EP1238280A2 - Agents et procedes pour le diagnostic de la borreliose de lyme et vaccin contre la borreliose - Google Patents

Agents et procedes pour le diagnostic de la borreliose de lyme et vaccin contre la borreliose

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Publication number
EP1238280A2
EP1238280A2 EP00991154A EP00991154A EP1238280A2 EP 1238280 A2 EP1238280 A2 EP 1238280A2 EP 00991154 A EP00991154 A EP 00991154A EP 00991154 A EP00991154 A EP 00991154A EP 1238280 A2 EP1238280 A2 EP 1238280A2
Authority
EP
European Patent Office
Prior art keywords
protein
borrelia
burgdorferii
fragment
borrelia burgdorferii
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00991154A
Other languages
German (de)
English (en)
Inventor
Peter Jungblut
Aydan Dilgimen
Sascha Thies
Brigitte Wittmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
WITA GmbH Technologiezentrum Teltow
Original Assignee
WITA GmbH Technologiezentrum Teltow
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by WITA GmbH Technologiezentrum Teltow filed Critical WITA GmbH Technologiezentrum Teltow
Publication of EP1238280A2 publication Critical patent/EP1238280A2/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • 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 invention relates to an agent and a method for diagnosing Lyme disease and a Lyme vaccine.
  • the following antigens of Lyme disease could be identified, which are characterized by high specificity and sensitivity: GAPDH, oligopeptide permease, oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment , (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein cp32-6 Borrelia burgdorferii, membrane assoc. protein p66 percursor Borrelia burgdoferii, oligopeptide ABC transporter periplasmic BP
  • RNA polymerase (rpoA) homologous, P66 protein (fragment), flagellin (fragment), DNA direct.
  • RNA polymerase integral outer membrane protein p66, pyruvate kinase (pyk) homolog, phosphoglycerate kinase (pgk) and / or BBU28760 NID.
  • pyk pyruvate kinase
  • pgk phosphoglycerate kinase
  • BBU28760 NID One of the most common transmitted by ticks
  • LM lymphadenosis cutis benigna
  • the species of the Borrelia burgdorferi complex (also known as Borrelia burgdorferi sensu lato) are divided into three main genus species, B. burgdorferi sensu stricto, B. garinii and B. afzelii.
  • OspA outer-surface lipoproteine
  • a test antigen known for Lyme disease a corresponding vaccine could be provided in the USA.
  • This polyvalent vaccine OspA protects mice against B. Burgdorferi sensu stricto, B. garinii and B. afzelii. (Gern, L. Vaccine 1997, 15 1551-1557, see also Appendix 1).
  • a vaccine must therefore contain other or further proteins and / or peptides in order to enable broad protection.
  • the object of the invention was to find further antigens for the diagnosis of LM and to provide a corresponding vaccine which allows a wide range of applications.
  • glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
  • Oligopeptide permease oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment, (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein cp32-6 Borrelia burgdorferii, membrane assoc.
  • fragment fragment
  • flagellin fragment
  • DNA direct.
  • RNA polymerase integral outer membrane protein p66
  • pyruvate kinase pyk homolog
  • the term pharmaceutical or diagnostic composition means a substance which is suitable for the diagnosis and / or treatment of diseases, in particular diseases related to Lyme disease, preferably in an amount sufficient to do so To achieve effect.
  • pharmaceutical or diagnostic composition used in the present description means both the active substance itself and the active substance in connection with pharmaceutical compatible carriers, adjuvants, other active ingredients, etc.
  • treatment means the prophylactic and / or therapeutic effect of a medicinal substance.
  • borreliosis antigens relates to both naturally occurring borreliosis antigens and all modifications, mutants or derivatives of the borreliosis antigens, borreliosis antigens produced by recombination techniques, the amino acid modifications, such as inversions, deletions, insertions, Deposits etc. are included, provided that at least some of the essential functions of the wild-type Lyme disease antigens are present.
  • Lyme disease antigens may also include unusual amino acids and / or modifications such as alkylation, oxidation, thiol modification, denaturation and oligomerization and the like.
  • Lyme disease antigens can in particular be proteins and / or peptides and / or fusion peptides which, in addition to other proteins, peptides or parts thereof, contain Lyme disease antigens in whole or in part.
  • the Lyme antigens are shortened forms of the naturally occurring Lyme antigens, such as small peptides.
  • promoter means a DNA sequence that is mostly upward (5 ') from the coding sequence of a structural gene and which controls expression of the coding region, in particular a Lyme antigen, by providing a recognition sequence for an RNA polymerase and / or for other factors which are required for the transcription to start in the correct place. Promoter sequences are necessary, but not always sufficient to control the expression of the gene.
  • nucleic acid means a large molecule which can be single-stranded or double-stranded and consists of monomers (nucleotides) which contain a sugar unit, a phosphate unit and either a purine or a pyrimidine residue.
  • the nucleic acid can be cDNA, genomic DNA or RNA, for example mRNA.
  • nucleic acid sequence means a natural or synthetic polymer of single- or double-stranded DNA or RNA which alternatively contains synthetic, non-natural or modified nucleotide bases which can be incorporated into DNA or RNA polymers.
  • gene means a DNA sequence encoding a specific protein and regulatory elements which control the expression of this DNA sequence.
  • the term coding sequence relates to the part of a gene which codes for a protein, a polypeptide or a part thereof, the regulatory sequences and / or elements which control the initiation or termination of the transcription.
  • the coding sequence and / or regulatory element can be one that is normally found in the cell, in which case it is referred to as autologous or endogenous, or one that is not normally found in the cell Cell is localized, in which case it is referred to as heterologous.
  • a heterologous gene can also consist of autologous elements that are arranged in an order and / or orientation that is normally not found in the cell to which the gene is transferred.
  • a heterologous gene may be derived in whole or in part from any source known in the art, including a bacterial or viral genome or episome, core eucaryotic or plasmid DNA, cDNA, or chemically synthesized DNA.
  • the structural gene can form a continuous coding region or can comprise one or more introns which are delimited by suitable splice connections.
  • the structural gene can be composed of sections that come from different, naturally occurring or synthetic sources.
  • a detectable gene product is a nucleotide or amino acid sequence, in particular a borreliosis antigen, which can be detected using a test.
  • expression of a detectable gene product imparts a feature to the cell that is a simple selection of the cell from other cells allowed that do not express the detectable gene product.
  • vector means a recombinant DNA construct, which can be a plasmid, virus or an autonomously replicating sequence, a phage or a nucleotide sequence, which is linear or circular, consisting of single or double stranded DNA or RNA, in which a series of nucleotide sequences have been linked or recombined into a unique construction and which can introduce a promoter fragment and a DNA sequence of a selected gene product in sense or antisense orientation together with suitable untranslated 3 'sequences into a cell.
  • Plasmids are genetic elements that are stably inherited without being part of the chromosome of their host cell. They can include DNA or RNA and be linear and circular. Plasmids encode molecules that ensure their replication and stable inheritance during cell replication and can encode products of considerable importance to medicine, agriculture and the environment. For example, they code for toxins that greatly increase the virulence of pathogenic bacteria. You can also encode genes that confer resistance to antibiotics. In molecular biology, plasmids are generally used as vectors for cloning and expression of recombinant genes. According to the rules of the standard designation familiar to the person skilled in the art, plasmids are generally designated with the lowercase letter p, the uppercase letter and / or numbers precede or follow.
  • plasmids disclosed in the present specification are either commercially available, open to the public, or can be constructed from available plasmids using routine, well-known, published methods. Many plasmids and other cloning and expression vectors that can be used in the present invention are well known and readily available to those skilled in the art. In addition, those skilled in the art can readily construct any number of other plasmids suitable for use in the invention. From the present disclosure, the properties, construction and use of both such plasmids and other vectors are readily apparent to the person skilled in the art.
  • expression used in the present description is intended to describe the transcription and / or coding of the sequence of the gene product, for example of Lyme antigens.
  • a DNA chain encoding the sequence of a gene product is first transcribed into a complementary RNA, which is often an mRNA, and then the mRNA so transcribed is translated into the gene product mentioned above, if it is the gene product is a protein.
  • the expression also includes the transcription of a DNA which has been inserted with respect to its regulatory elements in the antisense direction.
  • An expression that is constitutive and possibly further increased by an externally controlled promoter fragment can produce multiple copies of mRNA and large amounts of the selected gene product can also include the overproduction of a gene product.
  • the peptides or proteins of the invention are isolated.
  • isolated used in the present description means, in connection with proteins, a polypeptide which is present without the material with which it is associated in its natural state or at most with a part thereof. Based on the weight of the total protein in a particular sample, the isolated protein is at least 0.5%, preferably at least 5%, more preferably at least 25% and even more preferably at least 50%. Most preferably, the isolated protein is essentially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated and forms a single major band on a polyacrylamide gel and a single protein spot on a two-dimensional gel. Substantially free means that the protein is at least 75%, preferably at least 85%, more preferably at least 95% and most preferably at least 99% free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated ,
  • Antibody means a polypeptide that is essentially derived from an immunoglobulin gene or immunoglobin genes is encoded, or fragments thereof that specifically bind / bind and recognize an analyte (antigen).
  • Known immunoglobin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu genes for the constant region as well as the innumerable genes for the variable immunoglobulin region.
  • Antibodies occur, for example, as intact immunoglobulins or as a series of well-characterized fragments that are generated by cleavage with various peptidases.
  • Antibody also means modified antibodies (eg oligomeric, reduced, oxidized and labeled antibodies).
  • antibody used in the present description also includes antibody fragments which have been generated either by modification of whole antibodies or by means of de novo synthesis using recombinant DNA techniques.
  • the term antibody includes both intact molecules and fragments thereof, such as Fab, F (ab ') 2 and Fv, which can bind the epitope determinant.
  • the ability of the antibody to selectively bind its antigen or receptor has been partially retained in these fragments, the fragments being defined as follows:
  • Fab the fragment containing a monovalent antigen binding fragment of an antibody molecule
  • Fab can be produced by cleavage of an entire antibody with the enzyme papain, whereby an intact light chain and part of a heavy chain are obtained
  • the Fab 'fragment of an antibody molecule can be obtained by treating an entire antibody with pepsin and then reducing it, whereby an intact light chain and part of the heavy chain are obtained; two Fab 'fragments are obtained per antibody molecule;
  • F (ab ') 2 the fragment of the antibody which can be obtained by treating an entire antibody with the enzyme pepsin without subsequent reduction;
  • F (ab ') 2 is a dimer of two Fab' -
  • Fv defined as a genetically modified fragment that covers the variable range of light
  • Chain and the variable region of the heavy chain contains and is expressed in the form of two chains;
  • Single chain antibody defined as a genetically engineered molecule containing the light chain variable region and the heavy chain variable region, linked by a suitable polypeptide linker to a genetically fused single chain molecule
  • epitope used in the present invention means any antigen determinant on antigens, particularly those associated with Lyme disease to which the paratope of an antibody binds.
  • Epitope determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have both specific features of the three-dimensional structure and specific charge features.
  • the antibodies according to the invention have an additional benefit in that they can be used as reagents in immunoassays, such as RIA, ELISA and the like. They can also be used to isolate Lyme disease antigens or domains from cells or other biological samples.
  • the antibodies could e.g. B. to establish a tissue culture-based test to find, isolate or modify novel Lyme disease antigens or novel compounds that modify the interaction of Lyme disease antigens and receptors and / or targets.
  • the humanized or chimeric antibodies can include parts derived from two different types (e.g. human constant area and mouse binding area).
  • the parts originating from two different types can be chemically combined using conventional methods or can be produced as a single fusion protein using genetic engineering methods.
  • a DNA that contains the proteins of the two parts of the Chimeric antibody encoded can be expressed as a single fusion protein.
  • an antibody specifically binds to a protein, e.g. a different biological structure, or shows a specific immunoreactivity if the antibody performs its function in a binding reaction in the presence of a heterogeneous population of proteins and other biological substances, which can be used to decide whether the protein or there is another biological structure.
  • the specified antibodies preferably bind to a specific protein, while there is no significant binding to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that has been selected for a specific protein because of its specificity.
  • Various immunoassay embodiments can be used to select antibodies which show a specific immunoreactivity with a special protein.
  • Immune test refers to a test in which an antibody is used to specifically bind an analyte.
  • the immunoassay is characterized in that specific binding properties of a specific antibody are used to isolate the analyte, to test it in a targeted manner and / or to determine it quantitatively.
  • vaccination is a way of preventing diseases associated with Lyme disease.
  • the development of a vaccine involves the identification of factors that are decisive for virulence, or of structures or borreliosis antigens that are particularly accessible to the human immune system to eliminate a pathogen.
  • antigens are usually associated with the membrane of the borreliosis pathogen.
  • protein used in the present invention with the immunogenic properties of borreliosis antigens or borreliosis pathogens refers to any protein, polypeptide or peptide which (1) can serve as an antigen for antibodies that are linked to borreliosis antigens or borreliosis - Specifically bind pathogens or (2) when administered as a vaccine, have a protective effect against infection with Lyme disease antigens or Lyme pathogens.
  • the person skilled in the art can use conventional methods to determine proteins, peptides or polypeptides which have such properties.
  • these proteins, polypeptides or peptides have 50%, 60%, 70% or 80%, preferably 90%, more preferably 95% and most preferably 98% homology to the proteins or peptides which are identified as Lyme disease antigens, this homology, for example, using the “Smith-Waterman” homology search algorithm, for example using the “MPSRCH” program (Oxford Molecular) can be determined using an affinity gap search with the following parameters gap open penalty 12, gap extension penalty 1.
  • hybridize used in the present invention refers to conventional hybridization conditions in which 5x SSPE, 1% SDS, 1x Denhardts solution are used as the solution and the hybridization temperatures are between 35 ° C. and 70 ° C., preferably 65 ° C.
  • washing is preferably carried out first with 2xSSC, 1% SDS and then with 0.2x SSC at temperatures between 35 ° C and 70 ° C, preferably at 65 ° C (for the definition of SSPE, SSC and Denhardts solution see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)).
  • Fragment includes DNA and / or protein molecules that differ from the original sequence
  • the antigens and their fragments react specifically specifically with anti-B. garinii-, anti-B. burgdorferii- and / or anti-B. afzelii antibodies and have no cross-reactivity with other non-Lyme borreliosis (LM) specific antibodies. They have a high sensitivity and specificity (their signal frequency is more than 50%). All proteins (see Table 3) were not previously known as immune-relevant Lyme antigens.
  • LM non-Lyme borreliosis
  • LM which is known in Europe in particular through the widespread strains B. burgdoferii, B. garnii and B. afzelii are induced, diagnosed and treated.
  • the invention therefore relates to diagnostic agents or a diagnostic composition for Detection of Lyme disease, which antigens glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • Oligopeptide permease oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment, (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc.
  • RNA polymerase homologous, P66 protein (fragment), flagellin (fragment), DNA direct.
  • RNA polymerase integral outer membrane protein p66, pyruvate kinase (pyk) homologous, phosphoglycerate kinase (pgk) and / or BBU28760 NID and / or their fragments.
  • pyk pyruvate kinase
  • pgk phosphoglycerate kinase
  • BBU28760 NID / or their fragments.
  • antigens preferably react specifically with anti-B. garinii-, anti-B. burgdorferii- and / or anti-B. afzelii antibodies.
  • the subject matter of the invention also encompasses the nucleic acid sequences which code for the antigens and / or fragments and which express the antigens.
  • the antigens can be obtained from B.
  • B. garnii and / or B. afzelii can be isolated and purified or according to known techniques chemically or genetically as recombinant proteins or in the form of their genes (DNA, cDNA), including regulatory units known to the person skilled in the art, such as promoters and nucleic acids or nucleic acid sequences, in particular in the form of plasmids and vectors Are made available that represent coding sequences for the proteins or antigens and thus form detectable gene products.
  • the present invention therefore also relates to a diagnostic method for the detection of an acute, chronic or previous infection with Lyme pathogens, in which antibodies against Lyme disease from a sample, a fragment thereof or protein are brought into contact with its immunogenic properties and then determined whether these are bound to the antigen.
  • the detection takes place by detecting antibodies against Lyme pathogens produced by the host in the sample (by means of the antigens according to the invention).
  • a blood or lymph sample is taken, for example, the serum or the lymph is obtained and brought into contact with the antigens according to the invention and then it is determined whether antibodies from the serum are bound to the antigen.
  • the diagnostic method according to the invention can be designed as an ELISA, RIA or another common detection method, in which, for example, the antibody bound from the serum or the lymph is used of a second antibody.
  • the antigen or a fragment thereof can be immobilized, ie adsorbed, for example, on the wall of a plastic shell in such a way that the specific binding specificity is retained.
  • the identification of the antigens from B. garnii, B. burgdorferii and / or B. afzelii takes place e.g. by combining high-resolution two-dimensional electrophoresis with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).
  • MALDI-MS matrix-assisted laser desorption ionization mass spectrometry
  • the antigens according to the invention are preferably immobilized in the agent according to the invention on a solid support.
  • All solid phases known per se which are in the form of membranes, gels, test strips, paper, film, filters, plates or spheres, act as solid carriers.
  • Immobilization places the antigens in a reaction space-limited state.
  • immobilization is understood to mean all methods which lead to a restriction of the mobility of the antigens by biological, chemical or physical means.
  • the carrier or the solid phase on which the antigens are immobilized can also be membranes, braids and / or fibrils, for example, in addition to those already mentioned.
  • the antigens can be immobilized on a support directly or via spacers. Spacer in All spacers are within the meaning of the invention, which can form, for example, a short molecular chain between antigen and carrier. For example, hydroxylated chains can be used to avoid specific hydrophobic interactions. However, it is also possible to immobilize the antigens via selected acceptor molecules.
  • the acceptor molecules have the properties necessary for this, such as, for example, molecular charge, chemically modifiable groups and / or immune or nucleic acid, hybridization affinities, etc.
  • the acceptor molecules have the properties necessary for this, such as, for example, molecular charge, chemically modifiable groups and / or immune or nucleic acid, hybridization affinities, etc.
  • the immobilization can be carried out by different methods, such as, for example, the binding of the antigens to one another or to supports, by holding onto the network of a polymeric matrix or by means of membranes.
  • the immobilization not only makes the antigens reusable, but they can also be easily separated again after the process of interaction with the biological sample. They can be used in much higher local concentrations and in continuous flow systems.
  • the binding or immobilization of the antigens to the carrier can be by direct carrier connection and by Cross-linking takes place.
  • the carrier bond is either ionic / adsorptive or by covalent bond.
  • Cross-linking in the sense of the invention is, for example, cross-linking of the antigens with one another or with other polymers.
  • the inclusion of the antigens is also possible, for example, if the carriers are sintered glass sponges, which in particular enables the Lyme antigens to be concentrated. Furthermore, immobilization on the support is possible by the gel inclusion in carrageenan, by the inclusion in polyacrylamide, by the gel inclusion in alginate, in ENT polymers on ceramics with polyamine and the crosslinking by glutaraldehyde.
  • the presence of functional groups on the support can be a prerequisite for successful covalent fixation of the Lyme disease antigens.
  • a possible activation method for example in the case of dextran gel, is the reaction with cyanogen bromide.
  • various types of bonds can be formed, for example ethers, thioethers, esters etc.
  • Coupling methods for the covalent attachment of antigens to agar, agarose and Sephadex carriers and to silanized surfaces of porous glasses are also known to the person skilled in the art.
  • Possible changes in the activity of the antigens can be avoided or reduced by spacing the antigens onto the carrier be immobilized. The spacers give the antigens the necessary flexibility for optimal binding to the antibody and for optimal recognition of the binding partner to be analyzed.
  • the invention also relates to the use of the antigens glyceralaldehyde-3-phosphate dehydrogenase (GAPDH), oligopeptide permease, oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment, (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc.
  • GPDH glyceralaldehyde-3-phosphate dehydrogenase
  • oligopeptide permease oligopeptide ABC transporter periplasmic BP
  • oppA-2 oligopeptide ABC transporter periplasmic BP
  • oppA-2 oligopeptide ABC transporter periplasmic BP
  • GPDH
  • RNA polymerase homologous, P66 protein (fragment), flagellin (fragment), DNA direct.
  • RNA polymerase integral outer membrane protein p66, pyruvate kinase (pyk) homologous, phosphoglycerate kinase (pgk) and / or BBU28760 NID and / or its fragments and / or the nucleic acid sequences coding for the antigens and / or fragments for diagnosis and / or therapy from Lyme disease.
  • the invention also relates to the use of the antigens glyceraldehyde-3-phosphate dehydrogenase, oligopeptide permease, oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment, (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc.
  • RNA polymerase homologous, P66 protein (fragment), flagellin (fragment), DNA direct.
  • RNA polymerase integral outer membrane protein p66, pyruvate kinase (pyk) homologous, phosphoglycerate kinase (pgk) and / or BBU28760 NID and / or their fragments and / or the nucleic acid sequences coding for the antigens and / or fragments for the production of an agent for diagnosis and / or therapy for Lyme disease.
  • the invention further relates to a method for detecting Lyme disease, which is characterized in that the antigens GAPDH,
  • Oligopeptide permease oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment, (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc. protein p66 percursor Borrelia burgdoferii, oligopeptide ABC transporter periplasmic BP
  • RNA polymerase (rpoA) homologous, P66 protein (fragment), flagellin (fragment), DNA direct.
  • RNA polymerase integral outer membrane protein p66, pyruvate kinase (pyk) homologous, phosphoglycerate kinase (pgk) and / or BBU28760 NID and / or their fragments with a biological sample containing Lyme disease antibody under conditions that cause the formation of an antigen -Antibody complex allow, is brought into contact, and after an incubation period the complex formation of an antibody with the antigen is detected.
  • pyk pyruvate kinase
  • pgk phosphoglycerate kinase
  • BBU28760 NID fragments with a biological sample containing Lyme disease antibody under conditions that cause the formation of an antigen -Antibody complex allow
  • the detection of the Lyme antibodies is preferably carried out in an immunoassay, preferably in a solid phase immunoassay, with direct or indirect coupling of a reaction partner with an easily detectable labeling substance.
  • detection can preferably be carried out in an ELISA, a RIA or a fluorescence immunoassay.
  • the implementation of these detection methods is well known to the person skilled in the art.
  • the antigen in this case e.g. GAPDH, oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment, (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc.
  • GAPDH oligopeptide ABC transporter periplasmic BP
  • Bb glycosyl transferase IgtD homolog
  • heat shock protein 90 VLSE fragment
  • putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc.
  • RNA polymerase integral outer membrane protein p66, pyruvate kinase (pyk) homologous, phosphoglycerate kinase (pgk) and / or BBU28760 NID bound directly or indirectly to a carrier substance, such as polystyrene.
  • a carrier substance such as polystyrene.
  • antigen-bound antibodies become direct or indirect detected by means of enzyme-coupled substances.
  • These substances can be antibodies, fragments of antibodies or high-affinity ligands such as avidin, which binds to a biotin label.
  • Suitable enzymes are peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease or glucose oxidase.
  • the bound enzymes and thus for example the bound antibodies can be quantified by adding a chromogenic substrate.
  • the antigen e.g. GAPDH, oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment, (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc.
  • GAPDH oligopeptide ABC transporter periplasmic BP
  • oppA-2 oligopeptide ABC transporter periplasmic BP
  • IgtD homolog glycosyl transferase IgtD homolog
  • heat shock protein 90 VLSE fragment
  • putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc.
  • RNA polymerase homologous, P66 protein (fragment), flagellin (fragment), DNA direct.
  • RNA polymerase integral outer membrane protein p66, pyruvate kinase (pyk) homolog, phosphoglycerate kinase (pgk) and / or BBU28760 NID directly or indirectly bound to a carrier substance such as polystyrene.
  • a carrier substance such as polystyrene.
  • antigen-bound antibodies are detected by means of substances which carry a radioactive label, for example 125 l.
  • These substances can be antibodies, fragments of antibodies or high-affinity ligands such as avidin, which binds to a biotin label.
  • the bound radioactivity can be quantified using a suitable measuring device.
  • the antigen-bound antibodies are detected in a fluorescence immunoassay by means of substances which carry a fluorescence label, for example fluorescein isothiocyanates (FITC).
  • FITC fluorescein isothiocyanates
  • These substances can be antibodies, fragments of antibodies or high affinity ligands such as e.g. Avidin that binds to a biotin label.
  • the bound amount of fluorescent dye is then quantified using a suitable measuring device.
  • Antibodies of the patient and thus the infection can be detected directly.
  • animal or human liquids such as e.g. Blood, plasma, serum, urine, CSF and synovial fluid understood.
  • the detection of Lyme disease in humans is preferably carried out by detecting Lyme antibodies in the serum.
  • the invention further relates to a Lyme vaccine or a pharmaceutical composition, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)., Oligopeptide ABC transporter periplasmic BP (oppA-2) (Bb), Glycosyl transferase IgtD homolog, heat shock protein 90, VLSE fragment, (U76406) putative vls rec. casette Vls6 Borrelia burgdorferii, Flagellin Protein Borrelia garinii, (AE001578) conserved hypothetical protein Borrelia burgdorferii, membrane assoc.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • RNA polymerase homologous, P66 protein (fragment), flagellin (fragment), DNA direct.
  • RNA polymerase integral outer membrane protein p66, pyruvate kinase (pyk) homologous, phosphoglycerate kinase (pgk) and / or BBU28760 NID and / or its fragments or the DNA coding for these proteins and / or fragments.
  • the vaccine according to the invention can comprise defined pathogen products, dead vaccines, live vaccines, synthetic peptides, recombinant proteins, deletion mutants, recombinant vaccine strains and anti-idiotypic vaccines.
  • Vaccination with defined pathogen products advantageously represents a simple vaccination against the Lyme disease pathogen or Lyme disease.
  • the immune response can include both an immune response against the pathogen and against pathogen products formed by it.
  • Vaccination with dead vaccines in the sense of the invention comprises vaccination with killed Lyme pathogens, whereby pathogens can be both the complete organisms and individual antigenic structures thereof.
  • Vaccination with live vaccines advantageously leads to a strong induction of sufficient resistance to Lyme disease.
  • An antigen or a molecule that the protective Epitope responsible for the immune response can advantageously also contain further areas.
  • the peptide responsible for the protection can be used detached from the harmful molecular segments. It is advantageously possible to couple the peptides to a carrier molecule. Of course, it is also possible to produce artificial polypeptides which consist of repeating subunits of protective peptides. Vaccination with recombinant antigens, or peptides or proteins in the sense of the invention advantageously allows the suitable polypeptides to be produced on a large scale and thus ensures that sufficient amounts of vaccine are provided, even in the case of pathogen strains or pathogen modifications which are difficult or impossible to cultivate. In this way, any contamination from harmful pathogen products is advantageously excluded as long as they are not encoded by the same gene.
  • Appropriate measures can be taken to change the genome of the pathogen so that it is no longer able to cause disease in the host. This is advantageously achieved by deleting genes that are required for virulence or survivability in the host. Since these metabolites are essential for the pathogen, but are not provided by the patient's organism, the pathogen in the patient advantageously dies after consuming its own reserves. However, the survival time in the host is long enough to elicit a protective immune response. Recombinant vaccines are an advantage if this is the case protective antigen or the Lyme pathogen, which is in recombinant form, but alone is not able to induce protection. This is particularly important if the protection is primarily carried by different lymphocyte populations.
  • the gene responsible for the protective antigen can be cloned in a suitable carrier, which makes it possible to elicit a suitable immune response against the recombinant molecule.
  • the concept of an anti-idiotypic vaccine in the sense of the invention is based on the fact that anti-idiotypic antibodies, which are recognized by protective antibodies, are able to stimulate their synthesis.
  • anti-idiotypic antibodies which are recognized by protective antibodies, are able to stimulate their synthesis.
  • the production of molecular antibodies can advantageously be less expensive than the production of the antigen itself.
  • the immunity caused by anti-idiotypic antibodies can advantageously be controlled in a different way than that against the nominal antigen.
  • Anti-idiotypic vaccines or vaccines can therefore advantageously provide adequate protection even in small children.
  • the present invention thus also relates to an immunogenic composition, preferably a vaccine, which contains the above-described borreliosis antigens, a fragment thereof and / or protein with its immunogenic properties or antibodies which are directed against the antigens.
  • the vaccine according to the invention optionally additionally contains a pharmaceutically acceptable carrier.
  • Suitable carriers and the formulation of such vaccines are known to the person skilled in the art. Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions, etc.
  • the vaccine can be administered orally or parenterally, for example intradermally, subcutaneously or intramuscularly.
  • the appropriate dosage is determined by the attending physician and depends on various factors, for example the type of administration, the age and weight of the recipient, etc. It can range from 1 ⁇ g to 200 ⁇ g per patient. In children the dose is reduced to 5 ⁇ g and in hemodialysis patients the dose is increased to 50-300 ⁇ q.
  • the supernatants which contain the total protein mixture, were each determined by high-resolution two-dimensional polyacrylamide gel electrophoresis (2DE gels) (according to Jungblut P., Grabher, G., Stöffler, G., Electrophoresis 20, 3611-3622, 1999; and Klose and Kobaltz, 1995, Electrophoresis 16, 1043-1049).
  • 2DE gels high-resolution two-dimensional polyacrylamide gel electrophoresis
  • the proteins were separated in a preparative 2DE gel, stained with Coomassie, punched out and cleaved with trypsin. The resulting tryptic peptides were eluted and desalted and analyzed by MALDI mass spectrometry. By comparing the Mass data ("mass fingerprints" of the tryptic peptides) with the existing databases (SWISSPROT, PIR), the proteins were identified using the Mascot mass comparison program and provided with the protein spot numbers (see Tables 1, 2 and 3).
  • Table 2 shows which proteins reacted positively with which sera. Serum 5 was very weak and therefore gave a positive reaction only with protein 1, 3, 6, 19 from B.. Burgdorferii and with protein 1, 19 from B. garinii VS286 and with protein 24 from B. garinii 20047. It is It is likely that the identified proteins with a negative reaction with patient sera will also react positively if further sera are measured.
  • Table 3 lists the names of the identified proteins, or their associated genes, and the access numbers (“accession” numbers) of these proteins in the databases (S.Pt: SwissProt database; PIR BLS: PIR database) Table 3 lists the possible cell or plasmid positions of the genes and, where known, the functions of the proteins (Fraser et al., Nature 390, 580-586, 1997).
  • Proteins include:
  • RNA polymerase rpoA
  • P66 protein fragment
  • proteins are four hypothetical proteins which have so far only been characterized as ORFs / "open reading frames", which have thus far not been isolated and characterized at the protein level (protein spots 2, 4, spot 9, spot 16 and spot 38 ).
  • Antigens are of importance for diagnosis and vaccine development.
  • the positively identified antigens confirm the antigens identified according to Jungblut et al. , 1999 (proteins p83 / l00, flagellin,
  • these proteins were identified in the following way: They were punched out of small 2DE gels and identified by mass spectrometry or by special antibodies; the serum diagnosis was carried out by ELISA tests such as under routine laboratory conditions or on one-dimensional gels with rabbit antisera or human sera. Under these conditions, in addition to the known antigens (e.g. OspA, outer-surface lipoprotein), two new antigens were identified, namely GAPDH and ABC Transporter Oligopeptide Permease.
  • OspA outer-surface lipoprotein
  • Table 1 Important Borrelia proteins on 2DE gels Analytical 2DE gels were produced from the 5 different Borrelia strains and stained with silver.
  • Table 2 Immunological test against 9 patient sera. Analytical 2DE gels from the various Borrelia strains were transferred undyed to PVDF membranes. All of these membranes were incubated with the 9 different sera from neuroborreliosis patients.
  • Name of the identified protein (see Table 2)
  • Fiaser CM Casjens S, Hua ⁇ g WM, Sutton GG, Clayton R, Lathigra R, White O, Ketchum 1) "Zhang.J -R., Hardliam J., M., Barbour A., G. And Norris S. Antigenic Variation in lyme A, Dodson R, Hickey EK, G inn M, Dougherty B, Tomb JF, Fleischmann RD, Richardson disease borreliae by pro recombination of VMP-like sequence casettes. " Cell 89 (2), 275-285
  • Bui dorferi HSP70 homolog Characterization o immunoreactive stress protein "mfecUmmim 60 (9), 3704-3713 (1992).

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Abstract

L'invention concerne des agents pour le diagnostic et/ou le traitement de la Borréliose de Lyme comprenant les antigènes glycéraldéhyde-3-phosphat-déshydrogénase (GAPDH), oligopeptide perméase, oligopeptide ABC transporteur périplasmique BP(oppA-2)(Bb), glycolyse transférase IgtD homologue, protéine 90 de choc thermique, fragment VLSE, (U76406) putative v1s rec. cassette V1s6 <i>Borrelia burgdorferii</i>, flagelline <i>Borrelia garinii</i>, (AE001578) protéine hypothétique conservée cp32-6 <i>Borrelia burgdorferii</i>, protéine associée à la membrane p66 précurseur <i>Borrelia burgdoferii</i>, oligopeptide ABC transporteur périplasmique BP (oppA-4)(Bc), fructose-biphosphate aldose (fba) <i>Borrelia burgdorferii</i>, DNAK protéine de choc thermique protéine 70 <i>Borrelia burgdorferii</i>, orfE <i>Borrelia burgdorferii</i>, protéine B de surface extérieure précurseur <i>Borrelia burgdorferii</i>, L-lactate déshydrogénase (ldh), P83/100 gène <i>Borrelia burgdorferii</i>, énolase 2phosphoglycerate <i>Borrelia burgdorferii</i>, flagelline <i>Borrelia garinii</i>, protéine hypothétique BBE28 <i>Borrelia burgdorferii</i>, ADN direct. ARN polymérase (rpoA) homologue, protéine P66 (fragment), flagelline (fragment), ADN direct. ARN polymérase, protéine p66 de membrane extérieure intégrale, pyruvate kinase (pyk) homologue; phosphoglycerate kinase (pgk) et/ou BBU28760 NID et/ou des fragments de ceux-ci et/ou des séquences d'acide nucléique codant pour lesdits antigènes et/ou fragments.
EP00991154A 1999-12-08 2000-12-08 Agents et procedes pour le diagnostic de la borreliose de lyme et vaccin contre la borreliose Withdrawn EP1238280A2 (fr)

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DE19960113A DE19960113A1 (de) 1999-12-08 1999-12-08 Mittel und Verfahren zur Diagnose von Lyme Borreliose sowie Borreliose-Impfstoff
DE19960113 1999-12-08
PCT/EP2000/012454 WO2001042790A2 (fr) 1999-12-08 2000-12-08 Agents et procedes pour le diagnostic de la borreliose de lyme et vaccin contre la borreliose

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PL223175B1 (pl) 2012-10-22 2016-10-31 Inst Chemii Bioorganicznej Polskiej Akademii Nauk Szczepionka przeciw boreliozie, konstrukt genetyczny, rekombinowane białko, sposób otrzymywania konstruktu genetycznego, sposób otrzymywania szczepionki, sposób otrzymywania rekombinowanych białek, zastosowanie rekombinowanych białek do wytwarzania szczepionki przeciwko boreliozie
ES2885833T3 (es) 2016-04-20 2021-12-15 Centro De Investig Energeticas Medioambientales Y Tecnologicas O A M P Composición y métodos para potenciar la expresión génica de PKLR
CN105920593A (zh) * 2016-06-08 2016-09-07 山东省海洋生物研究院 一种水产迟钝爱德华氏菌亚单位口服微胶囊疫苗
WO2018083491A1 (fr) 2016-11-03 2018-05-11 University Of Leicester Détection de borréliose sur la base de phages et moyens associés
EP3410117A1 (fr) * 2017-06-02 2018-12-05 Université de Strasbourg Détection de borrelia burgdorferi sensu lato chez des patients atteints d'une infection de lyme disséminée et tardive
JP7403461B2 (ja) 2017-10-16 2023-12-22 セントロ デ インベスティガシオンス エネルジェチカス メディオアンビエンタゥス イェ テクノロジカス オー．エイ． エム．ピー． ピルビン酸キナーゼ欠損症を治療するためのｐｋｌｒ送達用レンチウイルスベクター

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US5324630A (en) * 1991-06-28 1994-06-28 The Regents Of The University Of California Methods and compositions for diagnosing lyme disease
US5620862A (en) * 1993-11-24 1997-04-15 University Of Connecticut Methods for diagnosing early Lyme disease
AU1836097A (en) * 1996-01-22 1997-08-11 Regents Of The University Of California, The Borrelia burgdorferi outer membrane proteins
EP0915977A1 (fr) * 1996-05-08 1999-05-19 Yale University Polypeptides du type b. burgdorferi exprimes in vivo
DE19629543C2 (de) * 1996-07-22 1999-02-11 Immuno Ag Immunassay zum Nachweis von anti-B. burgdorferi Antikörpern und Verfahren zur Serodiagnose bei Lyme Borreliose, diagnostische Mittel und Testkits zur Durchführung der Verfahren
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