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WO1994020536A1 - Methodes, compositions et trousses pour le diagnostic de la maladie de lyme - Google Patents

Methodes, compositions et trousses pour le diagnostic de la maladie de lyme Download PDF

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Publication number
WO1994020536A1
WO1994020536A1 PCT/US1994/002889 US9402889W WO9420536A1 WO 1994020536 A1 WO1994020536 A1 WO 1994020536A1 US 9402889 W US9402889 W US 9402889W WO 9420536 A1 WO9420536 A1 WO 9420536A1
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Prior art keywords
burgdorferi
polypeptide
antibody
antigen
app
Prior art date
Application number
PCT/US1994/002889
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English (en)
Inventor
Rance B. Lefebvre
Guey-Chuen Perng
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The Regents Of The University Of California
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Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU63668/94A priority Critical patent/AU6366894A/en
Publication of WO1994020536A1 publication Critical patent/WO1994020536A1/fr

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    • 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
    • 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
    • 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 a 22-26 Kdalton (Kd) antigenic peptide capable of eliciting protective immunity against Borrelia burgdorferi (B. burgdorferi) .
  • This invention further relates to the recombinant gene encoding the antigenic peptide of the invention, to a self- replicating DNA comprising the gene, to a transformed host and to antibodies specific for the antigenic peptide of the invention.
  • the present invention is useful for detecting the presence of B. burgdorferi in a biological sample, e.g., via diagnostic kits, and for vaccination against B. burgdorferi.
  • Lyme disease was first described in the late 1970's as a unique grouping of arthritic symptoms in patients from Lyme, Connecticut. The disease was shown to be caused by infection with Borrelia burgdorferi, a spirochete, following exposure to deer (ixodid) ticks. It is now known that lyme disease in humans is a multi-systemic disorder characterized by dermatologic, rheumatologic, cardiac, and neurologic manifestations. The existence of B. burgdorferi has now been demonstrated throughout the world. Several strains of B. burgdorferi from North -America and Europe have been characterized at both the genetic and antigenic level. However, the unequivocal diagnosis of lyme disease remains problematic.
  • B. burgdorferi has been reported to be cross-reactive with other related human pathogens, such as 3. hermsii (the causative agent of tick- born relapsing fever) and Treponema palladium (the causative agent of syphilis) .
  • hermsii the causative agent of tick- born relapsing fever
  • Treponema palladium the causative agent of syphilis
  • B. burgdorferi isolates are routinely characterized by specific monoclonal antibodies or by genetic analysis. Spirochete isolates have been shown to have great diversity at the genetic and antigenic levels, including chromosomal and plasmid levels. Multiple linear plasmids unique to the Borrelia species were shown to express the major surface antigens. In addition, linear plasmids in the relapsing fever Borrelia were shown to encode the genes responsible for antigenic variation. Various antigens from B. burgdorferi have been identified recently. A 20 to 22 Kd antigen expressed in B. burgdorferi, referred to as the pC protein, was described by Fuchs et al.
  • the antigen is described as a soluble protein present in the periplasmic space, which is easily extracted from damaged cells or cells which have lost their outer surface.
  • Coleman and Benach recently described a 22 Kd B. burgdorferi antigen which is immunologically highly cross-reactive with other bacterial antigens (Coleman and Benach, "Characterization of Antigenic Determinants of Borrelia burgdorferi Shared by Other Bacteria", J. Infect. Dis. 165:658-666 (1992)).
  • the existence of some immunodominant conserved antigens of B. burgdorferi that are recognized during the known stages of human infection was shown by immunochemical methods.
  • One immunodominant antigen was found to have a 79 to 83 Kd apparent molecular weight (app. MW) .
  • the gene encoding this antigen was described and characterized by LeFebvre et al. and Perng et al. (LeFebvre et al., "The 83- Kilodalton Antigen of Borrelia burgdorferi which Stimulates Immunoglobulin M (IgM) and IgG Responses in Infected Hosts is Expressed by a Chromosomal Gene", J. Clin. Microbiol.
  • B. burgdorferi antigens are either different from the present invention or have been obtained and described in an impure form that has, therefore, not being characterized to a point that a comparison with the present invention is possible.
  • This invention relates to a pure, isolated polypeptide comprising an antigenic peptide selectively binding to an antibody raised against a 22 to 26 Kd app. MW conserved B. burgdorferi antigen.
  • the polypeptide may be glycosylated or substantially free of carbohydrate.
  • This invention further relates to a composition of matter comprising the polypeptide of this invention, and a non-proteolytic carrier, and optionally, a pure, isolated polypeptide comprising a peptide selectively binding to an antibody raised against a 79 to 83 Kd app. MW conserved B. burgdorferi antigen.
  • This invention further relates to a diagnostic kit for detecting B. burgdorferi antibodies comprising the antigenic peptide of the invention, and instructions for its use, and optionally an anti-antigenic peptide antibody.
  • an anti-B. burgdorferi vaccine comprising in separate containers the antigenic peptide of the invention, a pharmaceutically acceptable, non-proteolytic carrier, and instructions for its use.
  • the polypeptide of the invention may be used for detecting the presence of antibodies selectively binding to the 22 to 26 Kd app. MW conserved B. burgdorferi antigenic peptide or fragments thereof disclosed herein by contacting it with a biological sample, and detecting the formation of any complexes formed by the polypeptide with sample components.
  • the present polypeptide may also be used to inhibit or counter B. burgdorferi infection by administering to a subject in need of the treatment an effective amount of the polypeptide of the invention.
  • the invention in another aspect, relates to a pure, isolated polynucleotide, comprising a DNA or RNA segment which encodes the polypeptide of the invention.
  • the deoxypolyribonucleotide of the invention may be contained in a self-replicating DNA which may, in turn, be used to transfect a host organism.
  • the polynucleotide is also provided in the form of a composition with a carrier, and optionally, a second pure, isolated polynucleotide comprising a DNA encoding a 79 to 83 Kd app. MW conserved B. burgdorferi antigen.
  • the invention relates to a diagnostic kit for detecting B. burgdorferi comprising the deoxypolyribonucleotide of the invention, and instructions for its use.
  • the deoxypolyribonucleotide may be used for detecting B. burgdorferi comprising contacting it with a biological sample under stringent hybridization conditions, and detecting the formation of any hybrids.
  • Another aspect of the invention is an antibody that selectively binds to the antigenic polypeptide described above.
  • the antibody may be polyclonal or monoclonal, and it may also be provided as a composition with a non- proteolytic carrier, optionally also containing other antibodies such as an antibody that selectively binds to a 79 to 83 Kd app. MW conserved B. burgdorferi antigen.
  • the antibody of the invention may be used in a diagnostic kit for detecting a 22 to 26 Kd app. MW B. burgdorferi conserved antigenic peptide with instructions for its use, and optionally, also an antibody selectively binding the 79 to 83 Kd app. MW B. burgdorferi conserved antigen.
  • the antibody provided herein may also be used for inhibiting or countering B. burgdorferi infection by administering to a subject in need of the treatment an effective amount of the antibody.
  • the invention moreover, relates to a vaccine for passive immunization against B. burgdorferi comprising in separate containers the antibody of the invention and a pharmaceutically acceptable, non-proteolytic carrier, and instructions for its use.
  • the antibody of the invention may be used for detecting the presence of B. burgdorferi or conserved antigenic peptides thereof by contacting it with a biological sample, and detecting the formation of any complexes formed by the antibody with sample components.
  • Spirochetal diseases have been medically difficult to approach given the difficulties of achieving reliable vaccine prophylaxis, accurate diagnosis, and successful therapy.
  • the time required and the difficulty involved in culturing the pathogenic organisms are responsible for these difficulties, as is the fact that cultured spirochetes may lose or no longer express key virulence factors and immunogens when cultured in vitro.
  • a further source of concern is the wide range of genetic and antigenic diversity in B. burgdorferi isolates.
  • the present inventors have unexpectedly found a system that utilizes a highly conserved characteristic of the spirochete without requiring the continuous growth of the spirochete in vitro.
  • the present invention relies on the finding by the inventors of a chromosomal gene producing an immunocloruinant antigen, which is generally conserved and expressed in B. burgdorferi, and which the inventors have successfully used to detect innumerable isolates of the spirochete.
  • chromosomal genes encoding immunodominant antigens are surprising since most genes encoding major Borrelia burgdorferi antigens were located on linear plasmids, and would therefore not be expected to be as widely conserved as chromosomal genes.
  • a computerized amino acid analysis of the conserved antigen provided herein indicates that it may be located either in the periplasmic space and/or the outer surface.
  • the antigen is shown by serological assays to be expressed by B. burgdorferi in naturally exposed hosts. Thus, it may be administered to a host to elicit protective immunity against B. burgdorferi infection.
  • the antibodies raised against the antigen of the invention are also suitable as diagnostic or prophylactic agents to detect or treat Lyme borreliosis in humans.
  • the present invention thus, arose from the cloning, characterizing, and sequencing of a B. burgdorferi highly conserved chromosomal gene and the isolation and characterization of a purified polypeptide encoded by the gene.
  • the present polypeptide comprises an antigenic peptide selectively binding to an antibody raised against a 22 to 26 Kd app. MW conserved B. burgdorferi antigen, any remaining sequence being unrelated to the peptide.
  • a molecular weight of about 22 Kd has been determined by denaturing gel electrophoresis.
  • This polypeptide represents the carbohydrate-free form of the antigen.
  • a molecular weight of about 26 Kd is calculated.
  • This may represent a polypeptide also containing the leader sequence which is detached from the 22 Kd segment observed by gel electrophoresis.
  • the peptide comprises the amino acid secjuence shown in Table 1 below Table l: Amino Acid Sequence of Preferred Peptide
  • the polypeptide of the invention may be obtained by expression of a deoxypolyribonucleotide or polyribonucleotide encoding it or by amino acid synthesis by methods known in the art.
  • the 22 to 26 Kd app. MW conserved B. burgdorferi antigen may be obtained by isolation from the microorganism as is shown in the exemplary disclosure hereinbelow.
  • the polypeptide comprises at least one determinant or epitopic site which is characteristic of the naturally-occurring 22 to 26 Kd app. MW conserved B. burgdorferi antigen.
  • characteristic it is meant that the determinant allows the immunological detection of the polypeptide in a biological sample with reasonable assurance, in most cases allowing the immunologically distinction of B.
  • the polypeptide of the invention finds applications in an anti-B. burgdorferi vaccine, in the preparation of polyclonal and monoclonal antibodies, and in the detection of antibodies selectively binding to it. Each of these uses is described in more detail hereinafter.
  • the polypeptide of the invention is preferably identical or equivalent to at least a portion of Seq ID No. 2.
  • equivalent it is meant that a substantial identity of amino acids exists over the sequence minus the leader peptide, or that each position of the corresponding sequences is either identical to, or has conservative substitutions in, at least about 60% of the residues, preferably at least about 70% of the residues, and more preferably at least about 80% of the residues.
  • the polypeptide sequences may be modified by occasional deletions, additions, or replacements in accordance with known methods. (Sequence Analysis Software Package, University of Wisconsin Biotechnology Center, Madison, WI) . Conservative substitutions or replacements within the following groups are contemplated, among others.
  • Synthetic polypeptides which are immunologically cross- reactive with the natural 22 to 26 Kd app. MW antigen may be produced by at least two general approaches.
  • polypeptides having fewer than about 100 amino acids, more usually having fewer than about 50 amino acids may be synthesized by the Merrifield solid-phase synthesis method where amino acids are sequentially added to a growing chain (Merrifield, J. Am. Chem. Soc. 85:2149-2146 (1963)).
  • the e-quipment for automatically synthesizing polypeptides is commercially available from suppliers such as Applied Biosystems, Inc., Foster City, California; DuPont Company, Biotechnology Systems, Wilmington, DE; and MilliGen/Biosearch, a division of Millipore, Burlington, MA.
  • An alternative for synthesizing the polypeptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a polypeptide comprising the 22 to 26 Kd app. MW conserved B. burgdorferi antigen, analogues thereof, or antigenic or immunogenic fragments thereof lacking the leader sequence encompassing amino acids 1 to 25 of Table 1 above. Expression can be established by standard procedures (Crowl et al.,
  • the gene itself may be natural or synthetic, as described above.
  • the natural gene is obtainable from cDNA or genomic libraries using standard techniques.
  • probes synthesized based on Seq. ID No. 1 provided below may be used to identify and isolate genes representing other alleles of the gene encoding the 22 to 26 Kd app. MW conserved antigen using well known methods.
  • the polypeptides are usually obtained in pure form. That is, the polypeptides are preferably at least about 50 wt% pure, and substantially free of interfering proteins and contaminants.
  • the polypeptides of the present invention are preferably isolated or synthesized to a purity of at least about 80 wt%, and more preferably to at least about 95 wt% or greater purity.
  • conventional protein purification techniques including denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) , homogeneous polypeptide compositions of at least about 99 wt% purity may be obtained.
  • the polypeptides may be purified with the antibodies described hereinafter by affinity chromatography.
  • affinity chromatography may be performed by first linking antibodies to a solid support and then contacting the solid support with the source of the polypeptides, e.g., lysates of B. burgdorferi, which naturally produce the 22 to 26 Kd app. MW conserved antigen or lysates of cultured cells in which the 22 to 26 Kd app. MW conserved antigen, analogues thereof, or antigenic or immunogenic fragments thereof have been recombinantly produced.
  • a fusion protein comprising the polypeptide of the invention and a second pure, isolated polypeptide comprising a peptide selectively binding to an antibody raised against a 79 to 83 Kd app. MW conserved B. burgdorferi antigen operatively bound thereto.
  • the fusion protein may be prepared by cloning different antigen or immunogenic portions of DNAs encoding the polypeptide of the invention and the 79 to 83 Kd polypeptide with or without the leader sequence.
  • the characteristics and sequence of the 79 to 83 Kd app. MW conserved B. burgdorferi antigen are disclosed in LeFebvre et al. and Perng et al. (LeFebvre et al. (1990), supra; Perng et al. (19S1) , supra) the entire text of which relating to the 79 to 83 Kd polypeptide is included herein by reference.
  • the polypeptide of the invention is also provided as a composition of matter with a non-proteolytic carrier.
  • Non-proteolytic carriers are known in the art and need not be further described herein.
  • the present composition may also comprise a second purified, isolated polypeptide such as a peptide selectively binding to an antibody raised against a 79 to 83 Kd app. MW conserved B. burgdorferi antigen.
  • Still part of this invention is a diagnostic kit for detecting B. burgdorferi antibodies that comprises the antigenic polypeptide of this invention and instructions for its use.
  • the diagnostic kit may also comprise an anti- 22 to 26 Kd B. burgdorferi antigen antibody, the 79 to 83 Kd app. MW conserved B. burgdorferi peptide or fragments thereof, and a solid substrate. Other components may also be included.
  • an anti-B. burgdorferi vaccine comprising in separate containers the polypeptide of the invention that is capable of eliciting an immunological response against B. burgdorferi when administered to a susceptible host, and a pharmaceutically acceptable non- proteolytic carrier, and instructions for its use.
  • the vaccine may also comprise other peptides conferring immunity against other pathogens and/or the polypeptide selectively binding to an antibody raised against the 79 to 83 Kd B. burgdorferi antigen.
  • the vaccine may be administered as is known in the art in a single dose or it may also be, later on, administered as a booster for increasing and maintaining the immunological response elicited in a subject.
  • the amount of the polypeptide administered to a subject such as a human, may be 50 to 450 ⁇ g/kg body weight/dose, and more preferably 150 to 300 ⁇ g/kg/dose. However, other amounts are also suitable and the dose may be repeated as deemed appropriate by the practitioner.
  • the polypeptide of this invention may be used for detecting the presence of anti-B. burgdorferi antibodies selectively binding to the 22 to 26 Kd app. MW B. burgdorferi conserved antigenic peptide by contacting it with the biological sample, and detecting the formation of any complexes formed by the polypeptide with sample components.
  • the polypeptide may also be provided as a mixture with other B. burgdorferi polypeptides such as the 79 to 83 Kd antigenic polypeptide, analogues thereof or fragments thereof.
  • the biological sample may be serum, urine, saliva, tears, mucose of various origins, and feces, among others. This method may be practiced with the diagnostic kit provided above.
  • the polypeptide of the invention may also be used for inhibiting or countering B. burgdorferi infection by administering to a subject in need of the treatment an effective amount of the polypeptide of the invention, optionally with the polypeptide selectively binding to the anti-79 to 83 Kd B. burgdorferi antigen antibody.
  • the polypeptide may be administered in an amount of about 100 to 400 ⁇ g/kg body weight/day, and more preferably about 200 to 300 ⁇ g/kg body weight/day. Other amounts may also be administered.
  • polynucleotide comprising a DNA or RNA segment encoding the polypeptide of the invention.
  • the polynucleotide of the invention may comprise a DNA segment about 961 base pairs long or about 936 base pairs long lacking the segment encoding the leader sequence.
  • the polynucleotide comprises a DNA segment that has the sequence shown in Table 2 below or redundant sequences thereof or variations thereof that correspond to the permutations, deletions or additions described above for the polypeptide.
  • Another aspect of the invention is a hybrid polynucleotide comprising the polynucleotide of the invention and a second pure, isolated polynucleotide comprising a DNA encoding the 79 to 83 Kd app. MW conserved antigen of B. burgdorferi, operatively linked thereto.
  • the polynucleotides may be prepared by methods known in the art, for example, by isolation of the corresponding DNA fragments from B. burgdorferi and enzyme restriction cutting and ligation.
  • the polynucleotides and hybrid derivatives may also be prepared by cloning DNA fragments and/or synthesis as is known in the art.
  • the polynucleotide of the invention may be provided in a diagnostic kit for B. burgdorferi that also comprises instructions for its use, and optionally a polynucleotide associated with the 79 to 83 Kd antigen of B. burgdorferi.
  • This kit may be utilized for DNA hybridization with B. burgdorferi present in a biological sample obtained from a patient under stringent conditions in order to identify in a selective manner the corresponding conserved genomic sequences of B. burgdorferi.
  • polynucleotide encoding the 22 to 26 Kd app. MW polypeptide of the invention is contained within a 1.65 kb Clal restriction fragment of the chromosomal DNA of B. burgdorferi, reference strain B31.
  • nucleic acids of the invention including deoxypolyribonucleotides and polyribonucleotides, encoding the polypeptides described above, may be expressed in cultured cells to provide isolatable quantities of the polypeptides.
  • Other useful nucleic acids are those that are substantially homologous to the sequences encoding the 22 to 26 Kd antigen, with or without the leader peptide encoding segment, or fragments thereof, and may be either natural or synthetic. Such homologous nucleic acids may find use as probes or primers for locating or characterizing natural or synthetic nucleic acids encoding the 22 to 26 Kd app. MW conserved antigen or fragmentary antigenic peptides thereof.
  • Nucleic acid sequences are considered to be substantially homologous when one of the following conditions is fulfilled.
  • Stringent conditions are known in the art and need not be further described herein.
  • the combination of temperature and salt concentration is more important in defining stringency than either the temperature or the salt concentration alone.
  • Other conditions which affect stringency include the GC content of the sequence, the extent of complementarity of the sequences, the length of the sequences involved in the hybridization, and the composition of buffer solution(s) used in the hybridization mixture. These and other factors affecting stringency are well described in the scientific and patent literature.
  • the nucleic acids of the present invention may be synthesized, based on the DNA sequence provided as Seq. ID No. 1, using well known synthetic techniques.
  • short, single-stranded DNA fragments oligonucleotides
  • oligonucleotides may be prepared by the phosphoramadite method described by Beaucage and Carruthers (Beaucage and Carruthers, Tett. Lett. 22:1859-1862 (1981), the corresponding section of which is incorporated herein by reference) .
  • a dcuble- stranded fragment may then be obtained by either synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase and an appropriate primer sequence.
  • the polymerase chain reaction ' technique may also be used for the production of probes and/or the amplification of polynucleotides for synthetic purposes.
  • Suitable primer pairs for use with the polymerase chain reaction may be obtained based on the DNA sequence provided as Seq. ID No. 1 as described by Innis et al. , the pertinent disclosure of which is incorporated herein by reference (Innis et al., Ed., PCR Protocols, Academic Press, New York, (1990).
  • composition of matter comprising the polynucleotide of this invention, and a carrier.
  • Typical carriers are known in the art such as saline, buffers, and the like and need not be further exemplified herein.
  • Other components may also be present such as salts and the like.
  • the composition of the invention may further comprise a second pure B. burgdorferi isolated polynucleotide comprising a DNA encoding a polypeptide comprising the 79 to 83 Kd app. MW conserved B. burgdorferi antigen, analogues thereof or fragments thereof of at least about 10 consecutive base pairs.
  • the combination of the two nucleotides affords the present technology a higher degree of specificity for B. burgdorferi than each one of them separately.
  • Also provided herein is a self-replicating DNA comprising the pure, isolated polynucleotide of the invention. Natural or synthetic DNA fragments encoding the polypeptide comprising the 22 to
  • DNA constructs may be incorporated into DNA constructs, usually self-replicating constructs, capable of introduction into, and in vitro expression in, cell culture.
  • the DNA constructs are capable of replication in a unicellular host, such as a bacteria or yeast, but may also be intended for introduction to, and in some instances integration within, the genome of cultured mammalian or other eukaryotic cell lines.
  • DNA constructs prepared for introduction into specified hosts may include a replication system recognized by the host, the desired polypeptide encoding sequence, e.g., the 22 to 26 Kd app.
  • the transcriptional regulatory sequences may typically include a heterologous promoter which is recognized by the host.
  • available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with an insertion site for the antigen coating region are known in the art and may be employed.
  • a host transformed with the self-replicating DNA of the invention may be as indicated above a eukaryotic or prokaryotic cell and, depending on the host cell, the self-replicating DNA may have different requirements for uptake, replication, transcription and translation sequences.
  • These transformed host cells may be utilized for the production of the DNA, RNA and polypeptide of the invention as is described above or is known in the art.
  • the polynucleotide of the invention may be used for detecting B. burgdorferi in a biological sample by contacting it with the biological sample under stringent hybridization conditions, and detecting the formation of any hybrids formed with B. burgdorferi polynucleotides present in the sample. Conditions suitable for conducting this detection method were described above and/or are known in the art.
  • This invention also provides an antibody that selectively binds to the 22 to 26 Kd app. MW B. burgdorferi conserved antigenic peptide with or without the leader segment.
  • This antibody may be polyclonal or monoclonal as it is prepared by the various methods known in the art. In order to prepare polyclonal antibodies the 22 to 26 Kd app. MW B. burgdorferi peptide is administered to an animal, e.g., rabbit and the like, and the animal's serum obtained thereafter. The antibodies may be purified from the serum or the serum utilized as is.
  • the monoclonal antibody may be prepared by methods known in the art such as the fusion of a B-cell having a certain specificity, such as producing an antibody selectively binding to the 22 to 26 Kd app. MW B. burgdorferi antigen, and a myeloma cell line by the K ⁇ hler and Milstein method known in the art.
  • polypeptides of the present invention may be produced using the purified or synthetic polypeptides of the present invention. Once a sufficient quantity of the polypeptide has been obtained by any of the methods described above, polyclonal antibodies specific for the determinant regions of the peptide may be produced by in vivo or in vitro techniques. In vivo techniques rely on the exposure of antigenic polypeptides or fragments thereof to any of a wide variety of vertebrates. Suitable vertebrates are typically non-human, such as mice, rats, rabbits, sheep, goats, and the like.
  • the polypeptide used as an immunogen is smaller than about 10 Kd, particularly smaller than about 6 Kd, it may be necessary to join the polypeptide to a larger molecule to elicit the desired immune response.
  • the immunogens may be injected into the animal host according to a predetermined schedule, and the animals bled periodically, with successive bleeds generally having improved titer and specificity. Such antisera are then purified for use in the production of the desired onospecific antibodies of the present invention.
  • the purification may be accomplished by affinity chromatography using the polypeptides of the invention coupled to a suitable solid phase. Such affinity chromatography for the purification of antibodies is well described, e.g., by Hudson & Hay.
  • Suitable in vitro techniques for producing the monospecific antibodies of the present invention involve the in vitro exposure of lymphocytes to the polypeptides corresponding the 22 to 26 Kd app. MW conserved B. burgdorferi antigen or antigenic fragments thereof. Such techniques produce monoclonal antibodies as described in Huse et al. and Ward et al. (Huse et al., Science 246:1275- 1281 (1989); Ward et al., Nature 341:544-546 (1989)), the pertinent texts of which are incorporated herein by reference.
  • Monoclonal antibodies may also be prepared by the now classic technique of K ⁇ hler and Milstein, as well as by improvements on their basic technique. Briefly, these methods rely on the preparation of immortalized cell lines capable of producing antibodies having the desired specificity, i.e., reactivity with the determinant regions of the 22 to 26 Kd app. MW conserved antigen or antigenic or immunogenic fragments thereof. Such immortalized cell lines may be produced by a variety of techniques. Conveniently, a small vertebrate, such as a mouse, may be hyperimmunized with the antigen by the method described in connection with the preparation of polyclonal antibodies.
  • the vertebrate may then be killed, usually several days after the final immunization, the spleen removed, and the spleen cells immortalized.
  • the manner of immortalization is not critical.
  • the most common technique is fusion with a myeloma cell fusion partner, as first described by K ⁇ hler and Milstein (K ⁇ hler and Milstein, Eur. J. Immunol. 6:511-519 (1976)), the pertinent text of which is recorporated herein by reference.
  • Other techniques include EVB transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, and the like, and any other method which provides for the stable maintenance of the cell line and production of monoclonal antibodies.
  • the manner of fusion is usually not critical and various techniques may be employed.
  • the spleen cells and myeloma cells are combined in the presence of a non-ionic detergent, usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's Medium, for a few minutes.
  • a non-ionic detergent usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's Medium, for a few minutes.
  • the non-ionic detergent may be rapidly removed by washing the cells.
  • the fused cells may then be placed in small culture wells, usually in a microtiter plate, at relatively low density, ranging from about 1 to 5xl0 5 per well, in a selective medium chosen to support the growth of the hybrid cells while being lethal to the myeloma cells.
  • the myeloma cell line has been mutated to be sensitive to a lethal agent, typically being HAT sensitive. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed and plates containing hybrid positive wells are identified. The plates and wells having only one colony per well are selected, and the supernatants from these wells are tested for binding activity against the desired 22 to 26 Kd app. MW conserved antigen or isolated antigenic or immunogenic fragments thereof. Once positive hybridomas are identified, the cell line may be maintained as a viable culture and/or by lyophilization and frozen storage. Depending on the desired use of the antibody, further screening of the hybridomas may be desirable. Hybridomas providing high titers are desirable.
  • Antibodies having very high specificity for corresponding determinants are desirable for use in immunodiagnostic assays as described in more detail hereinbelow.
  • monoclonal antibodies may be isolated from the supernatants of the growing colonies. The yield of antibodies obtained, however, is usually low. The yield may be enhanced by various techniques, such as injection of the hybridoma cell line into the peritoneal cavity of a vertebrate host which will accept the cells. The monoclonal antibodies may then be harvested from the ascites fluid or the blood. Proteinaceous and other contaminants may be removed from the monoclonal antibodies by conventional techniques, e.g. , chromatography, gel filtration, precipitation, extraction, or the like, prior to their use.
  • polypeptide used as the immunogen By properly selecting the polypeptide used as the immunogen, antibodies having high specificity and affinity for the desired 22 to 26 Kd app. MW conserved antigen epitope can be obtained.
  • the polypeptide selected should represent one or more epitopic sites which are unique to the antigen and which can distinguish B. burgdorferi from closely related pathogens and other potentially cross-reactive proteins. Such unique epitopes are found on the polypeptide expressed by cells containing sequences disclosed herein, such as Seq. ID No. 2, with or without the leader sequence, or fragments thereof.
  • polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles, biotin, and the like. Patents teaching the use of such labels include U.S. Patent Nos.
  • Antibodies and polypeptides prepared as described above may be used in various immunological techniques for detecting the conserved antigens of B. burgdorferi or fragments thereof in biological samples, particularly body fluids, including blood, plasma, serum, urine, stools, and the like as well as cell samples, such as tissue biopsy samples. The use of blood, plasma and serum samples is preferred.
  • an antibody mixture is also provided that comprises the antibody of the invention and a non-proteolytic carrier. Suitable carriers such as pharmaceutically acceptable carriers, and the like, are known in tlie art and need not be further described herein.
  • the mixture may also comprise a second antibody that selectively binds to the 79 to 83 Kd app. MW conserved B. burgdorferi antigen described previously by Perng, et al. (Perng et al. (1991) , supra) .
  • This antibody may be prepared by immunizing an animal as described above with the 79 to 83 Kd app.
  • MW B. burgdorferi antigen and obtaining the animal's serum.
  • the antibodies having the desired specificity may thus be isolated.
  • a diagnostic kit for B. burgdorferi that comprises the antibody of this invention and instructions for its use.
  • the kit of the invention may further comprise an antibody selectively binding to the 79 to 83 Kd app. MW B. burgdorferi antigen.
  • a mixture of the two antibodies may be utilized for greater sensitivity of the test.
  • the kit of the invention may be applied to the detection of the presence of B. burgdorferi or conserved antigenic peptides thereof that comprises contacting the biological sample with the antibody of the invention and detecting the formation of any complexes formed by the antibody and the sample's components.
  • the detection method may be conducted by using a mixture of the above- described antibody and an antibody raised against the 79 to 83 Kd app. MW B. burgdorferi antigen.
  • a first type of immunoassay combines the patient's sample with a monospecific antibody, either polyclonal or monoclonal, which is capable of specifically binding the 22 to 26 Kd app. MW antigen in the sample.
  • the antibody may be labeled or immobilized as described above.
  • the detection of any complex formed between the antibody and antigen in the patient's sample is indicative of the presence of B. burgdorferi in the sample.
  • the addition of an anti-79 to 83 Kd app. MW B. burgdorferi antibody provides higher sensitivity. Numerous protocols are available for performing such antibody-based immunoassays and are well described in the scientific and patent literature.
  • a second type of immunoassays relies on the combination of the biological sample with the polypeptide of the invention, e.g., the isolated 22 to 26 Kd app. MW peptide, and optionally 79 to 83 Kd app. MW peptide, analogues thereof, or antigenic or immunogenic fragments thereof.
  • the polypeptide binds to a patient's antibodies produced in response to infection with B. burgdorferi.
  • Protocols for detecting a patient's antibody with the polypeptide of the invention generally rely on the use of the immobilized polypeptide to capture and remove the antibody from the sample.
  • the captured antibody may be detected with labeled anti-human antibody in a sandwich assay or using a labeled polypeptide in a semi-competitive assay, among others.
  • An exemplary polypeptide-based assay for detecting infection by B. burgdorferi is an enzyme linked im unosorbent assay (ELISA) where the polypeptide is immobilized on a solid phase, usually a test well in a microliter plate, although polystyrene beads, glass beads, agarose beads, and other solid phases are also suitable. After exposure to the sample and binding of the antibody, if any is present, the solid phase is exposed to a labeled antibody directed against the patient's antibodies, e.g., labeled anti-human antibody. The label may be an enzyme which may be detected by exposure to appropriate substrates.
  • ELISA techniques are amply described in the patent and scientific literature. Of course, numerous other assay protocols exist which may combine use of the polypeptide antigens and antibodies of the present invention. The above descriptions of polypeptide-based and antibody-based assays are intended to be exemplary only.
  • the presence of B. burgdorferi in a patient's sample may be detected using nucleic acid hybridization probes prepared as described above.
  • the samples may be treated to release chromosomal DNA from any B. burgdorferi which may be present in the sample.
  • the patient's sample may be cultured to expand the amount of B. burgdorferi present prior to treatment for the release of chromosomal DNA.
  • the B. burgdorferi cells may be lysed to free their chromosomal DNA, and the released DNA fixed on a solid phase support.
  • the DNA may then be denatured so that single stranded fragments remain on the solid phase, and the solid phase contacted with the labeled oligonucleotide probes of the present invention under hybridizing conditions.
  • the hybridizing conditions should be sufficiently stringent to ensure high binding specificity. Any hybridization of the nucleic acid probes to the solid phase is thus diagnostic of the presence of the B. burgdorferi.
  • Such nucleic acid hybridization methods are described, e.g., in U.S. Patent No. 4,358,535, the pertinent disclosure of which is incorporated herein by reference.
  • An alternative protocol permits the B. burgdorferi to be lysed in a liquid phase without prior culturing to expand the amount of the microorganism present.
  • Lysing may be effected using a detergent to free the nucleic acids under denaturing conditions.
  • the lysed solution may then be contacted with the solid phase which selectively binds a single strand of nucleic acid.
  • the single stranded DNA may then be detected using oligonucleotides with binding specificity to DNA sequences of the polynucleotide of the invention.
  • hybridization assays Methods for performing such nucleic acid hybridization assays are described in general in Hanes and Higgins (Hanes ard Higgins, Nucleic Acid Hybridization: "A Practical Approach", IRL Press (1985)) the relevant disclosure of which is incorporated herein by reference.
  • the use of hybridization assays may be combined with the polymerase chain reaction to amplify any amount of the polynucleotide of the invention, e.g., the gene encoding the 22 to 26 Kd and/or 79 to 83 Kd app. MW conserved antigen(s) which may be present in a biological sample.
  • the polymerase chain reaction employs pairs of nucleotides, usually about 10 to 50 nucleotides in length, and which are spaced from about 50 to 500 nucleotides apart on either of the two complementary strands of the polynucleotide(s) of the invention, e.g., the 22 to 26 Kd or the 79 to 83 Kd app- MW DNAs.
  • Specimen preparation may include cellular lysis, typically followed by separation of the released nucleic acids on a solid phase, usually for example, glass beads or silicone particles such as those employed in the commercial kit GENECLEAN, (Bio 101, La Jolla, CA) .
  • the single stranded nucleic acids may be amplified by thermal cycling in the presence of the nucleotide primers and DNA polymerase. Typically, the temperature may be cycled between about 98°C and 60"C, for time periods as short as 1 or 2 minutes per cycle. Cycling may be continued for as long as it is desired to achieve amplification of a portion of the gene bounded by the two primers. The amplified gene fragment may then be detected using labeled oligonucleotide probes generally as described above.
  • Vaccines against B. burgdorferi infection may be prepared using the compositions of the present invention. These vaccines may be passive, consisting of Ig supplementation which interferes with B.
  • a vaccine for passive immunization against B. burgdorferi may comprise, in separate containers, the antibody of the invention and a pharmaceutically acceptable, non- proteolytic carrier, and instructions for its use. The components may be mixed prior to administration, and the vaccine may further comprise the antibody raised against the 79 to 83 Kd app. MW conserved B. burgdorferi antigen as described above. Alternatively, antigenic vaccines may be administered to produce humoral and/or cellular responses providing protective immunity by active suppression of B. burgdorferi infection.
  • One such vaccine prepared utilizing the polypeptides of the invention, e.g., the 22 to 26 Kd polypeptide antigen or immunogenic fragments thereof, usually comprises one of the following.
  • multiple vaccination may be achieved by fusing, e.g., the 22 to 26 Kd app. MW antigen or a segment thereof with other target antigens such as the 79 to 83 Kd app. MW B. burgdorferi antigen or an immunogenic fragment thereof or a non-B. burgdorferi antigen on a single protein.
  • target antigens such as the 79 to 83 Kd app.
  • fusion proteins induce protection against multiple infectant vectors.
  • a mixture of different i munogens may be simultaneously administered or inoculated.
  • These immunogens may be prepared in vaccine dosage form by well-known procedures. These vaccines may be provided in various forms. Preferred is the injectable form which may, for example, be administered intramuscularly, intravenously, or subcutaneously.
  • These vaccines may also be administered intranasally by aspiration, or orally by mixing the active components with food or water, when provided in tablet form, and the like.
  • Means for administering, or more typically inoculating, these vaccines should be apparent to those skilled in the art from the teachings herein. Accordingly, the scope of the invention is not limited to any particular delivery form.
  • these immunogens may be combined with a suitable physiologically acceptable non-proteolytic carrier, for example, water, saline, alcohol, fats, waxes, or buffered vehicles, with or without various adjuvants or immunomodulating agents.
  • a suitable physiologically acceptable non-proteolytic carrier for example, water, saline, alcohol, fats, waxes, or buffered vehicles, with or without various adjuvants or immunomodulating agents.
  • Suitable immunological adjuvants or agents include, but are not limited to, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum) , beryllium sulfate, silica, kaolin, carbon, water- in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Proplonobacterium acnes) , Bordetolla pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan) .
  • Other suitable adjuvants are Amphigen (oil-in-water) , Alhydrogel (aluminum hydroxide) , or a mixture of Amphigen and Alhydrogel.
  • the proportion of immunogen and adjuvant may be varied over a broad range as long as both are present in effective amounts.
  • the amount of the immunogen may range broadly from about 1.0 pg to about 100 mg per kg of host, usually at least about 10 pg, typically at least about 100 pg, and preferably at least about 1 ng per kg of host weight, and usually less than about 1 mg, typically less than about 10 ⁇ g, and more typically less than about 1 ⁇ g, and preferably less than about 100 ng per kg of host.
  • a preferable range is from about 10 pg to about 100 ng per dose.
  • a suitable dose volume is about 0.01 and 5 ml, preferably about 0.5 ml for a person of 20-59 kg body weight.
  • Comparable dose forms may also be prepared for parenteral administration to smaller or larger patients.
  • the amount of immunogen per dose will usually be smaller, for a smaller patient.
  • a regimen of 1 to 4 doses may be used, with the injections spaced over a 2 to 6-week period.
  • a two-dose regimen may be used.
  • the second dose of the vaccine may be administered some weeks after the first dose, for example, about 2 to 4 weeks later for patients that have been previously exposed to B. burgdorferi.
  • the vaccine may also be combined with other vaccines such as the 79 to 83 Kd B. burgdorferi antigen or vaccines for other diseases to produce multivalent vaccines. It may also be combined with other medicaments, for example, antibiotics.
  • a pharmaceutically effective amount of the vaccine may be employed with a pharmaceutically acceptable non-proteolytic carrier such as a viral capsid protein complex or diluent understood to be useful for the vaccination of animals.
  • the antibody of the invention may also be used for inhibiting or countering B. burgdorferi infection by administration of an effective amount thereof to a patient in need of the treatment.
  • the antibody may optionally be administered with an anti-79 to 83 Kd app. MW antigen antibody for further immunogenic effect.
  • the antibody is administered in an amount of about 100 to 500 ⁇ g/kg body weight/day, and more preferably about 250 to 350 ⁇ g/kg body weight/day.
  • a chromosomal DNA enriched preparation was obtained as described previously (LeFebvre et al. (1990) , supra) . Briefly, 20 ml of logarithmic growth-phase cells of B. burgdorferi, strain B31, were washed three times in phosphate-buffered saline, pH 7.2 with 5 mM MgCl 2 . The pellets were suspended in 100 ⁇ l of 15% sucrose in 50 mM Tris hydrochloride pH 8.0, 50 mM EDTA, to which an equal volume of 5 M NaCl was added. To this solution was added 1/10 volume 1% sodium deoxycholate.
  • the solution was then placed on ice for 20 min., followed by centrifugation in a microcentrifuge for 5 min.
  • the pellet containing the chromosomal DNA was suspended in 100 ⁇ l of 15% sucrose in 50 mM Tris hydrochloride, pH 8.0, and 50 mM EDTA.
  • 50 ⁇ l of 10% SDS and 25 ⁇ l of 20 mg/ml proteinase K were added 50 ⁇ l of 10% SDS and 25 ⁇ l of 20 mg/ml proteinase K.
  • the solution was incubated for 30 min at 37 a C, followed by phenol extraction and ethanol precipitation.
  • the chromosomal DNA was digested with Clal and ligated into a Clal-digested Pev-Vrf expression vector (Crowl et al. (1985) , supra) .
  • E. coli RRI was transformed with the recombinant plasmids.
  • the expression of cloned genes by this vector was controlled by heat shock at 42 a C for 2 hours as previously described (Crowl et al. (1985) , supra) .
  • Recombinant colonies were screened for expression of B. burgdorferi antigens by colony blot immunoassay using standard procedures.
  • Example 2 Antibody Treatment and ' Use for
  • Rabbit antiserum to whole B. burgdorferi was prepared as previously described (LeFebvre et al. (1990), supra). Rabbit antibodies specific for the 26 Kd antigen were affinity purified as follows. A lysate of transformed E. coli cells containing the recombinant pLP2 plasmid and expressing the antigen was fractionated in a 12% SDS-PAGE gel and the proteins were transferred to nitrocellulose by standard semi-dry blot procedures (Laemmli, U.K., "Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4", Nature 227:680-685 (1970), Towbin, H.
  • the sera were used at a dilution of 1:50 after pre-adsorption with an E. coli lysate. Immunoblot strips were probed ith the ELISA positive canine sera for B. burgdorferi.
  • the proteins represented on these strips consisted of lysed E. coli JM83 whole cells containing and expressing the pLP2 recombinant vector of the invention.
  • the 26 Kd antigen reacted strongly with the 8 canine sera tested in this experiment. Of 10 ELISA positive canine samples tested 9 reacted with the 26 Kd antigen and 1 did not.
  • the gene expressing the 26 Kd antigen was determined to be contained within a 1.65 kb fragment of a Clal insert.
  • the recombinant plasmid expressing this antigen was designated pLP2.
  • the gene was sequenced according to standard procedures (Sanger, F. , et al., "DNA Sequencing with Chain Terminating Inhibitors", Proc. Natl. Acad. Sci.
  • Example 4 Amino Acid Sequence and .Analysis of Antigen
  • the amino acid sequence of the 26 Kd antigen was derived from the Genetics Computer Group (GCG) software (GCG sequence analysis software package, Version 6.0, Genetics Computer Group, University of Wisconsin, Biotechnology Center, Madison, Wisconsin) .
  • Example 5 Butanol Extraction of B. burgdorferi
  • the outer surface proteins were prepared as an enriched sample as previously described (Gondolf, K.B., et al., "Isolation of an Outer-Membrane Protein Complex from Borrelia-burgdorferi by Normal Butanol Extraction and High Performance Ion-Exchange Chromatography", J. Chromat. 521:325-334 (1990)). Briefly, 500 ml of logarithmic phase B. burgdorferi B 31 cells were harvested, washed in phosphate buffered saline (PBS) and sonicated for a total of 5 min.
  • PBS phosphate buffered saline
  • the sonicate was centrifuged at 27,000 x g for 60 min at 4 S C, the pellet resuspended in 2.5 ml of PBS and 5 ml of n-butanol were added and the entire suspension mixed for 1 min at 4 B C. The solution was again centrifuged at 27,000 x g for 60 min at 4 a C. The supernatant was then harvested and used in subsequent experiments as an outer surface protein enriched fraction.
  • a gel was run with (a) the butanol extracted, outer membrane protein-enriched B. burgdorferi obtained in example 5, (b) lysed whole cell B31 B. burgdorferi, (c) lysed whole cell E. coli containing the pLP2 recombinant plasmid, (d) lysed whole cell E. coli, and (e) molecular weight standards and stained with Coomassie blue ( Figure not shown) .
  • SDS-PAGE electrophoresis was performed as described previously (Laemmli (1970), supra; Towbin et al. (1979), supra) . Approximately 20 ⁇ g of butanol extracted, outer membrane protein enriched B. burgdorferi, lysed whole cell B31 B. burgdorferi, lysed whole cell E. coli containing the pLP2 recombinant plasmid, lysed whole cell E. coli, and molecular weight standards were added to a 12% gel. The gel was run at constant voltage of 200 V for 45 min.
  • the electrophoresed proteins were transferred to nitrocellulose membranes with a Pharmacia/LKB semi-dry transfer system according to the manufacturer's specifications (Pharmacia/LKB, Uppsala, Sweden) .
  • the membranes were blocked and probed by standard procedures (Maniatis, T. , et al., "Molecular Cloning: a Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1982)).
  • the blots were screened with sera from rabbit anti-B. burgdorferi B31 polyclonal antiserum.
  • the major reacting proteins are the outer surface A and B (ospA and B) , the 26 Kd app. MW conserved antigen and some smaller molecular weight proteins by an ELISA assay.
  • the sera were diluted 1:50.
  • Protein A conjugated to alkaline phosphatase (1:10,000, Sigma, St. Louis, MO) was used as the secondary reactant in these assays.
  • the enzyme substrate for the alkaline phosphatase was 5-bromo-4-chloro-3- indolylphosphate.
  • a gel was run containing the same samples as in the previous gel but in reverse order of loading, and immunoblotted ( Figure not shown) .
  • the blot was reacted with rabbit anti-B. burgdorferi B31 polyclonal antiserum.
  • the major reacting proteins are the outer surface A and B (ospA and B) proteins, the 26 Kd antigen, and some smaller molecular weight proteins.
  • the 26 Kd antigen reacted strongly with the serum containing the butanol-extracted proteins. This indicates that the antigen is likely situated on the outer surface or in the periplasmic space of the microorganisms.
  • Example 7 Immunoblot of B. burgdorferi Protein Gel electrophoresis of several different protein preparations was performed as described in Example 6 above. The blot was probed with monospecific, polyclonal antibod ⁇ ies directed against the 26 Kd app. MW conserved antigen, which were purified as described above. The antibodies bound only to the 26 Kd antigen. This clearly indicates that the 26 Kd polypeptide is a unique protein that does not share cross-reactive epitopes with other B. burgdorferi proteins.
  • amino acid sequence of the 26 Kd polypeptide and the polynucleotide encoding it determined herein were placed with the GenBank/EMBL and were given Accession No. M90084.
  • Example 9 Characterization of 26 Kd Polypeptide and the Polynucleotide Encoding It
  • a gene encoding a 26 Kd antigen from B. burgdorferi was cloned and the sequence of both the gene and its expressed antigen were determined.
  • the gene was shown to be chromosomally encoded by hybridization studies using a specific probe and whole cell B. burgdorferi DNA in pulsed field gels (Data not shown) . The methods used were described previously (LeFebvre et al. (1990), supra, and Perng et al. (1991), supra).
  • the DNA sequence of the gene expressing the 26 Kd antigenic polypeptide is shown below in Table 2 above.
  • the deduced amino acid sequence is shown in Table 1 above.
  • the ATG start codon was found to be followed by 596 bases encoding a protein of 199 amino acids with an about 26 Kd molecular weight.
  • Putative -35' (ttgctct) and a -10' (tattat) sequences were identified and have been underlined in Table 2 above.
  • a putative signal sequence was identified in the protein consisting of a short, positively charged amino terminus sequence (M-G-K) followed by a hydrophobic region (amino acids 4 to 16) , a polar C- terminus, a valine at -3 (amino acid 23) , and a threonine at -1 (amino acid 25) .
  • M-G-K short, positively charged amino terminus sequence
  • hydrophobic region amino acids 4 to 16
  • valine at -3 amino acid 23
  • threonine at -1 amino acid 25
  • the 22 Kd protein resides in the periplasmatic space as evidenced by its not being degraded by proteinase K treatment of intact cells.
  • the 26 Kd protein expressed by the recombinant plasmid in E. coli cells reacted with a monoclonal antibody raised against the 22 Kd periplasmatic protein described by Simpson et al. (1991) , supra.
  • Example 10 Comparison with Other B-burgdorferi Antigens
  • the 22 to 26 Kd app. MW protein (and the gene encoding it) represents a significant conserved antigen expressed by B. burgdorferi in this molecular weight range.
  • Fuchs et al. (Fuchs et al. (1992), supra) reported the sequence of a gene expressing a 22 Kd antigen (pC) in B. burgdorferi. However, based on a comparison of the gene sequence and respective proteins the two are different and unrelated to each other.
  • the 22 to 26 Kd protein described herein is also different from the 22 Kd protein described by Lucas et al. (Luft et al.
  • the molecular weight of the protein of the invention, expressed by the pLP2 recombinant vector has been calculated to be 26 Kd based on the amino acid sequence deduced from the gene sequence.
  • the molecular weight was found to be about 22 Kd. This discrepancy may be due to lack of resolution in the electrophoretic system at this size range.
  • the protein visualized by SDS-PAGE may represent a processed version of the antigen after the removal of the signal sequence (amino acids 1-26) , which is cleaved off during processing. The removal of the signal sequence leaves a calculated protein of 23 Kd molecular weight, which is more consistent with the resolution of the protein in SDS polyacrylamide gels.
  • Lys Lys lie Ala Asp Thr Lys Asn lie Lys lie lie Ala 155 160 165

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Abstract

L'invention concerne une séquence nucléotidique et la séquence d'acides aminés dérivée d'une protéine Borrelia burdorferi de 22 à 26 kDa, utilisées pour le diagnostic, la génération d'anticorps et la production de vaccins sous-unitaires et passifs contre la maladie de Lyme.
PCT/US1994/002889 1993-03-11 1994-03-10 Methodes, compositions et trousses pour le diagnostic de la maladie de lyme WO1994020536A1 (fr)

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US7008625B2 (en) 1993-11-01 2006-03-07 Research Foundation Of The State University Of New York Recombinant constructs of Borrelia burgdorferi
US7605248B2 (en) 1993-11-01 2009-10-20 Research Foundation Of The State University Of New York Recombinant constructs of Borrelia burgdorferi
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WO1995012676A1 (fr) * 1993-11-01 1995-05-11 Associated Universities, Inc. Proteines chimeres comprenant des polypeptides de borrelia et leurs utilisations
US7179448B2 (en) 1993-11-01 2007-02-20 Research Foundation Of The State Of New York Recombinant constructs of Borrelia burgdorferi
WO1997015600A1 (fr) * 1995-10-26 1997-05-01 The Board Of Governors For Higher Education, State Of Rhode Island And Providence Plantations 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)
EP0880543A1 (fr) * 1995-10-26 1998-12-02 The Board Of Governors For Higher Education State Of Rhode Island And Providence Plantations 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)
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)
US6610301B1 (en) 1996-08-14 2003-08-26 Mikrogen Molekularbiologische Entwicklungs - Gmbh Immunologically active proteins from Borrelia burgdorferi, nucleic acids which encode them, and their use in test kits and as vaccines
DE19632862B4 (de) * 1996-08-14 2006-08-03 Mikrogen Molekularbiologische Entwicklungs-Gmbh Immunologisch aktive Proteine von Borrelia burgdorferi, dafür kodierende Nukleinsäuren sowie deren Verwendung in Testkits und als Impfstoffe
US6808711B2 (en) 1996-08-14 2004-10-26 Mikrogen Molekularbiologische Entwicklungs-Gmbh Immunologically active proteins from Borrelia burgdorferi, nucleic acids which encode them, and their use in test kits and as vaccines
DE19632862A1 (de) * 1996-08-14 1998-02-19 Mikrogen Molekularbiol Entw Immunologisch aktive Proteine von Borrelia burgdorferi, dafür kodierende Nukleinsäuren sowie deren Verwendung in Testkits und als Impfstoffe
US8680236B2 (en) 2000-08-18 2014-03-25 Brookhaven Sciences Associates, Llc Altered OspA of borrelia burgdorferi
US8992936B2 (en) 2000-08-18 2015-03-31 Research Foundation Of The State University Of New York Altered OspA of Borrelia burgdorferi

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