Isolated immunogenic polypeptide, immunogenic composition and its use

专利摘要:
POLYPEPTIDE COMPRISING A SEGMENT OF THE D-DOMAIN OF A VARIANT OF PROTEIN A(S PA), AS WELL AS A PHARMACEUTICAL COMPOSITION AND USES FOR INDUCING AN IMMUNE RESPONSE AGAINST A STAPHYLOCOCCUS BACTERIA. The present invention relates to compositions and their uses for treating or preventing a bacterial infection, particularly infection with a Staphylococcus bacteria, or for inducing an immune response against the bacteria. The invention further relates to a polypeptide comprising a segment of the D domain of a protein A (SpA) variant.
公开号:BR112013000097B1
申请号:R112013000097-0
申请日:2011-07-01
公开日:2022-02-01
发明作者:Olaf Schneewind；Alice G. Cheng；Dominique M. Missiakas；Hwan Kim
申请人:The University Of Chicago；
IPC主号:

专利说明:

[0001] This patent application claims the priority benefit of provisional patent applications serial numbers US 61/361,218, filed July 2, 2010, and 61/370,725, filed August 4, 2010, which are incorporated herein as a reference in its entirety.
[0002] This invention was developed with government sponsorship, as per AI057153, AI052474, and GM007281, granted by the National Institutes of Health. The government has certain rights in the invention. I. FIELD OF THE INVENTION
[0003] The present invention relates generally to the scientific fields of immunology, microbiology, and pathology. More particularly, it relates to methods and compositions involving bacterial Protein A variants, which can be used to invoke an immune response against bacteria. II. BACKGROUND
[0004] The number of community-acquired and hospital-acquired infections has increased during the past few years with the increasing use of intravascular devices. Hospital-acquired (nosocomial) infections are a major cause of morbidity and mortality, particularly in the United States, where they affect more than 2 million patients annually. The most frequent infections are urinary tract infections (33% of infections), followed by pneumonia (15.5%), infections at surgical sites (14.8%), and primary bloodstream infections (13%) (Emorl and Gaynes, 1993).
[0005] Major nosocomial pathogens include Staphylococcus aureus, coagulase-negative coagulase-negative staphylococci (mainly Staphylococcus epidermidis), Enterococcus spp., Escherichia coli, and Pseudomonas aeruginosa. Although these pathogens cause approximately the same number of infections, the severity of the disorders they can produce combined with often antibiotic-resistant isolates skew this ranking in the direction of S. aureus and S. epidermidis being the most significant nosocomial pathogens.
[0006] Staphylococci can cause a wide range of diseases in humans and other animals through the production or invasion of toxins. Staphylococcal toxins are also a common cause of food poisoning as bacteria can grow in improperly stowed food.
[0007] Staphylococcus epidermidis is a normal commensal of the skin, and is also an important opportunistic pathogen responsible for infections of impaired medical devices and infections at surgical sites. Medical devices infected with S. epidermidis include cardiac pacemakers, cerebrospinal fluid shunts, continuous ambulatory peritoneal dialysis catheters, orthopedic devices, and prosthetic heart valves.
[0008] Staphylococcus aureus is the most common cause of nosocomial infections with significant morbidity and mortality. It is the cause of some cases of osteomyelitis, endocarditis, septic arthritis, pneumonia, abscesses, and toxic shock syndrome. S. aureus can survive on dry surfaces, increasing the possibility of transmission. Any S. aureus infection can cause staphylococcal scalded skin syndrome, a skin reaction to exotoxin absorbed into the bloodstream. It can also cause a type of pyemic septicemia that can be lethal. Problematically, methicillin-resistant Staphylococcus aureus (MRSA) has become a major cause of hospital-acquired infections.
[0009] S. aureus and S. epidermidis infections are typically treated with antibiotics, with penicillin being the drug of choice, while vancomycin is used for methicillin-resistant isolates. The percentage of staphylococcal strains that show broad-spectrum antibiotic resistance has become increasingly prevalent, posing a threat to effective antimicrobial therapy. In addition, the recent emergence of a vancomycin-resistant strain of S. aureus has raised concerns that MRSA strains are emerging and widespread and for which no effective therapy is available.
[00010] An alternative to antibiotic treatment for staphylococcal infections is under investigation and it uses antibodies directed against staphylococcal antigens. This therapy involves administration of polyclonal antisera (WO00/15238, WO00/12132) or treatment with monoclonal antibodies against lipoteichoic acid (WO98/57994).
[00011] An alternative approach would be to use active vaccination to generate an immune response against staphylococci. The S. aureus genome has been sequenced and many coding sequences have been identified (documents no. WO02/094868, EP0786519), which can lead to the identification of potential antigens. The same is true for S. epidermidis (document no WO01/34809). As a refinement of this approach, other researchers have identified proteins that are recognized by hyperimmune sera from patients who have suffered staphylococcal infection (WO 01/98499, WO 02/059148).
[00012] S. aureus secretes the plethora of virulence factors within the extracellular milieu (Archer, 1998; Dinges et al., 2000; Foster, 2005; Shaw et al., 2004; Sibbald et al., 2006). Similar to most secreted proteins, these virulence factors are displaced by the Sec mechanism across the plasma membrane. Proteins secreted by the Sec mechanism carry a leader peptide at the N-terminus, which is removed by the leader peptidase after the preprotein is engaged in the Sec translocon (Dalbey and Wickner, 1985; van Wely et al., 2001). Recent genome analysis suggests that actinobacteria and members of Firmicutes encode an additional secretion system that recognizes a subset of proteins in a Sec-independent manner (Pallen, 2002). ESAT-6 (6 kDa early secreted antigenic target) and CFP-10 (10 kDa culture filtrate antigen) from Mycobacterium tuberculosis represent the first substrates of this unusual secretion system called ESX-1 or Snm in M. tuberculosis (Andersen et al. ., 1995; Hsu et al., 2003; Pym et al., 2003; Stanley et al., 2003). In S. aureus, two ESAT-6-like factors, designated EsxA and EsxB, are secreted via the Ess pathway (ESAT-6 secretion system) (Burts et al., 2005).
[00013] The first generation of vaccines targeted against S. aureus or against the exoproteins it produces met with limited success (Lee, 1996). There remains a need to develop effective vaccines against staphylococcal infections. Additional compositions to treat staphylococcal infections are also needed. SUMMARY OF THE INVENTION
[00014] Protein A (SpA) (SEQ ID NO:33), a surface protein anchored to the cell wall of Staphylococcus aureus, provides bacterial evasion from innate and adapted immune responses. Protein A binds to immunoglobulins in its Fc part, interacts with the VH3 domain of B cell receptors, which inappropriately stimulates B cell proliferation and apoptosis, binds to von Willebrand factor A1 domains to activate intracellular clotting, and also binds to TNF Receptor-1 to contribute to the pathogenesis of staphylococcal pneumonia. Due to the fact that Protein A captures immunoglobulin and has toxic attributes, the possibility that this surface molecule could function as a vaccine in humans has not been rigorously pursued. In this invention, it was demonstrated that Protein A variants are no longer able to bind immunoglobulins, which in this way are deprived of their toxigenic potential, that is, they are non-toxigenic, they stimulate humoral immune responses that protect against staphylococcal disease.
[00015] In certain embodiments, the SpA variant is a full-length SpA variant that comprises the A, B, C, D, and/or E domain. In certain aspects, the SpA variant comprises or consists of the amino acid sequence that is 80, 90, 95, 98, 99, or 100% identical to the amino acid sequence of SEQ ID NO:34. In other embodiments, the SpA variant comprises an SpA segment. The SpA segment may comprise at least or at most 1, 2, 3, 4, 5 or more IgG binding domains. The IgG domains can be at least or at most 1, 2, 3, 4, 5 or more A, B, C, D, or E variant domains. In certain aspects, the SpA variant comprises at least or at most 1, 2, 3, 4, 5, or more A variant domains. In another aspect, the SpA variant comprises at least or at most 1, 2, 3, 4, 5, or more B variant domains. In yet another aspect, the SpA variant comprises at least or at most 1, 2, 3, 4, 5, or more C variant domains. In yet another aspect the SpA variant comprises at least or at most 1, 2, 3, 4, 5, or more D variant domains. In certain aspects, the SpA variant comprises at least or at most 1, 2, 3, 4, 5, or more E variant domains. In another aspect the SpA variant comprises a combination of domains A, B, C, D, and And in various combinations and permutations. The combinations may include all or part of an SpA signal peptide segment, an SpA X region segment, and/or an SpA choice signal segment. In other aspects, the SpA variant does not include an SpA signal peptide segment, an SpA X region segment, and/or an SpA choice signal segment. In certain aspects a variant domain A comprises a substitution at positions 7, 8, 34, and/or 35 of SEQ ID NO:4. In another aspect a variant domain B comprises a substitution at positions 7, 8, 34, and/or 35 of SEQ ID NO:6. In yet another aspect a C-variant domain comprises a substitution at positions 7, 8, 34, and/or 35 of SEQ ID NO:5. In certain aspects a variant domain D comprises a substitution at positions 9, 10, 36, and/or 37 of SEQ ID NO:2. In another aspect an E variant domain comprises a substitution at positions 6, 7, 33, and/or 34 of SEQ ID NO:3.
[00016] In certain aspects, a D domain variant of SpA or its equivalent may comprise a mutation at positions 9 and 36; 9 and 37; 9 and 10; 36 and 37; 10 and 36; 10 and 37; 9, 36, and 37; 10, 36, and 37, 9, 10 and 36; or 9.10 and 37 of SEQ ID NO:2. In another aspect, analogous mutations may be included in one or more A, B, C, or E domains.
[00017] In other aspects, the amino acid glutamine (Q) at position 9 of SEQ ID NO:2 (or its amino acid analogue in other domains of SpA) may be substituted for an alanine (A), an asparagine (N), a aspartic acid (D), a cysteine (C), a glutamic acid (E), a phenylalanine (F), a glycine (G), a histidine (H), an isoleucine (I), a lysine (K), a leucine (L), a methionine (M), a proline (P), a serine (S), a threonine (T), a valine (V), a tryptophan (W), or a tyrosine (Y). In some aspects the glutamine at position 9 can be replaced by an arginine (R). In another aspect, the glutamine at position 9 of SEQ ID NO:2, or its equivalent, may be substituted for a lysine or a glycine. Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the substitutions can be explicitly excluded.
[00018] In another aspect, the amino acid glutamine (Q) at position 10 of SEQ ID NO:2 (or its amino acid analogue in other domains of SpA) may be replaced by an alanine (A), an asparagine (N), an aspartic acid (D), a cysteine (C), a glutamic acid (E), a phenylalanine (F), a glycine (G), a histidine (H), an isoleucine (I), a lysine (K), a leucine (L), a methionine (M), a proline (P), a serine (S), a threonine (T), a valine (V), a tryptophan (W), or a tyrosine (Y). In some aspects the glutamine at position 10 can be replaced by an arginine (R). In another aspect, the glutamine at position 10 of SEQ ID NO:2, or its equivalent, may be substituted for a lysine or glycine. Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the substitutions can be explicitly excluded.
[00019] In certain aspects, the aspartic acid (D) at position 36 of SEQ ID NO:2 (or its amino acid analogue in other domains of SpA) may be substituted for an alanine (A), an asparagine (N), a arginine (R), a cysteine (C), a phenylalanine (F), a glycine (G), a histidine (H), an isoleucine (I), a lysine (K), a leucine (L), a methionine ( M), a proline (P), a glutamine (Q), a serine (S), a threonine (T), a valine (V), a tryptophan (W), or a tyrosine (Y). In some aspects, the aspartic acid at position 36 can be substituted for a glutamic acid (E). In certain aspects, an aspartic acid at position 36 of SEQ ID NO:2, or its equivalent, may be substituted with an alanine or a serine. Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the substitutions can be explicitly excluded.
[00020] In another aspect, the aspartic acid (D) at position 37 of SEQ ID NO:2 (or its amino acid analogue in other domains of SpA) can be replaced by an alanine (A), an asparagine (N), an arginine (R), a cysteine (C), a phenylalanine (F), a glycine (G), a histidine (H), an isoleucine (I), a lysine (K), a leucine (L), a methionine ( M), a proline (P), a glutamine (Q), a serine (S), a threonine (T), a valine (V), a tryptophan (W), or a tyrosine (Y). In some aspects, the aspartic acid at position 37 can be substituted for a glutamic acid (E). In certain aspects, an aspartic acid at position 37 of SEQ ID NO:2, or its equivalent, may be substituted for an alanine or a serine. Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the substitutions can be explicitly excluded.
[00021] In a specific embodiment, the amino acid at position 9 of SEQ ID NO:2 (or an analogous amino acid in another domain of SpA) is replaced with an alanine (A), a glycine (G), an isoleucine (I) , a leucine (L), a proline (P), a serine (S), or a valine (V). In certain aspects, the amino acid at position 9 of SEQ ID NO:2 is replaced by a glycine. In another aspect the amino acid at position 9 of SEQ ID NO:2 is replaced by a lysine.
[00022] In a specific embodiment, the amino acid at position 10 of SEQ ID NO:2 (or an analogous amino acid in another domain of SpA) is replaced by an alanine (A), a glycine (G), an isoleucine (I) , a leucine (L), a proline (P), a serine (S), or a valine (V). In certain aspects, the amino acid at position 10 of SEQ ID NO:2 is substituted for a glycine. In another aspect the amino acid at position 10 of SEQ ID NO:2 is replaced by a lysine.
[00023] In a specific embodiment, the amino at position 36 of SEQ ID NO:2 (or an analogous amino acid in another domain of SpA) is replaced by an alanine (A), a glycine (G), an isoleucine (I) , a leucine (L), a proline (P), a serine (S), or a valine (V). In certain aspects, the amino acid at position 36 of SEQ ID NO:2 is substituted with a serine. In another aspect the amino acid at position 36 of SEQ ID NO:2 is replaced by an alanine.
[00024] In a specific embodiment, the amino at position 37 of SEQ ID NO:2 (or an analog amino acid from another domain of SpA) is replaced with an alanine (A), a glycine (G), an isoleucine (I) , a leucine (L), a proline (P), a serine (S), or a valine (V). In certain aspects, the amino acid at position 37 of SEQ ID NO:2 is substituted with a serine. In another aspect the amino acid at position 37 of SEQ ID NO:2 is replaced by an alanine.
[00025] In certain aspects, the SpA variant includes a substitution of (a) one or more amino acid substitutions in an IgG Fc-binding subdomain of the SpA, A, B, C, D, and/or E domain that disrupts or decreases binding to IgG Fc, and (b) one or more amino acid substitutions in a VH3-binding subdomain of the SpA, A, B, C, D, and/or E domain that disrupts or decreases binding to VH3 . In still other aspects the amino acid sequence of an SpA variant comprises an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical, including all values and ranges between they to the amino acid sequence of SEQ ID NOs:2-6.
[00026] In another aspect the SpA variant includes (a) one or more amino acid substitutions in an IgG Fc binding subdomain of the SpA D domain, or at a corresponding amino acid position in other IgG domains, that disrupts or diminishes binding to IgG Fc, and (b) one or more amino acid substitutions in a VH3-binding subdomain of the SpA D domain, or at a corresponding amino acid position in other domains of IgG, which disrupts or decreases binding to VH3 . In certain aspects the amino acid residue F5, Q9, Q10, S11, F13, Y14, L17, N28, I31, and/or K35 (SEQ ID NO:2,QQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLG EAKKLNES) of the IgG Fc binding subdomain of the D domain are modified or replaced. In certain aspects, amino acid residue Q26, G29, F30, S33, D36, D37, Q40, N43, and/or E47 (SEQ ID NO:2) of the VH3 binding subdomain of the D domain are modified or substituted, as so that binding to Fc or VH3 is attenuated. In other aspects, the corresponding modifications or substitutions can be manipulated at corresponding positions of domain A, B, C, and/or E. Corresponding positions are defined by aligning the amino acid sequence of domain D with one or more amino acid sequences of other IgG binding domains of SpA; see, for example, Figure 2A. In certain aspects, the amino acid substitution can be any of the other 20 amino acids. In another aspect, conservative amino acid substitutions may be specifically excluded from possible amino acid substitutions. In other respects, only non-conservative substitutions are included. In any case, any substitution or combinations of substitutions that reduce domain binding, such that the toxicity of SpA is significantly reduced, is contemplated. Significance of reduction in binding refers to a variant that produces minimal or no toxicity when introduced into an individual and can be assessed using in vitro methods described herein.
[00027] In certain embodiments, an SpA variant comprises at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more SpA D domain peptides. In certain aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more amino acid residues of the SpA variant are substituted or modified, including, but not limited to, amino acids F5, Q9, Q10, S11, F13, Y14, L17, N28, I31, and/or K35 (SEQ ID NO:2) of the IgG Fc-binding subdomain of the D, and amino acid residue Q26, G29, F30, S33, D36, D37, Q40, N43, and/or E47 (SEQ ID NO:2) of the VH3 binding subdomain of domain D. In one aspect of the invention, glutamine residues at position 9 and/or 10 of SEQ ID NO:2 (or corresponding positions in other domains) are mutated. In another aspect, aspartic acid residues 36 and/or 37 of SEQ ID NO:2 (or corresponding positions in other domains) are mutated. In another aspect, residues of glutamine 9 and 10, and aspartic acid 36 and 37 are mutated. Purified non-toxicogenic SpA or SpA-D mutants/variants described herein are no longer able to significantly bind (i.e. demonstrate attenuated or disrupted binding affinity) to FCY or F(ab)2 VH3 and also do not stimulate B cell apoptosis These non-toxigenic Protein A variants of Protein A can be used as subunit vaccines and generate humoral immune responses and confer protective immunity against S. aureus challenge. Compared to wild-type full-length Protein A or wild-type SpA Domain D, immunization with SpA-D variants resulted in an increase in Protein A-specific antibody. Using a mouse staphylococcal challenge model and formation It was observed that immunization with the non-toxicogenic Protein A variants provided significant protection against staphylococcal infection and abscess formation. As virtually all S. aureus strains express Protein A, immunization of humans with non-toxigenic Protein A variants can neutralize this virulence and thus establish protective immunity. In certain aspects, protective immunity protects or ameliorates infection by drug-resistant strains of staphylococcus, such as USA300 strains and other MRSA.
[00028] Modalities include the use of Protein A variants in methods and compositions for the treatment of bacterial and/or staphylococcal infection. This invention also provides an immunogenic composition comprising a Protein A variant or immunogenic fragment thereof. In certain aspects, the immunogenic fragment is a segment of the D domain of Protein A. In addition, the present invention provides methods and compositions that can be used to treat (e.g., limit staphylococcal abscess formation and/or persistence in a individual) or prevent bacterial infection. In some cases, methods of stimulating an immune response involve administering to the subject an effective amount of a composition that includes or encodes all or part 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) a secreted virulence factor, and/or a cell surface peptide or protein, or (ii) a recombinant nucleic acid molecule encoding a secreted virulence factor, and/or a cell surface protein or peptide.
[00029] In other aspects, the subject may receive administration of all or part of a Protein A variant, such as a segment of the variant Protein A D domain. The polypeptide of the invention can be formulated into a pharmaceutically acceptable composition. The composition may further comprise one or more of at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 additional staphylococcal antigens or their immunogenic fragment (e.g. Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla (e.g. H35 mutants) ), IsdC, SasF, vWbp, or vWh). Additional staphylococcal antigens that may be used in combination with a Protein A variant 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 exotoxins (WO 00/02523), SEB exotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC binding protein /MntC/saliva (US5,801,234), SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein (see PCT Publications Nos. WO2007/113222, WO2007/113223, WO2006/032472, WO2006/032475, WO2006/ 032500, each of which is incorporated herein by reference in its entirety). The Staphylococcal antigen or immunogenic fragment can be administered concomitantly 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 may also be a recombinant nucleic acid molecule encoding a Protein A variant. A recombinant nucleic acid molecule may encode the Protein A variant and for a staphylococcal antigen or immunogenic fragment thereof. As used herein, the term "modulate" or "modulate" encompasses the meanings of the words "enhance" or "inhibit". "Modulation" of activity can be an increase or decrease in activity. As used herein, the term "modulator" refers to compounds that effect the function of a group, including upregulation, induction, stimulation, potentiation, inhibition, downregulation, or suppression of a protein, nucleic acid, gene, organism or similar.
[00030] In certain embodiments, the methods and compositions use or include or encode all or part of the Protein A variant or antigen. In other aspects, the Protein A variant may be used in combination with secreted factors or surface antigens including, but not limited to, one or more of an Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, or isolated vWh or its immunogenic segment. Additional antigens that may be used in combination with a Protein A variant include, but are not limited to, 52 kDa vitronectin binding protein (WO 01/60852), Aaa, Aap, Ant, autolysine glucosaminidase, autolysine amidase, Cna, protein collagen binder (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 exotoxins (WO 00/02523), SEB exotoxins (WO 00 /02523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more among Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh, vitronectin binding protein 52 kDa (WO 01/60852), Aaa, Aap, Ant, autolysine glucosaminidase, autolysine 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 activator protein III (RAP), SasA, SasB, SasC, SasD , SasK,SBI, SdrF(WO 00/12689), SdrG / Fig (WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523), SEB exotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and /or Vitronectin binding protein may be specifically excluded from a formulation of the invention.
[00031] The following table lists the various combinations of SpA variants and various other staphylococcal antigens. Table 1. SpA and staphylococcal antigen combinations.









[00032] In still other aspects, the isolated Protein A variant is multimerized, for example, dimerized or a linear fusion of two or more polypeptides or peptide segments. In certain aspects of the invention, a composition comprises multimers or concatamers 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. Concatamers are linear polypeptides that have one or more repeating peptide units. The SpA polypeptides or fragments may be consecutive or separated by a spacer or other peptide sequences, for example, one or more additional bacterial peptides. In another aspect, the other polypeptides or peptides contained in the multimer or concatamer may include, but are not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 between Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh or their immunogenic fragments. Additional staphylococcal antigens that may be used in combination with a Protein A variant include, but are not limited to, 52 kDa vitronectin binding protein (WO 01/60852), Aaa, Aap, Ant, autolysine 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 exotoxins (WO 00/02523), SEB exotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein.
[00033] The term "Protein A variant" or "SpA variant" refers to polypeptides that include an IgG domain of SpA, having one or more amino acid substitutions that disrupt binding to Fc and VH3. In certain aspects, an SpA variant includes a variant D domain peptide, as well as variants of SpA polypeptides and segments thereof that are non-toxicogenic and stimulate an immune response against Protein A from staphylococcal bacteria and/or bacteria that express it.
[00034] Embodiments of the present invention include methods of eliciting an immune response against a staph bacterium or staphylococcus in a subject, comprising providing the subject with an effective amount of a Protein A variant or a segment thereof. In certain aspects, methods of eliciting an immune response against a staphylococcal bacterium or staphylococcus in a subject comprise providing the subject with 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 proteins and/or cell surface proteins or segments/fragments thereof. A secreted protein or cell surface protein includes, but is not limited to, Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF proteins , vWbp, and/or vWh and their immunogenic fragments. Additional staphylococcal antigens that can be used in combination with a Protein A variant 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 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 exotoxins (WO 00/02523), SEB exotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein.
[00035] Embodiments of the invention include compositions that include a polypeptide, peptide, or protein that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to Protein A, or a second protein or peptide that is a secreted bacterial protein or a cell surface protein. In another embodiment of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to a polypeptide of Protein A D domain (SEQ ID NO:2), E domain (SEQ ID NO:3), A domain (SEQ ID NO:4), C domain (SEQ ID NO: 5), domain B (SEQ ID NO:6), or a nucleic acid sequence encoding a domain D domain, domain E, domain A, domain C, or domain B polypeptide of Protein A. In certain aspects, a segment of Protein A polypeptide must have an amino acid sequence of SEQ ID NO:8. Similarity or identity, the preferred identity being, is known in these techniques and a number of 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 field, including, but not limited to, the sequence identity algorithm of Smith & Waterman (1981), the sequence identity algorithm of Needleman & Wunsch (1970) , by the similarity search method of Pearson & Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in 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 parameters, or by inspection. Preferably, the percent identity is calculated using alignment tools known and readily available to those skilled in these techniques. Percent identity is essentially the number of identical amino acids divided by the total number of amino acids compared multiplied by one hundred.
[00036] Still other embodiments include methods of stimulating a protective or therapeutic immune response in a subject against a staphylococcal bacterium, comprising administering to the subject an effective amount of a composition that includes (i) an SpA variant, for example, a domain polypeptide D of variant SpA or its peptide; or (ii) a nucleic acid molecule encoding a variant SpA polypeptide or peptide thereof, or (iii) administering a variant SpA D domain polypeptide with any combination or allowance of bacterial proteins described herein. In a preferred embodiment, the composition is not a staphylococcal bacterium. In certain respects, the individual is a human being or a cow. In another aspect the composition is formulated into a pharmaceutically acceptable formulation. Staphylococci can be Staphylococcus aureus.
[00037] Still other embodiments include vaccines comprising a pharmaceutically acceptable composition that has an SpA variant polypeptide, or any combination or permutation of protein(s) or peptide(s) described herein, wherein the composition is capable of stimulating an immune response. against a staphylococcal bacterium. The vaccine may comprise an isolated SpA variant polypeptide, or any combination or permutation of the described protein(s) or peptide(s). In certain aspects of the invention, the isolated SpA variant polypeptide, or any combination or permutation of described protein(s) or peptide(s) is multimerized, for example, dimerized or concatamerized. In another aspect, the vaccine composition is contaminated by less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.05% ( or any range between these values) of other staphylococcal proteins. A composition may further comprise an isolated non-SpA polypeptide. Typically, the vaccine comprises an adjuvant. In certain aspects, a protein or peptide of the invention is linked (covalently or non-covalently) to the adjuvant, preferably the adjuvant chemically conjugated to the protein.
[00038] In still other embodiments, a vaccine composition is a pharmaceutically acceptable composition that has a recombinant nucleic acid that encodes all or part of a variant SpA polypeptide, or any combination or permit of protein(s) or peptide(s) described herein, wherein the composition is capable of stimulating an immune response against staphylococcal bacteria. The vaccine composition may comprise a recombinant nucleic acid encoding all or part of a SpA variant polypeptide, or any combination or permutation of protein(s) or peptide(s) described herein. In certain embodiments, the recombinant nucleic acid contains 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 includes a recombinant non-staphylococcal bacterium that contains the nucleic acid. Recombinant non-staphylococci may be salmonella or other gram-positive bacteria. The vaccine may comprise a pharmaceutically acceptable excipient, more preferably an adjuvant.
[00039] Still other embodiments include methods of stimulating in a subject a protective or therapeutic immune response against a staphylococcal bacterium, comprising administering to the subject an effective amount of a composition of a variant SpA polypeptide or segment/fragment thereof, and further comprising one or more more between a protein Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, or vWh or its peptide. In a preferred embodiment, the composition comprises a non-staphylococcal bacterium. In another aspect the composition is formulated into a pharmaceutically acceptable formulation. The staphylococci against which an individual is treated may be Staphylococcus aureus. The methods of the invention also include compositions of the SpA variant, which contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 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 a 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, a combination of antigens may include (1) an SpA and IsdA variant; (2) SpA and ClfB variant; (3) SpA and SdrD variant; (4) SpA and Hla variant or Hla variant; (5) SpA and ClfB, SdrD, and Hla variant or Hla variant; (6) SpA, IsdA, SdrD, and Hla variant or Hla variant; (7) SpA, IsdA, ClfB, and Hla variant or Hla variant; (8) SpA, IsdA, ClfB, and SdrD variant; (9) SpA, IsdA, ClfB, SdrD and Hla variant or Hla variant; (10) SpA, IsdA, ClfB, and SdrD variant; (11) SpA, IsdA, SdrD, and Hla variant or Hla variant; (12) SpA, IsdA, and Hla variant or Hla variant; (13) SpA, IsdA, ClfB, and Hla variant or Hla variant; (14) SpA, ClfB, and SdrD variant; (15) SpA, ClfB, and Hla variant or Hla variant; or (16) SpA, SdrD, and Hla variant or Hla variant.
[00040] In certain aspects, a bacterium that delivers a composition of the invention must be limited or attenuated with respect to prolonged or persistent growth or abscess formation. In yet another aspect, SpA variant(s) can be overexpressed in the attenuated bacterium to further enhance or supplement an immune response or vaccine formulation.
[00041] The term "EsxA protein" refers to a protein that includes wild-type EsxA polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against EsxA proteins from staphylococcal bacteria.
[00042] The term "EsxB protein" refers to a protein that includes wild-type EsxB polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against EsxB proteins from staphylococcal bacteria.
[00043] The term "SdrD protein" refers to a protein that includes wild-type SdrD polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against SdrD proteins from staphylococcal bacteria.
[00044] The term "SdrE protein" refers to a protein that includes wild-type SdrE polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against SdrED proteins from staphylococcal bacteria.
[00045] The term "IsdA protein" refers to a protein that includes wild-type IsdA polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against IsdA proteins from staphylococcal bacteria.
[00046] The term "IsdB protein" refers to a protein that includes wild-type IsdB polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against IsdB proteins from staphylococcal bacteria.
[00047] The term "Eap protein" refers to a protein that includes wild-type Eap polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against Eap proteins from staphylococcal bacteria.
[00048] The term "Ebh protein" refers to a protein that includes wild-type Ebh polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against Ebh proteins from staphylococcal bacteria.
[00049] The term "Emp protein" refers to a protein that includes wild-type Emp polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against Emp proteins from staphylococcal bacteria.
[00050] The term "EsaB protein" refers to a protein that includes wild-type Esab polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against EsaB proteins from staphylococcal bacteria.
[00051] The term "EsaC protein" refers to a protein that includes wild-type EsaC polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against EsaC proteins from staphylococcal bacteria.
[00052] The term "SdrC protein" refers to a protein that includes wild-type SdrC polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against SdrC proteins from staphylococcal bacteria.
[00053] The term "ClfA protein" refers to a protein that includes wild-type CIfA polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against CIfA proteins from staphylococcal bacteria.
[00054] The term "ClfB protein" refers to a protein that includes wild-type ClfB polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against ClfB proteins from staphylococcal bacteria.
[00055] The term "Coa protein" refers to a protein that includes wild-type Coa polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against Coa proteins from staphylococcal bacteria.
[00056] The term "Hla protein" refers to a protein that includes wild-type Hla polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against Hla proteins from staphylococcal bacteria.
[00057] The term “IsdC protein” refers to a protein that includes wild-type IsdC polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against IsdC proteins from staphylococcal bacteria.
[00058] The term "SasF protein" refers to a protein that includes wild-type SasF polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against SasF proteins from staphylococcal bacteria.
[00059] The term "vWbp protein" refers to a protein that includes wild-type vWbp polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against vWbp proteins from staphylococcal bacteria.
[00060] The term "vWh protein" refers to a protein that includes wild-type vWh polypeptides isolated from staphylococcal bacteria and their segments, as well as variants that stimulate an immune response against vWh proteins from staphylococcal bacteria.
[00061] An immune response refers to a humoral response, a cellular response, or a humoral response and also a cellular response in an organism. An immune response can be measured by assays that include, but are not limited to, assays that measure the presence or amount of antibodies that specifically recognize a cell surface protein or protein, assays that measure T cell activation or proliferation, and/or assays that measure modulation in terms of the activity or expression of one or more cytokines.
[00062] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, or 99% identical or similar to an EsxA protein. In certain aspects, the EsxA protein must have all or part of the amino acid sequence of SEQ ID NO:11.
[00063] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an EsxB protein. In certain aspects, the EsxB protein must have all or part of the amino acid sequence of SEQ ID NO:12.
[00064] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an SdrD protein. In certain aspects, the SdrD protein must have all or all or part of the amino acid sequence of SEQ ID NO:13.
[00065] In other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to an SdrE protein. In certain aspects, the SdrE protein may have all or part of the amino acid sequence of SEQ ID NO:14.
[00066] In still other embodiments of the invention, a composition can include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an IsdA protein. In certain aspects, the IsdA protein must have all or part of the amino acid sequence of SEQ ID NO:15.
[00067] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an IsdB protein. In certain aspects, the IsdB protein must have all or part of the amino acid sequence of SEQ ID NO:16.
[00068] Embodiments of the invention include compositions that include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% , or 99% identical or similar to an EsaB protein. In certain aspects, the EsaB protein must have all or part of the amino acid sequence of SEQ ID NO:17.
[00069] In other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to a ClfB protein. In certain aspects, the ClfB protein must have all or part of the amino acid sequence of SEQ ID NO:18.
[00070] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an IsdC protein. In certain aspects, the IsdC protein must have all or part of the amino acid sequence of SEQ ID NO:19.
[00071] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to a SasF protein. In certain aspects, the SasF protein must have all or part of the amino acid sequence of SEQ ID NO:20.
[00072] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein which is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an SdrC protein. In certain aspects, the SdrC protein must have all or part of the amino acid sequence of SEQ ID NO:21.
[00073] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to a CIfA protein. In certain aspects, the CIfA protein must have all or part of the amino acid sequence of SEQ ID NO:22.
[00074] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an Eap protein. In certain aspects, the Eap protein must have all or part of the amino acid sequence of SEQ ID NO:23.
[00075] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an Ebh protein. In certain aspects, the Ebh protein must have all or part of the amino acid sequence of SEQ ID NO:24.
[00076] In still other embodiments of the invention, a composition can include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an Emp protein. In certain aspects, the Emp protein must have all or part of the amino acid sequence of SEQ ID NO:25.
[00077] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an EsaC protein. In certain aspects, the EsaC protein must have all or part of the amino acid sequence of SEQ ID NO:26. The EsaC polypeptide sequence can be found in protein databases and includes, but is not limited to, accession numbers ZP_02760162 (GI:168727885), NP_645081.1 (GI:21281993), and NP_370813.1 (GI:15923279) , each of which is incorporated herein by reference as of the priority date of this patent application.
[00078] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein which is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to a Coa protein. In certain aspects, the Coa protein must have all or part of the amino acid sequence of SEQ ID NO:27.
[00079] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to an Hla protein. In certain aspects, the Hla protein must have all or part of the amino acid sequence of SEQ ID NO:28.
[00080] In still other embodiments of the invention, a composition can include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to a vWa protein. In certain aspects, the vWa protein must have all or part of the amino acid sequence of SEQ ID NO:29.
[00081] In still other embodiments of the invention, a composition may include a polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical or similar to a vWbp protein. In certain aspects, the vWbp protein must have all or part of the amino acid sequence of SEQ ID NO:32.
[00082] In certain aspects, a polypeptide or segment/fragment may 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 sequence of amino acids of the reference polypeptide. The term "similarity" refers to a polypeptide that has a sequence that has about a percentage of amino acids that are either identical to the reference polypeptide or constitute conservative substitutions in the reference polypeptides.
[00083] The polypeptides described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more variant amino acids within at least, or in the maximum 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 I I I , 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, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 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 in between, def SEQ ID NO:2- 30, or SEQ ID NO:32-34.
[00084] A polypeptide segment as described herein may include 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, 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, 206, 207, 208, 209, 2 10, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 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 in between these values, from SEQ ID NO:2-30, or SEQ ID NO:33-34.
[00085] The compositions may be formulated into a pharmaceutically acceptable composition. In certain aspects of the invention, the staphylococcal bacteria is a S. aureus bacterium.
[00086] In other aspects, a composition may be administered more than once to the subject, and may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more times . Administration of the compositions includes, but is not limited to, oral, parenteral, subcutaneous, intramuscular, intravenous administration, or various combinations of these administrations, including inhalation or aspiration.
[00087] In still other embodiments, a composition comprises a recombinant nucleic acid molecule encoding a polypeptide described herein or segments/fragments thereof. Typically, a recombinant nucleic acid molecule encoding a polypeptide described herein contains a heterologous promoter. In certain aspects, a recombinant nucleic acid molecule of the invention is a vector, in still other aspects, the vector is a plasmid. In certain embodiments, the vector is a viral vector. In certain aspects, a composition includes a recombinant non-staphylococcal bacterium that contains or expresses a polypeptide described herein. In specific aspects, the non-sphylococcal recombinant bacterium is Salmonella or other gram-positive bacteria. A composition is typically administered to mammals, such as human subjects, but administration to other animals that are capable of eliciting an immune response is contemplated. In other aspects, a staphylococcal bacteria that contains or expresses the polypeptide is Staphylococcus aureus. In other embodiments, the immune response is a protective immune response.
[00088] In other embodiments, a composition comprises a recombinant nucleic acid molecule encoding all or part of one or more Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB proteins , ClfA, ClfB, Coa, Hla, IsdC, SasF, SpA, vWbp, or vWh or peptide or variant thereof. Additional staphylococcal antigens that may be used in combination with the polypeptides described herein include, but are not limited to, 52 kDa vitronectin-binding protein (WO 01/60852), Aaa, Aap, Ant, autolysine glucosaminidase, autolysin amidase, Cna, protein collagen binder (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 exotoxins (WO 00/02523), SEB exotoxins (WO 00 /02523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein. In specific respects, a bacterium is a non-staphylococcal recombinant bacterium such as salmonella or other gram-positive bacteria.
[00089] The compositions of the invention are typically administered to human subjects, but administration to other animals that are capable of eliciting an immune response against a staphylococcal bacterium is contemplated, particularly cattle, horses, goats, sheep and other domestic animals, i.e. i.e. mammals.
[00090] In certain respects, the staphylococcal bacterium is Staphylococcus aureus. In other embodiments, the immune response is a protective immune response. In still other aspects, the methods and compositions of the invention can be used to prevent, ameliorate, reduce, or treat infection of tissues or glands, for example, mammary glands, particularly mastitis and other infections. Other methods include, but are not limited to, prophylactically reducing the bacterial load in an individual who does not show signs of infection, particularly those individuals suspected or at risk of being colonized by a target bacterium, e.g., patients who are or will be at risk or susceptible to infection during hospitalization, treatment, and/or recovery.
[00091] Any embodiment discussed with respect to one aspect of the invention also applies to other aspects of the invention. In any specific embodiment discussed in the context of a polypeptide or peptide or SpA variant nicleic acid can be implemented with respect to other antigens such as Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh, vitronectin binding protein 52 kDa (WO 01/60852), Aaa, Aap, Ant, autolysine glucosaminidase, autolysine 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 activator protein III (RAP), SasA, SasB, SasC, SasD , SasK,SBI, SdrF(WO 00/12689), SdrG / Fig (WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523), SEB exotoxins (WO 00/0 2523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein (or nucleic acids), and vice versa back It should also be understood that any one or more of Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh, protein 52 kDa vitronectin ligand (WO 01/60852), Aaa, Aap, Ant, autolysine glucosaminidase, autolysine 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+ ytansport, MHC II analogue (US5648240), MRPII, Npase, RNA activator protein III (RAP), SasA, SasB, SasC, SasD, SasK,SBI, SdrF(WO 00/12689), SdrG / Fig ( WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523), SEB exotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5, 801,234), SsaA, SSP-1, SSP-2, and/or protein bind Vitronectin components may be specifically excluded from a claimed composition.
[00092] Embodiments of the invention include compositions that do or do not contain a bacterium. A composition may or may not include an attenuated or viable or intact staphylococcal bacterium. In certain aspects, the composition comprises a bacterium that is not a staphylococcal bacterium or does not contain staphylococcal bacteria. In certain embodiments, a bacterial composition comprises an isolated or recombinantly expressed Staphylococcal Protein A variant or a nucleotide encoding the same. The composition may be or include a recombinantly engineered staphylococcal bacterium that has been altered in such a way as to comprise altering the bacterium for a secreted cell surface virulence factor or a protein. For example, the bacterium can be recombinantly modified to express more virulence factor or cell surface protein than it would if unmodified.
[00093] The term “isolated” may refer to a nucleic acid or polypeptide that is substantially free from cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) or uses source of origin, or chemical precursors or other chemicals (when chemically synthesized). Furthermore, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel. Furthermore, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that is not naturally occurring and/or is not typically in a functional state.
[00094] Portions of the invention, such as polypeptides, peptides, antigens, or immunogens, may be covalently or non-covalently conjugated to other moieties such as adjuvants, proteins, peptides, supports, fluorescent moieties, or labels. The term "conjugate" or "immunoconjugate" is widely used to define the operational association of a moiety with another agent and is not intended to refer solely to any type of operational association, and is not particularly limited to chemical "conjugation". Recombinant fusion proteins are particularly contemplated. Compositions of the invention may further comprise a pharmaceutically acceptable adjuvant or excipient. An adjuvant may be covalently or non-covalently coupled to a polypeptide or peptide of the invention. In certain aspects, the adjuvant is chemically conjugated to a protein, polypeptide, or peptide.
[00095] The term “supply” is used in its normal meaning to indicate “supply or provide the use”. In some embodiments, the protein is delivered directly by delivering the protein, while in other embodiments, the protein is delivered efficiently by delivering a nucleic acid encoding the protein. In certain aspects, the invention contemplates compositions that comprise various combinations of nucleic acids, antigens, peptides, and/or epitopes.
[00096] The individual must have (eg, be diagnosed with a staphylococcal infection), must be suspected of having, or must be at risk of developing a staphylococcal infection. The compositions of the present invention include immunogenic compositions in which the antigen(s) or epitope(s) are contained in an amount effective to achieve the intended purpose. More specifically, an effective amount means an amount of active ingredients necessary to stimulate or elicit an immune response, or confer resistance, amelioration or mitigation of infection. In more specific aspects, an effective amount prevents, alleviates or ameliorates the symptoms of the disease or infection, or prolongs the survival of the subject being treated. Determination of the effective amount is well within the skill of those skilled in the art, especially in light of the detailed description provided herein. For any preparation used in the methods of the invention, an effective amount or dose can be estimated initially from in vitro studies, cell culture, and/or animal model tests. For example, a dose can be formulated in animal models to achieve a desired immune response or concentration or titration of circulating antibody. Such information can be used to more accurately determine useful doses in humans.
[00097] The embodiments in the examples section are embodiments of the invention applicable to all aspects of the invention.
[00098] The use of the term “or” in the claims is to mean “and/or” unless explicitly stated to refer to alternatives only or the alternatives are mutually exclusive, although the description grounds a definition that refers to alternatives only and "and/or." It is also contemplated that anything listed using the term “or” may be specifically excluded.
[00099] In this entire patent application, the term "about" is used to indicate a value that includes the standard deviation of error for the device or method being employed to determine the value.
[000100] Following long-standing patent law, the words “the”, “the”, “a” and “an”, when used in conjunction with the word “comprises” in the claims or specification, denotes a or more, unless specifically noted.
[000101] Other objects, features and advantages of the present invention will become apparent from the detailed description which follows. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are provided for illustrative purposes only, as various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description. DESCRIPTION OF DRAWINGS
[000102] In order that the subject of the features, advantages and objects of the invention stated above, as well as others are evident and can be understood in detail, more specific descriptions and certain embodiments of the invention summarized above are illustrated in the accompanying drawings. These drawings form part of the descriptive report. It should be noted, however, that the attached drawings illustrate certain embodiments of the invention and, therefore, should not be considered limiting in scope.
[000103] Figures 1A-1B. (Figure 1A) Primary structure of the Protein A precursor with a YSIRK template signal peptide at the N-terminus, five immunoglobulin binding domains as tandem repeats designated E, D, A, B, C, X region, and the selection signal LPXTG. (Figure 1B) After the synthesis of the Protein A precursor, staphylococcus secrete this product via the Sec pathway, and sortase A cleaves the selection signal LPXTG between T and G residues. Nucleophilic attack of amine groups within lipid II in the no intermediate linked to Protein A thioester-sortase forms the amide bond that binds Protein A to the cell wall envelope and allows its presentation on the bacterial surface.
[000104] Figure 2 is a three-dimensional model of the molecular interactions between the D domain of Protein A SpA, the VH3 Fab domain of the B cell receptor, and the FCY domain of immunoglobulin. The model is obtained from two crystal structures (Graille et al., 2000 and Gouda et al., 1992) that revealed side chain residues involved in the formation of ionic bonds that enable these complexes. Gln-9 and Gln-10 of SpA-D promote binding to FCY, while Asp-36 and Asp-37 enable complex formation with VH3 Fab.
[000105] Figure 3. Left panel - Coomassie Blue stained SDS-PAGE reveals the migratory position of purified His-tagged SpA, SpA-D, SpA-DQ9,10K;D36,37A, human IgG, and sortase A ( SrtA), a control protein. Right table - Coomassie Blue stained SDS-PAGE to reveal eluted Protein A immunoglobulin complexes eluted after human IgG affinity chromatography on Ni-NTA columns preloaded with His-tagged SpA, SpA-D, SpA- DQ9.10K; D36.37A or MissA.
[000106] Figure 4. ELISA analyzes to quantify human immunoglobulin (hIgG), F(ab)2 fragments of human IgG and human Fc fragments of immunoglobulin (hFc). Plates were coated with equal amounts of His-tagged SpA, SpA-D, SpA-DQ9.10K;D36.37A or SrtA. hIgG-HRP, F(ab)2-HRP and hFc-HRP were added onto the plates and incubated for one hour. The absorbance at 450nm was recorded and plotted to determine half-maximal titers.
[000107] Figure 5. Purified SpA-D, SpA-DQ9,10K;D36,37A or a sham control with PBS were injected into the peritoneum of mice and analyzed for their ability to reduce the B cell population in the spleen of BALB mice /c experimental. The animals were terminated 4 hours after injection, their spleen was removed, the tissue was homogenized and stained with CD19 antibodies directed against B cells. The number of B cells was quantified by FACS selection.
[000108] Figure 6. Generation of a non-toxicogenic protein A vaccine, a translation product of protein A (SpA) from S. aureus a. Newman and USA300 LAC with an N-terminal signal peptide (open bar), five immunoglobulin-binding domains (IgBDs designated E, D, A, B, and C), X variable region, and C-terminal selection signal (filled bar). B. Amino acid sequence of the five IgBDs as well as non-toxicogenic SpA-DKKAA, with the positions of α triple helix bundles (H1, H2 and H3) as well as glutamine (Q) 9, 10 and aspartate (D) 36, 37 indicated . ç. SDS-PAGE stained with Coomassie Blue of SpA, SpA-D, SpA-DKKAA or SrtA purified on Ni-NTA Sepharose in the presence or absence of human immunoglobulin (hIgG). d. ELISA that examines the association of immobilized SpA, SpA-D or SpA-DKKAA with human IgG, as well as their Fc or F(ab)2 von Willebrand factor (vWF) fragments. and, CD19+ B lymphocytes in splenic tissue from BALB/c mice that had been mock immunized or treated with SpA-D or SpA-DKKAA were quantified by FACS.
[000109] Figure 7. Non-toxicogenic Protein A vaccine prevents abscess formation. The histopathology of renal tissue isolated during autopsy from BALB/c mice that had been mock immunized (PBS) or vaccinated with SpA, SpA-D as well as SpA-DKKAA and challenged with S. aureus Newman. Thin sectioned tissues were stained with hematoxylin-eosin. The hollow arrows identify infiltrates of polymorphonuclear leukocytes (PMN). Filled arrows identify staphylococcal abscess communities.
[000110] Figure 8. Antibodies created by the non-toxigenic Protein A vaccine block the superantigenic function of SpA B cells. The. Rabbit antibodies raised against SpA-DKKAA were purified on a matrix with immobilized antigen and analyzed by SDS-PAGE stained with Coomassie Blue. Antibodies were cleaved with pepsin and F(ab)2 fragments were purified by a second round of affinity chromatography on the SpA-DKKAA matrix. B. SpA-DKKAA-specific F(ab)2 and F(ab)2 interfere with binding of SpA or SpA-D to human immunoglobulin (hIgG), or, c. to the von Willebrand Factor (vWF).
[000111] Figure 9. Full-length non-toxicogenic protein A generates improved immune responses. The. Full-length SpAKKAA was purified on Ni-NTA Sepharose if analyzed by SDS-PAGE stained with Coomassie Blue. B. CD19+ B lymphocytes in mouse splenic tissue from BALB/c mice that had been mock immunized or treated with SpA or SpAKKAA were quantified by FACS. ç. ELISA which examines the association of immobilized SpA or SpAKKAA with human IgG as well as their Fc or F(ab)2 fragments or von Willebrand factor (vWF). d. Antibody titers in human or mouse serum to diphtheria toxoid (CRM197) and non-toxicogenic SpAKKAA or SpA-DKKAA. Human volunteers with a history of DTaP immunization and staphylococcal infection (n=16) as well as mice (n=20) that had been infected with S. aureus Newman or USA 300 LAC or immunized with SpAKKAA or SpA-DKKAA were examined by dot blot. quantitative.
[000112] Figure 10. Staphylococcal infection does not generate protective immunity. BALB/c mice (n=20) were infected with S. aureus Newman or sham challenged (PBS) for thirty days and the infection was stopped with chloramphenic treatment. Both animal cohorts were then challenged with S. aureus Newman and bacterial load (CFU) in the renal tissue homogenizer was analyzed after autopsy on Day 4.
[000113] Figure 11. Comparison of abscess formation in mice treated with PBS, SpA, SpA-D, and SpA-DKKAA.
[000114] Figures 12A-12C (A) ELISA which examines the association of immobilized SpA, SpA-D, SpA-DKKAA or SpA-DGGSS with human IgG as well as their Fc or F(ab)2 and IgM fragments. The statistical significance of binding of SpA-DKKAA and SpA-DGGSS to each ligand was compared to binding of SpA-D; SpA-D binding was compared to SpA (n=3); * means P<0.05; ** means P<0.01. (B) ELISA that examines the level of cross-reactive antibodies of hyperimmune serum samples collected from mice actively immunized (n=5) with SpA-D, SpA-DKKAA and SpA-DGGSS. (C) Abscess formation in mice treated with PBS, SpA-D, SpA-DKKAA and SpA-DGGSS. DETAILED DESCRIPTION OF THE INVENTION
[000115] Staphylococcus aureus is a commensal of human skin and nostrils, and is a major cause of bloodstream, skin, and soft tissue infections (Klevens et al., 2007). Recent dramatic increases in mortality from staphylococcal diseases are attributed to the spread of methicillin-resistant strains of S. aureus (MRSA) that 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 health care costs for 94,300 MRSA-infected individuals in the United States exceeded $2.4 billion (Klevens et al., 2007). The current MRSA epidemic has precipitated a health services crisis, which needs to be addressed by the development of a preventive vaccine (Boucher and Corey, 2008). To date, an FDA-licensed vaccine that prevents disease caused by S. aureus is not available.
[000116] The inventors describe herein the use of Protein A, a surface protein anchored to the cell wall of staphylococcus, for the generation of variants that can serve as subunit vaccines. The pathogenesis of staphylococcal indections is initiated as bacteria invade the skin or bloodstream through trauma, surgical wounds, or medical devices (Lowy, 1998). Although the invading pathogen can be phagocytosed and killed, staphylococci can also evade immune defenses and latent infections in organ tissues, inducing inflammatory responses that attract macrophages, neutrophils, and other phagocytes (Lowy, 1998). Responsive invasion of immune cells to the site of infection is accompanied by liquefaction necrosis as the host seeks to prevent staphylococcal spread and allow removal of necrotic tissue debris (Lam et al., 1963). Such lesions can be observed by microscopy, as the hypercellular areas contain necrotic tissue, leukocytes, and a central nest of bacteria (Lam et al., 1963). Unless staphylococcal accesses are surgically drained and treated with antibiotics, disseminated infection and septicemia produce a lethal outcome (Sheagren, 1984). 1. Staphylococcal antigens A. Staphylococcal Protein A (SpA)
[000117] All strains of Staphylococcus aureus express the structural gene for Protein A (SpA) (Jensen, 1958; Said-Salim et al., 2003), a well-characterized virulence factor whose surface product is anchored to the cell wall (SpA). ) encompasses five highly homologous immunoglobulin-binding domains designated E, D, A, B, and C (Sjodahl, 1977). These domains show ~80% identity at the amino acid level, are 56 to 61 residues in length, and are organized as tandem repeats (Uhlen et al., 1984). SpA is synthesized as a precursor protein with a signal peptide YSIRK/GS at the N-terminus and a selection signal with the template LPXTG at the C-terminus (DeDent et al., 2008; Schneewind et al., 1992). Protein A anchored to the cell wall is present in great abundance on the staphylococcal surface (DeDent et al., 2007; Sjoquist et al., 1972). Each of its immunoglobulin-binding domains is made up of antiparallel α helices that resemble the three-helix bundle and bind to the Fc domain of immunoglobulin G (IgG) (Deisenhofer, 1981; Deisenhofer et al., 1978), the heavy chain VH3 (Fab) from IgM (i.e. the B cell receptor) (Graille et al., 2000), von Willebrand factor and its A1 domain [vWF AI is a ligand for platelets] (O'Seaghdha et al. , 2006) and tumor necrosis factor α (TNF-α) receptor I (TNFRI) (Gomez et al., 2006), which is presented on airway epithelial surfaces (Gomez et al., 2004; Gomez et al., 2006). al., 2007).
[000118] SpA prevents phagocytosis of staphylococcal neutrophils through its ability to bind to the Fc component of IgG (Jensen, 1958; Uhlen et al., 1984). Furthermore, SpA is able to activate intravascular coagulation through its binding to Willebrand factor AI domains (Hartleib et al., 2000). Plasma proteins such as fibrinogen and fibronectin act as bridges between staphylococci (CIfA and CIfB) and platelet integrin GPIIb/IIIa (O'Brien et al., 2002), an activity that is supplemented through the association of Protein A with vWF AI , which allows staphylococci to capture platelets via the platelet receptor GPIb-α (Foster, 2005; O'Seaghdha et al., 2006). SpA also binds to TNFRI and its interaction contributes to the pathogenesis of staphylococcal pneumonia (Gomez et al., 2004). SpA activates pro-inflammatory signaling through TNFR1-mediated activation of TRAF2, the p38/c-Jun kinase, mitogens activate protein kinase (MAPK) and the transcription factor Rel, NF-KB. Binding of SpA also induces the release of TNFR1, an activity that appears to require the TNF-converting enzyme (TACE) (Gomez et al., 2007). All of the aforementioned SpA activities are mediated through its five IgG-binding domains and can be perturbed by the same amino acid substitutions, initially defined by their need for interaction between Protein A and human IgG1 (Cedergren et al., 1993.
[000119] SpA also functions as a B cell superantigen by capturing the Fab region of IgM-bearing VH3, the B cell receptor (Gomez et al., 2007; Goodyear et al., 2003; Goodyear and Silverman, 2004; Roben et al., 2007; Goodyear et al., 2003; Goodyear and Silverman, 2004; Roben et al., 2007; Goodyear et al., 2003; al., 1995). After intravenous challenge, staphylococcal Protein A (SpA) mutations show a reduction in staphylococcal burden in organ tissues and dramatically decreased ability to form abscesses (described herein). During infection with wild-type S. aureus, abscesses form within forty-eight hours and are detectable by light microscopy of thinly sectioned renal tissue stained with hematoxylin-eosin, initially marked by an influx of polymorphonuclear leukocytes (PMNs). On the fifth day of infection, the abscesses increase in size and become enclosed in a central population of staphylococci, surrounded by a layer of amorphous eosinophilic material and a large fraction of PMNs. Histopathology revealed an uncountable necrosis of PMNs in the vicinity of the staphylococcal nest in the center of abscess lesions as well as a blanket of healthy phagocytes. The inventors also observed a rim of necrotic PMNs at the periphery of the abscess lesions, delimiting the eosinophilic pseudocapsule that separated healthy renal tissue from the infectious lesion. Staphylococcal variants lacking Protein A are unable to establish the histopathological features of abscesses and are cleared during infection.
[000120] In previous studies, Cedergren et al. (1993) concocted individual substitutions in the Fc fragment linking the domain B subdomain of SpA, L17D, N28A, I31A and K35A. Those authors created these proteins to test data collected from the three-dimensional structure of a complex between a domain of SpA and Fc1. Cedergren et al. determined the effects of these mutations on stability and binding, but did not consider the use of these substitutions for the production of a vaccine antigen.
[000121] Brown et al. (1998) describe studies to manipulate new proteins based on SpA that allow the use of more favorable elution conditions when used as affinity ligands. The mutations studied included individual mutations of Q13A, Q14H, N15A, N15H, F17H, Y18F, L21H, N32H, or K39H. Brown et al. report that Q13A, N15A, N15H, and N32H substitutions made little difference to the dissociation constant values and that the Y18F substitution resulted in a 2-fold decrease in binding affinity compared to wild-type SpA. Brown et al. also report that the L21H and F17H substitutions decrease binding affinity by five-fold and one hundred-fold, respectively. Those authors also studied analogous substitutions in two tandem domains. Therefore, studies by Brown et al. were directed to generate a more favorable SpA elution profile, and thus the use of His substitutions to produce a pH sensitive change in binding affinity. Brown et al. is silent on the use of SpA as a vaccine antigen.
[000122] Graille et al. (2000) describe a crystal structure of the D domain of SpA and the Fab fragment of the human IgM antibody. Graille et al. define by analysis of a crystal structure the amino acid residues of domain D that interact with the Fab fragment such as residues Q26, G29, F30, Q32, S33, D36, D37, Q40, N43, E47, or L51, as well as residues of amino acids that form the interface between the subdomains of the D domain. Graille et al. define the molecular interactions of these two proteins, but are silent on any use of substitutions at interacting residues when producing a vaccine antigen.
[000123] O'Seaghdha et al. (2006) describe studies aimed at elucidating which subdomain of the D domain binds to vWF. The authors generated individual mutations in the Fc or VH3 binding subdomains, i.e. amino acid residues F5A, Q9A, Q10A, F13A, Y14A, L17A, N28A, I31A, K35A, G29A, F30A, S33A, D36A, D37A, Q40A, E47A , or Q32A.. The authors find that vWF binds to the same subdomain that binds Fc. O'Seaghda et al. define the subdomain of the D domain responsible for binding vWF, but are silent on any use of substitutions at interacting residues to produce vaccine antigen.
[000124] Gomez et al. (2006) describe the identification of residues responsible for TNFR1 activation using individual mutations of F5A, F13A, Y14A, L17A, N21A, I31A, Q32A, and K35A. Gomez et al. are silent on any use of substitutions at interacting residues to produce a vaccine antigen.
[000125] Affinity-tagged recombinant Protein A, a polypeptide encompassing the five domains of IgG (EDCAB) (Sjodahl, 1977) but lacking the C-terminal Region X (Guss et al., 1984), was purified from from recombinant E. coli and used as a vaccine antigen (Stranger-Jones et al., 2006). Because of SpA's attributes in binding to the Fc part of IgG, a specific humoral response to Protein A could not be measured (Stranger-Jones et al., 2006). The inventors overcame this obstacle by generating SpA-DQ9,10K;D36,37A. BALB/c mice immunized with recombinant Protein A (SpA) showed significant protection against intravenous challenge with S. aureus strains: a 2.951 log reduction in staphylococcal burden compared to wild type (P > 0.005; Student's t test) ( Stranger-Jones et al., 2006). SpA-specific antibodies may cause phagocytic clearance prior to abscess formation and/or impact formation of the aforementioned eosinophilic barrier in abscesses that separate staphylococcal populations of immune cells, as they do not form during infection with mutant strains of Protein. Each of the five domains of SpA (i.e., domains formed from triple-helix bundles designated E, D, A, B, and C) exert similar binding properties (Jansson et al., 1998). The solution and crystal structure of the D domain was resolved with and without the Fc and VH3 (Fab) ligands, which bind Protein A in a non-competitive manner at distant sites (Graille et al., 2000). Mutations at residues that are known to be involved in IgG binding (FS, Q9, Q10, S11, F13, Y14, L17, N28, I31 and K35) are also required for the binding of vWF AI and TNFR1 (Cedergren et al., 1993; Gomez et al., 2006; O'Seaghdha et al., 2006), while residues important for VH3 interaction (Q26, G29, F30, S33, D36, D37, Q40, N43, E47) do not seem to have any impact on other bonding activities (Graille et al., 2000; Jansson et al., 1998). SpA specifically targets a subset of B cells that express VH3 family-related IgM on its surface, ie, VH3-type B cell receptors (Roben et al., 1995). After interacting with SpA, these B cells proliferate and commit apoptosis, leading to the preferential and prolonged deletion of innate-like B lymphocytes (ie, marginal zone B cells and follicular B2 cells) (Goodyear et al., 2003; Goodyear et al., 2003; Goodyear et al. al., 2004). Molecular basis of the appearance and surface function of Protein A.
[000126] Protein A is synthesized as a precursor in the bacterial cytoplasm and secreted via its signal peptide YSIRK into the cross-wall, i.e. the dividing septum of staphylococcus (Figure 1) (DeDent et al., 2007; DeDent et al., 2008). After cleavage of the C-terminal selection signal LPXTG, Protein A is anchored to bacterial peptidoglycan cross-bridges by sortase A (Mazmanian et al., 1999; Schneewind et al., 1995; Mazmanian et al., 2000). Protein A is the most abundant surface protein of staphylococci; the molecule is expressed by virtually all strains of S. aureus (Cespedes et al., 2005; Kennedy et al., 2008; Said-Salim et al., 2003). Staphylococci rotate more than 15-20% of their cell wall per division cycle (Navarre and Schneewind, 1999). Murine hydrolases cleave the glycan and peptide strands of the polyglycan wall, thereby releasing Protein A with its attached disaccharide tetrapeptide from the C-terminal cell wall into the extracellular medium (Ton-That et al., 1999). Thus, by physiological design, Protein A is anchored to the cell wall and presented on the bacterial surface, but also released into surrounding tissues during host infection (Marraffini et al., 2006).
[000127] Protein A captures immunoglobulins on the bacterial surface and this biochemical activity allows staphylococci to escape the host's innate and acquired immune responses (Jensen, 1958; Goodyear et al., 2004). Interestingly, the I X region of Protein A (Guss et al., 1984), a repeat domain tying IgG-binding domains to the LPXTG selection signal/cell wall anchor, is perhaps the most variable part of the staphylococcal genome. (Said-Salim, 2003; Schneewind et al., 1992). Each of the five immunoglobulin binding domains of Protein A (SpA), formed from bundles of three helices are designated E, D, A, B, and C, exert similar structural and functional properties (Sjodahl, 1977; Jansson et al. ., 1998). The solution and crystal structure of the D domain was resolved with and without the Fc and VH3 (Fab) ligands, which bind Protein A in a non-competitive manner at distinct sites (Graille 2000).
[000128] In the crystal structure complex, Fab interacts with helix II and helix III of the D domain through a surface consisting of four β-strands of the VH region (Graille 2000). The main axis of helix II of the D domain is approximately 50° to the orientation of the strands, and the interhelix part of the D domain is closer to the C0 strand. The interaction site on Fab is distant from the Ig light chain and the heavy chain constant region. The interaction involves the following D domain residues: Asp-36 of helix II, Asp-37 and Gln-40 in the loop between helix II and helix III, and several other residues (Graille 2000). Both interacting surfaces are predominantly made up of polar side chains, with three negatively charged residues in the D domain and two positively charged residues in the 2A2 Fab hidden by the interaction, producing a global electrostatic attraction between the two molecules. Of the five polar interactions identified between Fab and the D domain, three are between side chains. A salt bridge is formed between Arg-H19 and Asp-36 and two hydrogen bonds are made between Tyr-H59 and Asp-37 and between Asn-H82a and Ser-33. Because of the conservation of Asp-36 and Asp-37 in all IgG binding domains of Protein A, the inventors mutated these residues.
[000129] The SpA-D sites responsible for Fab binding are structurally separate from the surface domain that mediates Fcy binding. The interaction of FCY with the D domain mainly involves residues in helix I with less involvement of helix II (Gouda et al., 1992; Deisenhofer, 1981). With the exception of Gln-32, a minor contact in both complexes, none of the residues that mediate the FCY ETA interaction involved in Fab binding. To examine the spatial relationship between these different Ig binding sites, the SpA domains in these complexes were superimposed to build a model of a complex between Fab, the SpA D domain, and the Fcy molecule. In this ternary model, Fab and Fcy form a sandwich around opposite faces of helix II with no evidence of steric hindrance from any interaction. These findings illustrate how, despite its small size (i.e., 56-61 aa), an SpA domain can exhibit both activities simultaneously, explaining the experimental evidence that Fab interactions with an individual domain are non-competitive. Residues for interactions between SpA-D and FCY are Gln-9 and Gln-10.
[000130] In contrast, occupation of the Fc part of IgG in the D domain blocks its interaction with vWF A1 and probably also TNFR1 (O'Seaghdha et al., 2006). Mutations at residues essential for IgG Fc pigging (F5, Q9, Q10, S11, F13, Y14, L17, N28, I31 and K35) are also required for binding of vWF A1 and TNFR1 (O'Seaghdha et al. , 2006; Cedergren et al., 1993; Gomez et al., 2006), while the critical residues for the VH3 interaction (Q26, G29, F30, S33, D36, D37, Q40, N43, E47) do not have any impact on IgG Fc, vWF A1, or TNFR1 binding activities (Jansson et al., 1998; Graille et al., 2000). The binding activity of Protein A immunoglobulin Fab targets a subset of B cells that express VH3 family-related IgM on their surface, i.e., these molecules function as VH3-like B cell receptors (Roben et al., 1995) . After interacting with SpA, these B cells rapidly proliferate and then undergo apoptosis, leading to the preferential and prolonged deletion of innate-like B lymphocytes (i.e., marginal B cells and follicular B2 cells) (Goodyear and Silverman, 2004; Goodyear and Silverman) , 2003). More than 40% of circulating B cells are targeted by Protein A interaction and the VH3 family interaction represents the largest family of human B cell receptors to confer protective humoral immune responses against pathogens (Goodyear and Silverman, 2004; Goodyear and Silverman). , 2003). Thus, Protein A functions analogously to staphylococcal superantigens (Roben et al., 1995), although the latter class of molecules, e.g. SEB, TSST-1, TSST-2, form complexes with the T cell receptor to inappropriately stimulating host immune responses, and thus, precipitating disease-characteristic features of staphylococcal infections (Roben et al., 1995; Tiedemann et al., 1995). Together these findings document the contributions of Protein A in establishing staphylococcal infections and in modulating host immune responses.
[000131] In summary, Protein A domains can be considered to have two different interfaces for binding to host molecules and any development of Protein A-based vaccines must consider generating variants that do not disrupt host cell signaling, aggregation of platelets, sequestration of immunoglobulins or the induction of B cell proliferation and apoptosis. These Protein A variants should also be useful for analyzing vaccines for the ability to generate antibodies that block the aforementioned activities of SpA and occupy the five repeat domains at their double-binding interfaces. This goal is articulated and pursued in this invention for the first time and methods are described in detail for generating Protein A variants that can be used as safe vaccines for humans. To disrupt IgG Fcy, the binding of vWF AI and TNFR1, glutamine (Q) 9 and 10 [numbering derived from the SpA D domain as described in Uhlen et al., 1984] were mutated, and lysine substitutions were generated for both. glutamines with the expectation that they abolish ligand attributes at the first binding interface. To disrupt binding of IgM Fab VH3, aspartate (D) 36 and 37 were mutated, each of which was required for association with the B cell receptor. D36 and D37 were both replaced with alanine. The Q9,10K and D36,37A mutations are in this case combined into the recombinant molecule SpA-DQ9,10K;D36,37A and tested for Protein A binding attributes. Furthermore, SpA-D and SpA-DQ9,10K;D36 .37A are subjected to immunization studies in mice and rabbits and analyzed for [1] production of specific antibodies (SpA-D Ab); [2] ability of SpA-D Ab to block the association of Protein A and its four different ligands; and [3] attributes of SpA-D Ab to generate protective immunity against staphylococcal infections (see examples section below). B. Staphylococcal Coagulases
[000132] Coagulases are enzymes produced by Staphylococcus bacteria that convert fibrinogen to fibrin. Coa and vWh activate prothrombin without proteolysis (Friedrich et al., 2003). The coagulase^prothrombin complex recognizes fibrinogen as a specific substrate, converting it directly to fibrin. The crystal structure of the active complex revealed binding of the D1 and D2 domains to prothrombin and insertion of its Ile1-Val2 N terminus into the Ile16 pocket, inducing a functional active site on the zymogen through the conformation change (Friedrich et al., 2003). α-thrombin exosite I, the fibrinogen recognition site, and pro-exosite I on prothrombin are blocked by Coa D2 (Friedrich et al., 2003). However, the association of the tetrameric complex (Coa^prothrombin)2 binds fibrinogen at a new site with high affinity (Panizzi et al., 2006). This model explains the coagulant properties and efficient conversion of fibrinogen by coagulase (Panizzi et al., 2006).
[000133] Fibrinogen is a large glycoprotein (Mr ~340,000), formed by three pairs of A a, B β, and Y chains covalently linked to form a “trimer dimer”, where A and B designate the fibrinopeptides released by the thrombin cleavage (Panizzi et al., 2006). The elongated molecule folds into three separate domains, a central E fragment that contains the N-terminus of all six chains and two flanking D fragments formed primarily by the C-terminus of the Bβ and y chains. These globular domains are connected by three long triple helix structures. Coagulase-prothrombin complexes, which convert human briinogen to fibrin in autopolymerization, are not targeted by inhibitors of circulating thrombin (Panizzi et al., 2006). Therefore, staphylococcal coagulases bypass the physiological blood clotting pathway.
[000134] All strains of S. aureus secrete coagulase and vWbp (Bjerketorp et al., 2004; Field and Smith, 1945). Although earlier work has reported important contributions of coagulase to the pathogenesis of staphylococcal infections (Ekstedt and Yotis, 1960; Smith et al., 1947), more recent investigations with molecular genetic tools have challenged this view by noting that there are no virulence phenotypes at all. with endocarditis, skin abscess, and mouse models of mastitis (Moreillon et al., 1995; Phonimdaeng et al., 1990). Generating isogenic variants of S. aureus Newman, a fully virulent clinical isolate (Duthie et al., 1952), it is described here that coa mutants do show virulence defects in a lethal bacteremia and mouse renal abscess model. In the inventors' experience, S. aureus 8325-4 is not fully virulent and it is assumed that mutational lesions in this strain may not be able to reveal viruence defects in vivo. Furthermore, antibodies generated against Coa or vWbp perturb the pathogenesis of S. aureus Newman infections to a degree that mirrors the impact of gene deletions. Coa and vWbp contribute to the formation of staphylococcal abscesses and lethal bacteremia, and may also function as protective antigens in subunit vaccines.
[000135] Biochemical studies document the biological value of antibodies against Coa and vWbp. By binding to the antigen and blocking its association with clotting factors, the antibodies prevent the formation of Coa^prothrombin and vWbp^prothrombin complexes. Passive transfer studies revealed protection of guinea pigs against the formation of staphylococcal accessions and lethal challenge by Coa and vWbp antibodies. Therefore, Coa and vWbp neutralizing antibodies generate immune protection against staphylococcal disease.
[000136] Early studies revealed a need for coagulase to resist phagocytosis in the blood (Smith et al., 1947) and the inventors observed a similar phenotype for Δcoa mutants in the blood of lepirudin-treated mice (see Example 3 below). As vWbp has a higher affinity for human prothrombin than the mouse counterpart, it is suspected that the same thing may be true for ΔvWbp variants in human blood. Furthermore, the expression of Coa and vWbp in abscess lesions, as well as their attacking distribution in the surrounding eosinophilic pseudocapsule (populations of staphylococcal abscesses (SACs) or the peripheral fibrin wall, suggest that secreted coagulases contribute to the establishment of these lesions. This hypothesis was tested and, in fact, the Δcoa mutants were defective in establishing abscesses. A corresponding test, blocking Coa function with specific antibodies, produced the same effect. Consequently, it is proposed that fibrin clotting is an event critical in establishing staphylococcal abscesses that can be targeted for the development of protective vaccines. Because of their overlapping function over human prothrombin, Coa and vWbp are considered excellent candidates for vaccine development. C. Other Staphylococcal Antigens
[000137] Research over the past few decades has identified S. aureus exotoxins, surface proteins, and regulatory molecules as important virulence factors (Foster, 2005; Mazmanian et al., 2001; Novick, 2003). Much progress has been made in regulating these genes. For example, staphylococci carry out a bacterial census by secreting self-inducing peptides that bind to a cognate receptor at the threshold concentration, thereby activating phosphotransfer reactions and activating the transcription of many exotoxin genes (Novick, 2003). The pathogenesis of staphylococcal infections is based on these virulence factors (secreted exotoxins, exopolysaccharides, and surface adhesins). The development of staphylococcal vaccines is hampered by the multifaceted nature of staphylococcal invasion mechanisms. It is well established that live attenuated microorganisms are highly effective vaccines; the immune responses elicited by these vaccines are often of greater magnitude and of longer duration than those produced by non-replicating immunogens. One explanation for this may be that live attenuated strains establish limited infections in the host and mimic the early stages of natural infection. Embodiments of the invention are directed to compositions and methods that include SpA variant polypeptides and peptides, as well as other immunogenic extracellular proteins, polypeptides, and peptides (including secreted and cell surface proteins or peptides) from gram-positive bacteria for use in mitigate or immunize against infection. In specific embodiments, the bacterium is a staphylococcal bacterium. Extracellular proteins, polypeptides, or peptides include, but are not limited to, secreted and cell surface proteins from the target bacteria.
[000138] The human pathogen S. aureus secretes EsxA and EsxB, two ESAT-6-like proteins, through the bacterial envelope (Burts et al., 2005, which is incorporated herein by reference). Staphylococcal esxA and esxB are grouped with six other genes in the order of transcription: esxA esaA essA esaB essB essC esaC esxB. The acronyms esa, ess, and esx stand for accessory, system, and extracellular secretion of ESAT-6, respectively, depending on whether the encoded proteins play an accessory (esa) or direct (ess) role for secretion, or are secreted (esx) in the extracellular medium. The entire cluster of eight genes is referred to herein as the Ess cluster. EsxA, esxB, essA, essB, and essC are all necessary for the synthesis or secretion of EsxA and EsxB. Mutants that fail to produce EsxA, EsxB, and EssC show defects in the pathogenesis of murine S. aureus abscesses, suggesting that this specialized secretion system may be a generic strategy for human bacterial pathogenesis. Secretion of WXG100 non-substrates via 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 WXG100 and also not WXG100 proteins in pathogenic mycobacteria (Abdallah et al., 2007; Abdallah et al., 2006).
[000139] The Staphylococcus aureus Ess pathway can be considered as a secretion module equipped with specialized transport components (Ess), accessory factors (Esa) and cognate secretion substrates (Esx). EssA, EssB, and EssC are required for the secretion of EsxA and EsxB. Due to the fact that EssA, EssB and EssC are predicted to be transmembrane proteins, it is contemplated that these proteins form a secretion mechanism. Some of the proteins in the ess gene cluster can actively transport secreted substrates (acting as motors) while others can regulate transport (regulators). Regulation can be achieved, but need not be limited to transcriptional or post-translational mechanisms for secreted polypeptides, selection of substrates specific to defined sites (e.g., extracellular medium or host cells), or timing of secretion events during infection. . At this point, it is uncertain whether all secreted Esx proteins function as toxins or indirectly contribute to pathogenesis.
[000140] Staphylococci rely on surface protein-mediated adhesion to host cells or tissue invasion as a strategy to evade immune defenses. Furthermore, S. aureus uses surface proteins to sequester iron from the host during infection. Most surface proteins involved in staphylococcal pathogenesis carry C-terminal selection signals, that is, they are covalently linked to the cell wall envelope by sortase. Furthermore, staphylococcal strains lacking the genes necessary for anchoring surface proteins, ie, sortase A and B, show a dramatic defect in virulence in several different mouse models of disease. Therefore, surface protein antigens represent a validated vaccine target, as the corresponding genes are essential for the development of staphylococcal disease and can be exploited in various embodiments of the invention. The sortase superfamily of enzymes are gram-positive transpeptities responsible for surface protein virulence factors for anchoring in the peptidoglycan cell wall layer. Two sortase isoforms were identified in Staphylococcus aureus, SrtA and SrtB. These enzymes have been shown to recognize a template of LPXTG in substrate proteins. The SrtB isoform appears to be important in the acquisition of heme iron and iron homeostasis, while the SrtA isoform plays a critical role in the pathogenesis of gram-positive bacteria by modulating the ability of the bacteria to adhere to host tissue through covalent anchoring of adhesins. and other proteins to the cell wall peptidoglycan. In certain embodiments, the SpA variants described herein may 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.
[000141] Certain aspects of the invention include methods and compositions concerning proteinaceous compositions including polypeptides, peptides, or SpA variants encoding nucleic acids and other staphylococcal antigens such as other proteins transported via the Ess pathway, or substrates of sortases. These proteins can be modified by deletion, insertion, and/or substitution.
[000142] Esx polypeptides include the amino acid sequence of Esx proteins from bacteria in the genus Staphylococcus. The Esx sequence can be from a specific staphylococcal species, such as Staphylococcus aureus, and can be from a specific strain, such as Newman. In certain embodiments, the EsxA sequence is SAV0282 from strain Mu50 (which is the same amino acid sequence as Newman) and can be accessed using Genbank Accession Number Q99WU4 (gi|68565539), which is incorporated herein by reference. In other embodiments, the EsxB sequence is SAV0290 from strain Mu50 (which is the same amino acid sequence as Newman) and can be accessed using Genbank Accession Number Q99WT7 (gi|68565532), which is incorporated herein by reference. In other embodiments, other polypeptides transported via the Ess pathway can be used, the sequences of which can be identified by those skilled in these techniques using databases and resources accessible on the internet.
[000143] Sortase substrate polypeptides include, but are not limited to, the amino acid sequences of the SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, IsdC or SasF proteins of bacteria in the genus Staphylococcus. The sequence of the sortase substrate polypeptides can be from a specific staphylococcal species, such as Staphylococcus aureus, and can be from a specific strain, such as Newman. In certain embodiments, the SdrD sequence is from strain N315 and can be accessed using Genbank Accession Number NP_373773.1 (gi|15926240), which is incorporated herein by reference. In other embodiments, the SdrE sequence is from strain N315 and can be accessed using Genbank Accession Number NP_373774.1 (gi|15926241), which is incorporated by reference. In other embodiments, the IsdA sequence is SAV1130 from strain Mu50 (which is the same amino acid sequence as Newman) and can be accessed using Genbank Accession Number NP_371654.1 (gi|15924120), which is incorporated by reference. In other embodiments, the IsdB sequence is SAV1129 of the Mu50 strain (which is the same amino acid sequence as Newman) and can be accessed using Genbank Accession Number NP_371653.1 (gi|15924119), which is incorporated by reference. In other embodiments, other polypeptides transported via the Ess pathway or processed by sortase can be used, the sequences of which can be identified by those skilled in these techniques using databases and resources accessible on the internet.
[000144] Examples of the various proteins that may be used in the context of the present invention can be identified by analyzing bacterial genome database submissions, including, but not limited to, 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 CP000255), NC_007795 (GI:87201381 and GenBank CP000253) each of which is incorporated by reference.
[000145] As used herein, the term "protein" or "polypeptide" refers to a molecule comprising at least ten amino acid residues. In some embodiments, a wild-type version of a protein or polypeptide is employed; however, in many embodiments of the invention, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A "modified protein" or "modified polypeptide" or a "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered from 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 proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to an activity or function, yet still retain a wild-type activity or function in other respects, such as immunogenicity.
[000146] In certain embodiments, the size of a protein or polypeptide (wild-type or modified) may comprise, but are not limited to, 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, 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, 850 875 , 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or more amine molecules, and any range between these values, or derived from a corresponding amino acid sequence described herein or referenced. It is contemplated that polypeptides can be mutated by being truncated, making them shorter than their corresponding wild-type form, but they could also be altered by fusing or conjugating a heterologous protein sequence with a specific function (e.g., to setting or location, for increased immunogenicity, for purification purposes, etc.).
[000147] As used herein, the term "amine molecule" refers to any amino acid, amino acid derivative, or amino acid mimetic known in the art. In certain embodiments, the residues of the proteinaceous molecule are sequential, with no molecules other than amine interrupting the sequence of residues of the amine molecule. In other embodiments, the sequence may comprise one or more groups of different amine molecules. In specific embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more groups of non-amine molecules.
[000148] Accordingly, the term "proteinaceous composition" encompasses sequences of amine molecules comprising at least one of the 20 amino acids common in naturally synthesized proteins, or at least one modified or unusual amino acid.
[000149] Proteinaceous compositions may be prepared by any technique known to those skilled in the art, including (i) the expression of proteins, polypeptides, or peptides by standard molecular biology techniques, (ii) the isolation of proteinaceous compounds from sources or (iii) the chemical synthesis of proteinaceous materials. The nucleotide, as well as the protein, polypeptide, and peptide sequences for several genes have been described previously, and can be found in recognized computer databases. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (on the worldwide web at ncbi.nlm.nih.gov/). The coding regions for these genes can be amplified and/or expressed using the techniques described herein or as known to those skilled in the art.
[000150] Amino acid sequence variants of SpA, coagulases and other polypeptides of the invention may be substitution, insertion or deletion variants. A variation in a polypeptide 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 polypeptide amino acids, compared to wild-type. A variant may comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values in between, identical to any sequence provided or referenced herein, for example, SEQ ID NO: 2-8 or SEQ ID NO:11-30. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids. A polypeptide processed or secreted via the Ess pathway or other surface proteins (see Table 1) or sortase substrates of any staphylococcal species and strain are contemplated for use in the compositions and methods described herein.
[000151] Deletional variants typically lack one or more residues from the native or wild-type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon can be introduced (by substitution or insertion) into a coding nucleic acid sequence to generate a truncated protein. Insertion mutants typically involve the addition of material at a non-terminal point on the polypeptide. This may include the insertion of one or more residues. Terminal additions, called fusion proteins, can also be generated. These fusion proteins include multimers or concatamers of one or more peptides or polypeptides described or referenced herein.
[000152] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, i.e., an amino acid is replaced by one of similar shape and charge. Conservative substitutions are well known in these techniques and include, for example, changes from: alanine to serine; arginine to lysine; asparagine for glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine for asparagine; glutamate to aspartate; glycine to proline; histidine for asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine for tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine for isoleucine or leucine. Alternatively, the substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing a residue with one that is chemically dissimilar, such as a polar or electrically charged amino acid with a nonpolar or unaltered amino acid, and vice versa.Table 2. Exemplary surface proteins from S. aureus strains


[000153] The proteins of the invention may be recombinant, or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein can be isolated from bacteria. It is also contemplated that a bacterium that contains a variant can be implemented in compositions and methods of the invention. Consequently, a protein does not need to be isolated.
[000154] The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see Table 2). below). Table 3 Codon Table


[000155] It should also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially as stated in one of the sequences described herein, provided that the sequence meets the criteria stated above, including maintenance of the protein's biological activity (eg, immunogenicity), where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking the 5' or 3' parts of the coding region.
[000156] The following text is a discussion based on changing the amino acids of a protein to create a variant polypeptide or peptide. For example, certain amino acids can be substituted for other amino acids in a protein structure with or without marked loss of interactive binding ability with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. As it is the interactive ability and nature of a protein that defines the functional activity of the protein, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and yet produce a protein with a desirable property. . It is thus contemplated that various changes can be made in the DNA sequences of genes.
[000157] It is contemplated that in compositions of the invention, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml. The concentration of protein in a composition can be at least about or at most 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 in between) . Of these values, about, at least about, or at most 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% may be an SpA variant or a coagulase, and may be used in combination with other peptides or polypeptides, such as other peptides and/or bacterial antigens.
[000158] The present invention contemplates the administration of variant SpA polypeptides or peptides to effect a preventive therapy or therapeutic effect against the development of a disease or condition associated with a staphylococcal pathogen.
[000159] In certain aspects, combinations of staphylococcal antigens are used in the production of an immunogenic composition that is effective in treating or preventing staphylococcal infection. Staphylococcal infections progress through several different stages. For example, the staphylococcal life cycle involves commensal colonization, initiation of infection by accessing adjacent tissues or the bloodstream, and/or anaerobic multiplication in the blood. The interaction between determinants of S. aureus virulence 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. Combinations of certain antigens can elicit an immune response that protects against multiple stages of staphylococcal infection. The effectiveness of the immune response can be measured in animal model assays and/or using an opsonophagocytic assay. D. Polypeptides and Polypeptide Production
[000160] The present invention describes polypeptides, peptides, and proteins and immunogenic fragments thereof for use in various embodiments of the present invention. For example, specific polypeptides are tested or used to elicit an immune response. In specific embodiments, all or part of the proteins of the invention can be synthesized in solution or on a solid support according to conventional techniques. Several automatic synthesizers are available on the market and can be used according to known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference.
[000161] Alternatively, recombinant DNA technology may be employed, where a nucleotide sequence encoding a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultured under appropriate conditions for expression.
[000162] An embodiment of the invention includes the use of gene transfer into cells, including microorganisms, for the production and/or presentation of polypeptides or peptides. The gene for the polypeptide or peptide of interest can be transferred into appropriate host cells, and then the cells cultured under the appropriate conditions. The generation of recombinant expression vectors, and the elements included therein, are well known in these techniques and are briefly discussed here. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell which is isolated and purified.
[000163] Another embodiment of the present invention uses autologous B lymphocyte cell lines, which are transfected with a viral vector that expresses an immunogenic product, and more specifically, a protein that has 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, as well as Chinese hamster ovary cell lines. , W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or that modifies and processes the gene product in the desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important to protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure correct processing modification of the expressed foreign protein.
[000164] A number of selection systems can be used, including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes, in tk, hgprt, or aprt cells, respectively. Furthermore, resistance to antimetabolites can be used as the basis of selection: in the case of 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.
[000165] Animal cells can be advertised in vitro in two ways: as non-anchorage-dependent cells that grow in suspension in the entire volume of the culture, or as anchor-dependent cells that require attachment to a solid substrate for propagation (i.e., a monolayer type of cell growth).
[000166] Non-encouragement-dependent suspension cultures from established continuous cell lines are the most widely used means of large-scale production of cells and cell products. However, suspension cultured cells have limitations, such as tumorogenic potential and lower protein production than adherent cells.
[000167] When a protein is specifically mentioned herein, it is preferably a reference to a native or recombinant protein or optionally a protein in which any signal sequence has been removed. The protein can be isolated directly from the staphylococcal strain or produced by recombinant DNA techniques. Immunogenic fragments of the protein can be incorporated into the immunogenic composition of the invention. They are fragments that comprise at least 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, or 100 amino acids, including all values and ranges between them, taken contiguously from the protein amino acid sequence. Furthermore, such immunogenic fragments are immunologically reactive with antibodies generated against staphylococcal proteins or with antibodies generated by infection of a mammalian host with staphylococcus. Immunogenic fragments also include fragments which, when administered at an effective dose (either alone or as a hapten bound to a carrier), elicit a protective or therapeutic immune response against staphylococcal infection, in certain respects it is protective against S. aureus infection. and/or S. epidermidis. Such an immunogenic fragment may include, for example, the protein lacking an N-terminal leader sequence, and/or a transmembrane domain and/or a C-terminal anchor domain. In a preferred aspect, the immunogenic fragment according to the invention comprises substantially the entire extracellular domain of a protein that has 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 between these values, with a selected segment of the sequence of a polypeptide described or referenced herein.
[000168] Also included in immunogenic compositions of the invention are fusion proteins consisting of one or more staphylococcal proteins, or immunogenic fragments of staphylococcal proteins. Such fusion proteins may be recombinantly prepared and may comprise a part of at least 1, 2, 3, 4, 5, or 6 staphylococcal sproteins or segments. Alternatively, a fusion protein may comprise multiple parts of at least 1, 2, 3, 4 or 5 staphylococcal proteins. They can combine different staphylococcal proteins and/or multiples of the same protein or protein fragment, or immunogenic fragments in the same protein (forming a multimer or a concatamer). Alternatively, the invention also includes individual fusion proteins of staphylococcal proteins or immunogenic fragments thereof, such as a fusion protein with heterologous sequences such as a T cell epithelium supplier or purification markers, for example: β-galactosidase, glutathione-S - transferase, green fluorescent proteins (GFP), epitope tags such as FLAG, myc tag, polyhistidine, or viral surface proteins such as influenza virus hemagglutinin, or bacterial proteins such as tetanus toxoid, diphtheria toxoid, or CRM197. II. NUCLEIC ACIDS
[000169] In certain embodiments, the present invention relates to recombinant polynucleotides encoding the proteins, polypeptides, peptides of the invention. Nucleic acid sequences for SpA, coagulases and other bacterial proteins are included, all of which are incorporated by reference, and can be used to prepare peptides or polypeptides.
[000170] As used in this patent application, the term "polynucleotide" refers to a nucleic acid molecule that is recombinant or has been isolated free from total genomic nucleic acid. Included in the term "polynucleotide" are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein coding sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA, or synthetic), their analogues, or a combination of them. Additional coding or non-coding sequences may, but not necessarily, be present in a polynucleotide.
[000171] In this regard, the term "gene," "polynucleotide," or "nucleic acid" is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences necessary for transcription, post modification, -translation, or appropriate localization). As should be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and manipulated smaller nucleic acid segments that express, or can be adapted to express, proteins, polypeptides, domains, peptides, fusion, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a sequence of contiguous nucleic acids of: 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, 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, 940, 950, 960, 970, 980, 990, 1,000, 1,010, 1,020, 1,030, 1,040, 1,050, 1,060, 1,070, 1,080, 1,090, 1,095, 1,100, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 9,000, 10,000, or more nucleotides, nucleosides, or base pairs, including all values and ranges between these values polynucleotide encoding one or more amino acid sequences are described or referenced herein. It is also contemplated that a specific polypeptide may be encoded by nucleic acids that contain variations that have slightly different nucleic acid sequences, yet encode the same or substantially similar protein (see Table 3 above).
[000172] In specific embodiments, the invention relates to isolated nucleic acid segments and recombinant vectors that incorporate nucleic acid sequences encoding an SpA or coagulase variant. The term "recombinant" may be used in conjunction with a polynucleotide or polypeptide and refers generically to a polypeptide or polynucleotide produced and/or manipulated in vitro or is a product of replication of such a molecule.
[000173] In other embodiments, the invention relates to isolated nucleic acid segments and recombinant vectors that incorporate nucleic acid sequences encoding a SpA or coagulase variant polypeptide or peptide to generate an immune response in an individual. In various embodiments, the nucleic acids of the invention can be used in genetic vaccines.
[000174] The nucleic acid segments used in the present invention may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, of such that its overall length can vary considerably. It is contemplated, therefore, that a nucleic acid fragment of almost any length may be employed, with the full length preferably being limited by ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification, transport, secretion, post-translational modification, or therapeutic benefit of the polypeptide, such as attention or efficacy. As discussed above, a tag or other heterologous polypeptide can be added to the coding sequence of the modified polypeptide, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.
[000175] In certain other embodiments, the invention relates to isolated nucleic acid segments and recombinant vectors that include in their sequence a contiguous nucleic acid sequence of SEQ ID NO:1 (D domain of SpA) or SEQ ID NO: 3 (SpA) or any other nucleic acid sequences encoding coagulases or other secreted virulence factors and/or surface proteins including proteins transported via the Ess pathway, processed by sortase, or proteins incorporated herein by reference.
[000176] In certain embodiments, the present invention provides polynucleotide variants that have substantial identity to the sequences described herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges in between, compared to a polynucleotide sequence of this invention, using the methods described herein (e.g., BLAST analysis using default parameters). The invention also contemplates the use of polynucleotides that are complementary to all of the polynucleotides described above. A. Vectors
[000177] The polypeptides of the invention may be encoded by a nucleic acid molecule comprised in a vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic sequence can be inserted for introduction into a cell, where it can be replicated and expressed. A nucleic acid sequence can be "heterologous", meaning it is in a context foreign to the cell into which the vector is being introduced or the nucleic acid into which it is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid, but at a position within the host cell or nucleic acid where it is not normally found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (eg, YACs). Those skilled in these techniques should be well versed in constructing a vector by standard recombinant techniques (eg Sambrook et al., 2001; Ausubel et al., 1996, both 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, a marker, or an immunogenicity-enhancing peptide. Useful vectors that encode such fusion proteins include pIN vectors (Inouye et al., 1985), vectors that encode an extension of histidines, and pGEX vectors, for use in generating soluble glutathione S-transferase (GST) fusion proteins for purification. purification and further separation or cleavage.
[000178] The term "expression vector" refers to a vector that contains a nucleic acid sequence that encodes at least part of a gene product capable of being transcribed. In some cases, the RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors may contain a series of "control sequences" that refer to nucleic acid sequences necessary for the transcription and possibly translation of a coding sequence operably linked in a specific host organism. In addition to control sequences that direct transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also serve other functions and are described herein. 1. Promoters and Enhancers
[000179] A “promoter” is a control sequence. The promoter is typically a region of a nucleic acid sequence in which the initiation and rate of transcription are controlled. It may contain genetic elements to which proteins and regulatory molecules can bind, such as RNA polymerase and other transcription factors. The terms "operably positioned", "operably linked", "under control" and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation with respect to a nucleic acid sequence to control initiation and expression. transcription 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.
[000180] Of course, it may be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type or organism chosen for expression. Those skilled in the art of molecular biology are aware of the promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al., 2001, incorporated herein by reference). The promoters employed can be constitutive, tissue-specific, or inducible and in certain embodiments can direct high-level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides.
[000181] Various elements/promoters can be employed in the context of the present invention to regulate the expression of a gene. Examples of such inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus, include, but are not limited to, Immunoglobulin Heavy Chain (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 Chain (Queen et al., 1983; Picard et al., 1984), T Cell Receptor (Luria et al., 1987; Winoto et al., 1989; Redondo et al.; 1990), HLA DQ α and/or DQ β □ QSullivan et al., 1987), Interferon β (Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988), Interleukin-2 (Greene et al., 1989), Interleukin Receptor -2 (Greene et al., 1989; Lin et al., 1990), MHC Class II 5 (Koch et al., 1989), MHC Class II HLA-DRα (απSherman et al., 1989), β-Actin ( Kawamoto et al., 1988; Ng et al.; 1989), Muscle Creatine Kinase (MCK) (Jaynes et al., 1988; Horlick et al. I., 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), β-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-Antitrypain (Latimer et al., 1990), H2B (TH2B) Histone (Hwang et al., 1990), Mouse Collagen and/or Type I (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), Growth Factor then Platelet Derived (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., 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. 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), Papilloma Virus (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), Gibbon Simian Leukemia Virus (Holbrook et al., 1987; Quinn et al., 1989).
[000182] Inducible elements include, but are not limited to, MT II - Phosphoryl 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., 1984; Ponta et al., 1983; ., 1985; Sakai et al., 1988); Interferon-β - 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 - Phosphoryl Ester Ester (TPA) (Angel et al., 1987b); SV40 - Phorbol Ester 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 H-2i<b gene - 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 - Thyroid Hormone α Gene (Chatterjee et al., 1989).
[000183] The specific promoter employed to control the expression of the peptide or protein encoding a polynucleotide of the invention is not believed to be critical, provided it is capable of expressing the polynucleotide in an targeted cell, preferably a bacterial cell. When a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter could include a bacterial, human or viral promoter.
[000184] In embodiments in which a vector is administered to an individual for expression of the protein, it is contemplated that a desirable promoter for use with the vector is one that is not down-regulated by cytokines or one that is strong enough even if down-regulated, it produces an effective amount of an SpA variant to elicit an immune response. Non-limiting examples of them are CMV IE and RSV LTR. Tissue-specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells or macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters. 2. Internal Ribosome Binding Sites (IRES) Initiation Signals
[000185] A specific initiation signal may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG start codon, may need to be provided. Those skilled in these techniques should be able to easily determine this and provide the necessary signals.
[000186] In certain embodiments of the invention, the use of internal ribosome binding site elements (IRES) is to create multigenic or polycistronic messages. IRES elements are able to deviate from the methylated Cap 5'π-dependent translational scan model and begin translation at internal sites (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see US Patent Nos. 5,925,565 and 5,935,819, incorporated herein by reference). 3. Selectable and Triable Markers
[000187] In certain embodiments of the invention, cells that contain a 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, a marker imparts an identifiable change to the cell allowing easy identification of cells that contain the expression vector. Generally, a selectable marker is one that confers a property that allows selection. A positive selectable marker is one in which the presence of the marker allows its selection, whereas a negative selectable marker is one in which the presence of the marker prevents its selection. An example of a positive selectable marker is a drug resistance marker. B. Host Cells
[000188] As used herein, the terms "cell", "cell lineage" and "cell culture" may be used interchangeably. All of these terms also include their progeny, which are any and all subsequent generations. It should be understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can and has been used as a receptor for vectors or viruses. A host cell can be "transfected" or "transformed", which refers to a process by which an exogenous nucleic acid, such as a recombinant protein coding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary cell in question and its progeny.
[000189] Host cells can be derived from prokaryotes and eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for vector replication or expression of part or all of the sequence(s) of nucleic acid(s). A number of cell lines and cultures are available for use as a host cell, and they can be obtained from the American Type Culture Collection (ATCC), which is an organization that serves as an archive for live cultures and genetic materials (www.atcc.org). ). C. Expression Systems
[000190] There are numerous expression systems that comprise at least some or all of the compositions discussed above. Prokaryotic and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many of these systems are commercially and widely available.
[000191] The insect cell/baculovirus system can produce a high level of protein expression from a heterologous nucleic acid segment, as described in US patents 5,871,986 and 4,879,236, both incorporated herein by reference, and which can be purchased, for example, under the name MAXBAC® 2.0 by INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM by CLONTECH®.
[000192] In addition to the described expression systems of the invention, other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL® Inducible Mammalian Expression System, which involves a synthetic inducible ecdysone receptor, or its pET Expression System, an expression system in E. coli. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ System (Tetracycline Regulated Expression), a mammalian inducible expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system, called the Pichia methanolica Expression System, which is designed for the production of high levels of recombinant proteins in the methylotrophic yeast Pichia methanolica. Those skilled in these techniques should know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide. III. POLYSACCHARIDES
[000193] The immunogenic compositions of the invention may further comprise capsular polysaccharides including one or more PIA (also known as PNAG) and/or S. aureus Type V and/or Type VIII capsular polysaccharide and/or Type I capsular polysaccharide and /or Type II and/or Type III of S. epidermidis. A. PIA (PNAG)
[000194] It is now clear that various forms of staphylococcal surface polysaccharides identified as PS/A, PIA and SAA are the same chemical entity - PNAG (Maira-Litran et al., 2004). Therefore, the term PIA or PNAG encompasses all these polysaccharides or oligosaccharides derived therefrom.
[000195] PIA is an adhesin intercellular polysaccharide and consists of a polymer of β-(1^6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. This polysaccharide is present in S. aureus and also in S. epidermidis and can be isolated from any source (Joyce et al., 2003; Maira-Litran et al., 2002). For example, PNAG can be isolated from S. aureus strain MN8m (WO 04/43407). PIA isolated from S. epidermidis is an integral constituent of biofilm. It is responsible for mediating cell/cell adhesion and probably also functions to shield the growing colony from the host's immune response. The polysaccharide formerly known as poly-N-succinyl-β-(1^6)-glucosamine (PNSG) has recently been shown not to have the expected structure, as the identification of N-succinylation was incorrect (Maira-Litran et al., 2002) . Therefore, the polysaccharide formerly known as PNSG and now recognized as PNAG is also encompassed by the term PIA.
[000196] PIA (or PNAG) can have different sizes ranging from more than 400 kDa to between 75 and 400 kDa to between 10 and 75 kDa for oligosaccharides consisting of up to 30 repeat units (of β-(1^6 linked glucosamine) ) substituted with N-acetyl and O-succinyl constituents). Any size of PIA polysaccharide or oligosaccharide can be used in an immunogenic composition of the invention; in one aspect, the polysaccharide is greater than 40 kDa. Sizing can be achieved by any method known in the art, for example by microfluidization, ultrasonic irradiation or by chemical cleavage (documents WO 03/53462, EP497524, EP497525). In certain aspects, 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.
[000197] PIA (PNAG) can have different degrees of acetylation due to substitution in the amine groups by acetate. In vitro produced PIA is almost completely substituted at the amine groups (95-100%). Alternatively, a deacetylated PIA (PNAG) can be used having less than 60%, 50%, 40%, 30%, 20%, 10% acetylation. The use of a deacetylated PIA (PNAG) is preferred, as the non-acetylated epitopes of PNAG are efficient in mediating the opsonic killing of gram-positive bacteria, preferably S. aureus and/or S. epidermidis. In certain aspects, PIA (PNAG) is between 40 kDa and 300 kDa in size and is deacetylated such that less than 60%, 50%, 40%, 30% or 20% of amine groups are acetylated.
[000198] The term deacetylated PNAG (dPNAG) refers to a PNAG polysaccharide or oligosaccharide in which less than 60%, 50%, 40%, 30%, 20% or 10% of the amine groups are acetylated. In certain aspects, PNAG is deacetylated to form dPNAG by chemically treating the native polysaccharide. For example, native PNAG is treated with a basic solution such that the pH rises above 10. For example, PNAG is treated with 0.15M, 0.2-4M, 0.15M NaOH, KOH, or NH4OH. .3-3M, 0.5-2M, 0.75-1.5M or 1M. Treatment is for at least 10 to 30 minutes, or 1, 2, 3, 4, 5, 10, 15 or 20 hours at a temperature of 20-100, 25-80, 30-60 or 30-50 or 35-45°C. dPNAG can be prepared as described in WO 04/43405.
[000199] The polysaccharide(s) may be conjugated or unconjugated to a carrier protein. B. Type 5 and Type 8 Polysaccharides of S. aureus
[000200] Most strains of S. aureus that cause infection in man contain either Type 5 or Type 8 polysaccharides. Approximately 60% of human strains are Type 8 and approximately 30% are Type 5. Structures Type 5 capsular polysaccharide antigens and Type 8 are described in Moreau et al., (1990) and Fournier et al., (1984). Both have FucNAcp in their repeat unit as well as ManNAcA which can be used to introduce a sulfhydryl group. The 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> Recently (Jones, 2005) NMR spectroscopy has revised the structures to : 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>
[000201] Polysaccharides can be extracted from the appropriate strain of S. aureus using a method well known to those skilled in these techniques; see US patent 6,294,177. For example, ATCC 12902 is a Type 5 strain of S. aureus and ATCC 12605 is a Type 8 strain of S. aureus.
[000202] Polysaccharides are natively sized or alternatively can be sized, for example, by microfluidization, ultrasonic irradiation, or by chemical treatment. The invention also covers oligosaccharides derived from S. aureus type 5 and 8 polysaccharides. The type 5 and 8 polysaccharides included in the immunogenic composition of the invention are preferably conjugated to a carrier protein as described below or are alternatively unconjugated. The immunogenic compositions of the invention alternatively contain type 5 or type 8 polysaccharide. C. S. aureus antigen 336
[000203] In one embodiment, the immunogenic composition of the invention comprises the S. aureus 336 antigen described in US patent 6,294,177. The 336 antigen comprises β-linked hexosamine, contains O-acetyl groups, and specifically binds to antibodies to S. aureus Type 336 deposited under ATCC 55804. In one embodiment, the 336 antigen is a polysaccharide that is natively sized or alternatively can be dimensioned, for example, by microfluidization, ultrasonic irradiation, or by chemical treatment. The invention also covers oligosaccharides derived from the 336 antigen. The 336 antigen can be unconjugated or conjugated to a carrier protein. D. Type I, II and III Polysaccharides of S. epidermidis
[000204] Among the problems associated with the use of polysaccharides in vaccination is the fact that polysaccharides by themselves are deficient immunogens. It is preferred that the polysaccharides used in the invention are linked to a protein carrier which provides bystander T cell assistance to enhance immunogenicity. Examples of such carriers that can be conjugated to polysaccharide immunogens include the diphtheria and tetanus toxoids (DT, DT CRM197 and TT respectively), Lapa Californiana Hemocyanin (KLH), and the purified protein derivative of Tuberculin (PPD), exoprotein A. aeruginosa (rEPA), Haemophilus influenzae protein D, pneumolysin, or fragments of any of the above carriers. Suitable fragments for use include fragments that encompass T-helper epitopes. In particular, the H. influenza protein D fragment should preferably contain 1/3 of the N-terminal protein. Protein D is an IgD-binding protein of Haemophilus influenzae (document in EP 0 594 610 B1) and is a potential immunogen. In addition, staphylococcal proteins can be used as a carrier protein in the polysaccharide conjugates of the invention.
[000205] A carrier protein would be particularly advantageous for use in the context of a staphylococcal vaccine and would be staphylococcal alpha toxoid. The native form can be conjugated to a polysaccharide, as the conjugation process reduces toxicity. Preferably, genetically detoxified alpha toxins such as His35Leu or His35Arg variants are used as carriers, as residual toxicity is lower. Alternatively, the alpha toxin is chemically detoxified by treatment with a cross-linking reagent, formaldehyde or glutaraldehyde. A genetically detoxified alpha toxin is optionally chemically detoxified, preferably by treatment with a crosslinking reagent, formaldehyde or glutaraldehyde to further reduce toxicity.
[000206] Polysaccharides may be linked to one or more carrier proteins by any known method (for example, the methods described in US patents 4,372,945, 4,474,757, and 4,356,170). Preferably, CDAP conjugation chemistry is conducted (see WO 95/08348). In CDAP, the cyanyl introduction reagent, 1-cyano-dimethylaminopyridinium tetrafluoroborate (CDAP), is preferably used for the synthesis of polysaccharide/protein conjugates. The cyanylation reaction can be carried out under relatively mild conditions, which prevents the hydrolysis of alkali-sensitive polysaccharides. This synthesis allows direct coupling to a carrier protein.
[000207] Conjugation involves preferably. Produce a direct link between the carrier protein and the polysaccharide. Optionally, a spacer (such as adipic dihydride (ADH)) can be introduced between the Carrier protein and the polysaccharide. IV. IMMUNE RESPONSE AND ASSAYS
[000208] As discussed above, the invention pertains to concerns provoking or inducing an immune response in an individual against an SpA variant or coagulase peptide. In one embodiment, the immune response may protect against or treat an individual who has, is suspected of having, or is at risk of developing an infection or related disease, particularly those related to staphylococci. One of the immunogenic compositions of the invention is for preventing nosocomial infections by inoculating an individual prior to undergoing procedures in a hospital or other setting that has a higher risk of infection. A. Immunoassays
[000209] The present invention includes the implementation of serological assays to assess whether and to what degree an immune response is induced or provoked by the compositions of the invention. There are many types of immunoassays that can be implemented. Immunoassays encompassed by the present invention include, but are not limited to, those described in US patent 4,367,110 (monoclonal antibody double sandwich assay), and US patent 4,452,901 (Western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.
[000210] Immunoassays are generally binding assays. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. In one example, antibodies or antigens are immobilized on a selected surface, such as a well in a polystyrene microtiter plate, dipstick, or column holder. Then, a test composition suspected of containing the desired antigen or antibody, such as a clinical specimen, is added to the wells. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen or antibody can be detected. Detection is generally performed 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 accomplished by adding a second antibody specific for the desired antigen, and then adding a third antibody that has binding affinity for the second antibody, with the third antibody being bound to a detectable label.Competition ELISAs are also possible implementations in which test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the unknown sample is determined by mixing the sample with the 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 for binding to the well, and thus, reduces the final signal. Regardless of the format employed, ELISAs have certain features in common, such as coating, incubation or binding, washing to remove nonspecifically bound species, and detecting bound immune complexes.
[000211] Antigens or antibodies can also be attached to a solid support, such as in the form of a plate, beads, dipstick, membrane, or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. When coating a plate with antigen or antibody, one should generally incubate the plate wells with a solution of the antigen or antibody overnight or for a specified period. The plate wells will then be washed to remove incompletely adsorbed material. Any remaining surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral to the test antisera. They include bovine serum albumin (BSA), casein, and powdered bed solutions. The coating allows blocking of nonspecific adsorption sites on the immobilizing surface, and thus, reduces background caused by nonspecific binding of antisera on the surface. B. Diagnosis of Bacterial Infection
[000212] In addition to the use of proteins, polypeptides, and/or peptides, as well as antibodies that bind to these polypeptides, proteins, and/or peptides, to treat or prevent infection as described above, the present invention contemplates the use of these polypeptides. , proteins, peptides, and/or antibodies in a number of ways, including detecting the presence of staphylococci to diagnose an infection, whether in a patient or medical equipment that may also become infected. In accordance with the invention, a preferred method of detecting the presence of infections involves the steps of obtaining a sample suspected of being infected by one or more species or strains of staphylococcal bacteria, such as a sample taken from an individual, for example, from saliva, tissue, bone, muscle, cartilage or skin of the individual. After sample isolation, diagnostic analyzes utilizing the polypeptides, proteins, peptides, and/or antibodies of the present invention can be conducted to detect the presence of staphylococci, and these analytical techniques for determining this presence in a sample are well known to those who skilled in these techniques and include methods such as radioimmunoassay analysis, Western blot analysis, and ELISA. In general, in accordance with the invention, a method for diagnosing an infection is contemplated, in which a sample suspected of being infected with staphylococci receives the addition of the polypeptide, protein, peptide, antibody, or monoclonal antibody according to the present invention. , and staphylococci are indicated by antibody binding to polypeptides, proteins, and/or peptides, or polypeptides, proteins, and/or peptides binding to antibodies in the sample.
[000213] Accordingly, antibodies according to the invention can be used for the prevention of infection by staphylococcal bacteria (i.e. passive immunization), for the treatment of an ongoing infection, or for use as research tools. The term "antibodies" as used herein includes monoclonal, polyclonal, chimeric, single-chain, bispecific, "simianized", and humanized or "primatized" antibodies, as well as Fab fragments, such as those fragments that maintain binding specificity. antibodies, including the products of a Fab immunoglobulin expression library. Accordingly, the invention contemplates the use of single chains such as the variable heavy and light chains of antibodies. The generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art. Specific examples of generating an antibody to a bacterial protein can be found in patent application publication US 20030153022 , which is incorporated herein by reference in its entirety.
[000214] Any of the above described polypeptides, proteins, peptides and/or antibodies can be directly labeled with a detectable marker for identification and quantification of staphylococcal bacteria. Labels for use in radioimmunoassays 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 beads or latex. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA). C. Protective Immunity
[000215] In some embodiments of the invention, proteinaceous compositions confer protective immunity to a subject. Protective immunity refers to the ability of a body to mount a specific immune response that protects the individual against the development of a specific disease or condition, which involves the agent against which there is an immune response. An immunogenically effective amount is capable of conferring protective immunity on the individual.
[000216] As used herein in the specification and in the claim framework that follows, the term polypeptide or peptide refers to an extension of amino acids covalently linked together via peptide bonds. Different polypeptides have different functionalities in accordance with the present invention. While in one aspect, a polypeptide is derived from an immunogen designed to induce an active response in a recipient, in another aspect of the invention, a polypeptide is derived from an antibody, which results after the induction of an immune response active in, for example, an animal, and which serves to induce a passive immune response in the recipient. In both cases, however, the polypeptide is encoded by a polynucleotide according to any possible codon usage.
[000217] As used herein, the term "immune response" or its equivalent "immune response" refers to the development of a humoral (antibody-mediated), cellular (mediated by antigen-specific T cells or their secretory products) response. or a humoral as well as cellular response directed against a protein, peptide, carbohydrate, or polypeptide of the invention in a recipient patient. This response can be an active response induced by the administration of immunogen or a passive response induced by the administration of antibody, antibody-containing material, or coated (primed) T cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4(+) helper T cells and/or cytotoxic CD8(+) T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, or other components of innate immunity. As used herein, the term "active immunity" refers to any immunity conferred on an individual by the administration of an antigen.
[000218] As used herein, the term "passive immunity" refers to any immunity conferred on a subject without the administration of an antigen to the subject. "Passive immunity", therefore, includes, but is not limited to, administration of activated immune effectors including cellular mediators or protein mediators (eg, monoclonal and/or polyclonal antibodies) of an immune response. A monoclonal or polyclonal antibody composition can be used in passive immunization for the prevention or treatment of infection by organisms carrying the antigen recognized by the antibody. An antibody composition may include antibodies that bind to a number of antigens which may, in turn, be associated with various organisms. The antibody component may be a polyclonal antiserum. In certain aspects, the antibody or antibodies are affinity purified from an animal or second individual that has been challenged with one or more antigens. Alternatively, a mixture of antibodies can be used, which is a mixture of monoclonal and/or polyclonal antibodies to antigens present on the same, related or different microbes or organisms, such as gram-positive bacteria, gram-negative bacteria, including but not limited to without limitations, staphylococcal bacteria.
[000219] Passive immunity may be conferred on a patient or individual by administering to the patient immunoglobulins (Ig) and/or other immune factors obtained from a donor or source other than a patient having known immunoreactivity. In other aspects, an antigenic composition of the present invention may be administered to an individual who then acts as a source or donor for globulin, produced in response to a challenge with the antigenic composition ("hyperimmune globulin"), which contains antibodies directed against staphylococcus. or other organism. A subject so treated would donate plasma from which the hyperimmune globulin would then be obtained via conventional plasma fractionation methodology, and administered to another subject to confer resistance against or to treat staphylococcal infection. The hyperimmune blobulins according to the invention are particularly useful for immunodeficient individuals, for individuals undergoing invasive procedures or when time does not allow the individual to produce their own antibodies in response to vaccination. See US Patent 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 herein by reference in its entirety for methods and compositions. eemplifications related to passive immunity.
[000220] For the 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 contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are not typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-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 indicates the ability of one antibody to block the binding of another antibody to a target antigen. T cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by 3H-thymidine uptake by primed T cells in response to an epitope (Burke et al., 1994). , by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) or by cytokine secretion.
[000221] The presence of a cell-mediated immune response can be determined by proliferation assays (CD4 (+) T cells) or CTL (cytotoxic T lymphocyte). The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T cells from an isogenic immunized animal and measuring the protective or therapeutic effect in a second individual.
[000222] As used in this specification and 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 immune response. recipient, which proteins include IgG, IgD, IgE, IgA, IgM and related proteins.
[000223] Under normal physiological conditions, antibodies are found in plasma and other body fluids and in the membrane of certain cells, and are produced by lymphocytes of the type denoted B cells or their functional equivalent. IgG class antibodies are made up of up to four polypeptide chains linked together by disulfide bonds. The four chains of intact IgG molecules are two identical heavy chains referred to as H chains and two identical light chains referred to as L chains.
[000224] To produce polyclonal antibodies, a host, such as a rabbit or goat, is immunized with the antigen or antigen fragment, generically with an adjuvant and, if necessary, coupled to a carrier. Antibodies to the antigen are subsequently collected from the host sera. The polyclonal antibody can be affinity purified against the antigen making it monospecific.
[000225] Monoclonal antibodies can be produced by hyperimmunization of an appropriate donor with the antigen or ex vivo using primary splenic cell cultures or spleen-derived cell lines (Anavi, 1998; Huston et al., 1991; Johnson et al., 1991; Mernaugh et al., 1995).
[000226] As used in this specification and claims, the phrase "an immunological part of an antibody" includes a Fab fragment of an antibody, an Fv fragment of an antibody, a heavy chain of an antibody, a light chain of an antibody , a heterodimer consisting of an antibody heavy chain and a light chain, a variable fragment of an antibody light chain, a variable fragment of an antibody heavy chain, and a variant single chain of an antibody, which is also known as scFv. In addition, the term includes chimeric immunoglobulins which are the expression products of fused genes derived from different species, one of the species being a human, in which case the chimeric immunoglobulin is said to be humanized. Typically, an immunological part of an antibody competes with the intact antibody from which it was derived for specific binding to an antigen.
[000227] Optionally, an antibody, or preferably an immunological part of an antibody, can be chemically conjugated, expressed as a fusion protein with other proteins. For the purposes of this specification and the appended claims, all such fused proteins are included in the definition of antibodies or an immunological part of an antibody.
[000228] As used herein, the terms "immunogenic agent" or "immunogen" or "antigen" are used interchangeably to describe a molecule capable of inducing an immune response against itself after administration to a recipient, either alone, in in conjunction with an adjuvant, or presented in an exhibition vehicle. D. Treatment Methods
[000229] A method of the present invention includes treating a disease or condition caused by a staphylococcal pathogen. An immunogenic polypeptide of the invention can be given to induce a response in a person infected with staphylococcus or suspected of having been exposed to staphylococcus. The methods may be employed for individuals who have tested positively for exposure to staphylococcus or who are considered to be at risk of infection based on possible exposure.
[000230] Particularly, the invention encompasses a method of treatment for staphylococcal infection, particularly hospital-acquired nocosomial infections. The immunogenic compositions and vaccines of the invention are particularly advantageous for use in elective surgery. Such patients must know the date of surgery in advance and could be inoculated in advance. The immunogenic compositions and vaccines of the invention are advantageous also for use in inoculating public health professionals.
[000231] In some embodiments, treatment is administered in the presence of adjuvants or carriers or other staphylococcal antigens. Furthermore, in some instances, the treatment comprises the administration of other agents commonly used against bacterial infection, such as one or more antibiotics. The use of peptides for vaccination may require, but not necessarily, the conjugation of the peptide to an immunogenic carrier protein. , such as hepatitis B surface antigen, keyhole limpet hemocyanin, or bovine serum albumin. Methods for effecting this conjugation are well known in the art. V. VACCINE AND OTHER PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION A. Vaccines
[000232] The present invention includes methods for preventing or ameliorating staphylococcal infections, particularly hospital-acquired nosocomial infections. Accordingly, the invention contemplates vaccines for use in active and passive immunization modalities. Immunogenic compositions proposed to be suitable for use as a vaccine can be prepared from immunogenic SpA polypeptides, such as a SpA D domain variant, or immunogenic coagulases. In other embodiments, SpA or coagulases can be used in combination with other virulent secreted proteins, surface proteins or immunogenic fragments thereof. In certain aspects, the antigenic material is dialyzed extensively to remove unwanted low molecular weight molecules and/or lyophilized for easier formulation into a desired vehicle.
[000233] Other options for a protein/peptide based vaccine envelop introducing nucleic acids encoding the antigen(s) as DNA vaccines. In this regard, recent reports have described the construction of recombinant vaccinia virus that express 10 contiguous minimal CTL epitopes (Thomson, 1996) or a combination of B cell epitopes, cytotoxic T cell lymphocytes (CTL), and T helpers (Th ) of various microbes (An, 1997), and the successful use of these constructs to immunize mice for priming protective immune responses. Therefore, there is ample evidence in the literature for the successful use of peptides, peptide-pulsed antigen presenting cells (APCs), and peptide-encoding constructs for efficient in vivo priming of protective immune responses. The use of nucleic acid sequences as vaccines is exemplified in US patents 5,958,895 and 5,620,896.
[000234] The preparation of vaccines that contain polypeptide or peptide sequence(s) as active ingredients is generally well known in these techniques, as exemplified in US patents 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 injectable products as liquid solutions or suspensions: solid forms for solution or suspension in a liquid prior to injection can also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerin, ethanol, or the like and combinations thereof. Furthermore, if desired, the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers, or adjuvants which enhance the effectiveness of the vaccines. In specific embodiments, vaccines are formulated with a combination of substances, as described in US patents 6,793,923 and 6,733,754, which are incorporated herein by reference.
[000235] Vaccines can be conventionally administered parenterally, by injection, eg subcutaneous or intramuscular injection. Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides: such suppositories may be formed from mixtures containing an active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%. Oral formulations include commonly used excipients such as 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 from about 10% to about 95% active ingredient, preferably from about 25% to about 70%.
[000236] Polypeptides and DNA constructs that encode polypeptides can be formulated into a vaccine in neutral or or salt form. Pharmaceutically acceptable salts include 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 acid, or organic acids such as acetic, oxalic, tartaric. , mandelic, and the like.
[000237] Typically, vaccines are administered in a manner compatible with the dosage formulation, and in an amount that should be therapeutically effective and immunogenic. The amount to be administered depends on the subject being treated, including the ability of the subject's immune system to synthesize antibodies and the degree of protection desired. The precise amounts of active ingredient needed to be administered depend on professional judgment. However, appropriate dosage ranges are on the order of several hundred micrograms of active ingredient per vaccination. Appropriate schedules for initial administration and booster injections are also varied, but are typified by an initial administration followed by subsequent inoculations of further administrations.
[000238] The way of application can be varied widely. Any of the conventional methods for administering a vaccine are applicable. They are believed to include oral application in a physiologically acceptable stable solid base or in a physiologically acceptable dispersion, parenterally, by injection and the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the individual.
[000239] In certain cases, it will be desirable to have multiple administrations of the vaccine, for example, 2, 3, 4, 5, 6 or more administrations. Vaccinations can be at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 5, 6, 7, 8, 9, 10, 11, 12 of twelve weeks, including all ranges in between. Periodic boosts at intervals of 1-5 may be desirable to maintain protective antibody levels. The course of immunization may be followed by testing for antibodies against the antigens, as described in US patents 3,791,932; 4,174,384 and 3,949,064. 1. Carriers
[000240] A given composition may vary its immunogenicity. It is often necessary, therefore, to reinforce the host's immune system, as can be achieved by coupling a peptide or polypeptide to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as egg albumin, mouse serum albumin, or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxy-succinimide ester, carbodiimide, and bis-benzidine bis-benzidine. 2. Adjuvants
[000241] The immunogenicity of polypeptide or peptide compositions can be enhanced by the use of non-specific stimulants of the immune response, known as adjuvants. Suitable adjuvants include all immunostimulating compounds, such as cytokines, toxins, or synthetic compositions. A number of adjuvants can be used to enhance an antibody response against a variant SpA polypeptide or coagulase, or any other bacterial protein or combination contemplated herein. Adjuvants can (1) trap the antigen in the body to cause a slow release; (2) attracting cells involved in the immune response to the site of administration; (3) induce proliferation or activation of immune system cells; or (4) enhance the spread of the antigen throughout the individual's body.
[000242] Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that can be used include IL-1, IL-2, IL-4, IL-7, IL-12, interferon y, GMCSP, BCG, aluminum salts such as aluminum hydroxide or other aluminum compound, 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, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MHC antigens can still be used. Other adjuvants or methods are exemplified in US patents 6,814,971, 5,084,269, 6,656,462, which are incorporated herein by reference.
[000243] Various methods to achieve adjuvant effect for the vaccine include the use of agents such as aluminum hydroxide or phosphate (alum), commonly used as a solution at about 0.05 to about 0.1% in saline buffered with phosphate, mixing with synthetic polymers of sugars (Carbopol®) used as a solution at about 0.25%, aggregation of the protein in the vaccine by heat treatment at temperatures in the range between about 70°C and about 101°C for a period of 30 seconds to 2 minutes respectively. Aggregation reactivating with pepsin-treated antibodies (Fab) to albumin; admixture with bacterial cells (eg, C. parvum), endotoxin or lipopolysaccharide components of gram-negative batteries; emulsion in physiologically acceptable oily vehicles (e.g. manid monooletate (Aracel A)); or emulsion with a 20% solution of perfluorocarbon (Fluosol-DA®) used as a substitute block can also be used to produce an adjuvant effect.
[000244] Examples of frequently preferred adjuvants include complete Freund's adjuvant (a non-specific stimulant of the immune response, which contains killed Mycobacterium tuberculosis), incomplete Freunda's adjuvant, and aluminum hydroxide.
[000245] In some respects, it is preferred that the adjuvant be selected to be a preferential inducer of a Th1 or Th2 type of response. High levels of Th1-type cytokines tend to favor the induction of cell-mediated immune responses against a given antigen, while high levels of Th2-type cytokines tend to favor the induction of humoral immune responses against the antigen.
[000246] The distinction of Th1 and Th2-type immune response is not absolute. In reality, an individual will support an immune response that is described as being predominantly Th1 or predominantly Th2. However, it is often convenient to consider cytokine families in terms of that 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-Y and IL-2 cytokines by T lymphocytes. Other cytokines often directly associated with the induction of Th1-type immune responses are not produced by T cells, such as IL-12. In contrast, Th2-type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10.
[000247] In addition to adjuvants, it may be desirable to co-administer biological response modifiers (BRM) to enhance immune responses. BRMs have been shown to up-regulate T-cell immunity or down-regulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (MIC; 1200 mg/d) (Smith/Kline, PA); or low dose (CYP; 300 mg/m2) cyclophosphamity (Johnson/Mead, NJ) and cytokines such as interferon Y, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7 . B. Lipid Components and Portions
[000248] In certain embodiments, the present invention pertains to compositions comprising one or more lipids associated with a nucleic acid or a polypeptide/peptide. A lipid is a substance that is insoluble in water and extractable with an organic solvent. Compounds other than those specifically described herein are understood by those skilled in the art to be lipids, and are encompassed by the compositions and methods of the present invention. A lipid and a non-lipid component can be attached to each other, either covalently or non-covalently.
[000249] A lipid can be a naturally occurring lipid or a synthetic lipid. However, a lipid is usually a biological substance. Biological lipids are well known in these techniques, and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether- and ester-linked fatty acids, and polymerizable lipids, and combinations thereof. .
[000250] A nucleic acid molecule or a polypeptide/peptide, associated with a lipid may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, bound covalently to a lipid, contained as a suspension in a lipid, or otherwise associated with a lipid. A lipid/poxvirus associated composition of the present invention is not limited to any specific structure. For example, they may simply be smeared into a solution, possibly forming aggregates that are not uniform in size or shape. In another example, they can be present in a bilayer structure, like micelles, or with a “collapsed” structure. In another non-limiting example, a lipofectamine (Gibco BRL)/poxvirus or Superfect (Qiagen)/poxvirus complex is also contemplated.
[000251] In certain embodiments, a composition may comprise 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%, or any range between these values, of a specific lipid, lipid type, or non-lipid component such as an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid, or other material described herein or as would be known to those skilled in these techniques. In a non-limiting example, an oid composition will comprise about 10% to about 20% neutral lipids, and about 33% to about 34% of a cerebroside, and about 1% cholesterol. In another non-limiting example, a liposome may comprise about 4% to about 12% terpenes, where about 1% of the micelle is specifically lycopene, leaving about 3% to about 11% of the liposome as comprising other terpenes; and about 10% to about 35% phosphatidylcholine, and about 1% of a non-lipid component. Accordingly, it is contemplated that the compositions of the present invention may comprise any of the lipids, types of lipids or other components in any combination or percentage range. C. Combined Therapy
[000252] The compositions and related methods of the present invention, particularly the administration of a secreted virulence factor or surface protein, including a variant SpA polypeptide or peptide, and/or other bacterial peptides or proteins, to a patient/subject, it can also be used in combination with the administration of traditional therapies. They include, but are not limited to, the administration of antibiotics such as streptomycin, ciprofloxacin, doxycycline, gentamicin, chloramphenicol, trimethoprim, sulfamethoxazole, ampicillin, tetracycline or various other combinations of antibiotics.
[000253] In one aspect, it is contemplated that a polypeptide vaccine and/or therapy is used in conjunction with antibacterial treatment. Alternatively, therapy may precede or follow treatment with another agent at intervals ranging from minutes to weeks. In certain embodiments in which the other agents and/or proteins or polynucleotides are administered separately, it must generally be ensured that a significant period of time does not expire between the time of each delivery, such that the agent and the antigenic composition are capable of to exert a beneficially combined effect on the individual. 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 situations, it may be desirable to extend the time period for administration significantly, where a range of several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6 , 7 or 8) between the respective administrations.
[000254] Various combinations may be employed, e.g. antibiotic therapy is “A” and the immunogenic molecule given as part of an immune therapy regimen, such as an antigen, is “B”: A/B/AB/A /BB/B/AA/A/BA/B/BB/A/AA/B/B/BB/A/B/BB/B/B/AB/B/A/BA/A/B/BA/B /A/BA/B/B/AB/B/A/AB/A/B/AB/A/A/BA/A/A/BB/A/A/AA/B/A/AA/A/B /THE
[000255] Administration of the immunogenic compositions of the present invention to a patient/subject will follow generic protocols for the administration of these compounds, taking into account the toxicity, if any, of the SpA composition, or other compositions described herein. Treatment cycles are expected to be repeated as needed. It is also contemplated that various customary therapies, such as hydration, may be applied in combination with the therapy described. D. General Pharmaceutical Compositions
[000256] In some embodiments, the pharmaceutical compositions are administered to a subject. Different aspects of the present invention involve administering an effective amount of a composition to a subject. In some embodiments of the present invention, staphylococcal antigens, members of the Ess pathway, including Esa or Esx class polypeptides or peptides, and/or members of sortase substrates may be administered to the patient to protect infection by one or more staphylococcal pathogens. Alternatively, an expression vector encoding one or more of these polypeptides or peptides can be given to a patient as a preventative treatment. Additionally, such compounds may be administered in combination with an antibiotic or an antibacterial. Such compositions should generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
[000257] In addition to compounds formulated for parenteral administration, such as those for intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets, or other solids for oral administration; controlled release capsules; and any other form currently used, including creams, lotions, mouthwashes, inhalants and the like.
[000258] The active compounds of the present invention can be formulated for parenteral administration, for example, formulated for injection intravenously, intramuscularly, subcutaneously, or even intraperitoneally. The preparation of an aqueous composition which contains a compound or compounds which increase the expression of a class I MHC molecule should be known to those skilled in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms for use in preparing solutions or suspensions after the addition of a liquid prior to injection may also be prepared and the preparations may also be emulsified.
[000259] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerin, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[000260] Pharmaceutical forms suitable for injectable use include aqueous solutions or dispersions; formulations that include 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 must be fluid to the degree that it can be injected easily. It must also be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
[000261] The proteinaceous compositions may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are 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 obtained from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.
[000262] The carrier can also be a solvent or dispersion medium that contains, for example, water, ethanol, polyol (for example, glycerin, propylene glycol, and liquid polyethylene glycol, and the like), appropriate mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the proper particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be generated 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 isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents which delay absorption, for example, aluminum monostearate and gelatin.
[000263] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as needed, and then filtering under sterile conditions. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other necessary ingredients among those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from a solution of them previously filtered under sterile conditions.
[000264] Administration of the compositions according to the present invention should typically be via any conventional route. This includes, but is not limited to, oral, nasal, or buccal administration. Alternatively, administration may be ortopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In certain embodiments, a vaccine composition may be inhaled (e.g., US Patent No. 6,651,655, which is specifically incorporated herein by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically 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, suitable for contacting human and animal tissues without toxicity, irritation, excessive allerphic response, or other problematic complications commensurate with the risk/benefit ratio. The term "pharmaceutically acceptable carrier" means 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.
[000265] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isothenic with sufficient saline or glucose. These specific aqueous solutions are spatially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous medium that can be employed should be known to those skilled in the art in light of the present disclosure. For example, a dosage could be dissolved in isotonic NaCl solution and added to the hypodermoclysis fluid or injected into the proposed infusion site (see, for example, Remington's Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the individual's condition. The person responsible for administration will, in any event, determine the appropriate dose for the subject in question.
[000266] An effective amount of the therapeutic or prophylactic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically distinct units suitable for use in an individual, each unit containing a predetermined amount of the composition calculated to produce the desired responses discussed above in connection with its administration, i.e., the route and the appropriate scheme. The amount to be administered, according to the number of treatments and also the unit dose, depends on the desired protection.
[000267] The precise amounts of the composition also depend on the judgment of the medical professional and are peculiar to each individual. Factors that affect the dose include the individual's physical and clinical status, route of administration, the intended goal of treatment (symptom relief versus cure), and the potency, stability, and toxicity of the specific composition.
[000268] After formulation, the solutions should be administered in a manner compatible with the dosage formulation and in a therapeutically or prophylactically effective amount. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. E. In vitro, ex vivo or in vivo administration
[000269] As used herein, the term in vitro administration refers to manipulations performed on cells removed from or outside a subject, including, but not limited to, cells in culture. The term ex vivo administration refers to cells that have been manipulated in vitro, and are subsequently administered to a subject. The term in vivo administration includes all manipulations performed within an individual.
[000270] In certain aspects of the present invention, the compositions may be administered in vitro, ex vivo, or in vivo. In certain in vitro embodiments, autologous B lymphocyte cell lines are incubated with a viral vector of the present invention for 24 to 48 hours or with SpA variant and/or coagulase and/or any other composition described herein for two hours. The transduced cells can then be used for in vitro analysis, or alternatively for ex vivo administration. US Patent Nos. 4,690,915 and 5,199,942, both hereby incorporated by reference, describe methods for ex vivo manipulation of blood mononuclear cells and bone marrow cells for use in therapeutic applications. F. Antibodies and Passive Immunization
[000271] Another aspect of the invention is a method of preparing an immunoglobulin for use in preventing or treating staphylococcal infection, comprising the steps of immunizing a recipient or donor with the vaccine of the invention and isolating the immunoglobulin from the recipient or donor. An immunoglobulin prepared by this method is another aspect of the invention. A pharmaceutical composition comprising the immunoglobulin of the invention and a pharmaceutically acceptable carrier is another aspect of the invention, which could be used in the manufacture of a medicament for the treatment or prevention of staphylococcal disease. A method for treating or preventing staphylococcal infection which comprises the step of administering to a patient an effective amount of the pharmaceutical preparation of the invention is another aspect of the invention.
[000272] Inoculums for the production of polyclonal antibodies are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent such as saline or other adjuvants suitable for human use to form an aqueous composition. An immune-stimulating amount of the inoculum is administered to a mammal and the inoculated mammal is then held for a time sufficient for the antigenic composition to induce protective antibodies.
[000273] Antibodies can be isolated to the desired degree by well known techniques such as affinity chromatography (Harlow and Lane, 1988). Antibodies may include antiserum preparations from a number of commonly used animals such as, for example, goats, primates, donkeys, pigs, horses, guinea pigs, rats or humans.
[000274] An immunoglobulin produced in accordance with the present invention may include whole antibodies, antibody fragments or sub-fragments. 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 invention. They can also be fragments (e.g. F(ab')2, Fab', Fab, Fv and the like) including hybrid fragments. An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act as an antibody by binding to specific antigens to form a complex.
[000275] A vaccine of the present invention can be administered to a recipient which then acts as a source of immunoglobulin, produced in response to challenge from the specific vaccine. An individual thus treated would donate plasma from which the hyperimmune globulin would be obtained through conventional plasma fractionation methodology. The hyperimmune globulin would be administered to another individual to confer resistance against or treat staphylococcal infection. The hyperimmune globulins of the invention are particularly useful for treating or preventing staphylococcal disease in neonates, immunocompromised individuals, or when treatment is necessary and there is no time for the individual to produce antibodies in response to vaccination.
[000276] A further aspect of the invention is a pharmaceutical composition comprising two or more monoclonal antibodies (or fragment thereof; preferably human or humanized) reactive against at least two constituents of the immunogenic composition of the invention, which could be used to treat or preventing infection by gram-positive bacteria, preferably staphylococci, more preferably S. aureus or S. epidermidis. Such pharmaceutical compositions comprise monoclonal antibodies which may be integral immunoglobulins of any class, chimeric antibodies, or hybrid antibodies with specificity for two or more antigens of the invention. They can also be fragments (e.g. F(ab') 2 , Fab', Fab, Fv and the like) including hybrid fragments.
[000277] Methods for producing monoclonal antibodies are well known in these techniques and may include the fusion of splenocytes with myeloma cells (Kohler and Milstein, 1975; Harlow and Lane, 1988). Alternatively, monoclonal Fv fragments can be obtained by screening an appropriate phage library (Vaughan et al., 1998). Monoclonal antibodies can be humanized or in part humanized by known methods. SAW. EXAMPLES
[000278] The following examples are provided for the purpose of illustrating various embodiments of the invention and are not intended to limit the present invention in any way. Those skilled in these techniques should appreciate that the present invention is well adapted to carry out the objectives and obtain the aforementioned purposes and advantages, as well as the objectives, purposes and advantages inherent therein. The present examples, together with the methods described are presently representative of preferred, and exemplary, embodiments, and are not intended to limit the scope of the invention. Changes therein and other uses that are encompassed within the spirit of the invention as defined within the scope of the claims are expected to occur to those skilled in the art. EXAMPLE 1 PROTEIN A NON-TOXIGENIC VARIANTS AS SUB-UNITARY VACCINES TO PREVENT STAPHYLOCOCCUS AUREUS INFECTIONS A. RESULTS Animal model for S. aureus infection
[000279] BALB/c mice were infected by intravenous injection with 1x10 7 CFU of the human clinical isolate S. aureus Newman (Baba et al., 2007). Within 6 hours after infection, 99.999% of staphylococci disappeared from the bloodstream and were distributed through the vasculature. Staphylococcal spread to peripheral tissues occurred rapidly, as the bacterial load in the kidney and other organ tissues reached 1 x 105 CFU g-1 within the first three hours. The staphylococcal load in renal recurrences increased by 1.5 log CFU within twenty-four hours. Forty-eight hours after infection, the mice developed abscesses disseminated in multiple organs, detectable by light microscopy of thin sectioned kidney tissue stained with hematoxylin-eosin. The initial diameter of the abscesses was 524 μM (± 65 μM); the lesions were initially marked by an influx of polymorphonuclear leukocytes (PMNs) and harbor no discernible organization of staphylococci, the majority of which appeared to reside in PMNs. On the fifth day of infection, the abscesses increased in size and contained a central population of staphylococci, surrounded by a layer of eosinophilic amorphous material and a large sheath of PMNs. Histopathology revealed heavy necrosis of PMNs in the vicinity of a staphylococcal focus in the center of the abscess lesions, as well as a blanket of healthy phagocytes. A rim of necrotic PMNs was observed at the periphery of abscess lesions, bordering eosinophilic amorphous material that separates healthy renal tissue from lesions. Abscesses eventually reached a diameter of > 1524 μM on Day 15 or 36. At later time intervals, the staphylococcal burden increased to 104-106 CFU g-1 and lesions from growing abscesses migrated toward the organ capsule. Peripheral lesions were susceptible to rupture, thereby releasing necrotic material and staphylococci into the peritoneal cavity or retroperitoneal space. These events resulted in bacteremia as well as a secondary wave of abscesses, eventually precipitating a lethal outcome.
[000280] To count the staphylococcal load in the kidney tissue, the animals were killed, their kidneys were excised and the tissue homogenate was spread on agar medium to form colonies. On the fifth day of infection, an average of 1x106 CFU g-1 in renal tissue for S. aureus Newman was observed. To quantify abscess formation, the kidneys were visually inspected, and each individual organ was given a score of one or zero. The final sum was divided by the total number of kidneys to calculate the percentage of superficial abscesses (Table 4). In addition, randomly selected kidneys were formalin-fixed, embedded, thinly sectioned, and stained with hematoxylin-eosin. For each kidney, four sagittal sections at 200 μM intervals were visualized by microscopy. Lesion numbers were counted for each section and averaged to quantify the number of abscesses within the kidneys. S. aureus Newman caused 4.364 ± 0.889 abscesses per kidney, and superficial abscesses were observed in 14 of 20 kidneys (70%) (Table 4).
[000281] When examined by scanning electron microscopy, S. aureus Newman was located in tightly associated coverings in the center of abscesses. Staphylococci were contained by an amorphous pseudocapsule that separated the bacteria from the leukocyte sheath of abscesses. No immune cells were observed in these central foci of staphylococci; however, occasional erythrocytes were located among the bacteria. The bacterial populations in the abscess center, designated “staphylococcal abscess populations” (SAC), appeared homogeneous and coated with electron-dense granular material. The kinetics of the appearance of infectious lesions and the morphological attributes of the abscesses formed by S. aureus Newman were similar to those observed after the infection of mice with S. aureus USA300 (LAC), the clone of S. aureus (CA-MRSA) resistant to methicillin acquired by the current epidemic community in the United States (Diep et al., 2006).Table 4. Genetic requirements for S. aureus Newman abscess formation in mice#
S. aureus Protein A (SpA) mutants are avirulent and cannot form abscesses
[000282] Sortase A is a transpeptidase that immobilizes nineteen surface proteins on the envelope of the S. aureus Newman strain (Mazmanian et al., 1999; Mazmanian et al., 2000). Previous work identified sortase A as a factor of cirulence in multiple animal model systems; however, the contributions of this enzyme and its anchored surface proteins to the formation or persistence of abscesses have not yet been revealed (Jonsson et al., 2002; Weiss et al., 2004). Compared to the wild-type predecessor (Baba et al., 2007), an isogenic variant srtA (ΔsrtA) failed to form abscess lesions on gross or histopathological examination on Days 2, 5, or 15. In mice infected with the mutant strA, only 1x104 CFU g-1 was recovered from kidney tissue on Day 5 of infection, which is a reduction of 2.046 log10 CFU g-1 compared to the wild-type parental strain (P=6.73x10-6 ). A similar defect was observed for the srtA mutant of the MRSA USA300 strain (data not shown). Scanning electron microscopy indicated that srtA mutants were highly dispersed and often associated with leukocytes in healthy kidney tissue. On Day fifteen after infection, srtA mutants were cleared from renal tissues, a >3.5 logic reduction of CFU g-1 compared to wild type (Table 3). Thus, sortase A-anchored surface proteins allow the formation of abscess lesions and the persistence of bacteria in host tissues, where staphylococci replicate as populations embedded in an extracellular matrix and shielded against surrounding leukocytes by an amorphous pseudocapsule.
[000283] Sortase A anchors a large spectrum of proteins with LPXTG template selection signals in the cell wall envelope, thereby providing the surface presentation of many virulence factors (Mazmanian et al., 2002). To identify surface proteins necessary for the formation of staphylococcal abscesses, bursa aurealis insertions were introduced into 5' coding sequences of genes encoding polypeptides with LPXTG model proteins (Bae et al., 2004) and these mutations were transduced into S. aureus. Newman. Mutations in the structural gene for Protein A (spa) reduced the staphylococcal burden in kidney tissues of infected mice by 1.004 log10 (P=0.0144). When analyzed for their ability to form abscesses in renal tissues by histopathology, the spa mutants were observed to be unable to form abscesses compared to the wild-type parental strain S. aureus Newman (wild-type S. aureus Newman 4.364 ± 0.889 kidney abscesses versus the isogenic spa mutant with 0.375 ± 0.374 lesions; P = 0.0356). Protein A blocks both innate and adaptive immune responses.
[000284] Studies have identified Protein A as a critical virulence factor during the pathogenesis of S. aureus infections. Previous work has shown that Protein A prevents phagocytosis of staphylococci by binding the Fc component of immunoglobulin (Jensen 1958; Uhlén et al., 1984), activates platelet aggregation via von Willebrand factor (Hartleib et al., 2000). ), functions as a B cell superantigen by capturing the VH3-bearing IgM F(ab)2 region (Roben et al., 1995), and through its activation of TNFR1, can initiate staphylococcal pneumonia (Gomez et al., 2004). ). Due to the fact that Protein A captures immunoglobulin and has toxic attributes, the possibility that this surface molecule could function as a vaccine in humans has not been rigorously pursued. The inventors demonstrate for the first time that Protein A variants incapable of binding immunoglobulins, vWF and TNFR-1 lose their toxigenic potential and are able to stimulate humoral immune responses that protect against staphylococcal disease. Molecular basis of the surface presentation and function of Protein A
[000285] Protein A is synthesized as a precursor in the bacterial cytoplasm and is secreted through its signal peptide YSIRK in the transverse wall, ie, the dividing septum of the staphylococcal cell (Figure 1). (DeDent et al., 2007; DeDent et al., 2008). After cleavage of the C-terminal selection signal LPXTG, Protein A is anchored to bacterial peptidoglycan cross-bridges by sortase A (Schneewind et al., 1995; Mazmanian et al., 1999; Mazmanian et al., 2000). Protein A is the most abundant surface protein of staphylococci; the molecule is expressed by virtually all strains of S. aureus (Said-Salim et al., 2003; Cespedes et al., 2005; Kennedy et al., 2008). Staphylococci turn over 15-20% of their cell wall per division cycle (Navarre and Schneewind 1999). Murine hydrolases cleave glycan filaments and peptidoglycan wall peptides, thereby releasing Protein A with its C-terminal cell wall disaccharide tetrapeptide into the extracellular medium (Ton-That et al., 1999). Thus, by physiological design, Protein A is anchored to the cell wall and presented on the bacterial surface, but released into surrounding tissues during host infection (Marraffini et al., 2006).
[000286] Protein A captures immunoglobulins on the bacterial surface and this biochemical activity allows staphylococci to escape from the host's innate and acquired immune responses (Jensen 1958; Goodyear and Silverman 2004). Interestingly, the X region of Protein A (Guss et al., 1984), a repeat domain that ties IgG-binding domains to the LPXTG/cell wall selection signal anchor, is perhaps the most variable part of the staphylococcal genome. (Schneewind et al., 1992; Said-Salim et al., 2003). Each of the five immunoglobulin binding domains of Protein A (SpA), formed from triple-helix bundles and designated E, D, A, B, and C, exert similar structural and functional properties (Sjodahl 1977; Jansson et al. , 1998). The solution e and the crystal structure of the D domain were resolved with and without the Fc and VH3 (Fab) ligands, which bind to Protein A in a non-competitive manner at distinct sites (Graille et al., 2000).
[000287] In the crystal structure complex, Fab interacts with helix II and helix III of the D domain through a surface consisting of four β-strands of the VH region (Graille et al., 2000). The main axis of helix II of the D domain is approximately 50° to the orientation of the strands, and the interhelix part of the D domain is closer to the C0 strand. The interaction site on Fab is distant from the Ig light chain and the heavy chain constant region. The interaction involves the following D domain residues: Asp-36 of helix II as well as Asp-37 and Gln-40 in the loop between helix II and helix III, in addition to other residues with SpA-D (Graille et al., 2000). Both interacting surfaces are predominantly made up of polar side chains, with three negatively charged residues in the D domain and two positively charged residues in Fab 2A2 hidden by the interaction, producing a global electrostatic attraction between the two molecules. Of the five polar interactions identified between Fab and the D domain, three are between side chains. A salt bridge is formed between Arg-H19 and Asp-36 and two hydrogen bonds are made between Tyr-H59 and Asp-37 and between Asn-H82a and Ser-33. Because of the conservation of Asp-36 and Asp-37 in all five IgG binding domains of Protein A, these residues were selected for mutagenesis.
[000288] The SpA-D sites responsible for Fab binding are structurally separate from the surface domain that mediates Fcy binding. The interaction of Fcy with domain B mainly involves residues in helix I with less involvement of helix II (Deisenhofer 1981; Gouda et al., 1992). With the exception of Gln-32, a minor contact in both complexes, none of the residues that mediate the Fcy interaction are involved in Fab binding. To examine the spatial relationship between these different Ig binding sites, the SpA domains in these complexes were superimposed to build a model of a complex between Fab, the SpA D domain, and the Fcy molecule. In this ternary model, Fab and Fcy form a sandwich around opposite faces of the helix II helix with no evidence of steric hindrance from any interaction. These findings illustrate how, despite its small size (i.e., 56-61 aa), an SpA domain can simultaneously exhibit both activities, explaining the experimental evidence that Fab interactions with an individual domain are non-competitive. Residues for interaction between SpA-D and Fcy are Gln-9 and Gln-10.
[000289] In contrast, occupation of the Fc part of IgG in the D domain blocks its interaction with vWF A1 and probably also TNFR1 (O'Seaghdha et al., 2006). Mutations at residues essential for binding of IgG Fc (F5, Q9, Q10, S11, F13, Y14, L17, N28, I31 and K35) are also required for binding of vWF A1 and TNFR1 (Cedergren et al., 1993). ; Gómez et al., 2006; O'Seaghdha et al. 2006), while the critical residues for the VH3 interaction (Q26, G29, F30, S33, D36, D37, Q40, N43, E47) have no impact. on the binding activities of IgG Fc, vWF A1 or TNFR1 (Jansson et al., 1998; Graille et al., 2000). Protein A immunoglobulin binding activity targets a subset of B cells that express IgM related to the VH3 family on their surface, i.e., these molecules function as receptors of VH3-type B cells (Roben et al., 1995) . After interacting with SpA, these B cells rapidly proliferate and then commit apoptosis, leading to the preferential and prolonged deletion of similar, innate B lymphocytes (i.e., marginal zone B cells and follicular B2 cells) (Goodyear and Silverman 2003; Goodyear et al. Silverman 2004). Importantly, more than 40% of circulating B cells are supported by Protein A interaction, and the VH3 family represents the largest family of human B cell receptors to confer protective humoral responses against pathogens (Goodyear and Silverman 2003; Goodyear and Silverman). 2004). Thus, Protein A functions analogously to staphylococcal superantigens (Roben et al., 1995), although the latter class of molecules, e.g. SEB, TSST-1, TSST-2, form complexes with the T cell receptor to inappropriately stimulating host immune responses and thus precipitating disease-characteristic features of staphylococcal infections (Roben et al., 1995; Tiedemann et al., 1995). Together these findings document the contributions of Protein A in establishing staphylococcal infections and modulating host immune responses. Non-toxigenic Protein A variant
[000290] The inventors developed a non-toxigenic variant of Staphylococcal Protein A and, with this reagent available, aimed for the first time to measure the immune response of animals to immunization of Protein A. In addition, the inventors set out to find out if immunization from animals with a non-toxigenic variant of Protein A could generate immune responses that create protective immunity against staphylococcal infection.
[000291] To disrupt the binding activities of IgG Fc, vWF A1 and TNFR1 of Protein A, glutamine residues (Q) 9 and 10 [the numbering in this case is derived from that established for the D domain of SpA] were modified generating substitutions of either lysine or glycine in place of both glutamines with the expectation that these substitutions abolish the ionic bonds formed between wild-type Protein A and its ligands. The added effect of the double lysine substitutions may be that these positively charged residues institute a repellent charge for immunoglobulins. To disrupt binding of IgM Fab VH3, the inventors selected aspartate (D) residues 36 and 37 of SpA-D, each of which is required for the association of Protein A with the B cell receptor. D36 and D37 were both replaced by alanine. The Q9,10K and D36,37A mutations were combined into the recombinant molecule SpA-DQ9,10K;D36,37A and examined for Protein A binding attributes.
[000292] Briefly, the Staphylococcus aureus N315 Protein A (spa) genomic sequence was amplified by PCR with the primers (GCTGCACATATGGCGCAACACGATGAAGCTCAAC [5'primer](SEQ ID NO:35) and AGTGGATCCTTATGCTTTGTTAGCATCTGC [3'primer] (SEQ ID NO :36)), cloned into the pET15b vector (pYSJ1, codons 48-486) (Stranger-Jones, et al., 2006) and recombinant plasmid transformed into E. coli BL21(DE3) (Studier et al., 1990). The Protein A product derived from pYSJ1 harbors SpA residues 36-265 fused to the N-terminal His tag (MGSSHHHHHHSSGLVPRGS (SEQ ID NO:37)). After inducible expression of IPTG, recombinant SpA His6-tagged at the N-terminus was purified by affinity chromatography on Ni-NTA resin (Stranger-Jones et al., 2006). The SpA domain D (SpA-D) was amplified by PCR with a specific primer pair (AACATATGTTCAACAAAGATCAACAAAGC [5' primer](SEQ ID NO:38) and AAGGATCCAGATTCGTTTAATTTTTTAGC [3'primer] (SEQ ID NO:39)), subcloned into pET15b vector (pHAN1, spa codons 212-261) and E. coli transformed recombinant plasmid BL21(DE3) to express and purify recombinant N-terminal His6-tagged protein. To generate mutations in the SpA-D coding sequence , sets of primer pairs were synthesized (for D to A substitutions: CTTCATTCAAAGTCTTAAAGCCGCCCCAAGCCAAAGCACTAAC [5' primer] (SEQ ID NO:40) and GTTAGTGCTTTGGCTTGGGGCGGCTTTAAGACTTTGAATGAAG [3'primer] (SEQ ID NO:41); for Q to K substitutions CATATGTTCAACAAAGATAAAAAAAAGCGCCTTCTATGAAATC [5'primer] (SEQ ID NO:42) and GATTTCATAGAAGGCGCTTTTTTTATCTTTGTTGAACATATG [3'primer] (SEQ ID NO:43); for Q-to-G substitutions CATATGTTCAACAAAGATGGAGGAAGCGCCTTCTATGAAATC [5'primer] (SEQ ID NO:44) and GATTTCATAGA AGGCGCTTCCTCCATCTTTGTTGAACATATG' [3' primer] (SEQ ID NO:45). Primers were used for fast-changing mutagenesis protocols. After mutagenesis, the DNA sequences were confirmed for each of the recombinant proteins: SpA, SpA-D and SpA-DQ9,10G;D36,37A and SpA-DQ9,10K;D36,37A. All proteins were purified from recombinant E. coli lysates using Ni-NTA chromatography and subsequently dialyzed against PBS and stored at 4°C.
[000293] To measure immunoglobulin binding to Protein A and its variants, 200 μg of purified protein was diluted in a volume of 1 mL using column buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and then loaded onto a pre-equilibrated Ni-NTA column (bed volume 1 mL). Columns were washed with 10 ml of column buffer. 200 µg of purified human IgG was diluted in a total volume of 1 mL of column buffer and then applied to each of the columns loaded with Protein A and its variants. The columns were subsequently washed with 5 ml of wash buffer (10 mM imidazole in column buffer) and 5 ml of column buffer. Protein samples were eluted with 2 mL of elution buffer (500 mM imidazole in column buffer), fractions were collected and aliquots were subjected to SDS-PAGE gel electrophoresis, followed by Coomassie Blue staining. As illustrated in Figure 3, wild-type Protein A (SpA) and its SpA D domain retained immunoglobulin during chromatography. In contrast, the SpA-DQ9,10K;D36,37A variant did not bind immunoglobulin.
[000294] To quantify the binding of Protein A and its variants to the Fc part of immunoglobulin and the VH3 domain of Fab, HRP-conjugated human immunoglobulin G [hIgG], the Fc part of human IgG [hFc] and the F( human IgG ab)2 [hF(ab)2] as well as ELISA analyzes were used to quantify the relative amount of binding to Protein A and its variants. The data in Figure 4 demonstrate binding of SpA and SpA-D to hIgG and hFc, while SpA-DQ9,10G;D36,37A and SpA-DQ9,10K;D36,37A showed only background binding activities. Similar amounts bound SpA from hFc and hF(ab)2; however, binding of SpA-D to hF(ab)2 was reduced compared to full-length SpA. This result suggests that the presence of multiple IgG binding domains may cooperatively increase the ability of Protein A to bind to the B cell receptor. When compared to the reduced binding potency of SpA-D to hF(ab)2, of the two variants only SpA-DQ9,10K;D36,37A showed a significant reduction in the ability to bind the immunoglobulin VH3 domain. To examine the toxigenic attributes of SpA-D and its variants, the purified proteins were injected into mice, which were sacrificed after 4 hours to remove their spleens. Organ tissues were homogenized, capsular material was removed and B cells were stained with fluorescent CD19 antibodies. After FACS analysis to quantify B cell abundance in splenic tissues, it was observed that SpA-D caused a 5% drop in B cell count compared to a sham control (PBS) (Figure 5). In contrast, SpA-DQ9,10K;D36,37A did not cause a reduction in B cell counts, indicating that the mutant molecule had lost its toxigenic attributes of stimulating B cell proliferation and death (Figure 5). In summary, s amino acid substitutions at SpA-D residues Q9, Q10, D36, and D37 abolished the ability of Protein A domains to bind immunoglobulins or exert toxigenic functions in human and animal tissues. Non-toxigenic Protein A variants elicit vaccine protection
[000295] To test whether or not Protein A and its variants can function as vaccine antigens, SpA, SpA-D, SpA-DQ9,10K;D36,37A, and SpA-DQ9,10K;D36,37A were emulsified with adjuvant complete or incomplete Freund test and were used to immunize 4-week-old BALB/c mice on Day 1 and Day 11 with 50 μg of purified protein. Animal cohorts (n=5) were analyzed for humoral immune responses to immunization by bleeding the animals before (Day 0) and after the immunization schedule (Day 21). Table 5 indicates that the immunized mice generated only a sparse humoral response directed toward wild-type Protein A or its SpA-D module, whereas the amount of antibody generated after immunization with SpA-DQ9,10K;D36,37A or SpA-DQ9.10K;D36.37A was increased four to five times. After the intravenous challenge with 1 x 10 7 CFU of S. aureus Newman, the animals were sacrificed on day 4, their kidneys were removed and analyzed for staphylococcal load (plating the tissue homogenate on agar plates and counting colonic forming units, CFU) or histopathology. As expected, mice (n = 19) sham-immunized (PBS) harbored 6.46 log10 (± 0.25) CFU in renal tissue, and infectious lesions were organized into 3.7 (± 1.2) abscesses per organ. (n=10) (Table 5). Immunization of animals with SpA led to a reduction of 2.51 log10 of CFU on day 5 (P=0.0003) with 2.1 (± 1.2) abscesses per organ. These last data indicate that there was no significant reduction in abscess formation (P=0.35). Immunization with SpA-D generated similar results: a 2.03 log10 reduction of CFU on day 5 (P=0.0001) with 1.5 (± 0.8) abscesses per organ (P=0.15). In contrast, immunization with SpA-DQ9.10K;D36.37A or SpA-DQ9.10G;D36.37A created greater protection, with a reduction of 3.07 log10 and 3.03 log10 CFU on day 4, respectively (significance P<0.0001 statistic for both observations). Furthermore, immunization with SpA-DQ9,10K;D36,37A and also SpA-DQ9,10G;D36,37A generated significant protection against the formation of staphylococcal abscesses, as only 0.5 (± 0.4) and 0. 8 (± 0.5) infectious lesions per organ (P=0.02 and P=0.04) were identified. Therefore, immunization with non-toxigenic Protein A variants generates increased humoral immune responses to Protein A and provides protective immunity against staphylococcal challenge. These data indicate that Protein A is an ideal candidate for a human vaccine that prevents S. aureus disease.
[000296] These encouraging results have several implications for the design of a human vaccine. First, the generation of substitution mutations that affect the immunoglobulin's ability to bind to Protein A domains, either alone or in combination of two or more domains, can generate non-toxigenic variants suitable for vaccine development. It seems possible that a combination of IgG binding domains that closely resemble the structure of Protein A can generate even better humoral immune responses as reported here for the SpA D domain alone. Furthermore, a possible attribute of Protein A-specific antibodies may be that the interaction of antigen-binding sites with the microbial surface may neutralize the ability of staphylococci to capture immunoglobulins via their Fc part or to stimulate the B cell receptor via intermediary of VH3 binding activities.

Models of vaccine protection in murine abscess, murine lethal infection and murine pneumonia.
[000297] Three animal models were established for the study of infectious disease by S. aureus. These models are used here to examine the level of protective immunity provided through the generation of Protein A-specific antibodies.
[000298] Murine Abscess - BALB/c Mice (24 day old, female, 8-10 mice per group, Charles River Laboratories, Wilmington, MA) are immunized by intramuscular injection in the hind leg with purified protein (Chang et al., 2003; Schneewind et al., 1992). Purified SpA, SpA-D or SpA-DQ9,10K;D36,37A (50 μg protein) are administered on days 0 (emulsified 1:1 with complete Freund's adjuvant) and 11 (emulsified 1:1 with incomplete Freund's adjuvant) ). Blood samples were collected by retroorbital bleeding on days 0, 11, and 20. Sera are examined by ELISA for IgG titers to specific binding activity of SpA-D and SpA-DQ9,10K;D36,37A. Immunized animals are challenged at 21 by retroorbital injection of 100 µL of S. aureus Newman or S. aureus USA300 suspension (1 x 10 7 CFU). For this, night cultures of S. aureus Newman are diluted 1:100 in tryptic soy broth and grown for 3 h at 37°C. Staphylococci are centrifuged, washed twice, and diluted in PBS to yield an A600 of 0.4 (1 x 10 8 CFU per mL). Dilutions are verified experimentally by plating on agar and colony formation. Mice are anesthetized by intraperitoneal injection of 80-120 mg of ketamine and 36 mg of xylazine per kilogram of body weight and given retroorbital injection. On day 5 or 15 after challenge, mice are sacrificed by inhalation of compressed CO2. The kidneys are removed and homogenized in 1% Triton X-100. Fractions are diluted and plated on agar medium for triplicate CFU determination. For hostology, kidney tissue is incubated at room temperature in 10% formalin for 24 h. Tissues are embedded in paraffin, thinly sectioned, stained with hematoxylinleosin, and examined by microscopy.
[000299] Murine Lethal Infection - BALB/c mice (24 days old, females, 8-10 mice per group, Charles River Laboratories, Wilmington, MA) are immunized by intramuscular injection in the hind leg with SpA, SpA-D or SpA -DQ9.10K; D36.37A purified (50 μg protein). The vaccine is administered on days 0 (emulsified 1:1 with complete Freund's adjuvant) and 11 (emulsified 1:1 with incomplete Freund's adjuvant). Blood samples were collected by retroorbital bleeding on days 0, 11, and 20. Sera are examined by ELISA for IgG titers with specific binding activity of SpA-D and SpA-DQ9,10K;D36,37A. Immunized animals are challenged on day 21 by retroorbital injection of 100 µL of S. aureus Newman or S. aureus USA300 suspension (15 x 10 7 CFU) (34). For this, overnight cultures of S. aureus Newman are diluted 1:100 in fresh tryptic soy broth and grown for 3 h at 37°C. Staphylococci are centrifuged, washed twice, diluted in PBS to yield an A600 of 0.4 (1 x 10 8 CFU per mL) and concentrated. Dilutions are verified experimentally by plating on colony formation agar. Mice are anesthetized by intraperitoneal injection of 80-120 mg of ketamine and 36 mg of xylazine per kilogram of body weight. Immunized animals are challenged on day 21 by intraperitoneal injection with 2 x 10 10 CFU of S. aureus Newman or 3-10 x 10 9 CFU of clinical isolates of S. aureus. The animals are monitored for 14 days, and the lethal disease is recorded.
[000300] Murine pneumonia model - S. aureus Newman or USA300 (LAC) strains are grown at 37°C in tryptic soy/agar broth to an OD660 0.5. 50-mL fractions of the culture are centrifuged, washed in PBS, and suspended in 750 μL PBS for mortality studies (3-4 x 10 8 CFU per 30 μL volume), or 1,250 μL PBS (2 x 10 8 CFU per 30 μL volume) for bacterial load and histology experiments (2, 3). For pulmonary infection, 7-week-old C57BL/6J mice (The Jackson Laboratory) are anesthetized prior to inoculation of 30 μL of S. aureus suspension into the left nostril. Animals are caged in a supine position for recovery and observed for 14 days. For active immunization, 4-week-old mice are given 20 μg of SpA-D or SpA-DQ9,10K;D36,37A in CFA on day 0 via the intramuscular route, followed by a booster with 20 μg of SpA- D or SpA-DQ9,10K;D36,37A in incomplete Freund's adjuvant (IFA) on day 10. Animals are challenged with S. aureus on day 21. Sera were collected before immunization and on day 20 to assess production of specific antibody. For passive immunization studies, 7-week-old mice receive 100 μL of NRS (normal rabbit serum) or SpA-D-specific rabbit antisera via intraperitoneal injection 24 h prior to challenge. To assess the pathological correlates of pneumonia, infected animals are killed by forced inhalation of CO2 before both lungs are removed. The right lung is homogenized to count the pulmonary bacterial load. The left lung is placed in 1% formalin and embedded in paraffin, sectioned thinly, stained with hematoxylin-eosin, and analyzed by microscopy.
[000301] Rabbit antibodies - 200 μg SpA-D or purified SpA-DQ9,10K;D36,37A are used as an immunogen for the production of rabbit antisera. 200 µg of protein is emulsified with CFA for injection on day 0, followed by booster injections with 200 µg of protein emulsified with IFA on days 21 and 42. Rabbit antibody titers are determined by ELISA. Purified antibodies are obtained by affinity chromatography of rabbit serum on SpA-D or SpA-DQ9,10K;D36,37A Sepharose. The concentration of eluted antibodies is measured by absorbance at A280 and specific antibody titers are determined by ELISA.
[000302] Active immunization with SpA D domain variants - To determine vaccine efficacy, animals are actively immunized with SpA-D or SpADQ9.10K; D36,37A purified. As a control, animals are immunized with adjuvant alone. Antibody titers against Protein A preparations are determined using SpA-D or SpA-DQ9,10K;D36,37A as antigens; note that the SpA-DQ9,10K;D36,37A variant fails to bind to the Fc or Fab part of IgG. Using the infectious disease models described above, any reduction in bacterial burden (abscess and murine pneumonia), histopathological evidence of staphylococcal disease (abscess and mutin pneumonia), and protection against lethal disease (challenge and lethal murine pneumonia) are measured.
[000303] Passive immunization with affinity purified polyclonal rabbit antibodies generated against the SpA-domain D variant. To determine the protective immunity of Protein A-specific rabbit antibodies, mice are passively immunized with 5 mg/kg of rabbit-derived antibodies of purified SpA-D or SpA-DQ9,10K;D36,37A. Both antibody preparations are purified by affinity chromatography using immobilized SpA-D or SpA-DQ9,10K;D36,37A. As a control, animals are passively immunized with rV10 antibodies (a pest protective antigen that has no impact on the outcome of staphylococcal infections). Antibody titers against all Protein A preparations are determined using SpA-DQ9.10K; D36,37A as an antigen, as this variant cannot bind to the Fc or Fab part of IgG. Using the infectious disease models described above, reduction in bacterial burden (abscess and murine pneumonia), histopathological evidence of staphylococcal disease (abscess and murine pneumonia), and protection against lethal disease (challenge and lethal murine pneumonia) are measured. EXAMPLE 2 PROTEIN A NON-TOXIGENIC VACCINE FOR METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS INFECTION
[000304] Clinical isolates of S. aureus express protein A (Shopsin et al., 1999, whose primary translation product consists of an N-terminal signal peptide (DeDent et al., 2008), five Ig-BDs (designated E , D, A, B and C) (Sjodahl, 1977), X region with variable repeats of an eight-residue peptide (Guss et al., 1984), and C-terminal selection signal for SpA anchoring in the cell wall (Schneewind et al., 1992; Schneewind et al., 1995) (Figure 6) Guided by amino acid homology (Uhlen et al., 1984), the α triple helix bundle structure of IgBDs (Deisenhofer et al., 1978; Deisenhofer et al., 1981) and their atomic interactions with Fab VH3 (Graille et al., 2000) or Fcy (Gouda et al., 1998), glutamine 9 and 10 were selected as well as aspartate 36 and 37 as critical for the association of SpA with antibodies or B cell receptor, respectively. The Gln9Lys, Gln10Lys, Asp36Ala and Asp37Ala substitutions were introduced into the D domain to generate SpA-DKKAA (F figure 6). The ability of isolated SpA-D or SpA-DKKAA to bind human IgG was analyzed by affinity chromatography (Figure 6). Polyhistidine-labeled SpA-D as well as full-length SpA retained human IgG on Ni-NTA, whereas SpA-DKKAA and a negative control (SrtA) did not (Figure 6). A similar result was observed with von Willebrand factor (Hartleib et al., 2000), which, together with tumor necrosis factor receptor 1 (TNFR1) (Gomez et al., 2004), can also bind to the protein. A via glutamine 9 and 10 (Figure 6). Human immunoglobulin comprises 60-70% of VH3 type IgG. The inventors distinguish between Fc domain and Ig B cell receptor activation and the measured association of human Fcy and F(ab)2 fragments, both bound to full-length SpA or SpA-D, but not binding to SpA-DKKAA (Figure 6). Injection of SpA-D into the peritoneal cavity of mice resulted in B cell expansion, and then apoptotic collapse of CD19+ lymphocytes in the spleen tissue of BALB/c mice (Goodyear and Silverman, 2003) (Figure 6). B cell superantigen activity was not observed after injection with SpA-DKKAA, and TUNEL staining of splenic tissue failed to detect an increase in apoptotic cells following injection of SpA or SpA-D (Figure 6).
[000305] Six-week-old naive BALB/c mice were injected with 50 μg of purified SpA, SpA-D or SPA-DKKAA emulsified in CFA and boosted with the same antigen emulsified in IFA. In agreement with the hypothesis that SpA-D promotes apoptotic collapse of activated clonal B cell populations, the inventors observed a tenfold higher titer of SpA-DKKAA specific antibodies after immunization of mice with the non-toxigenic variant compared to the B cell superantigen (Spa-D versus SpA-DKKAA P<0.0001, Table 6). Antibody titers by immunization with full-length SpA were higher than those elicited by SpA-D (P=0.0022), which is possibly due to the larger size and structure of the reiterative domain of this antigen (Table 6) . However, even SpA elicited lower antibody titers than SpA-DKKAA (P=0.0003), which encompasses only 50 amino acids of protein A (520 residues, SEQ ID NO:33). Immunized mice were challenged by intravenous inoculation with S. aureus Newman, and the ability of staphylococci to seed abscesses in renal tissues was examined by autopsai four days after challenge. In the homogenizing kidney tissue of similarly immunized mice (PBS/adjuvant), an average staphylococcal load of 6.46 log10 CFU g-1 was counted (Table 6). Immunization of mice with SpA or SpA-D led to a reduction in staphylococcal burden; however, animals vaccinated with SpA-DKKAA showed an even greater reduction, 3.07 log10 CFU g-1 of S. aureus Newman in renal tissues (P < 0.0001, Table 6). The formation of kidney abscesses was analyzed by histopathology (Figure 7). Mock-immunized animals harbored an average of 3.7 (±1.2) abscesses per kidney (Table 6). Vaccination with SpA-DKKAA reduced the mean number of abscesses to 0.5 (±0.4)(P= 0.0204), whereas immunization with SpA or SpA-D did not cause a significant reduction in the number of abscesses. abscesses (Table 6). Lesions from animals vaccinated with SpA-DKKAA were smaller in size, with fewer infiltrating PMNs, and characteristically lacked populations of staphylococcal abscesses (Cheng et al., 2009) (FIG. 7). Abscesses in animals that had been immunized with SpA or SpA-D showed the same overall structure of lesions in mock-immunized animals (Figure 7).
[000306] The inventors examined whether immunization with SpA-DKKAA can protect mice against MRSA strains, and selected the USA300 LAC isolate for animal challenge (Diep et al., 2006). This highly virulent CA-MRSA strain spread rapidly in the United States, causing significant human morbidity and mortality (Kennedy et al., 2008). Compared with adjuvanted control mice, animals immunized with SpA-DKKAA harbored a 1.07 log10 CFU g-1 reduction in bacterial load from infected kidney tissues. Histopathology examination of renal tissue after challenge with S. aureus USA300 revealed that the mean number of abscesses was reduced from 4.04 (±0.8) to 1.6 (±0.6) (P=0.02774). ). In contrast, immunization with SpA or SpA-D did not cause a significant reduction in bacterial load or abscess formation (Table 6).
[000307] Rabbits were immunized with SpA-DKKAA and specific antibodies were purified on SpA-DKKAA affinity column, and then by SDS-PAGE (Figure 8). SpA-DKKAA specific IgG was cleaved with pepsin to generate Fcy and F(ab)2 fragments, and the latter were purified by SpA-DKKAA column chromatography (Figure 8). Binding of human IgG or vWF to SpA or SpA-D was perturbed by SpA-DKKAA-specific F(ab)2, indicating that SpA-DKKAA-derived antibodies neutralize protein A B cell siperatigen function as well as their interactions with Ig (Figure 8).
[000308] To further improve the properties of the vaccine for non-toxigenic protein A, the inventors generated SpAKKAA, which includes all five IgBDs with four amino acid substitutions - substitutions corresponding to Gln9Lys, Gln10Lys, Asp36Ala and Asp37Ala of the D domain - in each one of its five domains (E, D, A, B and C). SpAKKAA labeled with polyhistidine was purified by affinity chromatography and analyzed by SDS-PAGE stained with Coomassie Blue (Figure 9). Unlike full-length SpA, SpAKKAA did not bind to human IgG, Fc and F(ab)2 or vWF (Figure 9). SpAKKAA no longer showed B cell superantigen activity, as injection of the variant into BALB/c mice did not cause a depletion of CD19+ B cells in the splenic tissue (Figure 9). Vaccination with SpAKKAA generated higher specific antibody titers than immunization with SpA-DKKAA and produced mice with high protection against S. aureus USA300 challenge (Table 6). Four days after challenge, animals vaccinated with SpAKKAA harbored 3.54 log10 CFU g-1 less staphylococci in kidney tissues (P=0.0001) and also caused a greater reduction in the number of abscess lesions (P=0.0109 ) (Table 6).
[000309] SpAKKAA was used to immunize rabbits. Leporid antibodies specific for SpA-DKKAA or SpAKKAA were affinity purified on matrices with immobilized cognate antigen and injected at a concentration of 5 mg kg-1 body weight into the peritoneal cavity of BALB/c mice (Table 7). Twenty-four hours later, specific antibody titers were determined in the serum and the animals were challenged by intravenous inoculation with S. aureus Newman. Passive transfer reduced the staphylococcal burden in kidney tissues to SpA-DKKAA (P=0.0016) or SpAKKAA (P=0.0005) specific antibodies. On histopathology examination, both antibodies reduced the abundance of abscess lesions in the kidneys of mice challenged with S. aureus Newman (Table 7). Together these data reveal that vaccine protection after immunization with SpA-DKKAA or SpAKKAA is conferred by antibodies that neutralize protein A. Table 6. Immunization of mice with vaccines for protein A.

Table 7. Passive immunization of mice with antibodies against protein A.


[000310] After infection with virulent S. aureus, mice did not develop protective immunity against subsequent infection with the monoma strain (Burts et al., 2008) (Figure 10). The mean abundance of SpA-DKKAA-specific IgG in these animals was determined by dot blot as 0.20 μg mL-1 (±0.04) and 0.14 μg mL-1 (±0.01) for the Newman and USA300 LAC, respectively (Figure 9). The minimum protein A-specific IgG concentration required for disease protection in animals vaccinated with SpAKKAA or SpA-DKKAA (P .0.05 log10 reduction in staphylococcal load CFU g-1 in renal tissue) was calculated as 4.05 μg mL-1 (±0.88). The mean serum concentration of SpA-specific IgG in healthy adult human volunteers (n=16) was 0.21 μg mL-1 (± 0.02). Therefore, S. aureus infections in mice or humans are not associated with immune responses that generate significant levels of neutralizing antibodies directed against protein A, which is possibly due to the B-cell superantigen attributes of this molecule. In contrast, the mean serum concentration of diphtheria toxin-specific IgG in human volunteers, 0.068 μg mL-1 (±0.20), was within the range for protective immunity against diphtheria (Behring, 1890; Lagergard et al., 1992) .
[000311] Clinical isolates of S. aureus express protein A, an essential virulence factor whose B-cell superantigen activity and evasive attributes against opsonophagocytic clearance are absolutely necessary for staphylococcal abscess formation (Palmqvist et al., 2005; Cheng et al., 2005; Cheng et al. al., 2009; Silverman and Goodyear, 2006). Protein A can thus be considered as a toxin, essential for pathogenesis, whose molecular attributes must be neutralized to achieve protective immunity. By generating non-toxigenic variants incapable of binding to Igs via the Fcy or VH3-Fab domains, the inventors measure for the first time protein A neutralizing immune responses as a correlate for protective immunity against S. aureus infection. In contrast to many methicillin-sensitive strains, the CA-MRSA isolate from USA300 LAC is significantly more virulent (Cheng et al., 2009). For example, immunization of guinea pigs with the surface protein IsdB (Kuklin et al., 2006; Stranger-Jones et al., 2006) generates antibodies that confer protection against S. aureus Newman (Stranger-Jones et al., 2009), but not against challenge with USA300. The methods used include:
[000312] Bacterial strains and growth. Staphylococcus aureus Newman and USA300 strains were grown in tryptic soy broth (TSB) at 37°C. Escherichia coli strains DH5α and BL21 (DE3) were grown in Luria-Bertani (LB) broth with 100 μg mL-1 ampicillin at 37 °C.
[000313] Rabbit antibodies. The SpA coding sequence was amplified by PCR with two primers, gctgcacatatggcgcaacacgatgaagctcaac (SEQ ID NO:35) and agtggatccttatgcttgagctttgttagcatctgc (SEQ ID NO:36) using S. aureus Newman template DNA. SpA-D was amplified by PCR with two primers, aacatatgttcaacaaagatcaacaaagc (SEQ ID NO:38) and aaggatccagattcgtttaattttttagc (SEQ ID NO:39). The sequence was mutagenized to SPA- DKKAA with two sets of primers, catatgttcaacaaagataaaaaaagcgccttctatgaaatc (SEQ ID NO: 42) and gatttcatagaaggcgctttttttatctttgttgaacatatg (SEQ ID NO: 43) for Q9K, Q10K and cttcattcaaagtcttaaagccgccccaagccaaagcactaac (SEQ ID NO: 40) and gttagtgctttggcttggggcggctttaagactttgaatgaag (SEQ ID NO:41) for D36A,D37A. The SpAKKAA sequence was synthesized by Integrated DNA Technologies, Inc. PCR products were cloned into pET-15b generating His6-tagged recombinant protein at the N-terminus. Plasmids were transformed into BL21(DE3). Overnight cultures of transformants were diluted 1:100 in fresh medium and grown at 37°C to an OD600 of 0.5, at which point the cultures were induced with 1 mM isopropyl β-D-1-thiogalatopyranoside (IPTG) and grown for more three hours. Bacterial cells were pelleted by centrifugation, placed and suspended in column buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) and disrupted with a French pressure cell at 953 atm (14,000 psi). Lysates were removed from the membrane and insoluble components by ultracentrifugation at 40,000 xg. Proteins in the soluble lysate were subjected to nickel/nitiloacetic acid (Ni-NTA, Qiagen) affinity chromatography. Proteins were eluted in column buffer containing successively higher concentrations of imidazole (100-500 mM). Protein concentrations were determined by bicinconic acid (BCA) analysis (Thermo Scientific). For antibody generation, rabbits (6 months old, New Zealand breed, Caucasian, female, Charles River Laboratories) were immunized with 500 μg of protein emulsified in Complete Freund's Adjuvant (Difco) subscapular injection. For booster immunizations, proteins emulsified in Incomplete Freund's Adjuvant and injection 24 or 48 days after the initial immunization. On day 60, rabbits were bled and serum was collected.
[000314] Purified antigen (5 mg protein) was covalently bound to HP columns activated with HiTrap NHS (GE Healthcare). Antigen coupled to a matrix was used for affinity chromatography of 10-20 ml of rabbit serum at 4°C. The loaded matrix was washed with 50 column volumes of PBS, antibodies were eluted with elution buffer (1M glycine, pH 2.5, 0.5M NaCl) and immediately neutralized with 1M Tris-HCl, pH 8, 5. Purified antibodies were dialyzed overnight against PBS at 4°C.
[000315] Fragments F(ab)2. Affinity purified antibodies were mixed with 3 mg of pepsin at 37°C for 30 minutes. The reaction was quenched with 1 M Tris-HCl, pH 8.5, and the F(ab)2 fragments were affinity purified with HP columns activated with HiTrap NHS- conjugated with specific antigen. Purified antibodies were dialyzed overnight against PBS at 4°C, loaded onto SDS-PAGE gel and visualized with Coomassie Blue staining.
[000316] Active and passive immunization. BALB/c mice (3 weeks old, female, Charles River Laboratories) were immunized with 50 µg of protein emulsified in Complete Freund's Adjuvant (Difco) by intramuscular injection. For booster immunizations, proteins were emulsified in Incomplete Freund's Adjuvant and injection 11 days after the initial immunization. B At 20 after immunization, 5 mice were bled to obtain the sera for specific antibody titers by enzyme-linked immunosorbent analysis (ELISA).
[000317] Affinity purified antibodies in PBS were injected at a concentration of 5 mg kg-1 of animal body weight into the peritoneal cavity of BALB/c mice (6 weeks old, females, Charles River Laboratories) 24 hours before of the challenge with S. aureus. The animals' blood was collected by means of a periorbital vein puncture. Blood cells were removed with heparinized microhematocrit capillary tubes (Fisher), and Z-gel separation microtubes (Sarstedt) were used to collect and measure antigen-specific antibody titers by ELISA.
[000318] Mouse kidney abscess. Night cultures of S. aureus Newman or USA300 (LAC) were diluted 1:100 in fresh TSB and grown for 2 hours at 37°C. Staphylococci were sedimented, washed and suspended in PBS at an OD600 of 0.4 (~1 x 108 CFU mL-1). Inoculums were quantified by spreading sample fractions on TSA and counting colonies formed. BALB/c mice (6 weeks old, female, Charles River Laboratories) were anesthetized by intraperitoneal injection with 100 mg ketamine and 20 mg ml-1 xylazine per kilogram of body weight. Mice were infected by retro-orbital injection with 1 x 10 7 CFU of S. aureus Newman or 5 x 10 6 CFU of S. aureus USA300. On day 4 after challenge, mice were sacrificed by CO2 inhalation. Both kidneys were removed, and the staphylococcal burden on one organ was analyzed by homogenizing the kidney tissue with PBS, 1% Triton X-100. Serial dilutions of the homogenizer were spread over TSA and incubated for colony formation. The remaining organ was examined by histopathology. Briefly, the kidneys were fixed in 10% formalin for 24 hours at room temperature. Tissues were embedded in paraffin, thinly sectioned, stained with hematoxylin-eosin, and inspected by light microscopy to count abscess lesions. All experiments with mice were performed according to institutional guidelines following review of the experimental protocol and approval by the Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC) at the University of Chicago.
[000319] Binding of Protein A. For binding of human IgG, Ni-NTA affinity columns were preloaded with 200 µg of purified proteins (SpA, SpA-D, SpA-DKKAA, and SrtA) in column buffer. After washing, 200 µg of human IgG (Sigma) was loaded onto the column. Protein samples were collected from washes and elutions, and subjected to SDS-PAGE gel electrophoresis, followed by Coomassie Blue staining. The purified proteins (SpA, SpAKKAA, SpA-D and SpA-DKKAA) were coated onto MaxiSorp ELISA plates (NUNC) in 0.1 M carbonate buffer (pH 9.5) at a concentration of 1 μg ml-1 overnight at 4°C. The plates were then blocked with 5% whole milk, and then incubated with serial dilutions of peroxidase-conjugated human IgG, Fc or F(ab)2 fragments for one hour. Plates were washed and developed using OptEIA ELISA (BD) reagents. Reactions were quenched with 1M phosphoric acid, and the A450 readings were used to calculate half maximum titration and percent binding.
[000320] Von Willebrand factor (vWF) binding assays. Purified proteins (SpA, SpAKKAA, SpA D and SpA-DKKAA) were coated and blocked as described above. The plates were incubated with human vWF at a concentration of 1 μg mL-1 for two hours, then washed and blocked with human IgG for an additional hour. After washing, the plates were incubated with a serial dilution of peroxidase-conjugated antibody directed against human vWF for one hour. Plates were washed and developed using OptEIA ELISA (BD) reagents. Reactions were quenched with 1M phosphoric acid, A450 readings were used to calculate half maximum titration and percent binding. For binding assays, plates were incubated with SpA-DKKAA-specific affinity purified F(ab)2 fragments at a concentration of 10 μg ml-1 for one hour prior to ligand binding assays.
[000321] Splenocyte apoptosis. Affinity purified proteins (150 μg SpA, SpA-D, SPAKKAA, and SPA-DKKAA) were injected into the peritoneal cavity of BALB/c mice (6 weeks old, females, Charles River Laboratories). Four hours after injection, the animals were sacrificed by CO2 inhalation. Their spleens were removed and homogenized. Cell debris was removed using cell filter and suspended cells were transferred to ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3 10 mM, 0.1 mM EDTA) to lyse erythrocytes. Leukocytes were pelleted by centrifugation, suspended in PBS and stained with R-PE conjugated anti-CD19 monoclonal antibody diluted 1:250 (Invitrogen) on ice and in the dark for one hour. Cells were washed with 1% FBS and fixed with 4% formalin overnight at 4°C. The next day, cells were diluted in PBS and analyzed by flow cytometry. The remaining organ was examined by histopathology. Briefly, spleens were fixed in 10% formalin for 24 hours at room temperature. Tissues were embedded in paraffin, thinly sectioned, stained with the apoptosis detection kit (Millipore), and inspected by light microscopy.
[000322] Antibody quantification. Sera were collected from healthy human volunteers or BALB/c mice that had been infected with S. aureus Newman or USA300 for 30 days or that had been immunized with SpA-DKKAA/SpAKKAA as described. Human/murine IgG (Jackson Immunology Laboratory), SpAKKAA, and CRM197 were spotted onto nitrocellulose membrane. Membranes were blocked with 5% whole milk, and then incubated with human or mouse sera. Affinity purified anti-Huamana/Murine IgG conjugated to IRDye 700DX (Rockland) was used to quantify signal intensities using the OdysseyTM (Li-Color) infrared imaging system. The experiments with blood from human volunteers involved protocols that were reviewed, approved and performed under the controlling supervision of The University of Chicago's Institutional Review Board (IRB).
[000323] Statistical analysis. Two Student t tests were performed to analyze the statistical significance of kidney abscesses, ELISA, and B-cell superantigen data. BIBLIOGRAPHIC REFERENCES
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权利要求:
Claims (11)
[0001]
1. An isolated immunogenic polypeptide characterized in that it comprises a variant domain of protein A (SpA) having (a) two amino acid substitutions that interrupt binding to FC and (b) two amino acid substitutions that interrupt binding to VH3, wherein for (a) the SpA variant comprises a glycine residue substitution at each of the amino acid positions corresponding to amino acid positions 9 and 10 of SEQ ID NO: 2 and for (b) the SpA variant comprises a residue substitution of serine at each of the amino acid positions corresponding to amino acid positions 36 and 37 of SEQ ID NO: 2.
[0002]
2. Polypeptide according to claim 1, characterized in that it further comprises: (i) one or more variants of a SpA E domain, domain A, domain B or domain C; (ii) two or more segments of the D domain; or (iii) a non-protein A segment, preferably a second antigenic segment; wherein the second antigen segment is optionally a staphylococcal antigen segment, preferably an Emp, EsxA, EsxB, EsaC, Cap, Ebh, EsaB, Coa, vWbp, vWh, Ha, SdrC, SdrD, SdrE, IsdA, IsdB, IsdC, CYA, C fB and/or SasF segment.
[0003]
3. Immunogenic composition, characterized in that it comprises the recombinant polypeptide, as defined in claim 1 or 2, in a pharmaceutically acceptable formulation.
[0004]
4. Immunogenic composition according to claim 3, characterized in that it also comprises: a. at least one second staphylococcal antigen; preferably wherein the second antigen is selected from Esag, E-mp, ESXA, ESX8, Esac, E-ap, Ebh, coa, VWbP, VWh, Hia, Sdrc, scYD, SdrE, Peptide ISdA, SdB, ISdC, CIfA, cra and/or SasF; or b. an adjuvant, preferably wherein the SpA variant is coupled to an adjuvant.
[0005]
5. Composition according to claim 3 or 4, characterized in that it further comprises a PIA polysaccharide or oligosaccharide: or a type V and/or type Vlll capsular polysaccharide or oligosaccharide of S. aureus.
[0006]
6. Composition according to any one of claims 3 to 5, characterized in that a staphylococcal capsular polysaccharide is conjugated to a protein carrier, preferably wherein the protein carrier is selected from the group consisting of tetanus toxoid , diphtheria toxoid, CRM 197, Haemophilus influenzae protein D, pneumolysin and alpha toxoid.
[0007]
7. Composition according to any one of claims 3 to 6, characterized in that said composition is a vaccine.
[0008]
8. Composition according to claim 7, characterized in that it also comprises a pharmaceutically acceptable excipient.
[0009]
9. Composition according to any one of claims 3 to 8, characterized in that the composition is used to induce an immune response against a Staphylococcus bacterium in an individual.
[0010]
10. Use of a composition, as defined in any one of claims 3 to 6, characterized in that it is in the manufacture of a medicament to induce an immune response against a staphylococcal bacterium in an individual.
[0011]
11. Composition according to claim 9 or use according to claim 10, characterized in that: a. the composition comprises an adjuvant; B. the staphylococcus bacterium is a S. aureus bacterium; ç. the staphylococcal bacterium is resistant to one or more treatments, preferably wherein the staphylococcal bacterium is resistant to methicillin; d. the composition is formulated for oral, parenteral, subcutaneous, intramuscular or intravenous administration; and. wherein the composition comprises a recombinant non-staphylococcal bacterium that expresses the SpA variant, preferably wherein the recombinant non-staphylococcal bacterium is a Salmonella; f. the subject is a mammal, preferably a human; g. the immune response is a protective immune response; or h. the subject has, is suspected of having, or is at risk of developing a staphylococcal infection; preferably where the subject is diagnosed with a persistent staphylococcal infection.

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SCHNEEWIND et al.0|Patent 2757543 Summary

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法律状态:
2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-02| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-09-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-07-07| B06G| Technical and formal requirements: other requirements [chapter 6.7 patent gazette]|

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2021-07-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-08-10| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-11-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-02-01| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/07/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US36121810P| true| 2010-07-02|2010-07-02|

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US61/361,218|2010-07-02|

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US37072510P| true| 2010-08-04|2010-08-04|

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US61/370,725|2010-08-04|

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PCT/US2011/042845|WO2012003474A2|2010-07-02|2011-07-01|Compositions and methods related to protein avariants|

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