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EP0643586A1 - Compositions utilisees dans le diagnostic et la prophylaxie de la maladie de lyme - Google Patents

Compositions utilisees dans le diagnostic et la prophylaxie de la maladie de lyme

Info

Publication number
EP0643586A1
EP0643586A1 EP93914155A EP93914155A EP0643586A1 EP 0643586 A1 EP0643586 A1 EP 0643586A1 EP 93914155 A EP93914155 A EP 93914155A EP 93914155 A EP93914155 A EP 93914155A EP 0643586 A1 EP0643586 A1 EP 0643586A1
Authority
EP
European Patent Office
Prior art keywords
burgdorferi
antigen
tick
antibody
animal
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Withdrawn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
EP93914155A
Other languages
German (de)
English (en)
Other versions
EP0643586A4 (fr
Inventor
William T. Golde
John T. Roehrig
Thomnas Burkot
Joseph F. Piesman
Barbara J. B. Johnson
Leonard W. Mayer
Mark G. Keen
Ann R. Hunt
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.)
Centers of Disease Control and Prevention CDC
SmithKline Beecham Corp
Original Assignee
Centers of Disease Control and Prevention CDC
SmithKline Beecham Corp
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 Centers of Disease Control and Prevention CDC, SmithKline Beecham Corp filed Critical Centers of Disease Control and Prevention CDC
Publication of EP0643586A1 publication Critical patent/EP0643586A1/fr
Publication of EP0643586A4 publication Critical patent/EP0643586A4/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Withdrawn legal-status Critical Current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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • Patent Application SN 944,464 filed September 14, 1992, which is a continuation-in-part of U. S. Patent Application SN 07/889,015, filed May 26, 1992.
  • the present invention relates generally to the field of pharmaceutical and diagnostic compositions useful in the diagnosis, treatment and prophylaxis of Lyme borreliosis. More specifically, the invention provides isolated natural and recombinant proteins, polypeptides and macromolecular antigens that are derived from Borrelia burgdorferi and differentiated in the tick vector.
  • Lyme borreliosis i.e., Lyme disease
  • Lyme disease the bacterium Borrelia burgdorferi
  • the reservoir of the infection is most likely the white footed mouse, Peromy ⁇ cus leucopus, and the disease can be transmitted to many mammalian species including dogs, cats, and man [J. G. Donahue, et al.. Am. "J. TrOP. Med. Hv ⁇ .. 3j6:92-96 (1987); R. T. Green, et al.. J. Clin. Micro.. 26:648-653 (1988)].
  • the B The B .
  • burgdorferi strain B31 is believed to be responsible for about 90% of the known isolates of Borrelosis in the United States.
  • the diagnosis of Lyme disease in humans and animals has been compromised by the lack of definitive serology leading to rapid and accurate testing and the lack of suitable bacterial antigens capable of eliciting an immune response in vaccinates.
  • Cultured preparations of Borrelia burgdorferi have been used to generate whole cell sonicate for ELISA and western blot analysis, which has yielded only marginal results [M. Karlsson et al.. Eur. J. Clin. Microbiol. Infect. Pis.. 8.:871-877 (1990), S. W. Luger et al. , Arch. Intern. Med..
  • a number of bacterial antigens have been identified that may be more useful in serodiagnosis, including outer surface proteins A and B (OspA and OspB) , flagellin, and other proteins designated P21, P39, P66, and P83 according to their estimated molecular weights [A. G.
  • PCT/US91/01500 published September 19, 1991 refers to B. burgdorferi antigens having molecular weights of 28 and
  • PCT/EP90/02282 published July 11, 1991 refers to B . burgdorferi antigens having molecular weights of 17, 22,
  • PCT/DK89/00248, published May 3, 1990 refers to B . burgdorferi antigens having molecular weights spanning the range of 20 through 85 kDa.
  • PCT patent application No. WO92/00055, published January 9, 1992 discloses OspA and OspB polypeptides, fusion proteins and derivatives of B . burgdorferi strains N40 and 25015. This publication refers to any DNA sequence or polypeptide that elicits in a treated mammalian host an immune response effective to protect against Lyme Disease caused by infection with Borrelia burgdorferi . In fact, the polypeptides are only identified by immunizing a mouse of strain C3H/He with polypeptide.
  • the invention provides isolated B . burgdorferi antigens which are regulated and differentiated by growth of the B . burgdorferi in a tick vector. Novel antigens of the invention are listed below in Table I.
  • antigens are characterized as being B . burgdorferi B31 strain specific and major histocompatibility complex (MHC) nonrestricted. Certain other of these antigens are characterized as being MHC- restricted. Sera generated to these antigens (B31 MHC nonrestricted and B31 MHC restricted) are further characterized by the ability or lack of ability to react with B . burgdorferi JD-1 strain; the antigens themselves (B31 MHC nonrestricted and B31 MHC restricted) are further characterized by being homologous or heterologous with B . burgdorferi JD-1 strain antigens. The most preferred antigens of this invention, because of their ability to induce cross-strain immunity to B .
  • MHC major histocompatibility complex
  • the invention provides a B . burgdorferi antigen obtained from the microorganism which is isolated directly from a tick which has optionally been infested (feeding * ) on infected host animals.
  • the B . burgdorferi has been passaged only a minimum number of times in growth media to retain its virulence and antigenicity before the antigen is isolated.
  • a desirable minimum number of in vitro passages is between 1 and 6.
  • the invention provides a B . burgdorferi antigen which is prepared from B . burgdorferi DNA isolated from a tick vector or from low passage culture of the microorganism. Still a further aspect provides a B . burgdorferi antigen which is prepared from PCR gene banks derived from ticks infected with B . burgdorferi or from low passage culture.
  • the present invention provides antibodies to the novel B . burgdorferi antigens of the invention. Such antibodies may be monoclonal, polyclonal or recombinant.
  • the present invention provides diagnostic reagents, therapeutic compositions, and vaccinal compositions which comprise at least one of the novel antigens or antibodies of the invention.
  • the invention provides diagnostic assays and kits, methods of treating Lyme disease, and methods of preventing Lyme disease which utilize the reagents and compositions of the invention.
  • Fig. 1 illustrates the antibody response to whole-cell sonicate, purified flagella, and recombinant OspA in tick-infected hamsters.
  • Antibody response was determined by ELISA on whole-cell sonicate (•), purified flagella ( ⁇ ) , or purified recombinant OspA (A) .
  • Fig. 2 illustrates the antigenic dose-response analysis of hamsters inoculated with various concentrations of B . burgdorferi .
  • Hamsters were bled at 2 (3) and 4 (®) weeks post-infection (P.I.).
  • Antibody response was determined by ELISA on whole-cell sonicate (solid lines) or recombinant OspA (dotted lines) .
  • the present invention identifies novel isolated Borrelia burgdorferi antigens which are regulated and/or differentiated by transmission of the B . burgdorferi through a tick vector.
  • a tick vector refers to an arthropod organism of the Insect Family, which is capable of vectoring B . burgdorferi .
  • the tick vector is optimally of the Ixodes genus, and of the Ixodes complex J. datruni-ni, J. scapularu ⁇ , I . pacificus, or I. ricinus or related species or genera that can transmit the microorganism which causes Lyme disease. Because growth of B .
  • this invention uses response in genetically defined MHC haplotype mice to serologically identify the differences between tick borne B . burgdorferi antigens and those produced by culture grown B . burgdorferi .
  • One of the best indicators of differentiation and/or regulation is the appearance or loss of antigens as measured by antibody specificity produced when the tissue or cells are inoculated into a human or an animal. Antigens produced and vectored by ticks are distinct from prior art B . burgdorferi antigens which have been multiply passaged in vitro in growth media.
  • MHC major histocompatibility complex
  • proteins of all cells of an animal or man degrade or turn over as a natural progression of events.
  • the immune surveillance looks at each peptide removed from the cell. Typically, it is displayed through the MHC as it leaves the cell or is internalized and degraded to individual amino acids to re-enter the metabolite pool. Because an animal does not want to react to its own proteins, that animal's "self" proteins are restricted to response. If a protein looks like a self protein then the immune system of that animal will not react. If the immune system sees a protein or antigen as foreign, the MHC does not restrict the response.
  • antigens of this invention are significantly characterized as not restricted to any major histocompatibility complex (MHC) haplotype.
  • MHC restricted is meant that an antigen of this invention is capable of eliciting an immune response to Lyme disease in humans and animals regardless of their immunogenetic background (the human or animal's MHC does not restrict the immune response) .
  • the antigens of this invention describe those serological responses that occur in mice early after infestation with infected ticks, they may also represent the virulence (common) components associated with virulent B . burgdorferi infection.
  • the antigens are further characterized as crossreactive with more than one B . burgdorferi strain that is capable of inducing clinical symptomology in humans and/or animals associated with Lyme disease, as demonstrated in the examples below.
  • crossreactive indicates that sera generated to antigens derived from one strain is capable of reacting with antigens of another strain and that the antigens of the two strains are homologous. Sera generated to antigens derived from one strain which is not capable of reacting with antigens of another strain indicates that the antigens are heterologous.
  • Antigens of B . burgdorferi are identified below with reference to their molecular weight in kilodaltons. For example, a B . burgdorferi antigen of about 17 kd in molecular weight is identified as P17; an antigen of about 22 kd in molecular weight is identified as P22 and so on.
  • P41 (fla) refers to a flagella-associated antigen of molecular weight 41 kd. The other antigens of Table I are not associated with the flagella, outer surface protein (Osp) A, or Osp B. The characteristics of B .
  • burgdorferi low- passage antigens i.e., antigens obtained from the Borrelia microorganism which has been passaged in culture less than 6 times
  • Table I summarizes the data from Examples 11-13, in which western blot analysis was performed using sera from a panel of inbred strains of mice infected with B31 strain B . burgdorferi by exposure to Ixodes dammini ticks infected with the B31 isolate of the spirochete on proteins from B31 and JD-1 strains.
  • Table I shows that certain antigens are characterized by being B31 MHC nonrestricted, i.e. these antigens are capable of eliciting an immune response in all haplotypes.
  • B31 MHC nonrestricted antigens are further characterized by being (a) JD-l crossreactive and JD-1 MHC nonrestricted, (b) JD-1 crossreactive and JD-1 MHC restricted, or (c) JD-1 nonreactive.
  • the antigens of group (a) are capable of eliciting the broadest immune response in animals. Antigens which fall into categories (b) and (c) are also useful in vaccine compositions of this invention.
  • Table I also shows that the antibody response to a number of the novel protein antigens derived from B . burgdorferi strain B31 were MHC restricted. For example, until late in the infection, only the mouse strain BIO animals respond to the P83 protein. Since mice of the strains B10.BR and B10.D2 are perfect genetic matches except for the MHC locus, apparently, MHC haplotypes H-2 d and H-2 k are relatively inefficient at presenting this protein when compared to H-2 b . See, particularly, Examples 10 and 12 below.
  • these MHC-restricted antigens are also further characterized by being (a) JD-1 crossreactive and JD-1 MHC nonrestricted, (b) JD-1 crossreactive and JD-1 MHC restricted, or (c) JD-1 nonreactive.
  • the immune response elicited by these novel B . burgdorferi antigens is B31 MHC restricted, and thus, do not elicit an immune response in all animal haplotypes, they are also useful in vaccines to prevent Lyme Disease in humans and other animals and in diagnostic assays.
  • Another preferred characteristic of the novel antigens of this invention lies in their ability to produce antibodies in an infected human or animal early in the B . burgdorferi infection period, e.g., within the first two weeks post infection (P.I.). Still another preferred characteristic of antigens of this invention is that the antibodies directed thereto are stably produced in the human or animal throughout the infection period.
  • stably produced throughout the infection period is meant detectable production of antibody in the period post infection spanning from about two weeks P.I. through and beyond 60 days P. I. It is anticipated that such antibodies will remain detectable in a human or an animal for up to one year P.I. or longer because in most cases the spirochete will persist in the host for long periods of time.
  • antigens isolated from the organism and formulated with an appropriate delivery system are anticipated to provide an immune response that would predispose the animal to infection.
  • these antigens of this invention are preferably characterized by the ability to prevent or protect a human or an animal against Lyme Disease by eliciting a stable protective antibody response in a human or animal early during Borrelia burgdorferi infection.
  • these novel B . burgdorferi antigens are useful in diagnosing Lyme disease, and in vaccinal and therapeutic compositions.
  • the antigens of the invention may be isolated from any selected strain, variant, group or subunit of Borrelia .
  • antigens are isolated from B . burgdorferi, particularly from subgroups A and B which are associated most frequently with Lyme borreliosis.
  • the spirochete isolates may also be from subgroups C through F [A. G. Barbour et al. , J. Infect.
  • the antigens of this invention are obtained and isolated from the microorganism directly from the tick vector or from sera of a human or animal infected with B . burgdorferi via a tick bite.
  • the antigens nay also be obtained by serial tick to host animal infections and then obtained from the tick or the serum of a tick infested, B . burgdorferi infected animal. " None of the prior art references describe the need to maintain the B . burgdorferi by tick to host passage. While passaging in growth media is preferably avoided, optionally, the microorganism can be passaged at least once in BSK media simply to amplify the microorganism to provide the antigenic material. For example, where the B .
  • burgdorferi strain JP-1 one passage supplies sufficient amplification. Where the B31 strain is employed, passages up to 6 may be employed. However, to obtain the antigens of this invention, no high passage in vitro culturing is employed.
  • the term "high passage” is defined as greater than 6 passages for strain B31, and more than one passage for strain JP1.
  • one of skill in the art can determine the number of passages which constitute "high passage” for other strains of B . burgdorferi by assaying the passaged microorganism in hamsters, as described in Examples 5 through 8 below. Failure of the microorganism to cause clinical symptoms of Lyme Pisease, or to illustrate the growth characteristics of B .
  • the microorganism has been cultured in vitro sufficiently to lose most of its virulence and thus the antigens obtained therefrom differ from the antigens of the present invention.
  • the antigens of the invention are preferably isolated by immunoblot procedures according to their respective molecular weights, as described below in Example 4. Such isolation may provide the antigens in a form substantially free from other proteinaceous and non- proteinaceous materials of the microorganism and the tick vector.
  • the molecules comprising the B are preferably isolated by immunoblot procedures according to their respective molecular weights, as described below in Example 4. Such isolation may provide the antigens in a form substantially free from other proteinaceous and non- proteinaceous materials of the microorganism and the tick vector.
  • the molecules comprising the B are preferably isolated by immunoblot procedures according to their respective molecular weights, as described below in Example 4.
  • burgdorferi polypeptides and antigens of this invention may be isb ⁇ ated from the tick and further purified using any of a variety of conventional methods including: liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with inorganic ligands or monoclonal antibodies) ; size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like.
  • liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like
  • affinity chromatography such as with inorganic ligands or monoclonal antibodies
  • size exclusion chromatography size exclusion chromatography
  • immobilized metal chelate chromatography immobilized metal chelate chromatography
  • gel electrophoresis gel electrophoresis
  • polynucleotide and amino acid sequences of these antigens may be obtained by resort to conventional genetic engineering techniques, PCR, and the like.
  • the antigen of this invention may be prepared or isolated from B . burgdorferi DNA isolated from the tick vector using PNA probes and PCR techniques.
  • the antigen may be obtained from PCR gene banks derived from B . burgdorferi-infected ticks or from low passage of the microorganism in growth media.
  • B . burgdorferi is believed to need the environment of its vector, the tick, in order to differentiate as an organism or to process certain of its proteins and antigens into virulent and infective states.
  • the B . burgdorferi proteins or antigens of this invention which are obtained from the tick are either modified by the tick environment, or are completely different proteins from those obtained by repeated culturing of B . burgdorferi in growth media.
  • the detection of the P39 bands is consistent throughout the screen of all these different sources of antigen with all of the mouse sera.
  • Antigens of this invention are also preferably characterized by recognition by sera or T cells obtained from tick infested, B . burgdorferi infected, humans or animals. In experiments, sera from animals treated with Borrelia burgdorferi infected ticks failed to recognize a family of Borrelia burgdorferi proteins grown in culture media. See, for example, Examples 7 and 10 below.
  • the prior art fails to positively identify any of the Borrelia burgdorferi antigens on the basis of criteria which are able to discriminate between various reported Borrelia burgdorferi antigens, because the most frequently reported characteristic is molecular weight. However, despite similar molecular weights, the present inventors are able to discriminate between:
  • the antigens of this invention may be proteinaceous, such as a protein, a polypeptide or fragment thereof. These antigens may be isolated generally free from contamination with proteinaceous and other materials with which the antigens are associated in nature. Thus, antigens of this invention may be characterized by immunological measurements against the vector borne pathogen using MHC-restricted response in genetically inbred animals. These measurements include, without limitation, western blot, macromolecular weight determinations by biophysical determinations, such as SDS-PAGE/staining, HPLC and the like, antibody recognition assays, T-cell recognition assays, MHC binding assays, and assays to infer immune protection or immune pathology by... doptive transfer of cells, proteins or antibodies.
  • B . burgdorferi antigens, proteins and polypeptides of this invention may be part of a larger and/or multimeric protein.
  • Antigens of this invention may be in combination with B . burgdorferi outer surface proteins, such as OspA and OspB.
  • the antigen may be in the form of a fusion protein.
  • an antigen or polypeptide of this invention may be fused at its N-terminus or C-terminus to OspA polypeptide, or OspB polypeptide or to a non-OspA non-OspB polypeptide or combinations thereof.
  • OspA and OspB polypeptides which may be useful for this purpose include polypeptides identified by the prior art [see, e.g. PCT/US91/04056] and variants thereof.
  • Non-OspA, non-OspB polypeptides which may be useful for this purpose include polypeptides of the invention and those identified by the prior art, including, the B . burgdorferi , flagella-associated protein and fragments thereof, other B . burgdorferi proteins and fragments thereof, and non-B. burgdorferi proteins and fragments thereof.
  • fusion proteins comprising multiple polypeptides of this invention are constructed for use in the methods and compositions of this invention.
  • These fusion proteins or multimeric proteins may be produced recombinantly, or may be synthesized chemically. They also may include the polypeptides of this invention fused or coupled to moieties other than amino acids, including lipids and carbohydrates.
  • antigens of this invention may be employed in combination with other Borrelia vaccinal agents described by the prior art, as well as with other species of vaccinal agents, e.g, derived from parvovirus, coronavirus, leptosporidia.,,_rand rabies.
  • Such .proteins are effective in the prevention, treatment and diagnosis of Lyme disease as caused by a wide spectrum of B. -burgdorferi isolates.
  • Antigens of this invention are non-OspA, non- OspB B. burgdorferi antigens that can elicit a protective response in vaccinated humans or animals that are exposed to infestation with Borrelia burgdorferi infected ticks where any of the following circumstances apply:
  • antigens are derived from Borrelia burgdorferi taken from the host or tick without intervention of in vitro cultivation or with minimum numbers of passages in growth media (see Table I, and Example 9) ;
  • non-OspA and non-OspB antigen can elicit in a vaccinated human or animal antibody or a cell mediated immune (CMI) response that can eliminate or greatly reduce the ability to cultivate viable Borrelia burgdorferi from infected ticks used to infect host humans or animals (see Example 15) .
  • CMI cell mediated immune
  • antigens of this invention which induce a protective immune response in humans or animals and are not restricted to MHC include the following:
  • the antigen P18 is characterized by a molecular weight of about 18 kd as measured by immunoblot (See Example 4) .
  • This antigen is capable of eliciting an antibody response by one month post-infection and stably throughout infection in humans or animals infected with B. burgdorferi by a tick vector. It also elicits a response for mice of the H-2 k , H-2 d , and H-2 b haplotypes of the MHC, i.e., P18 is not MHC-restricted.
  • This antigen also demonstrates the ability to elicit a crossreactive response between strains of B.
  • the antigen P43 is characterized by a molecular weight of about 43 kD as measured by immunoblot (See Example 4) .
  • This antigen is capable of eliciting an antibody response in animals infected with B. burgdorferi by a tick vector by 12 days after infection, which response remains stable throughout infection.
  • P43 is capable of eliciting a response from hamsters and mice of the H-2 , H-2 b , and H-2 d haplotypes of the MHC, i.e., P43 is not MHC restricted.
  • This antigen is expressed by at least 2 different strains of B .
  • Another exemplary antigen of this invention is P39, which is characterized by a molecular weight of about 39 kd as measured by immunoblot. ⁇ Like the other antigens of this invention, it elicits an antibody response in humans and animals infected with B. burgdorferi by a tick vector, which response is stable throughout infection. It is_not MHC-restricted, and it can elicit a cross-reactive response between a variety of B. burgdorferi strains.
  • the isolated antigen P17 is characterized by a molecular weight of about 17 kD as measured by immunoblot (See Example 4) . This antigen is also characterized by the ability to elicit an antibody response by one month post-infection and stably throughout infection. P17 is also capable of eliciting an MHC-nonrestricted response when assayed on homologous strain B31, but responds to the heterologous strain JD-1 in a restricted fashion. (see Example 13) . P17 is not MHC restricted with respect to B31, but restricted with respect to JD-1.
  • This antigen also demonstrates the ability to elicit a crossreactive response between strains of B. burgdorferi , e.g., strains B31 and JD-1.
  • Other B31 MHC nonrestricted antigens are P24, P22 and P14, which are non-reactive with JD-1.
  • antigens of interest in the present invention are those which are B31 MHC restricted, but JD- l non-reactive, i.e., P29 and P32.
  • Other antigens of interest are those which are B31 MHC restricted, JD-1 crossreactive and non-MHC restricted, i.e., P30.
  • B. burgdorferi antigens, P83, P52-65, P41 (fla), P28 and P34 are B31 non-MHC restricted, JD-1 crossreactive but restricted.
  • the antigens of the present invention may also be modified to increase their immunogenicity.
  • the antigens may be coupled to chemical compounds or immunogenic carriers, -provided that the coupling does not interfere with the desired biological activity of either the antigen or the carrier.
  • Useful immunogenic carriers known in the art include, without limitation, keyhole limpet hemocyanin (KLH) ; bovine serum albumin (BSA) , ovalbumin, PPD (purified protein derivative of tuberculin) ; red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • PPD purified protein derivative of tuberculin
  • red blood cells tetanus toxoid
  • cholera toxoid agarose beads
  • activated carbon or bentonite.
  • Useful chemical compounds for coupling include, without limitation, dinitrophenol groups and arsonilic acid.
  • the antigens may also be modified by other techniques, such as denaturation with heat and/or SDS. Further, the antigens of the invention may be optionally fused to a selected polypeptide or protein, e
  • Borrelia antigens OspA and OspB other Borrelia antigens, and proteins or polypeptides derived from other microorganisms.
  • Any antigen of the present 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 of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.
  • the present invention also provides antibodies capable of recognizing and binding naturally-occurring antigens of this invention from the B. burgdorferi pathogen, when it is present in a biological fluid.
  • biological fluid includes blood, plasma, serum, tears, saliva, urine, vaginal secretions, synovial fluid, bladder wall, or ear puncture.
  • These antibodies are useful in diagnosis of Lyme disease and in therapeutic compositions for treating humans and/or animals that test positive for, or, prior to testing,, exhibit symptoms of, Lyme Disease.
  • the antibodies are useful in diagnosis alone or in combination with antibodies to other antigens of this invention as well as antibodies to other known B . burgdorferi antigens. These antibodies may be generated by conventional means utilizing the isolated antigens of this invention.
  • polyclonal antibodies may be generated by conventionally stimulating the immune system of a selected animal or human with one or more of the isolated antigens, allowing the immune system to produce natural antibodies thereto, and collecting these antibodies from the animal or human's blood or other biological fluid.
  • Table VI provides a list of monoclonal antibodies of the invention. However, it is also desirable to obtain and utilize other monoclonal antibodies (MAb) for the practice of the methods of this invention.
  • MAb monoclonal antibodies
  • hybridoma cell lines expressing desirable MAbs may be generated by well-known conventional techniques, e.g. Kohler and Milstein and the manner known modifications thereof, similarly desirable high titer antibodies may also be generated by applying known recombinant techniques to the monoclonal or polyclonal antibodies developed to these antigens [see, e.g., PCT Patent Application No. PCT/GB85/00392; British Patent Application Publication No. GB2188638A; Amit et al.. Science. 233:747-753 (1986); Queen et al., Proc. Nat'l. Acad. Sci. USA. 11:10029-10033 (1989); PCT Patent Application No.
  • the antigens may be assembled as multi-antigenic complexes [see, e.g., European Patent Application 0339695, published November 2, 1989] and employed to elicit high titer antibodies capable of binding the selected antigen as it appears in the biological fluids of an infected animal or human.
  • the antibodies may be associated with conventional labels which are capable, alone or in concert with other compositions or compounds, of providing a detectable signal.
  • the labels are desirably interactive to produce a detectable signal.
  • the label is detectable visually, e.g. colorimetrically.
  • enzyme systems have been described in the art which will operate to reveal a colorimetric signal in an assay. As one example, glucose oxidase (which uses glucose as a substrate) releases peroxide as a product.
  • Peroxidase which reacts with peroxide and a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized TMB that is seen as a blue color.
  • a hydrogen donor such as tetramethyl benzidine (TMB)
  • TMB tetramethyl benzidine
  • Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP) , and hexokinase in conjunction with glucose-6- phosphate dehydrogenase which reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 nm wavelength.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • hexokinase in conjunction with glucose-6- phosphate dehydrogenase which reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 n
  • a dye in which a dye is embedded may be used in place of enzymes to form conjugates with the antibodies and provide a visual signal indicative of the presence of the resulting complex in applicable assays, fluorescent compounds, radioactive compounds or elements, or immunoelectrodes.
  • Detectable labels for attachment to antibodies useful in diagnostic assays of this invention may be easily selected from among numerous compositions known and readily available to one skilled in the art of diagnostic assays. The methods and antibodies of this invention are not limited by the particular detectable label or label system employed. Diagnostic Methods and Assays
  • the present invention also provides methods of diagnosing Lyme disease. These diagnostic methods may be useful in treating humans or animals exhibiting the clinical symptoms of, or suspected of having, Lyme disease.
  • this diagnostic method involves detecting the presence of naturally occurring anti-B. burgdorferi antibodies which are produced by the infected human or animal patient's immune system in its biological fluids, and which are capable of binding to the antigens of this invention or combinations thereof, optionally including P39 protein.
  • This method comprises the steps of incubating at least one B. burgdorferi antigen of this invention and optionally at least one other antigen from B. burgdorferi with a sample of biological fluids from the patient.
  • Antibodies present in the fluids as a result of B. -burgdorferi infection will form an antibody-antigen complex with the antigen.
  • the reaction mixture is analyzed to determine the presence or absence of these antigen- antibody complexes.
  • the step of analyzing the reaction mixture comprises contacting the reaction mixture with a labeled specific binding partner for the antibody.
  • the method entails conjugating latex beads to the isolated antigens of this invention. Subsequently, the biological fluid is incubated with the bead/protein conjugate, thereby forming a reaction mixture. The reaction mixture is then analyzed to determine the presence of the antibodies.
  • the diagnostic method of the invention involves detecting the presence of the naturally occurring antigen itself in its association with the B. burgdorferi pathogen in the biological fluids of an animal or human infected by the pathogen.
  • This method includes the steps of incubating an antibody of this invention (e.g. produced by administering to a suitable human and/or animal at least one Borrelia burgdorferi antigen of this invention and optionally at least one other B. burgdorferi antigen) , preferably conventionally labelled for detection, with a sample of biological fluids from a human or an animal to be diagnosed.
  • an antigen-antibody complex is formed (specific binding occurs) .
  • excess labelled antibody is optionally removed, and the reaction mixture is analyzed to determine the presence or absence of the antigen-antibody complex and the amount of label associated with the antibody.
  • Assays employing a protein antigen of the invention can be heterogenous (i.e., requiring a separation step) or homogenous. If the assay is heterogenous, a variety of separation means can be employed, including centrifugation, filtration, chromatography, or magnetism.
  • an enzyme linked immunosorbent assay i.e., an ELISA.
  • the ELISA is an antigen-capture two-site ELISA capable of recognizing many strains of B. Jburgdorferi and capable of distinguishing B. burgdorferi- infected from uninfected J. daj-uni-ni (larvae, nymphs, or adults) , using a third of a tick extract, thereby leaving adequate materials for confirmational testing.
  • the isolated antigen of the invention is adsorbed to the surface of a microtiter well directly or through a capture matrix (i.e., antibody).
  • Residual protein-binding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA) , heat-inactivated normal goat serum (NGS) , or BLOTTO (a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent) .
  • BSA bovine serum albumin
  • NGS heat-inactivated normal goat serum
  • BLOTTO a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent
  • the well After incubating for a sufficient length of time to allow specific binding to occur, the well is washed to remove unbound protein and then incubated with labeled anti-human immunoglobulin ( ⁇ Hulg) .
  • the label can be chosen from a variety of enzyr.es, including horseradish peroxidase (HRP) , ⁇ - galactosidase, alkaline phosphatase, and glucose oxidase, as described above.
  • HRP horseradish peroxidase
  • ⁇ - galactosidase alkaline phosphatase
  • glucose oxidase glucose oxidase
  • the MAbs or other antibodies of this invention which are capable of binding to the antigen(s) can be bound to ELISA plates.
  • the biological fluid is incubated on the antibody-bound plate and washed. Detection of any antigen-antibody complex, and qualitative measurement of the labelled MAb is performed conventionally, as described above.
  • Such an "antigen capture" assay has been developed for the detection of B. burgdorferi in ticks using monoclonal antibodies specific for OspA and is currently the preferred method of detecting the presence of B. Jburgdorferi in biological samples according to this invention. This assay is described in more detail in Example 14 below.
  • the assay uses monoclonal antibodies for antigen capture and labelled rabbit polyclonal anti-B. burgdorferi antibodies for signal generation.
  • the assay has a sensitivity of between about 50 to about 200 B. burgdorferi spirochetes, more particularly about 100 spirochetes. Infections in nymphs and adults can be determined using less than 25% of a tick, thereby allowing confirmational testing.
  • Suitable assay formats include the filter cup and dipstick.
  • an antibody of this invention is fixed to a sinter glass filter to the opening of a small cap.
  • the biological fluid or sample (5 mL) is worked through the filter. If the antigen is present (i.e., B. burgdorferi infection), it will bind to the filter which is then visualized through a second antibody/detector.
  • the dipstick assay involves fixing an antigen or antibody to a filter, which is then dipped. in the biological fluid, dried and screened with a detector molecule.
  • any number of conventional assay formats may be designed to utilize the isolated antigens and antibodies of this invention for the detection of B. burgdorferi infection in animals and humans.
  • This invention is thus not limited by the selection of the particular assay format, and is believed to encompass assay formats which are known to those of skill in the art. Diagnostic Kits
  • kits are useful for diagnosing infection with B. burgdorferi in a human or an animal sample which contains at least one B. burgdorferi antigen of this invention and/or at least one antibody capable of binding at least one B. burgdorferi antigen of this invention.
  • kits can include microtiter plates to which the B. burgdorferi antigen proteins or antibodies of the invention have been pre-adsorbed, various diluents and buffers, labeled conjugates for the detection of specifically bound antigens or antibodies, and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens.
  • kits can easily be determined by one of skill in the art.
  • Such components may include polyclonal or monoclonal capture antibodies to a B31 non-MHC-restricted, JD-1 crossreactive and non-MHC restricted, antigen of this invention, e.g., P18, P39 or P43; to a B31 non-MHC restricted, JD-1 MHC-restricted antigen, P17; or a cocktail of two or more of the antibodies, purified or semi-purified extracts of these antigens as standards, MAb detector antibodies, an anti-mouse or anti-human antibody with indicator molecule conjugated thereto, an ELISA plate prepared for absorption, indicator charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, and a sample preparator cup.
  • B31 MHC restricted antigens such as those listed in Table I above, may also form part of this kit.
  • Suitable exemplary monoclonal antibodies of the invention are provided in Table VI. These kits provide a convenient, efficient way for a clinical laboratory to diagnose B. burgdorferi infection.
  • the antibodies of the invention may further be used in therapeutic compositions and in methods for treating humans and/or animals infected with B. jburgdorferi.
  • a therapeutic composition may be formulated to contain a carrier or diluent and one or more of the antibodies of the invention (see Table VI) .
  • Suitable pharmaceutically acceptable carriers facilitate administration of the proteins but are physiologically inert and/or nonharmful. Carriers may be selected by one of skill in the art. Exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate,, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, and water.
  • the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol di ⁇ tearate alone or with a wax.
  • a time delay material such as glycerol monostearate or glycerol di ⁇ tearate alone or with a wax.
  • slow release polymer formulations can be used.
  • this composition may also contain conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable ingredients which may be used in a therapeutic composition in conjunction with the antibodies include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried milk.
  • agents useful in treating Lyme disease e.g., antibiotics or immunostimulatory agents, antagonists to receptor mediated activity of antigens of this invention and cytokine regulation»elements are expected to be useful in reducing and eliminating disease symptoms.
  • Agents which can be used to suppress or counteract the immune suppressants released by the tick vector should act to assist the natural immunity of the infected human or animal. Thus, such agents operate in concert with the therapeutic compositions of this invention. The development of therapeutic compositions containing these agents is within the skill of one in the art in view of the teaching of this invention.
  • a human or an animal may be treated for B. burgdorferi infection by administering an effective amount of such a therapeutic composition.
  • a composition is administered parenterally, preferably intramuscularly or subcutaneously.
  • parenterally preferably intramuscularly or subcutaneously.
  • it may also be formulated to be administered by any other suitable route, including orally or topologically.
  • a therapeutic composition of the invention may contain between about 0.05 ⁇ g/mL to about 1000 ⁇ g/mL of an antibody of the invention. Such a composition may be administered 1 - 3 times per day over a 1 day to 12 week period. However, suitable dosage adjustments may be made by the attending physician or veterinarian depending upon the age, sex, weight and general health of the human or animal patient.
  • Vaccine Compositions may contain between about 0.05 ⁇ g/mL to about 1000 ⁇ g/mL of an antibody of the invention. Such a composition may be administered 1 - 3 times per day over a 1 day to 12 week period. However, suitable dosage adjustments may be made by the attending physician or veterinarian depending upon the age, sex, weight and general health of the human or animal patient.
  • the present invention provides a vaccine composition useful in protecting against Lyme disease associated with B. burgdorferi and a prophylactic method entailing administering to an animal or human an effective amount of such a composition.
  • This vaccine composition may contain one or more of the antigens of the invention, or mixtures of two or more of these isolated proteins, or combinations of these antigens with other antigens of B . burgdorferi , such as the 39kP protein, the OspA and OspB proteins and a pharmaceutically acceptable carrier or diluent.
  • Exemplary carriers are as described above.
  • the vaccine composition may further contain adjuvants, preservatives, chemical stabilizers, or other antigenic proteins.
  • stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the target human or animal.
  • Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallade, the par ⁇ bens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • One or more of the above described vaccine components may be admixed r or adsorbed-with a conventional adjuvant.
  • the adjuvant is used as a non-specific irritant to attract leukocytes or enhance an immune response.
  • adjuvants include, among others, mineral oil and water, aluminum hydroxide, Amphigen, Avridine, L121/squalene, P-lactide-polylactide/glycoside, pluronic plyois, muramyl dipeptide, killed Bordetella , and saponins, such as Quil A.
  • Suitable amounts of the active ingredient can be determined by one of skill in the art based upon the level of immune response desired.
  • the vaccine composition contains between 1 ng to 1000 mg antigen, and more preferably, 0.05 ⁇ g to 1 mg per mL of antigen.
  • a vaccine composition of the invention may further comprise other, non-B. burgdorferi antigens, including, Bordetella bronchiseptica, canine parvovirus, canine distemper, rabies, Leptosporidia , canine coronavirus, and canine adenovirus.
  • Other vaccinal antigens originating from other species animals may also be included in these compositions, e.g., feline coronavirus, etc.
  • Suitable doses of the vaccine composition of the invention can be readily determined by one of skill in the art. Generally, a suitable dose is between 0.1 to 5 mL of the vaccine composition. Further, depending upon the human patient or the animal species being treated, i.e. its weight, age, and general health, the dosage can also be determined readily by one of skill in the art.
  • the vaccine will be administered once on a seasonal basis. Each tick season, usually in the spring, a booster should be administered.
  • the vaccine may be administered by any suitable route. However, parenteral administration, particularly intramuscular, and subcutaneous, is the preferred route. Also preferred is the oral route of administration.
  • the following examples illustrate the preferred methods for obtaining protein antigens of the invention and preparing the assays and compositions of the invention. Significantly, these examples indicate that the antigens of this invention have the potential to be more accurate diagnostic reagents than those presently in use.
  • the P17, P18, P39, and P43 proteins induced a non- MHC restricted response (with respect to B31 at least) in these examples and were crossreactive between at least two strains of Borrelia burgdorferi .
  • a P39 protein has already been cloned and a monoclonal antibody specific for P39 has been generated (Simpson and Schwan, unpublished) .
  • Example 1 Bacterial Strains and Their Growth
  • the JD1 strain of B. burgdorferi [J. Piesman et al., J. Clin. Microbiol.. 25:557-558 (1987) and T. G. Schwan et al.. J. Clin. Microbiol.. 22:1734-1738 (1989)] was maintained by alternating B. burgdorferi-infected J. dammini tick to hamster to tick passage for four repetitions in golden Syrian hamsters (SASCO, Omaha, NE) .
  • B. burgdorferi organisms were cultured at 34°C in a modified BSK II culture media [R. J. Sinsky et al.., ' J. Clin. Microbiol..-.: 27:1723-1727 (1989)].
  • This BSK II broth was further modified by adding 20 mg of cycloheximide per liter to selectively inhibit fungal contamination of cultured hamster ear biopsies.
  • the outer tick surfaces were first decontaminated with soaks in Wescodyne (Amsco Medical Products, Erie, PA) and 70% ethanol, followed by soaking in BSK II medium. Homogenates of infected Ixodes dammini nymphs, 2 to 3 weeks post-repletion, were then prepared by grinding 10 to 20 nymphs in 1 to 2 ml of phosphate- buffered saline (PBS) in a Ten Broeck homogenizer. Hamsters were inoculated i.p. or i.d., at multiple sites, with 0.4 ml of homogenate.
  • PBS phosphate- buffered saline
  • hamsters were inoculated i.d. with 0.1 ml of varying concentrations of organisms (1 x 10 4 to 1 x 10 8 cells/ml) .
  • the following assay was used with both the hamster studies of Examples 5 through 8 and the mice studies of Examples 9 through 13.
  • the ELISA assay was performed essentially as described in J. T. Roehrig et al.. Virology. 118:269-278 (1982)-, Briefly, antigens were coated on Immulon 2 plates (Dynatech, Inc., Kensington, MD) overnight at 4°c at a concentration of 2 ⁇ g/well for whole-cell sonicate, 60 ng/well for purified flagella, or 1 ⁇ g/well for recombinant OspA. The whole-cell sonicate was prepared according to H. Russell et al.. J. Infect. Pis..
  • ELISA antigen concentrations were determined in a standard box titration of antigen with positive or negative control sera. The antigen concentrations used were the minimum amounts demonstrating maximal binding of positive control sera throughout the aritisera dilution range. After coating with antigen, wells were then blocked for 1 hour at 37°C with 3% goat serum in PBS. Plates were rinsed with PBS with 0.5% Tween 20 (rinse buffer) . Bound hamster antibody was detected with goat anti-hamster IgG (H+L) peroxidase diluted 1:20,000 or 1:40,000 (Cappel Laboratories, Malvern, PA).
  • Substrate used to detect bound conjugate was 3,3',5,5'- tetramethylbenzidine .(TMB) /H 2 0 2 [T. F. Tsai et al.. J. Clin. Microbiol.. 2.6:2620-2625 (1988)]. Reactivity was stopped after 30 min by adding 50 ⁇ l/well of 2N H 2 S0 4 . Absorbance was measured at 450 nm in a Titertek Multiskan MC spectrophotometer (ICN Flow Laboratories, Costa Mesa, CA) .
  • Proteins were electroblotted onto Immobilon-P membranes (Millipore Corporation, Bedford, MA) for 1 hour at 100 mA in a Semiphor blotter (Hoeffer Scientific).
  • Murine monoclonal antibody tissue culture supernatants specific for p41, H9724; OspA, H5332; and OspB, H6831 (Symbicom, Umea, Sweden) were used at a 1:5 or 1:20 dilutions. Bound hamster antibody was detected with a goat anti-hamster IgG (H+L) peroxidase-labeled antibody (Cappel Laboratories, Malvern, PA) . Bound murine MAb was detected with goat anti-mouse IgG (F c ) peroxidase-labeled antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
  • Substrate used to detect bound peroxidase conjugate was a modified histochemical TMB/H 2 0 2 substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) or a chemiluminescent substrate (Amersham International, Arlington Heights, IL) . Stained blots were allowed to develop for 1 to 5 minutes and the reaction was stopped by rinsing in water. For the chemiluminescent substrate, the blot image was recorded by fluorography using HyperfilmTM-ECL (Amersham International, Arlington Heights, IL) .
  • mice were infected either by needle inoculation or by tick feeding as described in Examples l and 2 above. Both the type and quantity of antigens used for infection were varied. Animals were inoculated i.p. or i.d. with culture-grown bacteria or with homogenates of B. burgdorferi-infected J. dammini ticks. The number of infected ticks used for hamster infection was also varied to simulate a natural dose response.
  • the immunization protocol for this animal consisted of subcutaneous inoculation in two locations with 50 ⁇ g antigen in Freund's complete adjuvant. Two weeks later animals were boosted with 50. ⁇ g antigen in Freund's incomplete adjuvant.
  • the preinfection titer of this animal as measured in whole- cell sonicate ELISA was 1:320,000.
  • column 4 indicates number of infected ticks used for feeding or homogenate production, or concentration (log 10 ) of pure spirochetes
  • column 5 indicates number of days postinfection when serum samples were taken
  • column 6 lists the reciprocal end-point ELISA titers on whole-cell sonicate (Son) or purified flagella antigen.
  • Pre-immunization titers for all animals were ⁇ 40 on whole-cell sonicate, ⁇ 20 on OspA, and ⁇ 10 on flagella antigen.
  • the serum specimens from hamsters having high whole-cell sonicate ELISA titers (3, 8, 9, 14, 15, 16 and 17) were diluted 1:100, while all other sera were diluted 1:50 to better compare antibody activity in immunoblots, which were performed as described in Example 4. Bound antibodies were detected with the TMB/H 2 0 2 histochemical substrate. Tick-infected hamsters were bled at weekly intervals.
  • Anti-P43 antibody was detected at one week by immunoblot and appeared to remain stable throughout the infection. Very little anti-OspA antibody and no anti- OspB antibody could be detected by immunoblot through 19 weeks P.I. At 42 weeks P.I., the single surviving hamster had very little demonstrable antibody to either OspA or OspB. This hamster was, however, still culture positive. Antibody to the P39 antigen was not detected until 2-4 weeks P.I. Anti-P39 was still present late in infection.
  • Example 7 Antibody Dose-Response to Various Amounts of Needle Inoculated Cultured B. burgdorferi
  • an antigen dose-response analysis was performed using needle inoculation of varying amounts of culture grown B. burgdorferi (3.0 to 7.0 log 10 cells). Animals were bled at 2 and 4 weeks post-immunization. Pre-immunization titers for all animals were ⁇ 40 on whole-cell sonicate and ⁇ 20 on OspA antigen.
  • All animals inoculated with 1 x 10 6 or 1 x 10 7 cells were both culture positive for B. burgdorferi and seropositive in whole-cell sonicate ELISA.
  • One of five animals inoculated with 1 x 10 5 cells was culture positive, but all five were seropositive in whole-cell sonicate ELISA.
  • Two of five animals inoculated with 1 x 10 4 cells were culture positive, but three animals were seropositive in whole-cell sonicate ELISA.
  • All animals inoculated with 1 x 10 3 bacteria were culture negative and failed to raise detectable antibody when measured in whole-cell sonicate ELISA.
  • the LD 50 of this experiment was 1.3 x 10 5 cells/ nimal as determined by ear biopsy isolation.
  • OspA antibody as measured in OspA ELISA could be detected only after inoculation of 1 x 10 6 or 1 x 10 7 cells (Fig. 2) .
  • the immunoblot therefore, appeared to be more sensitive in detecting OspA antibodies than the ELISA.
  • a direct comparison of antibody responses to P43, P41, P39, OspB and OspA was performed by comparing the immunoblot reactivities observed by quantitative densitometry. Amounts of antibody bound in immunoblots were determined by integration of peak areas from densitometer scans using a Gilford 2600 spectrophotometer. The amount of detected anti-P43 was standardized to 1.0. For comparison to anti-P43 antibody, the amounts of other protein specific antibodies were normalized to the P43 value of 1.0. All sera were used at a 1:40 dilution.
  • mice of ages 4-6 weeks were obtained from Jackson Laboratories, Bar Harbor, MA (BALB/c, BIO, B10.BR, and B10.D2) or from the specific pathogen free (SPF) colony of ICR outbred mice maintained at DVBID, NCID, CDC at Fort Collins, CO. These four recombinant inbred strains and one outbred strain of mice were used as infection targets for transmission of B. burgdorferi infection by Ixodes ticks. The mice used in this study were BALB/c (H-2 d ) ,
  • BALB/c and BIO mice differ in their immunoglobulin (Ig) gene allotypes (BALB/c has Ig allotype "a”; BIO have Ig allotype "b") and the outbred mice have mixed MHC and Ig haplotypes.
  • Ig immunoglobulin
  • mice from each strain as well as ICR outbred mice were numbered by ear punch and preimmune serum samples were drawn. Each mouse was exposed to ten to twelve B. burgdorferi , strain B31 infected ticks as described above in Example 9. Table IV shows the number of ticks that successfully attached and fed on each animal as well as the results of culturing ear punch biopsies for the Borrelia spirochete 30 days post- infection.
  • Example 11 Response of Inbred Strains of Mice to Infection with B. burgdorferi Resulting from Tick Bite using the Homologous Bacterial Strain as Antigen
  • Serum samples were drawn from each mouse on days 5, 12, 19, 26, and 60 post infection and these serums were used in western blot analysis using whole lysates of Borrelia burgdorferi , strain B31, low passage (80 ⁇ g) , as antigen.
  • Samples were run in a discontinuous SDS-PAGE system using a Hoefer Scientific Instruments model SE600 vertical slab gel electrophoresis system. The stacking and resolving gels were 4% and 10% acrylamide respectively.
  • Bacterial lysates were run at 80 ⁇ g/gel in a single preparative well. Molecular weight standards (14 to 106 kD) were run in flanking lanes.
  • mice were added for exactly 5 minutes to detect bound alkaline phosphatase.
  • Preimmune sera as well as day 5 sera from all animals were negative in western blot using B. burgdorferi strain B31.
  • all mice began to generate antibodies that can be found in serum to a variety of Borrelia proteins. Further, the inbred strains of mice react differentially to these antigens.
  • BIO animals all show a strong response to a protein migrating at a molecular weight of just above 80kD, most likely the P83 reported by Dorward et al., cited above. The balance of the animals in this study have not responded to this protein at this stage of infection.
  • Example 12 Early response induced bv proteins in the 39kD molecular weight range
  • OspA, OspB, or flagellin was detected, all proteins which induce an early response from these as well as other strains of mice when challenged by needle inoculation of cultured Borrelia burgdorferi [U. E. Schaible, et al. , Eur. J. Immunol.. .21:2397-2405 (1991), E. Fikrig, et al. , Infection and Immunity. 5_3.:713-714 (1986)].
  • mice also maintained the response to P43 and a band(s) migrated at about 52 to 55kD. BIO.BR, and to a lesser degree B10.D2, detected a band slightly higher, at 58kD, and as on day 12, only the BIO animals responded to the 83kD band.
  • Example 13 Response to a heterologous strain of Borrelia burgdorferi Western blot analysis of sera from -five mouse strains binding B. Jburgdorferi, a low passage isolate of strain JP-1, was performed. The results of this analysis showed the ability of these sera to detect the same antigens from a different strain of B . burgdorferi . Sera drawn on day 12 showed no response to the five low molecular weight bands and a similar response to the band at 30kP as with the B31 low passage material. All mice responded to the P39 bands and there was a very marginal detection of P43. There was also a good response to P58 band.
  • the antigen capture ELISA was evaluated by varying a number of different parameters in the test including plates, both polystyrene (Immulon II) and polyvinyl chloride plates (Dynatech, Costar, catalogue numbers 2597 and 2787) .
  • Blocking reagents tested included BSA (1%, 1.5%, 2.5%, 5%), Casein (0.5%, 1%), 0.5% Boiled Casein and 0.5% Boiled Casein with 0.05% Tween 20; powdered skim milk (2.5%, 5%) with or without 0.05% Tween 20.
  • Monoclonal antibodies tested for antigen capture included antibodies against OspA (184.1 and H5332), OspB [H6831], the 41 kd flagellin (H9724) , the p60 molecule (149.1). These antibodies are available commercially from Symbicom (Umea, Sweden) .
  • Polyclonal antisera against B. burgdorferi made in rabbits and hyperimmune ascites made in mice were biotin-labelled according to the manufacturer's recommendations (Amersha ) and evaluated for signal recognition together with both streptavidin-horseradish peroxidase conjugates and streptavidin-alkaline phosphatase conjugates with ABTS and TMB as substrates for horseradish peroxidase and Sigma 104-105 as substrate for alkaline phosphatase.
  • the protocol for the optimal antigen capture ELISA was as follows.
  • Wells of polyvinylchloride plates (Costar) were coated with 50 ⁇ l of protein-A purified Mab H5332 at a concentration of 10 ⁇ g/ml in phosphate buffered saline, pH 7.4 (PBS). Following an overnight incubation at 4°C, wells were washed 3 times with PBS with 0.05% Tween 20 (PBST) and 200 ⁇ l of 2.5% skim milk in PBS with 0.05% Tween 20 were added to each well for one hour at room temperature. Laboratory strains of Borrelia to be tested in the assay were grown in BSKII and washed 3 times with PBS prior to counting.
  • PBST 0.05% Tween 20
  • NP40-PBS nonidet P-40 in PBS
  • BSA-PBS bovine serum albumen
  • Ixodes dammini nymphs to be assayed were homogenized in 20 ⁇ l of NP40-PBS in microtubes (Biorad) using a pestle shaped to fit the tube and attached to a power drill. Homogenization was facilitated by the addition of several grains of sterile sand to the microtube. 130 ⁇ l of BSA-PBS was then added to nymph extracts.
  • Adult I . dammini were homogenized in 50 ⁇ l of NP40-PBS as described and 250 ⁇ l of BSA-PBS were added.
  • Wells were emptied by aspiration and 50 ⁇ l of Borrelia spp or tick extracts to be tested were added to individual wells for 2 hours at room temperature (RT) .
  • Wells were then washed 3 times with PBST and 50 ⁇ l of biotin-labelled polyclonal rabbit serum at a .concentration of 2.5 ⁇ g/mL was added to each well for one hour at RT.
  • streptavidin-horseradish peroxidase (Amersham) at a 1:3000 dilution in BSA-PBS was added to each well.
  • the sensitivity of the assay is approximately 50-200 spirochetes.
  • the ELISA was able to distinguish B. Jburgdorferi-infected from uninfected I . dammini (larvae, nymphs or adults) using a third of a tick extract, thereby leaving adequate material for confirmational testing.
  • the assay did not recognize tick antigens of I . dammini , I . ⁇ capularis , I . pacificus , I . kingi or Ambylomma americanum .
  • Infected I . dammini nymphs were estimated to harbor between 1000 and 10000 B . burgdorferi spirochetes.
  • the assay was able to detect B. burgdorferi in ticks that had been stored frozen, dried at room temperature, or in 70% ethanol.
  • mice For this preliminary experiment, four strains of outbred mice, BALB/c, B10, and C3H were used. These animals were infected by placing up to ten B31 infected ticks on each animal. Three weeks after tick infestation, the mice were assayed for infection by taking an ear punch biopsy and culturing the tissue in BSKII media for spirochetes. After one week, all mice were positive for Borrelia by culture, indicating a systemic infection in the corresponding mouse. Infected mice were then treated with tetracycline at l mg/mL in the drinking water for two weeks. Tetracycline water was changed three times per week because of the light sensitivity of the antibiotic.
  • mice cultured negative indicating that they had been cured of the infection.
  • mice were assayed for infection three weeks after the second tick challenge. This was done as before by taking an ear punch biopsy and culturing the tissue in BSKII media. The results of this experiment are shown in Table V below. The results indicated that reinfection is likely even though the mice were infected with the same strain of Borrelia . Further, mice that did not get reinfected showed 40% or more of the feeding ticks were cleared of the infection, presumably as a result of feeding on an immune animal. This conclusion is based on the observation that no significant clearing was observed in the ticks that fed on age matched naive control mice and all of those animals became infected.
  • the source of B cell blasts for B cell fusions for monoclonal antibodies to antigens of Borrelia burgdorferi were spleen cells from mice infected with B. burgdorferi by exposure to infected ticks. These mice were shown to be infected, cured by administering tetracycline (see Example 15) , and exposed a second time to infected ticks. The spleen cells were harvested five to seven days after the second exposure (post tick drop off) .
  • the first fusion used immune B cells from a BALB/c mouse fused to the B cell lymphoma, P3X63Ag8. This fusion was named BB31 for BALB/c anti-B31.
  • BB31 for BALB/c anti-B31.
  • Six monoclonal antibodies have been isolated from this fusion.
  • the second fusion was done by the same protocol using a C57B1/10 (BIO) mouse as a source of immune B cells. This fusion was designated B10B31 for BIO anti- B31. From this fusion five more monoclonal antibodies have been isolated. A summary of these antibodies and their specificity is given in Table VI.

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  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Communicable Diseases (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
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Abstract

La présente invention se rapporte à de nouveaux antigènes isolés de B. burgdorferi qui ont été régulés et différenciés dans une tique vecteur. Ces antigènes permettent de diagnostiquer la maladie de Lyme et peuvent être utilisés dans des compositions destinées à la prophylaxie de cette maladie.
EP93914155A 1992-05-26 1993-05-26 Compositions utilisees dans le diagnostic et la prophylaxie de la maladie de lyme. Withdrawn EP0643586A4 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US88901592A 1992-05-26 1992-05-26
US889015 1992-05-26
US94446492A 1992-09-14 1992-09-14
US944464 1992-09-14
US99289692A 1992-12-18 1992-12-18
US992896 1992-12-18
PCT/US1993/004984 WO1993024145A1 (fr) 1992-05-26 1993-05-26 Compositions utilisees dans le diagnostic et la prophylaxie de la maladie de lyme

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EP0643586A1 true EP0643586A1 (fr) 1995-03-22
EP0643586A4 EP0643586A4 (fr) 1996-06-26

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EP (1) EP0643586A4 (fr)
JP (1) JPH08503194A (fr)
AU (1) AU4392093A (fr)
CA (1) CA2135800A1 (fr)
WO (1) WO1993024145A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0880543A4 (fr) * 1995-10-26 2000-08-02 Rhode Island Education Antigenes servant de candidats vaccins et tires du spirochete (borrelia burgdorferi) de la maladie de lyme induit par la salive d'une tique vecteur (ixodes scapularis)
CA2668405C (fr) 2006-11-03 2015-05-05 Schering-Plough Ltd. Vaccin contre la maladie de lyme canine

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
AU7058691A (en) * 1989-12-22 1991-07-24 Mikrogen Molekularbiologische Entwicklungs-Gmbh Immunologically active proteines from borrelia burgdorferi, related test kits and vaccine

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
INFECTION AND IMMUNITY, vol. 56, no. 8, August 1988, WASHINGTON US, pages 2047-2053, XP002001271 K. HANSEN ET AL.: "IMMUNOCHEMICAL CHARACTERIZATION OF AND ISOLATION OF THE GENE FOR A BORRELIA BURGDORFERI IMMUNODOMINANT 60-KILODALTON ANTIGEN COMMON TO A WIDE RANGE OF BACTERIA." *
INFECTION AND IMMUNITY, vol. 57, no. 9, September 1989, WASHINGTON US, pages 2733-2741, XP002001273 V.G. BUNDOC ET AL.: "CLONAL POLYMORPHISMS OF THE OUTER MEMBRANE PROTEIN OspB OF BORRELIA BURGDORFERI." *
INFECTION AND IMMUNITY, vol. 58, no. 6, June 1990, WASHINGTON US, pages 1711-1719, XP002001272 R. WALLICH ET AL.: "THE BORRELIA BURGDORFERI FLAGELLUM-ASSOCIATED 41-KILODALTON ANTIGEN (FLAGELLIN): MOLECULAR CLONING, EXPRESSION, AND AMPLIFICATION OF THE GENE." *
INFECTION AND IMMUNITY, vol. 60, no. 2, February 1992, WASHINGTON US, pages 657-661, XP002001270 E. FIKRIG ET AL.: "ROLES OF OspA, OspB, AND FLAGELLIN IN PROTECTIVE IMMUNITY TO LYME BORRELIOSIS IN LABORATORY MICE." *
JOURNAL OF CLINICAL MICROBIOLOGY, vol. 27, no. 10, October 1989, WASHINGTON, D.C., US, pages 2344-2349, XP000568737 R.S. LANE ET AL.: "ANTIGENIC CHARACTERISTICS OF BORRELIA BURGDORFERI ISOLATES FROM IXODID TICKS IN CALIFORNIA." *
JOURNAL OF IMMUNOLOGY, vol. 140, no. 1, 1 January 1988, BALTIMORE US, pages 265-272, XP002001274 J.L. BENACH ET AL.: "A MURINE IgM MONOCLONAL ANTIBODY BINDS AN ANTIGENIC DETERMINANT IN OUTER SURFACE PROTEIN A, AN IMMUNODOMINANT BASIC PROTEIN OF THE LYME DISEASE SPIROCHETE." *
JOURNAL OF IMMUNOLOGY, vol. 149, no. 11, 1 December 1992, BALTIMORE US, pages 3648-3653, XP002001275 J.T. ROEHRIG ET AL.: "THE HAMSTER IMMUNE RESPONSE TO TICK-TRANSMITTED BORRELIA BURGDORFERI DIFFERS FROM THE RESPONSE TO NEEDLE-INOCULATED, CULTURED ORGANISMS." *
See also references of WO9324145A1 *
THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 177, no. 1, January 1993, NEW YORK, N.Y., US, pages 9-17, XP000568811 W.T. GOLDE ET AL.: "THE MAJOR HISTOCOMPATIBILITY COMPLEX-RESTRICTED RESPONSE OF RECOMBINANT INBRED STRAINS OF MICE TO NATURAL TICK TRANSMISSION OF BORRELIA BURGDORFERI." *

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WO1993024145A1 (fr) 1993-12-09
AU4392093A (en) 1993-12-30
EP0643586A4 (fr) 1996-06-26
CA2135800A1 (fr) 1993-12-09
JPH08503194A (ja) 1996-04-09

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