Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira

Definitions

• the present invention relates to proteins and/or fragments and derivatives thereof and their use as vaccines and in biotechno logical methods.
• the vaccines particularly include immunogenic proteins in Treponema spp. isolated from digital dermatitis in cattle.
• the present invention further relates to antibodies raised against said proteins or fragments thereof, and the use of said proteins in diagnostic methods in which antibodies are detected as a sign of digital dermatitis in cattle.
• Digital dermatitis is a contagious claw disease causing lameness in cattle, most commonly seen in intensive dairy production. The disease was first described in 1974 in Italy.
• Treponema The rapid response to antibiotic treatment of DD lesions strongly supports a bacterial cause.
• Treponema spp. are central in the aetiology of DD.
• Another early observation of spirochetes was made 1988 when DD was described for the first time in the UK (Blowey and Sharp, 1988).
• Treponema Several phylotypes of Treponema can be present in the same lesion. Different phylotypes have been isolated from the same animal (Walker et al., 1995; Evans et al, 2008) and by cloning and sequencing of 16S rRNA genes, five different phylotypes were identified in a pooled sample from four cows (Choi et al, 1997). It has also been demonstrated by fluorescence in situ hybridization on biopsies from DD lesions that the distribution in the dermal layers differs between phylotypes (Moter et al, 1998). The Treponema phagedenis- like phylotype was located mainly in the stratum corneum and stratum spinosum.
• Treponema phagedenis-like phylotype has been indicated in several studies to be a key agent in the pathogenesis of DD (Klitgaard et al. 2008, Nordhoff et al. 2008, Yano et al. 2009).
• footbaths containing antibiotics are often used. These footbaths rapidly become contaminated with faeces and dirt and hence function as large selective cultures of antibiotic resistant bacteria.
• tetracyclines are used, but only for topical treatment of individual animals since on herd level footbaths with copper sulphate are recommended.
• the present invention aims at providing efficient methods for diagnosis of and immuno- protection against dermatitis in animals, particularly digital dermatitis in ruminants, as well as products for said purposes.
• the present invention revolves around immunogenic proteins in Treponema spp. isolated from digital dermatitis in cattle, and more specifically to recombinant proteins.
• the present invention relates to isolated Treponema phagedenis-Uke proteins, TmpA, Ttm, and PrrA, with amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, respectively, to fragments and derivatives thereof capable of inducing an immune response to Treponema spp., and to fragments and derivatives capable of binding to antibodies produced by a subject in an immune response against said protein, as further defined below.
• the Treponema proteins, and fragments and derivatives thereof are recombinantly produced.
• the invention relates to nucleic acid molecules encoding the proteins, fragments and derivatives according to the invention.
• the invention also relates to the use of said proteins, fragments and derivatives thereof in veterinary medicine, specifically as a vaccine for prevention of digital dermatitis.
• the present invention provides a veterinary vaccine for protection against digital dermatitis comprising one or more of said recombinant proteins and/or active fragments thereof, and conventional and suitable adjuvants.
• a vaccine may or may not further include other Treponema immunogens or whole cell lysates of different Treponema spp. in a vaccine for a broader immune response.
• the present invention also relates to a method for prevention of digital dermatitis in animals comprising the step of administering said vaccine to an animal in need thereof.
• a method of detecting presence of antibodies against Treponema spp. in a sample in which said recombinant proteins and/or active fragments are used to detect the presence of antibodies against Treponema spp. in said sample is provided.
• a method for diagnosis of digital dermatitis in an animal in which said recombinant proteins and/or active fragments are used to detect the presence of antibodies against Treponema spp. in an animal.
• said recombinant proteins and/or active fragments are used in an ELISA (Enzyme-Linked ImmunoSorbant Assay) method.
• the present invention relates to antibodies raised against said immunogenic proteins, or immunogenic derivatives or fragments thereof. Such antibodies are useful in treatment of disease caused by Treponema spp. by way of passive immunization and also in various laboratory methods such as immunomagnetic separation of Treponema bacteria. Brief description of the figure
• FIG. 1 Enzyme-linked immunosorbant assay with recombinant Treponema phagedenis-like strain VI immunogenic proteins TmpA, Ttm, and PrrA as antigens.
• the assays were performed with sera from eight dairy cows with acute digital dermatitis (black bars), two cows with no known history of digital dermatitis and five calves 6-7 months of age (gray bars).
• Horse-radish peroxidase (HRP) conjugated rabbit anti-bovine IgG antibodies (Sigma) (A) or monoclonal 22:26 anti-bovine IgG-HRP antibodies (Svanova Biotech AB) (B) were used as secondary antibodies. Corrected optical density (COD) was measured at 450 nm.
• an “immunogenic agent”, or “immunogen” is capable of inducing an immunological response against itself on administration to a patient, optionally in conjunction with an adjuvant.
• An “active fragment” or “active derivative” as used in the present specification is a fragment or derivative of a native immunogenic agent, capable of inducing an immunological response against said native immunogenic agent on administration to a patient, optionally in conjunction with an adjuvant.
• An active fragment or derivative comprises or mimics at least one "epitope" or "antigenic determinant”.
• binding fragment or “binding derivative” as used in the present specification is a fragment or derivative of a native immunogenic agent, capable of immunospecific binding to antibodies produced by a subject in an immune response against said native immunogenic agent.
• a binding fragment or derivative comprises or mimics at least one "epitope” or "antigenic determinant”.
• a “derivative" of a protein may be a protein showing substantial sequence homology to the original protein.
• the sequence homology may be 50% identity or more, such as 65%, 80%>, 85%), 90%), 95%o or 99% identity in amino acid sequence.
• the substituted amino acids are preferably conservative substitutions.
• the substituted amino acids may be natural or non- natural amino acids.
• epitope or "antigenic determinant” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.
• Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
• An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e. g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
• Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target immunogen, or fragment or derivative thereof.
• the term "antibody” refers to an intact antibody, or a binding fragment thereof.
• An antibody may comprise a complete antibody molecule (including polyclonal, monoclonal or chimeric), or comprise an antigen binding fragment thereof.
• Antibody fragments include F(ab') 2 , Fab, Fab', Fv, Fc, and Fd fragments, and can be incorporated into single domain antibodies, single- chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v- NAR and bis-scFv (See e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126- 1136).
• the present invention relates to an isolated protein having the amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or fragments or derivatives thereof capable of inducing an immune response to said protein, or a fragment or derivative capable of binding to antibodies produced by a subject in an immune response against said protein.
• Said protein, fragment or derivate may be used in veterinary medicine, such as in prevention of a disease caused by Treponema spp., such as digital dermatitis.
• the present invention relates to a method for treatment or prevention of a disease caused by Treponema spp. comprising administering to a subject isolated protein having the amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a fragment or derivative thereof capable of inducing an immune response to said protein.
• Said method may be used for a disease such as digital dermatitis.
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising an isolated protein having the amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a fragment or derivative thereof capable of inducing an immune response to said protein, and optionally pharmaceutically acceptable adjuvants, carriers and/or diluents.
• the present invention relates to a method for detecting the presence of antibodies against proteins from Treponema spp. in a sample, comprising the steps:
• a further aspect of the invention relates to a method for in vitro diagnosis of a disease caused by Treponema spp. comprising the steps:
• Said method may be used for a disease such as digital dermatitis.
• a further aspect of the invention relates to an antibody, or binding fragment thereof, binding specifically to an isolated protein having the amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
• Another aspect of the invention relates to a method for treatment or prevention of a disease caused by Treponema spp. comprising administering said antibody to a subject.
• Digital dermatitis is one example of such a disease.
• a further aspect of the invention relates to a method for separation of Treponema bacteria from a sample, comprising the steps:
• the separation may be achieved by for example immunomagnetic separation.
• Another aspect of the invention relates to a nucleic acid molecule encoding the protein, fragment or derivative according to the present invention.
• the nucleic acid molecule encoding the protein has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and parts thereof.
• a further aspect of the invention relates to a vector comprising said nucleic acid molecule and optionally regulatory sequences for expression in a host cell.
• Another aspect of the invention relates to a transgenic host cell comprising said vector.
• the present invention also relates to a method for producing a protein, fragment or derivative according to the present invention, comprising the steps:
• Shotgun phage display was used to identify three immunogenic proteins in an isolate (VI) of the DD Treponema phylotype closely related to T. phagedenis. This phylotype has been indicated in several studies to be a key agent in the pathogenesis of DD (Klitgaard et al. 2008, Nordhoff et al. 2008, Yano et al. 2009).
• the phage library was selected against antibodies from a rabbit immunized with live bacteria.
• the proteins, fragments and derivatives according to the first aspect of the invention may be isolated from a culture of the Treponema phylotype closely related to T. phagedenis strain VI, or, preferably, recombinantly produced as described below.
• the immunogenic proteins according to the present invention can be used in vaccines against diseases caused at least in part by the Treponema phylotype closely related to T phagedenis, or other Treponema spp.
• the complete proteins are used in vaccines. In certain embodiments only fragments comprising the relevant epitopes are used.
• one or more epitopes of one or more proteins are combined in a single molecule and used in a vaccine.
• the recombinant proteins, derivatives or fragments thereof may be thus used alone or in different combinations or as fusion proteins of the binding epitopes.
• Phage R408 (Promega) was used as helper phage.
• Escherichia coli TGI A(lac-proAB)
• LA Luria-Bertani agar
• Treponema DNA was prepared by conventional phenol-chloroform extraction.
• Restriction and modification enzymes were from MBI Fermentas AB and used according to manufacturer's instructions. Plasmids were prepared using QIAprepTM Miniprep (QIAGEN).
• the phage library was constructed in the pG8SAET phagemid vector.
• Treponema sp. strain VI chromosomal DNA was fragmented by sonication until the majority of the fragments were between 0.4-1.5 kb in length.
• the fragments were made blunt-ended by T4 DNA polymerase and T4 DNA kinase treatment and then ligated into SnaBl -digested and dephosphorylated phagemid vector pG8SAET using Ready-To-GoTM T4 DNA ligase tubes (GE Healthcare).
• the final library was generated by electrotransformation of the ligated material into E.
• Phage displaying immunogenic polypeptides were isolated by panning against rabbit anti- Treponema sp. strain VI IgG. Three panning experiments were performed. Microwells (MaxiSorpTM, Nalge Nunc International) were coated with Zymed recombinant Protein G (Invitrogen) at a concentration of 10 ⁇ g in 200 ⁇ 50 mM sodium carbonate, pH 9.5.
• the eluate was immediately neutralised with 2 M Tris-buffer pH 8.0 and used to infect E. coli TGI, which were plated on LA-plates with ampicillin (LAamp).
• LAamp ampicillin
• elution was also carried out by direct infection of TGI cells added to the well by the bound phages. After incubation overnight, colonies were counted and 100 colonies transferred to an LAamp-plate. These colonies were then transferred to nitrocellulose-filters for screening of E-tag expression using mouse anti-Etag antibodies (GE Healthcare) and secondary horse radish peroxidase-labelled sheep anti-mouse antibodies (GE Healthcare).
• the chromosomal DNA of Treponema sp. strain VI was sequenced and assembled at the KTH Genome Center at KTH Royal Institute of Technology, Sweden, using the Genome Sequencer FLX System, with long-read GS FLX Titanium chemistry and the 454 de novo assembler, Newbler (454 Life Sciences, Branford, CT, USA). An additional De Novo assembly of the reads was made with CLC Genomics Workbench 3 (CLC bio) and for further sequence editing CLC Main Workbench 5 (CLC bio) was used.
• CLC Genomics Workbench 3 CLC Genomics Workbench 3
• CLC bio CLC Main Workbench 5
• the genome sequence was used to predict the full open reading frames and the corresponding amino acid sequences of the three immunogenic proteins. Homology searches were performed using the BLAST algorithm at the National Center for Biotechnology Information. The
• SignalP 3.0 Server with Gram-positive data was used for prediction of signal peptides.
• One protein was predicted as a lipoprotein according to Setubal et al. 2006.
• Genomic Treponema sp. strain VI DNA for PCR was prepared with the DNeasy Blood & Tissue Kit (QIAGEN) following the protocol for Gram-negative bacteria.
• the thermal cycling conditions were 95°C for 1 min, 30 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 3 min and a final extension at 72°C for 5 min.
• PCR products were analyzed by agarose gel electrophoresis and purified with the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare).
• Purified amplicons were digested with either BamHI and Xhol or Ndel and Sapl according to the manufacturer's instructions (Fast digest, Fermentas) and purified as described earlier.
• the digested amplicons were ligated into the respective vector - BamHI and Xhol digested pGEX-6P-l (bulk GST purification module, GE Healthcare) or Ndel and Sapl digested pTXBl (IMPACTTM Kit, New England BioLabs) - using the ReadyToGo T4DNA Ligase (GE Healthcare).
• Ligated material were electrotransformed into competent Escherichia coli strain BL21 (DE3) (GST) or ER2566 (IMPACT) and spread on LA supplemented with ampicillin (final cone. 5( ⁇ g/ml). The presence of inserts in a number of colonies was analyzed by PCR using the vector sequencing primers. Clones with a correct size insert were further analyzed by DNA sequencing. Production of recombinant immunogenic Treponema proteins
• the cells were harvested and resuspended in a buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1 mM EDTA, and 0.05% (v/v) TWEEN20] and lysed by freezing and thawing. After centrifugation, the supernatants were sterile filtrated and applied onto a chitin column. The columns were washed extensively using the same buffer and treated subsequently with cleavage buffer [20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, and 30 mM dithiothreitol (DTT)].
• a buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1 mM EDTA, and 30 mM dithiothreitol (DTT)].
• the eluted samples containing the antigens were dialysed against phosphate-buffered saline [PBS; 137 mM NaCl, 2.7 mM KC1, 10 mM Na 2 HP0 4 , 1.4 mM KH 2 P0 4 (pH 7.4)].
• the E. coli cells were suspended in PBS supplemented with TWEEN20, final cone. 0.1% (v/v) (PBST) whereupon the cells were lysed by freezing and thawing. After centrifugation, the supernatant was sterile filtrated and batch purified with Glutathione-sepharose beads. After extensive washing using PBST the fusion protein was eluted with glutathione elution buffer or treated with scissor protease to release the produced protein. Finally, the amounts of antigens obtained were determined using spectrophotometry and the quality analyzed by SDS-PAGE coomassie staining. The proteins were stored finally at -20°C. Enzyme-linked immunosorbant assays (ELISAs)
• Horse-radish peroxidase conjugated swine-anti rabbit (Dako) and rabbit-anti cow (Dako) antibodies were added to the relevant wells, diluted 1 :4000 and 1 :500, respectively, and plates were incubated for one hour at 37°C. The wells were washed three times with 400 ⁇ PBS-T after which 100 ⁇ solution consisting of 20 mM
• TMB tetramethylbenzidine
• IHLC ⁇ 230 ⁇ / ⁇ pH 4.25
• Treponema spp. are fastidious organisms that require complex culture media and anaerobic environments for growth. Additionally, samples from cattle claws have a plethora of other bacteria contaminating the cultures.
• An option to concentrate and purify Treponema spp. for culturing and DNA isolation is immunomagnetic separation (Demirkan et al. 1999, Demirkan et al. 2001, Choi et al. 1996).
• Immunomagnetic beads covalently coated with for example anti-rabbit IgG coupled with rabbit antibodies raised against said immunogenic proteins, derivatives or active fragments thereof can be used for specific separation of the DD
• Treponema phylotype closely related to T. phagedenis Treponema phylotype closely related to T. phagedenis.
• Vaccines Bacterial proteins stimulating the immune system to antibody production can also be used for vaccine development. Recombinant proteins can be combined with immune-stimulating complexes (ISCOMs) and/or whole cell lysates to increase the immune response in the animal and hence the protection against the disease-causing agent/s.
• ISCOMs immune-stimulating complexes
• the assays were performed with sera from eight dairy cows with acute DD from the herd from which Tpl strain VI was isolated, two cows from another herd with no known history of DD, and five calves 6-7 months of age. Digital dermatitis diagnosis was made by visual examination. Microplates (PolySorpTM, Nalge Nunc International) were coated with recombinant proteins at concentrations of 1 ⁇ g/ml TmpA, 0.8 ⁇ g/ml Ttm or 0.02 ⁇ g/ml PrrA in 100 ⁇ 50 mM sodium carbonate, pH 9.5, overnight at 2°C. Wells were washed twice with PBS-T and blocked with PBS-T for one hour at room temperature.
• HRP horse-radish peroxidase conjugated rabbit anti-bovine IgG antibodies (Sigma) diluted 1 :8000 or monoclonal 22:26 anti-bovine IgG-HRP antibodies (Svanova Biotech AB) diluted 1 :4000 were added to the wells and plates were incubated for one hour at 37°C.
• Tpl strain VI The three immunogenic proteins identified in Tpl strain VI were produced recombinantly; PrrA as a full-length mature protein from aa +1 relative the cysteine residue of the predicted lipoprotein signal peptide to the last aa before the stop codon (aa:s 22-251), TmpA from aa +7 relative the cystein residue of the predicted lipoprotein signal peptide to the last aa before the stop codon (aa:s 29-344), and Ttm as a partial polypeptide covering aa:s 689-970, which are the aa:s constituting the consensus sequence of the overlapping Ttm sequences from the panning experiments.
• Putative tail tape measure protein 1.30 1.47 1.08 1.20 1.59 0.75 0.70 2.14 1.66 1.91 1.61 1.86 2.06 1.76 1.74 1.5 fragment
• TmpA homolog + putative tail tape 1.36 1.23 0.63 0.90 0.91 0.83 0.60 2.47 1.83 1.84 1.40 1.73 2.17 1.87 2.65 2.0 measure protein fragment
• Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions. Microbiology 144, 2459-2467.

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