Title: Surface antigens and proteins useful in compositions for the diagnosis and prevention of lyme disease
United States Patent: 6,660,274
Issued: December 9, 2003
Inventors: Philipp; Mario T. (Mandeville, LA)
Assignee: The Administrators of the Tulane Educational Fund (New Orleans, LA)
Appl. No.: 445803
Filed: December 13, 1999
PCT Filed: June 29, 1998
PCT NO: PCT/US98/13551
PCT PUB.NO.: WO99/00413
PCT PUB. Date: January 7, 1999
A novel isolated Borrielia burgdorferi sensu lato surface antigen is characterized by a relative molecular mass of 39.5 kDa. This antigen is expressed in vitro by spirochetes of a B. burgdorferi sensu lato strain. This antigen induces antibodies which kill spirochetes of a B. burgdorferi sensu lato strain by ADCK in vitro. Novel Borrelia cassette string protein or fragments thereof are also useful, as is the P39.5 protein in diagnosing Lyme disease and in compositions for treatment or prophylaxis thereof.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel Borrelia surface antigen, P39.5, that is expressed by the spirochete when it resides in the vertebrate host ("in vivo"). This antigen is targeted by antibody-dependent, complement-mediated (ADCK) killing in vitro. This novel antigen, fragments thereof, antibodies developed thereto, the nucleic acid sequences encoding same, and the use of such antigens, antibodies and nucleic acid sequences in diagnostic, therapeutic and prophylactic compositions and methods for the treatment or prevention of Lyme Disease provide advantages over the use of other Borrelia proteins and antibodies in known compositions and methods for this purpose.
I. The P39.5 Antigen of the Invention
In one embodiment, the present invention provides a novel isolated Borrelia antigen, referred to as P39.5. This antigen is expressed both in vitro and in vivo by Borrelia spirochetes, e.g., spirochetes of B. garinii strain IP90. This antigen, or a homolog thereof, is also expressed in vivo by spirochetes of the B. burgdorferi sensu stricto strains JD1, B31 and NT1. This antigen has an apparent molecular mass of 39.5 kDa in IP90 spirochetes, and is functionally characterized by the ability to elicit antibody in animals during the course of a natural infection with Borrelia spirochetes. These induced antibodies kill IP90 spirochetes by ADCK in vitro. The elicited antibody also kills spirochetes of the NT1 strain of B. burgdorferi sensu lato, a strain isolated from the cerebrospinal fluid of a patient in the United States, but not further characterized within the sensu lato complex of species. This strain was provided by Dr. Patricia Coyle, State University of New York at Stony Brook, N.Y.
The gene fragment, designated 7-1, from Borrelia garinii strain IP90 inserted in pBluescript II plasmid was transformed in E. coli and deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. ("ATCC") on Jun. 27, 1997 under Accession No. 98478. When this gene fragment is expressed in E. coli, isolated as a pure protein and the protein used as an immunogen in mice, the antibody thus produced reacts with P39.5 of IP90. Other gene fragments, designated, 1-1, 3-1, 6-1, 9-1 and 12-1, from Borrelia garinii strain IP90 were similarly each inserted in pBluescript II plasmids, transformed in E. coli and deposited. These latter deposits were made with the ATCC on Jun. 10, 1998 under Accession Nos. 98768 for 1-1, 98769 for 3-1, 98770 for 6-1, 98771 for 9-1 and 98772 for 12-1. All deposits were all made to meet the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, and fully comply with the requirements of the United States Patent and Trademark Office for deposits for patent purposes. Sequences useful in the present invention may be obtained from these deposits.
A. Nucleic Acid Sequences
The present invention provides mammalian nucleic acid sequences encoding fragments of P39.5. The nucleic acid sequences of this invention are isolated from cellular materials with which they are naturally associated. As described in the examples below, a segment of 1190 bp which encodes a portion of the coding region of the Borrelia P39.5 gene was cloned and sequenced. It encompasses a single open reading frame that encodes a putative 37.7 kDa protein. This partial DNA sequence is reported in SEQ ID NOS: 13 and 1. SEQ ID NO: 13 is a sequence immediately 5' to the first base of SEQ ID NO: 1. Together these sequences from a partial protein of 1189 bp and about 396 amino acids. The sequenced fragment is composed of mostly hydrophilic domains that contain several internally repeated regions and a unique region spanning about bp 627 to about bp 712 of SEQ ID NO: 1. See, FIG. 2, which identifies the repeated sequences and identifies the homologies and percent identities of such sequences.
Where in this text, protein and/or DNA sequences are defined by their percent homologies or identities to identified sequences, the algorithms used to calculate the percent identities or percent similarities include the following: the Smith-Waterman algorithm [J. F. Collins et al, 1988, Comput. Appl. Biosci., 4:67-72; J. F. Collins et al, Molecular Sequence Comparison and Alignment, (M. J. Bishop et al, eds.) In Practical Approach Series: Nucleic Acid and Protein Sequence Analysis XVIII, IRL Press: Oxford, England, UK (1987) pp.417], and the BLAST and FASTA programs [E. G. Shpaer et al, 1996, Genomics, 38:179-191]. These references are incorporated herein by reference.
Also encompassed within this invention are other Borrelia nucleic acid fragments, e.g., cassette string fragments, as well as fragments of P39.5. Such fragments are referred to as 1-1 (which encodes a protein/peptide P1-1), 3-1 (which encodes a protein/peptide P3-1), 6-1 (which encodes a protein/peptide P6-1), 9-1 (which encodes a protein/peptide P9-1) and 12-1 (which encodes a protein/peptide P12-1). Preferably, such fragments are characterized by encoding a biologically active portion of P39.5, e.g., an epitope. Generally, these oligonucleotide fragments are at least 15 nucleotides in length. However, oligonucleotide fragments of varying sizes may be selected as desired. Such fragments may be used for such purposes as performing polymerase chain reaction (PCR), e.g., on a biopsied tissue sample. For example, useful fragments of P39.5 DNA and corresponding sequences comprise sequences occurring between bp 793-816 of SEQ ID NO: 1 and bp 59-85 of SEQ ID NO: 13. Other useful fragments are identified in FIG. 2. Fragments of 1-1 include sequences of SEQ ID NOS: 3, 4 and 5. Fragments of 3-1 include sequences of SEQ ID NOS: 6 and 7; fragments of 6-1 include sequences of SEQ ID NOS: 8 and 9. A fragment of 9-1 includes the sequence of SEQ ID NO: 10. Fragments of 12-1 include the sequences of SEQ ID NOS: 11 and 12. The complete sequences of 1-1, 3-1, 6-1, 9-1 and 12-1 and other useful fragments may be readily obtained by one of skill in the art by resort to conventional DNA sequencing techniques applied to the DNA deposited with the ATCC above.
The DNA sequences of SEQ ID NOS: 1 and 3-12 as well as the deposited materials identified above permit one of skill in the art to readily obtain the corresponding anti-sense strands of these DNA sequences. Further, using known techniques, one of skill in the art can readily obtain additional genomic and cDNA sequences which flank the illustrated DNA sequences or the corresponding RNA sequences, as desired. Similarly the availability of SEQ ID NOS: 1 and 3-12 and the deposited materials of this invention permits one of skill in the art to obtain other species P39.5 analogs, B. garinii peptides, and fragments thereof, by use of the nucleic acid sequences of this invention as probes in a conventional technique, e.g., polymerase chain reaction. Allelic variants of these sequences within a species (i.e., sequences containing some individual nucleotide differences from a more commonly occurring sequence within a species, but which nevertheless encode the same protein or a protein with the same function) such as other variants of P39.5 SEQ ID NO: 2, may also be readily obtained given the knowledge of this sequence provided by this invention.
The present invention further encompasses nucleic acid sequences capable of hybridizing under stringent conditions [see, J. Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory (1989)] to the sequences of SEQ ID NO: 1, their anti-sense strands, or biologically active fragments thereof. An example of a highly stringent hybridization condition is hybridization at 2xSSC at 65oC., followed by a washing in 0.1xSSC at 65oC. for an hour. Alternatively, an exemplary highly stringent hybridization condition is in 50% formamide, 4xSSC at 42oC. Moderately high stringency conditions may also prove useful, e.g., hybridization in 4xSSC at 55oC., followed by washing in 0.1xSSC at 37oC. for an hour. An alternative exemplary moderately high stringency hybridization condition is in 50% formamide, 4xSSC at 30oC.
According to the invention, the nucleic acid sequences may be modified. Utilizing the sequence data of SEQ ID NOS: 1 and 3-12, it is within the skill of the art to obtain or prepare synthetically or recombinantly other polynucleotide sequences, or modified polynucleotide sequences, encoding the full-length proteins or useful fragments of the invention. Such modifications at the nucleic acid level include, for example, modifications to the nucleotide sequences which are silent or which change the amino acids, e.g. to improve expression or secretion. Also included are allelic variations, caused by the natural degeneracy of the genetic code.
Also encompassed by the present invention are mutants of the P39.5 fragments and cassette string sequences 1-1, 3-1, 6-1, 7-1, 9-1 and 12-1 provided herein. Such mutants include amino terminal, carboxy terminal or internal deletions, which substantially retain the antigenicity of the full-length P39.5 or other proteins or fragments. Such a truncated, or deletion, mutant may be expressed for the purpose of affecting the activity of the full-length or wild-type gene or gene fragments.
These nucleic acid sequences are useful for a variety of diagnostic, prophylactic and therapeutic uses. Advantageously, the nucleic acid sequences are useful in the development of diagnostic probes and antisense probes for use in the detection and diagnosis of Lyme disease by utilizing a variety of known nucleic acid assays, e.g., Northern and Southern blots, polymerase chain reaction (PCR), and other assay techniques known to one of skill in the art. The nucleic acid sequences of this invention are also useful in the production of P39.5 proteins and homologs as well as other proteins of B. garinii.
The nucleotide sequences of the invention may be isolated by conventional uses of polymerase chain reaction or cloning techniques such as those described in conventional texts such as Sambrook et al, cited above. For example, the nucleic acid sequences of the antigen of this invention may be prepared or isolated from B. garinii DNA using DNA primers and probes and PCR techniques. Alternatively, the antigen may be obtained from gene banks derived from B. garinii whole genomic DNA. These sequences, fragments thereof, modifications thereto and the full-length sequences may be constructed recombinantly using conventional genetic engineering or chemical synthesis techniques or PCR, and the like by utilizing the information provided herein.
B. Protein Sequences
The present invention also provides B. burgdorferi sensu lato proteins, such as B. garinii proteins and peptides, including P39.5 polypeptides or proteins, and the peptides/proteins represented by the designations P1-1, P3-1, P6-1, P7-1, P9-1 and P12-1 of this invention. These proteins are free from association with other contaminating proteins or materials with which they are found in nature. Because the native P39.5 was fully extracted into the detergent phase in a Triton-X114 extraction, it is likely a lipoprotein. The P39.5 antigen has a relative molecular mass of 39,500 daltons as measured by Western immunoblot (See Example 2 and FIG. 6). In one embodiment, the invention provides a partial P39.5 [SEQ ID NO:2] polypeptide of about 354 amino acids, likely a lipoprotein, having a predicted relative molecular mass of about 37.7 kD (i.e., "the P7-1 fragment"). This deduced amino acid sequence [SEQ ID NO: 2] is up to 22% identical to members of the variable major protein (Vmp) family of outer surface lipoproteins of Borrelia hermsii (see Example 6). It is about 50% identical to the Vmp-like sequence (vls) VlsE of the B31 strain of B. burgdorferi [J.-R. Zhang et al, cited above] and to other vls sequences of B. burgdorferi [i.e., Vls-6 Genbank no. U76406 (53% identity over 190 aa); VlsE Genbank no. U84553 (57% over 210 aa) and U84566 (58% over 209 aa) and U84555 (58% over 210 aa)].
VlsE is part of an antigen of B. burgdorferi that undergoes antigenic variation by a mechanism of recombination whereby a central fragment of the expressed copy (VlsE) is recombined with fragments from a string of 15 "cassettes" located upstream from the expressed copy [J. Zhang, cited above]. Each cassette is on average 500 bp in length, and the whole string occupies a stretch of DNA of about 8 kb. One of the remarkable features of this string of cassettes is that in B. burgdorferi B31, the cassettes are arranged in a nearly contiguous open reading frame interrupted only by a stop codon in cassette vls11 and two frame shifts in cassettes vls14 and vls16. See, e.g., FIG. 6, which is a copy of a similar structure defined in Zhang et al, cited above.
A longer partial protein sequence of P39.5 is formed by the direct fusion of the partial sequence from clone 14 [SEQ ID NO: 14] to the 5' end of the sequence of SEQ ID NO: 2. The identification of the sequence from clone 14 as a sequence 5' to the sequence of P7-1 resulting from overlapping sequences between the two clones from which these sequences were obtained, which overlapping sequences demonstrated that they were part of the same open reading frame. Thus the Leu (amino acid 47 of SEQ ID NO: 14) is immediately followed by the Lys (amino acid 1 of SEQ ID NO: 2) in the larger P39.5 deduced amino acid sequence.
The present invention provides partial DNA and deduced amino acid sequences of several fragments which appear to be part of the cassette string of B. garinii IP90. These fragments, which may include 7-1, are named 1-1, 3-1, 6-1, 9-1, and 12-1, and are between 1 and 2 kb in length. Each of them expresses a peptide (off of the lacZ promoter of pBluescript) which is commensurate with the size of the insert and which reacts with antibody from infected monkeys. None of the 5' ends of these fragments [SEQ ID NOS: 1, 3, 6, 8, 10 and 11] contains a hydrophobic leader or a signal peptidase II consensus sequence of the type that is characteristic of bacterial lipoproteins. These fragments must therefore be part of the cassette string of IP90 and, like the cassette string of B31, they are in-frame. These vls-like proteins are identified by partial 5' and 3' sequences [see, e.g., SEQ ID NOS: 1, 3, 5 through 12]. One of skill in the art using conventional techniques, such as PCR, may readily use the partial 5' and 3' sequences provided herein and the DNA sequences of (or DNA library of) B. garinii or the materials deposited with the ATCC for each cassette string protein identified above, to identify the complete sequences thereof. Such methods are routine and not considered to require undue experimentation, given the information provided herein.
Antigens of this invention may be characterized by immunological measurements including, without limitation, western blot, macromolecular mass 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 adoptive transfer of cells, proteins or antibodies.
The proteinaceous P39.5 surface antigen of this invention (as well as its naturally occurring variants or analogs in other species of Borrelia or the other Vls-like proteins identified herein) may be isolated in the form of a complete intact protein, or a polypeptide or fragment thereof. In one embodiment, P39.5 is isolated by immunoblot procedures according to its respective molecular mass, as described below in Example 5. Such isolation provides the antigen in a form substantially free from other proteinaceous and non-proteinaceous materials of the microorganism and the tick vector. The molecules comprising the Borrelia polypeptides and antigens of this invention may be isolated from the spirochete 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. One of skill in the art may select the most appropriate isolation and purification techniques without departing from the scope of this invention.
Alternatively, the amino acid sequences of P39.5 or the other Borrelia proteins of this invention may be produced recombinantly following conventional genetic engineering techniques [see e.g., Sambrook et al, cited above and the detailed description of making the proteins below].
i. Analogs/Modified Antigens
Also included in the invention are analogs, or modified versions, of the P39.5 protein or vls-like proteins P1-1, P3-1, P6-1, P7-1, P9-1 and P12-1, provided herein. Typically, such analogs differ from the specifically identified proteins by only one to four codon changes. Examples include polypeptides with minor amino acid variations from the illustrated partial amino acid sequence of, for example, P7-1 (SEQ ID NO: 2), in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties. Also provided are homologs of the proteins of the invention which are characterized by having at least 85% identity with SEQ ID NO:2 or with the sequences of the vls-like proteins. Based on the sequence information provided herein, one of skill in the art can readily obtain full-length P7-1 or P7-1 homologs and analogs, as well as the full-length viselike 1-1, 3-1, 6-1, 9-1 and 12-1 protein homologs and analogs from other bacterial species.
An antigen of the present invention may also be modified to increase its immunogenicity. For example, the antigen 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. For a review of some general considerations in coupling strategies, see Antibodies , A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988). 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. Useful chemical compounds for coupling include, without limitation, dinitrophenol groups and arsonilic acid.
The antigen may also be modified by other techniques, such as denaturation with heat and/or SDS.
ii. Fragments/Deletion Mutants
Further encompassed by this invention are additional fragments of the P39.5 polypeptide, the P7-1 peptide or of the other vls-like proteins identified herein. Such fragments are desirably characterized by having a biological activity similar to that displayed by the complete protein, including, e.g., the ability to induce antibodies to the causative agent of Lyme Disease. These fragments may be designed or obtained in any desired length, including as small as about 5-8 amino acids in length. Such a fragment may represent an epitope of the protein.
The internal repeats of P39.5 (see FIG. 2) indicate that this protein, like OspA, may undergo homologous recombinations in Borrelia which may lead to mutants expressing shortened, partially-deleted versions of the gene. Thus, the P7-1 protein [SEQ ID NO: 2] of the invention may be modified to create deletion mutants, for example, by truncation at the amino or carboxy termini, or by elimination of one or more amino acids. Deletion mutants of P7-1 created by homologous recombination of its repeat sequences (FIG. 2) are also encompassed by this invention, as are the DNA sequences encoding them. In certain deletion mutants, portions of the P39.5 coding region, described above, e.g., regions IIA, IIB, and B, are deleted.
Deletion mutants of the P39.5 antigens that, like P39.5 and unlike OspA, are expressed in vivo, will likely be killed upon infection of the vertebrate host. Most OspA deletion mutants escape killing with anti-OspA antibody in the absence of complement, but are killed by this antibody and complement acting together. Since anti-OspA antibody kills spirochetes in the tick midgut (where OspA is expressed, but complement is likely not active), OspA deletion mutants may escape the anti-Osp A antibody elicited by the OspA vaccine. In contrast, since P39.5 is expressed in vivo, where complement and anti-P39.5 antibody are both present, P39.5 deletion mutants may not escape a P39.5 based vaccine.
Still other modified fragments of P39.5, P1-1, P3-1, P6-1, P7-1, P9-1, or P12-1 may be prepared by any number of now conventional techniques to improve production thereof, to enhance protein stability or other characteristics, e.g. binding activity or bioavailability, or to confer some other desired property upon the protein. Other useful fragments of these polypeptides may be readily prepared by one of skill in the art using known techniques, such as deletion mutagenesis and expression.
iii. Fusion or Multimeric Proteins and Compositions
The P39.5 protein of the present invention, or fragments of it, as well as the vls-like cassette string proteins or fragments thereof, may also be constructed, using conventional genetic engineering techniques as part of a larger and/or multimeric protein or protein compositions. Antigens of this invention may be in combination with B. burgdorferi outer surface proteins, such as OspA and OspB, or various fragments of the antigens described herein may be in combination with each other. In such a combination, the antigen may be in the form of a fusion protein. The antigen of the invention may be optionally fused to a selected polypeptide or protein, e.g. Borrelia antigens OspA and OspB, other Borrelia antigens, and proteins or polypeptides derived from other microorganisms. For example, 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.
Still another fusion protein of this invention is provided by expressing the DNA molecule formed by the P39.5 DNA sequence or a fragment thereof fused to DNA fragments that are homologous (25-95% identity) to P39.5. One example of such a protein comprises the amino acid sequence of SEQ ID NO: 2 to which is fused amino acid fragments that are up to 95% identical to that sequence. These fragments may be inserted in any order and may contain repeated sequences such as those depicted in FIG. 2. These long strings of DNA that form a single open reading frame may be constructed from P39.5 homologs, including P39.5, or be derived from the naturally occurring long open reading frames, such as one or more of the vls cassette proteins. The vls cassette proteins from B. garinii (e.g., the proteins designated P1-1, P3-1, P6-1, P9-1 and/or P12-1) may be fused to each other and/or inserted into the P39.5 or P7-1 DNA sequence, thereby creating a large DNA molecule which expresses a protein which may stimulate a variety of antibody specificities.
These 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. Further, 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 derived from other viruses. Such proteins are effective in the prevention, treatment and diagnosis of Lyme disease as caused by a wide spectrum of B. burgdorferi isolates.
A protein composition which may be a preferred alternative to the fusion proteins described above is a cocktail (i.e., a simple mixture) containing different P39.5 proteins or fragments, or different mixtures of the cassette string proteins of this invention. Such mixtures of these proteins or antigenic fragments thereof are likely to be useful in the generation of desired antibodies to B. garinii.
An antigen of the present invention may also 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.
II. Methods of Making Antigens and Nucleic Acid Sequences of the Invention
A. Expression In Vitro
To produce recombinant P7-1 and/or other cassette string proteins or other fragments of P39.5 of this invention, the DNA sequences of the invention are inserted into a suitable expression system. Desirably, a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding the selected protein, e.g., P7-1, is operably linked to a heterologous expression control sequence permitting expression of the protein. Numerous types of appropriate expression vectors are known in the art for protein expression, by standard molecular biology techniques. Such vectors are selected from among conventional vector types including insects, e.g., baculovirus expression, or yeast, fungal, bacterial or viral expression systems. Other appropriate expression vectors, of which numerous types are known in the art, can also be used for this purpose. Methods for obtaining such expression vectors are well-known. See, Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d edition, Cold Spring Harbor Laboratory, New York (1989); Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein.
Suitable host cells or cell lines for transfection by this method include bacterial cells. Preferably, at least for use of the P39.5 protein which is believed to be a lipoprotein (e.g., see Example 5), the selected protein is expressed in bacteria, which have the cellular machinery to attach lipids. This expression system is especially preferred where the protein is being expressed for use as a vaccine or therapeutic. For example, the various strains of E. coli (e.g., HB101, MC1061, and strains used in the following examples) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, Streptomyces, and other bacilli and the like are also be employed in this method.
Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Other fungal cells may also be employed as expression systems. Although yeast can also lipidate proteins, the lipidation pattern may be different from bacteria, i.e., different from the native protein.
Mammalian cells, such as human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived from Swiss, Balb-c or NIH mice are used. Another suitable mammalian cell line is the CV-1 cell line. Still other suitable mammalian host cells, as well as methods for transfection, culture, amplification, screening, production, and purification are known in the art. [See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446].
Alternatively, insect cells such as Spodoptera frugipedera (Sf9) cells may be used.
Thus, the present invention provides a method for producing a recombinant Borrelia protein, e.g., P7-1 or other cassette string proteins, which involves transfecting, e.g., by conventional means such as electroporation, a host cell with at least one expression vector containing a polynucleotide of the invention under the control of a transcriptional regulatory sequence. The transfected or transformed host cell is then cultured under conditions that allow expression of the protein. The expressed protein is recovered, isolated, and optionally purified from the cell (or from the culture medium, if expressed extracellularly) by appropriate means known to one of skill in the art.
For example, the proteins are isolated in soluble form following cell lysis, or extracted using known techniques, e.g., in guanidine chloride. If desired, the proteins or fragments of the invention are produced as a fusion protein. Such fusion proteins are those described above. Alternatively, for example, it may be desirable to produce fusion proteins to enhance expression of the protein in a selected host cell, to improve purification, or for use in monitoring the presence of the desired protein, e.g., P7-1, in tissues, cells or cell extracts. Suitable fusion partners for the proteins of the invention are well known to those of skill in the art and include, among others, .beta.-galactosidase, glutathione-S-transferase, and poly-histidine.
B. Expression In Vivo
Alternatively, where it is desired that the P7-1 or other cassette string protein of the invention (whether full-length or a desirable fragment) be expressed in vivo, e.g., to induce antibodies, or as a DNA vaccine, an appropriate vector for delivery is readily selected by one of skill in the art. Exemplary vectors for in vivo gene delivery are readily available from a variety of academic and commercial sources, and include, e.g., adeno-associated virus [International patent application No. PCT/US91/03440], adenovirus vectors [M. Kay et al, Proc. Natl. Acad. Sci. USA, 91:2353 (1994); S. Ishibashi et al, J. Clin. Invest., 92:883 (1993)], or other viral vectors, e.g., various poxviruses, vaccinia, etc. Methods for insertion of a desired gene, e.g., P7-1, and obtaining in vivo expression of the encoded protein, are well known to those of skill in the art.
III. Antibodies of the Invention
The present invention also provides antibodies capable of recognizing and binding the isolated, or modified, or multimeric antigens of this invention, including antibodies derived from mixtures of such antigens or fragments thereof. 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 are also useful in passive vaccine compositions.
The antibodies of this invention are generated by conventional means utilizing the isolated, recombinant or modified antigens of this invention, or mixtures of such antigens or antigenic fragments. For example, polyclonal antibodies are generated by conventionally stimulating the immune system of a selected animal or human with the isolated antigen or mixture of antigenic proteins or peptides of this invention, allowing the immune system to produce natural antibodies thereto, and collecting these antibodies from the animal or human's blood or other biological fluid.
For example, an antibody according to the invention is produced by administering to a vertebrate host the antigen or antigenic composition of this invention, e.g., P7-1. Preferably a recombinant version of P7-1 (rP7-1) is used as an immunogen. A suitable polyclonal antibody against the P7-1 antigen kills IP90 spirochetes in vitro by antibody-dependent, complement-mediated killing (ADCK) regardless of whether the antibody was obtained by: (1) affinity purification using as immunoabsorbant native P39.5 antigen of IP90 spirochetes (as separated on a Western blot; see FIG. 6) and as source of antibody the antiserum generated during an infection of rhesus monkeys with JD1 spirochetes or, (2) by immunizing mice with recombinant P7-1 from IP90 (rP7-1).
Thus, an antibody of the invention is isolated by affinity purifying antiserum generated during an infection of a vertebrate animal, e.g., a rhesus monkey, with JD1 spirochetes, using as immunoabsorbant the native P39.5 antigen of IP90, or one or more of the cassette string proteins identified herein. Similarly, an antibody of the invention is isolated by immunizing mice with a purified, recombinant antigen of this invention, or a purified, isolated P39.5 of native origin. Monoclonal antibodies (MAbs) directed against P39.5 are also generated. Hybridoma cell lines expressing desirable MAbs are generated by well-known conventional techniques, e.g. Kohler and Milstein and the many known modifications thereof Similarly desirable high titer antibodies are 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, 86:10029-10033 (1989); PCT Patent Publication No. WO9007861; Riechmann et al., Nature, 332:323-327 (1988); Huse et al, Science, 246:1275-1281 (1988)].
Given the disclosure contained herein, one of skill in the art may generate chimeric, humanized or fully human antibodies directed against P39.5 or the cassette proteins, or antigenic fragments thereof by resort to known techniques by manipulating the complementarity determining regions of animals or human antibodies to the antigen of this invention. See, e.g., E. Mark and Padlin, "Humanization of Monoclonal Antibodies", Chapter 4, The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology of Monoclonal Antibodies, Springer-Verlag (June, 1994).
Alternatively, the antigens are assembled as multi-antigenic complexes [see, e.g., European Patent Application No. 0339695, published Nov. 2, 1989] or as simple mixtures of antigenic proteins/peptides and employed to elicit high titer antibodies capable of binding the selected antigen(s) as it appears in the biological fluids of an infected animal or human.
Further provided by the present invention are anti-idiotype antibodies (Ab2) and anti-anti-idiotype antibodies (Ab3). Ab2 are specific for the target to which anti-P39.5 antibodies of the invention bind and Ab3 are similar to P39.5 antibodies (Ab1) in their binding specificities and biological activities [see, e.g., M. Wettendorff et al., "Modulation of anti-tumor immunity by anti-idiotypic antibodies." In Idiotypic Network and Diseases, ed. by J. Cerny and J. Hiernaux J, Am. Soc. Microbiol., Washington D.C.: pp. 203-229, (1990)]. These anti-idiotype and anti-anti-idiotype antibodies are produced using techniques well known to those of skill in the art. Such anti-idiotype antibodies (Ab2) can bear the internal image of P39.5, the cassette proteins, or fragments thereof and are thus useful for the same purposes as P39.5, the cassette proteins or the fragments.
In general, polyclonal antisera, monoclonal antibodies and other antibodies which bind to the selected antigen (Ab1) are useful to identify epitopes of P39.5 or the cassette proteins to separate P39.5 (or the cassette proteins) and analogs thereof from contaminants in living tissue (e.g., in chromatographic columns and the like), and in general as research tools and as starting material essential for the development of other types of antibodies described above. Anti-idiotype antibodies (Ab2) are useful for binding the same target and thus may be used in place of the original antigen, e.g., P39.5, to induce an immune response. The Ab3 antibodies are useful for the same reason the Ab1 are useful. Other uses as research tools and as components for separation of P39.5 or the cassette proteins, from other contaminants, for example, are also contemplated for the above-described antibodies.
For use in diagnostic assays, the antibodies are associated with conventional labels which are capable, alone or in concert with other compositions or compounds, of providing a detectable signal. Where more than one antibody is employed in a diagnostic method, the labels are desirably interactive to produce a detectable signal. Most desirably, the label is detectable visually, e.g. calorimetrically. A variety of 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. 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. Other label systems that may be utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles [Bangs Laboratories, Indiana] 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. Still other labels include fluorescent compounds, radioactive compounds or elements. 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.
IV. Diagnostic Methods and Assays
The present invention also provides methods of diagnosing Lyme disease. These diagnostic methods are useful for diagnosing humans or animals exhibiting the clinical symptoms of, or suspected of having, Lyme disease.
In one embodiment, this diagnostic method involves detecting the presence of naturally occurring anti-P39.5 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 This method comprises the steps of incubating a P39.5 antigen or a cassette string antigen of this invention 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. Subsequently 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.
In a similar embodiment, this diagnostic method involves detecting the presence of naturally occurring anti-P1-1, anti-P3-1, anti-P6-l, anti P7-i, anti-P9-1, and/or anti-P12-1 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 This method comprises the steps of incubating one or preferably, a mixture, of these antigen(s) of this invention with a sample of biological fluids from the patient. Antibodies present in the fluids as a result of B. burgdorferi infection will form antibody-antigen complexes with the antigen(s). Subsequently 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.
In one embodiment of the method, purified antigen, fragment or mixture of antigens is electro- or dot-blotted onto nitrocellulose paper. Subsequently, the biological fluid (e.g. serum or plasma) is incubated with the blotted antigen, and antibody in the biological fluid is allowed to bind to the antigen(s). The bound antibody is then detected by standard immunoenzymatic methods.
In another embodiment of the method, latex beads are conjugated to the antigen(s) 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.
In another embodiment, the diagnostic method of the invention involves detecting the presence of the naturally occurring P39.5 or cassette string antigen(s) itself in its association with the Borrelia 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 an antigen of this invention preferably conventionally labelled for detection) with a sample of biological fluids from a human or an animal to be diagnosed. In the presence of Borrelia infection of the human or animal patient, an antigen-antibody complex is formed (specific binding occurs). Subsequently, excess labeled 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 therewith.
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.
One preferred assay for the screening of blood products or other physiological or biological fluids is an enzyme linked immunosorbant assay, i.e., an ELISA. Typically in an ELISA, the isolated antigen(s) 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). The well is then incubated with a biological sample suspected of containing specific anti-B. burgdorferi antibody. The sample can be applied neat, or more often, it can be diluted, usually in a buffered solution which contains a small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO. 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 (.alpha. HuIg) or labeled antibodies to other species, e.g., dogs. The label can be chosen from a variety of enzymes, including horseradish peroxidase (HRP), .beta.-galactosidase, alkaline phosphatase, and glucose oxidase, as described above. Sufficient time is allowed for specific binding to occur again, then the well is washed again to remove unbound conjugate, and the substrate for the enzyme is added. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally.
Further, MAbs or other antibodies of this invention which are capable of binding to the antigen(s) can be bound to ELISA plates. In another diagnostic method, the biological fluid is incubated on the antibody-bound plate and washed. Detection of any antigen-antibody complex, and qualitative measurement of the labeled MAb is performed conventionally, as described above.
Other useful assay formats include the filter cup and dipstick. In the former assay, an antibody of this invention is fixed to a sintered 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.
Other diagnostic assays can employ the antigen(s) or fragments of this invention as nucleic acid probes or an anti-sense sequences, which can identify the presence of infection in the biological fluid by hybridizing to complementary sequences produced by the pathogen in the biological fluids. Such techniques, such as PCR, Northern or Southern hybridizations etc. are well known in the art.
It should be understood by one of skill in the art that any number of conventional protein assay formats, particularly immunoassay formats, or nucleic acid assay formats, may be designed to utilize the isolated antigens and antibodies or their nucleic acid sequences or anti-sense sequences of this invention for the detection of Borrelia 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.
V. Diagnostic Kits
For convenience, reagents for ELISA or other assays according to this invention may be provided in the form of kits. Such kits are useful for diagnosing infection with Borrelia in a human or an animal sample. Such a diagnostic kit contains an antigen of this invention and/or at least one antibody capable of binding an antigen of this invention, or the nucleic acid sequences encoding them, or their anti-sense sequences. Alternatively, such kits may contain a simple mixture of such antigens or sequences, or means for preparing a simple mixture.
These kits can include microtiter plates to which the Borrelia antigen proteins or antibodies or nucleic acid sequences of the invention have been pre-adsorbed, various diluents and buffers, labeled conjugates for the detection of specifically bound antigens or antibodies, or nucleic acids and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens. Other components of these kits can easily be determined by one of skill in the art. Such components may include polyclonal or monoclonal capture antibodies, antigen of this invention, 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 calorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, and a sample preparator cup. Such kits provide a convenient, efficient way for a clinical laboratory to diagnose Borrelia infection.
VI. Therapeutic Compositions
The antigens, antibodies, nucleic acid sequences or anti-sense sequences of the invention, alone or in combination with other antigens, antibodies, nucleic acid sequences or anti-sense sequences may further be used in therapeutic compositions and in methods for treating humans and/or animals with Lyme Disease. For example, one such therapeutic composition may be formulated to contain a carrier or diluent and one or more of the anti-P7-1 or other anti-cassette string protein antibodies of the invention. 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, dextran, agar, pectin, peanut oil, olive oil, sesame oil, and water. Additionally, the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol distearate alone or with a wax. In addition, slow release polymer formulations can be used.
Optionally, 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.
Alternatively, or in addition to the antibodies of the invention, other agents useful in treating Lyme disease, e.g., antibiotics or immunostimulatory agents and cytokine regulation elements, are expected to be useful in reducing or eliminating disease symptoms. Agents which can be used to suppress or counteract the immune suppressants released by the tick vector or the spirochete should act to assist the natural immunity of the infected human or animal. Thus, such agents may 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 teachings of this invention.
According to the method of the invention, a human or an animal may be treated for Lyme Disease by administering an effective amount of such a therapeutic composition. An "effective amount" may be between about 0.05 to about 1000 .mu.g/mL of an antibody of the invention. A suitable dosage may be about 1.0 mL of such an effective amount. 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. Preferably, such a composition is administered parenterally, preferably intramuscularly or subcutaneously. However, it may also be formulated to be administered by any other suitable route, including orally or topically.
VII. Vaccine Compositions
None of the potential problems of the prior art arise with a vaccine based on an antigen which is expressed in the vertebrate host. An improved surface-antigen-based vaccine for the prevention of Lyme disease in humans and other animals is provided by the inclusion of the P39.5 antigen of this invention, and a pharmaceutically acceptable carrier or diluent. This vaccine composition may contain one or more of the isolated, recombinant, modified or multimeric forms of the P39.5 antigen of the invention, or mixtures thereof. Similarly, salts of the antigenic proteins may be employed in such compositions.
Another embodiment of a composition of the present invention is based on the other cassette string antigens, i.e., P1-1, P3-1, P6-1, P7-1, P9-1 and P12-1. The inventor proposes that the mechanism of antigenic variation to which the vlsE antigen is subjected indicates that this antigen is crucially important for spirochetal survival in the vertebrate host. According to this invention, a novel vaccine method involves circumventing the spirochetal defense mechanism by immunizing a host with a large fraction of the epitopes that are expressed on the cassette string that is the very source of the variation. This is made possible by the unique fact that most of this cassette string is in frame. The inventors have demonstrated this concept, as described in the Examples below, by immunizing a host with the cassette string antigen, P7-1, and inducing antibody that kills the spirochetes. Thus, according to this invention, one method of vaccination thus involves administering to the host several or all of the cloned and purified proteins described above, which are derived from antigenically dissimilar sections of the IP90 cassette string. While a variety of immunogenic compositions may be prepared from such proteins, it is presently preferred that a simple mixture of two or more of the cassette string proteins or peptide fragments thereof be employed for this purpose.
Combinations of the antigen(s) of this invention with other antigens of B. burgdorferi, such as the OspA, OspB and OspC proteins, BmpA, B, C or D proteins, or fragments thereof are also encompassed by this invention.
Exemplary carriers are as described above for therapeutic compositions. Optionally, the vaccine composition may further contain adjuvants, preservatives, chemical stabilizers, or other antigenic proteins. Typically, 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 parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
One or more of the above described vaccine components may be admixed or adsorbed with a conventional adjuvant. The adjuvant is used to attract leukocytes or enhance an immune response. Such adjuvants include, among others, Ribi, mineral oil and water, aluminum hydroxide, Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic plyois, muramyl dipeptide, killed Bordetella, and saponins, such as Quil A. In addition, 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 may also be included in these compositions, e.g., feline coronavirus, etc.
The invention thus also encompasses a prophylactic method entailing administering to an animal or human an effective amount of such a composition. The P39.5 and/or cassette string protein antigenic compositions are administered in an "effective amount", that is, an amount of antigen that is effective in a route of administration to provide a vaccinal benefit, i.e., protective immunity. Suitable amounts of the antigen can be determined by one of skill in the art based upon the level of immune response desired. In general, however, the vaccine composition contains between 1 ng to 1000 mg antigen, and more preferably, 0.05 .mu.g to 1 mg per mL of antigen. 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.
In general, 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. Routes of administration may be combined, if desired, or adjusted.
Further, the vaccine may be a DNA vaccine, which includes the P7-1 DNA sequence or a fragment thereof, another cassette string protein, or a fragment thereof, optionally under the control of regulatory sequences. Thus, the antigen-encoding DNA may be carried in a vector, e.g., a viral vector. Generally, a suitable vector-based treatment contains between 1x10-3 pfu to 1x1012 pfu per dose. However, the dose, timing and mode of administration of these compositions may be determined by one of skill in the art. Such factors as the age, and physical condition of the vaccinate may be taken into account in determining the dose, timing and mode of administration of the immunogenic or vaccine composition of the invention.
VIII. Drug Screening and Development
The proteins, antibodies and polynucleotide sequences of the present invention may also be used in the screening and development of chemical compounds or proteins which have utility as therapeutic drugs or vaccines for the treatment or diagnosis or prevention of Lyme Disease. As one example, a compound capable of binding to P39.5 and preventing its biological activity may be a useful drug component for the treatment or prevention of Lyme Disease. The methods described herein may also be applied to fragments of P39.5. Similarly, a compound capable of binding to a cassette string protein, or fragment thereof and preventing its biological activity may be a useful drug component for the treatment or prevention of Lyme Disease.
Suitable assay methods may be readily determined by one of skill in the art. Where desired, and depending on the assay selected, the selected antigen(s), e.g., P39.5, may be immobilized directly or indirectly (e.g., via an anti-P39.5 antibody) on a suitable surface, e.g., in an ELISA format. Such immobilization surfaces are well known. For example, a wettable inert bead may be used. Alternatively, the selected antigen, e.g., P39.5, may be used in screening assays which do not require immobilization, e.g., in the screening of combinatorial libraries. Assays and techniques exist for the screening and development of drugs capable of binding to an antigen of this invention, e.g., P39.5. These include the use of phage display system for expressing the antigenic protein(s), and using a culture of transfected E. coli or other microorganism to produce the proteins for binding studies of potential binding compounds. See, for example, the techniques described in G. Cesarini, FEBS Letters, 307(1):66-70 (July 1992); H. Gram et al., J. Immunol. Meth., 161:169-176 (1993); C. Summer et al., Proc. Natl. Acad. Sci. . USA, 89:3756-3760 (May 1992), incorporated by reference herein.
Other conventional drug screening techniques may be employed using the proteins, antibodies or polynucleotide sequences of this invention. As one example, a method for identifying compounds which specifically bind to a protein of this invention, e.g., P39.5, can include simply the steps of contacting a selected P39.5 protein with a test compound to permit binding of the test compound to P39.5; and determining the amount of test compound, if any, which is bound to the P39.5 protein. Such a method may involve the incubation of the test compound and the P39.5 protein immobilized on a solid support. Similar methods may be employed for one or more of the cassette string proteins.
Typically, the surface containing the immobilized ligand is permitted to come into contact with a solution containing the protein and binding is measured using an appropriate detection system. Suitable detection systems include the streptavidin horse radish peroxidase conjugate, direct conjugation by a tag, e.g., fluorescein. Other systems are well known to those of skill in the art. This invention is not limited by the detection system used.
Another method of identifying compounds which specifically bind to P39.5 or another protein of this invention can include the steps of contacting the protein, e.g., P39.5, immobilized on a solid support with both a test compound and the protein sequence which is a receptor for P39.5 to permit binding of the receptor to the P39.5 protein; and determining the amount of the receptor which is bound to the P39.5 protein. The inhibition of binding of the normal protein by the test compound thereby indicates binding of the test compound to the P39.5 protein. Similar methods may be employed for one or more of the cassette string proteins.
Thus, through use of such methods, the present invention is anticipated to provide compounds capable of interacting with P39.5 or portions thereof, and/or the cassette string protein(s) or portions thereof and either enhancing or decreasing the protein's biological activity, as desired. Such compounds are believed to be encompassed by this invention. Further wherever, above or below, the P39.5 protein is mentioned, it may be substituted with P7-1 or one or more of the other cassette string proteins.
Claim 1 of 6 Claims
What is claimed is:
1. A recombinant or synthetic protein or peptide that binds with antibodies to the causative agent of Lyme Disease in infected humans or animals, said protein or peptide selected from the group consisting of:
(a) an amino acid sequence of SEQ ID NO: 2;
(b) an amino acid sequence of SEQ ID NO: 14;
(c) an amino acid sequence encoded by SEQ ID NO: 3;
(d) an amino acid sequence encoded by SEQ ID NO: 7;
(e) an amino acid sequence encoded by SEQ ID NO: 11; and
(f) an amino acid sequence of a fragment of (a) through (e) of at least five amino acids in length.