EP0915977A1 - Polypeptides du type b. burgdorferi exprimes in vivo - Google Patents

Polypeptides du type b. burgdorferi exprimes in vivo

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Publication number
EP0915977A1
EP0915977A1 EP96915650A EP96915650A EP0915977A1 EP 0915977 A1 EP0915977 A1 EP 0915977A1 EP 96915650 A EP96915650 A EP 96915650A EP 96915650 A EP96915650 A EP 96915650A EP 0915977 A1 EP0915977 A1 EP 0915977A1
Authority
EP
European Patent Office
Prior art keywords
burgdorferi
polypeptide
polypeptides
lys
antibodies
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Withdrawn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
EP96915650A
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German (de)
English (en)
Inventor
Erol Fikrig
Kyoungho Suk
Stephen W. Barthold
Richard A. Flavell
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Yale University
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Yale University
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Publication date
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Publication of EP0915977A1 publication Critical patent/EP0915977A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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

  • This invention relates to compositions and methods useful for the prevention, diagnosis and treatment of Lyme disease More particularly, this invention relates to novel B. burgdorferi polypeptides which are able to elicit in a
  • This invention also relates to novel B. burgdorferi polypeptides that are expressed during infection of a host but are not expressed by B. burgdorferi in in vitro culture
  • This invention also relates to multicomponent vaccines comprising one or more ofthe novel B. burgdorferi polypeptides Also within the scope of this
  • Lyme borreliosis is the most common vector-borne infection in the United States [S.W. Barthold, et al., "An Animal Model For Lyme Arthritis", Ann. N.Y Acad. Sci.. 539, pp. 264-73 (1988)] It has been reported in every continent except Antarctica The clinical hallmark of Lyme disease is an early expanding skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnormalities
  • the causative agent of Lyme disease is a spirochete known as Borreha burgdorferi, transmitted primarily by Ixodes ticks ofthe Ixodes acinus complex. B. burgdorferi has also been shown to be carried in other species of ticks and in mosquitoes and deer flies. But, it appears that only ticks ofthe / ricwus complex are able to transmit the disease to humans
  • Lyme disease generally occurs in three stages Stage one involves localized skin lesions (erythema migrans) from which the spirochete is cultured more readily than at any other time during infection [B W Berger et al , "Isolation And Characterization Of The Lyme Disease Spirochete From The Skin Of Patients With Erythema Chronicum Migrans", J. Am. Acad.
  • Stage two occurs within days or weeks, and involves spread ofthe spirochete through the patient's blood or lymph to many different sites in the body including the brain and joints Varied symptoms of this disseminated infection occur in the skin, nervous system, and musculoskeletal system, although they are typically intermittent Stage three, or late infection, is defined as persistent infection, and can be severely disabling.
  • sonicated whole cultured spirochetes are used as the antigen 5 in such assays to detect anti-5. burgdorferi antibodies formed in the serum of infected individuals [J E. Craft et al., "The Antibody Response In Lyme Disease. Evaluation Of Diagnostic Tests", J. Infect. Dis , 149, pp 789-95 (1984)].
  • false negative and, more commonly, false positive results are associated with currently available tests. 0 At present, all stages of Lyme disease are treated with antibiotics
  • Borrelia Like Treponema palhdum, which causes syphilis, and leptospirae, which cause an infectious jaundice, Borrelia belong to the eubacterial phylum of spirochetes [A G. Barbour and S F Hayes, "Biology Of Borrelia Species", o Microbiol. Rev., 50, pp. 381 -400 ( 1986)] Borrelia burgdorferi have a protoplasmic cylinder that is surrounded by a cell membrane, then by flagella, and then by an outer membrane.
  • the two major outer surface proteins are the 31 kDa outer-surface protein A (OspA) and the 34 kDa outer surface protein B (OspB). Both proteins have been shown to vary from 7/42325 PC17US96/06610
  • OspC is a 22 kDa membrane lipoprotein previously identified as pC [R Fuchs et al , "Molecular Analysis and Expression of a Borrelia burgdorferi Gene Encoding a 22 kDa Protein 5 (pC) in Escherichia coli", Mol. Microbiol.. 6, pp 503-09 (1992)] OspD is said to be preferentially expressed by low-passage, virulent strains of B.
  • OspE a 19 kD 0 protein
  • OspF a 26 kD protein
  • Non-Osp B. burgdorferi proteins identified to date include the 41 kDa flagellin protein, which is known to contain regions of homology with other 5 bacterial flagellins [G.S Gassman et al , "Analysis ofthe Borrelia burgdorferi
  • B. burgdorferi is known to alter the antigens on its outer surface during different stages of its life cycle
  • OspC is not expressed by spirochetes within unfed ticks. However, it is synthesized following engorgement and the introduction of a blood meal into the lumen ofthe tick's midgut
  • OspA is a prominent surface antigen on spirochetes within the midguts of resting ticks
  • OspA expression decreases The downregulation of OspA within ticks allows spirochetes to survive in the presence of an OspA antibody response, suggesting that selective antigen expression may be a mechanism by which B. burgdorferi evade immune destruction.
  • B. burgdorferi antigens that are expressed only within the vertebrate host may aid in the serologic diagnosis of Lyme disease
  • Such proteins are not present on spirochetes cultured in Barbour-Stoenner-Kelly (BSK) II medium
  • BNK Barbour-Stoenner-Kelly
  • Selective in vivo expression of some B. burgdorferi proteins may be one reason that current diagnostic tests for Lyme disease, based on whole- cell lysates of cultured B. burgdorferi, are unreliable Such tests cannot detect antibodies directed toward the in vivo expressed antigens Accordingly, there also exists a need to identify B. burgdorferi proteins that provide more reliable diagnostic tests for Lyme disease.
  • OspC has also been shown to have protective effects in a gerbil model of B. burgdorferi infection
  • the protection afforded by immunization with this protein appears to be only partial [V Preac-Mursic et al , "Active Immunization with pC Protein o ⁇ Borrelia burgdorferi Protects Gerbils against B. burgdorferi Infection", Infection. 20, pp 342-48 (1992)]
  • identification of additional B. 0 burgdorferi antigens may enable the development of more reliable diagnostic reagents which are useful in various stages of Lyme borreliosis
  • the present invention provides novel B. burgdorferi polypeptides which are substantially free of a B. burgdorferi spirochete or fragments thereof and, 5 thus, are useful in compositions and methods for the diagnosis, treatment and prevention of B. burgdorferi infection and Lyme disease
  • this invention provides P21 polypeptides and compositions and methods comprising those polypeptides
  • this invention provides K2 polypeptides and o compositions and methods comprising those polypeptides
  • this invention provides P35 polypeptides and compositions and methods comprising those polypeptides
  • this invention provides P37 polypeptides and compositions and methods comprising those polypeptides 5 In another embodiment, this invention provides M30 polypeptides and compositions and methods comprising those polypeptides In another embodiment, this invention provides V3 polypeptides and compositions and methods comprising those polypeptides
  • this invention provides Jl polypeptides compositions and methods comprising those polypeptides 5 In another embodiment, this invention provides J2 polypeptides compositions and methods comprising those polypeptides
  • compositions and methods of each ofthe o aforementioned embodiments are characterized by novel B. burgdorferi polypeptides which elicit in treated animals the formation of an immune response
  • this invention provides a multicomponent vaccine comprising one or more novel B. burgdorferi polypeptides of this invention in addition to one or more other immunogenic B. burgdorferi polypeptides Such a 5 vaccine is effective to confer broad protection against B. burgdorferi infection
  • this invention provides antibodies directed against the novel B. burgdorferi polypeptides of this invention, and compositions and methods comprising those antibodies
  • this invention provides diagnostic means 0 and methods characterized by one or more ofthe novel B. burgdorferi polypeptides, or antibodies directed against those polypeptides These means and methods are useful for the detection of Lyme disease and B. burgdorferi infection They are also useful in following the course of treatment against such infection In patients previously inoculated with the vaccines of this invention, the detection 5 means and methods disclosed herein are also useful for determining if booster inoculations are appropriate In yet another embodiment, this invention provides methods for the identification and isolation of additional B. burgdorferi polypeptides, as well as compositions and methods comprising such polypeptides.
  • this invention provides methods for identifying bacterial genes encoding an antigenic protein which is expressed during infection of a host but is not expressed during in vitro culture ofthe bacteria.
  • this invention provides DNA sequences that code for the novel B. burgdorferi polypeptides of this invention, recombinant DNA molecules that are characterized by those DNA sequences, unicellular hosts transformed with those DNA sequences and molecules, and methods of using those sequences, molecules and hosts to produce the novel B. burgdorferi polypeptides and multicomponent vaccines of this invention.
  • DNA sequences of this invention are also advantageously used in methods and means for the diagnosis of Lyme disease and B. burgdorferi infection.
  • Figure 1 depicts the DNA and amino acid sequences ofthe P21 polypeptide of B. burgdorferi strain N40.
  • Figure 2 depicts the DNA and amino acid sequences ofthe P35 polypeptide of B. burgdorferi strain N40.
  • Figure 3 depicts the DNA and amino acid sequences ofthe P37 polypeptide of B. burgdorferi strain N40.
  • Figure 4 depicts the DNA and amino acid sequences ofthe M30 polypeptide of B. burgdorferi strain N40.
  • Figure 5 depicts the DNA and amino acid sequences ofthe V3 polypeptide of B. burgdorferi strain N40.
  • Figure 6 depicts the hydrophilicity profiles of P35 and P37.
  • Figure 7 depicts a comparison ofthe amino acid sequences of P21 and B. burgdorferi strain N40 OspE
  • Figure 8 depicts a comparison ofthe control regions of transcription and translation among the DNA sequences encoding P21 and K2 and the DNA sequences of other known B. burgdorferi outer surface proteins
  • This invention relates to novel B. burgdorferi polypeptides, the DNA sequences which encode them, antibodies directed against those polypeptides, compositions comprising the polypeptides or antibodies, and methods for the detection, treatment and prevention of Lyme disease
  • this invention relates to P21 polypeptides and compositions and methods comprising those polypeptides
  • this invention relates to K2 polypeptides and compositions and methods comprising those polypeptides
  • this invention relates to P35 polypeptides and compositions and methods comprising those polypeptides
  • this invention relates to P37 polypeptides and compositions and methods comprising those polypeptides In another embodiment, this invention relates to M30 polypeptides and compositions and methods comprising those polypeptides
  • this invention relates to V3 polypeptides and compositions and methods comprising those polypeptides
  • this invention relates to Jl and compositions and methods comprising those polypeptides
  • this invention relates to J2 and compositions and methods comprising those polypeptides
  • the preferred polypeptides, compositions and methods of each of the aforementioned embodiments are characterized by novel B. burgdorferi polypeptides that are immunogenic B. burgdorferi polypeptides
  • this invention relates to a multicomponent 5 vaccine against Lyme disease comprising one or more ofthe novel B. burgdorferi polypeptides of this invention in addition to other immunogenic B. burgdorferi polypeptides Such vaccine is useful to protect against infection by a broad spectrum of B. burgdorferi organisms
  • All ofthe novel B. burgdorferi polypeptides provided by this i o invention, and the DNA sequences encoding them, may be produced substantially free of B. burgdorferi spirochete or fragments thereof, and thus may be used in a variety of applications without the risk of unintentional infection or contamination with undesired B. burgdorferi components Accordingly, the novel B. burgdorferi polypeptides of this invention are particularly advantageous in compositions and
  • this invention relates to compositions and methods comprising antibodies directed against the novel B. burgdorferi polypeptides of this invention
  • Such antibodies may be used in a variety of applications, including to detect the presence of B. burgdorferi, to screen for 0 expression of novel B. burgdorferi polypeptides, to purify novel B. burgdorferi polypeptides, to block or bind to the novel B. burgdorferi polypeptides, to direct molecules to the surface of B. burgdorferi, to prevent or lessen the severity, for some period of time, of B. burgdorferi infection, and to decrease the level of B.
  • this invention relates to diagnostic means and methods characterized by the novel B. burgdorferi polypeptides disclosed herein or antibodies directed against those polypeptides. In yet another embodiment, this invention relates to methods for identifying bacterial genes that are selectively expressed in vivo
  • an "immunogenic B. burgdorferi polypeptide” is any amino acid sequence having the following properties listed herein.
  • B. burgdorferi polypeptide that, when administered to an animal, is capable of eliciting an immune response
  • Immunogenic B. burgdorferi polypeptides are intended to include not only the novel B. burgdorferi polypeptides of this invention but also the OspA and OspB polypeptides disclosed in PCT patent application WO 92/00055, the OspC protein as described in R Fuchs et al , supra, the OspE and OspF polypeptides disclosed in PCT patent application WO 95/04145, other B. burgdorferi proteins, and fragments, serotypic variants and derivatives of any of the above
  • immunogenic B. burgdorferi polypeptides are intended to include additional B. burgdorferi polypeptides which are identified according to the methods disclosed herein
  • a polypeptide which is "substantially free of a B. burgdorferi spirochete or fragments thereof is a polypeptide that, when introduced into modified Barbour-Stoener-Kelly (BSK-II) medium and cultured at 37°C for 7 days, fails to produce any B. burgdorferi spirochetes detectable by dark field microscopy or a polypeptide that is detectable as a single band on an immunoblot probed with polyclonal anti-_9. burgdorferi anti-serum
  • a B. burgdorferi polypeptide that is "selectively expressed in vivo" is a polypeptide encoded by a DNA sequence that corresponds to a B. burgdorferi gene that is expressed during infection of a host but is not expressed du ⁇ ng in vitro culture of said B. burgdorferi
  • a DNA sequence that "corresponds to a B. burgdorferi gene” is a DNA sequence that encodes a polypeptide that is the same as, a fragment of or a derivative of a naturally occurring B. burgdorferi polypeptide
  • P21 polypeptide denotes a polypeptide which is selected from the group consisting of (a) a P21 polypeptide consisting of amino acids 1-182 of SEQ ID NO 2,
  • (1) is a derivative of a P21 polypeptide of (a), said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a),
  • polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi which antibodies are immunologically reactive with a P21 polypeptide of (a)
  • polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the P21 polypeptide of (a) and (4) polypeptides that are immunologically reactive with antibodies elicited by immunization with the P21 polypeptide of (a)
  • K2 polypeptide denotes a polypeptide which is selected from the group consisting of
  • (1) is a derivative of a polyeptide of (a), said derivative being at least 80% identical in ammo acid sequence to the corresponding polypeptide of (a), (2) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a polypeptide of (a),
  • a "P35 polypeptide” denotes a polypeptide which is selected from the group consisting of. (a) a P35 protein comprising the amino acid sequence set forth in SEQ ID NO: 1
  • polypeptides that are immunologically reactive with antibodies elicited by immunization with the P35 polypeptide of (a) or (b) or (c).
  • a "P37 polypeptide” denotes a polypeptide which is selected from the group consisting of:
  • polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with 5. burgdorferi, which antibodies are immunologically reactive with a P37 polypeptide of (a) or (b) or (c),
  • polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the P37 polypeptide of (a) or (b) or (c), and
  • polypeptides that are immunologically reactive with antibodies elicited by immunization with the P35 polypeptide of (a) or (b) or (c)
  • M30 polypeptide denotes a polypeptide which is selected from the group consisting of
  • polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a M30 polypeptide of (a) or (b) or (c),
  • V3 polypeptide denotes a polypeptide which is selected from the group consisting of (a) a V3 protein having an amino acid sequence encoded by SEQ ID NO 10 and serotypic variants thereof,
  • polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi which antibodies are immunologically reactive with a polypeptide of (a) or (b) or (c)
  • polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the polypeptide of (a) or (b) or (c) and
  • V3 polypeptide is intended to include a B. burgdorferi polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained in ATCC deposit No _, which cross-hybridizes to the DNA sequence of SEQ ID NO. 10
  • J polypeptide denotes a polypeptide which is selected from the group consisting of
  • polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a polypeptide of (a) or (b) or (c),
  • polypeptides that are capable of eliciting antibodies that are o immunologically reactive with B. burgdorferi and the polypeptide of (a) or (b) or
  • polypeptides that are immunologically reactive with antibodies elicited by immunization with the polypeptide of (a) or (b) or (c)
  • Jl polypeptide is intended to include a B. 5 burgdorferi polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit No (2A). which cross-hybridizes to the B. burgdorferi DNA sequence contained within ATCC deposit No __
  • J2 polypeptide denotes a polypeptide which is selected from the group consisting of: 0 (a) a polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit No. (3) and serotypic variants thereof,
  • polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with B. burgdorferi, which antibodies are immunologically reactive with a polypeptide of (a) or (b) or (c);
  • polypeptides that are capable of eliciting antibodies that are immunologically reactive with B. burgdorferi and the polypeptide of (a) or (b) or (c), and
  • polypeptides that are immunologically reactive with antibodies elicited by immunization with the polypeptide of (a) or (b) or (c)
  • J2 polypeptide is intended to include a B. burgdorferi polypeptide encoded in whole or in part by the B. burgdorferi DNA sequence contained within ATCC deposit Nos C3A and 3B). which cross-hybridize to the B. burgdorferi DNA sequence contained within ATCC deposit No ⁇ 3 ⁇
  • a "novel B. burgdorferi polypeptide” is a P21 polypeptide, a K2 polypeptide, a P35 polypeptide, a P37 polypeptide, an M30 polypeptide, a V3 polypeptide, a Jl polypeptide or a J2 polypeptide
  • a "serotypic variant" of a novel B. burgdorferi polypeptide according to this invention is any naturally occurring polypeptide which may be encoded in whole or in part, by a DNA sequence which hybridizes, at 20- 27 °C below Tm, to the DNA sequence encoding the novel B. burgdorferi polypeptide
  • serotypic variants of a novel B. burgdorferi polypeptide according to this invention include polypeptides encoded by DNA sequences of which any portion may be amplified by using the polymerase chain reaction and oligonucleotide primers derived from any portion of the DNA sequence encoding the novel B.
  • a "derivative" of a novel B. burgdorferi polypeptide according to his invention is a novel B. burgdorferi polypeptide in which one or more physical, chemical, or biological properties has been altered
  • modifications include, but are not limited to amino acid substitutions, modifications, additions or deletions; alterations in the pattern of lipidation, glycosylation or phosphorylation; reactions of free amino, carboxyl, or hydroxyl side groups ofthe amino acid residues present in the polypeptide with other organic and non-organic molecules; and other modifications, any of which may result in 5 changes in primary, secondary or tertiary structure
  • a “protective antibody” is an antibody that confers protection, for some period of time, against any one ofthe physiological disorders associated with B. burgdorferi infection.
  • a "protective B. burgdorferi polypeptide” is a l o polypeptide that comprises a protective epitope
  • a "protective epitope” is (1) an epitope which is recognized by a protective antibody, and/or (2) an epitope which, when used to immunize an animal, elicits an immune response sufficient to prevent or lessen the severity for some period of time, of B. burgdorferi infection
  • Preventing or lessening the severity of infection may be evidenced by a change in the physiological manifestations of erythema migrans, arthritis, carditis, neurological disorders, and other Lyme disease related disorders It may be evidenced by a decrease in the level of spirochetes in the treated animal And, it may also be evidenced by a decrease in the level of spirochetes in infected ticks
  • a protective epitope may comprise a T cell epitope, a B cell epitope, or combinations thereof.
  • a "T cell epitope” is an epitope which, when presented to T cells by antigen presenting cells, results in a T cell response such as clonal expansion or expression of lymphokines or other immunostimulatory 5 molecules.
  • a T cell epitope may also be an epitope recognized by cytotoxic T cells that may affect intracellular B. burgdorferi infection
  • a strong T cell epitope is a T cell epitope which elicits a strong T cell response
  • a "B cell epitope” is the simplest spatial conformation of an antigen which reacts with a specific antibody.
  • a "therapeutically effective amount" of a polypeptide or of an antibody is the amount that, when administered to an animal, elicits an immune response that is effective to prevent or lessen the severity, for some period of time, of B. burgdorferi infection.
  • an "antibody directed against a novel B. burgdorferi polypeptide” is an antibody directed against a P21 polypeptide, a K2 polypeptide, a P35 polypeptide, a P37 polypeptide, an M30 polypeptide, a V3 polypeptide, a Jl polypeptide or a J2 polypeptide It should be understood that an antibody directed against a novel B. burgdorferi polypeptide may also be a protective antibody
  • An antibody directed against a novel B. burgdorferi polypeptide may be an intact immunoglobulin molecule or a portion of an immunoglobulin molecule that contains an intact antigen binding site, including those portions known in the art as F(v), Fab, Fab' and F(ab')2 It may also be a genetically engineered or synthetically produced molecule.
  • novel B. burgdorferi polypeptides disclosed herein are immunologically reactive with antisera generated by infection of a mammalian host with B. burgdorferi. Accordingly, they are useful in methods and compositions to diagnose and protect against Lyme disease, and in therapeutic compositions to stimulate immunological clearance of B. burgdorferi during ongoing infection.
  • novel B. burgdorferi polypeptides disclosed herein are immunogenic surface proteins of B. burgdorferi, they are particularly useful in a multicomponent vaccine against Lyme disease, because such a vaccine may be formulated to more closely resemble the immunogens presented by replication-competent B.
  • Multicomponent vaccines according to this invention may also contain polypeptides which characterize other vaccines useful for immunization against diseases other than Lyme disease such as, for example, diphtheria, polio, hepatitis, and measles. Such multicomponent vaccines are typically inco ⁇ orated 5 into a single composition.
  • compositions and methods of this invention comprise novel B. burgdorferi polypeptides having enhanced immunogenicity.
  • Such polypeptides may result when the native forms ofthe polypeptides or fragments thereof are modified or subjected to treatments to enhance their immunogenic o character in the intended recipient.
  • the polypeptides may be modified by coupling to 5 dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS.
  • the polypeptides are small polypeptides synthesized chemically, it may be desirable to couple them to an immunogenic carrier The coupling of course, must not interfere with the ability of either the polypeptide or the carrier to function appropriately.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • PPD purified protein derivative of tuberculin
  • red blood cells tetanus toxoid, cholera toxoid
  • agarose 5 beads activated carbon, or bentonite
  • Modification ofthe amino acid sequence ofthe novel B. burgdorferi polypeptides disclosed herein in order to alter the lipidation state is also a method which may be used to increase their immunogenicity and biochemical properties.
  • the polypeptides or fragments thereof may be expressed with or without the signal sequences that direct addition of lipid moieties.
  • polypeptides may also be prepared with the objective of increasing stability or rendering the molecules more amenable to purification and preparation.
  • One such technique is to express the polypeptides as fusion proteins comprising other B. burgdorferi or non- B. burgdorferi sequences.
  • derivatives ofthe novel B. burgdorferi polypeptides may be prepared by a variety of methods, including by in vitro manipulation ofthe DNA encoding the native polypeptides and subsequent expression ofthe modified DNA, by chemical synthesis of derivatized DNA sequences, or by chemical or biological manipulation of expressed amino acid sequences.
  • derivatives may be produced by substitution of one or more amino acids with a different natural amino acid, an amino acid derivative or non-native amino acid, conservative substitution being preferred, e.g., 3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline, 5-hydroxylysine may be substituted for lysine, and the like Causing amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties.
  • substitutions would include for example, substitution of a hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge.
  • the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence ofthe desired characteristics
  • the novel B. burgdorferi polypeptides disclosed herein are prepared as part of a larger fusion protein For example, a novel B.
  • burgdorferi polypeptide of this invention may be fused at its N-terminus or C-terminus to a different immunogenic B. burgdorferi polypeptide, to a non-5, burgdorferi polypeptide or to combinations thereof, to produce fusion proteins comprising the novel B. burgdorferi polypeptide
  • fusion proteins comprising novel B. burgdorferi polypeptides are constructed comprising B cell and/or T cell epitopes from multiple serotypic variants of B. burgdorferi, each variant differing from another with respect to the locations or sequences ofthe epitopes within the polypeptide
  • fusion proteins are constructed which comprise one or more ofthe novel B. burgdorferi polypeptides fused to other immunogenic B. burgdorferi polypeptides
  • Such fusion proteins are particularly effective in the prevention, treatment and diagnosis of Lyme disease as caused by a wide spectrum of B. burgdorferi isolates
  • the novel B. burgdorferi polypeptides are fused to moieties, such as immunoglobulin domains, which may increase the stability and prolong the in vivo plasma half-life ofthe polypeptide
  • moieties such as immunoglobulin domains
  • Such fusions may be prepared without undue experimentation according to methods well known to those of skill in the art, for example, in accordance with the teachings of United States patent 4,946,778, or United States patent 5, 116,964
  • the exact site ofthe fusion is not critical as long as the polypeptide retains the desired biological activity Such determinations may be made according to the teachings herein or by other methods known to those of skill in the art.
  • the fusion proteins comprising the novel B. burgdorferi polypeptides be produced at the DNA level, e g , by constructing a nucleic acid molecule encoding the fusion, transforming host cells with the molecule, inducing the cells to express the fusion protein, and recovering the fusion protein from the cell culture
  • the fusion proteins may be produced after gene expression according to known methods
  • novel B. burgdorferi polypeptides may also be part of larger 5 multimeric molecules which may be produced recombinantly or may be synthesized chemically Such multimers may also include the polypeptides fused or coupled to moieties other than amino acids, including lipids and carbohydrates
  • the multimeric proteins will consist of multiple T or B cell epitopes or combinations thereof repeated within the same molecule, either o randomly, or with spacers (amino acid or otherwise) between them
  • novel B burgdorferi polypeptides of this invention which are also immunogenic B. burgdorferi polypeptides are incorporated into a multicomponent vaccine which also comprises other immunogenic B. burgdorferi polypeptides
  • a multicomponent vaccine which also comprises other immunogenic B. burgdorferi polypeptides
  • Such a 5 multicomponent vaccine by virtue of its ability to elicit antibodies to a variety of immunogenic B. burgdorferi polypeptides, will be effective to protect against Lyme disease as caused by a broad spectrum of different B burgdorferi isolates, even those that may not express one or more ofthe Osp proteins
  • the multicomponent vaccine may contain the novel B. burgdorferi o polypeptides as part of a multimeric molecule in which the various components are covalently associated. Alternatively, it may contain multiple individual components
  • a multicomponent vaccine may be prepared comprising two or more ofthe novel B. burgdorferi polypeptides, or comprising one novel B burgdorferi polypeptide and one previously identified B. burgdorferi polypeptide, wherein each 5 polypeptide is expressed and purified from independent cell cultures and the polypeptides are combined prior to or during formulation
  • a multicomponent vaccine may be prepared from heterodimers or tetramers wherein the polypeptides have been fused to immunoglobulin chains or portions thereof.
  • a vaccine could comprise, for example, a P35 polypeptide fused to an immunoglobulin heavy chain and an OspA polypeptide fused to an immunoglobulin light chain, and could be produced by transforming a host cell with DNA encoding the heavy chain fusion and DNA encoding the light chain fusion.
  • the host cell selected should be capable of assembling the two chains appropriately
  • the heavy and light chain fusions could be produced from separate cell lines and allowed to associate after purification
  • the multicomponent vaccine will comprise numerous T cell and B cell epitopes of immunogenic B. burgdorferi polypeptides, including the novel B. burgdorferi polypeptides of this invention.
  • This invention also contemplates that the novel B. burgdorferi polypeptides of this invention, either alone or with other immunogenic B. burgdorferi polypeptides, may be administered to an animal via a liposome delivery system in order to enhance their stability and/or immunogenicity Delivery ofthe novel B.
  • burgdorferi polypeptides via liposomes may be particularly advantageous because the liposome may be internalized by phagocytic cells in the treated animal Such cells, upon ingesting the liposome, would digest the liposomal membrane and subsequently present the polypeptides to the immune system in conjunction with other molecules required to elicit a strong immune response
  • the liposome system may be any variety of unilamellar vesicles, multilamellar vesicles, or stable plurilamellar vesicles, and may be prepared and administered according to methods well known to those of skill in the art, for example in accordance with the teachings of United States patents 5,169,637, 4,762,915, 5,000,958 or 5,185,154.
  • it may be desirable to express the novel B. burgdorferi polypeptides of this invention, as well as other selected B. burgdorferi polypeptides, as lipoproteins, in order to enhance their binding to liposomes.
  • Any ofthe novel B. burgdorferi polypeptides of this invention may be used in the form of a pharmaceutically acceptable salt.
  • Suitable acids and bases which are capable of forming salts with the polypeptides ofthe present invention are well known to those of skill in the art, and include inorganic and organic acids and bases
  • a method which comprises the steps of treating an animal with a therapeutically effective amount of a novel B. burgdorferi polypeptide, or a fusion protein or a multimeric protein comprising a novel B. burgdorferi polypeptide, in a manner sufficient to prevent or lessen the severity, for some period of time, of B. burgdorferi infection
  • the polypeptides that are preferred for use in such methods are those that contain protective epitopes.
  • Such protective epitopes may be B cell epitopes, T cell epitopes, or combinations thereof.
  • a method which comprises the steps of treating an animal with a multicomponent vaccine comprising a therapeutically effective amount of a novel B. burgdorferi polypeptide, or a fusion protein or multimeric protein comprising such polypeptide in a manner sufficient to prevent or lessen the severity, for some period of time, of B. burgdorferi infection
  • the polypeptides, fusion proteins and multimeric proteins that are preferred for use in such methods are those that contain protective epitopes, which may be B cell epitopes, T cell epitopes, or combinations thereof
  • the most preferred polypeptides, fusion proteins and multimeric proteins for use in these compositions and methods are those containing both strong T cell and B cell epitopes.
  • B cells expressing surface immunoglobulin that recognizes the B cell epitope(s).
  • the B cells will then process the antigen and present it to T cells.
  • the T cells will recognize the T cell epitope(s) and respond by proliferating and producing lymphokines which in turn cause B cells to differentiate into antibody producing plasma cells.
  • a closed autocatalytic circuit exists which will result in the amplification of both B and T cell responses, leading ultimately to production of a strong immune response which includes high titer antibodies against the novel B. burgdorferi polypeptide.
  • T H 2 T-helper cells type 2
  • T H 2 T-helper cells type 2
  • the induction of T H 2 cells may also be favored by the mode of administration ofthe polypeptide for example by administering in certain doses or with particular adjuvants and immunomodulators, for example with interleukin-4.
  • overlapping fragments ofthe novel B. burgdorferi polypeptides of this invention are constructed.
  • the polypeptides that contain B cell epitopes may be identified in a variety of ways for example by their ability to (1 ) remove protective antibodies from polyclonal antiserum directed against the polypeptide or (2) elicit an immune response which is effective to prevent or lessen the severity of B. burgdorferi infection
  • the polypeptides may be used to produce monoclonal antibodies which are screened for their ability to confer protection against B. burgdorferi infection when used to immunize naive animals Once a given monoclonal antibody is found to confer protection, the particular epitope that is recognized by that antibody may then be identified As recognition of T cell epitopes is MHC restricted, the polypeptides that contain T cell epitopes may be identified in vitro by testing them for their ability to stimulate proliferation and/or cytokine production by T cell clones generated from humans of various HLA types, from the lymph nodes, spleens, or peripheral blood lymphocytes of C3H/He mice, or from domestic animals Compositions comprising multiple T cell epitopes recognized by individuals with different Class II antigens are useful for prevention and treatment of Lyme disease in a broad spectrum of patients
  • a novel B. burgdorferi polypeptide containing a B cell epitope is fused to one or more other immunogenic B. burgdorferi polypeptides containing strong T cell epitopes
  • the fusion protein that carries both strong T cell and B cell epitopes is able to participate in elicitation of a high titer antibody response effective to neutralize infection with B.
  • T cell epitopes may also be provided by non-5, burgdorferi molecules
  • strong T cell epitopes have been observed in hepatitis B virus core antigen (HBcAg)
  • HBcAg hepatitis B virus core antigen
  • linkage of one of these segments to segments ofthe surface antigen of Hepatitis B virus, which are poorly recognized by T cells results in a major amplification ofthe anti-HBV surface antigen response, [D R Milich et al , "Antibody Production To The Nucleocapsid And Envelope Of The Hepatitis B Virus Primed By A Single Synthetic T Cell Site", Nature. 329, pp 547-49 (1987)]
  • B cell epitopes of the novel B. burgdorferi polypeptides are fused to segments of HBcAG or to other antigens which contain strong T cell epitopes, to produce a fusion protein that can elicit a high titer antibody response against B. burgdorferi
  • novel B. burgdorferi polypeptides of this invention may be prepared by recombinant means, chemical means, or combinations thereof
  • polypeptides may be generated by recombinant means using the DNA sequences of B. burgdorferi strain N40 as set forth in the sequence listings contained herein
  • DNA encoding serotypic variants ofthe polypeptides may likewise be cloned, e g , using PCR and oligonucleotide primers derived from the sequences herein disclosed
  • the genes encoding novel B. burgdorferi polypeptides from strain 25015 and other strains of B. burgdorferi that are known to differ antigenically from strain N40, in order to obtain a broad spectrum of different epitopes which would be useful in the methods and compositions of this invention
  • the OspA gene of B. burgdorferi strain 25015 is known to differ from the OspA gene of B. burgdorferi strain N40 to the extent that anti-OspA antibodies, which protect against subsequent infection with strain N40, appear ineffective to protect against infection with strain 25015
  • burgdorferi polypeptides may also be used to isolate and clone other related surface proteins from B. burgdorferi and related spirochetes which may contain regions of DNA sequence homologous to the DNA sequences of this invention.
  • the DNA sequences of this invention may also be used in PCR reactions to detect the presence of B. burgdorferi in a suspected infected sample
  • novel B. burgdorferi polypeptides of this invention may be expressed in unicellular hosts
  • the sequences are generally operatively linked to transcriptional and translational expression control sequences that are functional in the chosen host.
  • the expression control sequences, and the gene of 5 interest will be contained in an expression vector that further comprises a selection marker
  • the DNA sequences encoding the polypeptides of this invention may or may not encode a signal sequence If the expression host is eukaryotic, it generally is preferred that a signal sequence be encoded so that the mature protein is o secreted from the eukaryotic host
  • an amino terminal methionine may or may not be present on the expressed polypeptides of this invention If the terminal methionine is not cleaved by the expression host, it may, if desired, be chemically removed by standard techniques 5
  • a wide variety of expression host/vector combinations may be employed in expressing the DNA sequences of this invention
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, adeno- associated virus, cytomegalovirus and retroviruses including lentiviruses
  • Useful 0 expression vectors for bacterial hosts include bacterial plasmids, such as those from E.
  • plasmids such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g ⁇ GTIO and ⁇ GTl 1, and other phages
  • Useful expression vectors for yeast cells include the 2 ⁇ 5 plasmid and derivatives thereof
  • Useful vectors for insect cells include pVL 941
  • any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence when operatively linked to it — may be used in these vectors to express the DNA sequences of this invention
  • useful expression control sequences include the expression control sequences associated with structural genes ofthe foregoing expression vectors
  • useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the tq * » system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast ⁇ -mating system and other constitutive and inducible promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various
  • DNA sequences encoding the novel B. burgdorferi polypeptides of this invention are cloned in the expression vector lambda ZAP II (Stratagene, La Jolla, CA), in which expression from the lac promoter may be induced by IPTG.
  • DNA encoding the novel B. burgdorferi polypeptides of this invention is inserted in frame into an expression vector that allows high level expression ofthe polypeptide as a glutathione S- transferase fusion protein.
  • a fusion protein thus contains amino acids encoded by the vector sequences as well as amino acids ofthe novel B. burgdorferi polypeptide
  • a wide variety of unicellular host cells are useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant cells
  • SF9 Spodoptera frugiperda
  • animal cells such as CHO and mouse cells
  • African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10
  • human cells as well as plant cells
  • Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity ofthe product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the polypeptide correctly, their fermentation or culture requirements, and the ease of purification from them ofthe products coded for by the DNA sequences of this invention
  • the molecules comprising the novel B. burgdorferi polypeptides encoded by the DNA sequences of this invention may be isolated from the
  • novel B. burgdorferi polypeptides may be generated by any of several chemical techniques
  • they may be prepared using the solid-phase synthetic technique originally described by R B Merrifield, "Solid Phase Peptide Synthesis I The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc. 83, pp 2149-54 (1963), or they may be prepared by synthesis in solution
  • a summary of peptide synthesis techniques may be found in E Gross & H J Meinhofer, 4 The Peptides Analysis, Synthesis, Biology, Modern Techniques Of Peptide And Amino Acid Analysis, John Wiley & Sons, (1981) and M Bodanszky, Principles Of Peptide Synthesis, Springer- Verlag (1984)
  • antibodies directed against the novel B. burgdorferi polypeptides are generated Such antibodies are immunoglobulin molecules or portions thereof that are immunologically reactive with a novel B. burgdorferi polypeptide ofthe present invention It should be understood that the antibodies of this invention include antibodies immunologically reactive with fusion proteins and multimeric proteins comprising a novel B. burgdorferi polypeptide.
  • Antibodies directed against a novel B. burgdorferi polypeptide may be generated by a variety of means including infection of a mammalian host with B. burgdorferi, or by immunization of a mammalian host with a novel B. burgdorferi polypeptide ofthe present invention.
  • Such antibodies may be polyclonal or monoclonal, it is preferred that they are monoclonal Methods to produce polyclonal and monoclonal antibodies are well known to those of skill in the art. For a review of such methods, set Antibodies, A Laboratory Manual, supra, and D.E. Yelton, et al., Ann. Rev, of Biochem . 50, pp 657-80 (1981)
  • Determination of immunoreactivity with a novel B. burgdorferi polypeptide of this invention may be made by any of several methods well known in the art, including by immunoblot assay and ELISA
  • An antibody of this invention may also be a hybrid molecule formed from immunoglobulin sequences from different species (e g , mouse and human) or from portions of immunoglobulin light and heavy chain sequences from the same species. It may be a molecule that has multiple binding specificities, such as a bifunctional antibody prepared by any one of a number of techniques known to those of skill in the art including the production of hybrid hybridomas; disulfide exchange, chemical cross-linking, addition of peptide linkers between two monoclonal antibodies; the introduction of two sets of immunoglobulin heavy and light chains into a particular cell line, and so forth.
  • the antibodies of this invention may also be human monoclonal antibodies produced by any ofthe several methods known in the art
  • human monoclonal antibodies may produced by immortalized human cells, by SCID-hu mice or other non-human animals capable of producing "human” antibodies, by the expression of cloned human immunoglobulin genes, by phage- display, or by any other method known in the art.
  • the antibodies of this invention may be advantageous to couple to toxins such as diphtheria, pseudomonas exotoxin, ricin A chain, gelonin, etc., or antibiotics such as penicillins, tetracyclines and chloramphenicol
  • toxins such as diphtheria, pseudomonas exotoxin, ricin A chain, gelonin, etc.
  • antibiotics such as penicillins, tetracyclines and chloramphenicol
  • burgdorferi polypeptide may have utility in therapeutic and prophylactic compositions and methods directed against Lyme disease and B. burgdorferi infection
  • the level of B. burgdorferi in infected ticks may be decreased by allowing them to feed on the blood of animals immunized with the novel B. burgdorferi polypeptides of this invention
  • the antibodies of this invention also have a variety of other uses For example, they are useful as reagents to screen for expression ofthe B. burgdorferi polypeptides, either in libraries constructed from B. burgdorferi DNA or from other samples in which the proteins may be present Moreover, by virtue of their specific binding affinities, the antibodies of this invention are also useful to purify or remove polypeptides from a given sample, to block or bind to specific epitopes on the polypeptides and to direct various molecules, such as toxins, to the surface of B. burgdorferi
  • C3H/He mice are preferred as an animal model
  • any animal that is susceptible to infection with B. burgdorferi may be useful
  • C3H/He mice are not only susceptible to B. burgdorferi infection but are also afflicted with clinical symptoms of a disease that is remarkably similar to Lyme disease in humans.
  • a particular polypeptide or antibody to C3H/He mice one of skill in the art may determine without undue experimentation whether that polypeptide or antibody would be useful in the methods and compositions claimed herein
  • the administration ofthe novel B. burgdorferi polypeptide or antibody of this invention to the animal may be accomplished by any ofthe methods disclosed herein or by a variety of other standard procedures For a detailed discussion of such techniques, see Antibodies, A Laboratory Manual, supra
  • a polypeptide if a polypeptide is used, it will be administered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes)
  • adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components ofthe immune system
  • the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.
  • novel B. burgdorferi polypeptides or antibodies of this invention may then be used in a therapeutically effective amount in pharmaceutical compositions and methods to treat or prevent Lyme disease which may occur naturally in various animals
  • compositions of this invention may be in a variety of conventional depot forms These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusible solutions
  • solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusible solutions
  • the preferred form depends upon the intended mode of administration and prophylactic application.
  • Such dosage forms may include pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.
  • carriers and adjuvants include, for example, RIBI, ISCOM, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol
  • Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols
  • the vaccines and compositions of this invention
  • compositions comprising an antibody of this invention may be administered by a variety of dosage forms and regimens similar to those used for other passive immunotherapies and well known to those of skill in the art
  • novel B. burgdorferi polypeptides may be formulated and administered to the patient using methods and compositions similar to those employed for other pharmaceutically important polypeptides (e.g , the vaccine against hepatitis)
  • Any pharmaceutically acceptable dosage route including parenteral, intravenous, intramuscular, intralesional or subcutaneous injection, may be used to administer the polypeptide or antibody composition
  • the composition may be administered to the patient in any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, intramuscularly ⁇ including paravertebrally and periarticularly — subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intralesionally, periostally or by oral or topical
  • novel B. burgdorferi polypeptides or antibodies of this invention may be administered to the patient at one time or over a series of treatments
  • the most effective mode of administration and dosage regimen will depend upon the level of immunogenicity, the particular composition and/or adjuvant used for treatment, the severity and course ofthe expected infection, previous therapy, the patient's health status and response to immunization, and the judgment ofthe treating physician. For example, in an immunocompetent patient, the more highly immunogenic the polypeptide, the lower the dosage and necessary number of immunizations.
  • the dosage and necessary treatment time will be lowered if the polypeptide is administered with an adjuvant Generally, the dosage will consist of 10 ⁇ g to 100 mg ofthe purified polypeptide, and preferably, the dosage will consist of 10-1000 ⁇ g Generally, the dosage for an antibody will be 0 5 mg-3.0 g.
  • the novel B. burgdorferi polypeptide is administered with an adjuvant, in order to increase its immunogenicity
  • adjuvants include RIBI, and ISCOM, simple metal salts such as aluminum hydroxide, and oil based adjuvants such as complete and incomplete Freund's adjuvant
  • the polypeptide usually is administered in an emulsion with the adjuvant
  • E.coli expressing proteins comprising a novel B. burgdorferi polypeptide are administered orally to non- human animals to decrease or lessen the severity of B burgdorferi infection
  • a palatable regimen of bacteria expressing a novel B. burgdorferi polypeptide, alone or in the form of a fusion protein or multimeric protein may be administered with animal food to be consumed by wild mice or deer, or by domestic 5 animals Ingestion of such bacteria may induce an immune response comprising both humoral and cell-mediated components See J C Sadoff et al , "Oral Salmonella Typhimurium Vaccine Expressing Circumsporozoite Protein Protects against Malaria", Science.
  • the antibodies of this 0 invention as well as the novel B. burgdorferi polypeptides of this invention, and the DNA sequences encoding them are useful as diagnostic agents for detecting infection with B. burgdorferi, because the polypeptides are capable of binding to antibody molecules produced in animals, including humans that are infected with B. burgdorferi, and the antibodies are capable of binding to B. burgdorferi or 5 antigens thereof
  • Such diagnostic agents may be included in a kit which may also comprise instructions for use and other appropriate reagents, preferably a means for detecting when the polypeptide or antibody is bound
  • the polypeptide or antibody may be labeled with a detection means that allows for the detection of the polypeptide when it is bound to an antibody, or for the detection ofthe antibody when it is bound to B. burgdorferi or an antigen thereof.
  • the detection means may be a fluorescent labeling agent such as 5 fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and the like, an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like, a radioactive element such as I or Cr that produces gamma ray emissions, or a radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as C, O, or N o Binding may also be detected by other methods, for example via avidin-biotin complexes.
  • FIC fluorescein isocyanate
  • FITC fluorescein isothiocyanate
  • an enzyme such as horseradish peroxidase (HRP), glucose oxidase or the like
  • HRP horseradish peroxidase
  • I or Cr glucose oxidase
  • a radioactive element such as I or Cr that produces gamma
  • monoclonal antibody molecules produced by a hybridoma can be metabolically labeled by incorporation of radioisotope-containing amino acids in the 5 culture medium, or polypeptides may be conjugated or coupled to a detection means through activated functional groups.
  • the diagnostic kits ofthe present invention may be used to detect the presence of a quantity of B. burgdorferi or anti-5. burgdorferi antibodies in a body fluid sample such as serum, plasma or urine
  • a novel B. burgdorferi polypeptide or an antibody of the present invention is bound to a solid support typically by adsorption from an aqueous medium
  • Useful solid matrices are well known in the art, and include crosslinked dextran, agarose, polystyrene, polyvinylchloride, cross-linked polyacrylamide; nitrocellulose or nylon- based materials, tubes, plates or the wells of microtiter plates
  • the polypeptides or 5 antibodies of the present invention may be used as diagnostic agents in solution form or as a substantially dry powder, e g., in lyophilized form
  • Novel B. burgdorferi polypeptides and antibodies directed against those polypeptides provide much more specific diagnostic reagents than whole B. burgdorferi and thus may alleviate such pitfalls as false positive and false negative results.
  • novel B. burgdorferi polypeptides of this invention that are selectively expressed in the infected host and not in cultured B. burgdorferi, and antibodies directed against the polypeptides, allow detection of antigens and antibodies in samples that are undetectable by diagnostic methods using lysates of cultured spirochetes as the antigen
  • polypeptides and antibodies ofthe present invention may also be useful for detection, prevention, and treatment of other infections caused by spirochetes which may contain surface proteins sharing amino acid sequence or conformational similarities with the novel B. burgdorferi polypeptides ofthe present invention
  • spirochetes include Borrelia Hermsu and Borrelia Recunentis, Leptospira, and Treponema
  • Antisera may be generated by any ofthe wide variety of techniques that are well known to those of skill in the art
  • bacteria include any pathogenic or non-pathogenic bacteria that are capable of proliferating in a host
  • the bacteria are pathogenic bacteria
  • a host is any living organism that may be infected by bacteria, including plant and animal hosts. In a preferred embodiment, the host is a mammal
  • non-viable bacteria are bacteria that are incapable of synthesizing proteins
  • the bacteria are heat-killed bacteria
  • the bacteria may be rendered non-viable by any method known in the art
  • components of non-viable bacteria include lysates, homogenates, or subcellular fractions thereof such as cell membrane containing fractions.
  • binding ofthe antisera is detected with a secondary antibody coupled to a detection means.
  • detection means any ofthe wide variety of detection means known in the art is useful Examples of useful detection means are set forth supra In order that this invention may be better understood, the following examples are set forth These examples are for purposes of illustration only, and are not to be construed as limiting the scope ofthe invention in any manner
  • E. coli SURE bacteria (Stratagene) for phage infection as follows
  • mice We prepared "immune" mouse anti-Z?. burgdorferi N40 antiserum as follows
  • B. burgdorferi N40 in complete Freund's adjuvant (CFA) We were unable to infect mice with the heat inactivated preparation or to culture spirochetes from the preparation placed in BSKII medium, thus confirming that all ofthe heat-inactivated spirochetes were killed
  • T3 and T7 universal primers to obtain an intial sequence of the plasmid From that initial sequence of 100-300 bp, we made new primers which used to extend the sequence 100-300 bp at a time until we obtained the entire sequence
  • the p21 gene at the 5' end ofthe operon, contains a 546 nucleotide open reading frame capable of encoding a 182- amino acid protein (SEQ ID NO 2)
  • SEQ ID NO 2 The deduced amino acid sequence of P21 contains a typical prokaryotic signal sequence for posttranslational processing by cleavage and lipidation, suggesting that the gene product is a lipoprotein of approximately 20.7 kDa.
  • P21 has 71% amino acid sequence identity to B. burgdorferi OspE ( Figure 7)
  • a K2 polypeptide is a polypeptide that comprises the 1 l-amino acid sequence of SEQ ID NO. 3
  • the amino terminal amino acids of K2 are 64% homologous with the amino terminal sequence of OspF Therefore, we would expect that the full-length protein encoded by the k2 gene would have similar homology to full-length OspF protein
  • a consensus ribosome binding site with the sequence -GGAG- (Shine-Dalgarno sequence) is located 10 bp upstream of the p21 ATG start codon Further upstream of this translational initiation sequence are the promoter segments known as the "-10" region and the "-35" region, which are similar to those found in E. coli and other B. burgdorferi genes (See Figure 8 for a comparison of these regions between various B.
  • the protein encoded by thep21 gene appears to be a surface lipoprotein As shown in SEQ ID NO. 2, the protein begins with a basic N-terminal peptide of five amino acids, followed by an amino-terminal hydrophobic domain of about 20 amino acids that corresponds to the leader peptide found in typical prokaryotic lipoprotein precursors [M E Brandt et al., supra and C H Wu and M Tokunaga, "Biogenesis of Lipoproteins in Bacteria". Current Topics in Microbiology and Immunology. 125.
  • the carboxyl terminus ofthe hydrophobic domain contains a cleavage site presumably recognized by a B. burgdorferi signal peptidase In P21 , as in OspF, the potential cleavage site is located between Ser 17 and Cys j g
  • the signal sequences ofthe B. burgdorferi N40p21 gene contains three amino acids between the leucine and cysteine instead of two (See R S Fuchs et al and S J 5 Norris et al , supra )
  • OspA contains three amino acids between the leucine and cysteine instead of two (See R S Fuchs et al and S J 5 Norris et al , supra )
  • OspB and OspD have been shown to be lipoproteins by the established
  • P21 contains a long hydrophilic domain separated by short stretches of hydrophobic segments
  • RNA was isolated from cultured B. burgdorferi by acid guanidium thiocyanate/phenol/ chloroform extraction [cite]
  • the p2J and ospA probes contained the entire p21 and ospA sequences, respectively
  • RNA PCR to detect p21 RNA
  • To remove any residual DNA we treated 10 ⁇ g of pooled RNA with RNase-free DNase (Promega) for 3 hours at 37° C with HPRI and the Rnase inhibitor.
  • RNA PCR with and without reverse transcriptase to exclude the possibility that residual DNA might also be amplified
  • cDNA by reverse transcription with Moloney murine leukemia virus reverse transcriptase (Stratagene) and 3' primers for either p21 (murine tissue and cultured B. burgdorferi), ⁇ -actin (murine tissue control), or ospA (cultured B. burgdorferi control)
  • p21 murine tissue and cultured B. burgdorferi
  • ⁇ -actin murine tissue control
  • ospA cultured B. burgdorferi control
  • the other insert contained an open reading frame of 996 nucleotides encoding a 332 amino acid protein (SEQ ID NO 7)
  • GenBank GenBank (July 1995)revealed that we had isolated a second novel, B. burgdorferi gene which we designated p37.
  • P37 As is evident from SEQ ID NO 7, the deduced amino acid sequence of P37 reveals a leader peptide similar to those found in typical prokaryotic lipoprotein precursors At the carboxy terminus ofthe hydrophobic core is a potential signal peptidase II cleavage site between Ser 19 and Cys 20 P35, however, has a potential cleavage site with five amino acids intervening between the Leu and the Cys, making a lipoprotein less likely It will be necessary to look for further evidence of to confirm that P35 is a lipoprotein Finally, P37 contains a long hydrophilic domain separated by short hydrophobic segments The hydrophilicity profiles of P35 and P37, shown in Figure 6 suggest that both are hydrophilic proteins. We identified -35 and -10 regions as well as ribosome binding sites upstream ofthe respective open reading frames.
  • Tris-borate- EDTA (TBE) buffer 0.025 M Tris, 0.5 mM EDTA 0 025 M boric acid
  • Chef-DRII® system Bio-Rad Laboratories, Richmond, Calif.
  • the ospA and ospD probes hybridized to plasmids migrating at 49 kb and 38 kb, respectively [A G. Barbour and CF. Garon, "Linear Plasmids ofthe Bacterium Borrelia burgdorferi Have Covalently Closed Ends", Science. 237, pp. 409-41 1 (1987) and S.J. Norris et al , supra].
  • the p30 probe identified the chromosome.
  • the full-length p21 probe bound at three locations but a/?2/-specific probe (SEQ ID NO 14) recognized a circular plasmid
  • Example 6 and the primers used in Example 9 We confirmed that p35 and p37 are expressed in infected mice Therefore, p35 and p37 are selectively expressed in vivo
  • the pMX vector which is capable of directing expression of cloned inserts as glutathione S-transferase fusion proteins [see J Sears et al , "Molecular Mapping of Osp A-Mediated Immunity to Lyme Borreliosis", J Immunol.. 147, pp 1995- 2000 ( 1991 )]
  • the PMX vector also contains a thrombin cleavage site immediately following the GT protein, thus, allowing the recovery of recombinant proteins without the GT fusion partner.
  • burgdorferi polypeptide fusion proteins (GT-P21, GT-P35, GT-P37 and GT-M30) from cell lysates as follows l o
  • K2 polypeptide and DNA encoding it may be isolated by a variety of methods available to one of skill in the art For example, antiserum o raised against the peptide set forth in SEQ ID NO 3 may be used to screen a
  • B. burgdorferi expression library for clones capable of expressing the protein
  • an expression library could be constructed in which smaller fragments of B. burgdorferi DNA are cloned in frame into an expression vector from which they would be expressed as glutathione S-transferase fusion proteins, such as 5 pGEX-2T, pMX, or pGEMEX
  • glutathione S-transferase fusion proteins such as 5 pGEX-2T, pMX, or pGEMEX
  • Such a library would have a high likelihood of expressing the sequence as a fusion protein, even if it is normally linked to a promoter that is not transcriptionally active in E. coli
  • the DNA encoding the peptide set forth in SEQ ID NO 3 may be used as the basis of an oligonucleotide probe to screen a small cDNA library
  • mice Two weeks later we sacrificed the mice and cultured the tissues for spirochetes and examine the organs, as described above. Immunization with GT-P21 did not protect mice from infection or disease
  • Each mouse in the control and treatment group developed specific antibody titer of at least 1 5000 which have been found to be sufficient to protect mice from infection and disease in cases of protective antibodies like OspA (Fikrig et al , 1992) Mice were challenged with spirochetes at the peak antibody titer period which is a week after the final boost.
  • P21 is not expressed in high quantity in the early stages of infection
  • immunization with P21 did not produce sufficient protective antibodies in mice or that P21 was not expressed in sufficient quantity on the surface ofthe spirochete to make them vulnerable to antibody-mediated killing
  • mice were placed five Ixodes dammini ticks, infected as described in Example XVIII, on each of 12 control mice immunized with GT or 12 mice immunized with GT-P21. After feeding to repletion, the ticks are allowed to naturally detached over water. Only a portion ofthe ticks are recovered from each group, the remainder apparently having been ingested by the mice.
  • Another technique to identify protective epitopes is to use the various fragments to immunize mice, allow ticks infected with B. burgdorferi to feed on the mice, and then determine, as set forth in Example VIII, whether the immune response elicited by the fragments is sufficient to cause a decrease in the level of B. burgdorferi in the ticks Any epitopes which elicit such a response, even if they are not sufficient by themselves to confer protection against subsequent infection with B. burgdorferi, may be useful in a multicomponent vaccine
  • Stimulation in animals of a humoral immune response containing 5 high titer neutralizing antibodies will be facilitated by antigens containing both T cell and B cell epitopes.
  • antigens containing both T cell and B cell epitopes we infect C3H/HeJ mice with B. burgdorferi strain N40 in complete Freund's adjuvant, as described supra.
  • T cell lines Ten days after priming, we harvest the lymph nodes and generate m vitro T cell lines These T cell lines are then cloned using limiting o dilution and soft agar techniques We use these T cell clones to determine which polypeptides contain T cell epitopes The T cell clones are stimulated with the various polypeptides and syngeneic antigen presenting cells Exposure ofthe T cell clones to the polypeptides that contain T cell epitopes in the presence of antigen
  • T cell epitopes ofthe polypeptides recognized by human T cells we isolate T cell clones from B. burgdorferi-m ' ⁇ ected patients of multiple HLA types T cell epitopes are identified by stimulating the clones with the
  • the correlation is performed by utilization of B lymphoblastoid cell lines expressing various HLA genes When a given T cell clone is mixed with the appropriate B lymphoblastoid cell line and a novel B. burgdorferi polypeptide, the 5 B cell will be able to present the polypeptide to the T cell Proliferation is then
  • T cell epitopes may be identified by adoptive transfer of T cells from mice immunized with various ofthe novel B. burgdorferi polypeptides of this invention to naive mice, according to methods well known to those of skill in the art [See, for example, M S DeSouza et al , "Long-Term Study of Cell-Mediated Responses to Borrelia burgdorferi in the Laboratory Mouse", Infect. Immun . 61. pp 1814-22 (1993)]
  • T cell epitopes ofthe novel B. burgdorferi polypeptides After identifying T cell epitopes ofthe novel B. burgdorferi polypeptides, we construct recombinant proteins comprising these epitopes as well as the B cell epitopes recognized by neutralizing antibodies These fusion proteins, by virtue of containing both T cell and B cell epitopes, permit antigen presentation to T cells by B cells expressing surface immunoglobulin These T cells in turn stimulate B cells that express surface immunoglobin, leading to the production of high titer neutralizing antibodies
  • fusion proteins from the novel B. burgdorferi polypeptides by linking regions ofthe polypeptides determined to contain B cell epitopes to strong T cell epitopes of other antigens
  • the oligonucleotide is then ligated to the 5' and 3' ends of segments of DNA encoding the B cell epitopes recognized by neutralizing antibodies, as in Example XI
  • the recombinant DNA molecules are then used to express a fusion protein comprising a B cell epitope from the novel B. burgdorferi polypeptide and a T cell epitope from the core antigen, thus enhancing 5 the immunogenicity ofthe polypeptide.
  • fusion proteins comprising epitopes ofthe novel B. burgdorferi polypeptides as well as epitopes ofthe tetanus toxoid protein
  • Bacterial flagellin are potent stimulators of cellular and humoral responses, and can be used as vectors for protective antigens [S M C Newton, C Jacob, B. Stocker, "Immune Response To Cholera Toxin Epitope Inserted In Salmonella Flagellin", Science.
  • mice immunized with this agent are challenged with B. burgdorferi as described supra
  • fusion proteins comprising B cell epitopes from one ofthe novel B. burgdorferi polypeptides and T cell epitopes from a different novel B. burgdorferi polypeptide or other immunogenic B. burgdorferi 5 polypeptides
  • fusion proteins comprising T cell epitopes from novel B. burgdorferi polypeptides and B cell epitopes from a novel B. burgdorferi polypeptide and/or other immunogenic B. burgdorferi polypeptides Construction of these fusion proteins is accomplished by recombinant DNA techniques well known to those of skill in the art. Fusion proteins and antibodies directed against them, are used in methods and composition to detect, treat, and prevent Lyme disease as caused by infection with B. burgdorferi
  • MOLECULE TYPE DNA (genomic) (ill)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic) (111) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
  • AAA AGC TGG AGC TCC ACC GCG GTG GCG GCC GCT CTA GAA CTA GTG GAT 276 Lys Ser Trp Ser Ser Thr Ala Val Ala Ala Ala Leu Glu Leu Val Asp 30 35 40
  • GAG ACT AGA AAA GAA AAA ATA CAA AAA CAA CAA GAT GAA TAT AAA GGG 420 Glu Thr Arg Lys Glu Lys He Gin Lys Gin Gin Asp Glu Tyr Lys Gly 75 80 85 90
  • AAA GCC AAA GCT AAA GAA GAA GCT GCA AAG TTT ACA AAA GAA GAC CTT 852 Lys Ala Lys Ala Lys Glu Glu Ala Ala Lys Phe Thr Lys Glu Asp Leu 220 225 230
  • GAA AAA AAT TTC AAG ACC TTA TTA AAT TAT ATT CAA
  • GTA AGT GTA AAG 900 Glu Lys Asn Phe Lys Thr Leu Leu Asn Tyr He Gin Val Ser Val Lys 235 240 245 250
  • MOLECULE TYPE DNA (genomic) (in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
  • MOLECULE TYPE DNA (genomic) (111) HYPOTHETICAL: NO ( iv ) ANTI-SENSE : NO
  • AAG CTT CTT CAA CAC TTA TCA AAA AGT GAA GAC CAA GCC AAT AAA ACT 144 Lys Leu Leu Gin His Leu Ser Lys Ser Glu Asp Gin Ala Asn Lys Thr 365 370 375 380
  • MOLECULE TYPE DNA (genomic) (111) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
  • AAATCAGCTA CTCCACAACA TGATCCAATT GAACAAAGTA ATTTTAAAAA TAGCCTTACT 660
  • MOLECULE TYPE peptide (111) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 17: CATGATACTA GCAGACAA 18
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA (ill)
  • HYPOTHETICAL NO (IV)
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • Address ol deposilan institution t including postal code and tountm
  • ⁇ utho ⁇ zccljalticcr is . ⁇ ulli ⁇ n/cd olticer

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Abstract

L'invention porte: sur des procédés et compositions servant à la prévention, au traitement et au diagnostic de la maladie de Lyme; sur de nouveaux polypeptides du type B. burgdorferi, leurs variantes sérotypiques, leurs fragments et leurs dérivés; sur des protéines de fusion et des protéines multimères les incluant; sur des vaccins à plusieurs composants contenant les nouveaux polypeptides du type B. burgdorferi, ainsi que d'autres polypeptides immunogènes du type B. burgdorferi; sur des séquences d'ADN, des molécules d'ADN de recombinaison et des cellules hôtes transformées utiles dans les procédés et compositions ci-dessus; sur des anticorps dirigés contre les nouveaux polypeptides du type B. burgdorferi et des trousses de diagnostic comportant lesdits polypeptides ou anticorps; et sur une méthode d'identification de gènes de bactéries exprimés sélectivement in vivo.
EP96915650A 1996-05-08 1996-05-08 Polypeptides du type b. burgdorferi exprimes in vivo Withdrawn EP0915977A1 (fr)

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CA2283494A1 (fr) * 1998-01-08 1999-07-15 Biomerieux, Inc. P37/flaa de recombinaison en tant que reactif pour diagnostic
DE19960113A1 (de) * 1999-12-08 2001-06-13 Wita Proteomics Ag Mittel und Verfahren zur Diagnose von Lyme Borreliose sowie Borreliose-Impfstoff
FI112544B (fi) * 2001-11-26 2003-12-15 Bortech Oy Menetelmä varhais- ja myöhäisvaiheen Lymen borrelioosin diagnosoimiseksi

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DK590288D0 (da) * 1988-10-24 1988-10-24 Symbicom Ab Kemiske forbindelser

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See references of WO9742325A1 *

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