EP0942982A1 - Proteines d'origine bacterienne de fixation sur l'elastine, sequences d'acide nucleique codant ladite proteine et procedes diagnostiques et therapeutiques d'utilisation de cette proteine - Google Patents

Proteines d'origine bacterienne de fixation sur l'elastine, sequences d'acide nucleique codant ladite proteine et procedes diagnostiques et therapeutiques d'utilisation de cette proteine

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Publication number
EP0942982A1
EP0942982A1 EP97914810A EP97914810A EP0942982A1 EP 0942982 A1 EP0942982 A1 EP 0942982A1 EP 97914810 A EP97914810 A EP 97914810A EP 97914810 A EP97914810 A EP 97914810A EP 0942982 A1 EP0942982 A1 EP 0942982A1
Authority
EP
European Patent Office
Prior art keywords
mscramm
binding
elastin
seq
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97914810A
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German (de)
English (en)
Inventor
Robert Paul Mecham
Pyong Woo Park
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Washington
Washington University in St Louis WUSTL
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University of Washington
Washington University in St Louis WUSTL
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Publication date
Application filed by University of Washington, Washington University in St Louis WUSTL filed Critical University of Washington
Publication of EP0942982A1 publication Critical patent/EP0942982A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to a microbial surface component recognizing adhesive matrix molecules (MSCRAMM) and to active polypeptide fragments thereof, and to nucleotide sequences encoding the protein and active polypeptide fragments thereof. More specifically, the invention relates to a protein on the surface of bacteria which binds a component of the extracellular matrix (ECM), which component is termed elastin, and to diagnostic and therapeutic methods which relate to this protein-protein interaction.
  • ECM extracellular matrix
  • ECM extracellular matrix
  • ISA/EP pathogenic bacteria also interact specifically with the host ECM through cell surface ECM binding molecules.
  • Cell surface ECM binding molecules of pathogenic bacteria belong to a group of proteins known collectively as adhesins or microbial surface components recognizing adhesive matrix molecules (MSCRAMMS). and are widely believed to play important roles in key steps of disease pathogenesis (1 1,12).
  • S. aureus has been identified as one of the causative agents of diseases such as infective endocarditis, osteomyelitis, aortitis, pneumonia, and scalded skin syndrome (13-15). Furthermore, several strains of S. aureus have a propensity to extravasate into the circulation to cause bacteremia and subsequent formation of metastatic abscesses. These properties imply that S. aureus is capable of interacting with various components of the host expressed in respective target tissues. In agreement with this hypothesis, S. aureus has been found to associate with many host determinants including major ECM components such as collagen (16), fibronectin (17), laminin (18), proteoglycans (19), and elastin (20).
  • major ECM components such as collagen (16), fibronectin (17), laminin (18), proteoglycans (19), and elastin (20).
  • S. aureus Most clinical isolates of S. aureus bind specifically to fibronectin. and mutant strains defective in fibronectin binding have decreased ability to colonize damaged heart valves in animal models of endocarditis (21).
  • the S. ⁇ wrew ⁇ -collagen binding interaction has been implicated in osteomyelitis and septic arthritis by Switalski et al. (22), in which expression of the collagen adhesin has been found to be both necessary and sufficient for attachment of S. aureus to the type II collagen-rich cartilage.
  • elastin The primary physiologic role of elastin is to confer the property of reversible elasticity to tissues and organs (24). Elastin expression is highest in the lung, skin, and blood vessels, but the protein is widely expressed in mammalian hosts for S aureus. Since elastin binding may be a mechanism for S. aureus to target elastin-rich tissues of the host, the cellular and biochemical properties of this interaction (20) were investigated in a previous study. S. aureus binding to elastin was found to be rapid, reversible, saturable, of high affinity (low nM), and ligand specific.
  • EbpS for elastin binding protein of Staphylococcus aureus
  • EbpS has been proposed to mediate S. aureus binding to elastin-rich host ECM.
  • EbpS is structurally distinct from the mammalian cell surface elastin binding protein, and the two elastin binding proteins recognize different regions in elastin.
  • EbpS binds to a region in the N-terminal 30 kDa fragment of elastin, whereas the mammalian elastin receptor recognizes the hexapeptide sequence VGVAPG in the C-terminal half of elastin (25).
  • nucleotide sequence encodes a bacterial MSCRAMM, which is implicated in the attachment, colonization and invasion of host cells by the bacteria.
  • the invention provides the MSCRAMM protein, biologically active fragments thereof, and antibodies and binding partners thereto, which are useful in the diagnosis and treatment of bacterial infections.
  • These reagents are particularily useful in treating infections of elastin-containing host tissues, for example lung, skin and blood vessels.
  • Corresponding pharmaceutical compositions and therapeutic methods involving the MSCRAMMs of the present invention are contemplated, that may be directed to a broad range of diseases and other conditions, including by way of non-limiting illustration, such conditions as tumor cell metastasis, wound healing, infective endocarditis, osteomyelitis, aortitis, pneumonia and scalded skin syndrome.
  • One aspect of the present invention includes an MSCRAMM having the following characteristics: a) it binds to elastin; b) its activity is inhibited in the presence of SDS; and c) it has enhanced activity in the presence of thiol reductants.
  • the MSCRAMM has a predicted molecular weight of about 25 kDa, and a predicted PI of about 4.9. More particularly, the present MSCRAMM is isolated from Staphylococcus aureus.
  • polypeptide fragments of an MSCRAMM and related polypeptides which retain the property of binding elastin.
  • a polypeptide comprises an amino acid sequence that corresponds to the elastin binding site of a MSCRAMM and consists of between 8 and 80 amino acids.
  • the polypeptide has the amino acid sequence consisting of the N-terminal 59 amino acids of the MSCRAMM.
  • the polypeptide consists of between 10 and 46 amino acids.
  • the polypeptide consists of between 12 and 21 amino acids.
  • the polypeptide contains about 10 amino acids.
  • the MSCRAMM has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:2 comprising a conservative substitution thereof.
  • polypeptide corresponds to an elastin binding site of a MSCRAMM that comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 16 comprising a conservative substitution thereof (amino acids (AAs) 18-23 of SEQ ID NO:2: TNSHQD).
  • polypeptide has the amino acid sequence of SEQ ID NO: 10 (AAs 1-78 of SEQ ID NO:2) or SEQ ID NO: 10 comprising a conservative substitution thereof.
  • polypeptide has the amino acid sequence of SEQ ID NO: 12 (AAs 1- 34 of SEQ ID NO:2) or SEQ ID NO: 12 comprising a conservative substitution thereof.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 13 (AAs 14-34 of SEQ ID NO:2) or SEQ ID NO: 13 comprising a conservative substitution thereof.
  • polypeptide comprises the amino acid sequence of SEQ ID NO: 14 (AAs 14-23 of SEQ ID NO:2) or SEQ ID NO: 14 comprising a conservative substitution thereof.
  • polypeptide consists of the amino acid sequence of SEQ ID NO: 15 (AAs 14-59 of SEQ ID NO:2) or SEQ ID NO: 15 comprising a conservative substitution thereof.
  • polypeptide of comprises the amino acid sequence of SEQ ID NO: 18 (AAs 18-34 of SEQ ID NO:2) or SEQ ID NO: 18 comprising a conservative substitution thereof.
  • polypeptide further inhibits the binding of S. aureus to elastin.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a MSCRAMM or an active polypeptide fragment thereof; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene.
  • the nucleic acid encodes a MSCRAMM having the DNA sequence shown in FIGURE 3 (SEQ ID NO: l).
  • a nucleic acid encodes a polypeptide that binds elastin; consists of between 8 and 80 amino acids; and comprises an amino acid sequence that corresponds to the elastin binding site of a microbial surface component recognizing adhesive matrix molecules (MSCRAMM).
  • the nucleic acid has the nucleic acid sequence of SEQ ID NO:9.
  • the nucleic acid has the nucleic acid sequence of SEQ ID NO:l 1.
  • the bacterial DNA sequences of the MSCRAMM of the present invention or portions thereof may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species.
  • the present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the MSCRAMM.
  • probes may be degenerate probes based on SEQ ID NO:3.
  • primers directed to the 5' and 3' sequences of the MSCRAMM such as SEQ ID NOS: 4 and 5, may be used to amplify related sequences by PCR.
  • the probes may be prepared with a variety of known vectors, such as the phage ⁇ vector.
  • the present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences set forth in FIGURE 3 (SEQ ID NO:l, respectively).
  • the preparation of antisense RNA and ribozymes are included herein.
  • the present invention also includes MSCRAMM proteins having the activities noted herein, and that display the amino acid sequences set forth and described above and selected from SEQ ID NO:2.
  • the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host.
  • the invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present MSCRAM(s) active polypeptide fragments thereof and the polypeptides of the present invention. More particularly, a unicellular host is transformed with the complete DNA sequence determined from the sequences set forth above or a fragment of the DNA sequence, e.g. SEQ ID NO: 1, or SEQ ID NOs: 9 or 11.
  • a recombinant expression system is provided to produce biologically active animal or human MSCRAMMs or the active polypeptide fragments thereof, or the polypeptides of the present invention.
  • MSCRAMM contemplates that specific bacterial cell surface proteins exist for correspondingly specific components in host tissue, such as elastin and the like, as described earlier. Accordingly, the exact structure of each MSCRAMM will understandably vary so as to achieve this binding and activity specificity. It is this specificity and the direct involvement of the MSCRAMM in the chain of events leading to bacterial infection, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities.
  • the present invention naturally contemplates several means for preparation and isolation of the MSCRAMM, including isolation from a natural source and/or as illustrated herein, through known recombinant techniques.
  • the invention is accordingly intended to cover such preparations within its scope.
  • Subsequent isolation of the MSCRAMM, active polypeptide fragments thereof and polypeptides of the present invention as illustrated herein, is also included as part of the invention.
  • the isolation of the genomic DNA and amino acid sequences disclosed herein facilitates the reproduction of the MSCRAMM by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
  • the invention includes an assay system for screening of potential drugs effective to modulate binding activity to target mammalian cells by interrupting or potentiating the binding of the MSCRAMM to host tissue.
  • the test drug could be administered to a cellular sample with the MSCRAMM that binds the host tissue, or an extract containing the MSCRAMM, to determine its effect upon the binding activity of the MSCRAMM to any chemical sample (including DNA), or to the test drug, by comparison with a control.
  • the assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the MSCRAMM, thereby inhibiting or potentiating infectivity.
  • Such an assay would be useful in the development of drugs that would be specific against particular infections. For example, such drugs might be used to prevent infection, or to treat infection, as for example, in association with an antibiotic.
  • the invention contemplates antagonists of the activity of a MSCRAMM, in particular, an agent or molecule that inhibits MSCRAMM binding to elastin.
  • the antagonist can be a peptide having the sequence of a portion of an elastin-binding domain of a MSCRAMM.
  • One of the characteristics of the present MSCRAMM is that it binds to a 30 kDa N- terminal fragment of elastin, which is present in tissues which require elasticity, such as lungs, skin and blood vessels.
  • the diagnostic utility of the present invention extends to the use of the present MSCRAMM in assays to screen for bacterial infection.
  • the present invention likewise extends to the development of antibodies against the MSCRAMM(s) and active polypeptide fragments thereof and polypeptides of the invention, including naturally raised and recombinantly prepared antibodies.
  • the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the MSCRAMM(s).
  • Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities thereby suiting them for additional diagnostic use conjunctive with their capability of modulating bacterial infectivity.
  • the MSCRAMMs active polypeptide fragements thereof, polypeptides of the invention, their analogs, cognates and/or mimics, and any antagonists or antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the MSCRAMM that has been labeled by either radioactive addition, or radioiodination.
  • immunoassays such as a radioimmunoassay, using for example, an antibody to the MSCRAMM that has been labeled by either radioactive addition, or radioiodination.
  • a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 6 C1, 51 Cr, "Co, 58 Co, 59 Fe, 90 Y, 125 I, m I, and 186 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric. fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • the present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the MSCRAMM, or to identify drugs or other agents that may mimic or block their activity.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the MSCRAMMs, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the MSCRAMM(s), its (or their) subunits, or active polypeptide fragments thereof, or polypeptides of the present invention or upon agents or other drugs determined to possess the same activity.
  • a first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from the binding activity of the
  • MSCRAMM or its subunits comprises administering an agent capable of modulating the production and/or activity of the MSCRAMM or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host.
  • agents capable of modulating the production and/or activity of the MSCRAMM or subunits thereof either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host.
  • drugs or other binding partners to the MSCRAMM or proteins may be administered to inhibit bacterial infection. More particularly in the treatment of S. aureus infection.
  • the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise effective inhibitors or enhancers of the MSCRAMM or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • pharmaceutical compositions may comprise effective inhibitors or enhancers of the MSCRAMM or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • drugs or other binding partners to the MSCRAMM or proteins, as represented by SEQ ID NO:2 may be administered to inhibit bacterial infectivity.
  • MSCRAMS active polypeptide fragments thereof and peptides of the present invention could be prepared in pharmaceutical formulations for administration in instances wherein antibiotic therapy is appropriate.
  • compositions for use in therapeutic methods which comprise or are based upon the MSCRAMM, its subunits, active polypeptide fragments thereof, polypeptides of the present invention binding partner(s) to MSCRAMMs, or upon agents or drugs that control the production, or that mimic or antagonize the activities of the MSCRAMM.
  • FIG. 1 Southern analysis of genomic DNA from S. aureus. Genomic DNA isolated from S. aureus strain 12598 was digested with EcoR I (lanes A & C) or EcoR VHind ⁇ lVHinc II (lane D) and p ⁇ BPS-1 was digested with EcoR I (lane B). Samples were fractionated by 1 % TAE-agarose electrophoresis and Southern blotted to nitrocellulose. The membranes were hybridized to a degenerate oligonucleotide
  • FIG. 1 Physical map of the pKS-2.6 insert. Sites recognized by various restriction endonucleases are indicated. Location and direction of the ebpS open reading frame is shown by the hatched box and arrow, respectively.
  • Figure 3 Primary sequence of ebpS. Nucleotide and predicted amino acid sequences are numbered starting at the first nucleotide of the open reading frame and translation initiation codon, respectively. The putative -35 and -10 hexamers, and ribosomal binding site are indicated. The experimentally determined N-terminal sequence of cell surface EbpS is shown in bold letters, and the experimentally determined amino acid sequences of EbpS are underlined. The N-terminal amino acids of the recombinant construct were sequenced from both full length rEbpS and N-terminal fragment of CNBr-cleaved rEbpS. The in-frame termination codon is indicted by an asterisk.
  • rEbpS purified from three different positive clones by Ni++-NTA affinity chromatography was fractionated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (lanes B-D). Migration pattern of the size standard is shown in lane A.
  • FIG. 5 rEbpS binds specifically to immobilized elastin peptides. Approximately 10 6 cpm of radiolabeled rEbpS was incubated with 1 ml of the elastin peptide affinity resin in the absence (lane C) or presence (lane D) of 2 mg of unlabeled elastin peptides for 2 h at room temperature in 1.5 ml of binding buffer. After thorough washing, bound proteins were eluted with 1% SDS buffer and analyzed by 10% SDS- PAGE and autoradiography. The starting material contained a 40 kDa degradation product in addition to the intact 45 kDa rEbps (lane B). Migration of 14 C-labeled size standards is shown in lane A.
  • FIG. 6 rEbpS specifically inhibits S. aureus binding to radiolabeled elastin.
  • Radioiodinated elastin (20 ng) was incubated with 2 x 10 8 live S. aureus cells in the absence or presence of 1.0, 2.0, 5.9, 9.8, or 19.6 ⁇ M of unlabeled rEbpS or 26 ⁇ M of mouse DHFR for 1 h at room temperature in 200 ⁇ l of TSB. The cells were pelleted by centrifugation, and the supernatant was discarded. Pellets were resuspended in 1 ml of TSB, transferred to new tubes, and washed two more time with TSB. Radioactivity associated with cells was measured using a gamma counter. Results are presented as mean % binding ⁇ standard deviation of triplicate determinations.
  • FIG. 7 Cell surface labeled EbpS binds to an antibody against rEbps.
  • Cell surface labeled extracts (10 7 cpm) prepared by IODOGEN radioiodination and subsequent lysostaphin digestion (lane A) were pre-absorbed to 3 ml of pig IgG-Affi-Gel 10 to remove protein from the starting material.
  • Starting material devoid of surface labeled protein A (lane B) was incubated with 1 ml of the anti-rEbpS IgG affinity resin in the absence (lane C) or presence (lane D) of 2 mg unlabeled rEbpS in 2 ml of binding buffer for 2 h at room temperature.
  • Figure 8 Fab fragments of anti-rEbpS IgG inhibit S. aureus binding to elastin. Radiolabeled elastin was incubated with live S. aureus cells in the absence or presence of 6, 10, 20, 50, and lOO ⁇ g of immune rEbpS IgG Fab or 20 and 100 ⁇ g of pre- immune Fab fragments for 1 h at room temperature in 2 ml of binding buffer, and binding was quantified as previously described. Data are presented as mean % binding ⁇ standard deviation of triplicate measurements.
  • FIG. 9 The elastin binding site in EbpS is contained within residues 14-59. Elastin binding properties of various EbpS fragments and recombinant constructs (described in Example 2) were assessed by their capacity to specifically bind to tropoelastin. Inactivity of the amino terminal synthetic peptide was determined by an inability to inhibit binding. Residues 14-59 (shaded area) are common to all fragments with elastin binding activity.
  • FIG. 10 Expression of recombinant EbpS proteins.
  • Recombinant EbpS proteins were purified by Ni-NTA affinity chromatography, fractionated by 15% SDS-PAGE, and stained with Coomassie Brilliant Blue R-250 (a) or transferred to nitrocellulose membranes and reacted with anti-rEbpS IgG (b) or anti-rEbpS IgG that had been pre-absorbed to trEbpS-2 (c).
  • Lane A ovalbumin
  • lane B rEbpS
  • lane C trEbpS-1
  • lane D trEbpS-2
  • lane E lysozyme.
  • Molecular masses of the recombinant proteins were approximated from the migration pattern of ovalbumin, lysozyme, and pre-stained size standards.
  • FIG 11 Recombinant trEbpS-1 and trEbpS-2 bind to elastin.
  • Tropolastin (3 ⁇ g) that was fractionated by 10% SDS-PAGE and Western blotted to nitrocellulose membranes was reacted with 5 ⁇ M biotinylated trEbpS-1 (lanes A and B) or trEbpS-2 (lanes C and D) in the absence (lanes A and C) or presence (lanes B and D) of 3 mg/ml elastin peptides. Binding of truncated EbpS proteins was visualized by subsequent incubation with avidin-horse radish peroxidase and 4-chloro-naphthol.
  • FIG. 12 Truncated recombinant EbpS proteins inhibit binding of S. aureus cells to radiolabeled elastin.
  • Live S. aureus cells (2x10 8 ) were incubated with radioiodinated elastin (10 ng) in the absence or presence of increasing concentrations of rEbpS, trEbpS-1, or trEbpS-2 for 1 hour at room temperature in 200 ⁇ l of TSB. The assay was terminated by centrifugation, and cell pellets were washed twice with 1 ml of TSB. Extent of binding was quantified by measuring radioactivity associated with the pellets.
  • Results are presented as mean relative % binding ⁇ SD of triplicate determinations, with measurements obtained in the absence of recombinant EbpS proteins defined as 100%.
  • Figure 13 The inhibitory effect of anti-rEbpS antibody is neutralized by pre-absorption with trEbpS-2.
  • Fab fragments from the original (control) and anti-rEbpS IgGs that had been pre-absorbed with trEbpS-2 (trEbpS-2 negative) were prepared by papain digestion. Increasing concentrations of Fab fragments were incubated with live S. aureus cells and radiolabeled elastin as previously described. Data are shown as mean relative % binding ⁇ SD from triplicate determinations.
  • Figure 14 Elastin binding site defined by peptide inhibition studies. Seven overlapping synthetic peptides spanning residues 14-36 were tested for their ability to inhibit binding of S. aureus to tropoelastin. Inhibition activity, qualitatively scored as + or -, is indicated next to the peptide number. Some amino acids in peptides P2 and P7 were substituted as indicated. The shaded box indicates the predicted active sequence required for elastin binding.
  • Figure 16 A synthetic peptide corresponding to EbpS residues 14-23 specifically inhibits staphylococcal elastin binding. Overlapping synthetic lOmers corresponding to residues 14-23 (P3), 21-30 (P4), and 27-36 (P5) were generated as described. S. aureus cells were incubated with labeled elastin in the absence or presence of 0.5, 1.0, or 2.0 mg/ml of P3-P5 peptides. Binding assays were processed as described previously. Error bars represent SD calculated from triplicate determinations.
  • MSCRAMM bacterial cell surface protein
  • elastin binding protein (ebpS) any variants not specifically listed
  • proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in FIGURE 3 (SEQ ID NO:2), and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms “MSCRAMM,” “bacterial cell surface protein” and “elastin binding protein (ebpS)” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • an “active polypeptide fragment” of a MSCRAMM is a polypeptide fragment of an MSCRAMM that binds to elastin.
  • a “polypeptide of the present invention” is a polypeptide that comprises an amino acid sequence that corresponds to the elastin binding site of an MSCRAMM. Such polypeptides consist of between 8 and 80 amino acids.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired fractional property of immunoglobulin-binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • polypeptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other the bonds, e.g.. ester, ether, etc.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • An "origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy 1) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • RNA polymerase a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine- Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand.
  • non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • codons specified above are for RNA sequences.
  • the corresponding codons for DNA have a T substituted for U.
  • Mutations can be made in SEQ ID NO:l such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • Such a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting polypeptide. Accordingly, such conservative changes are defined herein as a "conservative substitution”.
  • Another grouping may be those amino acids with phenyl groups:
  • Another grouping may be according to molecular weight (i.e., size of R groups):
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced into a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816.567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al.
  • Fab' antibody molecule portions are also well-known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the S phase activity of a target cellular mass, or other feature of pathology such as for example, elevated blood pressure, fever or white cell count as may attend its presence and activity.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • operatively linked includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • an appropriate start signal e.g., ATG
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 °C for both hybridization and wash. However, one skilled in the art will appreciate that such “standard hybridization conditions” are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20°C below the predicted or determined T m with washes of higher stringency, if desired.
  • the present invention concerns the identification of a bacterial MSCRAMM which binds a component of the extracellular matrix, mediating the attachment, colonization and/or invasion of the bacterial into a host tissue.
  • the present invention relates to all members of the herein disclosed elastin binding proteins.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a MSCRAMM, or a fragment thereof, that possesses a predicted molecular weight of about 25 kD and an amino acid sequence set forth in FIGURE 3 (SEQ ID NO:2); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 25 kD has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURE 2 (SEQ ID NO: 1).
  • Initial steps for purifying an MSCRAMM or active polypeptide fragment thereof of the present invention include salting in or salting out, such as in ammonium sulfate fractionations; solvent exclusion fractionations, e.g., an ethanol precipitation; detergent extractions to free membrane bound proteins using such detergents as TRITON X-100, TWEEN-20 etc.; or high salt extractions. Solubilization of proteins or polypeptides may also be achieved using aprotic solvents such as dimethyl sulfoxide and hexamethylphosphoramide. In addition, high speed ultracentrifugation may be used either alone or in conjunction with other extraction techniques.
  • Solid phase binding may be performed through ionic bonding, with either an anion exchanger, such as diethylaminoethyl (DEAE), or diethyl [2-hydroxypropyl] aminoethyl (QAE) SEPHADEX or cellulose; or with a cation exchanger such as carboxymethyl (CM) or sulfopropyl (SP) SEPHADEX or cellulose.
  • an anion exchanger such as diethylaminoethyl (DEAE), or diethyl [2-hydroxypropyl] aminoethyl (QAE) SEPHADEX or cellulose
  • a cation exchanger such as carboxymethyl (CM) or sulfopropyl (SP) SEPHADEX or cellulose.
  • Solid phase binding includes the exploitation of hydrophobic interactions e.g., the using of a solid support such as phenyl Sepharose and a high salt buffer; affinity-binding, using, e.g., elastin on an activated support; immuno-binding, using e.g., an antibody to an MSCRAMM or active polypeptide fragment thereof bound to an activated support; as well as other solid phase supports including those that contain specific dyes or lectins etc.
  • a further solid phase support technique that is often used at the end of the purification procedure relies on size exclusion, such as SEPHADEX and SEPHAROSE gels, or pressurized or centrifugal membrane techniques, using size exclusion membrane filters.
  • Solid phase support separations are generally performed batch-wise with low-speed centrifugations or by column chromatography.
  • High performance liquid chromatography HPLC
  • FPLC FPLC
  • Size exclusion techniques may also be accomplished with the aid of low speed centrifugation.
  • size permeation techniques such as gel electrophoretic techniques may be employed. These techniques are generally performed in tubes, slabs or by capillary electrophoresis.
  • Typical buffers can be purchased from most biochemical catalogues and include the classical buffers such as Tris, pyrophosphate. monophosphate and diphosphate and the Good buffers [Good, N.E., et al.
  • the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the MSCRAMM is implicated, to modulate the activity initiated by the MSCRAMM.
  • an appropriate inhibitor of the MSCRAMM could be introduced to block the interaction of the MSCRAMM with those factors causally connected with bacterial adhesion thereby.
  • the MSCRAMM or their binding partners or other ligands or agents exhibiting either mimicry or antagonism to the MSCRAMM or control over their production may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated with specific bacterial infection for the treatment thereof.
  • a variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the MSCRAMM or their subunits may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the MSCRAMM and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as bacterial infection or the like.
  • the MSCRAMM or its subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • small molecules that mimic or antagonize the activity(ies) of the MSCRAMM of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies And T- cell Hybridomas” (1981); Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.
  • Panels of monoclonal antibodies produced against MSCRAMM peptides can be screened for various properties; i.e., isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that neutralize the binding activity of the
  • MSCRAMM or its subunits in particular the binding activity of the first 59 amino acids of the molecule to the amino terminal portion of elastin.
  • Such monoclonals can be readily identified in binding activity assays such as ELISA or WESTERN BLOT.
  • High affinity antibodies are also useful when immunoaffinity purification of native or recombinant MSCRAMM is possible.
  • the anti-MSCRAMM antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • the anti-MSCRAMM antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions of whole antibody molecules.
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to a MSCRAMM/protein, such as an anti-MSCRAMM antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • an antagonist to a MSCRAMM/protein such as an anti-MSCRAMM antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • the anti-MSCRAMM antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions of whole antibody molecules.
  • patients capable of benefiting from this method include those suffering from bacterial infections associated with cancer, a pre- cancerous lesion, a viral infection or other like pathological derangement.
  • Methods for isolating the MSCRAMM and inducing anti-MSCRAMM antibodies and for determining and optimizing the ability of anti-MSCRAMM antibodies to assist in the examination of the target cells are all well
  • a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with an elastin-binding portion thereof.
  • Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000.
  • Fused hybrids are selected by their sensitivity to HAT.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present MSCRAMM and their ability to inhibit specified MSCRAMM activity in target cells.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody- containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • DMEM Dulbecco's minimal essential medium
  • fetal calf serum An exemplary inbred mouse strain is the Balb/c.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a MSCRAMM, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient.
  • the composition comprises an antigen capable of modulating the specific binding of the present MSCRAMM to a target cell.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • 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. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • 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.
  • 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 hour intervals by a subsequent injection or other administration.
  • the therapeutic compositions may further include an effective amount of the MSCRAMM/MSCRAMM antagonist or analog thereof, and one or more of the following active ingredients: an antibiotic, a steroid.
  • active ingredients an antibiotic, a steroid.
  • Intravenous Formulation II Ingredient mg/ml ampicillin 250.0
  • Intravenous Formulation V Ingredient mg/ml
  • MSCRAMM antagonist 5.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml
  • pg means picogram
  • ng means nanogram
  • ug means microgram
  • mg means milligram
  • ul or “ ⁇ l” mean microliter
  • ml means milliliter
  • 1 means liter.
  • the polypeptides of the present invention can be can be chemically synthesized.
  • the synthetic polypeptides are prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
  • Amino acids used for peptide synthesis may be standard Boc (N ⁇ -amino protected N ⁇ -t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield [J Am. Chem.
  • polypeptides of the invention may comprise D-amino acids, a combination of D- and L-amino acids, and various "designer" amino acids (e.g., ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids, etc.) to convey special properties.
  • Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine. Additionally, by assigning specific amino acids at specific coupling steps, ⁇ -helices, ⁇ turns, ⁇ sheets, ⁇ -turns, and cyclic peptides can be generated. In a further embodiment, subunits of peptides that confer useful chemical and structural properties will be chosen. For example, peptides comprising D-amino acids will be resistant to L-amino acid-specific proteases in vivo.
  • peptides that have more well defined structural properties, and the use of peptidomimetics, and peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties.
  • a peptide may be generated that incorporates a reduced peptide bond, i.e., R,-CH 2 -NH-R 2 , where R, and R 2 are amino acid residues or sequences.
  • a reduced peptide bond may be introduced as a dipeptide subunit.
  • Such a molecule would be resistant to peptide bond hydrolysis, e.g., protease activity.
  • Such peptides would provide ligands with unique function and activity, such as extended half-lives in vivo due to resistance to metabolic breakdown, or protease activity. Furthermore, it is well known that in certain systems constrained peptides show enhanced functional activity [Hruby, Life Sciences, 31:189-199 (1982)]; [Hruby et al, Biochem J., 268:249-262 (1990)]; the present invention provides a method to produce a constrained peptide that incorporates random sequences at all other positions.
  • non-classical amino acids may be inco ⁇ orated in the peptide in order to introduce particular conformational motifs: l,2,3,4-tetrahydroisoquinoline-3- carboxylate [Kazmierski et al, J. Am. Chem.
  • amino acid analogs and peptidomimetics may be inco ⁇ orated into a peptide to induce or favor specific secondary structures: LL-Acp (LL-3-amino- 2-propenidone-6-carboxylic acid), a ⁇ -turn inducing dipeptide analog (Kemp et al, J. Org.
  • DNA sequences disclosed herein may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • phage DNAS e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single strande
  • any of a wide variety of expression control sequences ⁇ sequences that control the expression of a DNA sequence operatively linked to it ⁇ may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast ⁇ -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture. It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention.
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
  • MSCRAMM analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention.
  • Analogs, such as fragments may be produced, for example, by pepsin digestion of bacterial material.
  • Other analogs, such as muteins can be produced by standard site-directed mutagenesis of MSCRAMM coding sequences.
  • Analogs exhibiting "elastin binding activity" such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.
  • a DNA sequence encoding MSCRAMM can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the MSCRAMM amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).
  • Synthetic DNA sequences allow convenient construction of genes which will express MSCRAMM analogs or "muteins".
  • DNA encoding muteins can be made by site-directed mutagenesis of native MSCRAMM genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • the present invention extends to the preparation of antisense oligonucleotides and ribozymes that may be used to interfere with the expression of the MSCRAMM at the translational level.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. (See Weintraub, 1990; Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into MSCRAMM-producing cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it [Cech, J. Am. Med. Assoc, 260:3030 (1988)]. Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena- ype Tetrahymena- ype and "hammerhead"-type.
  • Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead "-type recognize eleven- to eighteen-base sequences.
  • the DNA sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave mRNAs for MSCRAMM and their ligands.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as the earlier referenced polypeptide ligands, by reference to their ability to elicit the activities which are mediated by the present MSCRAMM.
  • the MSCRAMM can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular MSCRAMM activity in suspect target tissues.
  • antibody(ies) to the MSCRAMM can be produced and isolated by standard methods including the well known hybridoma techniques.
  • the antibody(ies) to the MSCRAMM will be referred to herein as Ab, and antibody(ies) raised in another species as Ab 2 .
  • MSCRAMM The presence of MSCRAMM in cells can be ascertained by the usual immunological procedures applicable to such determinations.
  • a number of useful procedures are known. Three such procedures which are especially useful utilize either the MSCRAMM labeled with a detectable label, antibody Ab, labeled with a detectable label, or antibody Ab 2 labeled with a detectable label.
  • the procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "M" stands for the MSCRAMM:
  • Ab 2 will react with Ab,. This is because Ab, raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab 2 .
  • Ab 2 may be raised in goats using rabbit antibodies as antigens. Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Ab will be referred to as a primary or anti-MSCRAMM antibody, and Ab 2 will be referred to as a secondary or anti-Ab, antibody.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the MSCRAMM or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, ,4 C, 32 P, 35 S, 36 C1, 5, Cr, "Co, 58 Co, 59 Fe, 90 Y, ,25 I, 131 I, and , 86 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3.654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • a particular assay system developed and utilized in accordance with the present invention is known as a receptor assay.
  • the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.
  • a purified quantity of the MSCRAMM may be radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined MSCRAMM, and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • an assay useful and contemplated in accordance with the present invention is known as a "cis/trans” assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex.
  • one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted.
  • the compound under test is an agonist for the receptor
  • the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene.
  • the resulting chemi luminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands.
  • the foregoing protocol is described in detail in U.S. Patent No. 4,981,784 and PCT International Publication No. WO 88/03168, for which pu ⁇ ose the artisan is referred.
  • test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined MSCRAMM activity or predetermined elastin binding activity capability in suspected target cells.
  • one class of such kits will contain at least the labeled MSCRAMM or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g.. "competitive,” "sandwich,”
  • kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the presence or capability of cells for predetermined elastin binding activity, comprising:
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the pu ⁇ oses stated above, which operates according to a predetermined protocol (e.g. "competitive,” “sandwich,” “double antibody,” etc.), and comprises:
  • an assay system for screening potential drugs effective to modulate the activity of the MSCRAMM may be prepared.
  • the MSCRAMM may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the MSCRAMM activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known MSCRAMM.
  • EbpS staphylococcal elastin binding protein
  • DNA sequence data indicate that the ebpS open reading frame consists of 606 bp, and encodes a novel polypeptide of 202 amino acids.
  • ⁇ bpS protein has a predicted molecular mass of 23,345 daltons and pi of 4.9.
  • ⁇ bpS was expressed in E coli as a fusion protein with polyhistidine residues attached to the N-terminus.
  • a polyclonal antibody raised against recombinant ⁇ bpS interacted specifically with the 25 kDa cell surface ⁇ bpS and inhibited staphylococcal elastin binding.
  • recombinant ⁇ bpS bound specifically to immobilized elastin and inhibited binding of Staphylococcus aureus to elastin.
  • Restriction endonucleases calf intestinal phosphatase, T4 DNA ligase, T4 polynucleotide kinase, isopropyl- ⁇ -D-galactoside (X-gal), Wizard Miniprep plasmid purification kit, and Hind Ill-digested ⁇ DNA markers were purchased from Promega (Madison, WI). DNase-free RNase was obtained from Boehringer Mannheim (Indianapolis IN). Luria-Bertani (LB) medium and LB agar-medium capsules were from BIO 101 (La Jolla, CA). Tryptic soy broth (TSB) was obtained from Remel (Lenexa, KS).
  • High melting point agarose was purchased from Fisher (St. Louis, MO) and SeaPlaque GTG Agarose (low melting) was obtained from FMC BioProducts (Rockland. ME).
  • Na 125 I, ⁇ - 32 P-ATP, and ⁇ - 32 P-CTP were from ICN (Costa Mesa, CA).
  • Papain and protein A immobilized to cross linked agarose, and IODOGEN were purchased from Pierce (Rockford, IL). Rapid-hyb buffer and rediprime DNA labeling system were obtained from Amersham (Arlington Heights, IL). Chroma Spin- 10 columns were purchased from Clontech (Palo Alto, CA).
  • QIAexpress vector kit type IV and the midi-prep plasmid purification kit were obtained from Qiagen (Chatsworth, CA). Nitrocellulose membrane and blotting paper were from Schleicher & Schuell (Keene, NH). Affi-Gel-10 affinity support was from Bio-Rad (Melville, NY). All other materials were purchased from Sigma Chemical (St. Louis, MO). Bacterial, plasmids, and culture conditions
  • S. aureus strain 12598 (Cowan) was purchased from the American Type Culture Collection (Rockville, MD). E coli strains DH5 ⁇ competent cells (MAX Efficiency) and Ml 5 (pREP4) were from Gibco BRL (Gaithersburg, MD) and Qiagen, respectively. Ml 5 cells contain the plasmid pREP4 which constitutively expresses the lac repressor from the lad gene. S. aureus cells were grown in TSB, and E. coli strains in LB media supplemented with appropriate antibiotics as described below.
  • the low copy number cloning plasmid, pHSG575 (26), was kindly provided by Dr. Michael Caparon (Department of Molecular Microbiology, Washington University School of Medicine).
  • the plasmid pBluescript KS+ was purchased from Stratagene (La Jolla, CA) and used for subcloning and sequencing pu ⁇ oses.
  • the expression plasmid pQE-30 was obtained from Qiagen. All of these plasmids were propagated in DH5 ⁇ cells and purified using the Qiagen Plasmid Midi-Prep Kit for further applications.
  • High molecular weight genomic DNA was isolated from 400 ml of an overnight culture of S. aureus strain 12598 cells by lysostaphin lysis, followed by treatment with DNase-free RNase, and subsequent purification by phenol/chloroform and chloroform extractions. After the final chloroform extraction, DNA in the aqueous layer was precipitated with ethanol and lyophilized.
  • a degenerate 30mer oligonucleotide probe corresponding to the amino acid sequence NNFKDDFEKN was generated by chemical synthesis.
  • the oligonucleotide was end- labeled with T4 polynucleotide kinase and ⁇ - 32 P-ATP, and the radiolabeled oligonucleotide was separated from uninco ⁇ rated 32 P by Chroma Spin- 10 spin chromatography. The specific activity was approximately 5 x 10 8 cpm/ ⁇ g of oligonucleotide.
  • the 2.6 kb Hind III/Hinc II probe was generated as described below and radiolabeled with ⁇ - 32 P-CTP using the rediprime DNA labeling system.
  • Genomic and plasmid DNAs were digested to completion with restriction endonucleases. Restriction endonuclease-cleaved DNAs were separated by TAE- agarose gel electrophoresis, and Southern blotted to nitrocellulose membranes. The membranes were baked at 80 °C for 2h under vacuum, and pre-hybridization, hybridization, and washing of the membranes were performed according to instructions supplied with the Rapid-hyb buffer. Washed blots were air-dried and exposed to Kodak XAR-5 films at -70 °C with intensifying screens for 0.5-2 days.
  • a size selected genomic library in the 4.2 kb region was generated. Genomic DNA from S. aureus strain 12598 was digested with EcoR I and fractionated with 1% low melting agarose electrophoresis. The 4.2 kb region was excised from the gel and melted at 68 °C for 15 min. DNA in the melted agarose was ligated in situ with pHSG575 treated with EcoR I and alkaline phosphatase according to instructions provided by FMC Products.
  • Competent DH5 ⁇ cells were transformed with the ligated material, and different dilutions were plated out on LB agar-medium plates supplemented with chloramphenicol (20 ⁇ g/ml), IPTG (0.5 nM), and X-Gal (40 ⁇ g/ml) for antibiotic and blue/white selections. White colonies were collected, propagated overnight, and the Wizard plasmid mini-prep was used to isolate plasmid DNA from cells. Purified plasmids were digested with EcoR I and screened by Southern blotting using the radiolabeled oligonucleotide probe.
  • the cloned 4.2 kb fragment was digested with Hind III and Hinc II, yielding a 2.6 kb fragment, which was subcloned into pBluescript KS+ and pUC19.
  • the 2.6 kb fragment was also used as a probe in Southern analyses with S. aureus genomic DNA.
  • the insert was digested using the Exo III/mung bean nuclease system (Stratagene, La Jolla, CA) to generate two sets of nested deletions. Multiple clones covering both strands in their entirety were sequenced by the Sanger dideoxynucleotide chain termination method as modified for TAQ polymerase cycle sequencing using an ABI373A automated DNA sequencer. Sequence data were assembled and discrepancies resolved using the Wisconsin Package (Genetics Computer Group, Madison, WI). The primary sequence of ebpS as shown in Figure 3 has been assigned the GenBank accession number.
  • a 2.6 kb Hind III/Hinc II fragment in pBluescript KS+(30ng) served as the template, and PCR reactions were performed with a Perkin Elmer thermocycler using standard reagents.
  • the open reading frame of ebpS was PCR,amplified using the sense oligonucleotide, 5'-TGTGGATCCATAGAAAGGAAGGTGGCTGTG-3'. and the antisense oligonucleotide, 5'GCAAAGCTTGCTGTACCAGGACCAATT-3'.
  • the sense oligonucleotide contained a BamFI I site (underlined), and A of the two ATG codons were changed to G (in bold letters) to avoid internal initiation of translation as recommended by Qiagen.
  • the antisense oligonucleotide contained a Hind III cleavage site (underlined). The exact conditions for amplification were 90 °C for 1 min. followed by 30 cycles of 94°C for 30 s, 53 °C for 30 s, and 72°C for 60 s.
  • the PCR product was digested with BamH I and Hind III, and gel purified.
  • Competent Ml 5 cells were transformed with the ligation product, selected by ampicillin (100 ⁇ g/ml) and kanamycin (20 ⁇ g/ml), and antibiotic-resistant cells were screened for recombinant protein expression.
  • the cells were pelleted by centrifugation (5000 x g), resuspended in 15 ml of buffer A (8 M urea. 100 mM NaH 2 PO , 10 mM Tris-HCl, pH 8), and vortexed gently for 15 min.
  • the lysed cells were centrifuged at 15,000 x g for 20 min at 4°C, and the supernatant was transferred to a tube containing 4 ml of nickel nitriloacetic acid (Ni ++ - NT A) resin pre-equilibrated with buffer A. The mixture was incubated for 30 min at room temperature with gentle agitation.
  • the yield of purified rEbpS under these conditions was approximately 5 mg per 100 ml of culture.
  • rEbpS was incubated in the dark for 24 h at room temperature with 1 mg of CNBr in 200 ⁇ l of 70% formic acid. At the end of incubation, the sample was diluted with 14 ml of de-ionized H 2 0 and speed- vac dried. The dried material was resuspended in 10 ml of de-ionized H 2 O and re- dried in 100 ⁇ g aliquots.
  • rEbpS pre-immune sera were collected, and New England White rabbits were injected with highly purified rEbpS (20 ⁇ g) mixed 1 : 1 with complete Freunds adjuvant. Booster injections (20 ⁇ g) mixed 1 : 1 with incomplete Freunds adjuvant were given at 5, 7, 10, 14, and 19 weeks. Sera were tested by Western immunoblotting using rEbpS.
  • IgG fractions were purified from immune and pre-immune sera by either caprylic acid precipitation (27) or protein A affinity chromatography.
  • caprylic acid precipitation (27) or protein A affinity chromatography approximately 100 mg of anti-rEbpS IgG were covalently coupled to 5 ml of Affi-Gel-10 according to manufacturer's instructions.
  • anti-rEbpS Fab fragments 50 mg of lyophilized IgGs were reacted overnight at 37 °C with 2 ml of immobilized papain in 5 ml of papain digestion buffer (20 mM NaH 2 PO 4 , 20 mM cysteine-HCl, 10 mM EDTA, pH 6.5).
  • Fab fragments were separated from undigested IgGs and Free Fc fragments by protein A affinity chromatography.
  • rEbpS (20 ⁇ g) and CNBr-cleaved rEbpS (80 ⁇ g) were iodinated with 300 ⁇ Ci of Na 1 5 I by the IODOGEN method.
  • the specific activities were approximately 2.3 x 10 4 and 1.2 x 10 4 cpm/ng protein for rSEBP and CNBr- cleaved rEbpS fragments, respectively.
  • Radiolabeled rEbpS (45 ng) in 1.5 ml of binding buffer (50 mM Tris, 500 mM NaCl, 2 nM CaCl 2 ,0.1 mg/ml BSA, pH 7.5) was incubated with 1 ml of the elastin peptide affinity resin for 2 h at room temperature in the absence or presence of 2 mg unlabeled elastin peptides. The mixture was transferred to disposable polypropylene columns and washed with binding buffer by gravity flow until radioactivity of the flow through reached background. Bound rEbpS was eluted with 3 ml of 1% SDS buffer, spin concentrated, and analyzed by 10% SDS-PAGE and autoradiography.
  • binding buffer 50 mM Tris, 500 mM NaCl, 2 nM CaCl 2 ,0.1 mg/ml BSA, pH 7.5
  • Binding of radiolabeled CNBr-cleaved rEbpS to immobilized elastin was assessed similarly, except 80 ng of the starting material was used and bound material was visualized by 12% SDS-PAGE and autoradiography . Detection of the native 25 kDa cell surface labeled EbpS with anti-rEbpS antibodies
  • the N-terminal sequence of native EbpS expressed on the cell surface of S. aureus was determined previously to be ANNFKDDFEKNRQ (20).
  • a degenerate oligonucleotide corresponding to residues 2-11 of the determined N-terminal sequence was generated and used as a probe.
  • Southern blot analysis was first performed with S. aureus strain 12598 genomic DNA digested with restriction endonucleases to identify the hybridizing genomic fragment. As shown in Figure 1 , the oligonucleotide probe hybridized to a 4.2 kb EcoR I fragment (lane A). On the basis of this observation, a size-selected genomic plasmid library in the 4.2 kb region was constructed from EcoR I-digested S.
  • aureus genomic DNA was screened with the oligonucleotide probe by Southern blotting. Of 120 colonies screened, two positive clones with identical restriction enzyme digestion patterns were isolated. One of these clones, pEBPS-1, was used for further analysis.
  • the radiolabeled oligonucleotide was hybridized to the pEBPS-1 insert, and the cloned insert itself was used as a probe for Southern analyses with EcoR I-and EcoR I/Hind Ill/Hind II- digested genomic DNA.
  • the oligonucleotide probe hybridized to the 4.2 kb pEBPS-.l insert (Fig. 1 : lane B) and the insert recognized a 4.2 kb EcoR I genomic fragment (Fig. 1 : lane C).
  • the radiolabeled pEBPS-1 insert hybridized to a 2.6 kb fragment (Fig.
  • oligonucleotide and cloned insert probes consistently detected single fragments with identical size in Southern analyses using genomic DNA digested with various restriction endonucleases, indicating that ebpS is present as a single copy gene.
  • pEBPS-1 was digested with Hind III and Hinc II to yield a_2.6 kb fragment. This fragment was subcloned into pBluescript II KS+ to generate pKS-2.6 and sequenced to locate an open reading frame containing the N-terminal sequence of cell surface EbpS. A 606 bp open reading frame which starts with an ATG codon was identified about 0.9 kb 3' of the Hind III site.
  • the physical map of pKS-2.6 and the primary sequence of the open reading frame with up- and downstream sequences are shown in figures 2 and 3, respectively. Putative -10 and -35 hexamers were identified at positions -31 and -54, with a spacing of 17 bp.
  • a third AT-rich promoter sequence has been proposed recently to exist in a region about 20 bp upstream of the -35 hexamer in E coli (28), and this region for ebpS was 75% AT.
  • a potential ribosome binding sequence which complemented perfectly with the extreme 3' region of Bacillus subtilis 16S RNA (UCUUUCCUCC) (29), was found at position -7.
  • UCUUUCCUCC Bacillus subtilis 16S RNA
  • ebpS was 64% AT and 36% CG.
  • two ATG codons were found in the correct reading frame of ebpS, we have designated the second ATG as the initiation codon based on the location of the putative ribosome binding site.
  • the N-terminal sequence of cell surface ⁇ bpS determined from peptide sequencing was found to start at the second residue of the predicted sequence, suggesting that the initial Met residue is cleaved.
  • the deduced sequence matched perfectly with the determined sequence of cell surface ⁇ bpS except for the first amino acid (Ala in native, Ser in deduced). Since Ser residues are often misread because of its small peak in the peptide sequencing chromatogram, we reexamined the original sequencing chromatogram of cell surface ⁇ bpS and have identified clear Ser and Ser' peaks indicating that the residue in concern is a Ser and not Ala.
  • the mature protein has a predicted molecular mass of 23,344.7 daltons and an acidic pi of 4.9. Accordingly, the protein has a preponderance of acidic amino acids Asp (10.9%) and Glu (1 1.9%), but, is devoid of Cys residues. Gamier analysis predicts a secondary structure that is 58.4% ⁇ helical and 23.8% coiled coil.
  • the BLAST network service of the NIH on the internet was used to search for sequence homologies. The May 1, 1995 releases of the Brookhaven Protein Data Bank, GenBank, ⁇ MBL Data Library, SWISS-PROT protein sequence database, and the trasnlated coding sequence of GenBank were used for comparison. No significant homologies were found between reported sequences in these databases and the primary sequence of ebpS.
  • elastin peptide affinity chromatography was performed with radiolabeled rEbpS. Iodinated rEbpS was incubated with the elastin peptide affinity resin for 2 h at room temperature in the absence or presence of excess unlabeled elastin peptides. The mixture was then washed extensively with buffer until radioactivity of the flow through reached background. The bound material was eluted with 1% SDS buffer and analyzed by SDS-PAGE and autoradiography. The starting material for this experiment was stored for one week at 4°C after purification with Ni ++ -NTA chromatography. As can be seen in Figure 5.
  • EbpS is the cell surface molecule responsible for elastin binding at the cellular level
  • an active form of soluble EbpS should interfere with S. aureus binding to elastin.
  • Elastin labeled with ,25 I was incubated with S. aureus cells in the absence or presence of varying concentrations of unlabeled rEbpS for 1 h at room temperature in 200 ⁇ l of TSB. After washing three times with TSB, radioactivity associated with the cellular pellet was measured with a gamma counter.
  • rEbpS inhibited binding of labeled elastin in a concentration dependent manner. Furthermore, S aureus binding to radiolabeled elastin was abrogated at the highest concentration of rEbpS tested (19 ⁇ M). The control polyhistidine fusion protein mouse dihydrofolate reductase (DHFR) did not affect binding at 26 ⁇ M. These results demonstrate that rEbpS inhibition of cellular elastin binding is specific and that the polyhistidine domain of rEbpS is not affecting binding.
  • DHFR control polyhistidine fusion protein mouse dihydrofolate reductase
  • EbpS is the cell surface protein mediating S. aureus binding to elastin.
  • affinity chromatography was performed with surface labeled S. aureus extracts and immobilized anti-rEbpS IgGs. S. aureus cells were surface labeled by the IODOGEN method and extracts were prepared by lysostaphin digestion.
  • Cell surface components of pathogenic bacteria play important roles in surviving the hostile environment of the host. For gram positive bacteria these surface molecules are used in pathogenic processes such as evading host immune responses (30), digesting host carbohydrates to expose host attachment sites (31, 32), capturing host enzymes to digest host tissues (33), and binding host tissue determinants to establish a firm basis for colonization (34).
  • Cell surface adhesins and MSCRAMMS interact with host ECM components, and participate in the colonization of and extravasation through tissues and organs.
  • S. aureus binds specifically to elastin. Results from binding assays at the cellular level suggested the existence of a single type of cell surface elastin binding protein that mediates the S.
  • EbpS is the surface protein mediating cellular elastin binding.
  • 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.
  • an antibody raised against rEbpS recognizes a 25 kDa protein expressed on the cell surface of S. aureus cells.
  • the common structure is cleaved after the Thr residue of the hexapeptide sequence and the protein is anchored to the cell wall via amide linkage of the carboxyl group of Thr and free amino group of the pentaglycine peptide moiety of the staphylococcal peptidoglycan.
  • EbpS does not contain other common motifs. This observation, however, is not unique for EbpS in that several other gram positive surface proteins have been found to deviate from this conserved structure.
  • EbpS and other proteins not conforming to the common structure are generally smaller than the majority of proteins expressing the shared motifs.
  • the list includes streptococcal proteins such as the fibronectin/fibrinogen binding protein (54 kDa) (42), albumin binding protein (36 kDa)(38), and the plasmin receptor (36 kDa) (43).
  • streptococcal proteins such as the fibronectin/fibrinogen binding protein (54 kDa) (42), albumin binding protein (36 kDa)(38), and the plasmin receptor (36 kDa) (43).
  • the initial Met residue of the streptococcal plasmin receptor is cleaved in the mature protein (43). It is not known whether these correlations have a role in an alternative mechanism for surface expression.
  • C-terminal signal peptides have been identified in several bacterial proteins (44) and alternative means of anchoring proteins to the cells surface have been reported in gram positive bacteria (45).
  • the C-terminus of EbpS may be processed intracellularly, although where and how this cleavage occurs, and if this processing event is one of the signals required for surface expression of EbpS, is yet to be ascertained.
  • ECM Cell-extracellular matrix
  • mammalian cells typically use the integrins, an ⁇ / ⁇ heterodimeric receptor complex, to interact with the ECM [Hynes, Cell, 69:11-25 (1992); Albelda, et al, FASEB J., 4:2868-2880 (1990)].
  • Bacterial pathogens also interact with the host matrix through specific cell surface ECM binding molecules categorized collectively as adhesins or MSCRAMMS [Patti, et al, 1994, supra; Hook, et al, Cell Differ. Dev, 23:433-438 (1990)].
  • the gram positive bacterial pathogen Staphylococcus aureus (S. aureus) has been found to interact with many ECM macromolecules such as collagen [Holderbaum, et al, Infect. Immun., 54:359-364 (1986); Speziale. et al, J.
  • aureus adhesins bind host ECM, these interactions have different biological pu ⁇ oses and binding profiles from mammalian ECM receptors.
  • Staphylococcal ECM adhesins are used for pathological pu ⁇ oses such as in colonization of host tissues.
  • collagen Patti, et al, Infect. Immun., 62:152-161 (1994)
  • fibronectin Baddour, Infect. Immun., 62:2143-2148 (1994)] adhesin mutants show a reduced capacity to cause disease in in vivo models, but are otherwise phenotypically normal.
  • staphylococcal adhesins function as monomers, and endogenous bacterial ligands have not been identified. Available evidence suggests that ligand binding sites in staphylococcal ECM adhesins are contained within small regions of the extracellular domain.
  • the ligand binding site in the staphylococcal fibronectin binding protein for example, has been mapped to a repetitive 38 amino acid motif, and corresponding synthetic peptides have been found to possess direct binding activity and to inhibit bacterial binding to fibronectin [Signas, et al, Proc Natl. Acad. Sci. USA, 86:699-703 (1989); Raja, et al, Infect.
  • EbpS elastin binding protein
  • Papain and protein A immobilized to cross-linked agarose, Immunopure Sulfo-NHS-Biotinylation kit, and IODOGEN were purchased from Pierce (Rockford, IL).
  • QIAexpress vector kit type IV and the midi-prep plasmid purification kit were obtained from Qiagen (Chatsworth, CA).
  • Nitrocellulose membrane and blotting paper were from Schleicher & Schuell (Keene, NH).
  • Aff ⁇ -Gel-10 affinity support was from Bio-Rad (Melville, NY). All other materials were purchased from Sigma Chemical (St. Louis, MO).
  • Synthetic peptides containing the deduced primary sequence of EbpS were prepared by conventional solid phase synthesis on an Applied Biosystems model 431 A synthesizer using FastMoc chemistry. Peptides were purified by reverse phase high performance liquid chromatography (Beckman C18. 0-80% linear water-acetonitrile gradient containing 0.05% trifluoroacetic acid). Purity of the peptides were confirmed by either amino terminal sequencing or electron spray mass spectrometry. All synthetic peptides were soluble in the assay buffer at the concentrations tested.
  • EbpS full length recombinant EbpS
  • rEbpS-2 truncated recombinant EbpS-1 and 2
  • 5'-GTTCGAGCTCTGATTGGTCTTTTTC-3' served as the reverse primer.
  • the forward primer contained a BamH I site (underlined), and A of the two ATG codons was changed to G (in bold letters) to avoid internal initiation of translation as recommended by Qiagen.
  • the reverse primers contained a Sac I cleavage site
  • PCR amplification was performed with a Perkin Elmer thermocycler using standard reagents. Conditions for PCR amplification, and subsequent restriction enzyme digestion, ligation, transformation, expression, and purification of recombinant proteins were as previously described [Park, et al, 1996, supra].
  • trEbpS-1 and trEbpS-2 were biotinylated using a commercially available kit (Pierce). Recombinant proteins (1 mg) were incubated with sulfo-NHS-biotin reagent (2 mg) in 1 ml of PBS for 2 h at 4°C. Biotinylated proteins were then separated from free biotinylating reagent by PD-10 gel filtration chromatography.
  • the blots were washed twice with blocking buffer and incubated for 2 hours at room temperature with either 5 ⁇ M biotinylated trEbpS-1 or trEbpS-2 in the absence or presence of 3 mg/ml elastin peptides in blocking buffer. After washing twice with blocking buffer, the blots were incubated with a 1 :1000 dilution of avidin conjugated to horseradish peroxidase. Membranes were developed by 4-chloro-naphthol.
  • trEbpS-1 with a predicted molecular mass of 12.8 kDa, contains residues 1-78 of EbpS (SEQ ID NO: 10), whereas trEbpS-2 spans residues 1-34 (SEQ ID NO: 12) and has a predicted mass of 7.5 kDa.
  • EbpS EbpS SEQ ID NO:2.
  • Aberrant migration in SDS-PAGE appears to be a common characteristic of gram positive cell surface proteins [McDevitt, et al, 1994, supra; Signas, et al, 1989, supra; Mu ⁇ hy, et al, Biochem. J, 277:277-279 (1991); Sela, et al, Mol Microbiol, 10:1049-1055 (1993); Sj ⁇ bring, Infect. Immun., 60:3601-3608 (1992); Talay, et al, Mol. Microbiol, 13:531-539 (1994)].
  • biotinylated proteins did not bind to either ovalbumin or BSA under similar conditions, demonstrating that the binding interaction between the two polypeptides, i.e., trEbpS-1 and trEbpS-2, and tropoelastin is specific.
  • trEbpS constructs on elastin binding at the cellular level were tested by incubating S. aureus cells with radiolabeled elastin in the absence or presence of increasing amounts of either soluble full length (rEbpS) or truncated forms of the receptor. All three polypeptides inhibited binding of S. aureus cells to elastin in a concentration dependent manner (Fig. 12). rEbpS and trEbpS- 1 completely inhibited elastin binding at the highest concentration tested. trEbpS-2 was somewhat less effective as an inhibitor, with about 20% residual elastin binding activity at the highest inhibitor concentration.
  • Fab fragments of a polyclonal antibody raised against rEbpS inhibit binding of S. aureus to elastin [Park, et al, 1996, supra], suggesting that a population of antibodies in the immune serum recognize a region in EbpS critical for elastin binding.
  • anti-rEbpS IgGs were absorbed to the trEbpS-2 construct coupled to Affi-Gel 10, and unbound IgGs were collected. Immunoblotting revealed that the trEbpS-2-absorbed immunoglobulins retained the ability to interact with both trEbpS and the full length rEbpS-1 (Fig. IOC, lanes B and C), although with reduced activity.
  • the immunoglobulin fraction that was not absorbed to the trEbpS-2 construct did not react with trEbpS-2 on Western blot (Fig. 10C, lane D).
  • Fab fragments from both the nonabsorbed and trEbpS-2-absorbed immunoglobulins were generated by papain digestion and tested for their effects on staphylococcal elastin binding. Consistent with previous findings, Fab fragments from the original anti-rEbpS IgGs abrogated the binding of S. aureus to elastin (Fig. 13). In contrast, serum preabsorbed with trEbpS-2 inhibited the binding of S. aureus to elastin by only 30% at the highest concentration tested (300 ⁇ g/ml).
  • HQDHTEDVE 29 Although no identical repetitive sequences were identified, there are two related sequences, 21 HQDHTEDVE 29 , SEQ ID NO:20 and "HQDTIENTE 45 , SEQ ID NO:23, in the amino-terminal end of the molecule.
  • the sequence 2, HQDHTEDVE 29 is within the putative amino terminal elastin binding site and is contained in all active EbpS constructs.
  • the second sequence, 37 HQDTIENTE S is present only in full length EbpS, and in trEbpS- 1, which does not actively bind tropoelastin.
  • PI SEQ ID NO: 18
  • P2 SEQ ID NO: 19
  • the PI peptide was made according to the deduced sequence of EbpS. In the P2 peptide, Asp 23 , Glu 26 , and Glu 29 were substituted with Asn, Pro, and Gin, respectively.
  • the charged amino acids were targeted for substitution because staphylococcal elastin binding has been shown to involve electrostatic interactions [Park, Cell Biology, 1-161 (1993)].
  • the PI peptide inhibited the binding of S. aureus to elastin in a concentration dependent manner. Elastin binding was abrogated at the highest concentration of PI. whereas minimal inhibition (-15%) was observed with the P2 peptide.
  • ECM adhesins are important for bacterial colonization of and dissemination through host tissues.
  • the identification of the elastin binding site of EbpS is required for understanding the mechanism of S. aureus adhesion to elastin.
  • the elastin binding site in EbpS was mapped to the amino terminal domain of the molecule. Overlapping synthetic peptides spanning amino acids 14-34 were then used to better define the binding domain. Among these, only peptides corresponding to residues 14-23 and 18-34 specifically inhibited elastin binding by more than 95%.
  • EbpS The minimal requirements for elastin recognition by EbpS are unexpectedly similar to what has been observed for the interaction between S. aureus and fibronectin.
  • Fibronectin binding to S. aureus is mediated by a surface fibronectin binding protein, and the fibronectin binding site in this adhesin has been mapped to an extracellular 38 amino acid motif repeated three times and partially a fourth time [Signas, et al, 1989, supra].
  • McGavin et al. J. Biol. Chem., 266:8343- 8347 (1991)] has shown that essential amino acids are contained within residues
  • flanking residues are required to acquire a conformation that is favorable for fibronectin binding.
  • the TNSHQD synthetic peptide by itself could be inactive because it folds improperly and flanking residues are required to form a secondary structure that is necessary for activity.
  • the N-terminal region of full length EbpS is predicted to fold into amphipathic ⁇ helices except for regions including residues
  • Mammalian receptors bind to their respective ligands through the interaction of structural domains in the receptor and a short contiguous peptide sequence in the ligand. Structural domains formed by both the ⁇ and ⁇ integrin subunits, for example, interact with short peptide sequences such as RGD [Pierschbacher, et al, Nature (Lond), 309:30-33 (1984)], LDV [Mould, et al, J. Biol. Chem., 265:4020-4024 (1990)], REDV [Mould, et al, J.
  • the amino terminal third of the elastin protein is the sight of EbpS binding.
  • An antibody generated against a peptide encoded by exons 9 and 10 of elastin specifically inhibits staphylococcal elastin binding indicating that the EbpS binding site is localized to this particular region.
  • EbpS elastin binding protein of Staphylococcus aureus
  • ECM extracellular matrix
  • MSCRAMMS microbial surface components recognizing adhesive matrix molecules
  • rEbpS full length recombinant EbpS
  • trEbpS truncated recombinant EbpS
  • TBS Tris-buffered saline
  • TSB tryptic soy broth.
  • a cell surface 25 kDa elastin binding protein of Staphylococcus aureus mediates binding of this pathogen to elastin.
  • Results from binding assays examining the activity of fragments of EbpS suggested that residues 1-59 contain the elastin recognition site.
  • Functional analysis of recombinant truncated forms of EbpS and synthetic peptides have been used to localize the elastin binding site to within a 21 amino acid region contained within residues 14-34 of the binding protein.
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE
  • AATATACCAT AAACATATGT CATGTGGGTA TATTTTATGT AAAATCATTG TAATAGAATA 120
  • AAAAGGCGCA GCAATTGGTG CTGGAACAGC GGGTGTTGCA GGTGC 789
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE RNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • Gin Ser lie Asp Thr Asn Ser His Gin Asp 1 5 10
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal

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Abstract

L'invention décrit des molécules matricielles adhésives de reconnaissance d'un composant superficiel microbien (MSCRAMM), qui sont caractérisées par leur capacité à se fixer sur l'élastine, leur activité inhibée en présence de SDS et leur activité augmentée en présence de réductants thiol. Plus particulièrement la molécule MSCRAMM est d'origine bactérienne et est un membre de la famille des protéines de fixation sur l'élastine. Dans un mode de réalisation, la molécule MSCRAMM comprend un polypeptide ayant une séquence d'acides aminés définie dans l'invention comme étant la séquence SEQ ID NO 2.2, et s'étend à ses fragments actifs. Le rôle des molécules MSCRAMM dans une infection bactérienne et ses séquelles et états pathologiques associés est remarquable et cette molécule peut être préparée et utilisée dans des procédures diagnostiques et des tests, y compris des analyses de découverte de médicaments, ainsi que dans des compositions pharmaceutiques applicables à des méthodes thérapeutiques correspondantes. Tant les agonistes que les antagonistes de MSCRAMM sont proposés et illustrés.
EP97914810A 1996-02-29 1997-02-28 Proteines d'origine bacterienne de fixation sur l'elastine, sequences d'acide nucleique codant ladite proteine et procedes diagnostiques et therapeutiques d'utilisation de cette proteine Withdrawn EP0942982A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US60913496A 1996-02-29 1996-02-29
US609134 1996-02-29
PCT/US1997/003106 WO1998038312A1 (fr) 1996-02-29 1997-02-28 Proteines d'origine bacterienne de fixation sur l'elastine, sequences d'acide nucleique codant ladite proteine et procedes diagnostiques et therapeutiques d'utilisation de cette proteine

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EP (1) EP0942982A1 (fr)
JP (1) JP2001505061A (fr)
AU (1) AU2192497A (fr)
CA (1) CA2247072A1 (fr)
WO (1) WO1998038312A1 (fr)

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Publication number Priority date Publication date Assignee Title
US6692739B1 (en) 1998-08-31 2004-02-17 Inhibitex, Inc. Staphylococcal immunotherapeutics via donor selection and donor stimulation
WO2000012132A1 (fr) * 1998-08-31 2000-03-09 Inhibitex, Inc. Procede d'immunotherapie contre les infections staphylococciques comprenant la selection des donneurs et la stimulation des donneurs
US7892552B2 (en) 2001-08-08 2011-02-22 University Of Utah Research Foundation Group B Streptococcus polypeptides nucleic acids and therapeutic compositions and vaccines thereof
ES2286133T3 (es) * 2000-08-08 2007-12-01 St. Jude Children's Research Hospital Acidos nucleicos de polipeptidos de estreptococos del grupo b y composiciones terapeuticas y vacunas de los mismos.
CN100359327C (zh) * 2001-06-15 2008-01-02 英希比泰克斯公司 识别凝血酶阴性的葡萄球菌和金黄色葡萄球菌的表面蛋白的交叉反应性单克隆抗体和多克隆抗体
WO2013159021A1 (fr) * 2012-04-20 2013-10-24 The Texas A&M University System Mscramm de liaison au collagène modifié comportant une affinité augmentée pour le collagène

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9838312A1 *

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AU2192497A (en) 1998-09-18
WO1998038312A1 (fr) 1998-09-03
CA2247072A1 (fr) 1998-09-03

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