Title:  Staphylococcal immunotherapeutics via donor selection and donor stimulation

United States Patent:  6,692,739

Issued:  February 17, 2004

Inventors:  Patti; Joseph M. (Cumming, GA); Foster; Timothy J. (Dublin, IE); Hook; Magnus (Houston, TX)

Assignee:  Inhibitex, Inc. (Alpharetta, GA); The Provost Fellows and Scholars of The College of the Holy and Undivided (Dublin, IE); The Texas A&M University System (College Station, TX)

Appl. No.:  386960

Filed:  August 31, 1999

Abstract

A method and composition for the passive immunization of patients infected with or susceptible to infection from Staphylococcus bacteria such as S. aureus and S. epidermidis infection is provided that includes the selection or preparation of a donor plasma pool with high antibody titers to carefully selected Staphylococcus adhesins or MSCRAMMs, or fragments or components thereof, or sequences with substantial homology thereto. The donor plasma pool can be prepared by combining individual blood or blood component samples which have higher than normal titers of antibodies to one or more of the selected adhesins or other proteins that bind to extracellular matrix proteins, or by administering carefully selected proteins or peptides to a host to induce the expression of desired antibodies, and subsequently recovering the enhanced high titer serum or plasma pool from the treated host. In either case, the donor plasma pool is preferably purified and concentrated prior to intravenous introduction into the patient, and the present invention is advantageous in that a patient can be immunized against a wide variety of potentially dangerous staphylococcal infections. Kits for identifying potential donor with high titers of the selected adhesins are also provided. The present invention thus provides methods and compositions which can be highly effective against infections associated with Staphylococcus bacteria.

DETAILED DESCRIPTION OF THE INVENTION

A method and composition for the passive immunization of patients infected with or susceptible to Staphylococcus bacterial infection, such as those caused by S. aureus or S. epidermidis, is provided that includes the selection or preparation of a donor plasma pool with high antibody titers to carefully selected Staphylococcus adhesins, or fragments thereof or sequences with substantial homology thereto; purification, concentration, and treatment of the donor plasma pool as necessary to obtain immunoglobulin in a purified state that has a higher than normal antibody titer to the selected staphylococcal adhesins; and then administration of an effective amount of the purified immunoglobulin to the patient in need thereof. The donor plasma pool can be prepared, for example by, by combining individual blood samples which have higher than normal titers of antibodies to one or more of the selected adhesins or fragments or sequences with substantial homology thereto. Kits for the identification of donor plasma pools with high titers of the selected adhesins are also provided. In an alternative embodiment, a method for obtaining a donor plasma pool that is highly effective against Staphylococcus infection is provided that includes administering carefully selected proteins or peptides to a host to induce the expression of desired antibodies, recovering the enhanced high titer plasma pool from the host, optionally purifying and concentrating the immunoglobulin, and providing it to a patient in need thereof.

Donor plasma pools are selected or prepared, purified, treated, and then administered in an effective amount to a patient in need thereof, which include high titer antibodies to at least:

(i) a fibrinogen binding protein, such as Clumping factor A ("ClfA") or Clumping factor B ("ClfB"), or fragments or components thereof, or a protein or fragment with sufficiently high homology thereto;

(ii) a collagen binding protein or peptide (or an appropriate site directed mutated sequence thereof ), a fragment or component thereof, such as the collagen binding domain protein M55, or a protein or fragment with sufficiently high homology thereto.

(iii) a fibronectin binding protein or peptide (or an appropriate site directed mutated sequence thereof), or a protein or fragment with sufficiently high homology thereto, in combination with the fibrinogen binding protein A and B (ClfA and ClfB), or useful fragments thereof or a protein or fragment with sufficiently high homology thereto;

(iv) the fibrinogen binding protein A (ClfA) and the fibrinogen binding protein B (ClfB), or useful fragments thereof or a protein or fragment with sufficiently high homology thereto;

(v) fibronectin binding protein or peptide (or an appropriate site directed mutated sequence thereof), or a protein or fragment with sufficiently high homology thereto, in combination with (I) the fibrinogen binding protein A and B (ClfA and ClfB), or a useful fragment thereof or a protein or fragment with sufficiently high homology thereto; and (ii) a collagen binding protein or useful fragment thereof;

(vi) components of any of the above in combination with an elastin binding protein or peptide or a protein or fragment with sufficiently high homology thereto;

(vii) components of any of the above embodiments in combination with a MHC II type binding protein or peptide or a protein or fragment with sufficiently high homology thereto;

(viii) components of any of the above embodiments in combination with a the fibrinogen binding proteins SdrC, SdrD or SdrE, or useful fragments thereof or proteins or fragments with sufficiently high homology thereto;

(ix) the fibrinogen binding protein SdrC, the fibrinogen binding protein SdrD and the fibrinogen binding protein SdrE or useful fragments thereof or a protein or fragment with sufficiently high homology thereto; or

(x) proteins SdrF, SdrG and SdrH from coagulase-negative bacteria such as S. epidermidis or useful fragments thereof or a proteins or fragments with sufficiently high homology thereto.

Isolated peptide fragments from wild-type or naturally occurring variants and synthetic or recombinant peptides corresponding to wild-type, naturally occurring variants or introduced mutations that do not correspond to a naturally occurring binding domain of a binding protein can be used to select or produce donor plasma pools.

Fibronectin-binding MSCRAMMs

Fibronectin (Fn) is a 440-kDa glycoprotein found in the ECM and body fluids of animals. The primary biological function of fibronectin appears to be related to its ability to serve as a substrate for the adhesion of cells expressing the appropriate integrins. Several bacterial species have been shown to bind fibronectin specifically and to adhere to a fibronectin-containing substratum. Most S. aureus isolates bind Fn, but do so in varying extents, which reflects variations in the number of MSCRAMM molecules expressed on the bacterial cell surface. The interaction between Fn and S. aureus is highly specific (Kuusela, P., Nature, 276:718-20, 1978). Fn binding is mediated by two surface exposed proteins with molecular weights of 110 kDa, named FnBP-A and FnBP-B. The primary Fn binding site consists of a motif of 35-40 amino acids, repeated three to five times. The genes for these have been cloned and sequenced (Jonsson, K., et al., Eur. J Biochem., 202:1041-1048, 1991). Potential applications for vaccination with anti-FnBP antibodies include, but are not limited to, bovine mastitis, endocarditis and wound infections.

WO-A-85/05553 discloses bacterial cell surface proteins having fibronectin, fibrinogen, collagen, and or laminin binding ability.

U.S. Pat. Nos. 5,320,951 and 5,571,514 to Hook, et al., discloses the gene sequence of fibronectin binding protein A (fnbA), and biological products and methods based on this sequence.

U.S. Pat. No. 5,175,096 to Hook et al., discloses the gene sequence of fnbB, a hybrid DNA molecule (fnbB) and biological products and methods based on this sequence.

U.S. Pat. No. 5,652,217 discloses an isolated and purified protein having binding activity that is encoded by a hybrid DNA molecule from S. aureus of defined sequence.

U.S. Pat. No. 5,440,014 discloses a fibronectin binding peptide within the D3 homology unit of a fibronectin binding protein of S. aureus which can be used for vaccination of ruminants against mastitis caused by staphylococcal infections, for treatment of wounds, for blocking protein receptors, for immunization of other animals, or for use in a diagnostic assay.

U.S. Pat. No. 5,189,015 discloses a method for the prophylactic treatment of the colonization of a S. aureus bacterial strain having the ability to bind to fibronectin in a mammal that includes administering to the mammal in need of treatment a prophylactically therapeutically active amount of a protein having fibronectin binding properties, to prevent the generation of infections caused by a S. aureus bacterial strain having the ability to bind fibronectin, wherein the protein has a molecular weight of 87 kDa to 165 kDa.

U.S. Pat. No. 5,416,021 discloses a fibronectin binding protein encoding DNA from Streptococcus dysgalactiae, along with a plasmid that includes DNA encoding for fibronectin binding protein from S. dysgalactiae contained in E. coli, DNA encoding a fibronectin binding protein from S. dysgalactiae and an E. coli microorganism transformed by DNA encoding a fibronectin binding protein from S. dysgalactiae.

It has been observed that antibodies to wild type fibronectin binding protein do not substantially inhibit the ability of S. aureus to bind to fibronectin, and thus do not exhibit a significant therapeutic effect in vivo. PCT/US98/01222 discloses antibodies that block the binding of fibronectin to fibronectin binding proteins. The antibodies were raised against a site-directed mutated sequence of fibronectin binding protein that does not bind to fibronectin. It was identified that there is a rapid complexing of fibronectin with fibronectin binding proteins and fragments in vivo. Peptide epitopes that do not bind to fibronectin, even though based on a fibronectin binding domain of a fibronectin binding protein, do not form a complex with fibronectin in vivo. This allows antibodies to be made against the uncomplexed peptide epitope, which inhibit or block the binding of fibronectin to fibronectin binding proteins.

III. Collagen-binding MSCRAMMs

Collagen is the major constituent of cartilage. Collagen (Cn) binding proteins are commonly expressed by staphylococcal strains. The Cn binding MSCRAMM of S. aureus adheres to cartilage in a process that constitutes an important part of the pathogenic mechanism in staphylococcal infections. (Switalski, et al. Mol. Micro. 7(1), 99-107, 1993) Cn binding by staphylococcal bacteria such as S. aureus is found to play a role in at least, but not only, arthritis and septicemia. CNA proteins with molecular weights of 133, 110 and 87 kDa (Patti, J., et al., J. Biol. Chem.,267:4766-4772, 1992) have been identified. Strains expressing CNAs with different molecular weights do not differ in their Cn binding ability (Switalski, L. M., et al., Mol. Microbiol., 7:99-107, 1993).

Staphylococcal strains recovered from the joints of patients diagnosed with septic arthritis or osteomyelitis almost invariably express a CNA, whereas significantly fewer isolates obtained from wound infections express this adhesin. (Switalski, L. M., et al., Mol. Microbiol., 7:99-107, 1993) Similarly, S. aureus strains isolated from the bones of patients with osteomyelitis more often have an MSCRAMM recognizing the bone-specific protein, bone sialoprotein (BSP) (Ryden, C., et al, Lancet, 11:515-518, 1987). S. aureus colonization of the articular cartilage within the joint space appears to be an important factor contributing to the development of septic arthritis.

The cloning, sequencing, and expression of a gene CNA, encoding a S. aureus CNA protein has been reported (Patti, J., et al., J. Biol. Chem., 267:4766-4772, 1992). The CNA gene encodes an 133-kDa adhesin that contains structural features characteristic of surface proteins isolated from Gram-positive bacteria.

Recently, the ligand-binding site has been localized within the N-terminal half of the CNA (Patti, J. et al., Biochemistry, 32:11428-11435, 1993). By analyzing the Col binding activity of recombinant proteins corresponding to different segments of the MSCRAMM, a 168-amino-acid long protein fragment (corresponding to amino acid residues 151-318) that had appreciable Col binding activity was identified. Short truncations of this protein in the N or C terminus resulted in a loss of ligand binding activity but also resulted in conformational changes in the protein.

PCT WO 92/07002 discloses a hybrid DNA molecule which includes a nucleotide sequence from S. aureus coding for a protein or polypeptide having collagen binding activity and a plasmid or phage comprising the nucleotide sequence. Also disclosed are an E. coli strain expressing the collagen binding protein, a microorganism transformed by the recombinant DNA, the method for producing a collagen binding protein or polypeptide, and the protein sequence of the collagen binding protein or polypeptide.

Patti et al. (J of Biol Chem., 270, 12005-12011, 1995) disclose a collagen binding epitope in the S. aureus adhesin encoded by the CNA gene. In this study, the authors synthesized peptides derived from the sequence of the said protein and used them to produce antibodies. Some of these antibodies inhibit the binding of the protein to collagen.

PCT/US97/08210 discloses that certain identified epitopes of the collagen binding protein (M55, M33, and M17) can be used to generate protective antibodies. The application also discloses the crystal structure of the CNA which provides critical information necessary for identifying compositions which interfere with, or block completely, the binding of Col to CNAs. The ligand-binding site in the S. aureus CNA and a 25-amino-acid peptide was characterized that directly inhibits the binding of S. aureus to 125 I-labeled type II Col.

IV. Fibrinogen-binding MSCRAMMs

Fibrin is the major component of blood clots, and fibrinogen/fibrin is one of the major host proteins deposited on implanted biomaterials. Considerable evidence exists to suggest that bacterial adherence to fibrinogen/fibrin is important in the initiation of device-related infection. For example, as shown by Vaudaux et al, S. aureus adheres to in vitro plastic that has been coated with fibrinogen in a dose-dependent manner (J. Infect. Dis. 160:865-875 (1989)). In addition, in a model that mimics a blood clot or damage to a heart valve, Herrmann et al. demonstrated that S. aureus binds avidly via a fibrinogen bridge to platelets adhering to surfaces (J. Infect. Dis. 167: 312-322 (1993)). S. aureus can adhere directly to fibrinogen in blood clots formed in vitro, and can adhere to cultured endothelial cells via fibrinogen deposited from plasma acting as a bridge (Moreillon et al., Infect. Immun. 63:4738-4743 (1995); Cheung et al., J. Clin. Invest. 87:2236-2245 (1991)). As shown by Vaudaux et al. and Moreillon et al., mutants defective in the fibrinogen-binding protein clumping factor (ClfA) exhibit reduced adherence to fibrinogen in vitro, to explanted catheters, to blood clots, and to damaged heart valves in the rat model for endocarditis (Vaudaux et al., Infect. Immun. 63:585-590 (1995); Moreillon et al., Infect. Immun. 63: 4738-4743 (1995)).

An adhesin for fibrinogen, often referred to as "clumping factor," is located on the surface of S. aureus cells. The interaction between bacteria and fibrinogen in solution results in the instantaneous clumping of bacterial cells. The binding site on fibrinogen is located in the C-terminus of the gamma chain of the dimeric fibrinogen glycoprotein. The affinity is very high and clumping occurs in low concentrations of fibrinogen. Scientists have recently shown that clumping factor also promotes adherence to solid phase fibrinogen, to blood clots, and to damaged heart valves (McDevitt et al., Mol. Microbiol. 11: 237-248 (1994); Vaudaux et al., Infect. Immun. 63:585-590 (1995); Moreillon et al., Infect. Immun. 63: 4738-4743 (1995)).

Two genes in S. aureus have been found that code for two Fg binding proteins, ClfA and ClfB. The gene, clfA, was cloned and sequenced and found to code for a polypeptide of 92 kDa. ClfA binds the gamma chain of fibronectin, and ClfB binds the alpha and beta chains (Eidhin, et al., Mol Micro, awaiting publication, 1998). ClfB is a cell wall associated protein with a predicted molecular weight of 88 kDa and an apparent molecular weight of 124 kDa that binds both soluble and immobilized fibrinogen and acts as a clumping factor.

The gene for a clumping factor protein, designated ClfA, has recently been cloned, sequenced and analyzed in detail at the molecular level (McDevitt et al., Mol. Microbiol. 11: 237-248 (1994); McDevitt et al., Mol. Microbiol. 16:895-907 (1995)). The predicted protein is composed of 933 amino acids. A signal sequence of 39 residues occurs at the N-terminus followed by a 520 residue region (region A), which contains the fibrinogen binding domain. A 308 residue region (region R), composed of 154 repeats of the dipeptide serine-aspartate, follows. The R region sequence is encoded by the 18 basepair repeat GAY TCN GAY TCN GAY AGY in which Y equals pyrimidines and N equals any base. The C-terminus of ClfA has features present in many surface proteins of gram-positive bacteria such as an LPDTG motif, which is responsible for anchoring the protein to the cell wall, a membrane anchor, and positive charged residues at the extreme C-terminus.

The platelet integrin alpha IIb.beta.3 recognizes the C-terminus of the gamma chain of fibrinogen. This is a crucial event in the initiation of blood clotting during coagulation. ClfA and alpha IIb.beta.3 appear to recognize precisely the same sites on fibrinogen gamma chain because ClfA can block platelet aggregation, and a peptide corresponding to the C-terminus of the gamma chain (198-41 1) can block both the integrin and ClfA interacting with fibrinogen (McDevitt et al., Eur. J. Biochem. 247:416-424 (1997)). The fibrinogen binding site of alpha IIb.beta.3 is close to, or overlaps, a Ca2+ binding determinant referred to as an "EF hand". ClfA region A carries several EF hand-like motifs. A concentration of Ca2+ in the range of 3-5 mM blocks these ClfA-fibrinogen interactions and changes the secondary structure of the ClfA protein. Mutations affecting the ClfA EF hand reduce or prevent interactions with fibrinogen. Ca2+ and the fibrinogen gamma chain seem to bind to the same, or to overlapping, sites in ClfA region A.

The alpha chain of the leukocyte integrin, alpha MB2, has an insertion of 200 amino acids (A or I domain) which is responsible for ligand binding activities. A novel metal ion-dependent adhesion site (MIDAS) motif in the I domain is required for ligand binding. Among the ligands recognized is fibrinogen. The binding site on fibrinogen is in the gamma chain (residues 190-202). It was recently reported that Candida albicans has a surface protein, alpha Intlp, having properties reminiscent of eukaryotic integrins. The surface protein has amino acid sequence homology with the I domain of M.beta.2, including the MIDAS motif. Furthermore, Intlp binds to fibrinogen.

ClfA region A also exhibits some degree of sequence homology with alpha Intlp. Examination of the ClfA region A sequence has revealed a potential MIDAS motif. Mutations in putative cation coordinating residues in the DxSxS portion of the MIDAS motif in ClfA results in a significant reduction in fibrinogen binding. A peptide corresponding to the gamma-chain binding site for alpha M.beta.2 (190-202) has been shown by O'Connell et al. to inhibit ClfA-fibrinogen interactions (O'Connell et al., J. Biol. Chem., in press). Thus it appears that ClfA can bind to the gamma-chain of fibrinogen at two separate sites. The ligand binding sites on ClfA are similar to those employed by eukaryotic integrins and involve divalent cation binding EF-hand and MIDAS motifs. Despite the low level of identity between ClfA and ClfB, both proteins bind fibrinogen (on different chains) by a mechanism that is susceptible to inhibition by divalent cations, despite not sharing obvious metal binding motifs.

Other fibrinogen binding proteins are disclosed in co-pending U.S. patent application Ser. No. 09/200,650, incorporated herein by reference. This application discloses isolated fibrinogen binding proteins ClfB, SdrC, SdrD and SdrE as well as antibodies to the proteins and diagnostic kits that include the proteins or the antibodies. Also claimed are a method of preventing a S. aureus infection that includes administering to the patient an effective amount of ClfB, SdrC, SdrD, SdrE, or a binding fragment thereof and a method of inducing an immunological response comprising administering to a patient a pharmaceutical composition that includes ClfB, SdrC, SdrD, SdrE, or an active fragment thereof.

ClfB has a predicted molecular weight of approximately 88 kDa and an apparent molecular weight of approximately 124 kDa. ClfB is a cell-wall associated protein and binds both soluble and immobilized fibrinogen. In addition, ClfB binds both the alpha and beta chains of fibrinogen and acts as a clumping factor. The ClfB protein has been demonstrated to be a virulence factor in experimental endocarditis.

The SdrC, SdrD and SdrE proteins are related in primary sequence and structural organization to the ClfA and ClfB proteins and are localized on the cell surface. The SdrC, SdrD and SdrE proteins are cel wall-associated proteins, having a signal sequence at the N-terminus and an LPXTG (SEQ ID NO: 2) motif, hydroqhobic domain and positively charged residues at the C-terminus. Each also has an SD repeat containing region R of sufficient length to allow efficient expression of the ligand binding domain region A on the cell surface. With the A region of the SdrC, SdrD and SdrE proteins located on the cell surface, the proteins can interact with proteins in plasma, the extracellular matrix or with molecules on the surface of host cells. They share some limited amino acid sequence similarity with ClfA and ClfB. Additionally, SdrC, SdrD and SdrE also exhibit cation-dependent ligand binding to extracellular matrix proteins. For example, SdrC binds vitronectin and SrdE binds bone sialoprotein (BSP).

It has ben discovered that in the A region of SrdC, SrdD, SrdE, ClfA and ClfB there is a highly conserved amino acid sequence that can be used to derive a consensus TYTFTDYVD (SEQ ID NO: 3) motif. The motif can be used in multicomponent vaccines to impart broad spectrum immunity to bacterial infections, and also can be used to produce momoclonal or polyclonal antibodies that impart broad spectrum passive immunity. In an alternative embodiment, any combination of the variable sequence motif derived from the Sdr and Clf protein families, (T/I) (Y/F) (T/V) (F) (T) (D/N) (Y) (V) (D/N), can be used to impart immunity or produce protective antibodies.

ClfB, SdrC, SdrD and SdrE thus share a common consensus TYTFTDYVD (SEQ ID NO: 3) motif which overlaps the ligand binding/Ca2+ binding region of ClfA. Therefore the proteins interact with fibrinogen and other host components. ClfB, SdrC, SdrD and SdrE subdomains, depending on the protein, include subdomains A and B1-B5. Other information regarding extracellular matrix binding proteins has been disclosed in U.S. application Ser. No. 09/200,650, incorporated herein by reference.

V. Elastin-binding MSCRAMMs

The primary role of elastin is to confer the property of reversible elasticity to tissues and organs (Rosenbloom, J., et al., FASEB J., 7:1208-1218, 1993). Elastin expression is highest in the lung, skin and blood vessels, but the protein is widely expressed in mammalian hosts for S. aureus. S. aureus binding to elastin was found to be rapid, reversible, of high affinity and ligand specific. Furthermore, a 25 kDa cell surface elastin binding protein (EbpS) was isolated and proposed to mediate S. aureus binding to elastin-rich host ECM. EbpS binds to a region in the N-terminal 30 kDa fragment of elastin.

PCT/US97/03106 discloses the gene sequences for an elastin binding protein. DNA sequence data disclosed indicates that the ebps open reading frame consists of 606 bp, and encodes a novel polypeptide of 202 amino acids. EbpS protein has a predicted molecular mass of 23,345 daltons and pI of 4.9. EbpS was expressed in E. coli as a fusion protein with polyhistidine residues attached to the N-terminus. A polyclonal antibody raised against recombinant EbpS interacted specifically with the 25 kDa cell surface EbpS and inhibited staphylococcal elastin binding. Furthermore, recombinant EbpS bound specifically to immobilized elastin and inhibited binding of Staphylococcus aureus to elastin. A degradation product of recombinant EbpS lacking the first 59 amino acids of the molecule and a C-terminal fragment of CNBr-cleaved recombinant EbpS, however, did not interact with elastin. These results strongly suggest that EbpS is the cell surface molecule mediating binding of Staphylococcus aureus to elastin. The finding that some constructs of recombinant EbpS do not interact with elastin suggests that the elastin binding site in EbpS is contained in the first 59 amino acids of the molecule.

Several independent criteria indicate that EbpS is the surface protein mediating cellular elastin binding. First, rEbpS binds specifically to immobilized elastin and inhibits binding of S. aureus cells to elastin in a dose dependent manner. These results establish that EbpS is an elastin binding protein that is functionally active in a soluble form. Second, an antibody raised against rEbpS recognizes a 25 kDa protein expressed on the cell surface of S. aureus cells. In addition to the size similarity and antibody reactivity, further evidence that this 25 kDa protein is cell surface EbpS is provided by the experiment showing that binding of the 25 kDa protein to immobilized anti-rEbpS IgG is inhibited in the presence of excess unlabeled rEbpS. Finally, Fab fragments prepared from the anti-rEbpS antibody, but not from its pre-immune control, inhibit binding of S. aureus to elastin. This result suggests that the topology of surface EbpS is such that the elastin binding site is accessible to interact with ligands (i.e. elastin and the anti-rEbpS Fab fragment) and not embedded in the cell wall or membrane domains. The composite data demonstrate that EbpS is the cell surface protein responsible for binding S. aureus to elastin.

The present and previous findings suggest the existence of a functionally active 40 kDa intracellular precursor form of EbpS that requires processing at the C-terminus prior to surface expression. This notion is based on the following observations: i) there exists an intracellular 40 kDa elastin binding protein that is never detected during cell surface labeling experiments, ii) the 25 kDa EbpS and the 40 kDa elastin binding protein have an identical N-terminal sequence, and iii) a single gene exists for EbpS. Because the size of the ebps open reading frame is not sufficient to encode a 40 kDa protein, at first the inventors disregarded this hypothesis. However, their studies with rEbpS demonstrated that although the actual size of the recombinant protein is 26 kDa, it migrates aberrantly as a 45 kDa protein in SDS-30 PAGE. This finding suggests that full length native EbpS, with a predicted size of 23 kDa, may be migrating in SDS-PAGE as the 40 kDa intracellular precursor, and that the 25 kDa surface form of EbpS is actually a smaller form of the molecule processed at the C-terminus. Although EbpS lacks an N-terminal signal peptide and other known sorting and anchoring signals, this proposed intracellular processing event may explain some questions regarding how EbpS is targeted to the cell surface. In fact, C-terminal signal peptides have been identified in several bacterial proteins (Fath, M. J. and Kolter, R., Microbiol. Rev., 57:995-1017, 1993) and alternative means of anchoring proteins to the cells surface have been reported in gram positive bacteria (Yother, J. and White, J. M., J. Bacteriol., 176:2976-2985, 1994).

Using overlapping EbpS fragments and recombinant constructs, the elastin binding site in EbpS was mapped to the amino terminal domain of the molecule (PCT/US97/03106). Overlapping synthetic peptides spanning amino acids 14-34 were then used to better define the binding domain. Among these, peptides corresponding to residues 14-23 and 18-34 specifically inhibited elastin binding by more than 95%. Common to all active synthetic peptides and proteolytic and recombinant fragments of EbpS is the hexameric sequence ¹⁸ Thr-Asn-Ser-His-Gln-Asp²³. Further evidence that this sequence is important for elastin binding was the loss of activity when Asp²³ was substituted with Asn in the synthetic peptide corresponding to residues 18-34. However, the synthetic hexamer TNSHQD by itself did not inhibit staphylococcal binding to elastin. These findings indicate that although the presence of the TNSHQD sequence is essential for EbpS activity, flanking amino acids in the N- or C-terminal direction and the carboxyl side chain of Asp²³ are required for elastin recognition.

VI. MHC II-analogous Proteins, (MAP)

In addition to fibrinogen, fibronectin, collagen and elastin, S. aureus strains associate with other adhesive eukaryotic proteins, many of which belong to the family of adhesive matrix proteins, such as vitronectin. (Chatwal, G. S., et al., Infect. Immun., 55:1878-1883, 1987). U.S. Pat. No. 5,648,240, incorporated herein by reference, discloses a DNA segment comprising a gene encoding a S. aureus broad spectrum adhesin that has a molecular weight of about 70 kDa. The adhesin is capable of binding fibronectin or vitronectin and includes a MHC II mimicking unit of about 30 amino acids. Further analyses of the binding specificities of this protein reveal that it functionally resembles an MHC II antigen in that it binds synthetic peptides. Thus, in addition to mediating bacterial adhesion to ECM proteins, it may play a role in staphylococcal infections by suppressing the immune system of the host. The patent further claims a recombinant vector that includes the specified DNA sequence, a recombinant host cell transformed with the vector, and DNA which hybridizes with the DNA of specified sequence. Also disclosed is a composition that includes a protein or polypeptide encoded by the specified DNA sequence and a method of inducing an immune response in an animal that includes administering an immunogenic composition that includes the encoded protein or polypeptide. A method of making a MHC II antigen protein analog comprising the steps of inserting the specified DNA sequence in a suitable expression vector and culturing a host cell transformed with the vector under conditions to produce the MHC II antigen protein analog is additionally claimed in the patent.

VII. SDR Proteins From Staphylococcus Epidermidis

Staphylococcus epidermidis, a coagulase-negative bacterium, is a common inhabitant of human skin and a frequent cause of foreign-body infections. Pathogenesis is facilitated by the ability of the organism to first adhere to, and subsequently to form biofilms on, indwelling medical devices such as artificial valves, orthopedic devices, and intravenous and peritoneal dialysis catheters. Device-related infections may jeopardize the success of medical treatment and significantly increase patient mortality. Accordingly, the ability to develop vaccines that can control or prevent outbreaks of S. epidermidis infection is of great importance, as is the development of means that can prevent or treat infection from a broad spectrum of bacteria, including both coagulase-positive and coagulase negative bacteria.

Three Sdr (serine-aspartate (SD) repeat region) proteins that are expressed by S. epidermidis have been designated as SdrF, SdrG and SdrH, and the amino acid sequences of these proteins and their nucleic acid sequences are disclosed in co-pending U.S. patent application of Foster et al. which is based on U.S. provisional application Ser. Nos. 60/098,443 and 60/117,119. All of these applications are incorporated herein by reference.

In accordance with the present invention, the donor selection and donor stimulation methods described herein can also be performed with regard to the SdrF, SdrG or an SdrH protein. In these methods, individuals may be identified and selected who have higher than normal antibody titers to the SdrF, SdrG or an SdrH proteins, and a donor plasma pool can be prepared which will have higher than normal titers to one or more of these proteins. Accordingly, donor plasma can be prepared in accordance with the present invention which will be useful in methods to prevent or treat infection from coagulase-negative staphylococcal infections such as those associated with S. epidermidis.

VIII. Proteins and Peptides with Substantial Homology or Equivalent Function to Those Described Herein

Donor plasma pools can be screened or stimulated as desired, with full sequence proteins, peptides, protein or peptide fragments, isolated epitopes, fusion proteins, or any alternative which binds to the target ECM, whether in the form of a wild type, a site-directed mutant, or a sequence which is substantially homologous thereto.

When used in conjunction with amino acid sequences, the term "substantially similar" means an amino acid sequence which is not identical to published sequences, but which produces a protein or peptide having the same functionality and activities, either because one amino acid is replaced with another similar amino acid, or because the change (whether it be substitution, deletion or insertion) does not substantially effect the active site of the protein. Two amino acid sequences are "substantially homologous" when at least about 70%, (preferably at least about 80%, and most preferably at least about 90 or 95%) of the amino acids match over the defined length of the sequences.

It should also be understood that each of the MSCRAMM polypeptides of this invention may be part of a larger protein. For example, a ClfA polypeptide of this invention may be fused at its N-terminus or C-terminus to a ClfB polypeptide, or to a non-fibrinogen binding polypeptide or combinations thereof. Polypeptides which may be useful for this purpose include polypeptides derived any of the MSCRAMM proteins, and serotypic variants of any of the above.

Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to Table 1. In keeping with standard polypeptide nomenclature (J. Biol. Chem., 243:3552-3559, 1969), abbreviations for amino acid residues are shown in Table I. It should be understood by one skilled in the art that the codons specified in Table 1 are for RNA sequences. The corresponding codons for DNA have a T substituted for U.

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties which can stimulate the production of a substantially similar antibody. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, corresponding DNA sequences which encode said peptides or antibodies against said peptides without appreciable loss of the biological utility or activity of the donor plasma pool immunoglobulin that is recovered.

It is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+1.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (+0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The following non-classical amino acids may be incorporated in the peptide in order to introduce particular conformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., J Am. Chem. Soc., 113:2275-2283, 1991); (2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron Lett., 1991); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, Ph.D. Thesis, University of Arizona, 1989); hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al, J. Takeda Res. Labs., 43:53-76, 1989) .beta.-carboline (D and L) (Kazmierski, Ph.D. Thesis, University of Arizona, 1988); HIC (histidine isoquinoline carboxylic acid) (Zechel et al, Int. J. Pep. Protein Res., 43, 1991); and HIC (histidine cyclic urea) (Dharanipragada).

The following amino acid analogs and peptidomimetics may be incorporated into a peptide to induce or favor specific secondary structures: LL-Acp (LL-3-amino-2-propenidone-6carboxylic acid), a .beta.-turn inducing dipeptide analog (Kemp et al., J. Org. Chem., 50:5834-5838, 1985); .beta.-sheet inducing analogs (Kemp et al., Tetrahedron Lett., 29:5081-5082, 1988); .beta.-turn inducing analogs (Kemp et al., Tetrahedron Lett., 29:5057-5060, 1988); alpha-helix inducing analogs (Kemp et al., Tetrahedron Lett., 29:4935-4938, 1988); ?-turn inducing analogs (Kemp et al., J. Org. Chem., 54:109:115, 1989); and analogs provided by the following references: Nagai and Sato, Tetrahedron Lett., 26:647-650 (1985); DiMaio et al., J. Chem. Soc. Perkin Trans., p. 1687 (1989); also a Gly-Ala turn analog (Kahn et al., Tetrahedron Lett., 30:2317, 1989); amide bond isostere (Jones et al., Tetrahedron Lett., 29:3853-3856, 1988); tetrazol (Zabrocki et al., J. Am. Chem. Soc., 110:5875-5880, 1988); DTC (Samanen et al., Int. J. Protein Pep. Res., 35:501:509, 1990); and analogs taught in Olson et al., J. Am. Chem. Sci., 112:323-333 (1990) and Garvey et al., J. Org. Chem., 56:436 (1990). Conformationally restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.

IX. Preparation of Purified Immunoglobulin

In one embodiment, purified immunoglobulin (A, D, E, or G) is prepared that has a high titer of antibodies to the selected adhesins. The term "high titer" in this context means the presence of an antibody in an amount which is 2-fold or greater, e.g., up to 10-20 more times higher than that found in a normal population of 100 random samples of blood.

The blood product can be prepared by (i) selection and purification of the immunoglobulin of a donor which has naturally high titers of antibodies to the selected adhesins, (ii) the combination of donor immunoglobulin from several individuals which have a high titer of antibodies to one or more of the selected adhesins, to produce the desired composite profile; or (iii) stimulation of the desired antibodies in one or more donors to form the desired composite antibody profile by exposing the donor to the selected antigens and obtaining blood sample of the exposed donor after sufficient time to produce and accumulate the resulting immunoreactive antibodies. The first two embodiments are referred to as "donor select" programs and the third is referred to as a "donor stimulation" program.

Donor Stimulation

Using the peptide antigens described herein, the present invention also provides methods of stimulating high antibody levels in a donor, which includes administering to an animal, for example a human, a pharmaceutically-acceptable composition comprising an immunologically effective amount of an MSCRAMM-derived peptide composition. The composition can include partially or significantly purified MSCRAMM-derived peptide epitopes, obtained from natural or recombinant sources, which proteins or peptides may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such epitopes. Smaller peptides that include reactive epitopes, such as those between about 30 and about 100 amino acids in length will often be preferred. The antigenic proteins or peptides may also be combined with other agents, such as other staphylococcal or streptococcal peptide or nucleic acid compositions, if desired. The composition may also include staphylococcal produced bacterial components such as those discussed above, obtained from natural or recombinant sources, which proteins may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such peptides.

Further means contemplated by the inventors for generating an immune response in an animal includes administering to the animal, or human subject, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding a peptide epitope, or an immunologically effective amount of an attenuated live organism that includes and expresses such a nucleic acid composition. Antigenic functional equivalents of the proteins and peptides described herein also fall within the scope of the present invention. Antigenically functional equivalents, or epitopic sequences, may be first designed or predicted and then tested, or may simply be directly tested for cross-reactivity.

In the case of preventing bacterial adhesion, the preparation of epitopes which produce antibodies which inhibit the interaction of a specific gene product or proteoglycans which are structurally similar to the specific gene product are particularly desirable.

The identification or design of suitable MSCRAMM epitopes, and/or their functional equivalents, suitable for use in immunoformulations, vaccines, or simply as antigens (e.g., for use in detection protocols), is a relatively straightforward matter. For example, one may employ the methods of Hopp, as enabled in U.S. Pat. No. 4,554,101, incorporated herein by reference, that teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.

Plasmapheresis

The term plasmapheresis describes a technique in which blood is removed from an animal, separated into its cellular and plasma components, the cells are then returned to the animal, and the plasma retained. Large volume plasmapheresis requires the removed plasma to be replaced by a suitable fluid, and when this is done, the technique is often known as plasma exchange. Any components found in plasma can be removed by plasma exchange. Plasma exchange is the method still in use at most blood banks and public donation centers in the United States. Plasma extracted this way for commercial sale is available for use in a preferred embodiment of this invention.

During plasma donation, it is necessary to replace the fluid taken to prevent circulatory collapse. In most circumstances, the osmotic effect of the plasma needs to be replaced. A 5% solution of human albumin obtained from donor blood is a safe and effective replacement. It is standard practice in the medical community to add 2 ml of KCl solution and 2 ml of 10% calcium gluconate solution to the albumin. Most plasma exchange units replace every 2 liters of plasma removed with 1.5 liters of human albumin solution and 0.5 liters of normal saline.

The methods currently in use for plasma separation are centrifugation and filtration. The technique of U.S. Pat. No. 5,548,066 may be used to prepare the donor plasma pool if it is not commercially available, and is incorporated by reference herein. First, a plurality of blood donors are identified. These donors are mature mammals, typically mammals of the same species for which the serum will be employed. Where a specific ailment is to be treated or prevented, such as mastitis in mammals or other diseases caused by staphylococcal bacteria such as S. aureus, it is preferred that the donors have been exposed either naturally or through immunization to the causative organism or some antigenic portion thereof. Further, to achieve a consistent serum product, it is preferred that the donor group be relatively large. It is preferred to use human hosts to prepare the donor plasma pools. Once the donors have been identified, blood is drawn from the donors. Since the serum is refined directly from the blood, it is desired to obtain the maximum quantity of blood to thus obtain the maximum quantity of serum. For humans, an established limit of blood is drawn periodically over time.

It is preferred to identify and maintain a consistent donor group by repeated drawing of smaller quantities of blood, for example, drawing of blood once a month from humans. The frequency of the drawing will of course influence the quantity which may be safely drawn. In general, it is desired to draw the maximum amount of blood over the course of time without causing detriment to the health of the donor. This may dictate drawing small amounts with great frequency, or the maximum amount possible at a reduced frequency, depending upon the particular species. The blood volume of the donor may be estimated by standard formulas available from the Center for Disease Control.

The health of the donor is of course a consideration in this process if long-term bleeding is desired. Before donating, the donor will be checked for general good health, and if the donor is in poor health the bleeding may be deferred until the next scheduled date. Beyond this, it is preferred that long-term health records be kept, preferably including more detailed information. In this regard, it is noted that production quantities of the present serum is a good indicator of the health of the donor.

Typically, the serum is separated from the blood of the donor and consists of material from the immune system. In one method, detailed records are kept for the amount of serum produced from the blood as a yield percentage, such as 7 liters of serum from 14 liters of blood provides a yield of 50%. In the preferred method, records of the yield percentage are kept for each donor for each bleeding. These percentages may then be used to determine if the donor should be bled at the next scheduled time. In particular, if the action to be taken is expressed as a function of yield percentages, a guideline may be expressed as follows: yield percentage /=65%, rest. As may be seen, the donor is not bled if the serum yield is above or below the normal range. Such a yield percentage may indicate an underlying ailment. The subject may be bled, possibly in a reduced amount, in the caution ranges, depending upon the donor's history and/or further examination. In this regard, it has been found that a small percentage of individuals consistently produce yield percentages around 60-62%.

The method of blood and plasma collection is generally standard and well to known to those of skill in the art. Any method can be used that achieves the desired results. Once the blood has been collected, it is subjected to procedures for extracting the desired components. A first important step in this process is to permit each vessel of collected blood to sit at room temperature at least until substantial clotting has occurred, usually one hour. During this period the blood moves from body temperature to room temperature, and is exposed to air. This exposure to air permits the fibrinogen to change into fibrin, causing clotting of the blood.

This clotting period is an important aspect of serum retrieval. The clotting provides a rough separation of the cellular material from the liquid. Additionally, while the exact mechanism is not known, it is believed that the clotting period causes white blood cells to die and, for a percentage of such cells, to burst or rupture such that the chemical material, including antibody, therein is released from the cells. It is believed that this material remains within the serum and acts to provide "information" to the immune system of the recipient of the serum. This "information" may help to "program" white blood cells for particular microorganisms, similar to providing them with a memory of the microorganism, such that the white blood cells of the recipient respond quickly, and in a manner similar to a subject which has been vaccinated or is immune.

This period of non-refrigeration also causes a rough filtering of the collected blood. In particular, the clotted blood with the relatively heavy red blood cells will fall toward the bottom of the vessel, while the liquid plasma, immunoglobulins and chemical material will be pushed toward the top. To assist in this process, and a process described below, it is preferred that the collection vessel be tall and thin, having proportions similar to a standard test tube.

The liquid portion obtained at this stage is raw serum which, after being filtered and sterilized, can impart immunity. Further steps are optionally carried out. However, to increase the yield, various other steps prior to filtration are preferred.

A first of these steps, after the collected blood has had sufficient time to clot, is refrigeration to approximately 20-60^oC. This refrigeration reduces the temperature of the blood from room temperature to the refrigeration temperature. Such cooling of course prevents growth of bacteria, mold, etc. Additionally, during this cooling the clotted blood settles further, and the clotted blood contracts. This contraction (and possibly the cooling) may cause a further percentage of the white blood cells to rupture. Additionally, the contraction of the clotted blood serves to express from the clot immunoglobulins and chemical materials which have been trapped therein. This refrigeration should last at least until the blood has achieved the refrigeration temperature, and preferably for about 14-18 hours, or overnight.

A second preferred step is physical pressing of the clotted blood. This pressing is believed to cause yet more rupturing of white cells, thus yielding even more of the transfer factor. Additionally, in a manner similar to the cooling contraction, the pressing serves to force immunoglobulins and transfer factor from the clot.

The preferred method of pressing is to insert a sterile weight into the refrigerated vessel of collected blood. For example, a cylinder having a close sliding fit within the vessel and a weight of approximately two pounds. As may be envisioned, the liquid material will flow about the cylinder until the cylinder has come to rest upon the clotted blood settled at the bottom of the vessel. It is preferred that the pressing weight be maintained in place for about 6-24 hours.

It is noted that the pressing can serve as a first active filtration step. The close fit of the weight serves to separate the liquid raw serum above and the solid material below, although a precision fit of the weight in the vessel is not required. Since this may serve as a first, rough, filtration step, it may conveniently be used to determine the quantity of raw serum produced for calculation of the yield percentage. Specifically, noting the height of the column of raw serum and knowing the diameter of the vessel provides the volume of raw serum produced.

At this point the filtering process proper begins. This further processing includes filtration to remove all cellular material. This filtration is achieved in multiple steps. The first filtration step is a gross filtering. This may be achieved simply by pouring the contents of the vessel into a collection vat while holding a screen over the opening in the collection vessel. Where the high-yield steps of refrigeration and pressing have been used, the pressing cylinder still within the vessel may act in conjunction with the screen to filter, and the screen may mainly filter out the cylinder itself. Where these high-yield steps have not been taken, a finer filter screen may be desired. The clotted cells remaining within the vessel are properly disposed of, and the vessel sterilized for later use.

This is a preferred point for combining the serum from different donors. It is noted, however, that samples from multiple donors can be combined at any point subsequent to the initial gross filtration step.

The raw serum may still contain a large amount of cells and cellular debris. As the next filtration step, the reclaimed liquid is then placed into a continuous flow centrifuge. For example, the liquid may be placed in a Sharples AS16NF continuous flow centrifuge, which will operate at approximately 13,000 to 15,000 rpm. The liquid is drawn off during this process while yet more of the cells and cellular debris is removed.

Following the isolation of the plasma, the antibodies are purified away from other cell products. This can be accomplished by a variety of protein isolation procedures, known to those skilled in the art of immunoglobulin purification, such as ion exchange, affinity purification, etc. Means for preparing and characterizing antibodies are well known in the art. For example, serum samples can be passed over protein A or protein G sepharose columns to bind IgG (depending on the isotype). The bound antibodies are then eluted with, e.g. a pH 5.0 citrate buffer. The elute fractions containing the Abs, are dialyzed against an isotonic buffer. Alternatively, the eluate is also passed over an anti-immunoglobulin-sepharose column. The Ab is then eluted with 3.5 M magnesium chloride. Abs purified in this way can then tested for binding activity by, for example, an isotype-specific ELISA and immunofluorescence staining assay of the target cells.

In an alternative embodiment, the liquid is instead subjected to a further filtration step. This further step actually consists of several sub-steps, with the liquid being passed through several filters of progressively finer gauge. In particular, the liquid is passed through at least a 0.65 micron filter, then a 0.2 micron nominal filter, and then through a 0.2 micron absolute filter. By passing the liquid through the 0.2 nominal filter first, most of the bacteria, mold, and fibrin will be removed prior to passing through the 0.2 absolute filter.

At this point the liquid has had essentially all solid cellular material removed. The chemical materials and immunoglobulins, however, remain in the liquid, which is referred to as clarified serum.

The clarified serum can be used (after sterilization described below) as the final serum. However, it is preferred that the clarified serum be concentrated. This concentration reduces the volume and thus reduces the amount of material which must be shipped. Additionally, certain recipients, such as infant mammals, can not accept a large quantity of medication intravenously due to a lack of capacity. As such, concentration permits a full dosage of the serum to be administered. The concentration is preferably performed by repeated ultra-filtration to remove water molecules, as is known in the art. Such filtration has a cut-off filter of between 10,000 and 100,000 mol. wt. During this process, samples of the clarified serum may be taken to determine if the serum has been sufficiently concentrated. It is preferred that the final serum be concentrated to about 2 to 6 times the clarified serum, and most preferably 2 to 4 times.

Determination of the concentration level is made by testing the amount of IgG (or other immunoglobulin) within the serum. An initial test may be made of the clarified serum, and this result compared with the tests made upon the serum during the ultra-filtration process. For example, if the initial test results in the clarified serum having an IgG concentration of 1 g/100 ml, then the concentration process may be stopped when later tests report an IgG concentration of between about 2-6 g/100 ml, and preferably about 3 g/100 ml. The determination of the IgG amount may be made by the radial immunodiffusion test. However, it is preferred that serum protein electrophoresis be performed on the whole serum to obtain an entire gamma globulin result. This is believed to be more accurate, and provides a clear indication of the IgG level. Once the concentration process has been completed the concentrated unsterilized serum is bottled or packaged using standard procedures.

Upon completion of the concentration and packaging process, the result is unsterilized serum. The next step is to sterilize the serum. While this sterilization is effected, it is important that the unsterilized serum not be denatured. To provide sterilization without denaturing, the unsterilized serum is frozen to a hard freeze condition. For the unsterilized serum, this is approximately -29^oC. (-21^oF.). While still frozen, the material is then subjected to sufficient gamma irradiation that the material is sterilized, but is not denatured. This level may vary among various species, but may be determined without undue experimentation. It is important that the material be sufficiently cold (hard frozen) such that the material remains frozen during the irradiation step, otherwise denaturing will occur. It is for this reason that the material is frozen to the relatively low temperature. If it is found that if the irradiation process is sufficiently short, or refrigeration is provided during irradiation, then a higher temperature (though still below freezing) could be tolerated.

At this point the final serum has been obtained, although it is frozen. The packages of the serum are thus placed in refrigeration and allowed to thaw to the refrigeration temperature, where they are stored until use.

After administration, the serum has been found to provide cellular immunity similar to a vaccine, and can be used with or without the introduction of the virulent. In general, the present serum should provide protection against bacteria for which the donor group has immunity. In humans, a wide variety of vaccination uses are possible, including general vaccination for individuals with impaired immunity, such as is caused by diabetes, and vaccination for individuals preparing to undergo surgery due to the of nosocomial infection. In addition to humans, the inventive serum should also be of utility for many mammals, such as farm and domestic mammals and humans. For cattle, one particular use would be to avoid bovine mastitis, a common ailment which costs the dairy industry millions of dollars per year.

X. Uses for MSCRAMM and Antibody Compositions

The immunotherapeutic product of the present invention is a purified and concentrated extract of plasma, or serum from a purified donor pool. The serum contains antibodies released from the white blood cells in the extracted blood, and possibly other chemical materials present in the extracted blood. This serum is believed to provide information which is "read" by the immune system of the recipient to provide an extended period of immunity, typically on the order of six to eight weeks. Purified donor plasma pools can be used for the treatment of wounds, for blocking protein receptors or for immunization (vaccination).

The plasma pools comprise antibodies which are useful for interfering with the initial physical interaction between a pathogen and mammalian host responsible for infection, such as the adhesion of bacteria, particularly gram positive bacteria, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block protein-mediated mammalian cell invasion; to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial proteins that mediate tissue damage; and, to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or surgical techniques.

In general, both poly- and monoclonal antibodies against MSCRAMM peptides may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding the peptides discussed herein or related proteins. They may also be used in inhibition studies to analyze the effects of MSCRAMM-derived peptides in cells or animals. Anti-MSCRAMM epitope antibodies will also be useful in immunolocalization studies to analyze the distribution of MSCRAMMs during various cellular events, for example, to determine the cellular or tissue-specific distribution of the MSCRAMM peptides under different physiological conditions. A particularly useful application of such antibodies is in purifying native or recombinant MSCRAMMS, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.

Immunological compositions, including vaccine, and other pharmaceutical compositions containing the selected donor pool plasma concentrate are included within the scope of the present invention. The combination of immunoglobulins against binding proteins, or active or antigenic fragments thereof, or fusion proteins thereof, can be formulated and packaged, alone or in combination with other antibodies, using methods and materials known to those skilled in the art for vaccines. The immunological response may be used therapeutically or prophylactically and may provide passive immunity.

XI. Preparation of Proteins and Antibodies

The skilled reader can employ conventional molecular biology, microbiology, and recombinant DNA techniques to prepare the proteins, peptides, and antibody compositions described herein. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III (Ausubel, R. @-I ed., 1994); "Cell Biology: A Laboratory Handbook" Volumes I-III (J. E. Celis, ed., 1994); "Current Protocols in Immunology" Volumes I-III([Coligan, J. E., ed., 1994); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins eds., 1985); "Transcription And Translation" (B. D. Hames & S. J. Higgins, eds., 1984); "Animal Cell Culture" (R. I. Freshney, ed, (1986); "Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

The antibody obtained through this invention may be labeled directly with a detectable label for identification and quantification of staphylococcal bacterial such as S. aureus, S. epidermidis, etc. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances including colored particles such as colloidal gold and latex beads. Suitable immnunoassays include enzyme-linked immunosorbent assays (ELISA).

Alternatively, the antibody can be labeled indirectly by reaction with labeled substances that have an affinity for immunoglobulin, such as protein A or G or second antibodies. The antibody may be conjugated with a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate detected using labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten and the antibody-hapten conjugate detected using labeled anti-hapten antibody. These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art. Antibodies to the binding proteins may also be used in production facilities or laboratories to isolate additional quantities of the protein, such as by affinity chromatography.

In another identification embodiment, microliter plates pre-treated with poly-L-lysine are used to bind one of the target cells to each well, the cells are then fixed, e.g. using 1% glutaraldehyde, and the antibodies are tested for their ability to bind to the intact cell. In addition, FACS, immunofluorescence staining, idiotype specific antibodies, antigen binding competition assays, and other methods common in the art of antibody characterization may be used in conjunction with the present invention to identify preferred donors.

Humanized antibodies are antibodies of animal origin that have been modified using genetic engineering techniques to replace constant region and/or variable region framework sequences with human sequences, while retaining the original antigen specificity.

Such antibodies are commonly derived from rodent antibodies with specificity against human antigens. Such antibodies are generally useful for in vivo therapeutic applications. This strategy reduces the host response to the foreign antibody and allows selection of the human effector functions.

The techniques for producing humanized immunoglobulins are well known to those of skill in the art. For example, U.S. Pat. No. 5,693,762 discloses methods for producing, and compositions of, humanized immunoglobulins having one or more complementarily determining regions (CDR's). When combined into an intact antibody, the humanized immunoglobulins are substantially non-immunogenic in humans and retain substantially the same affinity as the donor immunoglobulin to the antigen, such as a protein or other compound containing an epitope. Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present invention include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 which describes recombinant immunoglobin preparations and U.S. Pat. No. 4,867,973 which describes antibody-therapeutic agent conjugates.

U.S. Pat. No. 5,565,332 describes methods for the production of antibodies, or antibody fragments, which have the same binding specificity as a parent antibody but which have increased human characteristics. Humanized antibodies may be obtained by chain shuffling, perhaps using phage display technology, in as much as such methods will be useful in the present invention the entire text of U.S. Pat. No. 5,565,332 is incorporated herein by reference.

XII. Production of High Titer MSCRAMM-specific IgG from Biological Fluids Via Affinity Purification

In accordance with the present invention, it is also possible to utilize modes of affinity isolation and purification in order to produce high titer MSCRAMM-specific immunoglobulins from biological fluids such as blood or plasma. In the preferred modes of this aspect of the invention, recombinant or wild-type/native MSCRAMMs can be covalently coupled to a substrate or resin, such as Sepharose.TM. or agarose, to form an affinity matrix. The MSCRAMM affinity matrix can be used to selectively isolate antibodies from serum, plasma, or other biological fluids. In the preferred embodiment, the biological fluid is passed over the MSCRAMM affinity matrix, and the matrix is then washed to remove non-specifically bound antibodies. The washed matrix is then subjected to conditions, such as low pH or high salt, so that MSCRAMM specific antibodies remaining on the matrix are eluted. The anti-MSCRAMM titer of the eluted material will be considerably higher than that of the original biological fluid, and the eluted material can then be utilized in the same manner as the other donor-selected or donor-stimulated compositions of the present invention.

XIII. Pharmaceutical Compositions

Pharmaceutical compositions for immunization of donors containing the MSCRAMM proteins, nucleic acid molecules, antibodies, or fragments thereof may be formulated in combination with a pharmaceutical carrier such as saline, dextrose, water, glycerol, ethanol, other therapeutic compounds, and combinations thereof. The formulation should be appropriate for the mode of administration. Suitable methods of administration include, but are not limited to, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.

The preferred route is by intravenous administration.

The pharmaceutical composition for treatment of any of the conditions described herein, should comprise, in a pharmaceutically acceptable excipient, an effective amount of immunoglobulin to treat or prevent the target disorder.

Compositions which contain immunoglobulins as active ingredients are well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. The therapeutic donor immunoglobulin pool compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of MSCRAMM binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.

The immunological compositions, such as vaccines, and other pharmaceutical compositions can be used alone or in combination with other blocking agents to protect against human and animal infections caused by staphylococcal bacterial including S. aureus and others. In particular, the compositions can be used to protect humans against endocarditis or to protect humans or ruminants against mastitis caused by staphylococcal infections. The vaccine can also be used to protect canine and equine animals against similar staphylococcal infections.

To enhance immunogenicity, the donor plasma pool concentrate proteins may be conjugated to a carrier molecule. Suitable immunogenic carriers include proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and derivatives thereof, particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solid phases. Other protein derived or non-protein derived substances are known to those skilled in the art. An immunogenic carrier typically has a molecular weight of at least 1,000 daltons, preferably greater than 10,000 daltons. Carrier molecules often contain a reactive group to facilitate covalent conjugation to the hapten. The carboxylic acid group or amine group of amino acids or the sugar groups of glycoproteins are often used in this manner. Carriers lacking such groups can often be reacted with an appropriate chemical to produce them. Preferably, an immune response is produced when the immunogen is injected into animals such as mice, rabbits, rats, goats, sheep, guinea pigs, chickens, and other animals, most preferably mice and rabbits. Alternatively, a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide may be sufficiently antigenic to improve immunogenicity without the use of a carrier.

In a preferred embodiment, a donor stimulating vaccine is packaged for immunization by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. The vaccine is most preferably injected intramuscularly into the deltoid muscle. The vaccine is preferably combined with a pharmaceutically acceptable carrier to facilitate administration. The preferred carrier is usually water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.

The carrier to which the protein may be conjugated may also be a polymeric delayed release system. Synthetic polymers are particularly useful in the formulation of a vaccine to effect the controlled release of antigens. For example, the polymerization of methyl methacrylate into spheres having diameters less than one micron has been reported by Kreuter, J., "Microcapsules and Nanoparticles in Medicine and Pharmacology," M. Donbrow, Ed., CRC Press, p. 125-148.

The amount of immunogen composition used in the production of the polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization. The preferred dose for human administration is from 0.01 mg/kg to 10 mg/kg, preferably approximately 1 mg/kg. Based on this range, equivalent dosages for heavier body weights can be determined. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The vaccine may additionally contain stabilizers such as thimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, Mo.) or physiologically acceptable preservatives.

The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the process may continue.

The compositions preferably further comprise an adjuvant. Many adjuvants are known for use in vaccinations in animals and are readily adapted to this composition. At this time, the only adjuvant widely used in humans has been alum (aluminum phosphate or aluminum hydroxide). Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines.

The isolated peptide can be linked to a selected amino acid sequence to make a fusion protein. As a nonlimiting example, a fusion protein can be made that comprises at least a first peptide of a fibronectin binding domain of fibronectin binding protein operatively linked to a selected amino acid sequence, wherein the first peptide does not specifically bind to fibronectin. In preferred aspects, the first peptide is linked to a selected carrier molecule or amino acid sequence, including, but not limited to, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).

One of the important features provided by the donor stimulation embodiment of the present invention is a polyclonal sera that is relatively homogenous with respect to the specificity of the antibodies therein. Typically, polygonal antisera is derived from a variety of different "clones," i.e., B-cells of different lineage. Monoclonal antibodies, by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality.

When peptides are used as antigens to stimulate the production of polyclonal sera, one expects considerably less variation in the clonal nature of the sera than if a whole antigen were employed. Unfortunately, if incomplete fragments of an epitope are presented, the peptide may very well assume multiple (and probably non-native) conformations. As a result, even short peptides can produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that does not react or reacts poorly with the native molecule.

Polyclonal antisera according to the present invention is produced against peptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore, more stable in an immunologic sense and thus express a more consistent immunologic target for the immune system. Under this model, the number of potential B-cell clones that will respond to this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be higher. In various embodiments, the present invention provides for polyclonal antisera where the clonality, i.e., the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonality--90%, 95% or greater--is contemplated.

XIV. Kits

This invention also includes a kit for the identification of blood or plasma with high titers of desired antibodies. The preferred kit contains sufficient antigen to bind substantially all of the antibody in the sample in about ten minutes or less, or sufficient antibody which can target an antibody in the sample that is to be detected. The antigen or antibody in the kit, e.g., any of the MSCRAMMs or their binding domains as described above, is preferably immobilized on a solid support, and can be labeled with a detectable agent such as those described above or commonly known in the art. The kit optionally contains a means for detecting the detectable agent. If the antigen or antibody in the kit is labeled with a fluorochrome or radioactive label, no means for detecting the agent will typically be provided, as the user will be expected to have the appropriate spectrophotometer, scintillation counter, or microscope. If the detectable agent is an enzyme, a means for detecting the detectable agent can be supplied with the kit, and would typically include a substrate for the enzyme in sufficient quantity to detect all of the antigen-antibody complex. One preferred means for detecting a detectable agent is a substrate that is converted by an enzyme into a colored product. A common example is the use of the enzyme horseradish peroxidase with 2,2'-azino-di-[3-ethyl-benzothiazoline sulfonate] (ABTS).

The invention includes a method for detecting biological samples with an elevated titer of antibodies to selected staphylococcal MSCRAMMs. As used herein the term biological sample refers to a sample of tissue or fluid isolated from a host, typically a human, including, but not limited to, plasma or serum. To confirm that a factor within donor plasma is immunologically cross-reactive with one or more epitopes of the disclosed peptides is a straightforward matter. This can be readily determined using specific assays, e.g., of a single proposed epitopic sequence, or using more general screens, e.g., of a pool of randomly generated synthetic peptides or protein fragments. The screening assays may be employed to identify-either equivalent antigens or cross-reactive antibodies. In any event, the principle is the same, i.e., based upon competition for binding sites between antibodies and antigens.

Any test which measures the binding of an antigen to an antibody can be used to evaluate the level of antigen or antibody in the host's biological sample according to the present invention. A number of other such tests are known and commonly used commercially.

Immunocytochemistry and immunohistochemistry are techniques that use antibodies to identify antigens on the surface of cells in solution, or on tissue sections, respectively. Immunocytochemistry is used to quantitate individual cell populations according to surface markers. Immunohistochemistry is used to localize particular cell populations or antigens. These techniques are also used for the identification of autoantibodies, using tissues or cells that contain the presumed autoantigen as substrate. The antibodies are usually identified using enzyme-conjugated antibodies to the original antibody, followed by a chromogen, which deposits an insoluble colored end product on the cell or tissue.

Another common method of evaluation is a radioimmunoassay, in which radiolabeled reagents are used to detect the antigen or antibody. Antibody can be detected using plates sensitized with antigen. The test antibody is applied and detected by the addition of a radiolabeled ligand specific for that antibody. The amount of ligand bound to the plate is proportional to the amount of test antibody. This test can be reversed to test for antigen. Variations of radioimmunoassays are competition RIA, direct binding RIA, capture RIA, sandwich RIA, and immunoradiometric assay (RMA).

Enzyme linked immunoabsorbent assays (ELISA) are a widely used group of techniques for detecting antigen and antibodies. The principles are analogous to those of radioimmunoassays except that an enzyme is conjugated to the detection system rather than a radioactive molecule. Typical enzymes used are peroxidase, alkaline phosphatase and 2-galactosidase. These can be used to generate colored reaction products from colorless substrates. Color density is proportional to the amount of reactant under investigation. These assays are more convenient than RIA, but less sensitive.

The Western blotting (immunoblotting) method is used to characterize unknown proteins. Components of the biological sample are separated by gel electrophoresis. SDS gets separate according to molecular weight and IEF gels separate the samples according to charge characteristics. The separated proteins are transferred to membranes (blotted) and identified by immunocytochemistry.

Less often used but suitable methods of evaluation include the Farr assay (in which radiolabeled ligands bind to and detect specific antibody in solution which are precipitated and quantified), precipitin reactions (in which antibodies and antigens crosslink into large lattices to form insoluble immune complexes; only works if antigen and antibody are present in sufficient amounts, at near equivalence, and when there are enough epitopes available to form a lattice); nephelometry (measures immune complexes formed in solution by their ability to scatter light); immunodiffusion (detects antigens and antibodies in agar gels); counter-current electrophoresis (similar to immunodiffusion, except that an electric current is used to drive the antibody and antigen together; useful for low concentrations of antigen or antibody); single radial immunodiffusion (SRID)(quantitates antigens by allowing them to diffuse outward from a well into an antibody containing gel; technique can be reversed by diffusing unknown antibody solutions into an antigen-containing well); rocket electrophoresis (similar to SRID, except that the test antigen is moved into the gel by an electric field); and immunofluorescence (similar to immunochemistry, except that it used fluorescence rather than enzyme conjugates). The antibody used to contact the sample of body fluid is preferably immobilized onto a solid substrate. The antibody can be immobilized using a variety of means, as described in Antibodies: A Laboratory Manual, cited supra. Suitable solid substrates include those having a membrane or coating supported by or attached to sticks, synthetic glass, agarose beads, cups, flat packs, or other solid supports. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes.

Means for labeling antibodies with detectable agents are also described in Antibodies: A Laboratory Manual. The amount of antigen in the host biological sample can be determined by any means associated with the selected assay. For example, the selected immunoassay can be carried out with known increasing amounts of antigen to produce a standard curve or color chart, and then the amount of test antigen can be determined by comparing the result of the test to the standard curve or chart that correlates the amount of antigen-antibody complex with known amounts of antigen. The amount of antigen determined to be present in the host biological sample can be used to evaluate the patient's condition in a number of ways. First, the level of antigen can be compared to a population norm based on statistical data. Second, the level of antigen can be considered in light of the patient's own history of antigen level.

The kit can optionally contain a lysing agent that lyses cells present in the sample of body fluid. Suitable lysing agents include surfactants such as Tween-80, Nonidet P40, and Triton X-100. Preferably, the lysing agent is immobilized onto the solid support along with the antibody.

The kit can also contain a buffer solution for washing the substrate between steps. The buffer solution is typically a physiological solution such as a phosphate buffer, physiological saline, citrate buffer, or Tris buffer.

The kit can optionally include different concentrations of a preformed antigen to calibrate the assay. The kit can additionally contain a visual or numeric representation of amounts of antigen in a calibrated standard assay for reference purposes. For example, if an assay is used that produces a colored product, a sheet can be included that provides a depiction of increasing intensities associated with differing amounts of antigen.

The kit can optionally include two antibodies in the detection system. The first antibody which is present in small amounts is specific for the antigen being assayed for. The second antibody provided in higher amounts is used to detect the first antibody. For example, a rabbit antibody can be used to detect the LOOH/amine antigen, and then an anti-rabbit IgG antibody can be used to detect the bound rabbit antibody. Goat antibodies and anti-antibodies are also commonly used.

As one nonlimiting example, a kit for the detection of the lipid peroxidation state of a patient is provided that includes a rabbit antibody specific for desired antibody, anti-rabbit IgG antibody in sufficient amounts to detect the bound first antibody, an enzyme conjugated to the second antibody and a substrate for the enzyme which changes color on exposure to the enzyme.

Claim 1 of 26 Claims

What is claimed is:

1. A selected purified human donor immunoglobulin composition comprising an antibody titer to an S. aureus serine-aspartate repeat (Sdr) protein in combination with an antibody titer to an S. epidermidis serine-asparate repeat (Sdr) protein wherein both antibody titers are higher than that found in pooled intravenous immunoglobulin obtained from unselected human donors, said composition obtained by a method comprising obtaining blood or plasma samples from human donors, screening said samples so as to select those samples having an antibody titer to an S. aureus Sdr protein and an antibody titer an S. epidermidis Sdr protein that are both in an amount that is higher than that found in pooled intravenous immumoglobulin obtained from unselected donors, recovering blood or plasma from the selected high-titer donors, and treating the donor blood plasma to obtain immumoglobulin in a purified state having an antibody titer to an S. aureus Sdr protein and an antibody titer to an S. epidermidis Sdr protein that are both in an amount which is higher than that found in pooled intravenous immunoglobulin obtained from unselected human donors.

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