Methods and Compositions Involving Staphylococcus Protein A (SpA) Variants

Abstract

Embodiments relate to methods and compositions for preventing or treating bacterial infections, particularly infections caused by Staphylococcus.Embodiments relate to improved non-toxigenic Protein A (SpA) variants.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 847,832, filed May 14, 2019, which is incorporated by reference in its entirety.

[0002] This invention was made with government support under Grant Nos. AI038897 and AI052474 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] I. FIELD OF THEINVENTION The present invention relates generally to the fields of immunology, microbiology, and pathology. More specifically, the present invention relates to methods and compositions involving bacterial Protein A variants that can be used to elicit an immune response against bacteria. [Background technology]

[0004] II. Background The number of both community-acquired and hospital-acquired infections has increased in recent years with the increased use of intravascular devices. Hospital-acquired (nosocomial) infections are a major cause of morbidity and mortality, and more specifically, in the United States, hospital-acquired infections affect more than 2 million patients annually. The most frequent infections are urinary tract infections (33% of infections), followed by pneumonia (15.5%), surgical site infections (14.8%) and major bloodstream infections (13%) (Emorl and Gaynes, 1993).

[0005] The main nosocomial pathogens include Staphylococcus aureus, coagulase-negative staphylococci (mostly Staphylococcus epidermidis), enterococcus spp., Escherichia coli, and Pseudomonas aeruginosa. Although these pathogens cause roughly equal numbers of infections, the severity of the damage they can produce, combined with the frequency of antibiotic-resistant isolates, ranks S. aureus and S. epidermidis as the most important nosocomial pathogens.

[0006] Staphylococci can cause a wide variety of diseases in humans and other animals, either through toxin production or invasion. Staphylococcal toxins are also a common cause of food poisoning, as the bacteria can grow in improperly stored foods.

[0007] S. epidermidis is a normal skin commensal that is also an important opportunistic pathogen involved in infection of malfunctioning medical devices and at surgical sites. Medical devices infected by S. epidermidis include cardiac pacemakers, cerebrospinal fluid shunts, continuous ambulatory peritoneal dialysis catheters, orthopedic devices, and artificial heart valves.

[0008] S. aureus is the most common cause of hospital-acquired infections with significant morbidity and mortality. It is responsible for some cases of osteomyelitis, endocarditis, septic arthritis, pneumonia, abscesses, and toxic shock syndrome. S. aureus can survive on dry surfaces, increasing the chances of infection. Any S. aureus infection can cause staphylococcal scalded skin syndrome, a skin reaction to exotoxins absorbed into the bloodstream. This can also lead to a type of sepsis called pyemia, which can be life-threatening. Of concern is that methicillin-resistant S. aureus (MRSA) has become the leading cause of hospital-acquired infections.

[0009] Staphylococcus aureus and Staphylococcus epidermidis infections are typically treated with antibiotics, with penicillin being the drug of choice, and vancomycin being used for methicillin-resistant isolates. The proportion of Staphylococcus strains that exhibit broad resistance to antibiotics is becoming increasingly widespread and poses a threat to effective antibacterial therapy. Furthermore, the recent emergence of vancomycin-resistant S. aureus strains has stoked fears of the emergence and spread of MRSA strains for which no effective treatment is available.

[0010] Alternatives to antibiotic treatment for staphylococcal infections are under investigation using antibodies directed against staphylococcal antigens, which involve administration of polyclonal antisera (WO00 / 15238, WO00 / 12132) or treatment with monoclonal antibodies against lipoteichoic acid (WO98 / 57994).

[0011] An alternative approach would be the use of active vaccination to generate an immune response against staphylococci. The genome of S. aureus has been sequenced and many of the coding sequences identified (WO02 / 094868, EP0786519), which may lead to the identification of potential antigens. The same applies to S. epidermidis (WO01 / 34809). As an elaboration of this approach, others have identified proteins recognized by hyperimmune sera from patients suffering from staphylococcal infections (WO01 / 98499, WO02 / 059148).

[0012] S. aureus secretes a large number of virulence factors into the extracellular environment (Archer, 1998; Dinges et al., 2000; Foster, 2005; Shaw et al., 2004; Sibbald et al., 2006). Like most secreted proteins, these virulence factors are translocated in and out of the plasma membrane by the Sec machinery. Proteins secreted by the Sec machinery have an N-terminal leader peptide that is removed by a leader peptidase when the preprotein is bound to the Sec translocon (Dalbey and Wickner, 1985; van Wely et al., 2001). Recent genome analyses suggest that members of the phyla Actinobacteria and Firmicutes encode additional secretion systems that recognize a subset of proteins in a Sec-independent manner (Pallen, 2002). ESAT-6 (early secreted antigen target 6 kDa) and CFP-10 (culture filtrate antigen 10 kDa) of Mycobacterium tuberculosis represent the first substrates of this novel secretion system, named ESX-1 or Snm in M. tuberculosis (Andersen et al., 1995; Hsu et al., 2003; Pym et al., 2003; Stanley et al., 2003). In Staphylococcus aureus, two ESAT-6-like factors, named EsxA and EsxB, are involved in the Ess pathway ( E SAT-6 secretion system ( E SAT-6 s ecretion s The enzyme is secreted by the mitochondrial system (Burts et al., 2005).

[0013] First generation vaccines targeting S. aureus or exoproteins produced by S. aureus have met with limited success (Lee, 1996). There remains a need to develop effective vaccines against staphylococcal infections. There is also a need for additional compositions for treating staphylococcal infections.

[0014] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the art of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method utilized to determine the value.

[0015] The use of the words "a" and "an," when used in conjunction with the term "comprising," can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0016] As used herein, the terms "or" and "and / or" are used to describe multiple components in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0017] The words "comprising" (and any form of including, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), "characterized by" (and any form of including, e.g., "characterized as"), or "containing" (and any form of including, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0018] The compositions and methods for their use can "comprise", "consist essentially of", or "consist of" any of the components or steps disclosed throughout the specification. The phrase "consisting of" excludes any element, step, or ingredient not specified. The phrase "consisting essentially of" limits the scope of the described subject matter to those materials or steps specified and which do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term "comprising" can also be practiced in the context of the term "consisting of" or "essentially consisting of".

[0019] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may be applied to any other embodiment of the invention. Moreover, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or utilize any composition of the invention. Aspects of the embodiments described in the examples are also embodiments that may be contemplated in the context of embodiments discussed elsewhere in different examples or elsewhere in this application, e.g., in the Summary of the Invention, Detailed Description of the Invention, Claims, Brief Description of the Drawings.

[0020] Other objectives, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. Summary of the Invention

[0021] Compositions and methods are provided for improved Protein A variants with reduced binding to VH3 of immunoglobulins that reduce their toxin-producing capacity and stimulate a humoral immune response that protects against staphylococcal disease. In certain embodiments, the improved Protein A variants stimulate a humoral immune response that can (i) prevent colonization and (ii) cause decolonization of actively colonized individuals.

[0022] In some embodiments, compositions and methods involve an isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution of a glutamine residue in each of domains A-E corresponding to positions 9 and 10 in domain D, and (ii) a glutamic acid substitution in each of domains A-E corresponding to position 33 in domain D, wherein the polypeptide does not detectably crosslink IgG and IgE in blood or activate basophils or mast cells compared to a negative control. The variant SpA region is contemplated in some embodiments to crosslink IgG and / or IgE and / or activate basophils in blood, and not pose significant safety or toxicity issues or significant risk of anaphylactic shock in human patients. In some embodiments, the K for VH3 from human IgG is A The binding affinity was improved by SpA variants (SpAKKAA In some embodiments, the SpA variant is KKAA K for VH3 from human IgG that is at least two-fold reduced compared to A In some embodiments, the KA binding affinity can be measured by any method disclosed herein. In some embodiments, the SpA variant has a binding affinity to SpA. KKAA 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold or more (or any range derivable therein) reduced K for VH3 from human IgG compared to A In some embodiments, the SpA variant has a binding affinity to SpA KKAA , 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300% or more (or any range derivable therein) reduced K for VH3 from human IgG compared to A It has binding affinity. Of course, it is understood that the comparison is made using the same or equivalent assay. In some embodiments, the SpA variant is about 1×10 5 M -1 K for VH3 from human IgG is less than A In some embodiments, the SpA variant has a binding affinity of about 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 x 10 5 M -1 K for human IgG VH3 is less than or equal to (or any arrangement derivable therein). AIn some embodiments, the isolated polypeptide comprising an SpA variant has no substitutions in any of domains A-E corresponding to positions 36 and 37 in domain D. In some embodiments, the only substitutions in the SpA variant are (i) a lysine substitution for a glutamine residue in each of domains A-E corresponding to positions 9 and 10 in domain D, and (ii) a glutamic acid substitution in each of domains A-E corresponding to position 33 in domain D (SpA Q9,10K / S33E In some embodiments, the isolated polypeptide comprises an SpA variant. In some embodiments, the isolated polypeptide comprises an SpA variant. Q9,10K / S33E It consists of:

[0023] In some embodiments, there is an isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution of a glutamine residue in each of domains A-E corresponding to positions 9 and 10 in domain D, and (ii) a threonine substitution in each of domains A-E corresponding to position 33 in domain D, wherein the polypeptide does not detectably crosslink IgG and IgE in blood or activate basophils or mast cells relative to a negative control. The variant SpA region is contemplated in some embodiments such that it does not crosslink IgG and / or IgE in blood or activate basophils, pose significant safety or toxicity concerns in human patients, or pose a significant risk of anaphylactic shock in human patients. In some embodiments, the KA binding affinity to VH3 from human IgG is greater than or equal to that of an SpA variant (SpA KKAA In some embodiments, the SpA variant is KKAA K for VH3 from human IgG that is at least two-fold reduced compared to AIn some embodiments, the KA binding affinity can be measured by any method disclosed herein. In some embodiments, the SpA variant has a binding affinity to SpA. KKAA 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold or more (or any range derivable therein) reduced K for VH3 from human IgG compared to A In some embodiments, the SpA variant has a binding affinity to SpA KKAA , 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300% or more (or any range derivable therein) reduced K for VH3 from human IgG compared to A It has binding affinity. Of course, it is understood that the comparison is made using the same or equivalent assay. In some embodiments, the SpA variant is about 1×10 5 M -1 K for VH3 from human IgG is less than A In some embodiments, the SpA variant has a binding affinity of about 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 x 10 5 M -1 K for human IgG VH3 is less than or equal to (or any arrangement derivable therein). AIn some embodiments, the isolated polypeptide comprising an SpA variant has no substitutions in any of domains A-E corresponding to positions 36 and 37 in domain D. In some embodiments, the only substitutions in the SpA variant are (i) a lysine substitution for a glutamine residue in each of domains A-E corresponding to positions 9 and 10 in domain D, and (ii) a threonine substitution in each of domains A-E corresponding to position 33 in domain D (SpA Q9,10K / S33T In some embodiments, the isolated polypeptide comprises an SpA variant. In some embodiments, the isolated polypeptide comprises an SpA variant. Q9,10K / S33T It consists of:

[0024] In some embodiments, the compositions and methods are Q9,10K / S33E Any of the embodiments discussed below in the context of SpA variants may be used in particular with SpA Q9,10K / S33E This has the significant advantage of reducing toxicity and / or activity associated with anaphylactic shock. Q9,10K / S33E In some embodiments, there is provided a method for inducing a safe immune response against Staphylococcus aureus in a human patient, comprising administering to the human patient an effective amount of a composition comprising: Q9,10K / S33E There is a method for inducing a safe immune response against Staphylococcus in a subject, comprising administering to the subject an effective amount of a composition comprising: The subject may be a laboratory animal, or an industrial animal, such as a cow, pig, sheep, goat, or any other animal raised for food, and may be suffering from a Staphylococcus infection, such as mastitis.

[0025] In some embodiments, the compositions and methods are Q9,10K / S33E Any of the embodiments discussed below in the context of SpA variants may be used in particular with SpA Q9,10K / S33T This has the significant advantage of reducing toxicity and / or activity associated with anaphylactic shock. Q9,10K / S33TIn one embodiment, there is provided a method for inducing a safe immune response against Staphylococcus aureus in a human patient comprising administering to the human patient an effective amount of a composition comprising:

[0026] In some embodiments, the compositions and methods include an isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution of the glutamine residues corresponding to positions 9 and 10 in each of Domains A-E, and (ii) at least one other amino acid substitution corresponding to positions 29 and / or 33 in SEQ ID NO:2 in each of Domains A-E, wherein the SpA variant has a lysine substitution of 1.0×10 -4 K for VH3 from human IgG exceeds M D In the same or additional embodiments, the SpA variant has a binding affinity of 1.0×10 -6 K for VH3 from human IgE exceeds M D It has binding affinity.

[0027] Embodiments include the creation of Protein A variants that are safe for human use that are unable to release immune mediators such as histamine that cause vascular leakage and anaphylactic reactions, particularly variants that are unable to bind to crosslink variant heavy chains of VH3 idiotype IgG and IgE molecules that bind to Fc receptors on the surface of immune cells.

[0028] Amino acid substitutions are made in each of domains A, B, C, D, and E (Domains A-E) of variant SpA to substitute amino acids corresponding to glutamine residues at positions 9 and 10 as numbered in Domain D, which is SEQ ID NO:2; the same amino acid substitutions are made to corresponding amino acids in Domains A, B, C, and E. The embodiment relates to SpA in which in each of Domains A-E, a lysine residue is substituted for the glutamic acid residues corresponding to positions 9 and 10 in Domain D.

[0029] In certain embodiments, the SpA variant is about 1.0×10 -4 M, 5.0×10 -4 M, 1.0×10 -3 M, 5.0×10 -3 M, 1.0×10 -2 M, 5.0×10 -2 M, 1.0×10 -1 M, 5.0×10 -1 K for VH3 from human IgG exceeds M (or any range derivable therein) D In some embodiments, the binding affinity to a VH3 from human IgG is about 1.0×10 -2 M or 5.0×10 -2 In the same or additional embodiments, the SpA variant has a genotype greater than about 1.0×10 -6 M, 5.0×10 -6 M, 1.0×10 -5 M, 5.0×10 -5 M, 1.0×10 -4 M, 5.0×10 -4 M, 1.0×10 -3 M, 5.0×10 -3 M, 1.0×10 -2 M, 5.0×10 -2 M, 1.0×10 -1 M, or 5.0 x 10 -1 K for VH3 from human IgE that exceeds M (or any range derivable therein) D The affinity of SpA and its variants to IgG may be measured using purified human IgG (from pooled serum) and purified human monoclonal antibodies (Trastuzumab human IgG1 VH3 clone antibody and IgE Trastuzumab). The assays used may be ELISA and / or surface plasmon resonance.

[0030] In certain embodiments, the variant SpA comprises an amino acid substitution corresponding to position 29 in SEQ ID NO:2 in each of domains A-E. In some cases, the amino acid substitution corresponding to position 29 is alanine, leucine, proline, phenylalanine, glutamic acid, arginine, lysine, serine, threonine, or glutamine. In some embodiments, the variant SpA comprises an amino acid substitution corresponding to position 29 that is alanine, phenylalanine, or arginine. In other embodiments, the substitution is not with one of the following: leucine, proline, glutamic acid, lysine, serine, threonine, or glutamine. In some embodiments, the variant SpA comprises an amino acid substitution corresponding to position 33 in SEQ ID NO:2 in each of domains A-E. In certain embodiments, the amino acid substitution corresponding to position 33 is with alanine, phenylalanine, glutamic acid, lysine, or glutamine. In certain cases, the substitution is with phenylalanine, glutamic acid, or glutamine. In other embodiments, the substitutions are not with alanine or lysine. In some SpA variants, there are substitutions as described herein corresponding to positions 29 and 33 in SEQ ID NO:2 in each of domains A-E. These may or may not be combined with amino acid substitutions corresponding to one or both of positions 36 and 37 in SEQ ID NO:2 in each of domains A-E. In some cases, there are substitutions at both positions 36 and 37. In some cases, the amino acid substitutions corresponding to positions 36 and 37 are alanine residues for aspartic acid residues.

[0031] In some embodiments, a variant SpA polypeptide comprises variant A-E domains (meaning a polypeptide having variant Domain A, variant Domain B, variant Domain C, variant Domain D, and variant Domain E), and each of the variant domains has at least 70%, 75%, 80%, 85%, 90%, 95%, or 96% identity to Protein A Domain D polypeptide (SEQ ID NO:2), Domain E (SEQ ID NO:3), Domain A (SEQ ID NO:4), Domain C (SEQ ID NO:5), Domain B (SEQ ID NO:6). Alternatively, a variant SpA polypeptide comprises variant A-E domains (meaning a polypeptide having variant Domain A, variant Domain B, variant Domain C, variant Domain D, and variant Domain E), and each of the variant domains has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to Domain D polypeptide (SEQ ID NO:2), Domain E (SEQ ID NO:3), Domain A (SEQ ID NO:4), Domain C (SEQ ID NO:5), Domain B (SEQ ID NO:6) of Protein A, other than those specifically identified as having amino acid substitutions. In other words, an SpA variant having substitutions at positions 9, 10, and 29 can have Domains A-E that are 100% identical to the non-variant sequences except for the substitutions. In some embodiments, for example, variant SpaA comprises variant A-E domains that do not contain any amino acid substitutions in SEQ ID NO:2 except for corresponding positions 9, 10, 29, 33, 36, and / or 37.

[0032] In certain embodiments, the variant SpaA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, and 29 in SEQ ID NO:2. In certain other embodiments, the variant SpaA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, and 33 in SEQ ID NO:2. In additional embodiments, the variant SpaA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, and 33 in SEQ ID NO:2. In addition, other embodiments relate to variant SpaA comprising variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, 36, and 37 in SEQ ID NO:2. In further embodiments, the variant SpaA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 33, 36, and 37 in SEQ ID NO:2. In other embodiments, the variant SpaA comprises variant AE domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, 33, 36, and 37 in SEQ ID NO:2.

[0033] In additional other embodiments, the variant SpA is part of an isolated polypeptide. The isolated polypeptide may or may not include a region from protein A. In some cases, the non-protein A segment is a second antigen segment, which may or may not be a Staphylococcus antigen segment. In some embodiments, the segment may include an Emp, EsxA, EsxB, EsaC, Eap, Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, IsdC, ClfA, ClfB, and / or SasF segment.

[0034] Embodiments include the use of Protein A variants in methods and compositions for the treatment of bacterial and / or staphylococcal infections. The application also provides immunogenic compositions comprising Protein A variants or immunogenic fragments thereof. In certain aspects, the immunogenic fragment is a Protein A domain D segment. Additionally, the present invention provides methods and compositions that can be used to treat (e.g., limit staphylococcal abscess formation and / or persistence in a subject) or prevent bacterial infections. In some cases, the method for stimulating an immune response involves administering to a subject an effective amount of a composition comprising or encoding all or a portion of a Protein A variant polypeptide or antigen, and in certain aspects, other bacterial proteins. Other bacterial proteins include, but are not limited to, (i) secreted virulence factors and / or cell surface proteins or peptides, or (ii) recombinant nucleic acid molecules encoding secreted virulence factors and / or cell surface proteins or peptides.

[0035] In some embodiments, there are methods involving SpA variants.Methods include treating Staphylococcus infection in a subject, inducing immune response against Staphylococcus in a subject, preventing Staphylococcus infection in a subject, preventing Staphylococcus colonization or recolonization in a subject, reducing Staphylococcus colonization or recolonization in a subject, treating Staphylococcus infection in a subject without significant toxicity, and / or inducing Staphylococcus killing in a subject.Steps can include administering to the subject an effective amount of SpA variant or a composition comprising SpA variant.The subject may be in need of treatment or prevention due to existing infection or risk of infection.

[0036] In some embodiments, the subject does not show any signs of toxicity from the isolated polypeptide or composition. In further embodiments, the subject does not show any signs of anaphylactic shock from the isolated polypeptide or composition. In some embodiments, the subject is evaluated for signs of toxicity, which may include signs of anaphylactic shock.

[0037] The polypeptide can be formulated into a pharma- ceutically acceptable composition, which can further comprise one or more of at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 additional staphylococcal antigens or immunogenic fragments thereof (e.g., Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla (e.g., H35 mutant), IsdC, SasF, vWbp, or vWh).Additional staphylococcal antigens that can be used in combination with the Protein A variants include, but are not limited to, 52 kDa vitronectin binding protein (WO 01 / 60852), Aaa (GenBank CAC80837), Aap (GenBank accession AJ249487), Ant (GenBank accession NP_372518), autolysin glucosaminidase, autolysin amidase, Cna, collagen binding protein (US6288214), EFB (FIB), elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein (US6008341), fibronectin binding protein (US5840846), FnbA, FnbB, GehD (US 2002 / 0169288), HarA, HBP, immunodominant ABC transporter, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activator protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00 / 12689), SdrG / Fig (WO 00 / 12689), SdrH (WO 00 / 12689), SEA exotoxin (WO 00 / 02523), SEB exotoxin (WO 00 / 02523), SitC and Ni These include ABC transporters, SitC / MntC / saliva binding proteins (US5,801,234), SsaA, SSP-1, SSP-2, and / or vitronectin binding proteins (see PCT Publications W02007 / 113222, W02007 / 113223, W02006 / 032472, W02006 / 032475, W02006 / 032500, each of which is incorporated herein by reference in its entirety). The staphylococcal antigen or immunogenic fragment can be administered simultaneously with the Protein A variant. The staphylococcal antigen or immunogenic fragment and the Protein A variant can be administered in the same composition. The Protein A variant can also be a recombinant nucleic acid molecule encoding the Protein A variant. The recombinant nucleic acid molecule can encode the Protein A variant and at least one staphylococcal antigen or immunogenic fragment thereof.As used herein, the term "modify" or "modification" encompasses the meaning of the words "enhance" or "inhibit." "Modification" of an activity can be either an increase or decrease in activity. As used herein, the term "modulator" refers to a compound that effects the function of a moiety, including upregulation, induction, stimulation, enhancement, inhibition, downregulation, or suppression of a protein, nucleic acid, gene, or organism, etc.

[0038] In certain embodiments, the methods and compositions use or comprise or encode all or part of a Protein A variant or antigen. In other aspects, the Protein A variant may be used in combination with a secreted factor or surface antigen, including, but not limited to, one or more of an isolated Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, or vWh polypeptide, or immunogenic segments thereof. Additional staphylococcal antigens that may be used in combination with the Protein A variants include, but are not limited to, 52 kDa vitronectin binding protein (WO 01 / 60852), Aaa, Aap, Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen binding protein (US6288214), EFB (FIB), elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein (US6008341), fibronectin binding protein (US5840846), FnbA, FnbB, GehD (US 2002 / 0169288), HarA, HBP, immunodominant ABC transporters, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00 / 12689), SdrG / Fig (WO 00 / 12689), SdrH (WO 00 / 12689), SEA exotoxin (WO 00 / 02523), SEB exotoxin (WO 00 / 02523), SitC and Ni ABC transporters, SitC / MntC / saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and / or vitronectin binding protein.In certain embodiments, the proteins selected from the group consisting of Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh, vitronectin binding protein of 52 kDa (WO 01 / 60852), Aaa, Aap, Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen binding protein (US6288214), EFB (FIB), elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein (US6008341), fibronectin binding protein (US5840846), FnbA, FnbB, GehD (US 2002 / 0169288), HarA, HBP, immunodominant ABC transporter, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activator protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00 / 12689), SdrG / Fig (WO 00 / 12689), SdrH (WO 00 / 12689), SEA exotoxin (WO 00 / 02523), SEB exotoxin (WO 00 / 02523), SitC and Ni One, two, three, four, five, six, seven, eight, nine, ten or more of ABC transporters, SitC / MntC / saliva binding proteins (US Pat. No. 5,801,234), SsaA, SSP-1, SSP-2, and / or vitronectin binding proteins may be specifically excluded from the formulation.

[0039] In yet further aspects, the isolated protein A variant is multimerized, e.g., dimerized, or is a linear fusion of two or more polypeptides or peptide segments. In certain aspects of the invention, the composition comprises multimers or concatemers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more isolated cell surface proteins or segments thereof. Concatemers are linear polypeptides with one or more repeating peptide units. The SpA polypeptides or fragments may be contiguous or separated by spacers or other peptide sequences, e.g., one or more additional bacterial peptides. In further aspects, other polypeptides or peptides included in the multimer or concatemer can include, but are not limited to, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen of Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh, or immunogenic fragments thereof.Additional staphylococcal antigens that may be used in combination with the Protein A variants include, but are not limited to, 52 kDa vitronectin binding protein (WO 01 / 60852), Aaa, Aap, Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen binding protein (US6288214), EFB (FIB), elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein (US6008341), fibronectin binding protein (US5840846), FnbA, FnbB, GehD (US 2002 / 0169288), HarA, HBP, immunodominant ABC transporters, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00 / 12689), SdrG / Fig (WO 00 / 12689), SdrH (WO 00 / 12689), SEA exotoxin (WO 00 / 02523), SEB exotoxin (WO 00 / 02523), SitC and Ni ABC transporters, SitC / MntC / saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and / or vitronectin binding protein.

[0040] The term "Protein A variant" or "SpA variant" refers to a variant of the Fc and V H 3. In certain aspects, SpA variants include variant domain D peptides, as well as variants of SpA polypeptides and segments thereof that are non-toxigenic and stimulate an immune response against staphylococcal protein A and / or bacteria expressing it.

[0041] The embodiments also include a method for inducing an immune response against Staphylococcus in a subject, comprising providing an effective amount of a Protein A variant or a segment thereof to the subject. In certain aspects, the method for inducing an immune response against Staphylococcus in a subject comprises providing an effective amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more secreted and / or cell surface proteins or segments / fragments thereof to the subject. Secreted or cell surface proteins include, but are not limited to, Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, and / or vWh proteins, and immunogenic fragments thereof. Additional staphylococcal antigens that may be used in combination with the Protein A variants include, but are not limited to, 52 kDa vitronectin binding protein (WO 01 / 60852), Aaa, Aap, Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen binding protein (US6288214), EFB (FIB), elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein (US6008341), fibronectin binding protein (US5840846), FnbA, FnbB, GehD (US 2002 / 0169288), HarA, HBP, immunodominant ABC transporters, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00 / 12689), SdrG / Fig (WO 00 / 12689), SdrH (WO 00 / 12689), SEA exotoxin (WO 00 / 02523), SEB exotoxin (WO 00 / 02523), SitC and Ni ABC transporters, SitC / MntC / saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and / or vitronectin binding protein.

[0042] Embodiments, including method, composition, and polypeptide embodiments, include certain embodiments in which the Staphylococcus comprises the WU1 or JSNZ strain of Staphylococcus aureus. In some embodiments, the Staphylococcus comprises an ST88 isolate.

[0043] In some embodiments, the subject or patient described herein, e.g., human patient, is a pediatric patient. A pediatric patient is a patient defined as being under 18 years old. In some embodiments, the patient is at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, or 90 years old (or any range derivable therein). In some embodiments, the pediatric patient is 2 years old or younger. In some embodiments, the pediatric patient is under 1 year old. In some embodiments, the pediatric patient is under 6 months old. In some embodiments, the pediatric patient is 2 months of age or younger. In some embodiments, the human patient is 65 years of age or older. In some embodiments, the human patient is a healthcare worker. In some embodiments, the patient is a patient scheduled to undergo surgery.

[0044] In some embodiments, the isolated polypeptide of the patient, composition is administered in four doses, with an interval between doses of at least four weeks. In some embodiments, the isolated polypeptide is given in four doses or exactly four doses. In some embodiments, the isolated polypeptide or composition is given at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, or 8 doses. In some embodiments, the first dose is administered at 6-8 weeks of age. In some embodiments, all four doses are administered at or before age 2 years. In some embodiments, the polypeptide or composition should be administered as a set of four doses at 2, 4, 6, and 12-15 months of age. The first dose may be given as early as 6 weeks of age. The interval between doses may be about 4-8 weeks. In some embodiments, the fourth dose is administered at approximately 12-15 months of age and at least 2 months after the third dose.

[0045] Embodiments include compositions comprising a polypeptide, peptide, or protein having or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or similarity to Protein A, or a second protein or peptide that is a secreted bacterial protein or a bacterial cell surface protein. In further embodiments, a composition may comprise a nucleic acid sequence encoding a polypeptide, peptide, or protein having, or at least, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or similarity to a Domain D polypeptide (SEQ ID NO:2), Domain E (SEQ ID NO:3), Domain A (SEQ ID NO:4), Domain C (SEQ ID NO:5), Domain B (SEQ ID NO:6) of Protein A, or a Domain D, Domain E, Domain A, Domain C, or Domain B polypeptide of Protein A. In certain aspects, a Protein A polypeptide segment has the amino acid sequence of SEQ ID NO:8. Similarity or identity, preferably identity, is known in the art and several different programs can be used to identify whether a protein (or nucleic acid) has sequence identity or similarity to a known sequence.Sequence identity and / or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman (1981), the sequence identity alignment algorithm of Needleman & Wunsch (1970), the similarity search method of Pearson & Lipman (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al. (1984), preferably using default settings, or by visual inspection. Preferably, percent identity is calculated by using alignment tools known and easily ascertainable by those skilled in the art. Percent identity is essentially 100 times the number of identical amino acids divided by the total number of amino acids compared.

[0046] Yet further embodiments include a method for stimulating a protective or therapeutic immune response in a subject against staphylococci, comprising administering to the subject an effective amount of (i) an SpA variant, e.g., a variant SpA domain A-E polypeptide or peptide thereof; or (ii) a composition comprising a nucleic acid molecule encoding such an SpA variant polypeptide or peptide thereof, or (iii) an SpA variant domain D polypeptide in any combination or permutation with the bacterial proteins described herein. In a preferred embodiment, the composition is not staphylococci. In certain aspects, the subject is a human or a cow or a pig or a sheep or a goat or any other animal raised for food, and may be suffering from a staphylococcal infection, such as mastitis. In a further aspect, the composition is formulated into a pharmaceutically acceptable preparation. The staphylococcus may be Staphylococcus aureus.

[0047] Yet further embodiments include vaccines comprising pharma- ceutically acceptable compositions having isolated SpA variant polypeptides or any other combination or permutation of proteins or peptides described herein, wherein the compositions are capable of stimulating an immune response against Staphylococcus. The vaccine may comprise isolated SpA variant polypeptides or any other combination or permutation of proteins or peptides described. In certain aspects of the invention, the isolated SpA variant polypeptides or any other combination or permutation of proteins or peptides described are multimerized, e.g., dimerized or concatemerized. In further aspects, the vaccine composition is contaminated with less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.05% (or any range derivable therein) of other Staphylococcus proteins. The composition may further comprise an isolated non-SpA polypeptide. Typically, the vaccine comprises an adjuvant. In certain aspects, the protein or peptide of the present invention is linked (covalently or non-covalently) to an adjuvant, preferably the adjuvant is chemically conjugated to the protein. In some embodiments, the adjuvant comprises alum. In some embodiments, the adjuvant comprises an adjuvant described herein.

[0048] In yet further embodiments, the vaccine composition is a pharma- ceutically acceptable composition having a recombinant nucleic acid encoding an SpA variant polypeptide, or all or part of any other combination or permutation of the proteins or peptides described herein, which composition is capable of stimulating an immune response against Staphylococcus. The vaccine composition may comprise a recombinant nucleic acid encoding an SpA variant polypeptide, or all or part of any other combination or permutation of the proteins or peptides described herein. In certain embodiments, the recombinant nucleic acid comprises a heterologous promoter. Preferably, the recombinant nucleic acid is a vector. More preferably, the vector is a plasmid or a viral vector. In some aspects, the vaccine comprises a recombinant non-staphylococcus comprising the nucleic acid. The recombinant non-staphylococcus may be Salmonella or another Gram-positive bacterium. The vaccine may comprise a pharma- ceutically acceptable excipient, more preferably an adjuvant. In some embodiments, the adjuvant comprises alum or an adjuvant described herein.

[0049] Yet further embodiments include a method for stimulating a protective or therapeutic immune response against staphylococci in a subject, comprising administering to the subject an effective amount of a composition of SpA variant polypeptide or a segment / fragment thereof, further comprising one or more of Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, or vWh proteins or peptides thereof. In a preferred embodiment, the composition comprises a non-staphylococcus. In a further aspect, the composition is formulated into a pharma- ceutically acceptable formulation. The staphylococcus to which the subject is being treated may be Staphylococcus aureus. The methods of the invention also include SpA variant compositions that include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or more secreted virulence factors and / or cell surface proteins, such as Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, or vWh, in various combinations. In certain aspects, the vaccine formulation includes Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, and vWh.In certain aspects, the antigen combinations include: (1) SpA variant and IsdA; (2) SpA variant and ClfB; (3) SpA variant and SdrD; (4) SpA variant and Hla or Hla variant; (5) SpA variant and ClfB, SdrD, and Hla or Hla variant; (6) SpA variant, IsdA, SdrD, and Hla or Hla variant; (7) SpA variant, IsdA, ClfB, and Hla or Hla variant; (8) SpA variant, IsdA, ClfB, and SdrD; (9) SpA variant, IsdA, ClfB, and SdrD; (10) an SpA variant, IsdA, ClfB, and SdrD; (11) an SpA variant, IsdA, SdrD, and an Hla or Hla variant; (12) an SpA variant, IsdA, and an Hla or Hla variant; (13) an SpA variant, IsdA, ClfB, and an Hla or Hla variant; (14) an SpA variant, ClfB, and SdrD; (15) an SpA variant, ClfB, and an Hla or Hla variant; or (16) an SpA variant, SdrD, and an Hla or Hla variant.

[0050] In certain aspects, the bacteria delivering the compositions of the invention are restricted or attenuated with respect to long-term or persistent growth or abscess formation. In yet further aspects, SpA variants can be overexpressed in the attenuated bacteria to further enhance or supplement the immune response or vaccine formulation.

[0051] The term "EsxA protein" refers to isolated wild-type EsxA polypeptide from staphylococci and proteins comprising segments thereof, as well as variants that stimulate an immune response against the staphylococcal EsxA protein.

[0052] The term "EsxB protein" refers to isolated wild-type EsxB polypeptide from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against the staphylococcal EsxB protein.

[0053] The term "SdrD protein" refers to isolated wild-type SdrD polypeptides from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against staphylococcal SdrD proteins.

[0054] The term "SdrE protein" refers to isolated wild-type SdrE polypeptides from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response to the staphylococcal SdrE protein.

[0055] The term "IsdA protein" refers to isolated wild-type IsdA polypeptide from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against the staphylococcal IsdA protein.

[0056] The term "IsdB protein" refers to isolated wild-type IsdB polypeptides from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response to the staphylococcal IsdB protein.

[0057] The term "Eap protein" refers to isolated wild-type Eap polypeptides derived from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response to the Staphylococcus aureus Eap protein.

[0058] The term "Ebh protein" refers to isolated wild-type Ebh polypeptides derived from staphylococci and proteins comprising segments thereof, as well as variants that stimulate an immune response to staphylococcal Ebh proteins.

[0059] The term "Emp protein" refers to proteins comprising isolated wild-type Emp polypeptides derived from Staphylococcus aureus and segments thereof, as well as variants which stimulate an immune response to the Staphylococcal Emp protein.

[0060] The term "EsaB protein" refers to isolated wild-type EsaB polypeptide from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against the Staphylococcus aureus EsaB protein.

[0061] The term "EsaC protein" refers to isolated wild-type EsaC polypeptide from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against the Staphylococcus aureus EsaC protein.

[0062] The term "SdrC protein" refers to isolated wild-type SdrC polypeptides from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response to the staphylococcal SdrC protein.

[0063] The term "ClfA protein" refers to isolated wild-type ClfA polypeptide from staphylococci and proteins comprising segments thereof, as well as variants that stimulate an immune response to the staphylococcal ClfA protein.

[0064] The term "ClfB protein" refers to isolated wild-type ClfB polypeptide from staphylococci and proteins comprising segments thereof, as well as variants that stimulate an immune response to the staphylococcal ClfB protein.

[0065] The term "Coa protein" refers to isolated wild-type Coa polypeptides derived from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against staphylococcal Coa proteins.

[0066] The term "Hla protein" refers to isolated wild-type Hla polypeptides derived from staphylococci and proteins comprising segments thereof, as well as variants that stimulate an immune response against staphylococcal Hla proteins.

[0067] The term "IsdC protein" refers to isolated wild-type IsdC polypeptide from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against the staphylococcal IsdC protein.

[0068] The term "SasF protein" refers to isolated wild-type SasF polypeptides derived from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against the staphylococcal SasF protein.

[0069] The term "vWbp protein" refers to isolated wild-type vWbp (von Willebrand factor binding protein) polypeptide from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response against the staphylococcal vWbp protein.

[0070] The term "vWh protein" refers to isolated wild-type vWh (von Willebrand factor binding protein homologue) polypeptides derived from Staphylococcus aureus and proteins comprising segments thereof, as well as variants that stimulate an immune response to the Staphylococcus aureus vWh protein.

[0071] Immune response refers to humoral response, cellular response, or both humoral and cellular response in an organism. Immune response can be measured by assays including, but not limited to, assays that measure the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays that measure T cell activation or proliferation, and / or assays that measure modifications in the activity or expression of one or more cytokines.

[0072] In certain aspects, a polypeptide or segment / fragment can have a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% or more identical to the amino acid sequence of a reference polypeptide. The term "similarity" refers to a polypeptide having a sequence that has a certain percentage of amino acids that are either identical to the reference polypeptide or that constitute conservative substitutions with the reference polypeptide.

[0073] The polypeptides described herein are those having SEQ ID NO:1-6 positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 1 48, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 2 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids, or any range derivable therein.

[0074] The polypeptide segments described herein are represented by SEQ ID NO: NO: 1-6, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 8 2, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 6, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 2 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids, or any range derivable therein.

[0075] The composition may be formulated into a pharma- ceutically acceptable composition.In certain aspects of the invention, the Staphylococcus is Staphylococcus aureus.

[0076] In further aspects, the composition may be administered to a subject more than once, and may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more times. Administration of the composition includes, but is not limited to, oral administration, including inhalation or aspiration, parenteral administration, subcutaneous administration, intramuscular administration, intravenous administration, or any combination thereof.

[0077] In yet further embodiments, the composition comprises a recombinant nucleic acid molecule encoding a polypeptide as described herein or a segment / fragment thereof. Typically, the recombinant nucleic acid molecule encoding a polypeptide as described herein comprises a heterologous promoter. In certain aspects, the recombinant nucleic acid molecule of the present invention is a vector, while in other aspects the vector is a plasmid. In certain embodiments, the vector is a viral vector. In certain aspects, the composition comprises a recombinant non-staphylococcus that contains or expresses a polypeptide as described herein. In certain aspects, the recombinant non-staphylococcus is Salmonella or another Gram-positive bacterium. The composition is typically administered to a mammal, such as a human subject, although administration to other animals capable of eliciting an immune response is contemplated. In a further aspect, the staphylococcus that contains or expresses the polypeptide is Staphylococcus aureus. In a further embodiment, the immune response is a protective immune response.

[0078] In a further embodiment, the composition comprises a recombinant nucleic acid molecule encoding all or a portion of one or more of the Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, SpA, vWbp or vWh proteins or peptides or variants thereof. Additional staphylococcal antigens that can be used in combination with the polypeptides described herein include the 52 kDa vitronectin binding protein (WO 01 / 60852), Aaa, Aap, Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen binding protein (US6288214), EFB (FIB), elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein (US6008341), fibronectin binding protein (US5840846), FnbA, FnbB, GehD (US 2002 / 0169288), HarA, HBP, immunodominant ABC transporters, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activator protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00 / 12689), SdrG / Fig (WO 00 / 12689), SdrH (WO 00 / 12689), SEA exotoxin (WO 00 / 02523), SEB exotoxin (WO 00 / 02523), SitC and Ni ABC transporters, SitC / MntC / saliva binding protein (US 5,801,234), SsaA, SSP-1, SSP-2, and / or vitronectin binding protein. In certain aspects, the bacterium is a recombinant non-staphylococcus, such as Salmonella or other gram-positive bacteria.

[0079] The compositions of the invention are typically administered to human subjects, but administration to other animals capable of eliciting an immune response against Staphylococcus aureus is contemplated, particularly cattle, horses, goats, sheep and other domestic animals, i.e., mammals.

[0080] In certain aspects, the staphylococcus is Staphylococcus aureus. In further embodiments, the immune response is a protective immune response. In further aspects, the methods and compositions of the present invention can be used to prevent, ameliorate, reduce or treat infections of tissues or glands, such as mammary glands, particularly mastitis and other infections. Other methods include, but are not limited to, prophylactically reducing bacterial load in subjects who do not show signs of infection, particularly subjects suspected of being infected by or at risk of being infected by target bacteria, such as patients who are or may be at risk of infection or susceptible to infection during hospitalization, treatment and / or recovery.

[0081] Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well. In particular, any of the embodiments discussed in the context of an SpA variant polypeptide or peptide or nucleic acid may also be used in conjunction with any of the following polypeptides or peptides: Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh, vitronectin binding protein of 52 kDa (WO 01 / 60852), Aaa, Aap, Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen binding protein (US6288214), EFB (FIB), elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein (US6008341), fibronectin binding protein (US5840846), FnbA, FnbB, GehD, (US 2002 / 0169288), HarA, HBP, immunodominant ABC transporter, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activator protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00 / 12689), SdrG / Fig (WO 00 / 12689), SdrH (WO 00 / 12689), SEA exotoxin (WO 00 / 02523), SEB exotoxin (WO 00 / 02523), SitC and Ni It is possible to carry out this with respect to other antigens, such as ABC transporters, SitC / MntC / saliva binding proteins (US Pat. No. 5,801,234), SsaA, SSP-1, SSP-2, and / or vitronectin binding proteins (or nucleic acids), and vice versa.Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh, vitronectin binding protein of 52 kDa (WO 01 / 60852), Aaa, Aap, Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen binding protein (US6288214), EFB (FIB), elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein (US6008341), fibronectin binding protein (US5840846), FnbA, FnbB, GehD (US 2002 / 0169288), HarA, HBP, immunodominant ABC transporter, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activator protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO 00 / 12689), SdrG / Fig (WO 00 / 12689), SdrH (WO 00 / 12689), SEA exotoxin (WO 00 / 02523), SEB exotoxin (WO 00 / 02523), SitC and Ni It is also understood that any one or more of ABC transporters, SitC / MntC / saliva binding proteins (US 5,801,234), SsaA, SSP-1, SSP-2, and / or vitronectin binding proteins may be specifically excluded from the claimed compositions.

[0082] Embodiments of the invention include compositions that contain or do not contain bacteria. The compositions may or may not contain attenuated or viable or intact staphylococci. In certain aspects, the compositions contain bacteria that are not staphylococci or do not contain staphylococci. In certain embodiments, the bacterial composition comprises an isolated or recombinantly expressed staphylococcal protein A variant or a nucleotide encoding the same. The composition may be or include recombinantly engineered staphylococci that have been altered in a manner that includes specifically altering the bacteria with respect to secreted virulence factors or cell surface proteins. For example, the bacteria may be recombinantly modified to express more virulence factors or cell surface proteins than they would express if unmodified.

[0083] The term "isolated" can refer to a nucleic acid or polypeptide that is substantially free of its original cellular, bacterial, viral or media material (if produced by recombinant DNA technology), or chemical precursors or other chemicals (if chemically synthesized). Furthermore, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, a compound is not considered to be "isolated" simply if it is attached to a column or embedded in an agarose gel. Furthermore, an "isolated nucleic acid fragment" or an "isolated peptide" is a nucleic acid or protein fragment that is not naturally occurring as a fragment and / or is not typically in a functional state.

[0084] Moieties of the invention, such as polypeptides, peptides, antigens, or immunogens, can be covalently or non-covalently conjugated or linked to other moieties, such as adjuvants, proteins, peptides, supports, fluorescent moieties, or labels. The terms "conjugate" or "immunoconjugate" are used broadly to define a functional association between one moiety and another agent, and are not intended to simply refer to any type of functional association, and are not specifically limited to chemical "conjugation". Recombinant fusion proteins are specifically contemplated. The compositions of the invention may further comprise an adjuvant or a pharma- ceutically acceptable excipient. In some embodiments, the adjuvant comprises alum or an adjuvant, as described herein. The adjuvant may be covalently or non-covalently coupled to the polypeptide or peptide of the invention. In certain aspects, the adjuvant is chemically conjugated to the protein, polypeptide, or peptide.

[0085] The term "providing" is used in accordance with its ordinary meaning to indicate "supplying or giving for use." In some embodiments, proteins are provided directly by administering the protein, while in other embodiments, proteins are effectively provided by administering a nucleic acid that encodes the protein. In certain aspects, the present invention contemplates compositions comprising various combinations of nucleic acids, antigens, peptides and / or epitopes.

[0086] The subject may have (e.g., have been diagnosed with) a staphylococcal infection, be suspected of having a staphylococcal infection, or be at risk of developing a staphylococcal infection. The compositions of the present invention include immunogenic compositions in which the antigen or epitope is contained in an amount effective to achieve the intended purpose. More specifically, an effective amount refers to the amount of active ingredient necessary to stimulate or induce an immune response, or to bring about resistance to, amelioration of, or remission of an infection. In more specific aspects, an effective amount prevents, alleviates, or ameliorates symptoms of disease or infection, or prolongs the survival of the subject being treated. Determination of an effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the present invention, the effective amount or dose can be initially estimated from in vitro studies, cell culture, and / or animal model assays. For example, a dose can be formulated in an animal model to achieve a desired immune response or concentration or titer of circulating antibodies. Such information can be used to more accurately determine a dose useful in humans.

[0087] In some embodiments, the polypeptides (SpA variants) of the disclosure provide a histamine release of 20, 19.5, 19, 18.5, 18, 17.5, 17, 16.5, 16, 15.5, 15, 14.5, 14, 13.5, 13, 12.5, 12, 11.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, 0.1, or 0% or less (or any range derivable therein) in a basophil histamine release assay.

[0088] It is understood that the embodiments in the Examples section are embodiments of the invention that are applicable to all aspects of the invention.

[0089] Although the use of the term "or" in the appended claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or." It is also contemplated that anything described with the term "or" can be expressly excluded.

[0090] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being utilized to determine the value.

[0091] In accordance with long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the appended claims or specification, mean one or more, unless specifically noted otherwise.

[0092] [The present invention 1001] 1. An isolated polypeptide comprising a variant protein A (SpA) having (i) a lysine substitution of a glutamine residue in each of domains A-E corresponding to positions 9 and 10 in domain D, and (ii) a glutamic acid substitution in each of domains A-E corresponding to position 33 in domain D, wherein the isolated polypeptide does not detectably crosslink IgG and IgE in blood or activate basophils or mast cells compared to a negative control. [The present invention 1002] K against VH3 from human IgG A The binding affinity of SpA variants (SpA) consisting of lysine substitutions of glutamine residues in domains A to E corresponding to positions 9 and 10 in domain D and alanine substitutions of aspartic acid residues in domains A to E corresponding to positions 36 and 37 in domain D, respectively, was KKAA 1001. The isolated polypeptide of the present invention, wherein the expression level of the isolated polypeptide is reduced compared to that of the [The present invention 1003] SpA KKAA K for VH3 from human IgG that is at least two-fold reduced compared to A 1003. The isolated polypeptide of claim 1001 or 1002 having binding affinity. [The present invention 1004] 1×10 5 M -1 K for VH3 from human IgG is less than A An isolated polypeptide according to any one of claims 1001 to 1003, having binding affinity. [The present invention 1005] The isolated polypeptide of any of claims 1001 to 1004, wherein the SpA variant does not have a substitution in any of domains A to E corresponding to positions 36 and 37 in domain D. [The present invention 1006] The isolated polypeptide of any of claims 1001 to 1005, wherein the only substitutions in the SpA variant are (i) and (ii). [The present invention 1007] 7. The isolated polypeptide of any of claims 1001 to 1006, which comprises an SpA variant. [The present invention 1008] A composition comprising the isolated polypeptide of any of claims 1001 to 1007, which contains less than 1% by weight of the total protein amount of a protein other than the isolated polypeptide. [The present invention 1009] The composition of the present invention further comprising an adjuvant. [The present invention 1010] The composition of the present invention 1009, wherein the isolated polypeptide is coupled to an adjuvant. [The present invention 1011] The composition of any one of claims 1009 to 1010, wherein the adjuvant comprises alum. [The present invention 1012] The composition of any one of claims 1008 to 1011, further comprising a pharma- ceutically acceptable excipient. [The present invention 1013] 1. An isolated polypeptide comprising a variant protein A (SpA) having (i) a lysine substitution for a glutamine residue in each of domains A to E corresponding to positions 9 and 10 in domain D, and (ii) a threonine substitution in each of domains A to E corresponding to position 33 in domain D, wherein the isolated polypeptide does not detectably crosslink IgG and IgE in blood or activate basophils or mast cells compared to a negative control. [The present invention 1014] K against VH3 from human IgG A The binding affinity of SpA variants (SpA) consisting of lysine substitutions of glutamine residues in domains A to E corresponding to positions 9 and 10 in domain D and alanine substitutions of aspartic acid residues in domains A to E corresponding to positions 36 and 37 in domain D, respectively, was KKAA ) is reduced compared to 1013 of the isolated polypeptide of the present invention. [The present invention 1015] SpA KKAA K for VH3 from human IgG that is at least two-fold reduced compared to A The isolated polypeptide of the invention 1013 or 1014 having binding affinity. [The present invention 1016] 1×10 5 M -1 K for VH3 from human IgG is less than A An isolated polypeptide according to any one of claims 1013 to 1015, having binding affinity. [The present invention 1017] 17. The isolated polypeptide of any of claims 1013 to 1016, wherein the SpA variant does not have a substitution in any of domains A to E corresponding to positions 36 and 37 in domain D. [The present invention 1018] The isolated polypeptide of any of claims 1013 to 1017, wherein the only substitutions in the SpA variant are (i) and (ii). [The present invention 1019] An isolated polypeptide according to any one of claims 1013 to 1018, which comprises an SpA variant. [The present invention 1020] A composition comprising the isolated polypeptide of any of claims 1013 to 1019, which contains less than 1% by weight of the total protein amount of protein other than the isolated polypeptide. [The present invention 1021] The composition of the present invention further comprising an adjuvant. [The present invention 1022] The composition of the invention 1021, wherein the isolated polypeptide is coupled to an adjuvant. [The present invention 1023] The composition of claim 1021 or 1022, wherein the adjuvant comprises alum. [The present invention 1024] The composition of any one of claims 1020 to 1023, further comprising a pharma- ceutically acceptable excipient. [The present invention 1025] A method for inducing a safe immune response against Staphylococcus in a human patient, comprising administering to the human patient an effective amount of any of the compositions of the present invention 1008-1012 or 1020-1024. [The present invention 1026] The method of claim 1025, wherein the staphylococcus comprises the WU1 or JSNZ strain of Staphylococcus aureus. [The present invention 1027] The method of any one of claims 1025 to 1026, wherein the Staphylococcus aureus comprises an ST88 isolate or any other human Staphylococcus aureus currently adapted to animals. [The present invention 1028] The method of any one of claims 1025 to 1027, wherein the human patient is a pediatric patient. [The present invention 1029] The method of claim 1028, wherein the pediatric patient is 2 years of age or younger. [The present invention 1030] The method of any one of claims 1025 to 1027, wherein the human patient is 65 years of age or older. [The present invention 1031] The method of any of claims 1025-1027 or 1030, wherein the human patient is a healthcare professional. [The present invention 1032] 1032. The method of any of claims 1025 to 1031, wherein the isolated polypeptide of the composition is administered in four doses, and the interval between doses is at least four weeks. [The present invention 1033] Use of any of the compositions of the invention 1008-1012 or 1020-1024 for the treatment of Staphylococcus aureus in a human patient to induce a safe immune response against Staphylococcus aureus. [The present invention 1034] The use of the present invention 1033, wherein the Staphylococcus comprises the WU1 or JSNZ strain of Staphylococcus aureus. [The present invention 1035] Use of 1033 or 1034 of the invention, wherein the Staphylococcus aureus comprises an ST88 isolate or any other human Staphylococcus aureus currently adapted to animals. [The present invention 1036] The use of any of claims 1033 to 1035, wherein the human patient is a pediatric patient. [The present invention 1037] The use of the present invention 1036, wherein the pediatric patient is 2 years of age or younger. [The present invention 1038] The use of any of claims 1033 to 1035, wherein the human patient is 65 years of age or older. [The present invention 1039] The use of any of claims 1033 to 1035 or 1038, wherein the human patient is a medical professional. [The present invention 1040] The use of any of claims 1033 to 1039, wherein the isolated polypeptide or composition is administered in four doses and the interval between doses is at least four weeks. [The present invention 1041] 1. An isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution for the glutamine residues corresponding to positions 9 and 10 in each of Domains A-E, and (ii) at least one other amino acid substitution corresponding to positions 29 and / or 33 in SEQ ID NO:2 in each of Domains A-E, wherein the SpA variant has a lysine substitution of 1.0×10 -4 K for VH3 from human IgG exceeds MD Binding affinity, and / or 1.0 x 10 -6 K for VH3 from human IgE exceeds M D The isolated polypeptide having binding affinity. [The present invention 1042] 1041. An isolated polypeptide of the invention, wherein variant SpA comprises an amino acid substitution corresponding to position 29 in SEQ ID NO:2 in each of domains A-E. [The present invention 1043] 1041. An isolated polypeptide of the invention, wherein variant SpA comprises an amino acid substitution corresponding to position 33 in SEQ ID NO:2 in each of domains A-E. [The present invention 1044] 1042. An isolated polypeptide of the invention, wherein variant SpA comprises amino acid substitutions corresponding to positions 29 and 33 in SEQ ID NO:2 in each of domains A to E. [The present invention 1045] The isolated polypeptide of any of claims 1041 to 1044, wherein variant SpA comprises an amino acid substitution corresponding to one or both of positions 36 and 37 in SEQ ID NO:2 in each of domains A to E. [The present invention 1046] An isolated polypeptide of the invention 1045, wherein variant SpA comprises amino acid substitutions corresponding to both positions 36 and 37 of SEQ ID NO:2 in each of domains A through E. [The present invention 1047] The isolated polypeptide of claim 1044 or 1045, wherein the amino acid substitutions corresponding to positions 36 and 37 are alanine residues for aspartic acid residues. [The present invention 1048] The isolated polypeptide of any of claims 1041 to 1046, wherein variant SpA comprises variant A-E domains having at least 70% identity to the amino acid sequence of SEQ ID NO:2. [The present invention 1049] 1048. The isolated polypeptide of the invention, wherein variant SpA comprises variant A-E domains having at least 80% identity to the amino acid sequence of SEQ ID NO:2. [The present invention 1050] 1049. The isolated polypeptide of the invention, wherein variant SpA comprises variant A-E domains having at least 90% identity to the amino acid sequence of SEQ ID NO:2. [The present invention 1051] The isolated polypeptide of the present invention 1050, wherein variant SpA comprises variant A-E domains that do not contain any amino acid substitutions in SEQ ID NO:2 except for corresponding positions 9, 10, 29, 33, 36, and / or 37. [The present invention 1052] 1051. An isolated polypeptide of the invention, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, and 29 in SEQ ID NO:2. [The present invention 1053] 1051. An isolated polypeptide of the invention, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, and 33 in SEQ ID NO:2. [The present invention 1054] 1051. An isolated polypeptide of the invention, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, and 33 in SEQ ID NO:2. [The present invention 1055] 1051. An isolated polypeptide of the invention, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, 36, and 37 in SEQ ID NO:2. [The present invention 1056] 1051. An isolated polypeptide of the invention, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 33, 36, and 37 in SEQ ID NO:2. [The present invention 1057] 1051. An isolated polypeptide of the invention, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, 33, 36, and 37 in SEQ ID NO:2. [The present invention 1058] The isolated polypeptide of any of claims 1042 to 1057, wherein the amino acid substitution corresponding to position 29 is alanine, leucine, proline, phenylalanine, glutamic acid, arginine, lysine, serine, threonine, or glutamine. [The present invention 1059] 1058. The isolated polypeptide of the invention, wherein the amino acid substitution corresponding to position 29 is alanine, phenylalanine, or arginine. [The present invention 1060] The isolated polypeptide of any of claims 1043 to 1057, wherein the amino acid substitution corresponding to position 33 is alanine, phenylalanine, glutamic acid, lysine, or glutamine. [The present invention 1061] The isolated polypeptide of any of claims 1042 to 1057, wherein the amino acid substitution corresponding to position 33 is phenylalanine, glutamic acid, or glutamine. [The present invention 1062] SpA variants are 1.0 × 10 -2 K for VH3 over M D An isolated polypeptide of any one of claims 1041 to 1061 having binding affinity. [The present invention 1063] The isolated polypeptide of any of claims 1041 to 1062, further comprising a non-protein A segment. [The present invention 1064] The isolated polypeptide of the invention 1063, wherein the non-protein A segment is a second antigenic segment. [The present invention 1065] The isolated polypeptide of the invention 1064, wherein the second antigenic segment is a Staphylococcus antigenic segment. [The present invention 1066] The isolated polypeptide of the invention 1065, wherein the staphylococcal antigen segment is an Emp, EsxA, EsxB, EsaC, Eap, Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, IsdC, ClfA, ClfB, and / or SasF segment. [The present invention 1067] A composition comprising any of the isolated polypeptides of the present invention 1041 to 1066, capable of stimulating an immune response in a subject in need thereof. [The present invention 1068] The composition of the invention 1067, comprising less than 1% by weight of the total protein, other than the isolated polypeptide. [The present invention 1069] The composition of any one of claims 1067 to 1068, further comprising an adjuvant. [The present invention 1070] The composition of the invention 1069, wherein the isolated polypeptide is coupled to an adjuvant. [The present invention 1071] The composition of the present invention 1070, wherein the adjuvant comprises alum. [The present invention 1072] The composition of any one of claims 1067 to 1071, further comprising a pharma- ceutically acceptable excipient. [The present invention 1073] A method for preventing or treating a staphylococcal infection comprising administering to a patient in need thereof any of the isolated polypeptides of the present inventions 1041 to 1066 or any of the compositions of the present inventions 1067 to 1072. [The present invention 1074] The method of claim 1073, wherein the Staphylococcus comprises the WU1 or JSNZ strain of Staphylococcus aureus. [The present invention 1075] The method of any one of claims 1073 to 1074, wherein the Staphylococcus aureus comprises an ST88 isolate or any other human Staphylococcus aureus currently adapted to animals. [The present invention 1076] The method of any one of claims 1073 to 1075, wherein the human patient is a pediatric patient. [The present invention 1077] The method of claim 1076, wherein the pediatric patient is 2 years of age or younger. [The present invention 1078] The method of any one of claims 1073 to 1075, wherein the human patient is 65 years of age or older. [The present invention 1079] The method of any of claims 1073 to 1075 or 1078, wherein the human patient is a healthcare professional. [The present invention 1080] 1079. The method of any of claims 1073 to 1079, wherein the isolated polypeptide or composition is administered in four doses and the interval between doses is at least four weeks. [The present invention 1081] 23. Use of an isolated polypeptide of any of claims 1041 to 1066 or a composition of any of claims 1067 to 1072 in the manufacture of a vaccine for the treatment or prevention of a staphylococcal infection. [The present invention 1082] The use of the present invention 1081, wherein the Staphylococcus comprises the WU1 or JSNZ strain of Staphylococcus aureus. [The present invention 1083] Use of 1081 or 1082 of the present invention, wherein the Staphylococcus aureus comprises an ST88 isolate or any other human Staphylococcus aureus currently adapted to animals. [The present invention 1084] The use of any of claims 1081 to 1083, wherein the human patient is a pediatric patient. [The present invention 1085] The use of invention 1084, wherein the pediatric patient is 2 years of age or younger. [The present invention 1086] The use of any of claims 1081 to 1083, wherein the human patient is 65 years of age or older. [The present invention 1087] The use of any of claims 1081 to 1083 or 1086, wherein the human patient is a medical professional. [The present invention 1088] The use of any of claims 1081 to 1087, wherein the isolated polypeptide or composition is administered in four doses and the interval between doses is at least four weeks. [The present invention 1089] A method for inducing an immune response against Staphylococcus in a subject, comprising the step of providing to the subject an effective amount of any of the isolated polypeptides of the present inventions 1041 to 1066 or any of the compositions of the present inventions 1067 to 1072. [The present invention 1090] The method of claim 1089, wherein the subject is also administered an adjuvant. [The present invention 1091] The method of any one of claims 1089 to 1090, wherein the composition comprises an adjuvant. [The present invention 1092] The method of the present invention 1091, wherein the SpA variant is coupled to an adjuvant. [The present invention 1093] The method of claim 1092, wherein the adjuvant comprises alum. [The present invention 1094] The method of any of claims 1089 to 1093, wherein the composition is formulated into a pharma- ceutically acceptable composition. [The present invention 1095] The method according to any one of claims 1089 to 1094, wherein the Staphylococcus is Staphylococcus aureus. [The present invention 1096] The method of claim 1095, wherein the Staphylococcus aureus is resistant to one or more treatments. [The present invention 1097] The method of claim 1096, wherein the bacteria is methicillin resistant. [The present invention 1098] The method of any of claims 1089 to 1097, further comprising administering the isolated polypeptide or composition to the subject two or more times. [This invention 1099] The method of any of claims 1089 to 1098, wherein the composition is administered orally, parenterally, subcutaneously, intramuscularly, or intravenously. [The present invention 1100] The method of any of claims 1089 to 1099, further comprising administering to the subject a composition comprising a second Staphylococcus antigen. [The present invention 1101] The method of claim 1100, wherein the second staphylococcal antigen is one or more of Emp, EsxA, EsxB, EsaC, Eap, Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, IsdC, ClfA, ClfB, and SasF. [The present invention 1102] The method according to any one of claims 1089 to 1101, wherein the subject is a mammal. [The present invention 1103] The method of claim 1102, wherein the subject is a human. [The present invention 1104] The method of any of claims 1089 to 1103, wherein the immune response is a protective immune response. [The present invention 1105] The method of any of claims 1089 to 1103, wherein the subject does not exhibit any signs of toxicity from the isolated polypeptide or composition. [The present invention 1106] The method of claim 1105, wherein the subject does not show any signs of anaphylactic shock from the isolated polypeptide or composition. [The present invention 1107] The method of any one of claims 1089 to 1106, wherein the Staphylococcus comprises the WU1 or JSN4Z strain of Staphylococcus aureus. [The present invention 1108] The method of any of claims 1089 to 1107, wherein the Staphylococcus aureus comprises an ST88 isolate or any other human Staphylococcus aureus currently adapted to animals. [The present invention 1109] The method of any one of claims 1103 to 1108, wherein the human patient is a pediatric patient. [The present invention 1110] The method of claim 1109, wherein the pediatric patient is 2 years of age or younger. [The present invention 1111] The method of any one of claims 1103 to 1108, wherein the human patient is 65 years of age or older. [The present invention 1112] The method of any of claims 1103-1108 or 1111, wherein the human patient is a healthcare professional. [The present invention 1113] The method of any of claims 1089 to 1112, wherein the isolated polypeptide or composition is administered in four doses and the interval between doses is at least four weeks. [The present invention 1114] providing to a subject having, suspected of having, or at risk of developing a Staphylococcal infection an effective amount of a composition comprising an isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution for the glutamine residues corresponding to positions 9 and 10 in each of Domains A-E, and (ii) at least one other amino acid substitution corresponding to positions 29 and / or 33 in SEQ ID NO:2 in each of Domains A-E. 1. A method for treating a Staphylococcus aureus infection in a subject, comprising administering to a subject a staphylococcal infection having ... -5 K for IgG exceeds M D The method, wherein the antibody has binding affinity. [The present invention 1115] The method of claim 1114, wherein the variant SpA comprises an amino acid substitution corresponding to position 29 in SEQ ID NO:2 in each of domains A to E. [The present invention 1116] The method of claim 1114, wherein the variant SpA comprises an amino acid substitution corresponding to position 33 in SEQ ID NO:2 in each of domains A to E. [The present invention 1117] The method of claim 1114, wherein the variant SpA comprises amino acid substitutions corresponding to positions 29 and 33 in SEQ ID NO:2 in each of domains A to E. [The present invention 1118] The method of any of claims 1114 to 1117, wherein the variant SpA comprises an amino acid substitution corresponding to one or both of positions 36 and 37 in SEQ ID NO:2 in each of domains A to E. [The present invention 1119] The method of claim 1118, wherein the variant SpA comprises amino acid substitutions corresponding to both positions 36 and 37 of SEQ ID NO:2 in each of domains A to E. [The present invention 1120] The method of any one of claims 1117 to 1118, wherein the amino acid substitutions corresponding to positions 36 and 37 are alanine residues for aspartic acid residues. [The present invention 1121] The method of any of claims 1114 to 1119, wherein variant SpA comprises variant A-E domains having at least 70% identity to the amino acid sequence of SEQ ID NO:2. [The present invention 1122] The method of claim 1121, wherein variant SpA comprises variant A-E domains having at least 80% identity to the amino acid sequence of SEQ ID NO:2. [The present invention 1123] The method of claim 1122, wherein variant SpA comprises variant A-E domains having at least 90% identity to the amino acid sequence of SEQ ID NO:2. [The present invention 1124] The method of claim 1123, wherein variant SpA comprises variant A-E domains that do not contain any amino acid substitutions in SEQ ID NO:2 except at corresponding positions 9, 10, 29, 33, 36, and / or 37. [The present invention 1125] The method of claim 1124, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, and 29 in SEQ ID NO:2. [The present invention 1126] The method of claim 1124, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, and 33 in SEQ ID NO:2. [The present invention 1127] The method of claim 1124, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, and 33 in SEQ ID NO:2. [The present invention 1128] The method of claim 1124, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, 36, and 37 in SEQ ID NO:2. [The present invention 1129] The method of claim 1124, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 33, 36, and 37 in SEQ ID NO:2. [The present invention 1130] The method of claim 1124, wherein variant SpA comprises variant A-E domains consisting only of amino acid substitutions corresponding to positions 9, 10, 29, 33, 36, and 37 in SEQ ID NO:2. [The present invention 1131] The method according to any one of claims 1115 to 1130, wherein the amino acid substitution corresponding to position 29 is alanine, leucine, proline, phenylalanine, glutamic acid, arginine, lysine, serine, threonine, or glutamine. [The present invention 1132] The method of claim 1131, wherein the amino acid substitution corresponding to position 29 is alanine, phenylalanine, or arginine. [The present invention 1133] The method according to any one of claims 1116 to 1130, wherein the amino acid substitution corresponding to position 33 is alanine, phenylalanine, glutamic acid, lysine, or glutamine. [The present invention 1134] The method according to any one of claims 1115 to 1130, wherein the amino acid substitution corresponding to position 33 is phenylalanine, glutamic acid, or glutamine. [This invention 1135] SpA variants are 1.0 × 10 -2 K for VH3 over M D The method according to any one of claims 1114 to 1134, having binding affinity. [The present invention 1136] The method of any one of claims 1114 to 1135, further comprising a non-protein A segment. [The present invention 1137] The method of any one of claims 1136 to 1137, wherein the non-protein A segment is a second antigen segment. [The present invention 1138] The method of claim 1137, wherein the second antigen segment is a Staphylococcus antigen segment. [This invention 1139] The method of the present invention 1138, wherein the staphylococcal antigen segment is an Emp, EsxA, EsxB, EsaC, Eap, Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, IsdC, ClfA, ClfB, and / or SasF segment. [The present invention 1140] The method of any one of claims 1114 to 1139, wherein the subject is diagnosed with a persistent Staphylococcus aureus infection. [This invention 1141] The method of any of claims 1114 to 1140, wherein the SpA variant induces the production of antibodies in the subject that bind to Protein A. [This invention 1142] The method of any of claims 1114 to 1141, wherein the isolated polypeptide is administered together with an adjuvant. [This invention 1143] The method of claim 1142, wherein the isolated polypeptide is coupled to an adjuvant. [This invention 1144] The method of any of claims 1114 to 1143, wherein the isolated polypeptide is formulated in a pharma- ceutically acceptable composition. [This invention 1145] The method of any of claims 1114 to 1144, further comprising administering a second Staphylococcus antigen. [This invention 1146] The method of claim 1145, wherein a second staphylococcal antigen is administered simultaneously with the isolated polypeptide. [This invention 1147] The method of claim 1146, wherein the second staphylococcal antigen and the isolated polypeptide are administered in the same composition. [This invention 1148] The method of claim 1147, wherein a second Staphylococcal antigen is fused to the SpA variant. [This invention 1149] The method of any of claims 1145 to 1148, wherein the second staphylococcal antigen is one or more of Emp, EsxA, EsxB, EsaC, Eap, Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, IsdC, ClfA, ClfB, and SasF peptides. [The present invention 1150] Any of the methods according to claims 1114 to 1149, wherein the Staphylococcus infection is Staphylococcus aureus infection. [This invention 1151] The method of any of claims 1114 to 1150, wherein the peptide is administered orally, parenterally, transdermally, transmucosally, subcutaneously, intramuscularly, or by inhalation. [This invention 1152] The method of any of claims 1114 to 1151, further comprising administering the isolated polypeptide to the subject two or more times. [This invention 1153] The method according to any one of claims 1114 to 1152, wherein the subject is a mammal. [This invention 1154] The method of claim 1153, wherein the subject is a human. [This invention 1155] The method of any of claims 1114 to 1152, wherein the subject does not exhibit any signs of toxicity from the isolated polypeptide or composition. [This invention 1156] The method of the present invention 1155, wherein the subject does not show any signs of anaphylactic shock from the isolated polypeptide or composition. [This invention 1157] The method of any one of claims 1114 to 1156, wherein the Staphylococcus comprises the WU1 or JSN4Z strain of Staphylococcus aureus. [This invention 1158] The method of any of claims 1106 to 1157, wherein the Staphylococcus aureus comprises an ST88 isolate or any other human Staphylococcus aureus currently adapted to animals. [This invention 1159] The method of any one of claims 1154 to 1158, wherein the human patient is a pediatric patient. [The present invention 1160] The method of claim 1159, wherein the pediatric patient is 2 years of age or younger. [The present invention 1161] The method of any one of claims 1154 to 1158, wherein the human patient is 65 years of age or older. [The present invention 1162] The method of any of claims 1154 to 1158 or 1161, wherein the human patient is a healthcare professional. [The present invention 1163] The method of any of claims 1114 to 1162, wherein the isolated polypeptide or composition is administered in four doses and the interval between doses is at least four weeks. [The present invention 1164] A method for decolonization or for preventing colonization or recolonization of Staphylococcus aureus in a subject, comprising administering to the subject an effective amount of any of the isolated polypeptides of the present inventions 1041 to 1066 or any of the compositions of the present inventions 1067 to 1072. [The present invention 1165] 1. An isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution of a glutamine residue in each of domains A through E corresponding to positions 9 and 10 in domain D, and (ii) a glutamic acid substitution in each of domains A through E corresponding to position 33 in domain D, wherein the polypeptide does not detectably crosslink IgG and IgE in blood or activate basophils or mast cells compared to a negative control; wherein the SpA variant does not have a substitution in any of domains A through E corresponding to positions 36 and 37 in domain D. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since it is believed that various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0093] So that the above features, advantages and objectives of the present invention, as well as others which will become apparent, may be attained and understood in detail, a more particular description of the invention briefly summarized above and certain specific embodiments thereof are set forth in the accompanying drawings, which form a part of the specification, and it should be noted, however, that the accompanying drawings illustrate certain specific embodiments of the invention and therefore should not be considered as limiting its scope. [Figure 1] Figure 1A-E. S. aureus ST88 isolate WU1, a mouse pathogen. (A) Domain structure and sequence homology of the vwb gene product from human clinical isolates S. aureus WU1 and S. aureus Newman. Percent amino acid (aa) identity of vWbp for its signal peptide (S), D1 and D2 domains (responsible for binding and activating host prothrombin), linker (open box), and C-terminal fibrinogen binding domain (C) are presented. (B) S. aureus whole culture samples of strain Newman (WT, wild type), and its Δcoa, Δvwb, Δcoa-vwb, and ΔclfA variants, strains WU1, JSNZ, USA300 LAC, and its Δvwb variants were analyzed for the production of vWbp (αvWbp), Coa (αCoa), Hla (αHla), and ClfA (aClfA) using polyclonal rabbit antibodies. (C) Polyclonal antibodies against the vWbp-C domain identify vWbp allelic variants from strains JSNZ and WU1 as well as vWbp from strain USA300 LAC. (D-E) Aggregation of Syto-9 stained S. aureus strains in human (D) or mouse (E) plasma was measured by means of aggregated bacteria in 12 microscopic fields as mean size and standard error, and statistical significance was assessed in pairwise comparisons with WT using two-way ANOVA with Sidak's multiple comparison test. ****, p<0.0001. [Diagram 2]Figure 2A-B. S. aureus WU1 persistently colonizes the nasopharynx of C57BL / 6 mice. Cohorts of C57BL / 6 mice (n=10) were inoculated intranasally with 1x108 CFU of the indicated S. aureus WU1 or PBS control, and weekly pharyngeal swabs were taken and bacterial loads were counted. Each dot represents the number of CFU per mouse. The median and standard deviation for each group of animals on a particular day are indicated by the horizontal line and error bars. [Diagram 3] Figure 3A-B. S. aureus WU1 expression of staphylococcal protein A (SpA) is required for persistent colonization of C57BL / 6 mice. (A) Immunoblots of S. aureus lysates from strains USA300 LAC, Newman, WU1, and the Δspa variant of WU1 without and with a plasmid for spa expression (pspa) were probed with SpA-specific antibody (αSpA) and sortase A-specific antibody (αSrtA). (B) Cohorts of C57BL / 6 mice (n=10) were inoculated intranasally with 1 × 108 CFU of S. aureus WU1 or its Δspa variant, and swabs were taken from the oropharynx of the animals at weekly intervals and bacterial loads were counted. Each dot indicates the number of CFU per mouse. The median and standard deviation of each group of animals on a particular day are displayed by horizontal lines and error bars. Bacterial colonization data sets were analyzed with two-way ANOVA and Sidak multiple comparison test; statistically significant differences between the two groups of animals (***p=0.0003; ****p<0.0001) are indicated by asterisks. [Figure 4]Immunization of C57BL / 6 mice with SpAKKAA promotes decolonization with S. aureus WU1. C57BL / 6 mice were immunized with 50 μg of purified recombinant SpAKKAA emulsified in CFA or PBS mock in CFA and boosted 11 days later with 50 μg of recombinant SpAKKAA emulsified in IFA or PBS mock in IFA. On day 0 of the colonization experiment, cohorts of C57BL / 6 mice (n=10) were inoculated intranasally with 1×108 CFU of S. aureus WU1. Swabs were taken from the oropharynx of the animals at weekly intervals and bacterial loads were counted. Each dot represents the number of CFU per mouse. The median and standard deviation of each group of animals on a particular day are displayed by the horizontal line and error bars. Bacterial colony formation data sets were analyzed with two-way ANOVA and Sidak multiple comparison test; statistically significant differences between the two groups of animals (*p<0.05; **p<0.01) are indicated by asterisks. [Diagram 5] Immunization of BALB / c mice with SpAKKAA promotes decolonization of S. aureus WU1. BALB / c mice were immunized with 50 μg of purified recombinant SpAKKAA emulsified in CFA or PBS mock in CFA and boosted 11 days later with 50 μg of recombinant SpAKKAA emulsified in IFA or PBS mock in IFA. On day 0 of the colonization experiment, a cohort of BALB / c mice (n=10) mice was inoculated intranasally with 1×108 CFU of S. aureus WU1. Swabs were taken from the oropharynx of the animals at weekly intervals and the bacterial load was counted. Each dot represents the number of CFU per mouse. The median and standard deviation of each group of animals on a particular day are shown by the horizontal line and error bars. Bacterial colony formation data sets were analyzed with two-way ANOVA and Sidak multiple comparison test; statistically significant differences between groups of animals (*p<0.05; **p<0.01; ****p<0.0001) are indicated by asterisks. [Figure 6]Immunization of BALB / c mice with SpAKKAA promotes clearance of S. aureus JSNZ from the nasopharynx. BALB / c mice were immunized with 50 μg of purified recombinant SpAKKAA emulsified in CFA or PBS mock in CFA and boosted 11 days later with 50 μg of recombinant SpAKKAA emulsified in IFA or PBS mock in IFA. On day 0 of the colonization experiment, a cohort of BALB / c mice (n=10) mice was inoculated intranasally with 1×108 CFU of S. aureus JSNZ. Swabs were taken from the oropharynx of the animals at weekly intervals and the bacterial load was counted. Each dot represents the number of CFU per mouse. The median and standard deviation of each group of animals on a particular day are shown by the horizontal line and error bars. Bacterial colony formation data sets were analyzed by two-way ANOVA with Sidak multiple comparison test; statistically significant differences (*p<0.05; **p<0.01) between the two groups of animals are indicated by asterisks. [Figure 7] Figure 7A-C. Improved SpA vaccine. A: Illustrative representation of SpAKKAA, SpAKKAA / A, and SpAKKAA / F variants. B: Binding affinity of the variants to human IgG. C: Binding affinity of the variants to human IgE. [Figure 8A] Binding assays. Western blots of SpA variants. [Figure 8B] Binding assays. ELISA of variants against the indicated molecules. [Figure 9] FIG. 9A-B. Protein A is required for persistent S. aureus nasal colonization of mice. [Figure 10] Protein A amino acid sequence alignment. Dark grey: amino acids that interact with the human Fcγ fragment; Light grey: amino acids that interact with the human Fab fragment; Asterisks represent amino acids that interact with both the human Fcγ fragment and the Fab fragment. Red: amino acids that interact with the human Fcγ fragment. [Figure 11] Surface plasmon resonance (SPR) analysis showing that the Z domain (G29A in SpA B domain) does not bind to the F(ab)2 fragment. [Figure 12A] Novel SpA* variants targeting G29. [Figure 12B] Novel SpA* variants targeting G29. [Figure 13] Novel SpA* variants targeting G29. [Figure 14] Novel SpA* variants targeting G29. [Figure 15] Novel SpA* variants targeting G29. [Figure 16] FIG. 1 is a diagram of the basil histamine release assay, as further described in Example 2. [Figure 17] FIG. 17A-B Staphylococcal protein A (SpA). (A) Schematic illustrating the primary structure of the SpA precursor (with an N-terminal signal peptide cleaved by signal peptidase, five immunoglobulin binding domains (designated IgBD-E, D, A, B, C), a cell wall spanning domain designated region Xr, a LysM domain for peptidoglycan binding, and a C-terminal LPXTG sorting signal cleaved by sortase A), cell wall SpA displayed on the bacterial surface, and released SpA molecules liberated from the cell wall envelope and released into host tissues. (B) Secretion and sortase A-mediated cell membrane anchoring of SpA, and release of peptidoglycan-linked SpA by S. aureus. [Figure 18] FIG. 18A-B Binding of SpA to the Fcγ domain of human IgG blocks antibody effector function (association of Fc receptors with complement receptors) and opsonogenic killing of S. aureus by phagocytes. Immune evasion properties of staphylococcal protein A. (A) Cell-wall-anchored SpA on the surface of S. aureus binds to the Fcγ of human IgG (IgG1, IgG2, and IgG4), blocking antibody effector functions that trigger opsonogenic killing of the bacteria. (B) Schematic diagram illustrating the primary structure of human IgG, its antigen-binding paratope (purple) effectors (C1q, FcγRs, FcRn), and the SpA binding site. [Figure 19]FIG. 19A-B Immune evasion properties of Staphylococcal protein A. (A) Immune evasion function of SpA during S. aureus infection. Cell-wall-anchored SpA on the surface of S. aureus binds to Fcγ of human IgG, blocking the effector function of antibodies that trigger opsonogenic killing of the bacteria. Shedding SpA crosslinks VH3 idiotypic variant heavy chains of human IgG and IgM (B cell receptors), activating B cell proliferation, class switching, somatic hypermutation, and secretion of VH3 idiotypic antibodies that can be crosslinked by SpA but do not recognize S. aureus antigens, thereby blocking the development of an adaptive immune response against S. aureus and the establishment of protective immunity. (B) Schematic diagram illustrating SpA binding to and crosslinking of VH3 idiotypic B cell receptor (IgM) and activation of CD79AB signaling. [Figure 20] FIG. 20A-B Immunoglobulin binding domains (IgBDs) of recombinant SpA, SpAKKAA, SpAAA, and SpAKKAA. (A) Schematic diagram illustrating the primary structure of the IgBD of recombinant SpA with an N-terminal polyhistidine tag for purification via affinity chromatography on Ni-NTA from the cytoplasm of E. coli. The amino acid sequence of the IgBD-E domain is shown below. The positions of the three α-helices (H1, H2, and H3) in each IgBD are indicated. SpAKK and SpAKKAA carry an amino acid substitution at Q9,10K (Gln9,10Lys). SpAKK and SpAKKAA carry an amino acid substitution at D36,37A (Asp36,37Lys). Numbering refers to the position of the amino acid in B-IgBD. (B) Amino acid sequence alignment of the five IgBDs of SpA. Conserved amino acids are indicated by a period (.). Gaps in the alignment are indicated by a dash (-). Nonconserved amino acids are listed in single letter code. SpA residues involved in IgG Fcγ binding are highlighted in red as reported by Graille et al. (138). SpA residues responsible for VH3 heavy chain binding are highlighted in green. The pink residue (Q32) contributes to both Fcγ and VH3 binding. [Figure 21]21A-B SpA-associated VH3 cross-linking activity and anaphylaxis. (A) Schematic illustrating the structure of human activating Fcγ and Fcε receptors and their VH3 idiotypic IgG and IgE ligands. (B) SpA cross-linking of VH3 idiotypic IgG or IgE binding to FcγR and FcεR receptors, respectively, on basophils or mast cells triggers the release of histamine, inflammatory mediators, and cytokines that promote anaphylactic reactions, vasodilation, and shock. Although not shown in (B), both mast cells and basophils express FcγR and FcεR receptors and respond to SpA cross-linking of VH3 idiotypic IgG bound to FcγR or SpA cross-linking of VH3 idiotypic IgE bound to FcεR receptors with histamine, pro-inflammatory mediators, and cytokines. [Figure 22] Anaphylactic activity of SpA vaccine candidates in mice. μMT mice (n=5) were sensitized with VH3 IgG by intradermal injection in the ear. Candidate vaccine antigens or PBS control were injected intravenously 24 hours later, followed by Evans Blue. Dye extravasation was quantified after extraction from ear tissue 30 minutes later by spectrophotometric measurement at 620 nm. Data were obtained from three independent experiments. For statistical analysis of the data, one-way ANOVA with Bonferroni's multiple comparison test was performed. Symbols: ns, not significant; *, P<0.05; **, P <O.01;***、P<0.001;****、p<0.0001。 [Diagram 23]FIG. 23A-B Mast cell degranulation. Cultured human mast cells (LAD2) were sensitized overnight with VH3 IgE, washed, and either left untreated (PBS) or exposed for 1 h to SpA or test articles SpAKKAA, SpAQ9,10K / S33E, SpAQ9,10K / S33T, or SpA-KR as positive control. β-hexosaminidase and histamine levels were measured in cell pellets and supernatants. The percentage of β-hexosaminidase release (A) and the amount of histamine release (B) are shown. For statistical analysis of the data, one-way ANOVA with Bonferroni's multiple comparison test was performed. Symbols: ns, not significant; *, P<0.05; **, P <O.01;***、P<0.001;****、P<0.0001。 [Figure 24A]FIG. 24A-E Immunization with SpAKKAA or SpAQ9,10K / S33E or SpAQ9,10K / S33T promotes progressive decolonization. Cohorts of C57BL / 6 mice (n=10) were inoculated intranasally with 1×108 CFU of S. aureus WU1. (A,B,D) Mice were swabbed weekly in the throat and bacterial load was counted. (C,E) Fecal samples were collected weekly after inoculation and bacterial load was counted. In panel (A), animals were immunized with adjuvant-PBS or -SpAKKAA. In panels (B-C), animals were immunized with adjuvant-SpAKKAA or -SpAQ9,10K / S33E; the same cohorts of animals were monitored for bacterial load in throat samples (B) and fecal samples (C). In panels (D-E), animals were immunized with adjuvants -PBS or -SpAKKAA or -SpAQ9,10K / S33E or -SpAQ9,10K / S33T; the same cohort of animals was monitored for bacterial load in throat samples (D) and fecal samples (E). Each square indicates the number of CFU per milliliter of throat swab or per gram of feces. The median and standard deviation for each group of animals on a particular day are displayed by horizontal lines and error bars. Data were examined by two-way ANOVA with Sidak multiple comparison test (*, p<0.05). In panels (D-E), data for each group (1-8 each) represent data from mock, SpAKKAA, SpAQ9,10K / S33E, or SpAQ9,10K / S33T, respectively. No statistically significant differences were noted between the two groups in panels B and C. [Figure 24B] Please see the legend to Figure 24A. [Figure 24C] Please see the legend to Figure 24A. [Figure 24D] Please see the legend to Figure 24A. [Figure 24E] Please see the legend to Figure 24A. [Diagram 25]Figure 25A-C Protective activity of SpA vaccine candidates in a mouse model of bloodstream infection. Three-week-old BALB / c mice (n=15) were immunized with SpAKKAA or SpAQ9,10K / S33E or SpAQ9,10K / S33T or PBS control. Mock or booster immunizations were performed on day 11. On day 20, mice were bled to assess serum half-maximal antibody titers against the vaccine candidates, denoted SpA* on the y-axis. The three bars in each group represent, from left to right, SpAKKAA, SpAQ9,10K / S33E, and SpaAQ9,10K / S33T. (A) On day 21, mice were challenged with 5x106 CFU of S. aureus USA300 (LAC) into the periorbital sinus of the right eye. Fifteen days after challenge, the animals were euthanized and the staphylococcal load in the kidneys (B) was counted and the abscess lesions (C) were counted. For statistical analysis of the data, one-way ANOVA with Bonferroni's multiple comparison test was performed. Symbols: ns, not significant; *, P<0.05; **, P <O.01;***、P<0.001;****、P<0.0001。 [Figure 26] Figure 26A-C Interaction between SpA vaccine candidates and SpA neutralizing monoclonal antibody 3F6. 3F6 antibody, recombinant rMAb 3F6 derived from HEK293 F cells (A, rMAb 3F6) or murine hybridoma monoclonal antibody (B, hMAb 3F6), was serially diluted across enzyme-linked immunosorbent assay plates coated with either SpAKKAA or SpAQ9,10K / S33E or SpAQ9,10K / S33T or PBS control. (C) Association constants were calculated using GraphPad Prism software. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] Detailed Description Staphylococcus aureus is a commensal bacterium of human skin and nares and a major cause of infection of the bloodstream, skin, and soft tissues (Klevens et al., 2007). The dramatic increase in mortality from staphylococcal disease in recent years is due to the spread of methicillin-resistant S. aureus (MRSA) strains, which are often not susceptible to antibiotics (Kennedy et al., 2008). In a large retrospective study, the incidence of MRSA infections was 4.6% of all hospitalizations in the United States (Klevens et al., 2007). Annual medical costs for 94,300 MRSA-infected individuals in the United States exceed $2.4 billion (Klevens et al., 2007). The current MRSA epidemic has created a public health crisis that must be addressed by the development of a preventive vaccine (Boucher and Corey, 2008). To date, no FDA-approved vaccine is available to prevent S. aureus disease.

[0095] The inventors herein describe the use of Protein A, a cell wall-anchored surface protein of Staphylococcus aureus, for the creation of variants that can function as subunit vaccines. The pathogenesis of Staphylococcus aureus infection begins when bacteria invade the skin or bloodstream via trauma, surgical wounds, or medical devices (Lowy, 1998). Invading pathogens can be phagocytosed and killed, but Staphylococcus aureus can also evade innate immune defenses and seed infections in organ tissues, inducing inflammatory responses that attract macrophages, neutrophils, and other phagocytes (Lowy, 1998). Responsive infiltration of immune cells into the site of infection is accompanied by liquefaction necrosis as the host attempts to block Staphylococcus aureus spread and allow for the removal of necrotic tissue debris (Lam et al., 1963). Such lesions can be observed microscopically as hypercellular areas containing necrotic tissue, leukemia, and a central focus of bacteria (Lam et al., 1963). Unless staphylococcal abscesses are surgically drained and treated with antibiotics, disseminated infection and sepsis can lead to a fatal outcome (Sheagren, 1984).

[0096] II. Staphylococcal antigens A. Staphylococcal Protein A (SpA) Staphylococcal protein A (SpA) binds immunoglobulins, thereby allowing S. aureus to evade the host immune response. SpA binding to Fcγ blocks the effector functions of IgG antibodies and opsonization killing of staphylococci by immune cells. H 3 SpA cross-linking of variant heavy chains of idiotypic IgM inhibits B cell proliferation and V that cannot bind antigenic determinants of Staphylococcus aureus H It induces the secretion of 3 clonal antibodies. It also induces the V H 3 SpA cross-linking of idiotypic IgG and IgE promotes histamine release and anaphylaxis. Previous studies have demonstrated that SpA with a lack of Fcγ and variant heavy chain binding KKAA When tested as a vaccine in preclinical trials, it KKAA elicits SpA neutralizing antibodies and protects animals against S. aureus colonization and invasive disease. KKAA V H 3, which retains cross-linking activity against idiotype IgG and IgE and has been shown to cause anaphylaxis, suggesting that it is unsafe for use in humans. H We demonstrate that an SpA variant lacking triple-stranded cross-linking and anaphylactic activity elicits SpA-neutralizing antibodies and protects animals from S. aureus colonization and bloodstream infection. H SpA variants unable to cross-link 3 idiotypic immunoglobulins can be used to prevent S. aureus colonization and invasive disease in humans.

[0097] B. Colonization and Disease Caused by Staphylococcus aureus S. aureus colonizes the human nasopharynx and gastrointestinal tract and is found as a persistent colonizer in approximately one-third of the human population (14). The remainder of the human population is intermittently colonized by S. aureus (14). Virtually all humans develop antibodies to some of the molecular determinants of S. aureus during childhood (99). However, these immune responses do not affect S. aureus colonization or protect against invasive disease (99). Colonization is a significant risk factor for S. aureus community- or hospital-acquired invasive disease, including infections of soft tissues, wounds, lungs, skeleton, bloodstream, and surgical sites (14, 100, 58, 6). In the United States, 3.4 million cases of S. aureus community-acquired infections (CAI) and 340,000 cases of hospital-acquired infections (HAI) require antibiotic and / or surgical therapy (49–51). Infections with antibiotic-resistant S. aureus strains, named MRSA (methicillin-resistant S. aureus), occur in 7% of CAIs and 22% of HAIs and are associated with poor disease outcomes, including treatment failure and death (85). All S. aureus diseases are characterized by their recurrence rate (i.e., in 4 out of 5 cases, recurrent infections are caused by the same strain) and the failure of the infected host to establish protective immunity (16). The development of a staphylococcal vaccine that can inhibit colonization and prevent invasive disease is an urgent and unmet clinical need. Although multiple vaccine candidates have been subjected to clinical efficacy trials, the products have failed to achieve their clinical endpoints (59–60, 101).

[0098] C. Staphylococcal Protein A (SpA) Is a Critical Immune Evasion Determinant Staphylococcal protein A (SpA), a sortase A-anchored surface protein, functions as a key immune evasion determinant that prevents the development of protective immunity during S. aureus colonization and infection (75, 33, 102, 48). All S. aureus clinical isolates carry the gene for staphylococcal protein A (spa), which gives rise to a precursor product composed of an N-terminal YSIRK / GxxS signal peptide followed by four to five immunoglobulin-binding domains, region X repeats (Xr), a LysM domain, and an LPXTG sorting signal (29, 32, 103) (Figure S17A). The SpA precursor resides in the bacterial cytoplasm and enters the secretory pathway at the septal membrane via its N-terminal YSIRK / GxxS signal peptide (68, 104). Upon translocation to the septal membrane, the C-terminal LPXTG sorting signal is cleaved by sortase A to form a thioester intermediate between its active site cysteine ​​residue and the carboxyl of the threonine (T) in the LPXTG motif (105–108) (Figure 17B). The thioester intermediate is cleaved by nucleophilic attack of the amino group (NH2-) in lipid II [C55-(PO4)2-MurNAc(L-Ala-D-iGlu-L-Lys(NH2-Gly5)-D-Ala-D-Ala)-GlcNAc], generating an amide bond between the C-terminal threonine and the pentaglycine bridge (107, 109). The SpA-lipid II intermediate is then incorporated into the transverse wall peptidoglycan and displayed on the bacterial surface (108, 70).

[0099] Cell-wall-anchored SpA on the bacterial surface binds to the Fcγ domain of IgG, disabling the effector function of antibodies and thereby protecting staphylococci from opsonization-mediated killing (OPK) by host immune cells (48, 76) (Figure 18). During replication, staphylococci release a portion of their peptidoglycan and peptidoglycan-linked SpA (herein named shed protein A or shed SpA) into the host environment due to muralytic enzyme activity that degrades the transverse wall in the vicinity of the septal membrane (110, 111) (Figure 17B). Shedding SpA is tethered to the cell-wall peptide by the structure L-Ala-D-iGln-L-Lys(SpA-LPET-Gly5)-D-Ala-Gly4 (46). Shedding SpA activates the VH3 idiotypic B cell receptor (BCR or IgM), thereby promoting the secretion of VH3 idiotypic IgG, IgA, IgD, IgE, and IgM via B cell proliferation and the generation of activated plasmablasts (75, 33) (Figure 19).

[0100] D. Immunoglobulin Binding and Toxicity Properties of SpA The N-terminal immunoglobulin binding domains (IgBDs) of SpA (134–137, triple-helical folds of 72 amino acid residues, designated E, D, A, B, and C) bind to the Fcγ domains of human IgG1, IgG2, and IgG4, respectively (112–116, 71) (Figure 20). SpA does not bind to the Fcγ domain of human IgG3, and only this IgG subclass has a short plasma half-life (113, 117). Each of the five IgBDs also binds to variant heavy chains of VH3-cloned human immunoglobulins, including IgM(BCR), IgG, IgE, IgD, and IgA (75, 48, 76, 118–135, 43) (Figure 19). The binding sites of the IgBDs are non-overlapping, and each SpA module can simultaneously bind to the Fcγ and VH3 heavy chains of antibodies (48) (Figure 20). A crucial property of SpA is its ability to interact with antibody effector functions (Fcγ), including binding sites for Fcγ receptors (FcγR) and C1q, which are required for OPK by immune cells (71, 72, 136, 137) (Figure 18B). In the human immune system, 54% of IgM (lacking Fcγ) and peripheral blood B cells interact with SpA (75, 73, 74). SpA binding is restricted to variant heavy chain products of the VH3 gene family, which contains 22 genes, compared to other major and minor VH genes, which have fewer genes: VH1 [11 genes], VH2 [2 genes], VH4 [11 genes], VH5 [2 genes], VH6 [1 gene], and VH7 [1 gene] (138). During colonization and invasive disease, shed SpA crosslinks VH3 clonal B cell receptors, triggering the secretion of antibodies (IgG, IgA, IgD, and IgE) that are adapted via somatic hypermutation for improved binding of variant heavy chains to SpA but are unable to recognize staphylococcal determinants as antigens (15) (Figure 19). This B cell superantigen activity (BCSA, i.e., VH3 binding activity of shed SpA) is responsible for impeding the development of protective immunity against S. aureus during colonization or invasive disease (102, 48, 139) (Figure 19).

[0101] The immunoglobulin-binding properties of SpA are also relevant to toxicity when the purified protein is injected into humans or animals. For example, 20–30% of guinea pig IgM interact with SpA via their variant heavy chains (VH3 idiotype) (76); injection of purified SpA (500 μg) into the bloodstream of guinea pigs results in anaphylactic shock, including restlessness, respiratory distress, and death (140). Even at a dose of 0.01 μg, SpA causes vascular leakage in guinea pigs (140). In mice, only 5–10% of IgM interact with SpA via their variant heavy chains (VH3 idiotype) (76). Mice are resistant to SpA-induced anaphylaxis, but these animals, when treated with intravenous infusion of human IgG, can succumb to SpA infusion due to histamine release from altered and activated basophils and mast cells (141, 142) (Figure 21). SpA-induced anaphylactic shock is prevented by pretreatment of animals with mepyramine, an H1 receptor histamine antagonist (140). Intravenous infusion of purified SpA (0.3–0.45 μg / kg) in humans is associated with severe adverse events, including headache, nausea, myalgia, chest pain, fever, acute lymphopenia, and leukopenia (143). Thus, purified SpA is a bacterial toxin and is not considered safe for use in humans (143, 144). The toxic activity of SpA is associated with its VH3 cross-linking activity against human IgG and IgE binding to their cognate receptors on basophils and mast cells, thereby causing histamine and cytokine release, vasodilation, vascular leakage, and shock (140, 142, 144, 145) (Figure 21).

[0102] E. SpA KKAA When immunostimulated with aluminum hydroxide or Freund's adjuvant and injected into mice or guinea pigs, purified SpA does not induce antibody responses in animals directed against its five IgBDs (76, 43). Similarly, S. aureus colonization or invasive disease is not accompanied by the development of SpA IgBD-specific antibodies in mice, guinea pigs, or humans (75, 102, 48, 43). Previous studies developed recombinant SpA variants by first analyzing the D immunoglobulin-binding domain of SpA. Kim and coworkers replaced glutamine (Q or Gln) 9 and 10 with lysine (K or Lys) and aspartic acid (D or Asp) 36 and 37 with alanine (A or Ala) to disrupt the association of SpA-D with immunoglobulins (43). The substitutions Q9K, Q10K, D36A, and D37A were introduced into recombinant polyhistidine-tagged SpA-D to generate SpA-D KKAA SpA-D or SpA-D purified and retained on nickel-nitrilotriacetic acid-agarose (Ni-NTA) were prepared. KKAA The ability of SpA-D to bind and subsequently co-elute with human IgG or IgM was finally analyzed by ELISA. As expected, SpA-D retained human IgG and human IgM on Ni-NTA. Compared to SpA-D, SpA-D KKAA captured 5.6 (±0.6)% (P<0.0001) of human IgG and 75.6 (±4.6)% (P=0.008) of human IgM (numbers in parentheses are standard error of the mean; statistical significance was calculated by unpaired Student's t test) (43). Recombinant polyhistidine-tagged SpA (lacking the signal peptide, Xr, LysM, and LPXTG sorting signals of the full-length SpA precursor) was engineered to introduce Q9K, Q10K, D36A, and D37A into each of the five IgBDs to generate SpA. KKAA (Figure 19) (42). When analyzed in comparison with polyhistidine-tagged recombinant SpA, SpA KKAAcaptured 2.3 (±0.1)% of human IgG (P = 0.0001), 2.2 (±0.1)% of the Fcγ fragments of human IgG (Fcγ binding activity, P < 0.0001), 4.4 (±1.0)% of the F(ab) fragments of human IgG (VH3 binding activity, P < 0.0001), and 4.2 (±0.1)% of human IgM (VH3 binding activity, P < 0.0001) on a Ni-NTA column (43). KKAA demonstrates the lack of two binding activities of SpA with human immunoglobulins, namely the Fcγ domain of IgG (IgG1, IgG2, and IgG4) and its association with variant heavy chains of VH3 idiotypic immunoglobulins.

[0103] F. SpA as a Vaccine Antigen KKAA Polyhistidine-tagged recombinant SpA and SpA KKAA was purified by immobilized metal affinity chromatography (IMAC) on Ni-NTA (43). The eluate was dialyzed against PBS, extracted with Triton X-114 to remove endotoxin, dialyzed, and the protein concentration was determined (BCA assay), and purity (>90%) was confirmed by Coomassie-stained SDS-PAGE and HPLC (43). KKAA was adsorbed on aluminum hydroxide [SpA KKAA Al(OH)3] or completely [SpA KKAA CFA] or incomplete [SpA KKAA [IFA] emulsified with either Freund's adjuvant (43). 50 μg SpA KKAA Prime-boost (11-day interval) immunization of C57BL / 6 and BALB / c mice with CFA / IFA was performed to prevent SpA KKAA Induces high-titer serum IgG antibodies to the antigen [9.2 μg / ml (±1.1); 1:6,000 ELISA half-maximal titer] (43). KKAA Prime-boost immunization of C57BL / 6 and BALB / c mice with Al(OH)3 produced low titer antibodies [1:500, half-maximal titer]; higher titers of IgG were observed in SpA KKAAA half-maximal titer of 1:4,000 was observed in CD1 mice immunized with Al(OH)3 (76). A prime-2 booster regimen (14 days apart) and 100 μg SpA KKAA Immunization of guinea pigs with Al(OH)3 inhibited SpA KKAA It produces high titers of serum IgG against SpA [1:50,000 half-maximal titer] (76). KKAA Specific mouse and guinea pig IgG bind to each of the five IgBDs of SpA and neutralize its ability to bind mouse, guinea pig, or human IgG via the Fcγ or VH3 idiotypic heavy chains (76). KKAA Specific mouse and guinea pig IgG neutralize SpA binding to immunoglobulins on the bacterial surface and promote OPK of S. aureus Newman and MRSA USA300 LAC in anticoagulated mouse, guinea pig, and human blood (48, 76, 43). KKAA Immunization of mice and guinea pigs with SpA protects the animals from S. aureus bloodstream infection (76, 43). Protection is based on SpA-neutralizing antibodies, since intraperitoneal or intravenous administration of purified polyclonal or monoclonal antibodies (mouse monoclonal antibody 3F6) directed against SpA IgBD is sufficient to protect against S. aureus bloodstream infection (increased survival, reduced bacterial load in organ tissues, and reduced abscess formation) (76, 84, 146, 147). Furthermore, by neutralizing the BCSA (VH3 cross-linking activity) of SpA, SpA-IgBD-specific antibodies boost IgG responses of mice and guinea pigs against secreted antigens during S. aureus bloodstream infection, and thus enhance the IgG response of SpA. KKAA This allows immunized animals to eliminate bacterial evaders and establish protective immunity against recurrent disease (48, 76, 43, 147), whereas naive or SpA-immunized animals are unable to establish protective immunity against S. aureus (48, 43).

[0104] G. Role of SpA in Staphylococcus aureus Colonization Previous studies have attempted to identify S. aureus factors that contribute to colonization by measuring bacterial adherence to human exfoliated nasal epithelial cells and in vivo colonization in mice (17, 18, 20). These include D-Ala and GlcNAc modifications of cell wall teichoic acid (WTA) that bind to the F-type scavenger receptor (SREC-I) in nasal epithelium (18, 27, 28). In addition, clumping factor B (ClfB) promotes staphylococcal adherence to loricrin and cytokeratin-10 in nasal epithelium (21). Compared to wild-type S. aureus, an isogenic clfB mutant showed reduced adherence to the nasal epithelium of human volunteers (7). Iron-regulated surface determinant A (IsdA) contributes to iron removal from host heme proteins, binds lactoferrin, and inhibits the antistaphylococcal activity of lactoferrin in nasal secretions (23, 24). S. aureus surface protein G (SasG) mediates zinc-dependent adhesion between bacterial cells during biofilm formation on nasal tissues (25, 26). Thus, multiple factors are required for staphylococcal attachment, biofilm formation, and colonization of nasopharyngeal tissues (78). Importantly, nasal colonization of mice with human clinical isolates requires prior antibiotic treatment of the animals to deplete the resident flora and result in the selection of antibiotic-resistant S. aureus (19). Thus, mice are not persistently colonized by human clinical S. aureus isolates (20). The role of staphylococcal protein A (SpA) during nasal colonization of S. aureus is enigmatic (29). When analyzed in 6,110 individuals in Oxfordshire (UK), all colonizing S. aureus strains express spa (32, 31). The tandem repeat structure of the spa gene promotes frequent recombination, and human colonization selects for spa alleles whose products maintain the five IgBDs, conferring a strong BCSA on staphylococci (33, 32). Analysis of human volunteers whose nasal carriage had been eliminated via mupirocin treatment demonstrated that S. aureus spa expression was not required for bacterial adherence to human nasal tissue (34).

[0105] S. aureus WU1, a member of the multilocus sequence ST88 clade, was isolated from an outbreak of preputial gland abscesses in male C57BL / 6 mice (102). Unlike human clinical isolates, S. aureus WU1 persistently colonizes the nasopharynx of C57BL / 6 and BALB / c mice without antibiotic selection, is inherited from dams, and persistently colonizes their offspring (102). Compared to wild-type S. aureus WU1, the ΔsrtA mutant is unable to anchor any of its surface proteins to the bacterial envelope and is unable to colonize the nasopharynx of C57BL / 6 mice (102) (unpublished observations). In contrast, the Δspa mutant does not show a defect in early colonization of mice (102). Nevertheless, the Δspa mutant fails to persist and is cleared from the nasopharynx after the first 3 weeks of early colonization (102). Decolonization is associated with an increase in serum IgG against ClfB, IsdA, SasG, and other staphylococcal surface proteins (102). KKAA Immunization of mice with SpA produces SpA-neutralizing antibodies that increase pathogen-specific IgG (including anti-ClfB, anti-IsdA, anti-SasG, anti-FnbA, anti-FnbB, and anti-Coa), thereby promoting decolonization of S. aureus WU1 (102). Similarly, intraperitoneal administration of the SpA-IgBD neutralizing mouse monoclonal antibody 3F6 promotes pathogen-specific increases in IgG and secretory IgA and clearance of S. aureus from the nasopharynx and gastrointestinal tract (146). Taken together, these data suggest that S. aureus colonization is associated with the release of SpA into host tissues and diversion of B cell responses, thereby allowing pathogen persistence on mucosal surfaces (102). SpA KKAA Vaccine-induced anti-SpA antibodies counter this mechanism by promoting IgG responses to multiple different secreted staphylococcal antigens that together reduce S. aureus colonization ( 102 ).

[0106] H. Procedure SpA for Affinity Chromatography of Immunoglobulins and Monoclonal Antibodies (MAbs) Recombinant SpA, full-length or individual domains (E, D, A, B, C), have been widely used as ligands for affinity chromatography purification of monoclonal antibodies (MAbs) (147, 148). Many human MAbs developed for clinical use are V H 3 idiotype IgG1 class. Recombinant SpA belongs to the V H Such antibodies can bind via both the trivariant framework and Fcγ (Figures 18 and 19), thereby requiring low pH (pH 3.1) for MAb elution from SpA affinity resins (e.g., Mab Select™, which encompasses all five IgBDs E, D, A, B, C). The use of low pH for MAb elution is accompanied by antibody unfolding and aggregation (149). Furthermore, recombinant wild-type SpA is sensitive to alkaline cleavage at Asn-Gly residues, limiting the utility of SpA affinity columns during cleaning-in-place protocols using alkali (≥0.1 M NaOH) to remove contaminating proteins and lipids and kill contaminating microorganisms (147, 149). The Z domain is modified by two amino acid substitutions, Ala 1 Val and Gly 29 The latter amino acid substitution results in an alkaline-sensitive Asn 28 -Gly 29 It removes peptide bonds (151, 152). Tandem repeats of two (ZZ), five (ZV), and ten (ZX) Z domains have been shown to bind antibodies and have been used in the purification of MAbs (130, 150). 29 Ala substitutions have also been reported in V of IgG1 antibodies. H It also reduces binding of Z, ZZ, and ZZZZ (MabSelectSure™) to the 3 framework, thereby lowering the pH required for IgG1 elution (pH 3.7) (148, 151, 152). 28 -Gly 29 Binding is mediated by the IgBD-C domain (Thr28 -Gly 29 ), which is intrinsically resistant to alkali (151). Computational analysis of the free energy change (ΔG) of folded native IgBD-C versus unfolded IgBD-C variants (harboring one of eight amino acid substitutions at Ser33 or Asp36) was hypothesized to correlate with changes in thermal stability, although this assumption has not been experimentally verified (149). In silico, reduced ΔG was further hypothesized to identify reduced binding to the VH3 variant heavy chain; this assumption has also not been experimentally verified (149). Furthermore, the assumption that in silico predictions can identify amino acid substitutions with the weakest binding and greatest stability could not be verified either (149). Nevertheless, the single amino acid substitutions Gly29Ala, Ser, and Asp36, respectively, were not able to be used to identify the weakest binding and greatest stability amino acid substitutions. 33 Glu, and Asp 36 One mutant with an intermediate change in ΔG, Ser, was created by creating a tandem repeat of five IgBD-C domains with Arg, thereby obtaining C-G29A.5d, C-S33A.5d, and C-D36A.5d, respectively. 33 Glu and Asp 36 Arg was chosen for experimental testing (149). The three proteins C-G29A.5d, C-S33E.5d, and C-D36A.5d all showed 3–5 × 10 8 M -1 Association constant K a The association constant of wild-type Protein A for human IgG is K a 1.4×10 8 M -1 (153). To measure binding to VH3 idiotypic IgG, Yoshida et al. used papain treatment to generate Fab fragments from trastuzumab, a VH3 idiotypic IgG1 MAb produced in Chinese hamster ovary (CHO) cells that binds to Her2 on some breast cancer cells (149). The affinity of C-G29A.5d for trastuzumab Fab was K a 4.4×10 5 M -1C-S33E.5d and C-D36A.5d showed an almost 100-fold decrease in affinity (K a 5×10 3 M -1 ) (149).

[0107] Herein, the inventors demonstrate that, specifically by removing its VH3 cross-linking activity, it is possible to prevent SpA in future clinical trials. KKAA We sought to improve vaccine safety. SpA IgBD residues Gly29, Ser33, and Asp36 are located along the edge of helix 2, where they contact VH3 framework residues on human immunoglobulin heavy chains to establish critical bonds, thereby bridging the VH3 idiotypic B cell receptor (138, 154). Previous studies have shown that SpA KKAA At the same time as developing vaccine antigens, 36,37 We have previously investigated Ala amino acid substitutions (43, 154). 29 Position and Ser 33 We focused on a systematic analysis of the amino acid at position 1, which is a Gln 9 Lys and Gln 10 Their contribution to binding to human immunoglobulins in the context of Lys was examined (48, 43). The ability of such SpA vaccine constructs to elicit SpA-specific antibodies and to confer protection against S. aureus colonization and invasive disease was investigated. To assess safety, the inventors used human V H SpA vaccine candidates were analyzed in in vitro and in vivo preclinical models for their efficacy in stimulating anaphylaxis in the presence of 3 idiotypic IgG.

[0108] I. Staphylococcal coagulase Coagulase is an enzyme produced by staphylococci that converts fibrinogen into fibrin. Coa and vW hcoagulase-prothrombin complex recognizes fibrinogen as a specific substrate and converts it directly to fibrin. The crystal structure of the activated complex has revealed the binding of the D1 and D2 domains to prothrombin and its Ile1-Val domain. 2 N-terminal Ile 16 Insertion into a pocket, resulting in the induction of a functional active site in the zymogen precursor through a conformational change, was revealed (Friedrich et al., 2003). Exosite I of α-thrombin, the fibrinogen recognition site, and proexosite I on prothrombin are blocked by D2 of Coa (Friedrich et al., 2003). Nevertheless, binding of the tetrameric (Coa·prothrombin)2 complex binds fibrinogen at the new site with high affinity (Panizzi et al., 2006). This model explains the clotting and efficient fibrinogen conversion by coagulase (Panizzi et al., 2006).

[0109] Fibrinogen is a large glycoprotein (Mr approx. 340,000) formed by three pairs of Aα-, Bβ- and γ-chains covalently linked to form a “dimer of trimers”, where A and B designate the fibrinopeptides released by thrombin cleavage (Panizzi et al., 2006). This elongated molecule folds into three distinct domains: a central fragment E containing the N-terminus of all six chains and two adjacent fragments D formed mainly by the C-terminus of the Bβ- and γ-chains. These globular domains are connected by a long triple-helical structure. The coagulase-prothrombin complex, which converts human fibrinogen into self-polymerizing fibrin, is not targeted by circulating thrombin inhibitors (Panizzi et al., 2006). Thus, staphylococcal coagulase bypasses the physiological blood clotting pathway.

[0110] All S. aureus strains secrete coagulase and vWbp (Bjerketorp et al., 2004; Field and Smith, 1945). Previous efforts have reported an important contribution of coagulase to the pathogenesis of staphylococcal infections (Ekstedt and Yotis, 1960; Smith et al., 1947), but more recent studies using molecular genetic tools have challenged this notion by finding a lack of virulence phenotypes in mouse endocarditis, skin abscess, and mastitis models (Moreillon et al., 1995; Phonimdaeng et al., 1990). By generating isogenic mutants of a fully virulent clinical isolate of S. aureus Newman (Duthie et al., 1952), we herein describe that coa mutants indeed display virulence defects in lethal bacteremia and renal abscess models in mice. In our experience, S. aureus 8325-4 is not sufficiently virulent, and we speculate that mutational lesions in this strain may not reveal virulence defects in vivo. Furthermore, antibodies raised against Coa or vWbp disrupt the pathogenesis of S. aureus Newman infection to an extent that reflects the effects of gene deletion. Coa and vWbp contribute to staphylococcal abscess formation and fatal bacteremia, and can also function as protective antigens in subunit vaccines.

[0111] Biochemical studies demonstrate the biological value of antibodies against Coa and vWbp. By binding to the antigen and blocking its binding to the coagulation factors, the antibodies prevent the formation of Coa·prothrombin and vWbp·prothrombin complexes. Passive transfer studies have demonstrated that Coa and vWbp antibodies protect experimental animals from staphylococcal abscess formation and lethal challenge. Thus, Coa and vWbp neutralizing antibodies generate immune protection against staphylococcal disease.

[0112] Previous studies have revealed that coagulase is required to resist phagocytosis in blood (Smith et al., 1947), and we observed a similar phenotype for Δcoa mutants in lepirudin-treated mouse blood (see Example 3 below). As vWbp shows a higher affinity for human prothrombin than its mouse counterpart, we suspect that the same may be true for ΔvWbp variants in human blood. Furthermore, the expression of Coa and vWbp in abscess lesions and their prominent distribution in the eosinophilic pseudocapsule or peripheral fibrin wall surrounding the staphylococcal abscess community (SAC) suggest that secreted coagulase contributes to the establishment of these lesions. When this hypothesis was tested, Δcoa mutants were indeed defective in the establishment of abscesses. A corresponding study blocking Coa function with a specific antibody produced the same effect. As a result, we propose that fibrin clotting is a crucial event in the establishment of staphylococcal abscesses that can be targeted for the development of a prophylactic vaccine. Both Coa and vWbp are believed to be excellent candidates for vaccine development because of their overlapping functions with human prothrombin.

[0113] J. Other Staphylococcal Antigens Research over the past decades has identified S. aureus exotoxins, surface proteins and regulatory molecules as important virulence factors (Foster, 2005; Mazmanian et al., 2001; Novick, 2003). Considerable progress has been achieved in regulating these genes. For example, staphylococci survey the bacterial population through the secretion of autoinducing peptides that bind to their cognate receptors at threshold concentrations, thereby activating the phosphorylation relay reaction and activating the transcription of many of the exotoxin genes (Novick, 2003). The pathogenesis of staphylococcal infections depends on these virulence factors (secreted exotoxins, exopolysaccharides and surface adhesins). The development of staphylococcal vaccines is hampered by the versatility of the staphylococcal invasion machinery. It is well established that live attenuated microorganisms are highly effective vaccines. The immune responses elicited by such vaccines are often larger and of longer duration than those induced by nonreplicating immunogens. One explanation for this may be that live attenuated strains establish a limited infection in the host, mimicking the early stages of natural infection.Embodiments of the present invention relate to compositions and methods comprising variant SpA polypeptides and peptides, as well as other immunogenic extracellular proteins, polypeptides and peptides (including both secreted and cell surface proteins or peptides) of Gram-positive bacteria for use in mitigating or immunizing against infection.In certain embodiments, the bacteria are Staphylococcus bacteria.The extracellular proteins, polypeptides or peptides include, but are not limited to, secreted and cell surface proteins of the target bacteria.

[0114] The human pathogen Staphylococcus aureus secretes two ESAT-6-like proteins, EsxA and EsxB, across the bacterial envelope (Burts et al., 2005, incorporated herein by reference). Staphylococcal esxA and esxB are clustered with six other genes in order of transcription: esxA esaA essA esaB essB essC esaC esxB. The acronyms esa, ess and esx stand for auxiliary, system and extracellular for ESAT-6 secretion, respectively, depending on whether the encoded protein plays an auxiliary (esa) or direct (ess) role in secretion or is secreted into the extracellular environment (esx). The entire cluster of eight genes is referred to herein as the Ess cluster. EsxA, esxB, essA, essB and essC are all required for the synthesis or secretion of EsxA and EsxB. Mutants unable to produce EsxA, EsxB and EssC show defects in pathogenesis of murine abscesses by S. aureus, suggesting that this specialized secretion system may represent a general strategy for human bacterial pathogenesis. Secretion of non-WXG100 substrates by the ESX-1 pathway has been reported for several antigens, including EspA, EspB, Rv3483c and Rv3615c (Fortune et al., 2005; MacGurn et al., 2005; McLaughlin et al., 2007; Xu et al., 2007). The alternative ESX-5 pathway has also been shown to secrete both WXG100 and non-WXG100 proteins in pathogenic mycobacteria (Abdallah et al., 2007; Abdallah et al., 2006).

[0115] The S. aureus Ess pathway can be viewed as a secretion module with specialized transport components (Ess), accessory factors (Esa) and cognate secreted substrates (Esx). EssA, EssB and EssC are required for secretion of EsxA and EsxB. As EssA, EssB and EssC are predicted to be transmembrane proteins, these proteins are thought to form the secretion apparatus. Some proteins in the ess gene cluster actively transport secreted substrates (acting as motors), whereas others regulate transport (regulators). Regulation can be achieved by, but is not necessarily limited to, transcriptional or post-translational mechanisms for secreted polypeptides, sorting of specific substrates to defined sites (e.g., extracellular medium or host cells), or timing of secretion events during infection. At present, it is unclear whether all secreted Esx proteins function as toxins or indirectly contribute to pathogenesis.

[0116] Staphylococci rely on surface protein-mediated adhesion to host cells or tissue invasion as a strategy for evading immune defenses. In addition, S. aureus utilizes surface proteins to sequester iron from the host during infection. The majority of surface proteins involved in staphylococcal pathogenesis possess C-terminal localization signals, i.e., they are covalently linked to the cell wall outer membrane by sortases. Furthermore, staphylococcal strains lacking genes required for surface protein anchoring, i.e., sortases A and B, show dramatic defects in virulence in several different mouse models of disease. Thus, surface protein antigens are effective vaccine targets since their corresponding genes are essential for the expression of staphylococcal disease and are available in various embodiments of the present invention. The sortase enzyme superfamily is a Gram-positive transpeptidase involved in anchoring surface protein virulence factors to the peptidoglycan cell wall layer. Two sortase isoforms, SrtA and SrtB, have been identified in S. aureus. These enzymes have been shown to recognize LPXTG motifs in substrate proteins. While the SrtB isoform appears to be important in heme iron acquisition and iron homeostasis, the SrtA isoform plays an important role in the pathogenesis of Gram-positive bacteria by modifying the ability of the bacteria to adhere to host tissues via covalent anchoring of adhesins and other proteins to the cell wall peptidoglycan. In certain embodiments, the SpA variants described herein can be used in combination with other staphylococcal proteins, such as Coa, Eap, Ebh, Emp, EsaC, EsaB, EsxA, EsxB, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, IsdC, SasF, vWbp and / or vWh proteins.

[0117] Certain aspects of the invention include methods and compositions relating to proteinaceous compositions comprising SpA variants, and other staphylococcal antigens, such as other proteins delivered by the Ess pathway, or polypeptides, peptides, or nucleic acids encoding sortase substrates. These proteins may be modified by deletion, insertion, and / or substitution.

[0118] The Esx polypeptide comprises the amino acid sequence of the Esx protein from bacteria in the genus Staphylococcus. The Esx sequence may be from a particular Staphylococcus species, such as Staphylococcus aureus, or from a particular strain, such as Newman. In a particular embodiment, the EsxA sequence is SAV0282 from strain Mu50 (this is the same amino acid sequence as in Newman), which can be accessed using Genbank accession number Q99WU4 (gi|68565539), which is incorporated herein by reference. In another embodiment, the EsxB sequence is SAV0290 from strain Mu50 (this is the same amino acid sequence as in Newman), which can be accessed using Genbank accession number Q99WT7 (gi|68565532), which is incorporated herein by reference. In further embodiments, other polypeptides transported by the Ess pathway can be used, and the sequences can be identified by one of skill in the art using databases and internet-accessible sources.

[0119] Sortase substrate polypeptides include, but are not limited to, the amino acid sequences of SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, IsdC or SasF proteins from bacteria in the genus Staphylococcus. Sortase substrate polypeptide sequences may be from a particular Staphylococcus species, such as Staphylococcus aureus, or from a particular strain, such as Newman. In certain embodiments, the SdrD sequence is from strain N315 and can be accessed using GenBank Accession No. NP_373773.1 (gi|15926240), which is incorporated by reference. In other embodiments, the SdrE sequence is from strain N315 and can be accessed using GenBank Accession No. NP_373774.1 (gi|15926241), which is incorporated by reference. In other embodiments, the IsdA sequence is SAV1130 from strain Mu50 (this is the same amino acid sequence as in Newman) and can be accessed using GenBank Accession No. NP_371654.1 (gi|15924120), which is incorporated by reference. In other embodiments, the IsdB sequence is SAV1129 from strain Mu50 (this is the same amino acid sequence as in Newman) and can be accessed using GenBank Accession No. NP_371653.1 (gi|15924119), which is incorporated by reference. In further embodiments, other polypeptides transported by the Ess pathway or processed by sortases can be used, and those of skill in the art can identify their sequences using databases and internet-accessible sources.

[0120] Examples of various proteins that can be used in the context of the present invention are those having accession numbers NC_002951 (GI:57650036 and GenBank CP000046), NC_002758 (GI:57634611 and GenBank BA000017), NC_002745 (GI:29165615 and GenBank BA000018), NC_003923 (GI:21281729 and GenBank BA000033), NC_002952 (GI:49482253 and GenBank BX571856), NC_002953 (GI:49484912 and GenBank BX571857), NC_007793 (GI:87125858 and GenBank BA000046), each of which is incorporated by reference. The genomes of the present invention can be identified by analysis of database deposits of bacterial genomes, including, but not limited to, NC_007795 (GI:87201381 and GenBank CP000255), NC_007795 (GI:87201381 and GenBank CP000253).

[0121] As used herein, a "protein" or "polypeptide" refers to a molecule that comprises at least 10 amino acid residues. In some embodiments, the wild type of a protein or polypeptide is utilized, however, in many embodiments of the present invention, a modified protein or polypeptide is utilized to generate an immune response. The above terms can be used interchangeably. A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to the wild type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide can have multiple activities or functions). Specifically, a modified / variant protein or polypeptide may be altered with respect to one activity or function, yet still retain the wild type activity or function in other respects, such as immunogenicity.

[0122] In certain embodiments, the size of a (wild type or modified) protein or polypeptide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 1 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 8 The amino acid sequence may include, but is not limited to, 75, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or more, and any range of amino acids derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that the polypeptides may be mutated by truncation to make them shorter than their wild-type counterparts, but also altered by fusing or conjugating them to heterologous protein sequences having specific functions (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).

[0123] As used herein, "amino molecule" refers to any amino acid, amino acid derivative, or amino acid mimic known in the art. In certain embodiments, the residues of a proteinaceous molecule are sequential and do not include any non-amino molecule that interrupts the sequence of amino molecule residues. In other embodiments, the sequence can include one or more non-amino molecule moieties. In certain embodiments, the sequence of residues of a proteinaceous molecule can be interrupted by one or more non-amino molecule moieties.

[0124] Thus, the term "proteinaceous composition" encompasses amino acid sequences which contain at least one of the 20 common amino acids found in naturally synthesized proteins, or at least one modified or unusual amino acid.

[0125] Proteinaceous compositions can be produced by any technique known to those skilled in the art, including (i) expression of proteins, polypeptides or peptides through standard molecular biology techniques, (ii) isolation of proteinaceous compounds from natural sources, or (iii) chemical synthesis of proteinaceous substances. Nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed and may be found in recognized computerized databases. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information (at www.ncbi.nlm.nih.gov / ). The coding regions for these genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art.

[0126] Amino acid sequence variants of SpA, coagulase, and other polypeptides of the invention can be substitutional, insertional, or deletion variants. Alterations in the polypeptides of the invention can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more non-contiguous or contiguous amino acids of the polypeptide compared to the wild-type. Variants may include an amino acid sequence that is at least 50%, 60%, 70%, 80% or 90% identical to any of the sequences provided or referenced herein, e.g., SEQ ID NOs:2-8 or SEQ ID NOs:11-30, including all values ​​and ranges therebetween. Variants may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids. Polypeptides or other surface proteins processed or secreted by the Ess pathway (see Table 1) or sortase substrates from any Staphylococcus species and strains are contemplated for use in the compositions and methods described herein.

[0127] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues may be deleted, or several adjacent amino acids may be deleted. A stop codon may be introduced (by substitution or insertion) into the encoding nucleic acid sequence to generate a truncated protein. Insertion variants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of one or more residues. Terminal additions, called fusion proteins, may also be generated. These fusion proteins comprise multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0128] Substitutional variants typically involve the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modify one or more properties of the polypeptide, with or without losing other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, changing alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartate to glutamate, cysteine ​​to serine, glutamine to asparagine, glutamate to aspartate, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine. Alternatively, the substitution may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing one residue with a chemically different residue, such as substituting a polar or charged amino acid for a non-polar or uncharged amino acid, and vice versa.

[0129] Table 10. Exemplary surface proteins of Staphylococcus aureus strains TIFF0007643730000001.tif131165

[0130] The proteins of the present invention may be recombinant or in vitro synthesized. Alternatively, non-recombinant or recombinant proteins may be isolated from bacteria. It is contemplated that bacteria containing such variants may be practiced in the compositions and methods of the present invention. As a result, the proteins may not be isolated.

[0131] The term "functionally equivalent codon" is used herein to refer to codons that code for the same amino acid, such as the six codons for arginine or serine, as well as codons that code for biologically equivalent amino acids (see Table 11 below).

[0132] Table 11: Codon table TIFF0007643730000002.tif97160

[0133] It will also be understood that the amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially as set forth in one of the sequences disclosed herein, so long as they meet the above criteria, including maintenance of the biological protein activity (e.g., immunogenicity) to which expression of the protein is associated. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0134] The following is a discussion based on changing the amino acids of a protein to create variant polypeptides or peptides. For example, certain amino acids can be substituted for other amino acids in a protein structure with significant or minor loss of interactive binding ability with structures such as, for example, the antigen-binding region of an antibody or the binding site on a substrate molecule. Since it is the interacting ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in the protein sequence and in the underlying DNA coding sequence and still produce a protein with desired properties. Thus, the inventors contemplate that various changes can be made in the DNA sequence of a gene.

[0135] It is contemplated that the compositions of the present invention have from about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The protein concentration in the composition may be about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein), at least about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein), or up to about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).Of these, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be an SpA variant or coagulase and may be used in combination with other peptides or polypeptides, such as other bacterial peptides and / or antigens.

[0136] The present invention contemplates the administration of variant SpA polypeptides or peptides to affect prophylactic or therapeutic efficacy against the development of diseases or conditions associated with infection with staphylococcal pathogens.

[0137] In certain aspects, combinations of staphylococcal antigens are used in the creation of immunogenic compositions that are effective in treating or preventing staphylococcal infections. Staphylococcal infections progress through several different stages. For example, the life cycle of staphylococci includes commensal colonization, initiation of infection by accessing adjacent tissues or the bloodstream, and / or anaerobic growth in the blood. Interactions between S. aureus virulence determinants and host defense mechanisms can induce complications such as endocarditis, metastatic abscess formation, and septic syndrome. Different molecules on the bacterial surface are involved in different stages of the infection cycle. Certain antigen combinations may induce immune responses that protect against multi-stage staphylococcal infections. The effectiveness of the immune response can be measured in animal model assays and / or using opsonization assays.

[0138] K. Polypeptides and Polypeptide Production The present invention describes polypeptides, peptides and proteins and their immunogenic fragments for use in various aspects of the present invention. For example, a particular polypeptide is used to induce or assay an immune response or to induce an immune response. In certain embodiments, all or part of the protein of the present invention can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, (1984); Tarn et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference.

[0139] Alternatively, recombinant DNA technology can be used in which a nucleotide sequence encoding the peptide of the invention is inserted into an expression vector, which is transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression.

[0140] In one embodiment of the present invention, the present invention includes the use of gene transfer into cells, including microorganisms, for the production and / or display of polypeptides or peptides. The gene for the polypeptide or peptide of interest can be transferred into a suitable host cell, followed by culturing the cell under suitable conditions. The construction of recombinant expression vectors and the elements contained therein are well known in the art and are briefly discussed herein. Alternatively, the protein produced can be an endogenous protein that is normally synthesized by the cell to be isolated and purified.

[0141] Another embodiment of the present invention uses an autologous B-lymphocyte cell line transfected with a viral vector expressing an immunogenic product, more specifically a protein with immunogenic activity. Other examples of mammalian host cell lines include, but are not limited to, Vero and HeLa cells, other B- and T-cell lines such as CEM, 721.221, H9, Jurkat, Raji, and Chinese hamster ovary cell lines, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. In addition, a host cell line can be selected that regulates the expression of the inserted sequence or modifies and processes the gene product in the desired manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms of post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.

[0142] Several selection systems can be used, including, but not limited to, the HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes in tk-, hgprt-, or aprt- cells, respectively. Similarly, antimetabolite resistance can be used as the basis of selection: dhfr, which confers resistance to trimethoprim and methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G418; and hygro, which confers resistance to hygromycin.

[0143] Animal cells can be grown in vitro in two ways: as non-anchorage-dependent cells, which grow in suspension throughout the majority of the culture, or as anchorage-dependent cells, which require attachment to a solid substrate for growth (i.e., monolayer-type cell growth).

[0144] Anchorage-independent or suspension cultures from continuous established cell lines are the most widely used means for large-scale production of cells and cell products. However, suspension cultured cells have limitations such as tumorigenicity and lower protein production than adherent cells.

[0145] When a protein is specifically mentioned herein, it is preferably a reference to a natural or recombinant protein, or a protein with any signal sequence removed, if necessary. The protein may be directly isolated from a staphylococcus strain, or may be produced by recombinant DNA technology. Immunogenic fragments of a protein may be included in the immunogenic composition of the present invention. These are fragments that include at least 10, 20, 30, 40, 50, or 100 amino acids consecutively taken from the amino acid sequence of the protein, including all values ​​and ranges therebetween. Moreover, such immunogenic fragments are immunologically reactive with antibodies generated against staphylococcus proteins, or with antibodies generated by infection of a mammalian host with staphylococci. Immunogenic fragments also include fragments that induce a protective or therapeutic immune response against staphylococcus infection when administered in an effective amount (either alone or as a hapten bound to a carrier), and in certain aspects, it prevents S. aureus and / or S. epidermidis infection. Such immunogenic fragments can include, for example, proteins lacking an N-terminal leader sequence, and / or a transmembrane domain and / or a C-terminal anchor domain. In preferred aspects, immunogenic fragments according to the invention include substantially all of the extracellular domain of a protein having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 97-99% identity, including all values ​​and ranges therebetween, to a selected segment of the sequence of a polypeptide described or referenced herein.

[0146] Also included in the immunogenic compositions of the invention are fusion proteins composed of one or more staphylococcal proteins, or immunogenic fragments of staphylococcal proteins. Such fusion proteins can be recombinantly produced and can include portions of at least one, two, three, four, five, or six staphylococcal proteins or segments. Alternatively, the fusion proteins can include portions of at least one, two, three, four, or five staphylococcal proteins. These can combine (to form multimers or concatemers) different staphylococcal proteins and / or multiple of the same protein or protein fragment, or immunogenic fragments of the same protein. Alternatively, the present invention also includes individual fusion proteins of staphylococcal proteins or immunogenic fragments thereof as fusion proteins with heterologous sequences such as donors of T cell epitopes or purification tags, e.g. β-galactosidase, glutathione-S-transferase, green fluorescent protein (GFP), epitope tags such as FLAG, myc tags, polyhistidine, or viral surface proteins such as influenza virus hemagglutinin, or bacterial proteins such as tetanus toxoid, diphtheria toxoid or CRM197.

[0147] II. Nucleic acids In certain embodiments, the present invention relates to recombinant polynucleotides encoding the proteins, polypeptides, and peptides of the present invention, including nucleic acid sequences for SpA, coagulase, and other bacterial proteins, all of which are incorporated by reference, that can be used to prepare peptides or polypeptides.

[0148] As used in this application, the term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated free of total genomic nucleic acid. Included within the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. A polynucleotide, in certain aspects, includes a regulatory sequence that is substantially isolated from a natural gene or protein coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may or may not be present in a polynucleotide.

[0149] In this regard, the terms "gene," "polynucleotide," or "nucleic acid" are used to refer to a nucleic acid (including any sequences required for proper transcription, post-translational modification, or localization) that encodes a protein, polypeptide, or peptide. As will be understood by those of skill in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or can be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide includes any of the following polynucleotides that encode one or more amino acid sequences described or referenced herein, including all values ​​and ranges between: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720 20, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620 , 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 10 It may comprise a contiguous nucleic acid sequence of 30, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000 or more nucleotides, nucleosides or base pairs.It is also contemplated that a particular polypeptide may be encoded by a nucleic acid, including variants that have slightly different nucleic acid sequences but still encode the same or a substantially similar protein (see Table 11 above).

[0150] In certain embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding variant SpA or coagulase. The term "recombinant" can be used in conjunction with a polynucleotide or polypeptide, which generally refers to a polypeptide or polynucleotide that has been produced and / or manipulated in vitro, or that is the product of replication of such a molecule.

[0151] In other aspects, the invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding variant SpA or coagulase polypeptides or peptides for generating an immune response in a subject. In various aspects, the nucleic acids of the invention can be used in genetic vaccines.

[0152] The nucleic acid segments used in the present invention can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and therefore their total length can vary considerably. Therefore, it is considered that nucleic acid fragments of almost any length can be utilized, with the total length being preferably limited by the ease of purification and the intended use in the recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can code for a polypeptide sequence with additional heterologous coding sequences, for example, to allow purification, transport, secretion, post-translational modification of the polypeptide, or to allow therapeutic utility, such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides can be added to the sequence encoding the modified polypeptide, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0153] In certain other aspects, the present invention relates to isolated nucleic acid segments and recombinant vectors that contain within their sequence a contiguous nucleic acid sequence derived from SEQ ID NO:2 (SpA domain D) or SEQ ID NO:4 (SpA), or any other nucleic acid sequence encoding a coagulase or other secreted virulence factor and / or surface protein, such as a protein delivered by the Ess pathway and processed by a sortase, or a protein incorporated herein by reference.

[0154] In certain embodiments, the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein, i.e., those that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity, including all values ​​and ranges therebetween, compared to the polynucleotide sequences of the invention using the methods described herein (e.g., BLAST analysis with standard parameters).

[0155] The present invention also contemplates the use of polynucleotides complementary to all of the polynucleotides described above.

[0156] A. Vector The polypeptides of the present invention may be encoded by a nucleic acid molecule contained in a vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted to introduce it into a cell where it can be replicated and expressed. A nucleic acid sequence may be "heterologous", which in context means that the nucleic acid sequence is foreign to the cell into which the vector is being introduced or to the nucleic acid into which it is being incorporated, including sequences that are homologous to the sequence in the cell or nucleic acid, but are at a location within the host cell or nucleic acid where they are not normally found. Vectors include DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well equipped to construct vectors through standard recombinant techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both of which are incorporated herein by reference). In addition to encoding a variant SpA polypeptide, the vector may encode other polypeptide sequences, such as one or more other bacterial peptides, tags, or immunogenicity enhancing peptides. Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al., 1985), vectors encoding stretches of histidines, and pGEX vectors for use in generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage.

[0157] The term "expression vector" refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that serve other functions and are described herein.

[0158] 1. Promoters and Enhancers A "promoter" is a regulatory sequence. A promoter is typically a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. It may include genetic elements to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation relative to a nucleic acid sequence to control the transcription initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0159] Of course, it may be important to use a promoter and / or enhancer that efficiently directs the expression of the DNA segment in the cell type or organism selected for expression.Those skilled in the art of molecular biology are generally aware of the use of a combination of promoter, enhancer, and cell type to express proteins (see Sambrook et al., 2001, incorporated herein by reference).The promoter used may be constitutive, tissue-specific, or inducible, and in certain embodiments, may direct high-level expression of the introduced DNA segment under specific conditions, such as large-scale production of recombinant protein or peptide.

[0160] A variety of elements / promoters may be used in the context of the present invention to regulate expression of the genes. Examples of such inducible elements, which are regions of nucleic acid sequence that can be activated in response to specific stimuli, include immunoglobulin heavy chains (Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al., 1990), immunoglobulin light chains (Queen et al., 1983; Picard et al., 1984), T cell receptors (Luria et al., 1987; Winoto et al., 1989; Redondo et al., 1990), HLA DQα and / or DQβ (Sullivan et al., 1992), and the IL-11 receptors (Lupin et al., 1987; Winoto et al., 1989; Redondo et al., 1990). 1987), β-interferon (Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988), interleukin-2 (Greene et al., 1989), interleukin-2 receptor (Greene et al., 1989; Lin et al., 1990), MHC class II 5 (Koch et al., 1989), MHC class II HLA-DRα (Sherman et al., 1989), β-actin (Kawamoto et al., 1988; Ng et al., 1989), muscle creatine kinase (MCK) (Jaynes et al., 1988; Horlick et al., 1989; Johnson et al., 1989), prealbumin (transthyretin) (Costa et al., 1988), elastase I (Ornitz et al., 1987), metallothionein (MTII) (Karin et al., 1987; Culotta et al., 1989), collagenase (Pinkert et al., 1987; Angel et al., 1987), albumin (Pinkert et al., 1987; Tronche et al., 1989, 1990); α-fetoprotein (Godbout et al., 1988; Campere et al., 1989), γ-globin (Bodine et al., 1987; Perez-Stable et al., 1990), β-globin (Trudel et al., 1987), c-fos (Cohen et al., 1987), c-Ha-Ras (Triesman, 1986; Deschamps et al., 1985), insulin (Edlund et al., 1985), neural cell adhesion molecule (NCAM) (Hirsh et al., 1990); α1-antitripain (Latimer et al., 1990); H2B (TH2B) histones (Hwang et al., 1990); mouse and / or type I collagen (Ripe et al., 1989), glucose-regulated proteins (GRP94 and GRP78) (Chang et al., 1989), rat growth hormone (Larsen et al., 1986), human serum amyloid A (SAA) (Edbrooke et al., 1989), troponin I (TN I) (Yutzey et al., 1989), platelet-derived growth factor (PDGF) (Pech et al., 1989), Duchenne muscular dystrophy (Klamut et al., 1990), SV40 (Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1997), and mitochondrial protein 1 (MMP1) (Pech et al., 1989). al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988), polyoma (Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell et al., 1988), retroviruses (Kriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989), papillomaviruses (Campo et al., 1983; Lusky et al., 1983; Spandidos and Wilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987), hepatitis B virus (Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988), human immunodeficiency virus (Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989), cytomegalovirus (CMV) IE (Weber et al., 1984; Boshart et al., 1985; Foecking et al., 1986), and gibbon ape leukemia virus (Holbrook et al., 1987; Quinn et al., 1989).

[0161] Inducible elements include MT II-phorbol ester (TFA) / heavy metals (Palmiter et al., 1982; Haslinger et al., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989); MMTV (mouse mammary tumor virus)-glucocorticoids (Huang et al., 1981; Lee et al., 1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985; Sakai et al., 1988); β-interferon-poly(rI)x / poly(rc) (Tavernier et al., 1983); adenovirus 5 E2-E1A (Imperiale et al., 1984); collagenase-phorbol ester (TPA) (Angel et al., 1987a); stromelysin-phorbol ester (TPA) (Angel et al., 1987b); SV40-phorbol ester (TPA) (Angel et al., 1987b); murine MX gene-interferon, Newcastle disease virus (Hug et al., 1988); GRP78 gene-A23187 (Resendez et al., 1988); α-2-macroglobulin-IL-6 (Kunz et al., 1989); vimentin-serum (Rittling et al., 1989); MHC class I gene H-2κb-interferon (Blanar et al., 1989); HSP70-E1A / SV40 large T antigen (Taylor et al., 1989, 1990a, 1990b); proliferin-phorbol ester / TPA (Mordacq et al., 1989); tumor necrosis factor-PMA (Hensel et al., 1989); and thyroid-stimulating hormone alpha gene-thyroid hormone (Chatterjee et al., 1989), but are not limited to these.

[0162] The particular promoter utilized to control the expression of the polynucleotide encoding the peptide or protein of the present invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in the target cell, preferably a bacterial cell. When a human cell is targeted, it is preferred to position the polynucleotide coding region adjacent to and under the control of a promoter capable of being expressed in a human cell. Generally speaking, such promoters may include either bacterial promoters, human promoters or viral promoters.

[0163] In the embodiment in which the vector is administered to a subject for the expression of a protein, it is contemplated that the promoter that is desirable to use with the vector is one that is not downregulated by cytokines, or even if it is downregulated, is strong enough to produce an effective amount of variant SpA to induce an immune response.Non-limiting examples of these are CMV IE and RSV LTR.Tissue-specific promoters can be used, especially if expression is in a cell in which expression of an antigen is desired, such as a dendritic cell or macrophage.Mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters.

[0164] 2. Initiation signals and internal ribosome binding sites (IRES) Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would be readily able to determine this and provide the necessary signals.

[0165] In certain embodiments of the present invention, internal ribosome entry site (IRES) elements are used to create multigenic or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can link heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to create polycistronic messages. Multiple genes can be efficiently expressed using a single promoter / enhancer and a single message can be transcribed (see U.S. Patent Nos. 5,925,565 and 5,935,819, which are incorporated herein by reference).

[0166] 3. Selectable and screenable markers In certain embodiments of the present invention, cells containing the nucleic acid construct of the present invention can be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, the marker confers an identifiable change to the cell, allowing easy identification of cells containing the expression vector. Generally, a selectable marker confers a property that allows for selection. A positive selectable marker is one whose presence allows for its selection, whereas a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0167] B. host cell As used herein, the terms "cell", "cell line" and "cell culture" can be used interchangeably. All of these terms include any and all subsequent generations of their progeny. It will be understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, a "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by the vector. A host cell can be and has been used as a recipient of a vector or virus. A host cell may be "transfected" or "transformed", which refer to the process by which exogenous nucleic acid, such as a sequence encoding a recombinant protein, is transferred or introduced into a host cell. A transformed cell includes the primary subject cell and its progeny.

[0168] Host cells can be of prokaryotic or eukaryotic origin, including bacteria, yeast cells, insect cells, and mammalian cells for replicating the vector or expressing part or all of the nucleic acid sequence. Numerous cell lines and cultures are available for use as host cells and can be obtained through the American Type Culture Collection (ATCC) (www.atcc.org), an organization that serves as a repository for living cultures and genetic material.

[0169] C. Expression Systems There are numerous expression systems that contain at least some or all of the compositions discussed above. Prokaryotic and / or eukaryotic based systems can be utilized for use in the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially available and widely available.

[0170] The insect cell / baculovirus system can provide high levels of protein expression of heterologous nucleic acid segments, as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both of which are incorporated herein by reference, and can be purchased, for example, from INVITROGEN® under the name MAXBAC® 2.0 and from CLONTECH® under the name BACPACK™ BACULOVIRUS EXPRESSION SYSTEM.

[0171] In addition to the expression systems disclosed in the present invention, other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL™ Inducible Mammalian Expression System with a synthetic ecdysone-inducible receptor or its pET expression system, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which has the T-REX™ (Tetracycline Regulated Expression) System, an inducible mammalian expression system using a full-length CMV promoter. INVITROGEN® also offers a yeast expression system called the Pichia methanolica Expression System, designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. Those skilled in the art will know how to express a vector, such as an expression construct, and how to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[0172] III. Polysaccharides The immunogenic compositions of the invention may further comprise capsular polysaccharides comprising one or more of PIA (also known as PNAG), and / or Staphylococcus aureus type V and / or type VIII capsular polysaccharides, and / or Staphylococcus epidermidis type I and / or type II and / or type III capsular polysaccharides.

[0173] A. PIA (PNAG) It is now clear that the various forms of staphylococcal surface polysaccharides identified as PS / A, PIA and SAA are the same chemical entity, namely PNAG (Maira-Litran et al., 2004). Therefore, the terms PIA or PNAG encompass all these polysaccharides or oligosaccharides derived from them.

[0174] PIA is a polysaccharide intercellular adhesin, composed of a polymer of β-(1→6)-linked glucosamine substituted with N-acetyl and O-succinyl moieties. This polysaccharide is present in both S. aureus and S. epidermidis and can be isolated from both sources (Joyce et al., 2003; Maira-Litran et al., 2002). For example, PNAG can be isolated from S. aureus strain MN8m (WO04 / 43407). PIA isolated from S. epidermidis is a complex component of biofilms. It is involved in mediating cell-cell adhesion and probably also functions to protect the growing colony from the host immune response. A polysaccharide known as poly-N-succinyl-β-(1→6)-glucosamine (PNSG) was recently shown not to have the expected structure due to an incorrect identification of N-succinylation (Maira-Litran et al., 2002). Therefore, the polysaccharide formerly known as PNSG, and now known to be PNAG, is also encompassed by the term PIA.

[0175] PIA (or PNAG) can be of various sizes, from over 400 kDa, to 75-400 kDa, to 10-75 kDa, to oligosaccharides composed of up to 30 repeating units (of β-(1→6) linked glucosamine substituted with N-acetyl and O-succinyl moieties). PIA polysaccharides or oligosaccharides of any size can be used in the immunogenic compositions of the invention, but in one aspect the polysaccharide is greater than 40 kDa. Sizing can be performed by any method known in the art, for example by microfluidization, sonication or chemical cleavage (WO 03 / 53462, EP497524, EP497525). In certain aspects, the PIA (PNAG) is at least or at most 40-400 kDa, 40-300 kDa, 50-350 kDa, 60-300 kDa, 50-250 kDa, and 60-200 kDa.

[0176] PIA (PNAG) can have different degrees of acetylation due to substitution on amino groups with acetate. PIA produced in vitro is almost completely (95-100%) substituted on amino groups. Alternatively, deacetylated PIA (PNAG) can be used, having less than 60%, 50%, 40%, 30%, 20%, 10% acetylation. The use of deacetylated PIA (PNAG) is preferred, since the nonacetylated epitopes of PNAG are efficient in mediating opsonized killing of Gram-positive bacteria, preferably Staphylococcus aureus and / or Staphylococcus epidermidis. In certain aspects, PIA (PNAG) has a size of 40 kDa to 300 kDa and is deacetylated such that less than 60%, 50%, 40%, 30% or 20% of amino groups are acetylated.

[0177] The term deacetylated PNAG (dPNAG) refers to PNAG polysaccharides or oligosaccharides in which less than 60%, 50%, 40%, 30%, 20% or 10% of the amino agroups are acetylated. In certain aspects, the native polysaccharide is chemically treated to deacetylate the PNAG to form dPNAG. For example, the native PNAG is treated with a basic solution such that the pH is raised to above 10. For example, the PNAG is treated with 0.1-5 M, 0.2-4 M, 0.3-3 M, 0.5-2 M, 0.75-1.5 M or 1 M NaOH, KOH or NH4OH. The treatment is at a temperature of 20-100°C, 25-80°C, 30-60°C or 30-50°C or 35-45°C for at least 10-30 minutes, or for 1, 2, 3, 4, 5, 10, 15 or 20 hours. dPNAG can be prepared as described in WO 04 / 43405.

[0178] The polysaccharide may or may not be conjugated to a carrier protein.

[0179] B. Type 5 and Type 8 polysaccharides from Staphylococcus aureus Most strains of S. aureus causing infections in humans contain either type 5 or type 8 polysaccharides. Approximately 60% of human strains are type 8 and approximately 30% are type 5. The structures of type 5 and type 8 capsular polysaccharide antigens have been described by Moreau et al. (1990) and Fournier et al. (1984). Both have FucNAcp in their repeating units and ManNAcA which can be used to introduce sulfhydryl groups. Their structures are: Type 5 →4)-β-D-ManNAcA(3OAc)-(1→4)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→ Type 8 →3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→ and recently (Jones, 2005) their structures have been elucidated by NMR spectroscopy. Type 5 →4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-β-D-FucNAc-(1→ Type 8 →3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-α-D-FucNAc(1→ has been corrected to.

[0180] Polysaccharides can be extracted from suitable S. aureus strains using methods well known to those of skill in the art, see U.S. Patent No. 6,294,177. For example, ATCC 12902 is a type 5 S. aureus strain and ATCC 12605 is a type 8 S. aureus strain.

[0181] The polysaccharides may be of native size or may be sized, for example, by microfluidization, sonication or chemical treatment. The invention also encompasses oligosaccharides derived from the type 5 and type 8 polysaccharides of Staphylococcus aureus. The type 5 and type 8 polysaccharides included in the immunogenic compositions of the invention are preferably conjugated to a carrier protein, as described below, or are unconjugated. The immunogenic compositions of the invention alternatively comprise either type 5 or type 8 polysaccharides.

[0182] C. Staphylococcus aureus 336 antigen In one embodiment, the immunogenic composition of the invention comprises the S. aureus 336 antigen described in U.S. Patent No. 6,294,177. The 336 antigen contains β-linked hexosamines, does not contain O-acetyl groups, and specifically binds to antibodies against S. aureus type 336 deposited under ATCC 55804. In one embodiment, the 336 antigen is a polysaccharide, which may be of native size or may be sized, for example, by microfluidization, sonication, or chemical treatment. The invention also encompasses oligosaccharides derived from the 336 antigen. The 336 antigen may be unconjugated or conjugated to a carrier protein.

[0183] D. Type I, II, and III polysaccharides from Staphylococcus epidermidis Among the problems associated with using polysaccharides for vaccination is the fact that polysaccharides themselves are poor immunogens. The polysaccharides utilized in the present invention are preferably linked to a protein carrier that provides bystander T cell help to improve immunogenicity. Examples of such carriers that can be combined with the polysaccharide immunogens include diphtheria and tetanus toxoids (DT, DT CRM 197, and TT, respectively), keyhole limpet hemocyanin (KLH), as well as tuberculin purified protein derivative (PPD), Pseudomonas aeruginosa exoprotein A (rEPA), protein D from Haemophilus influenzae, pneumolysin, or fragments of any of these. Suitable fragments for use include those that encompass T helper epitopes. In particular, a protein D fragment from Haemophilus influenzae would preferably include the N-terminal third of the protein. Protein D is an IgD-binding protein from Haemophilus influenzae (EP 0 594 610 B1) and is a potential immunogen. Additionally, staphylococcal proteins can be used as carrier proteins in the polysaccharide conjugates of the present invention.

[0184] A carrier protein that is considered to be particularly advantageous for use with staphylococcal vaccines is staphylococcal alpha toxoid. This native form may be conjugated to polysaccharides, as the conjugation process reduces toxicity. Preferably, genetically detoxified alpha toxin, such as His35Leu or His35Arg variants, is used as the carrier, as it has lower residual toxicity. Alternatively, the alpha toxin is chemically detoxified by treatment with the cross-linking reagents formaldehyde or glutaraldehyde. The genetically detoxified alpha toxin may be optionally chemically detoxified, preferably by treatment with the cross-linking reagents formaldehyde or glutaraldehyde, to further reduce toxicity.

[0185] Polysaccharides can be linked to carrier proteins by any known method, for example those described in U.S. Pat. Nos. 4,372,945, 4,474,757 and 4,356,170. CDAP conjugation chemistry is preferably performed (see WO 95 / 08348). In CDAP, the cyanylating reagent 1-cyano-dimethylaminopyridinium tetrafluoroborate (CDAP) is preferably used to synthesize polysaccharide-protein conjugates. The cyanylation reaction can be carried out under relatively mild conditions to avoid hydrolysis of alkali-sensitive polysaccharides. This synthesis allows direct coupling to carrier proteins.

[0186] Preferably, the conjugation involves the creation of a direct link between the carrier protein and the polysaccharide. Optionally, a spacer (such as adipic dihydride (ADH)) may be introduced between the carrier protein and the polysaccharide.

[0187] IV. Immune Responses and Assays As mentioned above, the present invention relates to eliciting or inducing an immune response against variant SpA or coagulase peptides in a subject. In one embodiment, the immune response can protect against or treat a subject having, suspected of having, or at risk of developing an infection or related disease, particularly a Staphylococcus-related disease. One use of the immunogenic compositions of the present invention is to prevent nosocomial infections by vaccinating subjects prior to receiving medical attention in a hospital or other environment with an increased risk of infection.

[0188] A. Immunoassays The present invention includes carrying out serological assays to evaluate whether and to what extent an immune response is induced or elicited by the compositions of the present invention. There are many types of immunoassays that can be carried out. Immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Pat. No. 4,367,110 (double monoclonal antibody sandwich assay) and those described in U.S. Pat. No. 4,452,901 (Western blot). Other assays include immunoprecipitation and immunocytochemistry of labeled ligands, both in vitro and in vivo.

[0189] Immunoassays are generally binding assays. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. In one example, an antibody or antigen is immobilized on a selected surface, such as a well in a polystyrene microtiter plate, a dipstick, or a column support. A test composition suspected of containing the desired antigen or antibody, such as a clinical sample, is then added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen or antibody can be detected. Detection is generally achieved by the addition of another antibody specific for the desired antigen or antibody, which is linked to a detectable label. This type of ELISA is known as a "sandwich ELISA". Detection can also be achieved by the addition of a second antibody specific for the desired antigen, followed by a third antibody having binding affinity for the second antibody, which is linked to a detectable label.

[0190] Competitive ELISA may also be performed, in which the test sample competes with a known amount of labeled antigen or antibody for binding. The amount of reactive species in an unknown sample is measured by mixing the sample with a known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the wells, thus reducing the final signal. Regardless of the format used, ELISAs have certain features in common, such as coating, incubation or binding, washing to remove non-specifically bound species, and detection of bound immune complexes.

[0191] The antigen or antibody may be linked to a solid support, such as in the form of a plate, bead, dipstick, membrane or column substrate, and the sample to be analyzed is applied to the immobilized antigen or antibody. When coating a plate with either antigen or antibody, the wells of the plate are generally incubated with a solution of the antigen or antibody overnight or for a certain period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Any available surface remaining in the wells is then "coated" with a non-specific protein that is antigenically neutral with respect to the test antiserum. These include solutions of bovine serum albumin (BSA), casein and milk powder. The coating allows for blocking of non-specific adsorption sites on the immobilizing surface, thus reducing the background caused by non-specific binding of antisera onto the surface.

[0192] B. Diagnosis of Bacterial Infection In addition to the use of proteins, polypeptides and / or peptides, and antibodies that bind these polypeptides, proteins and / or peptides to treat or prevent the above infections, the present invention contemplates the use of these polypeptides, proteins, peptides and / or antibodies in various ways, including detection of the presence of Staphylococcus to diagnose infections, whether in a patient or on a medical device, which may also be present. According to the present invention, a preferred method of detecting the presence of an infection involves obtaining a sample from an individual suspected of being infected with one or more Staphylococcus species or strains, such as a sample taken from the individual's blood, saliva, tissue, bone, muscle, cartilage or skin. Following isolation of the sample, a diagnostic assay utilizing the polypeptides, proteins, peptides and / or antibodies of the present invention can be performed to detect the presence of Staphylococcus, and such assay techniques for determining such presence in a sample are well known to those of skill in the art and include methods such as radioimmunoassays, Western blot analysis and ELISA assays. Generally, the present invention contemplates a method for diagnosing infection in which a sample suspected of being infected with Staphylococcus aureus is added to a polypeptide, protein, peptide, antibody or monoclonal antibody according to the present invention, and Staphylococcus aureus is indicated by an antibody that binds to the polypeptide, protein and / or peptide, or a polypeptide, protein and / or peptide that binds to the antibody, in the sample.

[0193] Thus, the antibodies according to the invention can be used for the prevention of Staphylococcus infection (i.e., passive immunization), for the treatment of ongoing infection, or as a research tool. The term "antibody" as used herein includes monoclonal, polyclonal, chimeric, single-chain, bispecific, simianized and humanized or primatized antibodies, as well as Fab fragments, such as fragments that maintain the binding specificity of the antibody, including the products of a Fab immunoglobulin expression library. Thus, the present invention contemplates the use of single chains, such as the variable heavy and light chains of an antibody. The generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art. Specific examples of the generation of antibodies against bacterial proteins can be found in U.S. Patent Application Publication No. 20030153022, which is incorporated herein by reference in its entirety.

[0194] Any of the above polypeptides, proteins, peptides and / or antibodies can be directly labeled with a detectable label for identification and quantification of staphylococci. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).

[0195] C. Protective immunity In some embodiments of the present invention, the proteinaceous composition confers protective immunity to a subject. Protective immunity refers to the body's ability to mount a specific immune response that prevents the subject from developing a particular disease or condition associated with the agent against which the immune response is generated. An immunogenically effective amount is capable of conferring protective immunity to a subject.

[0196] As used herein and in the appended claims, the term polypeptide or peptide refers to a sequence of amino acids covalently linked via peptide bonds. Different polypeptides have different functionalities according to the invention. According to one aspect, the polypeptide is derived from an immunogen designed to induce an active immune response in a recipient, whereas according to another aspect of the invention, the polypeptide is derived from an antibody that arises after eliciting an active immune response, for example in an animal, and can act to induce a passive immune response in a recipient. However, in both cases, the polypeptide is encoded by a polynucleotide according to any possible codon usage.

[0197] As used herein, the phrase "immune response" or its equivalent "immunological response" refers to the occurrence in a recipient patient of a humoral (antibody-mediated), cellular (mediated by antigen-specific T cells or their secretory products), or both humoral and cellular responses directed to a protein, peptide, carbohydrate, or polypeptide of the invention. Such responses can be active responses induced by administration of an immunogen or passive responses induced by administration of antibodies, antibody-containing materials, or primed T cells. Cellular immune responses are elicited by presentation of polypeptide epitopes in association with class I or class II MHC molecules to activate antigen-specific CD4 (+) T helper cells and / or CD8 (+) cytotoxic T cells. The response can also involve monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or other components of innate immunity. As used herein, "active immunity" refers to any immunity conferred to a subject by administration of an antigen.

[0198] As used herein, "passive immunity" refers to any immunity conferred to a subject without administration of an antigen to the subject. "Passive immunity" therefore includes, but is not limited to, administration of activating immune effectors, including cellular or protein mediators of the immune response (e.g., monoclonal and / or polyclonal antibodies). Monoclonal or polyclonal antibody compositions can be used for passive immunization to prevent or treat infections with organisms bearing the antigen recognized by the antibodies. The antibody composition can include antibodies that bind to different antigens that may be associated with different organisms. The antibody component can be a polyclonal antiserum. In certain aspects, the antibodies are affinity purified from an animal or a second subject inoculated with the antigen. Alternatively, an antibody mixture can be used, which is a mixture of monoclonal and / or polyclonal antibodies against antigens present in the same, related, or different microorganisms or organisms, such as gram-positive and gram-negative bacteria, including but not limited to Staphylococcus.

[0199] Passive immunity can be conferred to a patient or subject by administering to the patient immunoglobulins (Ig) and / or other immune factors obtained from a donor or other non-patient source with surrounding immune reactivity. In another aspect, the antigen composition of the invention can be administered to a subject that serves as a source or donor of globulins ("hyperimmune globulins") produced in response to exposure to the antigen composition, including antibodies made against staphylococci or other organisms. A subject thus treated can provide plasma from which hyperimmune globulins can be obtained by conventional plasma fractionation methods and administered to another subject to confer resistance to or treat staphylococcal infection. Hyperimmune globulins according to the invention are particularly useful for immunocompromised individuals, for individuals undergoing invasive procedures, or for individuals who do not have time to produce their own antibodies in response to vaccination. For exemplary methods and compositions related to passive immunization, see U.S. Pat. Nos. 6,936,258, 6,770,278, 6,756,361, 5,548,066, 5,512,282, 4,338,298, and 4,748,018, each of which is incorporated by reference herein in its entirety.

[0200] For purposes of this specification and the appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to a site on an antigen to which B and / or T cells respond or recognize. B cell epitopes can be formed from adjacent or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from adjacent amino acids are usually retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are usually lost on treatment with denaturing solvents. Epitopes usually contain at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial structure. Methods for determining the spatial structure of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay that demonstrates the ability of one antibody to block the binding of another antibody to a target antigen. T cells recognize continuous epitopes of approximately 9 amino acids in the case of CD8 cells, or approximately 13-15 amino acids in the case of CD4 cells. T cells that recognize an epitope react to the epitope by cloning the primed T cells. 3 They can be identified by in vitro assays measuring antigen-dependent proliferation as determined by H-thymidine incorporation (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996), or by cytokine secretion.

[0201] The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 (+) T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be discerned by separately isolating IgG and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0202] As used herein and in the appended claims, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an animal's or recipient's immune response, including IgG, IgD, IgE, IgA, IgM and related proteins.

[0203] Under normal physiological conditions, antibodies are found in plasma and other body fluids and in the membranes of certain cells and are produced by a type of lymphocyte designated B cells or their functional equivalents. Antibodies of the IgG class are composed of four polypeptide chains linked together by disulfide bonds. The four chains in an intact IgG molecule are two identical heavy chains, called H chains, and two identical light chains, called L chains.

[0204] To produce polyclonal antibodies, a host such as a rabbit or goat is immunized with an antigen or an antigen fragment, typically with an adjuvant and, if necessary, coupled to a carrier. Antibodies against the antigen are then recovered from the host's serum. Polyclonal antibodies can be affinity purified against the antigen making them monospecific.

[0205] Monoclonal antibodies can be produced ex vivo by hyperimmunization of a suitable donor with antigen or by using primary cultures of splenocytes or cell lines derived from the spleen (Anavi, 1998; Huston et al., 1991; Johnson et al., 1991; Mernaugh et al., 1995).

[0206] As used herein and in the appended claims, the phrase "immunological portion of an antibody" includes an antibody Fab fragment, an antibody Fv fragment, an antibody heavy chain, an antibody light chain, a heterodimer of an antibody heavy and light chain, a variable fragment of an antibody light chain, a variable fragment of an antibody heavy chain, and a single chain variant of an antibody, also known as scFv. In addition, the term also includes chimeric immunoglobulins that are the expression product of fusion genes from different species, one of which may be human, in which case the chimeric immunoglobulin is said to be humanized. Typically, an immunological portion of an antibody competes with the intact antibody from which it was derived for specific binding to an antigen.

[0207] Optionally, an antibody, or preferably an immunological portion of an antibody, may be chemically conjugated to or expressed as a fusion protein with other proteins, and for purposes of this specification and the appended claims, all such fusion proteins are included within the definition of an antibody or immunological portion of an antibody.

[0208] As used herein, the terms "immunogenic agent" or "immunogen" or "antigen" are used interchangeably to describe a molecule that can induce an immunological response against itself when administered to a recipient, either together with an adjuvant, alone, or presented on a display medium.

[0209] D. Treatment Method The methods of the invention include the treatment of diseases or conditions caused by staphylococcal pathogens. The immunogenic polypeptides of the invention can be administered to induce an immune response in individuals suffering from or suspected of being exposed to staphylococci. The methods can be utilized on individuals who have tested positive for exposure to staphylococci or who are believed to be at risk for infection based on potential exposure.

[0210] Specifically, the present invention encompasses methods of treating staphylococcal infections, particularly hospital-acquired nosocomial infections. The immunogenic compositions and vaccines of the present invention are particularly convenient for use in elective surgery cases. Such patients will know the date of surgery in advance and could be vaccinated in advance. The immunogenic compositions and vaccines of the present invention are also convenient for use to vaccinate medical personnel.

[0211] In some embodiments, the treatment is administered in the presence of an adjuvant or carrier or other staphylococcal antigen. Additionally, in some instances, the treatment includes administration of other drugs commonly used against bacterial infections, such as one or more antibiotics.

[0212] Use of peptides for vaccination may, but does not necessarily, require conjugation of the peptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin, or bovine serum albumin, and methods for accomplishing this conjugation are well known in the art.

[0213] V. Vaccines and Other Pharmaceutical Compositions and Administration A. Vaccines The present invention includes methods for preventing or ameliorating staphylococcal infections, particularly hospital-acquired nosocomial infections. Thus, the present invention contemplates vaccines for use in both active and passive immunization modes. Immunogenic compositions proposed to be suitable for use as vaccines can be prepared from immunogenic SpA polypeptides, such as SpA domain D variants, or immunogenic coagulase. In other embodiments, SpA or coagulase can be used in combination with other secreted virulence proteins, surface proteins, or immunogenic fragments thereof. In certain aspects, the antigenic material is extensively dialyzed to remove undesired low molecular weight molecules and / or lyophilized for easier formulation into the desired medium.

[0214] Another option for protein / peptide-based vaccines involves the introduction of nucleic acids encoding antigens as DNA vaccines. In this regard, recent reports describe the construction of recombinant vaccinia viruses expressing 10 minimal contiguous CTL epitopes (Thomson, 1996) or a combination of B cell, cytotoxic T lymphocyte (CTL) and T-helper (Th) epitopes from several microorganisms (An, 1997), and the successful immunization of mice with such constructs to prime a protective immune response. Thus, there is ample evidence in the literature of the success of utilizing peptides, peptide-pulsed antigen-presenting cells (APCs), and peptide-encoding constructs for efficient in vivo prime of a protective immune response. The use of nucleic acid sequences as vaccines is exemplified in U.S. Pat. Nos. 5,958,895 and 5,620,896.

[0215] The preparation of vaccines containing polypeptide or peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patent Nos. 4,608,251, 4,601,903, 4,599,231, 4,599,230, 4,596,792, and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared as injectables, either as liquid solutions or liquid suspensions: solid dosage forms suitable for liquid solution or suspension prior to injection can also be prepared. The preparations may be emulsified. The active immunogenic ingredient is often mixed with an excipient that is pharma- ceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Further, if desired, the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine. In certain embodiments, the vaccine is formulated using a combination of substances as described in U.S. Patent Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference.

[0216] Vaccines can usually be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include suppositories and, optionally, oral formulations. For suppositories, traditional binders and carriers can include, for example, polyalkalene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%. Oral formulations contain commonly employed excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain about 10% to about 95%, preferably about 25% to about 70%, of the active ingredient.

[0217] The polypeptides and DNA constructs encoding the polypeptides can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like.

[0218] Typically, vaccines are administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective and immunogenic. The amount administered depends on the subject to be treated, including the antibody synthesis capacity of the individual's immune system and the degree of protection desired. The exact amount of active ingredient required to be administered depends on the judgment of the physician. However, suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination. Suitable regimes for primary and booster vaccinations are similarly diverse, but typically include a primary vaccination followed by subsequent vaccinations or other administrations.

[0219] The mode of application may vary widely. Any of the conventional methods for administration of vaccines are applicable. These would include oral application in a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection, etc. The dosage of the vaccine will depend on the route of administration and will vary with the size and health of the subject.

[0220] In some cases, it may be desirable to have multiple administrations of the vaccine, for example, 2, 3, 4, 5, 6 or more administrations. Vaccinations may be at intervals of 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11, 12, 12 weeks, including all ranges between. Periodic booster immunizations at intervals of 1 to 5 years may be desirable to maintain protective levels of antibodies. The immunization course may be followed by assays for antibodies to the antigen, as described in U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064.

[0221] 1. Carrier A given composition may vary in its immunogenicity. Therefore, it is often necessary to boost the host immune system, which can also be achieved by coupling the peptide or polypeptide to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

[0222] 2. Adjuvants The immunogenicity of a polypeptide or peptide composition can be enhanced by using non-specific stimulators of immune responses, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions. Several adjuvants can be used to enhance the antibody response to variant SpA polypeptides or coagulase, or any other bacterial protein or combination contemplated herein. Adjuvants can (1) trap antigens in the body and cause sustained release; (2) attract cells involved in immune responses to the site of administration; (3) induce proliferation or activation of immune system cells; or (4) improve the spread of antigens throughout the subject's body.

[0223] Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, inorganic salts, polynucleotides, and natural substances. Specific adjuvants that can be used include IL-1, IL-2, IL-4, IL-7, IL-12, gamma-interferon, GMCSP, BCG, aluminum salts such as aluminum hydroxide or other aluminum compounds, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, namely MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion. MHC antigens can also be used. Other adjuvants or methods are exemplified in US Pat. Nos. 6,814,971, 5,084,269, and 6,656,462, each of which is incorporated herein by reference.

[0224] In some embodiments, the adjuvant comprises an immunostimulant, including but not limited to activators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLRs), inorganic salts, such as alum, alum in combination with monophosphoryl lipid (MPL) A of Enterobacteria, such as E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri, in particular MPL.RTM. (ASO4), individual MPL A of the aforementioned bacteria, saponins, such as QS-21, Quil-A, ISCOM, ISCOMATRIX, emulsions, such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21 + squalene + MPL.), liposomes and liposomal formulations such as AS01, synthetic or specially tailored microparticles and microcarriers such as bacterial derived outer membrane vesicles (OMVs) from N. gonorrhea and Chlamydia trachomatis, or chitosan particles, depot forming agents such as pluronic block copolymers, specially modified or tailored peptides such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphate such as RC529, or proteins such as bacterial toxins or toxin fragments immunostimulants.

[0225] Various methods of achieving vaccine adjuvant action include using agents such as aluminum hydroxide or aluminum phosphate (alum), typically used as about 0.05 to about 0.1% solutions in phosphate buffered saline, mixing with synthetic sugar polymers (Carbopol®) used as about 0.25% solutions, and aggregating proteins in the vaccine by heat treatment at temperatures ranging from about 70° C. to about 101° C. for 30 seconds to 2 minutes. Adjuvant action can also be achieved using agglutination by reactivation with pepsin-treated antibodies (Fab) against albumin, mixtures with bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsions in physiologically acceptable oily vehicles (e.g., mannide mono-oleate (Aracel A)); or emulsions with 20% perfluorocarbon solutions used as block substitutes (Fluosol-DA®).

[0226] Exemplary and often preferred adjuvants include Freund's complete adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis bacteria), Freund's incomplete adjuvant, and aluminum hydroxide.

[0227] In some aspects, it is preferable to select an adjuvant to be a preferential inducer of either a Th1 or Th2 type response: high levels of Th1 type cytokines tend to favor the induction of a cellular immune response to a given antigen, whereas high levels of Th2 type cytokines tend to favor the induction of a humoral immune response to the antigen.

[0228] The distinction between Th1 and Th2 type immune responses is not absolute. In practice, individuals support immune responses that are described as predominantly Th1 or predominantly Th2. However, it is often convenient to think of the family of cytokines in terms of those described in murine CD4+ T cell clones by Mosmann and Coffman (Mosmann, and Coffman, 1989). Traditionally, Th1 type responses are associated with the production of INF-γ and IL-2 cytokines by T lymphocytes. Other cytokines that are often directly associated with the induction of Th1 type immune responses, such as IL-12, are not produced by T cells. In contrast, Th2 type responses are associated with the secretion of IL-4, IL-5, IL-6, and IL-10.

[0229] In addition to adjuvants, it may be desirable to co-administer a biological response modifier (BRM) to enhance the immune response. BRMs have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); or low-dose cyclophosphamide (CYP; 300 mg / m 2 ) (Johnson / Mead, NJ) and genes encoding cytokines such as gamma interferon, IL-2 or IL-12, or proteins involved in immune helper function, such as B-7.

[0230] B. Lipid Components and Moieties In certain embodiments, the present invention relates to compositions comprising one or more lipids associated with nucleic acid or polypeptide / peptide. Lipids are substances that are insoluble in water and can be extracted with organic solvents. Compounds other than those specifically described herein are understood by those skilled in the art as lipids and are encompassed by the compositions and methods of the present invention. Lipid components and non-lipids may be attached to each other covalently or non-covalently.

[0231] Lipids can be natural lipids or synthetic lipids.However, lipids are usually biological substances.Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingoglycolipids, glycolipids, sulfatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.

[0232] The nucleic acid molecules or polypeptides / peptides bound to lipids may be dispersed in a solution containing lipids, dissolved by lipids, emulsified by lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained as a suspension in lipids, or otherwise bound to lipids. The lipid or lipid-poxvirus-bound compositions of the present invention are not limited to any particular structure. For example, they may simply be dispersed in the solution, forming aggregates that are possibly not uniform in either size or shape. In another example, they may exist in a bilayer structure, as micelles, or in a "collapsed" structure. In another non-limiting example, Lipofectamine (Gibco BRL)-poxvirus or Superfect (Qiagen)-poxvirus complexes are also contemplated.

[0233] In certain embodiments, the composition is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, ,About 30%,About 31%,About 32%,About 33%,About 34%,About 35%,About 36%,About 37%,About 38%,About 39%,About 40%,About 41%,About 42%,About 43%,About 44%,About 45%,About 46%,About 47%,About 48%,About 49%,About 50%,About 51%,About 52%,About 53%,About 54%,About 55%,About 56%,About 57%,About 58%,About 59%,About 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% %, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or any range therebetween of specific lipids, lipid types, or non-lipid components such as adjuvants, antigens, peptides, polypeptides, sugars, nucleic acids, or other materials as disclosed herein or known to those of skill in the art. In a non-limiting example, the composition can include about 10% to about 20% neutral lipids, and about 33% to about 34% cerebrosides, and about 1% cholesterol. In another non-limiting example, the liposomes can include about 4% to about 12% terpenes, with about 1% of the micelles being specifically lycopene, and about 3% to about 11% of the liposomes including other terpenes, as well as about 10% to about 35% phosphatidylcholine, and about 1% non-lipid components. Thus, it is contemplated that the compositions of the present invention may include any of the lipids, lipid types, or other components in any combination or percentage range.

[0234] C. Combination Therapy The compositions and related methods of the invention, particularly the administration of secreted virulence factors or surface proteins, including variant SpA polypeptides or peptides, and / or other bacterial peptides or proteins to a patient / subject, can also be used in conjunction with the administration of conventional therapies, including but not limited to the administration of antibiotics such as streptomycin, ciprofloxacin, doxycycline, gentamicin, chloramphenicol, trimethoprim, sulfamethoxazole, ampicillin, tetracycline, or various combinations of antibiotics.

[0235] In one aspect, it is contemplated that the polypeptide vaccine and / or treatment is used together with an antibacterial treatment. Alternatively, the treatment can precede or follow the treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and / or protein or polynucleotide is administered separately, it is generally ensured that no significant time passes between each delivery, so that the agent and antigen composition can still exert their beneficially combined effect on the subject. In such cases, it is contemplated that both modalities can be administered within about 12-24 hours of each other, or within about 6-12 hours of each other. In some cases, it may be desirable to extend the period of administration considerably, in which case the interval between each administration can be from a few days (2, 3, 4, 5, 6, or 7 days) to a few weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0236] For example, various combinations may be utilized, with antibiotic treatment being "A" and an immunogenic molecule, such as an antigen, being administered as part of an immunotherapeutic regime being "B." TIFF0007643730000003.tif22128

[0237] The administration of the immunogenic composition of the present invention to a patient / subject follows the general protocol for administration of such compounds, taking into account the toxicity, if any, of SpA compositions or other compositions described herein. It is expected that treatment cycles will be repeated as necessary. It is also contemplated that various standard treatments, such as hydration, can be applied in conjunction with the described treatment.

[0238] D. General Pharmaceutical Compositions In some embodiments, the pharmaceutical composition is administered to the subject. Various aspects of the invention involve administering an effective amount of the composition to the subject. In some embodiments of the invention, a staphylococcal antigen, a member of the Ess pathway, such as a polypeptide or peptide of the Esa or Esx class, and / or a member of a sortase substrate can be administered to the patient to protect against one or more staphylococcal pathogens. Alternatively, an expression vector encoding one or more such polypeptides or peptides can be given to the patient as a preventative treatment. Furthermore, such compounds can be administered in conjunction with an antibiotic or antibacterial agent. Such compositions are generally dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0239] In addition to compounds formulated for parenteral administration, such as for intravenous or intramuscular injection, other pharma- ceutically acceptable forms include, for example, tablets or other solids for oral administration; sustained release capsules; and any other form currently in use, including creams, lotions, mouthwashes, inhalants, and the like.

[0240] The active compound of the present invention can be formulated for parenteral administration, for example, for injection by intravenous, intramuscular, subcutaneous or even intraperitoneal route.The preparation of aqueous compositions containing compounds that increase the expression of MHC class I molecules will be known to those skilled in the art in light of this disclosure.Typically, such compositions can be prepared as injections, either as liquid solutions or liquid suspensions: solid dosage forms suitable for use in preparing solutions or suspensions by adding liquid prior to injection can also be prepared; and the preparation can be emulsified.

[0241] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils.Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0242] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that it can be easily syringable. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0243] Proteinaceous compositions may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and those formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0244] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. 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 dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use in the composition of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0245] Sterile injectable solutions are prepared by incorporating the required amount of active compound into a suitable solvent, optionally with various other ingredients as listed above, followed by filtration sterilization. In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium that contains the basic dispersion medium and other ingredients as required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients from its solution that has been previously sterilized by filtration.

[0246] Administration of the compositions according to the invention will typically be by any common route, including, but not limited to, oral, nasal or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal or intravenous injection. In certain embodiments, the vaccine composition can be inhaled (e.g., U.S. Patent No. 6,651,655, specifically incorporated by reference). Such compositions are usually administered as pharmaceutically acceptable compositions including pharmacologically acceptable carriers, buffers or other excipients. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problem or complication, according to a reasonable benefit-risk ratio. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.

[0247] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, available sterile aqueous media will be known to those skilled in the art in light of this disclosure. For example, a dose may be dissolved in isotonic NaCl solution and added to a subcutaneous infusion or injected at the proposed injection site (see, for example, Remington's Pharmaceutical Sciences, 1990). Some dosage variation will necessarily occur depending on the subject's condition. In any event, the person responsible for administration will determine the appropriate dose for the individual subject.

[0248] The effective amount of a therapeutic or prophylactic composition is determined based on the intended purpose. The term "unit dose" or "dosage" refers to a physically distinct unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired response described above in relation to its administration, i.e., the appropriate route and administration schedule. The amount administered, both by number of treatments and unit dose, depends on the protection desired.

[0249] The exact amount of the composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the subject, the route of administration, the intended purpose of the treatment (palliative versus curative), and the efficacy, stability, and toxicity of the particular composition.

[0250] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0251] E. In Vitro, Ex Vivo, or In Vivo Administration As used herein, the term in vitro administration refers to manipulations performed on cells removed from a subject or outside the subject's body, including but not limited to cultured cells. The term ex vivo administration refers to cells that have been manipulated in vitro and then administered to a subject. The term in vivo administration includes any manipulations performed within the subject's body.

[0252] In certain aspects of the invention, the compositions can be administered in vitro, ex vivo, or in vivo. In certain in vitro embodiments, autologous B-lymphocyte cell lines are incubated with the viral vectors of the invention for 24-48 hours, or with variant SpA and / or coagulase and / or any other composition described herein for 2 hours. The transduced cells can then be used for in vitro analysis or for ex vivo administration. U.S. Patent Nos. 4,690,915 and 5,199,942, both of which are incorporated herein by reference, disclose methods for ex vivo manipulation of blood mononuclear cells and bone marrow cells for therapeutic use.

[0253] F. Antibodies and Passive Immunization Another aspect of the invention is a method for preparing an immunoglobulin for use in the prevention or treatment of staphylococcal infections, comprising immunizing a recipient or donor with a vaccine of the invention and isolating the immunoglobulin from the recipient or donor. The immunoglobulin prepared by this method is a further aspect of the invention. A pharmaceutical composition comprising an immunoglobulin of the invention and a pharma- ceutically acceptable carrier is a further aspect of the invention that can be used in the manufacture of a medicament for the treatment or prevention of staphylococcal disease. A method for the treatment or prevention of staphylococcal infections comprising administering to a patient an effective amount of a pharmaceutical preparation of the invention is a further aspect of the invention.

[0254] Inocula for the production of polyclonal antibodies are typically prepared by dispersing the antigenic composition in a physiologically acceptable diluent, such as saline or other adjuvant suitable for human use, to form an aqueous composition. An immunostimulatory amount of the inoculum is administered to a mammal, and the inoculated mammal is then maintained for a period of time sufficient for the antigenic composition to induce protective antibodies.

[0255] The antibodies can be isolated to the extent desired by well-known techniques such as affinity chromatography (Harlow and Lane, 1988). Antibodies can include antiserum preparations from a variety of commonly used animals, e.g., goats, primates, donkeys, pigs, horses, guinea pigs, rats, or humans.

[0256] The immunoglobulins produced by the present invention can include whole antibodies, antibody fragments, or antibody subfragments. The antibodies can be whole immunoglobulins of any class (e.g., IgG, IgM, IgA, IgD, or IgE), chimeric antibodies, or hybrid antibodies with dual specificity for two or more antigens of the present invention. They can also be fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.), including hybrid fragments. Immunoglobulins also include natural, synthetic, or genetically engineered proteins that act like antibodies by binding to specific antigens to form complexes.

[0257] The vaccine of the present invention can be administered to a recipient, who will act as a source of immunoglobulin produced in response to exposure with a particular vaccine. A subject treated in this manner will provide plasma from which hyperimmune globulin can be obtained by conventional plasma fractionation methods. The hyperimmune globulin will be administered to another subject to confer resistance to or treat staphylococcal infection. The hyperimmune globulin of the present invention is particularly useful for the treatment or prevention of staphylococcal disease in young children, immunocompromised individuals, or when treatment is required and the individual does not have time to produce antibodies in response to vaccination.

[0258] A further aspect of the present invention is a pharmaceutical composition comprising two or more monoclonal antibodies (or fragments thereof; preferably human or humanized monoclonal antibodies) reactive against at least two components of the immunogenic composition of the present invention, which can be used to treat or prevent infections with Gram-positive bacteria, preferably Staphylococcus aureus or Staphylococcus epidermidis. Such pharmaceutical compositions contain monoclonal antibodies, which can be whole immunoglobulins, chimeric antibodies or hybrid antibodies of any class having specificity for two or more antigens of the present invention. They can also be fragments, including hybrid fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.).

[0259] Methods for producing monoclonal antibodies are well known in the art and can include fusion of splenocytes with myeloma cells (Kohler and Milstein, 1975; Harlow and Lane, 1988). Alternatively, monoclonal Fv fragments can be obtained by screening appropriate phage display libraries (Vaughan et al., 1998). Monoclonal antibodies can be humanized or partially humanized by known methods. EXAMPLES

[0260] VI. Working Examples The following examples are given for the purpose of illustrating various aspects of the present invention and are not intended to limit the present invention in any manner. Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the objects and advantages mentioned, in addition to those objects, purposes and advantages set forth herein. The examples, together with the methods described herein, are representative of presently preferred embodiments, are illustrative, and are not intended as limitations on the scope of the present invention. Modifications thereof and other uses encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

[0261] Example 1 Staphylococcal Protein A Contributes to Persistent Colonization of Mice by Staphylococcus aureus Staphylococcus aureus persistently colonizes the nasopharynx of approximately one-third of the human population, thereby promoting community-acquired and nosocomial infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, antibiotic effectiveness is limited by recolonization and selection with drug-resistant strains. Nasal colonization elicits IgG responses to staphylococcal surface antigens, but these antibodies fail to prevent subsequent colonization or disease. This example describes S. aureus WU1, a multilocus sequence type ST88 isolate, which persistently colonizes the nasopharynx of mice. In this example, it is reported that Staphylococcus protein A (SpA) is required for the persistence of S. aureus WU1 in the nasopharynx. Compared to animals colonized with wild-type S. aureus, mice colonized with Δspa variants have increased IgG responses to staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant that is unable to crosslink B cell receptors and bypass the antibody response elicits protein A-neutralizing antibodies that promote IgG responses to S. aureus colonization and reduce pathogen persistence.

[0262] A. Results 1. Staphylococcus aureus WU1 An outbreak of preputial gland infections in male C57BL / 6 mice was observed in an animal breeding colony. Samples were collected from the preputial gland adenitis (PGA) and nasopharynx of male and female C75BL / 6J mice and analyzed by growth on mannitol salt agar (MSA) and Baird-Parker agar (BPA). Multilocus sequence typing and spa genotyping revealed that animals were colonized with S. aureus ST88 spa genotype t186, which also carries PGA in male mice. S. aureus CC88, which carries spa genotype t186, was previously reported as a stably colonizing isolate from laboratory mice in the United States (37). Other spa genotypes include t325, t448, t690, t755, t786, t2085, t2815, t5562, t11285, and t12341 (37). The New Zealand JSNZ isolate harbors a distinct spa genotype, t729 (37). Nevertheless, both S. aureus JSNZ and WU1 share the type 8 capsular polysaccharide gene and lack the mecA gene and the T cell superantigen encoded by the mobile genetic element (MGE) (37). Furthermore, the hlb-converting phage expressing the human-specific immune evasion cluster 1 (IEC1) genes sak (staphylokinase), chp (CHIPS, S. aureus chemotaxis inhibitory protein), and scn (SCIN-A, staphylococcal complement inhibitor A) are absent in the genome of WU1 resulting in an intact α-hemolysin-encoding gene (hlb) (38). Of note, WU1-encoded IEC2 harbors the scn homologs scb / scc (SCIN-B / -C) along with hla (α-hemolysin) and ssl12-14 (staphylococcal superantigen-like 12-14) (39). Unlike other CC88 isolates that stably colonize mice (37), the genome of WU1 harbors the blaZ gene. When analyzed for genes encoding sortase-anchored surface proteins, we observed that S. aureus WU1 harbors genes for determinants previously associated with nasal colonization, including ClfB, IsdA, SdrC, SdrD, and SasG (Table 1).

[0263] Table 1. Conservation of protein products of selected open reading frames in the genomes of S. aureus WU1, JSNZ and Newman. TIFF0007643730000004.tif142136a. Comparison with Staphylococcus aureus strain 04-02981 b. Comparison with Staphylococcus aureus USA300 strain

[0264] S. aureus abscess formation is associated with determinants of bacterial agglutination by fibrin (40, 41). Agglutination requires two S. aureus secretory products: coagulase (Coa) and von Willebrand factor binding protein (vWbp), which activate host prothrombin and convert fibrinogen to fibrin (40). Clumping factor A (ClfA) binds fibrinogen and coats staphylococci with coagulase-generated fibrin fibrils, thereby preventing S. aureus uptake and killing by host phagocytes (41, 42). The clfA gene is identical in S. aureus WU1 and JSNZ but shows allele-specific differences from clfA from S. aureus Newman, the CC8 human clinical isolate routinely used in laboratory challenge experiments in mice (Table 1) (43). On the other hand, the differences observed in clfA are clade specific, since they could be seen in CC88 strains isolated from either human or murine hosts (data not shown). The coa gene products of S. aureus WU1, JSNZ, and Newman were virtually identical (Table 1). In contrast, the products of the vwb genes of S. aureus WU1 and JSNZ were significantly divergent from S. aureus Newman with the greatest sequence diversity in the prothrombin-binding D1 and D2 domains (Fig. 1A) and were not recognized by polyclonal antibodies raised against Newman vWbp (Fig. 1B). Secreted vWbp from the two CC88 strains could be recognized by serum raised against the conserved C-terminal domain of vWbp from strain USA300 (Fig. 1C). In contrast to S. aureus Newman, which secretes large amounts of Coa and rapidly clumps human and mouse plasma, S. aureus WU1 and JSNZ secrete less Coa and clump mouse plasma more rapidly than human plasma compared to strain Newman (Fig. 1B, D, E). The coagulase activity of S. aureus Newman is dependent on coa and vwb expression, since the corresponding Δcoa, Δvwb, and ΔcoaΔvwb mutants showed a lack of clumping in mouse and human plasma (Fig. 1D, E).Taken together, these data suggest that the ST88 allele of the vwb gene in S. aureus WU1 and JSNZ may promote efficient prothrombin-mediated coagulation and fibrin aggregation in mouse plasma, which may support the pathogenesis of invasive diseases such as PGA.

[0265] 2. Staphylococcus aureus WU1 persistently colonizes the nasopharynx of mice To analyze S. aureus WU1 for its ability to colonize mice, a cohort of female C57BL / 6 animals (n = 10) was analyzed by spreading throat swabs and fecal material on BPA. Naive mice, lacking bacterial growth on BPA, were anesthetized and 1 × 10 6 cells in phosphate-buffered saline (PBS) were injected into the right nostril. 8 Animals were inoculated by pipetting a 10 μl suspension of CFU S. aureus WU1. Animals were analyzed for colonization by taking swabs from the oropharynx at weekly intervals, i.e., 7, 14, 21, 28, 35, and 42 days after inoculation. Swabs were spread on BPA, incubated for colonization, and counted (Figure 2A). Even without prior antibiotic treatment or antibiotic selection, S. aureus WU1 increased by 1.2–2.9 log per swab over 42 days. 10Experimental animals were colonized with loads ranging from 0.1 to 1.2 CFU (Figure 2A). To assess persistent colonization by S. aureus WU1, colonies obtained after 42 days were analyzed by MLST and spa genotyping. Data showed that mice were still colonized by ST88 spa t186, suggesting that S. aureus WU1 persistently colonizes the nasopharynx of C57BL / 6 mice. As a control, mock PBS inoculation of a cohort of C57BL / 6J animals in separate cages maintained in the same animal facility room and on the same cage rack as S. aureus WU1-colonized animals did not result in staphylococcal colonization of the nasopharynx (Figure 2A). Day 42 fecal samples from mice were homogenized in PBS and plated on mannitol salt agar (MSA) for CFU enumeration (Figure 2B). Fecal samples from S. aureus WU1-colonized mice harbored 5.1-7.3 log1O CFU g-1 feces, suggesting that the gastrointestinal (GI) tract was also colonized with S. aureus WU1 strains. As a control, mock (PBS)-inoculated mice did not harbor S. aureus in their fecal samples (Figure 2B).

[0266] S. aureus WU1 colonization elicits serum IgG responses in mice. A previous study generated an S. aureus antigen matrix composed of 25 conserved secreted proteins. Each of the 25 recombinant affinity-tagged proteins was purified and immobilized on a membrane filter (44). To measure the host immune response upon colonization, blood was collected from naive or S. aureus WU1-colonized animals 15 days after inoculation and serum IgG responses were analyzed by incubation with S. aureus antigen matrix. IgG binding was detected with goat anti-mouse IgG conjugated to IRDye 680 (LI-COR) and quantified by infrared imaging. This experiment revealed that S. aureus WU1 colonization resulted in an increase in serum IgG directed against the sortase-anchored surface proteins ClfA, ClfB, IsdA, and IsdB, as well as against the large extracellular matrix-binding protein (Ebh), a cell size and peptidoglycan synthesis determinant of S. aureus ( 45 ) (Table 2 ).

[0267] Table 2. Serum IgG responses in C57BL / 6J mice colonized with S. aureus WU1 or its Δspa variant. TIFF0007643730000005.tif142169a. A cohort of C57BL / 6J mice was 8 CFU-labeled S. aureus strains were inoculated intranasally. Fifteen days after inoculation, animals were bled and serum samples were analyzed for antibody responses to staphylococcal antigens. b. Fold changes were calculated by dividing the mean signal intensity of the inoculated mice group by the mean signal intensity of the naive mice group. Data are presented as mean ± standard deviation. c. p-values ​​were calculated using two-way ANOVA with Tukey's multiple comparison test. ns = not significant.

[0268] 3. S. aureus WU1 requires staphylococcal protein A for persistent colonization. Similar to S. aureus Newman SpA, the spa gene product of S. aureus WU1 is composed of five IgBDs and carries only one amino acid substitution within the 278-residue domain. Immunoblotting experiments revealed that S. aureus strains Newman and WU1 produced comparable amounts of SpA (Figure 3A). Using allelic recombination, we generated a Δspa mutant of S. aureus WU1. As measured by immunoblotting, SpA production was lost in the Δspa mutant, and this loss was restored by plasmid-borne expression of wild-type spa (pSpA) (Figure 3A). Immunoblotting with an antibody against sortase A (SrtA) was used as a loading control (Figure 3A). When introduced into the right nostril of mice and analyzed for colonization by oropharyngeal swabs on day 7, the Δspa mutant initially colonized C57BL / 6J animals similarly to the wild-type strain WU1 (Figure 3B). However, at later time points, especially days 35 and 42, the Δspa mutant colonized fewer animals than the wild-type strain WU1 (Figure 3B). During bacterial growth, S. aureus releases SpA linked to peptidoglycan fragments into the surrounding environment (46). In a mouse model of intravenous S. aureus challenge, the released SpA activated B cell proliferation and enhanced secretion of VH3 idiotypic IgM and IgG molecules (33). However, expanded VH3 idiotypic IgG does not recognize staphylococcal antigens (33). The molecular mechanism of this B cell superantigen activity is based on SpA-mediated crosslinking of VH3 idiotypic B cell receptors, which induces B cell proliferation in a CD4 T helper cell- and RIPK2 kinase-dependent manner (33, 47). Animals infected with Δspa mutant staphylococci lack the expansion of VH3 idiotypic immunoglobulins and show an increased abundance of pathogen-specific IgG, thereby eliciting an immune response that is protective against subsequent S. aureus infection (48). We next wondered whether colonization with the Δspa mutant of WU1 was associated with an altered serum IgG response. Sera from animals that had been colonized for 15 days were analyzed for IgG binding to components of the S. aureus antigenic matrix (Table 2).This experiment revealed that antibodies against ClfB, IsdA, and SasG were subsequently increased in decolonized animals, but not in animals that remained colonized with the Δspa mutant (Table 2). Together, these data suggest that colonization of the nasopharynx of C57BL / 6 mice with Δspa mutant staphylococci is associated with increased IgG responses to key colonization determinants that appear to promote clearance of Δspa mutant S. aureus from the nasopharynx.

[0269] 4. Protein A neutralizing antibodies affect persistent colonization by Staphylococcus aureus Immunization of mice with wild-type protein A does not elicit IgG serum antibodies that bind to the five IgBDs and neutralize the ability of the five IgBDs to bind either the Fcγ domain of IgG molecules or the variant heavy chain of the VH3 idiotypic immunoglobulin (44). KKAA is a variant of SpA with 20 amino acid substitutions across the five IgBDs that abolishes Fcγ binding and also reduces association with VH3 idiotypic immunoglobulins ( 44 ). KKAA retains the overall α-helical content and antigenic structure of Protein A. As a result, adjuvanted SpA KKAA Immunization of mice with IgG induces high titers of protein A-neutralizing IgG (44). These antibodies block the anti-opsonic activity of protein A and B cell superantigens during S. aureus infection, broadly enhancing IgG responses to staphylococcal antigens and promoting the development of protective immunity (44). To test whether protein A-neutralizing antibodies affect S. aureus colonization, C57BL / 6 mice were immunized with adjuvanted SpA KKAA or adjuvant alone. KKAA Treated animals induced high titers of protein A neutralizing antibodies (Table 3). Upon inoculation with S. aureus WU1, oropharyngeal swabs revealed mean colonization loads that were not significantly different at 7 and 14 days postinoculation, indicating that mock and SpA KKAABoth immunized animals were initially colonized similarly (Fig. 4). However, starting at day 21, SpA KKAA Immunized mice decolonized at a higher frequency than mock-immunized animals (Figure 4). S. aureus WU1 colonization in mock-treated animals resulted in antibody responses to ClfB, IsdA, IsdB, SasD, and SasF when tested for serum IgG responses and compared with naive mice (Table 3). In animals that maintained S. aureus WU1 colonization, SpA KKAA Immunization resulted in antibody responses against ClfA, Coa, vWBP, and Hla (Table 3). KKAA Compared with vaccinated C57BL / 6J mice, subsequently decolonized animals showed elevated serum IgG against ClfA, ClfB, fibronectin-binding proteins A (FnBPA) and B (FnBPB), IsdB, Coa, and SasG (Table 3). Collectively, these data support the notion that SpA KKAA These results suggest that vaccination induces enhanced serum IgG responses in mice colonized with S. aureus. KKAA The vaccine induced antibodies against a number of different staphylococcal antigens, including known colonization factors (ClfB, IsdA, and SasG). Taken together, these SpA antibodies against colonizing staphylococci KKAA The vaccine-induced IgG response appears to promote decolonization of the nasopharynx.

[0270] Table 3. SpA versus serum IgG responses in S. aureus WU1-colonized C57BL / 6 mice KKAA Immune effects TIFF0007643730000006.tif174164a. Cohorts of C57BL / 6J mice were incubated with 50 μg of recombinant SpA emulsified in CFA. KKAA or PBS mock immunized in CFA and 50 μg of recombinant SpA emulsified in IFA on day 11. KKAA On day 24, mice were injected with 10 8CFU-labeled S. aureus strains were inoculated intranasally, and weekly pharyngeal swabs were taken to enumerate the bacterial load. Fifteen days after inoculation, animals were bled and serum samples were analyzed for antibody responses to staphylococcal antigens. b. SpA KKAA Fold changes were calculated by dividing the mean signal intensity of the immunized group by the mean signal intensity of the PBS mock-immunized group. Data are presented as mean ± standard deviation.

[0271] S. aureus WU1 colonization of BALB / c mice. To test whether S. aureus WU1 colonization is restricted to C57BL / 6 mice, we inoculated a cohort of naive BALB / c mice (n=20) with 1 × 10 8 CFU S. aureus WU1 was inoculated into the right nasal cavity, and nasopharyngeal colonization was measured by swab culture. Similar to C57BL / 6 mice, S. aureus WU1 persistently colonized BALB / c mice (Fig. 5). KKAA Immunization of BALB / c mice with IgG did not affect early colonization by S. aureus WU1. However, compared with mock-immunized animals, SpA KKAA Vaccination with Vaccination with Vaccinia gracilis promoted decolonization of BALB / c mice (Fig. 5 ).

[0272] 5. SpA KKAA Vaccine affects mouse colonization by Staphylococcus aureus JSNZ We next wondered whether protein A neutralizing antibodies also affect mouse colonization by S. aureus JSNZ. Unlike strains Newman and WU1, the spa gene product of S. aureus JSNZ contains only four IgBDs (37). Previous studies have demonstrated that SpA variants with four IgBDs are associated with reduced B cell superantigen activity compared to the five IgBDs commonly associated with S. aureus colonization of the human nasopharynx (33). When inoculated into the right nostril of anesthetized mice, S. aureus JSNZ effectively colonized the nasopharynx of BALB / c mice for 42 days (Figure 6). SpAKKAA Vaccination did not affect initial colonization by S. aureus JSNZ. However, compared to mock-immunized mice, BALB / c mice with serum neutralizing protein A antibodies decolonized S. aureus JSNZ at a higher frequency starting at day 21 (Figure 6). Taken together, these data suggest that S. aureus JSNZ also requires protein A-mediated B cell superantigen activity for persistent colonization of mice.

[0273] B. Materials and Methods 1. Media and bacterial growth conditions S. aureus strains were grown in tryptic soy broth (TSB) or tryptic soy agar (TSA) at 37°C. For experiments investigating mouse nasopharyngeal colonization, throat swab samples were grown on Baird-Parker agar at 37°C as indicated. For experiments investigating S. aureus GI tract colonization, fecal samples were grown on mannitol salt agar at 37°C as indicated. E. coli strains DH5α and BL21(DE3) were grown in Luria broth (LB) or agar at 37°C. Ampicillin (100 μg / ml for E. coli) and chloramphenicol (10 μg / ml for S. aureus) were used for plasmid selection.

[0274] 2. Staphylococcus aureus genotyping S. aureus isolate WU1 was obtained from abscess lesions of the nasopharynx and preputial gland of mice in our animal facility. Mouse S. aureus strain JSNZ was provided by Dr. Siouxsie Wiles (36). Staphylococcal genomic DNA was isolated using the Wizard Genomic DNA Purification kit (Promega). Spa genotyping and multilocus sequence typing (MLST) were performed as previously described (85). Briefly, for spa typing, genomic DNA of S. aureus strain WU1 was purified using the primers TIFF0007643730000007.tif5169 (86). PCR products were purified using Nucleospin Gel and PCR Clean-up kits, sequenced using primers 1095F and 1517R, and analyzed using Ridom software. For MLST typing, genomic DNA of S. aureus strain WU1 was amplified using the primers TIFF0007643730000008.tif90160 (see for example: saureus.mlst.net / misc / info.asp). PCR products were purified using Nucleospin Gel and PCR Clean-up kits, PCR amplified, sequenced and analyzed using online software (see for example: saures.mlst.net / ). The whole genome sequence file of S. aureus strain JSNZ was provided by Dr. Silva Holtfreter. Truseq DNA-seq library preparation Illumina MiSeq sequencing was performed with genomic DNA of S. aureus WU1 by the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory. Sequences were analyzed using Geneious software.

[0275] 3. S. aureus Mutants The spa gene of S. aureus WU1 was deleted using allelic recombination with the plasmid pKOR1 (87). To construct the Δspa mutant, two 1-kb DNA fragments, one upstream and one downstream of the spa gene, were cloned using primers The two flanking regions were amplified from the chromosome of S. aureus WU1 using TIFF0007643730000009.tif56160. The two flanking regions were fused together in a subsequent PCR, and the final PCR product was cloned into pKOR1 using the BP Clonase II kit (Invitrogen). The resulting plasmid was transferred successively into E. coli DH5α, S. aureus strain RN4220, and finally S. aureus strain WU1, and the temperature was shifted to 40°C to block plasmid replication and promote their insertion into the chromosome (87). Growth at 30°C was used to promote allelic replacement. Mutations in the spa genes were verified by DNA sequencing of the PCR amplified products.

[0276] 4. Aggregation assay Agglutination assays were performed as previously described (88). Briefly, overnight cultures of S. aureus strains were diluted 1:100 in fresh TSB and grown for 6 h at 37°C. 1 ml cultures (OD 600 Bacteria from 1000 cells / ml (normalized to 4.0) were incubated with SYTO 9 (1:500) (Invitrogen) for 15 min, washed twice with 1 ml PBS, and suspended in 1 ml PBS. Bacteria were mixed 1:1 with citrated human or mouse plasma on a glass microscope slide and incubated for 30 min. Samples were observed and images were captured with an IX81 live-cell total internal reflection fluorescence microscope using a 20× objective (Olympus). At least 10 images were acquired for each sample. The area of ​​the aggregation complexes in each image was measured and quantified using ImageJ software.

[0277] 5. Immunoblotting Overnight cultures of S. aureus strains were diluted 1:100 in fresh TSB (with chloramphenicol in the presence of plasmids) and incubated at OD at 37 °C. 600Cells from 1 ml cultures were centrifuged, suspended in PBS, and incubated with 20 μg / ml lysostaphin (AMBI) for 1 h at 37°C. Proteins in whole-cell lysates were precipitated with 10% trichloroacetic acid and 10 μg deoxycholate, washed with ice-cold acetone, air-dried, suspended in 100 μl of 0.5 M Tris HCl (pH 6.8) and 100 μl of SDS-PAGE sample buffer [100 mM Tris HCl (pH 6.8), 4% SDS, 0.2% bromophenol blue, 200 mM dithiothreitol], and boiled for 10 min. Proteins were separated by 12% SDS-PAGE and electrophoretically transferred to a PVDF membrane. PVDF membranes were blocked with 5% milk in Tris-buffered saline with Tween-20 (TBST) [20 mM Tris HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20]. ClfA was detected using mouse anti-ClfA 2A12.12 monoclonal antibody (1:2,000 dilution) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cell Signaling, 1:10,000 dilution). Coa was detected using rabbit anti-Coa polyclonal antibody (1:1,000 dilution) and HRP-conjugated anti-rabbit IgG (1:10,000 dilution). vWbp was detected using two different rabbit anti-vWbp polyclonal antibodies (1:1,000 dilution), which recognize either full-length vWbp or the C-terminal domain of vWbp from S. aureus Newman, respectively, and HRP-conjugated anti-rabbit IgG (1:10,000 dilution). SpA was detected using HRP-conjugated human IgM (1:10,000 dilution) in TBST. SrtA was detected using rabbit anti-SrtA polyclonal antibody (1:10,000 dilution) and HRP-conjugated anti-rabbit IgG (1:10,000 dilution).Antibody-stained membranes were washed with TBST, incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific), and developed on Amersham Hyperfilm ECL high-performance chemiluminescent film (GE Healthcare).

[0278] 6. Purification of recombinant proteins His-tagged SpA KKAA E. coli BL21(DE3) harboring pET15b+ plasmids for expression of 24 staphylococcal antigens (ClfA, ClfB, FnBPA, FnBPB, IsdA, IsdB, SasA, SasB, SasD, SasF, SasG, SasI, SasK, SdrC, SdrD, SdrE, EsxA, EsxB, SCIN, Eap, Efb, Hla, Coa, vWbp, and Ebh) were grown overnight, diluted 1:100 in fresh medium, and incubated at 37°C to an OD of approximately 0.5. 600 Cultures were induced with 1 mM isopropyl-β-d-thiogalactopyranoside and grown for an additional 3 h. Cells were pelleted, resuspended in column buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl), and cultured at 14,000 lb / in 2 The lysates were disrupted using a French pressure cell at 400 x g. Membranes and insoluble components were removed from the lysates by ultracentrifugation at 40,000 x g. The cleared lysates were subjected to Ni-NTA affinity chromatography and proteins were eluted with column buffers containing successively higher concentrations of imidazole (100-500 mM). The eluates were dialyzed against PBS and protein purity was confirmed by Coomassie-stained SDS-PAGE. Protein concentration was determined by bicinchoninic acid assay (Thermo Scientific).

[0279] 7. Mouse nasopharyngeal colonization Overnight cultures of S. aureus strain WU1 and its Δspa mutant were diluted 1:100 in fresh TSB and grown for 2 h at 37°C. Cells were centrifuged, washed, and suspended in PBS. Seven-week-old female BALB / c mice, C57BL / 6J mice, or B6.129S2-Ighm mice were cultured at 37°C for 2 h. tmlCgn 1 × 10 1 × 10 2 × 10 3 × 10 4 × 10 5 × 10 6 × 10 7 × 10 8 × 10 9 × 10 10 × 10 11 × 10 2 × 10 3 × 10 4 × 10 5 × 10 6 × 10 11 × 10 7 × 10 8 × 10 11 × 8 CFU of S. aureus (in a 10 μl volume) were pipetted into the right nostril of each mouse. On days 7, 14, 21, 28, 35, and 42 post-inoculation, swabs were taken from the oropharynx of the mice, and swab samples were spread on Baird-Parker agar and incubated for bacterial enumeration. On day 15 post-inoculation, mice were bled by periorbital vein puncture to obtain serum for antibody response analysis using a staphylococcal antigen matrix. On day 42 post-inoculation, fecal samples were collected and homogenized in PBS. Homogenates were plated on mannitol salt agar and incubated for bacterial enumeration. All mouse experiments were performed with review and approval of the experimental protocols in accordance with institutional guidelines by the Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC) at the University of Chicago. Animal experiments were repeated at least once to ensure reproducibility of the data.

[0280] 8. Active immunity Four-week-old mice were immunized with 50 μg of SpA emulsified in complete Freund's adjuvant (CFA; Difco). KKAA The mice were immunized by subcutaneous injection with 1 × 10 mAb and boosted with 50 μg of the same antigen emulsified in incomplete Freund's adjuvant (IFA) 11 days after the first immunization. On day 21, immunized mice were bled by periorbital vein puncture to obtain serum for ELISA. On day 24, mice were injected with 1 × 10 mAb. 8CFU of S. aureus strains WU1 or JSNZ were inoculated intranasally and nasopharyngeal colonization was monitored.

[0281] 9. Staphylococcal antigen matrix Nitrocellulose membranes were blotted with 2 μg of affinity purified Staphylococcus aureus antigens. The membranes were blocked with 5% degranulated milk and incubated with diluted mouse serum (1:10,000 dilution) and IRDye 680-conjugated goat anti-mouse IgG (LI-COR). Signal intensity was quantified using an Odyssey infrared imaging system (LI-COR).

[0282] 10. Statistical analysis Two-way ANOVA with Sidak multiple comparison test (GraphPad Software) was performed to analyze the statistical significance of nasopharyngeal colonization, ELISA, and antigen matrix data.

[0283] Example 2 Staphylococcal Protein A Variants The following assays can be used to evaluate the SpA variants described herein for their effectiveness in the methods and compositions of the disclosure.

[0284] A. Assay Vaccine protection in murine abscess, murine lethal infection, and murine pneumonia models. Three animal models have been established for the study of S. aureus infectious disease. These models can be used herein to test the level of protective immunity provided through the development of Protein A-specific antibodies.

[0285] Murine abscess-BALB / c mice (24-day-old females, 8-10 mice per group, Charles River Laboratories, Wilmington, MA) can be immunized with purified proteins by intramuscular injection into the hind leg (Chang et al., 2003; Schneewind el al., 1992). Purified SpA and / or SpA variants can be administered on days 0 (emulsified 1:1 with Freund's complete adjuvant) and 11 (emulsified 1:1 with Freund's incomplete adjuvant). Blood samples can be collected by retroorbital bleeding on days 0, 11, and 20. Serum can be tested by ELISA for IgG titers at specific binding activity of the variants. Immunized animals are immunized on day 21 with 100 μl of S. aureus Newman or S. aureus USA300 suspension (1 × 10 7 For this, an overnight culture of S. aureus Newman can be diluted 1:100 in fresh tryptic soy broth and grown for 3 hours at 37°C. The staphylococci are centrifuged, washed twice, and incubated at an A of 0.4. 600 can be diluted in PBS to obtain a concentration of 1 × 10 per ml 8 cfu). Dilution may be experimentally confirmed by plating and colony formation on agar media. Mice may be anesthetized by intraperitoneal injection of 80-120 mg ketamine and 3-6 mg xylazine per kilogram of body weight and infected by retro-orbital injection. On the 5th or 15th day after challenge, mice may be euthanized by compressed CO2 inhalation. Kidneys may be removed and homogenized in 1% Triton X-100. For triplicate determination of cfu, aliquots may be diluted and plated on agar media. For histological examination, kidney tissue may be incubated in 10% formalin at room temperature for 24 hours. Tissue may be embedded in paraffin, thin-sectioned, stained with hematoxylin-eosin, and examined by microscopy.

[0286] Murine lethal infection - BALB / c mice (24-day-old females, 8-10 mice per group, Charles River Laboratories, Wilmington, MA) can be immunized by intramuscular injection into the hind leg with purified SpA or SpA variants. Vaccine can be administered on days 0 (emulsified 1:1 with Freund's complete adjuvant) and 11 (emulsified 1:1 with Freund's incomplete adjuvant). Blood samples can be collected by retroorbital bleeding on days 0, 11, and 20. Serum is tested by ELISA for IgG titer at variant specific binding activity. Immunized animals are immunized on day 21 with 100 μl of S. aureus Newman or S. aureus USA300 suspension (15 × 10 7 For this, an overnight culture of S. aureus Newman can be diluted 1:100 in fresh tryptic soy broth and grown for 3 hours at 37°C. The staphylococci are centrifuged, washed twice, and incubated at an A of 0.4. 600 The solution was diluted in PBS to obtain a concentration of 1 × 10 per ml. 8 cfu), the dilution can be experimentally confirmed by plating and colony formation on agar media. Mice can be anesthetized by intraperitoneal injection of 80-120 mg ketamine and 3-6 mg xylazine per kilogram of body weight. Immunized animals can be immunized on day 21 with 2 × 10 10 cfu of Staphylococcus aureus Newman or 3–10 × 10 9 Animals can be challenged by intraperitoneal injection with cfu of a clinical S. aureus isolate. Animals can be monitored for 14 days and fatal disease can be recorded.

[0287] Murine pneumonia model - S. aureus strains Newman or USA300 (LAC) were cultured at OD 660 Up to 0.5 can be grown at 37°C in tryptic soy broth / agar medium. Aliquots of 50-ml cultures were centrifuged, washed with PBS, and then diluted with 750 μl PBS (3–4 × 10 8CFU / 30-μl volume), or 1,250 μl PBS (2 × 10 8 For pulmonary infection, 7-week-old C57BL / 6J mice (The Jackson Laboratory) can be anesthetized prior to inoculation of 30 μl of S. aureus suspension into the left nostril. Animals can be placed in a cage in a supine position for recovery and observed for 14 days. For active immunization, 4-week-old mice can receive 20 μg SpA variant in CFA via the im route on day 0, followed by a boost with 20 μg variant in incomplete Freund's adjuvant (IFA) on day 10. Animals can be challenged with S. aureus on day 21. Serum can be collected pre-immunization and on day 20 to assess specific antibody production. For passive immunization studies, 7-week-old mice can receive 100 μl of either NRS (normal rabbit serum) or SpA variant-specific rabbit antiserum via ip 24 hours prior to challenge. To assess pathological correlates of pneumonia, infected animals can be sacrificed via forced CO2 inhalation prior to removal of both lungs. The right lung can be homogenized for enumeration of pulmonary bacterial burden. The left lung can be placed in 1% formalin, paraffin embedded, thin sectioned, stained with hematoxylin-eosin, and analyzed by microscopy.

[0288] Rabbit antibodies - purified SpA variants can be used as immunogens for the production of rabbit antisera. Proteins can be emulsified in CFA for injection on day 0, followed by booster injections with protein emulsified in IFA on days 21 and 42. Rabbit antibody titers can be determined by ELISA. Purified antibodies can be obtained by affinity chromatography of rabbit serum on SpA variant Sepharose. The concentration of eluted antibodies can be determined by A 280 The antibody titer can be measured by absorbance at 37° C., and specific antibody titers can be determined by ELISA.

[0289] Active immunization with SpA variants. -To determine vaccine efficacy, animals can be actively immunized with purified SpA variants. As a control, animals can be immunized with adjuvant alone. Antibody titers to Protein A preparations can be determined using the SpA variants as antigens. Using the infectious disease models described above, any reduction in bacterial load (murine abscesses and pneumonia), histopathological evidence of staphylococcal disease (murine abscesses and pneumonia), and protection from lethal disease (murine lethal challenge and pneumonia) can be measured.

[0290] Passive immunization with affinity purified rabbit polyclonal antibodies raised against SpA variants. To determine the protective immunity of Protein A-specific rabbit antibodies, mice are passively immunized with rabbit antibodies from purified SpA variants. Both of these antibody preparations are purified by affinity chromatography using immobilized SpA variants. As a control, animals are passively immunized with rV10 antibody (a plague protective antigen that has no effect on the outcome of staphylococcal infection). Antibody titers against all Protein A preparations are determined using SpA variants as antigen. Using the infectious disease models described above, reduction in bacterial load (murine abscesses and pneumonia), histopathological evidence of staphylococcal disease (murine abscesses and pneumonia), and protection from lethal disease (murine lethal challenge and pneumonia) can be measured.

[0291] Bacterial strains and growth. Staphylococcus aureus strains Newman and USA300 can be grown in tryptic soy broth (TSB) at 37°C. E. coli strains DH5α and BL21(DE3) can be grown at 100 μg ml -1 It can be grown at 37° C. in Luria-Bertani (LB) broth with ampicillin.

[0292] Rabbit antibodies. SpA variants can be produced according to standard recombinant or synthetic protocols and purified antigens can be covalently coupled to HiTrap NHS-activated HP columns (GE Healthcare). The antigen matrix can be used for affinity chromatography of 10-20 ml of rabbit serum at 4°C. The charged matrix can be washed with 50 column volumes of PBS and the antibodies can be eluted with elution buffer (1 M glycine, pH 2.5, 0.5 M NaCl) and immediately neutralized with 1 M Tris-HCl, pH 8.5. The purified antibodies can be dialyzed overnight at 4°C against PBS.

[0293] F(ab)2 fragments. Affinity purified antibodies can be mixed with 3 mg of pepsin for 30 minutes at 37°C. The reaction can be quenched with 1 M Tris-HCl, pH 8.5, and F(ab)2 fragments can be affinity purified on a HiTrap NHS-activated HP column conjugated with a specific antigen. Purified antibodies can be dialyzed overnight at 4°C against PBS, loaded onto an SDS-PAGE gel, and visualized by Coomassie blue staining.

[0294] Active and passive immunization. BALB / c mice (3 weeks old, female, Charles River Laboratories) can be immunized with 50 μg protein emulsified in Freund's complete adjuvant (Difco) by intramuscular injection. For booster immunizations, protein can be emulsified in Freund's incomplete adjuvant and injected 11 days after the first immunization. On the 20th day after immunization, five mice can be bled to obtain serum for specific antibody titers by enzyme-linked immunosorbent assay (ELISA).

[0295] Affinity purified antibodies in PBS were administered at 5 mg / kg 24 h prior to challenge with S. aureus. -1The antibody may be injected intraperitoneally into BALB / c mice (6 weeks old, female, Charles River Laboratories) at a concentration of 100 mg / kg / day of experimental animal weight. Animal blood may be collected via periorbital vein puncture. Blood cells may be removed with heparinized microhematocrit capillary tubes (Fisher), and Z-gel serum separator microtubes (Sarstedt) may be used to collect and measure antigen-specific antibody titers by ELISA.

[0296] Mouse kidney abscesses. Overnight cultures of S. aureus Newman or USA300 (LAC) can be diluted 1:100 into fresh TSB and grown for 2 hours at 37°C. Staphylococci can be pelleted, washed, and cultured to an OD of 0.4. 600 (about 1×10 8 CFU ml -1 The inoculum can be quantified by spreading a sample aliquot on TSA and counting the colonies formed. BALB / c mice (6 weeks old, female, Charles River Laboratories) were incubated with 100 mg ml per kilogram of body weight. - Ketamine and 20 mg ml -1 Mice can be anesthetized via intraperitoneal injection with xylazine. 7 CFU of Staphylococcus aureus Newman or 5 x 10 6Mice can be infected by retro-orbital injection with CFU of S. aureus USA300. Four days after challenge, mice can be killed by CO2 inhalation. Both kidneys can be removed and the staphylococcal load in one organ can be analyzed by homogenizing the kidney tissue in PBS, 1% Triton X-100. Serial dilutions of the homogenate can be spread on TSA and incubated for colony formation. The remaining organs can be examined by histopathological examination. Briefly, kidneys can be fixed in 10% formalin for 24 hours at room temperature. Tissues can be embedded in paraffin, thin-sectioned, stained with hematoxylin-eosin, and examined by light microscopy to count abscess lesions. All mouse experiments can be performed with review and approval of the experimental protocols following institutional guidelines by the Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC) of the University of Chicago.

[0297] Protein A binding. For human IgG binding, a Ni-NTA affinity column can be preloaded with 200 μg of purified protein (SpA variant) in column buffer. After washing, 200 μg of human IgG (Sigma) can be loaded on top of the column. Protein samples can be collected from the washes and eluates and subjected to SDS-PAGE gel electrophoresis followed by Coomassie blue staining. The purified protein (SpA variant) can be preloaded at 1 μg ml in 0.1 M carbonate buffer (pH 9.5). -1 The antibody may be coated onto MaxiSorp ELISA plates (NUNC) overnight at 4° C. at a concentration of 100 mM NaCl. The plates may then be blocked with 5% whole milk and subsequently incubated with serial dilutions of peroxidase-conjugated human IgG, Fc or F(ab)2 fragments for 1 hour. The plates may be washed and developed using OptEIA ELISA reagents (BD). The reaction may be quenched with 1 M phosphoric acid and incubated for 1 hour at 4° C. for 1 hour at 4° C. 450The readings were used to calculate half-maximal titers and percent binding.

[0298] von Willebrand factor (vWF) binding assay. Purified proteins (SpA variants) can be coated and blocked as described above. Plates are loaded with 1 μg ml -1 The plate may be incubated with human vWF at a concentration of 0.5% for 2 hours, then washed and blocked with human IgG for an additional hour. After washing, the plate may be incubated with serial dilutions of peroxidase-conjugated antibodies directed against human vWF for 1 hour. The plate may be washed and developed using OptEIA ELISA reagents (BD). The reaction may be quenched with 1 M phosphoric acid and incubated for 2 hours with human IgG for 1 ... 450 The readings can be used to calculate half-maximal titers and percent binding. For inhibition assays, plates were pre-filled with 10 μg ml -1 The cells can be incubated for 1 hour with affinity purified F(ab)2 fragments specific for the SpA variants at a concentration of 0.1 mM NaCl.

[0299] Splenocyte apoptosis. Affinity purified protein (150 μg of SpA variant) can be injected intraperitoneally into BALB / c mice (6 weeks old, female, Charles River Laboratories). Four hours after injection, the animals were killed by CO2 inhalation. Their spleens can be removed and homogenized. Cell debris can be removed using a cell strainer, and suspended cells can be transferred into ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) to lyse red blood cells. White blood cells can be sedimented by centrifugation, suspended in PBS, and stained with 1:250 diluted R-PE conjugated anti-CD19 monoclonal antibody (Invitrogen) for 1 hour on ice in the dark. Cells can be washed with 1% FBS and fixed with 4% formalin overnight at 4°C. The next day, cells can be diluted in PBS and analyzed by flow cytometry. The remaining organs can be examined by histopathology. Briefly, the spleen can be fixed in 10% formalin for 24 hours at room temperature. The tissue can be embedded in paraffin, thin sectioned, stained with the Apoptosis Detection Kit (Millipore), and examined by light microscopy.

[0300] Antibody quantification. Serum can be collected from healthy human volunteers or BALB / c mice that have been infected for 30 days with either S. aureus Newman or USA300 or immunized with SpA variants as described above. Human / Mouse IgG (Jackson Immunology Laboratory), SpA variants, and CRM 197can be blotted onto a nitrocellulose membrane. The membrane can be blocked with 5% whole milk followed by incubation with either human or mouse serum. IRDye 700DX conjugate affinity purified anti-human / mouse IgG (Rockland) can be used to quantify signal intensity using the Odyssey™ Infrared Imaging System (Li-cor). Experiments with blood from human volunteers involved protocols that were reviewed, approved, and conducted under the regulatory oversight of the Institutional Review Board (IRB) of the University of Chicago.

[0301] Statistical Analysis. Two-tailed Student's t-tests can be performed to analyze the statistical significance of renal abscess, ELISA, and B-cell superantigen data.

[0302] These assays can be used to test the variants described herein (e.g., those shown in Figures 12-15). Additional assays, such as SPR analysis, can be used to test SpA, SpA / KKAA, and SpA / KKAA / F (SpA * 31) can be performed to determine the binding affinity of the novel SpA variants to human VH3-IgG and human VH3-IgE compared to controls. * The yield of the variant / gram of E. coli paste may also be tested. CD spectroscopy may be performed to test the α-helical content compared to SpA and SpA / KKAA. Protein stability upon purification and storage at various temperatures (4°C, 25°C and 37°C for 1-7 days) may also be determined.

[0303] To test drug safety and efficacy, the basil histamine release assay may be performed (Figure 16). This test is known in the art (see, for example, Kowal, K. et al, 2005. Allergy and Asthma Proc. Vol. 26, No. 6). Briefly, human serum and / or basophils may be incubated at 37°C for 60 minutes. Histamine release may be measured from stimulated (by addition of SpA variants) and unstimulated cells, and results may be expressed as histamine release as a percentage of total histamine content. In some aspects, histamine release >16.5% is a positive test result in both pediatric and adult patients.

[0304] Example 3 SpA vaccine variants with improved safety B. Results 1. SpA vaccine candidate Gly 29 Amino acid substitutions in The present inventors have developed a novel antibody that blocks the interaction between SpA and Fcγ, which binds human IgG and SpA. KK The SpA-IgBD that results in the greatest decrease in affinity between 9,10 Gly of five IgBDs (EDABC) that also carry Lys 29 (48) Toward this goal, we developed an N-terminal polyhistidine-tagged SpA Q9,10K / G29X Nineteen different plasmids encoding SpA were constructed. Q9,10K / G29XProteins were purified via affinity chromatography on Ni-NTA resin, eluted, dialyzed, concentration determined via BCA assay, and bound to Bio-Rad ProteOn HTG chips at the same concentration (250 nM). Each chip was subjected to surface plasmon resonance testing with serial dilutions of human IgG or PBS control. Association of human IgG with SpA vaccine candidates loaded on the chip was recorded, and the data was transformed to derive association constants for each protein (Table 4). As a control, we used wild-type SpA (K A 1.081×10 8 M -1 ) and SpA KKAA (K A 5.022×10 5 M -1 The association...

Claims

1. 1. An isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution of a glutamine residue in each of Domains A-E corresponding to positions 9 and 10 in Domain D, and (ii) a glutamic acid substitution in each of Domains A-E corresponding to position 33 in Domain D, wherein the polypeptide does not detectably crosslink IgG and IgE in blood or activate basophils or mast cells compared to a negative control containing phosphate buffered saline (PBS), wherein the positions corresponding to positions 9, 10, and 33 in Domain D are - positions 7, 8, and 31 in SEQ ID NO:4 for domain A; - positions 7, 8, and 31 in SEQ ID NO:6 for domain B; - positions 7, 8, and 31 in SEQ ID NO:5 for domain C; - positions 9, 10, and 33 in SEQ ID NO:2 for domain D; and - positions 6, 7, and 30 in SEQ ID NO:3 for domain E; and Further, the isolated polypeptide wherein domains A to E of variant SpA have at least 85% sequence identity to SEQ ID NOs: 4, 6, 5, 2, and 3, respectively.

2. 1. An isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution of a glutamine residue in each of Domains A-E corresponding to positions 9 and 10 in Domain D, and (ii) a threonine substitution in each of Domains A-E corresponding to position 33 in Domain D, wherein the polypeptide does not detectably crosslink IgG and IgE in blood or activate basophils or mast cells compared to a negative control containing phosphate buffered saline (PBS), wherein the positions corresponding to positions 9, 10, and 33 in Domain D are - positions 7, 8, and 31 in SEQ ID NO:4 for domain A; - positions 7, 8, and 31 in SEQ ID NO:6 for domain B; - positions 7, 8, and 31 in SEQ ID NO:5 for domain C; - positions 9, 10, and 33 in SEQ ID NO:2 for domain D; and - positions 6, 7, and 30 in SEQ ID NO:3 for domain E; and Further, the isolated polypeptide wherein domains A to E of variant SpA have at least 85% sequence identity to SEQ ID NOs: 4, 6, 5, 2, and 3, respectively.

3. (A) K against VH3 from human IgG A The binding affinity of SpA variants (SpA KKAA ) and / or (B) The isolated polypeptide is KKAA K for VH3 from human IgG that is at least two-fold reduced compared to A and / or (C) The isolated polypeptide was diluted to 1 × 10 5 M -1 K for VH3 from human IgG is less than A having binding affinity 3. An isolated polypeptide according to claim 1 or 2.

4. The SpA variant has no substitutions in any of domains A through E corresponding to positions 36 and 37 in domain D, wherein the positions corresponding to positions 36 and 37 in domain D are - positions 34 and 35 in SEQ ID NO:4 for domain A; - positions 34 and 35 in SEQ ID NO:6 for domain B; - positions 34 and 35 in SEQ ID NO:5 for domain C; - for domain D, positions 36 and 37 in SEQ ID NO:2; and - positions 33 and 34 in SEQ ID NO:3 for domain E; 4. The isolated polypeptide of claim 1, wherein

5. An isolated polypeptide described in any one of claims 1 to 3, wherein the only substitutions in the SpA variant are (i) and (ii).

6. An isolated polypeptide described in any one of claims 1 to 4, wherein the isolated polypeptide consists of an SpA variant.

7. A composition comprising an isolated polypeptide described in any one of claims 1 to 6, wherein the composition contains less than 1 wt% of the total protein amount, other than the isolated polypeptide.

8. The composition of claim 7, further comprising: (A) an adjuvant; and / or (B) A pharma- ceutically acceptable excipient.

9. 9. The composition of claim 7 or 8 for inducing a safe immune response against Staphylococcus aureus in a human patient.

10. (A) the staphylococci include ST88 isolates or any other human staphylococci currently adapted to animals; and / or (B) the staphylococcus includes the WU1 or JSNZ strain of Staphylococcus aureus; The composition of claim 9.

11. (A) A human patient comprising: (a) the patient is a pediatric patient, or (b) is 65 years of age or older; and / or (c) is a healthcare professional; and / or (B) the isolated polypeptide of the composition is administered in four doses with an interval between doses of at least four weeks; 11. The composition of claim 9 or 10.

12. A composition comprising an isolated polypeptide according to any one of claims 1 to 6 or a composition according to claim 7 or 8 for preventing or treating a staphylococcal infection.

13. (A) the staphylococci include ST88 isolates or any other human staphylococci currently adapted to animals; and / or (B) the staphylococcus includes the WU1 or JSNZ strain of Staphylococcus aureus; 13. The composition of claim 12.

14. (A) A human patient comprising: (a) the patient is a pediatric patient, or (b) is 65 years of age or older; and / or (c) is a healthcare professional; and / or (B) the isolated polypeptide or composition is administered in four doses with an interval between doses of at least four weeks; 14. The composition of claim 12 or 13.

15. A composition comprising an isolated polypeptide according to any one of claims 1 to 6 or a composition according to claim 7 or 8 for decolonization or prevention of colonization or recolonization of Staphylococcus aureus in a subject.

16. 1. An isolated polypeptide comprising a variant Protein A (SpA) having (i) a lysine substitution of a glutamine residue in each of Domains A-E corresponding to positions 9 and 10 in Domain D, and (ii) a glutamic acid substitution in each of Domains A-E corresponding to position 33 in Domain D, wherein the polypeptide does not detectably crosslink IgG and IgE in blood or activate basophils or mast cells compared to a negative control containing phosphate buffered saline (PBS); - positions 7, 8, 31, 34, and 35 in SEQ ID NO:4 for domain A; - positions 7, 8, 31, 34, and 35 in SEQ ID NO:6 for domain B; - positions 7, 8, 31, 34, and 35 in SEQ ID NO:5 for domain C; - for domain D, positions 9, 10, 33, 36, and 37 in SEQ ID NO:2; and - positions 6, 7, 30, 33, and 34 in SEQ ID NO:3 for domain E; and Further, the isolated polypeptide wherein domains A to E of variant SpA have at least 85% sequence identity to SEQ ID NOs: 4, 6, 5, 2, and 3, respectively.

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