WO2000077041A2 - Peptides essentiels a proteine de liaison de la decorine et procedes d'utilisation - Google Patents

Peptides essentiels a proteine de liaison de la decorine et procedes d'utilisation Download PDF

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Publication number
WO2000077041A2
WO2000077041A2 PCT/US2000/016713 US0016713W WO0077041A2 WO 2000077041 A2 WO2000077041 A2 WO 2000077041A2 US 0016713 W US0016713 W US 0016713W WO 0077041 A2 WO0077041 A2 WO 0077041A2
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Prior art keywords
dbpa
decorin
binding
protein
peptide
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PCT/US2000/016713
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English (en)
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WO2000077041A3 (fr
Inventor
Magnus Höök
Eric L. Brown
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The Texas A & M University System
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Priority to AU54955/00A priority Critical patent/AU5495500A/en
Publication of WO2000077041A2 publication Critical patent/WO2000077041A2/fr
Publication of WO2000077041A3 publication Critical patent/WO2000077041A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
    • 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
    • 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
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to the fields of molecular biology and microbiology. More particularly, it concerns a method of use of residues necessary for ligand binding in a decorin binding protein, DbpA. More particularly, certain embodiments concern methods and compositions comprising DNA segments, and peptides derived from bacterial species.
  • Lyme disease is a chronic, multisystemic disease caused by the spirochete Borrelia burgdorferi (1). It is transmitted to humans and other mammals during the blood meal of Ixodes ticks (2, 3) and it is the most common vector-borne disease in the United States (1). This initial skin infection is often accompanied by a local rash (erythema migrans) which can be followed by a general flu-like illness. Untreated Lyme borreliosis can develop into a chronic, multisystemic disorder that may affect the joints (Lyme arthritis), skin, heart, and central nervous system (1).
  • Microbial adhesion to and colonization of host tissue is an early, critical event in an infection process.
  • host tissue adherence appears to be of importance during different stages of the disease process. Initially, during an infected tick's blood meal, a small number of spirochetes are deposited in the dermis of the host where the bacteria appear to colonize collagen fibers (32,33). As the infection disseminates to other tissues, bacteria may colonize additional extracellular matrix (ECM) structures and host cells may be involved.
  • ECM extracellular matrix
  • Adherence of Borrelia burgdorferi to collagen fibers involves a specific binding of the spirochete to decorin, a dermatan sulfate proteoglycan which is associated with and "decorates" collagen fibers (17, 18).
  • a dermal route of entry into the host appears to be important for the development of disease. Spirochetes administered intravenously are rapidly and effectively cleared by Kupffer cells in the liver (34), whereas those inoculated intradermaly consistently establish infection (35). Perhaps the initial dermal colonization allows the organism to adapt to in vivo conditions before blood stream dissemination.
  • DbpA and DbpB are capable of binding to decorin and DbpA effectively inhibits the adherence of Borrelia burgdorferi to a decorin substrate (17).
  • DbpA and DbpA antiserum are independently protected against challenge with B. burgdorferi (10,11).
  • DbpA sequences vary significantly among different Borrelia strains. Nevertheless, antibodies to one recombinant form of DbpA can confer broad protection against various strains, suggesting that at least some immunoprotective epitopes are conserved.
  • OspA has been the most widely studied Borrelia protein, and vaccine trials in both murine models of LD and in humans indicate that this protein can confer protection to infection against the Lyme spirochete (6-8).
  • OspA-derived vaccine formulations exist. Since OspA expression occurs primarily in the tick vector, high anti-OspA titers need to be maintained to inhibit spirochete transmission (9-11) and anti-OspA antibodies lose efficacy against host- adapted Borrelia (12). Furthermore, protection against heterologous Borrelia strains is poor to nonexistent (13-16). Additionally, modulation of OspA expression by B.
  • burgdorferi may limit the efficacy of anti-OspA antibodies to spirochetes residing in the midgut, and cross-reactivity of OspA with human lymphocyte function-associated antigen- 1 (hLFA-1) may be a critical factor in treatment-resistant Lyme arthritis (5, 9, 10, 24).
  • hLFA-1 human lymphocyte function-associated antigen- 1
  • the decorin binding proteins (DbpA and DbpB) of Borrelia burgdorferi are adhesins of the MSCRNMM family (17) which recognize the proteoglycan decorin and may play a role in the colonization of the skin and establishment of disease (17, 18).
  • DbpA expression on B. burgdorferi is expressed in the vertebrate host (19).
  • Recent work testing DbpA as a vaccine candidate against heterologous Borrelia strains demonstrated that DbpA-based formulations are capable of conferring protection against challenge with B. burgdorferi (19), and may be more effective than OspA formulations. (11).
  • Applicants' discovery of antigenic determinants on proteins capable of eliciting protective immunity are used to design novel peptides that may have the potential of mimicking the protective effects of native proteins.
  • the risk of cross-reactivity against the host is reduced since peptide vaccines are chemically defined structures.
  • peptides are stable and no infectious materials are used in their production (25).
  • the present invention provides several novel methods and related biological compositions for protecting against infection with Borrelia burgdorferi. Also provided are methods of identifying substances that alter or modulate the interaction of various decorin binding proteins with decorin, and the substances so identified.
  • the invention first provides a protein fragment or related peptide that spans the critical
  • the invention also provides a method of inhibiting the binding of B. burgdorferi to
  • biomaterials and thus be employed in order to treat or prevent B. burgdorferi infections.
  • the isolated B. burgdorferi proteins of the present invention can thus be utilized in methods of treating or preventing B. burgdorferi infection through the inhibition of the ability of the bacteria to bind to decorin, or through the development of antibodies thereto which will prevent or inhibit the bacteria's ability to bind to host cells.
  • antisera and antibodies generated against the decorin binding proteins of the present invention which also can be utilized in methods of treatment which involve inhibition of the attachment of the DbpA proteins to decorin.
  • antisera and antibodies raised against the DbpA proteins, or immunogenic portions thereof may be employed in vaccines, and other pharmaceutical compositions containing the proteins for therapeutic purposes are also provided herein.
  • diagnostic kits containing the appropriate proteins, or antibodies or antisera raised against them, are also provided so as to detect bacteria expressing these proteins.
  • the protein fragment or related peptide composition is dispersed in a pharmaceutically-acceptable carrier.
  • the decorin to be contacted is comprised within an animal, and the protein fragment composition is administered to the animal.
  • the animal is a human subject.
  • the invention also provides a method of inhibiting decorin binding protein from binding decorin in a blood sample, the method comprising contacting the blood sample with an amount of a the DbpA-derived peptide fragment or related peptide composition effective to inhibit decorin/DbpA binding in the sample.
  • the DbpA-derived peptide fragment or related peptide composition is dispersed in a pharmaceutically-acceptable carrier.
  • the blood sample containing decorin is found within an animal, and the
  • protein, fragment or related peptide composition is administered to the animal.
  • the animal is a human subject.
  • application include, but are not limited to, topical, oral, anal, vaginal, intravenous,
  • intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration are intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.
  • the preferred dose for human administration will be determined based on the needs of
  • the vaccine may additionally contain stabilizers or
  • preservatives such as thimerosal (ethyl(2-mercaptobenzoate-
  • composition for topical administration, is formulated in the form of an ointment,
  • Wound or surgical dressings, sutures and aerosols may be impregnated with the composition.
  • composition may contain conventional additives, such as preservatives, solvents to promote
  • Topical formulations may also contain conventional carriers such as
  • cream or ointment bases ethanol, or oleyl alcohol.
  • the invention additionally provides a method of preventing Lyme disease in an animal
  • the invention provides a method of inhibiting the binding of DbpA to decorin in an animal, comprising providing to an animal an amount of a decorin binding DbpA- derived protein fragment or related peptide pharmaceutical composition effective to bind to decorin in the animal.
  • a method for identifying a candidate substance that alters the binding of a DbpA-derived protein fragment or related peptide to decorin comprising the steps of admixing a composition comprising a DbpA-derived protein fragment or related peptide with a decorin preparation and a candidate substance, and determining the ability of the DbpA-derived protein fragment or related peptide to bind to the decorin preparation in the presence and in the absence of the candidate substance, wherein the ability of the candidate substance to alter the binding of the DbpA-derived protein fragment or related peptide to the decorin preparation is indicative of a candidate substance that alters the binding of a DbpA- derived protein or peptide fragment to decorin.
  • the DbpA- derived protein or peptide fragment is prepared by recombinant means.
  • the method is further defined as a method for identifying a candidate substance that promotes the binding of a DbpA-derived protein fragment or related peptide to decorin, comprising determining the ability of a candidate substance to increase the binding of a DbpA-derived protein fragment or related peptide to decorin upon admixing with a composition that comprises a DbpA-derived protein fragment or related peptide.
  • the method is further defined as a method for identifying a candidate substance that inhibits the binding of a DbpA-derived protein fragment or related peptide to decorin, comprising determining the ability of a candidate substance to decrease the binding of a DbpA-derived protein fragment or related peptide to decorin upon admixing with a composition that comprises a DbpA-derived protein fragment or related peptide and a decorin preparation.
  • a modulator of DbpA-derived protein or peptide fragment binding to decorin prepared by a process comprising the steps of admixing a composition comprising a DbpA-derived protein or peptide fragment with a decorin preparation and a candidate modulator, identifying a modulator that alters the binding of the DbpA-derived protein or peptide fragment to decorin by determining the ability of the DbpA-derived protein or peptide to bind to the decorin preparation in the presence and in the absence of the candidate modulator, wherein the ability of the candidate modulator to alter the binding of the DbpA-derived protein or peptide fragment to the decorin preparation is indicative of a candidate modulator that alters the binding of a DbpA-derived protein or peptide fragment to decorin, and obtaining the modulator so identified.
  • the invention also provides a method of modulating DbpA-derived protein or peptide fragment binding to decorin, comprising contacting a composition comprising decorin and a DbpA-derived protein or DbpA-derived peptide fragment with an effective amount of a substance that modulates DbpA-derived protein or peptide fragment binding to decorin.
  • the method may be further defined as a method for promoting the binding of a DbpA-derived protein or peptide fragment to decorin, comprising contacting the composition with an effective amount of a substance that increases the binding of a DbpA- derived protein or peptide fragment to decorin.
  • the method may be further defined as a method for inhibiting the binding of a DbpA-derived protein or peptide fragment to decorin, comprising contacting the composition with an effective amount of a substance that decreases the binding of a DbpA-derived protein or peptide fragment to decorin.
  • the composition is comprised within an animal.
  • the animal is a human subject.
  • the invention also provides a method for identifying a candidate DbpA-derived peptide fragment with improved decorin binding, that may be characterized as comprising the steps of admixing a composition comprising a candidate DbpA-derived peptide fragment with a decorin preparation, and determining the ability of the candidate DbpA-derived peptide fragment to bind to the decorin preparation, wherein a candidate DbpA-derived peptide fragment that exhibits improved binding to decorin as compared to wild-type DbpA is indicative of a candidate DbpA- derived peptide fragment with improved decorin binding.
  • Applicants provide that at least two peptides, P4 and P5, have the ability of eliciting both DTH to DbpA and a protective immune response to Borrelia infection. Combinations of these peptides as well as multimeric formulations have similar potential in human use.
  • the DbpA-derived protein or peptide fragments used in the present invention may be utilized in many applications for the treatment, identification, or prevention of Borrelia infections.
  • compositions containing isolated DbpA-derived protein or peptide fragments, specifically the fragments or portions containing the decorin-binding domain may be used as blocking agents to bind to decorin-binding sites in a patient, or in implanted biomaterials or other instruments used in surgical operations, and thus be able to inhibit the binding of Borrelia bacteria to decorin and thereby treat or prevent Borrelia infection.
  • the DbpA-derived protein or peptide fragments of the invention may be utilized to generate antibodies which can treat or prevent Borrelia infection, either when generated directly in the patient through the use of vaccines, or through therapeutic compositions containing antibodies to the DbpA-derived protein or its active portions or fragments.
  • a method of inhibiting the attachment of Borrelia bacteria to decorin comprises administering an DbpA-derived protein or peptide fragment, in an amount sufficient to inhibit the attachment of Borrelia bacteria to decorin, and such administration may be utilized to block the sites for Borrelia attachment in a patient, a medical device, or a bioimplant.
  • a method is also provided for treating or preventing Borrelia infection in a patient comprising administering an DbpA-derived protein or peptide fragment, such as in a pharmaceutical composition, in an amount sufficient to treat or prevent an Borrelia infection.
  • the precise treatment regimen will be dependent upon the circumstances surrounding the need for treatment, including, e.g., the nature and condition of the patient, the extent and the seriousness of the afflicted area, and the amenability of the patient to particular forms of treatment.
  • the method involves other objects such as biomedical instruments or implants made from biological materials, an appropriate amount and treatment form will be determined based on the circumstances and the materials involved.
  • the isolated decorin-binding DbpA- derived protein or peptide fragments of the present invention may be used in combination with other peptides.
  • the isolated decorin-binding DbpA-derived protein or peptide fragments of the present invention are used in combination with adhesins.
  • the isolated decorin-binding DbpA- derived protein or peptide fragments of the present invention may be obtained through conventional isolation or recombination methods well known in the art.
  • a cloning vector such as a plasmid or phage DNA is cleaved with a restriction enzyme, and the DNA sequence encoding the DbpA-derived protein or peptide fragment, or its binding or other active fragments thereof, such as consensus or variable sequence amino acid motifs, is inserted into the cleavage site and ligated.
  • the cloning vector is then inserted into a host to produce the protein or fragment.
  • Suitable hosts include bacterial hosts such as Escherichia coli, Bacillus subtilis, yeasts and other cell cultures. Production and purification of the gene product may be achieved and enhanced using known molecular biology techniques.
  • pharmaceutical compositions are also provided which contain the DbpA-derived protein or peptide fragments, or active portions as described herein, and which may be formulated in combination with a suitable pharmaceutical vehicle, excipient or carrier well know in the art. Examples of some suitable vehicles, carriers and excipients would include saline, dextrose, water, glycerol, ethanol, other therapeutic compounds, and combinations thereof. The formulation should be appropriate for the mode of administration.
  • the DbpA-derived protein or peptide fragment compositions of the present invention will thus be useful for interfering with, modulating, or inhibiting binding interactions between Borrelia bacteria and decorin on host cells.
  • DbpA-derived protein or peptide fragments as described herein, as would be recognized by one of ordinary skill in this art, modification and changes may be made in the structure of the proteins and peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules.
  • amino acid substitutions are also possible without affecting the decorin binding ability of the isolated proteins of the invention, provided that the substitutions provide amino acids having sufficiently similar properties to the ones in the original sequences.
  • acceptable amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the isolated proteins of the present invention can be prepared in a number of suitable ways known in the art including typical chemical synthesis processes to prepare a sequence of polypeptides.
  • the synthetic polypeptides of the invention can thus be 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 a -amino protected N a -t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield, or the base-labile N a -amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han.
  • Both Fmoc and Boc N a -amino protected amino acids can be obtained from Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or Peninsula Labs or other chemical companies familiar to those who practice this art.
  • the method of the invention can be used with other N a -protecting groups that are familiar to those skilled in this art.
  • Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis. Second Edition, Pierce Chemical Co., Rockford, IL; Fields and Noble, 1990, Int. J. Pept Protein Res. 35:161-214, or using automated synthesizers, such as sold by ABS.
  • polypeptides of the invention may comprise D-amino acids, a combination of D- and L-amino acids, and various "designer" amino acids (e.g., H-methyl amino acids, CH -methyl amino acids, and Nu-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, .H -helices, a turns, a sheets, f -turns, and cyclic peptides can be generated.
  • subunits of peptides that confer useful chemical and structural properties may be used in accordance with the invention.
  • peptides comprising D-amino acids will be resistant to L-amino acid-specific proteases in vivo.
  • the present invention envisions preparing 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 -NH-R , where Ri and R 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. It is also 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 isolated natural, recombinant or synthetic proteins of the present invention, or antigenic portions thereof (including epitope-bearing fragments), or fusion proteins including the DbpA-derived protein or peptide fragments as described above can be administered to animals as immunogens or antigens, alone or in combination with an adjuvant, for the production of antibodies reactive with the DbpA-derived protein or peptide fragments or active portions thereof.
  • the DbpA-derived protein or peptide fragments of the present invention, or active domains thereof may be useful as vaccines to generate an immune response in a patient, or in methods of generating antibodies in a host organism which can then be introduced into a patient in order to prevent or treat an Borrelia infection.
  • the DbpA-derived protein or peptide fragments can be used to screen antibodies or antisera for hyperimmune patients from whom can be derived specific antibodies having a very high affinity for the proteins.
  • Antibodies to DbpA, or its binding domain can also be used in accordance with the invention for the specific detection of decorin-binding Borrelia proteins, for the prevention of infection from the Borrelia, for the treatment of an ongoing infection, or for use as research
  • antibodies as used herein includes monoclonal, polyclonal, chimeric, single
  • chromogenic substances including colored particles such as colloidal gold or latex beads.
  • Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).
  • the antibody may be labeled indirectly by reaction with labeled substances
  • the antibody may be conjugated with a second
  • the antibody may be conjugated to biotin and the
  • decorin-binding protein DbpA-derived protein or peptide fragment or its binding domain may also be used to isolate additional amounts of decorin.
  • the isolated DbpA-derived protein or peptide fragments of the present invention, or active fragments thereof, and antibodies to the proteins may thus be utilized in many applications involving the treatment, prevention and diagnosis of Borrelia bacterial infections as described above, or for the development of anti- Borrelia vaccines for active or passive immunization.
  • both the proteins and the antibodies are useful as blocking agents to prevent or inhibit the binding of Borrelia to decorin at the wound site or the biomaterials themselves.
  • the antibody is modified so that it is less immunogenic in the patient to whom it is administered.
  • the antibody may be "humanized” by transplanting the complimentarity determining regions of the hybridoma-derived antibody into a human monoclonal antibody as described, e.g., by Jones et al, Nature 321 :522-525 (1986) or Tempest et al. Biotechnology 9:266-273 (1991).
  • Medical devices or polymeric biomaterials to be coated with the antibodies, proteins and active fragments described herein include, but are not limited to, staples, sutures, replacement heart valves, cardiac assist devices, hard and soft contact lenses, intraocular lens implants (anterior chamber or posterior chamber), other implants such as corneal inlays, kerato- prostheses, vascular stents, epikeratophalia devices, glaucoma shunts, retinal staples, scleral buckles, dental prostheses, thyroplastic devices, laryngoplastic devices, vascular grafts, soft and hard tissue prostheses including, but not limited to, pumps, electrical devices including stimulators and recorders, auditory prostheses, pacemakers, artificial larynx, dental implants, mammary implants, penile implants, cranio/facial tendons, artificial joints, tendons, ligaments, menisci, and disks, artificial bones, artificial organs including artificial pancreas, artificial hearts, artificial limbs, and heart valves;
  • coated means to apply the protein, antibody, or active fragment to a surface of the device, preferably an outer surface that would be exposed to Borrelia bacterial infection.
  • the surface of the device need not be entirely covered by the protein, antibody or active fragment.
  • the present invention may be utilized as immunological compositions, including vaccines, and other pharmaceutical compositions containing the DbpA-derived protein or peptide fragment or its active regions, are included within the scope of the present invention.
  • Either the DbpA-derived protein or peptide fragment, or its decorin-binding domain, or other active or antigenic fragments thereof, or fusion proteins thereof, can be formulated and packaged, alone or in combination with other antigens, using methods and materials known to those skilled in the art for vaccines.
  • the immunological response may be used therapeutically or prophylactically and may provide antibody immunity or cellular immunity, such as that produced by T lymphocytes.
  • the immunological compositions, such as vaccines, and other pharmaceutical compositions can be used alone or in combination with other blocking agents to protect against human and animal infections caused by or exacerbated by Borrelia bacteria.
  • the proteins may be conjugated to a carrier molecule.
  • suitable immunogenic carriers include proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and derivatives thereof, particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solid phases. Other protein derived or non-protein derived substances are known to those skilled in the art.
  • An immunogenic carrier typically has a molecular weight of at least 1 ,000 Daltons, preferably greater than 10,000 Daltons. Carrier molecules often contain a reactive group to facilitate covalent conjugation to the hapten.
  • the carboxylic acid group or amine group of amino acids or the sugar groups of glycoproteins are often used in this manner. Carriers lacking such groups can often be reacted with an appropriate chemical to produce them.
  • an immune response is produced when the immunogen is injected into animals such as mice, rabbits, rats, goats, sheep, guinea pigs, chickens, and other animals, most preferably mice and rabbits.
  • a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide may be sufficiently antigenic to improve immunogenicity without the use of a carrier.
  • the isolated DbpA-derived protein or peptide fragment may be administered with an adjuvant in an amount effective to enhance the immunogenic response against the conjugate.
  • an adjuvant widely used in humans has been alum (aluminum phosphate or aluminum hydroxide).
  • chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. J. Immunol.
  • encapsulation of the conjugate within a proteoliposome as described by Miller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) may also be useful.
  • vaccine includes DNA vaccines in which the nucleic acid molecule encoding for a decorin-binding DbpA-derived protein or peptide fragment is used in a pharmaceutical composition is administered to a patient.
  • suitable delivery methods known to those skilled in the art include direct injection of plasmid DNA into muscles (Wolff et al, Hum. Mol. Genet. 1 :363, 1992), delivery of DNA complexed with specific protein carriers (Wu et al., J. Biol. Chem. 264:16985, 1989), coprecipitation of DNA with calcium phosphate (Benvenisty and Reshef, Proc. Natl. Acad. Sci.
  • the invention is a method of using a polynucleotide which comprises contiguous nucleic acid sequences capable of being expressed to produce an DbpA gene product upon introduction of said polynucleotide into eukaryotic tissues in vivo.
  • the encoded gene product preferably either acts as an immunostimulant or as an antigen capable of generating an immune response.
  • the nucleic acid sequences in this embodiment encode an immunogenic epitope, and optionally a cytokine or a T-cell costimulatory element, such as a member of the B7 family of proteins.
  • the first is the relative simplicity with which native or nearly native antigen can be presented to the immune system. Mammalian proteins expressed recombinantly in bacteria, yeast, or even mammalian cells often require extensive treatment to ensure appropriate antigenicity.
  • a second advantage of DNA immunization is the potential for the immunogen to enter the MHC class I pathway and evoke a cytotoxic T cell response. Immunization of mice with DNA encoding the influenza A nucleoprotein (NP) elicited a CD8 + response to NP that protected mice against challenge with heterologous strains of flu. (See Montgomery, D. L. et al.. Cell Mol Biol, 43(3):285-92, 1997 and Ulmer, J. et al, Vaccine, 15(8):792-794, 1997.)
  • DNA immunization can evoke both humoral and cell-mediated immune responses, its greatest advantage may be that it provides a relatively simple method to survey a large number of B. burgdorferi genes for their vaccine potential.
  • the amount of expressible DNA or transcribed RNA to be introduced into a vaccine recipient will have a very broad dosage range and may depend on the strength of the transcriptional and translational promoters used.
  • the magnitude of the immune response may depend on the level of protein expression and on the immunogenicity of the expressed gene product.
  • effective dose ranges of roughly about 1 ng to 5 mg, 100 ng to 2.5 mg, 1 €g to 750 Gg, and preferably about 10 €g to 300 €g, of DNA may be suitable, e.g., if administered directly into muscle tissue.
  • Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, or inhalation delivery may also be suitable as would be recognized by one skilled in this art.
  • booster vaccinations may be provided.
  • boosting with protein immunogens such as the isolated ACE protein or the isolated A domain is also contemplated.
  • the polynucleotide may be "naked", that is, unassociated with any proteins, adjuvants or other agents which affect the recipient's immune system.
  • the DNA may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture, or the DNA may be associated with an adjuvant known in the art to boost immune responses, such as a protein or other carrier.
  • Agents which assist in the cellular uptake of DNA such as, but not limited to, calcium ions, may also be used. These agents are generally referred to herein as transfection facilitating reagents and pharmaceutically acceptable carriers.
  • Techniques for coating microprojectiles coated with polynucleotide are known in the art and are also useful in connection with this invention.
  • Pharmaceutically acceptable carriers or buffer solutions are known in the art and include those described in a variety of texts such as Remington's Pharmaceutical Sciences.
  • an optimal dosing schedule for a vaccination regimen as set forth above will vary according to the needs of the particular patient, but may include as many as one to six or more administrations of the immunizing entity given at intervals of as few as two to four weeks, to as long as five to ten years, or occasionally at even longer intervals, as needed.
  • Suitable methods of administration of any pharmaceutical composition disclosed in this application include, but are not limited to, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.
  • the composition is formulated in the form of an ointment, cream, gel, lotion, drops (such as eye drops and ear drops), or solution (such as mouthwash). Wound or surgical dressings, sutures and aerosols may be impregnated with the composition.
  • the composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol.
  • a vaccine is packaged in a single dosage for immunization by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. If intramuscularly introduced, the vaccine is preferably injected directly intramuscularly into the deltoid muscle.
  • the vaccine is preferably combined with a pharmaceutically acceptable carrier to facilitate administration.
  • the carrier is usually water or a buffered saline, with or without a preservative.
  • the vaccine may be lyophilized for resuspension at the time of administration or in solution.
  • Microencapsulation of the protein will give a controlled release.
  • a number of factors contribute to the selection of a particular polymer for microencapsulation.
  • the reproducibility of polymer synthesis and the microencapsulation process, the cost of the microencapsulation materials and process, the toxicological profile, the requirements for variable release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered.
  • useful polymers are polycarbonates, polyesters, polyurethanes, polyorthoesters, polyamides, poly (D,L-lactide-co-glycolide) (PLGA) and other biodegradable polymers.
  • PLGA poly (D,L-lactide-co-glycolide)
  • the use of PLGA for the controlled release of antigen is reviewed by Eldridge et al., CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, 146:59-66 (1989).
  • the preferred dose for human administration will be determined based on the needs of the individual patient and the nature of the disorder being treated, for example ranging 0.01 mg/kg to 10 mg/kg. Based on this range, equivalent dosages for heavier body weights can be determined.
  • the dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual.
  • the vaccine may additionally contain stabilizers or pharmaceutically acceptable preservatives, such as thimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, MO).
  • thimerosal ethyl(2-mercaptobenzoate-S)mercury sodium salt
  • the DbpA-derived protein or peptide fragment described herein is useful for purposes such in vitro detection of decorin in a sample.
  • label conjugates may also be facilitated through use of such protein-label conjugates.
  • Various types of labels and methods of conjugating the labels to the proteins are well known to those skilled in the art.
  • Several specific labels are set forth below.
  • the labels are particularly useful when conjugated to a protein such as an antibody or receptor.
  • the protein can be conjugated to a radiolabel such as, but not restricted to, 32 P, 3 H, I4 C, 33 S, l25 I, or 131 I.
  • Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography.
  • Bioluminescent labels such as derivatives of firefly luciferin, are also useful.
  • the bioluminescent substance is covalently bound to the protein by conventional methods, and the labeled protein is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light.
  • Fluorogens may also be used to label proteins. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red. The fluorogens are generally detected by a fluorescence detector.
  • the DbpA-derived protein can alternatively be labeled with a chromogen to provide an enzyme or affinity label.
  • the protein can be biotinylated so that it can be utilized in a biotin-avidin reaction, which may also be coupled to a label such as an enzyme or fluorogen.
  • the protein can be labeled with peroxidase, alkaline phosphatase or other enzymes giving a chromogenic or fluorogenic reaction upon addition of substrate.
  • Additives such as 5-amino-2,3-dihydro-l,4-phthalazinedione (also known as Luminol ) (Sigma Chemical Company, St.
  • rate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol) (Sigma Chemical Company, St. Louis, MO) can be used to amplify enzymes such as horseradish peroxidase through a luminescent reaction; and luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used.
  • enzymes such as horseradish peroxidase through a luminescent reaction
  • luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used.
  • Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer.
  • proteins may be labeled with colloidal gold for use in immunoelectron microscopy in accordance with methods well known to those skilled in the art.
  • the location of a ligand in cells can be determined by labeling an antibody as described above and detecting the label in accordance with methods well known to those skilled in the art, such as immunofluorescence microscopy using procedures such as those described by Warren and Nelson (Mo/. Cell. Biol, 7: 1326-1337, 1987).
  • the DbpA- derived protein or peptide fragments, nucleic acid molecules or antibodies may also be useful for interfering with the initial physical interaction between a pathogen and mammalian host responsible for infection, such as the adhesion of bacteria, to mammalian extracellular matrix proteins such as decorin on in-dwelling devices or to extracellular matrix proteins in wounds; to block DbpA-mediated mammalian cell invasion; to block bacterial adhesion between decorin and bacterial DbpA proteins or portions thereof that mediate tissue damage; and, to block the normal progression of pathogenesis in infections initiated other than by the implantation of in- dwelling devices or surgical techniques.
  • a pathogen and mammalian host responsible for infection such as the adhesion of bacteria
  • mammalian extracellular matrix proteins such as decorin on in-dwelling devices or to extracellular matrix proteins in wounds
  • decorin on in-dwelling devices or to extracellular matrix proteins in wounds
  • DbpA-mediated mammalian cell invasion to block bacterial adh
  • the DbpA-derived protein or peptide fragments are useful in a method for screening compounds to identify compounds that inhibit decorin binding of Borrelia to host molecules.
  • the compound of interest is combined with one or more of the DbpA-derived protein or peptide fragments and the degree of binding of the protein to decorin or other extracellular matrix proteins is measured or observed. If the presence of the compound results in the inhibition of protein-decorin binding, for example, then the compound may be useful for inhibiting Borrelia in vivo or in vitro.
  • the method could similarly be used to identify compounds that promote interactions of Borrelia with host molecules.
  • the method is particularly useful for identifying compounds having bacteriostatic or bacteriocidal properties.
  • a synthetic reaction mixture a cellular compartment (such as a membrane, cell envelope or cell wall) containing one or more of the DbpA-derived protein or peptide fragments and a labeled substrate or ligand of the protein is incubated in the absence or the presence of a compound under investigation.
  • the ability of the compound to agonize or antagonize the protein is shown by a decrease in the binding of the labeled ligand or decreased production of substrate product.
  • Compounds that bind well and increase the rate of product formation from substrate are agonists.
  • Detection of the rate or level of production of product from substrate may be enhanced by use of a reporter system, such as a colorimetric labeled substrate converted to product, a reporter gene that is responsive to changes in ACE nucleic acid or protein activity, and binding assays known to those skilled in the art. Competitive inhibition assays can also be used.
  • a reporter system such as a colorimetric labeled substrate converted to product, a reporter gene that is responsive to changes in ACE nucleic acid or protein activity, and binding assays known to those skilled in the art.
  • binding assays known to those skilled in the art.
  • Competitive inhibition assays can also be used.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to nucleic acid molecules coding for DbpA-derived protein or peptide fragments and thereby inhibit their activity or bind to a binding molecule (such as collagen to prevent the binding of the DbpA nucleic acid molecules or proteins to its ligand.
  • a compound that inhibits DbpA activity may be a small molecule that binds to and occupies the binding site of the DbpA protein, thereby preventing binding to cellular binding molecules, to prevent normal biological activity.
  • small molecules include, but are not limited to, small organic molecule, peptides or peptide-like molecules.
  • Other potential antagonists include antisense molecules.
  • Preferred antagonists include compounds related to and variants or derivatives of the DbpA proteins or portions thereof.
  • the nucleic acid molecules described herein may also be used to screen compounds for antibacterial activity.
  • the invention further contemplates a kit containing one or more DbpA-specific nucleic acid probes, which can be used for the detection of decorin-binding proteins from Borrelia in a sample, or for the diagnosis of Borrelia infections.
  • a kit can also contain the appropriate reagents for hybridizing the probe to the sample and detecting bound probe.
  • the kit contains antibodies specific to either or both the DbpA-derived protein or peptide fragments which can be used for the detection o ⁇ Borrelia.
  • the kit contains either or both the DbpA-derived protein or peptide fragments which can be used for the detection o ⁇ Borrelia bacteria or for the presence of antibodies to decorin-binding DbpA proteins in a sample.
  • kits described herein may additionally contain equipment for safely obtaining the sample, a vessel for containing the reagents, a timing means, a buffer for diluting the sample, and a colorimeter, refiectometer, or standard against which a color change may be measured.
  • the reagents including the protein or antibody, are lyophilized, most preferably in a single vessel. Addition of aqueous sample to the vessel results in solubilization of the lyophilized reagents, causing them to react. Most preferably, the reagents are sequentially lyophilized in a single container, in accordance with methods well known to those skilled in the art that minimize reaction by the reagents prior to addition of the sample.
  • Fig. Peptide Map of DbpA. Overlapping synthetic peptides (P2-P9) (25 amino acids each) spanning the length of recombinant DbpA (underlined) were constructed. Lysine modifications performed on mutants K14-K170 are indicated in bold.
  • Fig. 3 Synthetic peptide inhibition of decorin binding.
  • Biotin-conjugated decorin (0.006 ⁇ g) was pre-incubated with either 5 ug of DbpA or P2-P9 for 1 hour and then added to DbpA-coated microtiter wells. The plate was developed and O.D. read at 410 nm. The data are expressed as +
  • mice were immunized with various peptide formulations or recombinant DbpA.
  • mice were challenged with 2 ⁇ g of the noted peptide formulations or recombinant DbpA (50 ⁇ l).
  • Specific footpad swelling was determined by subtracting the thickness of the footpad before challenge from the thickness measured 24 hours post challenge. The values are means + SE of 5 mice/group.
  • Fig. 5 Blood and joint cultures from mice immunized with DbpA or DbpA-peptides. Blood (A) or joints (B) were taken from mice immunized as described in the legend for Fig. 4. Joints and 50 ⁇ l of blood were used to inoculate BSK II media 7 and 14 days post infection, respectively. The data are presented as percent positive cultures of 5 mice/group.
  • Fig. 6 SDS-PAGE and Western ligand blot analysis of DbpA and chemically modified DbpA (K-mod). Purified DbpA and K-mod were subjected to SDS-PAGE (12%) under reducing conditions and stained with Coomassie brilliant blue (A) or transferred to a nitrocellulose membrane (B). After blocking additional protein-binding sites, proteins on the membrane were probed with digoxigenin-labeled decorin and visualized by alkaline phosphatase reactivity.
  • Fig. 7 conserved lysine residues on DbpA. conserveed lysine residues were determined by protein sequence alignment of DbpA (strain 297) with DbpA sequences from various Borrelia genotypes (26). Bb, B. burgdorferi; Ba, B. afzelii; Bg, B. garinii. Numbers above the alignment indicate the numerical designation of each mutant when the corresponding lysine was mutated to alanine. Critical lysine residues involved in decorin binding are indicated in bold-face.
  • Fig. 8 SDS-PAGE and Western ligand blot analysis of DbpA 549 and lysine modified proteins.
  • Purified recombinant DbpA 549, OspC, and lysine mutants of DbpA were subjected to either SDS-PAGE ( 12%) under reducing conditions and stained with Coomassie brilliant blue (A) or transferred to a nitrocellulose membrane (B). The membrane was probed with digoxigenin-labeled decorin and visualized by alkaline phosphatase reactivity.
  • Three of the peptides selected (P4, P5, and P9) contain lysine residues critical for decorin binding and were also recognized by serum raised against DbpA.
  • P4 and P5 which map to the K82 region of DbpA, are capable of inhibiting decorin attachment to DbpA- coated wells (64 and 92%, respectively).
  • the K163/K170 peptide, P9, inhibited 43% and P2 showed an unexpected inhibition of 42%. This however, may be due in part to covalent interactions between the P2 cystine residue and decorin, which result in reduced binding.
  • DbpA-immunized mice develop significant inflammation compared to controls, and P5- and P4/P5-immunized mice also develop significant responses compared to their lysine-modified equivalents and other control groups.
  • P4- and P9-immunized mice also develop a response greater than that of control groups but with less intensity and P2-immunized mice developed no response.
  • the DbpA-immunized mice required more time to develop a response capable of controlling the disseminating Borrelia, as seen in part by joint cultures taken 14 days post infection.
  • 20% of DbpA- and P4/P 5 -immunized mice and 60% of joints from P5-immunized mice were positive for Borrelia when compared to joints from the other groups (100% positive).
  • Arthritis incidence and severity was lowest in mice immunized with DbpA, P4/P5 and
  • DbpA and the DbpA-peptides may drive the development of humoral/cellular immunity against Borrelia with different efficiency and intensity, which accounts for some of the variability in the numbers of positive cultures observed following immunization among the different groups.
  • mice Specific-pathogen free female C3H/HeJ mice were purchased from Harlan Sprague Dawley (Houston, TX). The animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and the National Institutes of Health. The Institutional Animal Care and Use Committee approved all animal procedures. Female mice were 8-10 weeks old at the start of each experiment. Synthesis of DbpA-peptides
  • Peptides (Fig. 1) were synthesized by a solid phase method on a p-benzyloxybenzyl alcohol resin using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and a model 396 Multiple Peptide synthesizer from Advanced Chem Tech Inc. (Louisville, KY) as described previously (21). Peptides were analyzed by reverse-phase HPLC on a Waters 625 Liquid Chromatography system using a C 18 analytical column. The purity of the peptides as assessed by HPLC was greater than 90% (21). Modification of lysine residues on DbpA-peptides resulting in P2 Mod-P9 Mod were performed as described (20).
  • DbpA Anti-Serum for DbpA-peptides Immulon-1 microtiter plate wells (Dynatech Labs, Chantilly, VA) were coated with 2.0 ⁇ g of each peptide in 50 ⁇ l of PBS overnight at 4°C. The wells were washed and then blocked with 400 ⁇ l of Super Block (Pierce, Rockford, IL). All incubations were done at 37°C and all dilutions were made using Super Block. After washing, 100 ⁇ l of a 1 :1000 dilution of polyclonal, rabbit anti-DbpA (625) (17) was added for 1 hr.
  • Immulon-1 microtiter plate wells were coated with 0.4 ⁇ g of DbpA in 50 ⁇ l of PBS over night at 4°C. The wells were washed and then blocked as described above. During the blocking step, 0.12 ⁇ g/ml of biotin-conjugated decorin was pre-incubated at 37°C with 5 ⁇ g of each DbpA-derived peptide or DbpA. After washing, 100 ⁇ l of the biotin conjugated decorin inhibitor mixture was added to the wells and incubated for 1 h. After washing, the wells were incubated with AP-conjugated streptavidin and developed as described above. Immunization of mice with DbpA-derived peptide
  • mice were immunized with recombinant DbpA, P2, P4, P5, P4/P5, P9, or the corresponding lysine modified counterparts (P2 Mod, P4 Mod, P5 Mod or P9 Mod) using the immunization schedule established by Hanson (11). Briefly, mice were immunized with 20 ⁇ g of protein or peptide in complete Freund's adjuvant (CFA) (Week 1), boosted at week 4 with the same dose in incomplete Freund's adjuvant (IF A) and infected at week 6.
  • CFA complete Freund's adjuvant
  • IF A incomplete Freund's adjuvant
  • B. burgdorferi strain B31 (passage 5) was used in this study and cultured in BSK II (Barbour-Stoenner-Kelly) medium at 34° C (22).
  • Bacterial cultures were incubated in CO -enriched atmosphere in a GasPak chamber (BBL, Baltimore, MD) containing BBL GasPak Plus envelopes and a GasPak anaerobic indicator (Beckton dickinson, Cockeysville, MD) until the cells reached log phase. Bacterial counts were determined using dark field microscopy and a Petroff-Hausser chamber.
  • Escherichia coli strain JM101 (Qiagen, Chatsworth, CA) were grown at 37°C in Lennox broth (LB) (Difco, Detroit, MI), containing the appropriate antibiotics.
  • Human fibroblast skin cells (ATCC #CRL-1475) were cultured on 16-well chamber slides (Nunc, Naperville, IL) in Dulbecco's Modified Essential Media containing 10% fetal bovine serum (DMEM) at 37°C.
  • DMEM Dulbecco's Modified Essential Media containing 10% fetal bovine serum
  • mice/group mice were anesthetized with Metofane (methoxyflurane; Pitman-Moore, Mundelein, IL) and 10 mice/ group were infected i.d. at the base of the tail with 10 4 Borrelia in 100 ⁇ l of media 6 six weeks after the first immunization. The remaining 5 mice/group were tested for a DTH response to recombinant DbpA. Following infection, mice were bled 7 and 14 days postinfection. Serum was collected for antibody analysis and blood cultured for the detection of live spirochetes by inoculating 5 ml of BSK II medium with 50 ⁇ l of blood from each infected mouse and cultured for 2 weeks at 34°C as described above.
  • mice were sacrificed and one hind tibiotarsal joint, heart, bladder, and a 3 mm skin biopsy from the ear were aseptically removed and inoculated into separate tubes containing 6 ml of BSK II medium with antibiotics (50 ⁇ g/ml rifampacin and 100 ⁇ g/ml phosphomycin (Sigma), and incubated for 2 weeks at 34°C as described above.
  • antibiotics 50 ⁇ g/ml rifampacin and 100 ⁇ g/ml phosphomycin (Sigma)
  • footpads were measured before and 24 h after challenge using a spring-loaded micrometer
  • mice were anesthetized as described above during footpad measurements and injections. Swelling was determined by subtracting the footpad measurements before challenge from those taken 24 h after challenge.
  • infiltrate 0 no arthritis, 1, minimal or rare
  • DbpA was constructed using polymerase chain reaction (PCR).
  • DbpA lysine mutants K14-K170 were constructed using extension overlap PCR and involved single lysine to alanine substitutions of the residues indicated in Fig. 7.
  • Oligonucleotides used for PCR, listed in Table II, were purchased from Life Technologies, Inc. (Gaithersburg, MD). Lysine to alanine verification for each mutant was analyzed by nucleotide sequencing with the Sequenase version 2.0 sequencing kit (US Biochemicals). Expression and Purification of Recombinant Proteins
  • DbpA, OspC (outer surface protein) and DbpA site-directed mutants from B. burgdorferi strain 297 were expressed in E. coli (JM 101 ) harboring the appropriate plasmid.
  • E. coli was grown in LB until they reached an A 6 oo of 0.6.
  • IPTG Isopropyl-a-D-thiogalactopyranoside
  • BB binding buffer
  • the filtered supernatant was applied to the column and washed with 10 volumes of BB, then 10 volumes of BB containing 60 mM imidazole.
  • the bound proteins were eluted with BB containing 200 mM imidazole, dialyzed against PBS containing 10 mM EDTA, then dialyzed against PBS.
  • the protein concentration was determined by the Bicinchoninic Acid (BCA) Protein Assay (Pierce) and proteins were stored at -20°C.
  • BCA Bicinchoninic Acid
  • SDS-PAGE, Western Ligand Blots and Western Blot Proteins (purified DbpA, DbpA site-directed mutants and OspC) were subjected to SDS-
  • Immulon-1 microtiter plate wells (Dynatech Labs, Chantilly, VA) were coated with 0.4 €g of DbpA, DbpA mutants and K-mod in 50 €1 PBS overnight at 4°C. The wells were washed
  • biotin-conjugated decorin in 100 Gl of Super Block was added to the wells and incubated for lh. After washing, 100 €1 of a 1 :10,000 dilution of alkaline phosphatase (AP)-conjugated streptavidin (1 U/ml) (Boehringer Mannheim) was added and incubated for 1 h. The wells were washed and incubated for 30 min with 100 €1 of a 1 mg/ml Sigma 104 phosphatase substrate (Sigma) dissolved in 1 M diethanolamine, 0.5 mM MgCl , pH 9.8.
  • AP alkaline phosphatase
  • streptavidin 1 U/ml
  • Immulon-1 microtiter plate wells were coated with 0.4 €g of DbpA in 50 €1 of PBS overnight at 4°C. The wells were washed and then blocked as described above.
  • Biotin- conjugated decorin 100 ⁇ l of 0.12 ⁇ g/ml solution was pre-incubated at 37°C with various amounts ( 1.0, 0.5, 0.25, 0.12 and 0.0625 ⁇ g/well) of unlabeled DbpA, DbpA site-specific mutants, or K-mod. After washing, 100 ⁇ l of the biotin-conjugated decorin/inhibitor mixture was added to the wells and incubated for 1 h. After washing, the wells were incubated with streptavidin AP and developed as described above. All dilutions were made using Super Block. Attachment B. burgdorferi to Decorin Substrates anal Inhibition by Recombinant Proteins
  • Immulon- I microtiter plate wells were coated with 1.0 ⁇ g of decorin in 50 €1 PBS
  • OspA was added and incubated for 1 h.
  • the wells were washed and incubated with 100 €1 of a 1 :3000 dilution of AP-conjugated goat anti-mouse IgG for 1 h. After washing, the wells were developed as described above. All incubations took place at 37°C unless otherwise specified. All dilutions were made using Super Block. Immunofluorescence
  • Human skin fibroblasts were cultured and fixed. Cells were plated onto 16-well chamber slides (Nunc) at a density of 2.5 x 10 5 cells per ml, grown for 2-3 days then fixed with -20°C acetone and washed twice in PBS. All subsequent washes were done by immersing the slides in a staining dish filled with PBS 3 times, 10 minutes each wash unless otherwise specified. All incubations were done at room temperature. Unoccupied protein-binding sites on the slides were blocked with 80 €1 of Super Block (Pierce) containing 1% goat and 5% horse serum (blocking
  • rhodamine-conjugated goat anti-rabbit IgG was added for 30 min in the dark. The slides were examined after washing using fluorescence microscopy. Photographs were taken at 20X magnification using Fuji Film 1600 ASA slide film. Anti-DbpA and rhodamine-conjugated goat anti-rabbit IgG antibodies were adsorbed against CRL-1475 cells prior to use. An equal mixture of 1 x 10 6 sonicated and whole CRL 1475 fibroblasts in 300 €1 of PBS were incubated end over end at 37°C for 1 h with an equal volume of each antibody. Adsorbed antiserum was collected after centrifugation of each tube at 5000 rpm for 5 min.
  • Arginine residues on DbpA were chemically modified. Briefly, 1 mg of DbpA (2 ml) was dialyzed against 0.2 M sodium borate (pH 9.0) overnight at 4 ⁇ C. The dialyzed protein was transferred to an eppendorf tube and incubated in the dark at 37°C with 0.05 M cyclohexanedione. DbpA was then passed through a 0.02 M sodium borate-primed PD-10 column (Amersham Pharmacia Biotech. Piscataway, NJ) and four 0.5 ml aliquots were collected. Circular Dichroism Spectroscopy
  • Rabbit antiserum R625 (BioDesign International, Kennebunkport, ME) was raised against DbpA.
  • Monoclonal IgG2a antibody against OspA (4.5 mg/ml) (BOR-018-48316) was purchased from Capricorn Products, Inc. (Scarborough, ME).
  • Rhodamine-conjugated goat affinity purified antibody (1 mg/ml) to rabbit IgG (whole molecule) and alkaline phosphatase- conjugated (enzyme activity 1 191 U/ml) goat affinity purified antibody to murine IgG (whole molecule) were purchased from Cappel (Durham, NC). Examples:
  • Immunizations utilizing peptide formulations require that immune responses to the peptide(s) result in recognition of native-protein epitopes.
  • DbpA peptides containing the lysine residues previously demonstrated to be critical for decorin binding were also antigenic (20)
  • a panel of overlapping peptides 25 amino acids in length was constructed (Fig. 1) and probed with polyclonal anti-DbpA serum (17).
  • Cross-reactivity of peptide epitopes with native protein was examined by ELISA. As demonstrated in Fig.
  • DbpA peptides P2-P9, chemically modified peptides P2 Mod-P9 Mod, and an irrelevant peptide from the Staphylococcus aureus collagen-binding adhesin, CNA4 (OSKITVDNTKNTIDVTIQG) were used as inhibitors of decorin attachment to DbpA-coated wells.
  • P9 which contains K163 and K170 moderately inhibited decorin binding (42%) and P2 inhibited binding by 44%.
  • DTH is an in vivo method of testing T cell-dependent immune responses that are manifested by inflammation at the site of antigenic challenge.
  • Mice immunized with DbpA, P2, P4, PS, P4/P5 combined, P9, or the corresponding lysine-modified peptides were challenged in the footpad with 2 ⁇ g of recombinant DbpA. Footpad measurements were taken before and after challenge as described above.
  • DbpA-immunized mice developed a significant footpad swelling of 53.3+3.5 (not shown) compared to mice immunized with adjuvant alone or naive mice receiving only the DbpA challenge.
  • mice were immunized with DbpA, DbpA-peptides, or the lysine-modified DbpA- peptides and then infected as described above. Seven days post infection blood was examined for the presence of spirochetes. Mice immunized with P4, P5, P4/P5, or P9 had fewer positive cultures ( ⁇ 20%) when compared to P2, lysine-modified peptide-, DbpA, or adjuvant-immunized controls (Fig. 5).
  • DbpA and DbpB the 20 kDa molecular weight range
  • DbpA binds decorin in both ELISA and Western ligand blot analysis.
  • DbpA recognizes decorin in an organized extracellular matrix produced by cultured skin fibroblasts and mediates attachment of DbpA- coated beads to fibroblast cultures. To further characterize this interaction, residues within DbpA that are necessary for binding to decorin are identified.
  • Lysine residues are critical for the decorin binding activity of DbpA. To evaluate the importance of individual lysine residues, selected residues were changed to alanine and decorin- binding activity of the resulting mutants was analyzed. Of the 27 lysine residues present in the native DbpA protein sequence, three are present in the leader sequence. The recombinant form of DbpA used in this study contained part of the leader sequence, including one of the lysine residues (K14). By comparing the DbpA sequences of various B. burgdorferi sensu lato strains to the DbpA sequence of B. burgdorferi 297 (Fig.
  • Mutant K51 showed reduced decorin binding activity and a recombinant, unrelated His-tag
  • DbpA-coated microtiter wells The chemically modified DbpA (Kmod) was essentially unable
  • T helper cell response in Lyme arthritis Differential recognition o ⁇ Borrelia burgdorferi outer surface protein A in patients with treatment-resistant or treatment-responsive Lyme arthritis. J. Exp. Med.

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Abstract

Borrelia burgdorferi, l'agent étiologique de la maladie de Lyme exprime sur sa surface une protéine de liaison de la décorine, DbpA et DbpB. L'invention concerne des résidus de lysine nécessaires pour la DbpA se liant à la décorine et des peptides DbpA contenant ces résidus vitaux essentiels pour la liaison de la décorine. L'invention concerne également le fait que la vaccination utilisant les peptides qui comprennent ces domaines de liaison de Dbpa vitaux peut conférer une réaction d'hypersensibilité de type différé à la DbpA et réduire le nombre d'organismes Borrelia burgdorferi, présents chez les animaux infectés.
PCT/US2000/016713 1999-06-16 2000-06-16 Peptides essentiels a proteine de liaison de la decorine et procedes d'utilisation WO2000077041A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU54955/00A AU5495500A (en) 1999-06-16 2000-06-16 Decorin binding protein essential peptides and methods of use

Applications Claiming Priority (2)

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US13952399P 1999-06-16 1999-06-16
US60/139,523 1999-06-16

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WO2000077041A2 true WO2000077041A2 (fr) 2000-12-21
WO2000077041A3 WO2000077041A3 (fr) 2001-05-25

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WO2003046561A1 (fr) * 2001-11-26 2003-06-05 Bortech Oy Methode de diagnostic de la borreliose de lyme aux stades precoce et avance
EP1853289A2 (fr) * 2005-01-31 2007-11-14 Vaxinnate Corporation Nouveaux ligands de polypeptides destines a un recepteur de type toll 2 (tlr2)
WO2020086758A1 (fr) 2018-10-23 2020-04-30 Dragonfly Therapeutics, Inc. Protéines hétérodimères fusionnées à un fragment fc
WO2021216916A1 (fr) 2020-04-22 2021-10-28 Dragonfly Therapeutics, Inc. Formulation, schéma posologique et procédé de fabrication de protéines fusionnées avec fc hétérodimères

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DE19632862A1 (de) * 1996-08-14 1998-02-19 Mikrogen Molekularbiol Entw Immunologisch aktive Proteine von Borrelia burgdorferi, dafür kodierende Nukleinsäuren sowie deren Verwendung in Testkits und als Impfstoffe

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046561A1 (fr) * 2001-11-26 2003-06-05 Bortech Oy Methode de diagnostic de la borreliose de lyme aux stades precoce et avance
EP1853289A2 (fr) * 2005-01-31 2007-11-14 Vaxinnate Corporation Nouveaux ligands de polypeptides destines a un recepteur de type toll 2 (tlr2)
EP1853289A4 (fr) * 2005-01-31 2008-04-09 Vaxinnate Corp Nouveaux ligands de polypeptides destines a un recepteur de type toll 2 (tlr2)
WO2020086758A1 (fr) 2018-10-23 2020-04-30 Dragonfly Therapeutics, Inc. Protéines hétérodimères fusionnées à un fragment fc
WO2021216916A1 (fr) 2020-04-22 2021-10-28 Dragonfly Therapeutics, Inc. Formulation, schéma posologique et procédé de fabrication de protéines fusionnées avec fc hétérodimères

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