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WO1996004380A1 - Polypeptide d2d3 tire de staphylococcus aureus et utilisations dudit polypeptide - Google Patents

Polypeptide d2d3 tire de staphylococcus aureus et utilisations dudit polypeptide Download PDF

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Publication number
WO1996004380A1
WO1996004380A1 PCT/EP1995/003039 EP9503039W WO9604380A1 WO 1996004380 A1 WO1996004380 A1 WO 1996004380A1 EP 9503039 W EP9503039 W EP 9503039W WO 9604380 A1 WO9604380 A1 WO 9604380A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
dna
extracellular matrix
bacteria
matrix proteins
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PCT/EP1995/003039
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English (en)
Inventor
Ian Alfred Critchley
Ian Dodd
Paul Barnett
Danuta Ewa Irena Mossakowska
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Smithkline Beecham Plc
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Application filed by Smithkline Beecham Plc filed Critical Smithkline Beecham Plc
Priority to AU32238/95A priority Critical patent/AU3223895A/en
Publication of WO1996004380A1 publication Critical patent/WO1996004380A1/fr

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • D2D3 polypept ide from Staphylococcus aureus and uses thereof .
  • the present invention relates to novel polypeptides and monoclonal antibodies thereto, their preparation and their use to combat infection at the site of wounds, surgical implants and other in-dwelling devices such as catheters.
  • the present invention also relates to the use of such novel polypeptides and monoclonal antibodies as antiadherent agents in oral hygiene.
  • the present invention further relates to isolated nucleic acids encoding the polypepride and to recombinant host cells transformed with DNA encoding the polypepride.
  • this layer includes the plasma protein fibronectin and it is believed that staphylococci are able to bind to fibronectin through bacterial cell surface receptor proteins known as fibronectin binding protein (Fbp).
  • Fbp fibronectin binding protein
  • some studies have suggested that blood proteins do not promote adherence of staphylococci to biomaterial (eg. Muller et al 1991, Infect.Immun. 59: 3323-3326) thereby discouraging research into the interaction of these bacteria with these proteins as an approach in the prevention of adhesion to biomaterials.
  • Fibronectin binding proteins have been isolated from Staphylococcus aureus and the nucleotide sequence subsequently established [ Signas, C. et al. (1989) Proc. Nat. Acad. Sci 86, 699-703 : Jonsson. K. et al. (1991) Eur. J. Biochem. 202, 1041 - 1048] (FbpA and FbpB respectively).
  • the primary fibronectin binding domain of Fbp has been identified as a homologous unit (usually of 38 amino acids) that is repeated three times (D1-D3 region) and partially repeated a fourth time (D4 region).
  • EP0163623, EP0294349, EP0397633 and WO92/02555 disclose certain fibronectin binding polypeptides from S. aureus.
  • Fibronectin binding protein of Staphylococcus aureus is known to exist in at least two variants FbpA and FbpB [Jonsson et al.(1991), op. cit.].
  • Staphylococcus aureus J2385 has been deposited at the National Collection of Industrial and Marine Bacteria Ltd. (NCIMB), Aberdeen, Scotland under number NCIMB 40532 on 18 th December 1992.
  • the D1-D4 region has the amino acid sequence set out in Table 2 below.
  • pathogenic organisms bacteria and fungi
  • Streptococcus mutans Streptococcus mutans
  • Lactobacillus species pathogenic organisms in the oral cavity, in particular Streptococcus mutans, Lactobacillus species,
  • Antinobacillus actinomycetem comitans Actinomyces viscosus, Fusobacterium nucleatum, Porphyromonas intermedia, Eikenella corrodens, Wolinella recta, Bacteroides forsythus, Porphyromonas gingivalis and Candida albicans are responsible for a variety of problems.
  • Oral bacteria adhere to various surfaces in the oral cavity, such as teeth, gingiva and soft mucosal tissues, and form plaque on tooth surfaces. The latter can lead to the development of carious lesions, gingivitis, calculus and periodontal disease.
  • Oral fungi can also give rise to infections in the oral cavity, for instance thrush.
  • a suitable thrush for instance thrush.
  • an anti-microbial agent such as chlorhexidine, cetyl pyridinium chloride or rriclosan which have a bacteriocidal or bacteriostatic effect.
  • an alternative approach may be to use an antiadherent agent, to stop pathogenic organisms from adhering to surfaces within the oral cavity. Teeth become coated with an acquired pellicle which comprises an extracellular protein matrix. This includes specific and non-specific binding sites which are recognised by bacteria and fungi, in particular by receptors located on the surface of the bacterial and fungal cells. If these binding sites are blocked, then cells will be unable to adhere to the extracellular matrix.
  • WO 84/04546 discloses the use of a combination of polymers for preventing the adherence of cariogenic bacteria to tooth surfaces whilst EP0182523-A (Imperial Chemical Industries pic) describes certain novel polymers comprising hydrocarbyl groups with pendant carboxyl and pendant polyalkylene oxide groups which are said to prevent the adherence of cariogenic bacteria on teeth.
  • EP0182523-A Imperial Chemical Industries pic
  • the invention provides an isolated D2D3 polypepride from a Staphylococcus aureus Fbp and its use in the prevention of adhesion of bacteria, in particular gram positive bacteria, to extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds and also oral pathogens, to extracellular matrix proteins on surfaces in the oral cavity, in particular tooth surfaces.
  • the invention further relates to the manufacture of a medicament for such uses.
  • the gram positive bacteria include staphylococci such as S.
  • aureus and coagulase-negative staphylococci such as S. epidermidis.
  • oral pathogen' refers to bacteria and fungi which have been implicated in causing various disease states in the oral cavity, such as caries, gingivitis, periodontitis, calculus and thrush, for instance, Streptococcus mutans, Lactobacillus species, kctinobacillus actinomycetem comitans, Actinomyces viscosus, Fusobacterium nucleatum, Porphyromonas intermedia, Eikenella corrodens, Wolinella recta, Bacteroides forsythus, Porphyromonas gingivalis and Candida albicans. Examples of non-pathogenic early colonisers include Streptococcus sanguis and Streptococcus gordonii.
  • In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of a patient and remain in position for an extended time.
  • Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters, ophthalmic implants, etc.
  • isolated D2D3 polypepride is meant a polypepride consisting of the entire D2 and D3 regions of Staphylococcus aureus Fbp, in sequence.
  • the polypepride may include an
  • a preferred embodiment consists of residues corresponding to residues 747 to 823 of Staphylococcus aureus FbpA.
  • the Fbp is preferably from Staphylococcus aureus J2385 with sequence given in Table 2.
  • This invention also provides isolated nucleic acid molecules encoding the polypepride, including mRNAs, DNAs and cDNAs.
  • This invention also provides recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of the polypeptides as well as recombinant prokaryotic and/or eukaryotic host cells comprising the novel nucleic acid sequence.
  • This invention also provides transgenic non-human animals comprising a nucleic acid molecule encoding the polypepride.
  • 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 a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • double-stranded DNA molecule refers to the polymeric form of deoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in a double- stranded helix, both relaxed and supercoiled. 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 e.g., viruses, 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 sense strand of DNA.
  • a DNA "coding sequence of or a "nucleotide sequence encoding" a particular protein is a DNA sequence which is transcribed and translated into a polypepride when placed under the control of appropriate regulatory sequences.
  • 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. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), 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.
  • control sequences refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (i.e., the transcription and translation) of a coding sequence in a host cell.
  • a control sequence "directs the expression" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • a "host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence.
  • a cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the 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 exogenous 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.
  • a "heterologous" region of a DNA construct is an identifiable segment of
  • DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature is not found in association with the other molecule in nature.
  • This invention provides an isolated nucleic acid molecule encoding the polypeptide.
  • the isolated nucleic acids particularly the DNAs can be introduced into expression vectors by operatively linking the DNA to the necessary expression control regions (e.g. regulatory regions) required for gene expression.
  • the vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial), or eukaryotic (e.g. yeast, insect or mammalian) cells by methods well known in the art ("Current Protocols in Molecular Biology", Ausubel, F.M., et al. (eds.) Greene Publishing Assoc. and John Wiley Interscience, New York, 1989,1992).
  • the coding sequences for the desired proteins having been prepared or isolated can be cloned into a suitable vector or replicon.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage ⁇ (E. col ⁇ ), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram- negative bacteria), pGV1106 (gram- negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • the polypeptides of the present invention can be expressed using, for example, the E. coli tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739;
  • regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell.
  • Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence).
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • Modification of the coding sequences may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • prokaryotic expression vectors are known in the art. See, e.g., U.S. Patent Nos. 4,578,355; 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; see also U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395.
  • Yeast expression vectors are also known in the art. See, e.g., U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent
  • pSV2neo (as described in J. Mol. Appl. Genet. 1:327-341) which uses the SV40 late promoter to drive expression in mammalian cells or pCDNAlneo, a vector derived from pCDNA1(Mol. Cell Biol. 7:4125-29) which uses the CMV promoter to drive expression. Both these latter two vectors can be employed for transient or stable (using G418 resistance) expression in mammalian cells. Insect cell expression systems, e.g., Drosophila and Spodoptera, are also useful, see for example, PCT applications WO 90/06358 and WO 92/06212 as well as EP application EP0290261.
  • the polypeptide of the present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide of interest is expressed. The polypeptide is then isolated from the host cells and purified. If the expression system secretes the polypeptide into growth media, the polypeptide can be purified directly from the media. If the polypeptide is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the invention also extends to a process for preparing a polypeptide of the invention comprising expressing DNA encoding said polypeptide and recovering the expression product.
  • the D2D3 polypeptide, or an antigenically or immunologically equivalent polypeptide or a fusion protein thereof may be used as an antigen to immunize a mouse or other animal such as a rat or chicken.
  • 'antigenically equivalent derivative' encompasses a peptide or its equivalent which will be specifically recognised by certain antibodies which, when raised to peptides according to the present invention, block adhesion of staphylococci to in-dwelling medical devices.
  • 'immunologically equivalent derivative' as used herein encompasses a polypeptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to block adhesion of staphylococci to indwelling medical devices.
  • polypeptides in which one or more of the amino acid residues are modified before or after the polypeptide is synthesised may be used.
  • Such polypeptides may, for example, be prepared by substitution, addition, or rearrangement of amino acids or by chemical modification thereof. All such substitutions and modifications are generally well known to those skilled in the art of polypeptide chemistry.
  • the fusion protein may provide stability to the polypeptide.
  • the antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comprising multiple copies of the the D2D3 polypeptide , or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
  • antibody-producing cells from the immunised mammal are fused with myeloma cells to create hybridoma cells secreting monoclonal antibodies.
  • the hybridomas are screened to select a cell line with high binding affinity and favorable cross reaction with other staphylococcal species using one or more of the D2D3 polypeptide and/or the fusion protein.
  • the selected cell line is cultured to obtain the desired Mab.
  • Hybridoma cell lines secreting the monoclonal antibody are another aspect of this invention.
  • phage display technology could be utilised to select antibody genes with binding activities towards D2D3 either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Fbp or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).
  • the antibody or derivative thereof is modified to make it less immunogenic in the patient.
  • the antibody may most preferably be 'humanised' ; where the complimentarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P. et al (1986), Nature 321, 522-525 or Tempest et al.,(1991) Biotechnology 9, 266-273.
  • the modification need not be restricted to one of 'humanisation' ; other primate sequences (for example Newman, R. et al .1992, Biotechnology,10, 1455- 1460) may also be used.
  • the antibody should be screened again for high affinity to D2D3 polypeptide and/or fusion protein.
  • novel monoclonal antibodies and their fragments form a further aspect of the invention.
  • the antibody may be either intact antibody of M r approx 150,000 or a derivative of it, for example a Fab fragment or a Fv fragment as described in Skerra, A and Pluckthun, A (1988) Science 240 1038-1040. If two antigen binding domains are present each domain may be directed against a different epitope - termed
  • the antibody or derivative thereof may be prepared by conventional means for example by established monoclonal antibody technology (Kohler, G. and Milstein, C. (1975), op. cit.) or using recombinant means e.g. combinatorial libraries, for example as described in Huse, W.D. et al., (1989) Science 246,1275- 1281.
  • the antibody or derivative is prepared by expression of a DNA polymer encoding said antibody in an appropriate expression system.
  • the choice of vector for the expression system will be determined in part by the host, which may be a prokaryotic cell, such as E. coli or Streptomyces species, or a eukaryotic cell, such as a mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect cell.
  • the host may also be a transgenic animal or a transgenic plant [for example as described in Hiatt, A et ⁇ /.,(1989) Nature 34, 76-78].
  • Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses, derived from, for example, baculoviruses or vaccinia.
  • the Fab fragment may also be prepared from its parent monoclonal antibody by enzyme treatment, for example using papain, to cleave the Fab portion from the Fc portion.
  • the present invention further provides a monoclonal antibody (Mab), or a fragment thereof as above defined, in the prevention of adhesion of bacteria, in particular gram positive bacteria, to extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds and of oral pathogens to extracellular matrix proteins on surfaces in the oral cavity, in particular the acquired pellicle on tooth surfaces.
  • the invention particularly relates to the manufacture of a medicament for such uses.
  • Another aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising the above polypeptide or Mab or active fragment and a pharmaceutically acceptable carrier.
  • the polypeptide or Mab or active fragment may be administered to a patient as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
  • the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, sprays, swabs, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams.
  • Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
  • Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
  • composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device.
  • composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial infections, such as staphylococcal wound infections.
  • the daily dosage level of the active agent will be from 0.01 to 10 mg/kg, typically around 1 mg/kg.
  • the physician in any event will determine the actual dosage and interval of dosing that will be most suitable for an individual patient and will vary with the age, weight and response of the particular patient.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins, especially fibronectin, exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.
  • composition of the invention may be used to bathe an indwelling device immediately before insertion.
  • the active agent will preferably be present at a concentration of l ⁇ g/ml to 10mg/ml for bathing of wounds or indwelling devices.
  • a method according to the present invention is preferably carried out in a prophylactic manner, as part of a normal oral hygiene regime.
  • the polypeptide or Mab or active fragment is thus of use in oral hygiene, in particular in combating the plaque - related development of carious lesions, gingivitis, calculus or periodontal disease and in combating oral cavity infections such as candidiasis.
  • the fibronectin binding protein or polypeptide may also have a professional use at the time of oral surgery eg in guided tissue regeneration
  • periodontal disease or osseointegrated implants to prevent subsequent bacterial infection. It may also be used for irrigation of periodontal pockets and/or incorporated into controlled release devices for administration to the periodontal pocket to prevent or treat periodontal disease progression. It may also be used as a denture cleaner to prevent candidiasis.
  • the mechanism by which oral pathogens adhere to an oral cavity surface can occur in a number of ways including direct attachment to a surface or by indirect attachment to a surface which may be already coated with a pellicle and/or
  • the fibronectin binding protein or polypeptide can thus prevent the binding of non-pathogenic early colonisers and binding and co- aggregation of an oral pathogen.
  • preventing the adherence of oral pathogens to surfaces in the oral cavity therefore means preventing the direct adherence of oral pathogens or preventing the adherence of non-pathogenic early colonisers thereby preventing the subsequent binding and co-aggregation of oral pathogens or even preventing the adherence of pathogenic oral organisms to non-pathogenic early colonisers.
  • the present invention provides an oral hygiene composition comprising the above mentioned polypeptide or Mab or active fragment and an orally acceptable carrier.
  • Suitable oral hygiene compositions are well known in the art and include dentifrices, including opaque and transparent/translucent toothpastes, liquid
  • dentifrices non-abrasive gels, mouthwashes, mouthrinses and gargles, mouthsprays, gingival irrigating devices, paint-on varnishes, formulations to be sucked or chewed by the user such as gums and lozenges and dental flosses.
  • compositions will contain appropriate formulating agents such as abrasives, surfactants, humectants, thickening agents, flavouring agents, sweetening agents, opacifying agents, preservatives and water, selected from those conventionally used in the oral hygiene composition art for such purposes and which are compatible with the polypeptide or monoclonal antibodies hereinbefore described.
  • appropriate formulating agents such as abrasives, surfactants, humectants, thickening agents, flavouring agents, sweetening agents, opacifying agents, preservatives and water, selected from those conventionally used in the oral hygiene composition art for such purposes and which are compatible with the polypeptide or monoclonal antibodies hereinbefore described.
  • Suitable surfactants for use in compositions according to the present invention include, for instance, anionic, nonionic, cationic and amphoteric surfactants or mixtures thereof.
  • Suitable anionic surfactants include alkali metal (C 12-18 )alkyl sulphates, for instance sodium lauryl sulphate, and N-acyl sarcosinates and N-acyl taurines in which the acyl moiety has from 12 to 16 carbon atoms, for instance, N-lauroyl, N-myristoyl and N-palmitoyl sarcosine alkali metal salts.
  • Suitable nonionic surfactants include, for example, polyethoxylated sorbitol esters, in particular polyethoxylated sorbitol monoesters, for instance, PEG(40) sorbitan di-isostearate, and the products marketed under the trade name Tween' by ICI; polycondensates of ethylene oxide and propylene oxide (poloxamers), for instance the products marketed under the trade name 'Pluronic' by BASF-Wyandotte; condensates of propylene glycol; polyethoxylated hydrogenated castor oil, for instance, cremophors; and sorbitan fatty esters.
  • polyethoxylated sorbitol esters in particular polyethoxylated sorbitol monoesters, for instance, PEG(40) sorbitan di-isostearate, and the products marketed under the trade name Tween' by ICI
  • Suitable amphoteric surfactants include, for example, long chain imidazoline derivatives such as the product marketed under the trade name 'Miranol C2M' by Miranol; long chain alkyl betaines, such as the product marketed under the tradename 'Empigen BB' by Albright + Wilson, and long chain alkyl amidoalkyl betaines, such as cocamidopropylbetaine, and mixtures thereof.
  • Suitable cationic surfactants include the D,L-2-pyrrolidone-5-carboxylic acid salt of ethyl-N-cocoyl-L-arginate, marketed under the trade name CAE by Ajinomoto Co. Inc., and cocamidopropyl PG dimonium chloride phosphate and lauramidopropyl PG dimonium chloride phosphate, available under the trade names Monaquat PTC and Monaquat PTL, respectively, from Mona Corporation.
  • the surfactant is present in the range 0.005 to 20%, preferably 0.1 to 10%, more preferably 0.1 to 5% by weight of the dentifrice.
  • Suitable thickening agents include, for instance, nonionic thickening agents such as, for example, (C 1 -6 )alkylcellulose ethers, for instance methylcellulose;
  • hydroxy(C 1 -6 )alkylcellulose ethers for instance hydroxyethylcellulose and hydroxypropylcellulose
  • (C 2-6 )alkylene oxide modified (C 1 -6 )alkylcellulose ethers for instance hydroxypropyl methylcellulose
  • Other thickening agents such as natural and synthetic gums or gum like material such as Irish Moss, gum tragacanth, sodium carboxymethylcellulose, polyvinyl pyrrolidone, starch, xantham, carrageenan and thickening silicas may also be used.
  • the thickening agent is present in the range 0.01 to 30%, preferably 0.1 to 15%, more preferbly 1 to 5%, by weight of the composition.
  • Suitable humectants for use in compositions of the invention include for instance, glycerine, sorbitol, propylene glycol or polyethylene glycol, or mixtures thereof; which humectant may be present in the range from 5 to 90%, preferably 5 to 70%, more preferably 10 to 50% by weight of the dentifrice.
  • Suitable abrasives for use in dentifrice compositions of the present invention include calcium carbonate, calcium phosphates, calcium pyrophosphate, insoluble sodium metaphosphate, sodium aluminosilicate, alumina, hydra ted alumina, zinc orthophosphate, plastic particles, and silica, of which silica is the preferred abrasive.
  • Suitable silicas include natural amorphous silicas, such as, for instance, diatomaceous earth, and synthetic amorphous silicas, such as precipitated silicas and silica gels, including silica xerogels. Suitable silica xerogels are described in US 3,538,230. Suitable grades of precipitated silicas have BET surface areas in the range 20 to 300, preferably 20 to 100 m 2 /g and median agglomerate sizes in the range 2 to 50, preferably 5 to 30 ⁇ .
  • Suitable precipitated silicas and silica xerogels are those marketed under the trade names Sident and Syloblanc, by Degussa and W R Grace Corporation Davison Chemical Division, respectively.
  • the silica is a "low anion" silica.
  • the term "low-anion" silicas refers to those in which anionic impurities such as sodium sulphate and sodium silicate which normally arise during the course of the
  • Low anion silicas suitably have less than 1%, preferably less than 0.5% advantageously less than 0.25% by weight of anionic impurities.
  • Suitable such "low anion" silicas are described in EP 0 368 130 (Proctor & Gamble), EP 0 315 503 and EP 0 396 459 (Rhone-Poulenc) and WO 90/05113 (J.M. Huber Corp).
  • grades of commercially available silica with ionic impurities may be rendered suitable by washing thereof with deionised water.
  • Conductivity measurements on the water after washing may be used to monitor the efficacy of such washing.
  • the conductivity of the water after washing is reduced to less than 200 ⁇ Siemens/cm.
  • Suitable "low anion" silicas include the grade RP93 available from Rhone-Poulenc.
  • compositions will have from 5 to 80%, preferably from 10 to 60% by weight of the abrasive.
  • Suitable mouthwash formulations will have an aqueous base comprising water or aqueous ethanol, and optionally a further liquid such as glycerin or propylene glycol.
  • a surfactant may also be included, to improve the sensory properties of the composition.
  • Mouthwash compositions may be provided in a "ready to use” form; as a concentrated solution, for dilution by the user immediately prior to use; or in solid form, such as a tablet or in a sachet, for dissolution by the user immediately prior to use. Tablets may suitably be prepared using xylitol and/or sorbitol as the major ingredient.
  • the sachets and tablets may be formulated to provide, on dissolution, a still mouthwash, or, by the incorporation of a suitable effervescent couple, for instance sodium carbonate/bicarbonatre and citric acid, an effervescent mouthwash.
  • a suitable effervescent couple for instance sodium carbonate/bicarbonatre and citric acid
  • Oral hygiene compositions of the present invention may usefully further comprise an anti-caries agent, for instance a source of fluoride ions such as an alkali metal or amine fluoride salt, for example sodium fluoride, tin (II) fluoride.
  • an anti-caries agent for instance a source of fluoride ions such as an alkali metal or amine fluoride salt, for example sodium fluoride, tin (II) fluoride.
  • the fluoride ion source may be an alkali metal monofluorophosphate salt, for example sodium monofluorophosphate, optionally used in combination with an agent such as calcium glycerophosphate which is known to enhance the activity of monofluorophosphate (GB 1 384 375, Beecham Group).
  • an agent such as calcium glycerophosphate which is known to enhance the activity of monofluorophosphate (GB 1 384 375, Beecham Group).
  • the composition will comprise between 100 and 2500ppm, preferably 200 and 1500ppm of fluoride ions.
  • Oral hygiene compositions of the present invention may also comprise other active agents conventionally used in oral hygiene compositions, for instance:
  • an anti-plaque agent such as chlorhexidine, cetyl pyridinium chloride, triclosan, histatin or nisin (particularly in the purified form available as Ambicin N from Applied Microbiology Inc., New York);
  • an anti-calculus agent such as a tetra- or a di-alkali metal pyrophosphate salt, or a mixture thereof, an alkali metal tripolyphosphate salt or an azacycloheptane diphosphonate salt; or
  • an anti-sensitivity agent such as strontium acetate, strontium chloride or a potassium salt such as potassium nitrate, potassium chloride or potassium citrate.
  • an anti-sensitivity agent such as strontium acetate, strontium chloride or a potassium salt such as potassium nitrate, potassium chloride or potassium citrate.
  • Such agents will be included at levels to provide the desired therapeutic effect.
  • Oral hygiene compositions according to the present invention will have a pH which is orally acceptable, for instance in the range pH 5 to 10.
  • Oral hygiene compositions according to the present invention may be prepared by admixing the ingredients in the appropriate relative amounts in any order that is convenient and thereafter and if necessary adjusting the pH to give the final desired value.
  • the polypetide or Mab or active fragment will be incorporated into oral hygiene compositions according to the present invention at concentrations sufficient to provide the aforementioned daily rate eg 0.002% to 2.3%, preferably 0.23% (w/w).
  • residues of FbpA corresponds to the residues of FbpA according to Signas et al., (1989) op. cit.
  • Residues 709-838 of FbpA correspond to residues 1-130 of the sequence of 5.
  • aureus J2385 given in Table 2 and residues 709-886 correspond to residues 1-174.
  • PCR products with 3' deoxyadenosine overhangs were ligated into pGEM-T using the pGEM-T kit from Promega Corporation, Madison, USA according to the manufacturer's protocol.
  • DNA fragments were excised from agarose gels and DNA extracted using the QIAEX gel extraction kit according to the manufacturer's instructions (QIAGEN Inc., USA).
  • a single colony from a plate containing E. coli was selected at random and inoculated into a universal containing 10 ml of L-Broth and 50 ⁇ g/ml of ampicillin. This was incubated at 37°C, 220 rpm overnight. L-Broth was 1% (w/v)
  • Bactotryptone 0.5% (w/v) Bactoyeastextract, 0.5% (w/v) NaCl.
  • the alkaline lysis mini-plasmid DNA protocol is as follows:- 3 ml of overnight culture was spun down, the supernatant removed and the pellet resuspended in 100 ⁇ l GTE buffer (TE buffer/50 mM Glucose). 200 ⁇ l of 0.2M NaOH/1% Sodium Dodecyl Sulphate (SDS) were added and incubated for 5 min at room temperature, followed by addition of 150 ⁇ l 5 M potassium acetate (pH 4.8).
  • Plasmids were transformed into E. coli HB101 or E. coli BL21(DE3)
  • E. coli JM109 was purchased as a frozen competent culture as part of the Promega pGEM-T kit (see above).
  • the DNA fragment was excised and purified from agarose gels and then amplified by PCR from two primers complementary to the 5' ends of the DNA fragment.
  • Approximately 0.1 - 1 ⁇ g of template DNA (pBROC519a fragment) was added to reaction buffer (10mM Tris pH8.3, 50mM KCl, 0.1% gelatin, 1.5mM MgCl 2 ), 0.2mM of each dNTP, 1 ⁇ M each of primer oligonucleotides 1 and 2 in a total reaction volume of 99 ⁇ l.
  • the PCR reaction was set up so that DNA was heated to 94°C for 5 min, 54°C for 3 min, then 1 ⁇ l of Taq DNA polymerase (purchased from a commercial source e.g. Boehringer Mannheim) was added.
  • the reaction was cycled at 72°C for 2min, 94°C for lmin, 54°C for 2min a total of 35 times.
  • Plasmid DNA was from the alkaline lysis method and was denatured prior to sequencing by adding 1M NaOH/lmM EDTA and incubating at room temperature for 5 min.
  • a column was prepared by taking a 1.5 ml micro tube, removing the cap and piercing the bottom with a 0.8 x 40 mm sterile needle.
  • 20 ⁇ l of glass beads (Sigma, 425 - 600 microns) were added followed by 400 ⁇ l of CL-4B Sepharose (Sigma) in a 2: 1 slurry in TE buffer.
  • the micro tube was placed in the top of a second tube also with the cap removed and spun ( ⁇ 2000 g for 2 mins) at ambient temperature .
  • the bottom tube was discarded and replaced with a clean one.
  • the denatured DNA was added to the CL-4B resin, the spin repeated, the supernatant collected and used for sequencing. Sequencing was carried out using "SequenaseTM" (United States
  • Oligonucleotides were synthesised using a 381 A synthesiser (Applied Biosystems) according to the manufacturer's instructions or were obtained from Promega.
  • pT7-7 was digested with Lspl and Bglll and the protruding 5' ends filled in with the Klenow fragment of DNA Polymerasel.
  • the plasmid DNA fragment was purified by agarose gel electrophoresis, the blunt ends ligated together and transformed into E. coli DH1 by electroporation using a Bio-Rad Gene Pulser and following the manufacturers recommended conditions.
  • the resultant plasmid pBROC413 was identified by restriction enzyme analysis of plasmid DNA.
  • aureus J2385 is strain B described in Cookson et al. [1987] THE LANCET of August 15th. page 387. It is a clinical strain derived from a skin lesion.
  • Chromosomal DNA was prepared by treating cells collected from an overnight shake flask culture with lysostaphin to lyse them and phenol/chloroform to remove cell protein. From this unpurified DNA preparation the fragment of DNA encoding the fibronectin binding domains of Fibronectin Binding Protein was obtained by a PCR amplification reaction.
  • the oligonucleotide primers used in the PCR reaction were:-
  • FIB 1 5'-GGGAATTCATATGGGCCAAAATAGCGGTAACCAGTC-3'
  • FIB 2 5'-GCGGATCCTTACGTTGGTGGCACGATTGGAGGTG-3'
  • PCR amplification was carried out using 5. aureus J2385 chromosomal DNA (10ng.) FIB 1 (1 micromolar), FIB 2 (1 micromolar), Tris-HCl pH8.3 (10 mM), KCl (50mM), MgCl 2 (1.5 mM), gelatin (0.001%), Na dGTP (200 micromolar) Na dATP (200 micromolar), Na dTTP (200 micromolar), Na dCTP (200 micromolar) and Taq DNA Polymerase (2.5 units) in a final volume of 100 microlitres made up with distilled water.
  • the aqueous solution was overlaid with 80 microlitres of liquid paraffin and subjected to 30 cycles of 94 °C (1min.), 60°C (1 min.) and 72°C (2 min.) to enable the amplification to occur.
  • 10 microlitres of the aqueous reaction were examined after amplification on 1.5% agarose gel electrophoresis in the presence of ethidium bromide (0.5 micrograms/ml.) it was observed by comparison to a sample of DNA fragments of known size that a single species of DNA fragment of approximately 500 basepairs was obtained.
  • pBROC 519a was sequenced in both strands using the SEQUENASE II kit obtained from United States Biochemical. The sequence obtained revealed the cloned fragment encoded 524bp of S. aureus J2385 DNA.
  • aureus J2385 DNA was compared to the corresponding regions in 5.
  • aureus Fibronectin binding protein type A (Signas et al. [1989]) and S. aureus type B protein (Jonsson et al. [1991] EUR. J. BIOCHEM. vol 202 pages 1041-1048) it was clear that the fibronectin binding domains of the 5. aureus J2385 protein was similar to the respective domains of both the type A and type B proteins but was identical to neither (Table 2). This suggested that the S. aureus J2385 protein should be denoted type C.
  • Fibronectin was purified from a human plasma fraction (a gift from Dr.
  • EXAMPLE 1 Preparation of plasmid pDB1020 encoding D2 D3 protein.
  • pBROC519a was digested with restriction endonucleases to remove as much of the DNA as possible that was extraneous to that coding for the D2 and D3 domains.
  • the amino acid sequence corresponding to the D2D3 domains is given in SEQ ID No. 1.
  • PCR oligonucleotides were designed such that the forward primer (SEQ ID No 3; oligo 1) contained 6 bases of random sequence followed by an Ndel restriction endonuclease site followed by sequence coding for part of D2D3.
  • the Ndel site encompasses the ATG codon for methionine which is the initiating amino acid of protein translation .
  • the reverse primer (SEQ ID No 4; oligo 2) started with 6 bases of random sequence followed by the restriction endonuclease site for BamHl followed by a stop codon, followed by sequence coding for part of D2D3.
  • oligonucleotides 1 and 2 as the forward and reverse primers.
  • the PCR product was loaded onto a 3% agarose gel, made using SepRate-SDF agarose (Amersham
  • the DNA was cut from the gel on a minimal gel slice and eluted using the QIAEX kit (Qiagen Inc. USA).
  • thermostable polymerases can lead to non-template dependent addition of a single deoxyadenosine to the 3' end of PCR products.
  • the PCR product was cloned directly into pGEM-T as described in the General Methods to generate plasmid pDB1023.
  • the plasmid was transformed into E. coli JM109 and recombinants analysed by restriction
  • pDB1023 was digested with Ndel and BamHI. This resulted in the removal of the extra nucleotides introduced by PCR.
  • the approx. 240 bp DNA fragment coding for D2D3 was purified from agarose using the QIAEX kit and ligated into pBROC413 that had been cut with the same restriction endonucleases and purified in the same way.
  • the resulting plasmid pDB1020 was transformed into calcium chloride competent E. coli HB101 and DNA from recombinants analysed by restriction mapping with Ndel and BamHI. The resulting restriction map indicated correct incorporation of the D2D3-encoding region.
  • NCYZM 100 ⁇ g/ml ampicillin; cultures were grown at 37°C, 220 rpm until the A500 reached approx. 0.45.
  • Cells were induced with 1 mM isopropylthio- ⁇ -D-galactoside (IPTG) and allowed to grow for a further 3hrs. The cells were then centrifuged ( ⁇ 8000 x g, 4°C, 15min) and the supernatant discarded. The cell pellets were stored at -80°C.
  • Frozen cell pellet of E. coli BL21(DE3) (pDB1020) was allowed to thaw at 4°C for 2hrs and was then resuspended in 50 mM Tris/50 mM NaCl/1 mM EDTA/0.1 mM PMSF pH 8.0 at a ratio of 33 ml for pellet derived from one litre of culture.
  • the sonicate was immediately centrifuged (6000 x g / 4°C/ 10 min) and the pellet discarded.
  • the supernatant, containing the soluble D2D3, was adjusted to pH 7.4 and retained at -40°C.
  • D2D3 supernatant (from a 1L culture) was diluted 1: 1 in 0.1M NaH 2 PO 4 pH 7.6 (final pH adjusted to 7.6) and applied to a Q Sepharose (Pharmacia) column (i.d., 4.4cm; h, 3.1cm) equilibrated in 0.1 M NaH 2 PO 4 pH 7.6.
  • D2D3 adsorbed to the column and was eluted using 0.1M NaH 2 PO 4 / 0.5 M NaCl pH 7.6. At this pH, some D2D3 did not adsorb to Q Sepharose. Therefore a higher pH, e.g. 8.0, is preferred for the purification of D2D3.
  • the solubility of the D2D3 lyophilised product was 10-20mg/ml when reconstituted in H 2 O, 50mM formic acid or PBS.
  • PMMA polymethylmethacrylate
  • Staphylococcus aureus 8325-4 standard laboratory strain
  • Staphylococcus aureus 120 (a virulent clinical strain)
  • Staphylococcus epidermidis SE 902, a clinical isolate from a foreign body infection
  • Staphylococcal strains were grown overnight at 37°C in Mueller-Hinton Broth (MHB, Difco Laboratories). A total of 2 x 10 7 cfu from the overnight culture was incubated with 100 ⁇ Ci [m ethyl- 3 H]-thymidine (Amersham) in 1ml of MHB and grown for 3 hours at 37°C up to 1 x 10 8 - 2 x 10 8 cfu/ml. After removal of the unbound radioactivity by two washes (3,000 x g, 10 minutes) the labelled strain was suspended in 1ml of 0.15M NaCl.
  • Coverslips were immersed in solutions containing purified human fibronectin at two concentrations, 0.5 ⁇ g/ml for S. aureus strains and 4 ⁇ g/ml for S. epidermidis strains, for 60 minutes at 37°C. Coverslips were transferred to fresh tubes containing PBS and washed.
  • D2D3 polypeptide was diluted to the required concentration in PBS supplemented with cations (Gibco Laboratories) and albumin (20%, Sigma)
  • D2D3 was effective at inhibiting S. aureus 8325-4 and 5.
  • D2D3 also inhibited the adhesion of S. aureus 120; at 10 ⁇ g/ml D2D3 84% inhibition of adhesion was observed.
  • the dentifrice base may be used to provide a dentifrice comprising a D2D3 polypeptide 0.2, 0.5, 1, 1.5 or 2%.
  • the dentifrice base may be used to provide a dentifrice comprising a D2D3 polypeptide 0.2, 0.5, 1, 1.5 or 2%.
  • Staphylococcus aureus (as published) and Staphylococcus aureus J2385.
  • the Staphylococcus aureus sequence a comprises amino acid residues 709-886 (as in Signas et al. loc. cit.)

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Abstract

Polypeptide D2D3 isolé d'un Staphylococcus aureus Fbp, procédé pour sa préparation et utilisation dudit polypeptide dans la prévention de l'adhérence des bactéries, en particulier les bactéries Gram positif, à des protéines de matrice extracellulaire sur des dispositifs implantés à demeure ou à des protéines de matrice extracellulaire dans des blessures, et également des organismes pathogènes buccaux à des protéines de matrice extracellulaire dans la cavité buccale, en particulier la surface des dents.
PCT/EP1995/003039 1994-08-05 1995-07-28 Polypeptide d2d3 tire de staphylococcus aureus et utilisations dudit polypeptide WO1996004380A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0832973A2 (fr) * 1996-09-24 1998-04-01 Smithkline Beecham Corporation Protéine de liaison de GTP
WO1998031389A2 (fr) * 1997-01-21 1998-07-23 The Texas A & M University System Compositions de proteines se liant a la fibronectine, anticorps utilises et leurs procedes d'utilisation
EP0885297A1 (fr) * 1996-02-20 1998-12-23 Smithkline Beecham Corporation Nouveaux polypeptides era
US6685943B1 (en) 1997-01-21 2004-02-03 The Texas A&M University System Fibronectin binding protein compositions and methods of use
US7115264B2 (en) 2001-11-05 2006-10-03 Inhibitex Monoclonal antibodies to the fibronectin binding protein and method of use in treating or preventing infections

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002555A1 (fr) * 1990-08-10 1992-02-20 Alfa-Laval Agri International Aktiebolag Peptide liant de la fibronectine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002555A1 (fr) * 1990-08-10 1992-02-20 Alfa-Laval Agri International Aktiebolag Peptide liant de la fibronectine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHRISTER SIGNÄS ET AL.: "Nucleotide sequence of the gene for a fibronectin-bimding protein from Staphylococcus aureus: Use of this peptide sequence in the synthesis of biologically active peptides", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 86, no. 2, WASHINGTON US, pages 699 - 703 *
SHEELA HUFF ET AL.: "Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 22, 3 June 1994 (1994-06-03), MD US, pages 15563 - 15570 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0885297A1 (fr) * 1996-02-20 1998-12-23 Smithkline Beecham Corporation Nouveaux polypeptides era
EP0885297A4 (fr) * 1996-02-20 1999-11-24 Smithkline Beecham Corp Nouveaux polypeptides era
EP0832973A2 (fr) * 1996-09-24 1998-04-01 Smithkline Beecham Corporation Protéine de liaison de GTP
EP0832973A3 (fr) * 1996-09-24 1999-11-03 Smithkline Beecham Corporation Protéine de liaison de GTP
WO1998031389A2 (fr) * 1997-01-21 1998-07-23 The Texas A & M University System Compositions de proteines se liant a la fibronectine, anticorps utilises et leurs procedes d'utilisation
WO1998031389A3 (fr) * 1997-01-21 1999-01-21 Texas A & M Univ Sys Compositions de proteines se liant a la fibronectine, anticorps utilises et leurs procedes d'utilisation
AU744723B2 (en) * 1997-01-21 2002-02-28 The Texas A & M University System Fibronectin binding protein compositions, and methods of use
US6685943B1 (en) 1997-01-21 2004-02-03 The Texas A&M University System Fibronectin binding protein compositions and methods of use
US7115264B2 (en) 2001-11-05 2006-10-03 Inhibitex Monoclonal antibodies to the fibronectin binding protein and method of use in treating or preventing infections

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