicate the presence of Staphylococcus epidermidis in the sample. A method for the detection and / or identification of antibodies to Staphylococcus epidermidis in a biological sample comprising: (a) contacting the sample with a polypeptide comprising an amino acid sequence selected from one of SEQ ID NO: 1 to SEQ ID NO: 32, or a biological equivalent thereof, or a fragment thereof, under conditions that allow the formation of immune complexes; and (b) detect the presence of immune complexes in the sample, where the immune complexes indicate the presence of Staphylococcus epidermidis in the sample. A composition according to any one of claims 1 to 29 for use in a method for inducing an immune response against Staphylococcus epidermidis.