WO1997027301A9 - Compositions de proteines de liaison de la decorine et modes d'utilisation - Google Patents

Compositions de proteines de liaison de la decorine et modes d'utilisation

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Publication number
WO1997027301A9
WO1997027301A9 PCT/US1996/017081 US9617081W WO9727301A9 WO 1997027301 A9 WO1997027301 A9 WO 1997027301A9 US 9617081 W US9617081 W US 9617081W WO 9727301 A9 WO9727301 A9 WO 9727301A9
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WIPO (PCT)
Prior art keywords
seq
dbpb
protein
nucleic acid
peptide
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PCT/US1996/017081
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English (en)
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WO1997027301A1 (fr
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Priority claimed from US08/589,711 external-priority patent/US5853987A/en
Application filed filed Critical
Priority to EP96937742A priority Critical patent/EP0877808A1/fr
Priority to US09/117,257 priority patent/US6214355B1/en
Priority to JP9526823A priority patent/JP2000503842A/ja
Priority to AU75212/96A priority patent/AU720349B2/en
Publication of WO1997027301A1 publication Critical patent/WO1997027301A1/fr
Publication of WO1997027301A9 publication Critical patent/WO1997027301A9/fr

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  • the present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising DNA segments, and proteins derived from bacterial species. More particularly, the invention provides gene compositions encoding the decorin (Den) binding proteins (DBPs) from Borrelia burgdorferi and the corresponding peptide epitopes and protein sequences comprising native and synthetically-modified Den binding site domains.
  • DBPs decorin binding proteins
  • DNA segments encoding synthetically-modified ligand binding site domains and native and synthetic DbpA and DbpB proteins are disclosed, such as, for example, the use of dbp ⁇ and dbpB DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various pharmacological and immunological applications.
  • Lyme disease (Steere, 1989), or Lyme borreliosis, is transmitted by ticks, particularly of the genus Ixodes, and caused by spirochetes of the genus Borrelia.
  • Lyme disease agents that is borrelias isolated from humans or animals with clinical Lyme disease, are currently classified into at least four phylogenetic groups: B. burgdorferi sensu stricto, B. garinii, B. andersonii, and B. ajzelii. Strains potentially representing other phylogenetic groups of Lyme disease agents as well, such as group 25015, have been also isolated from ixodid ticks. Collectively these spirochetes are referred to as B.
  • Lyme disease is transmitted through the bite of a tick which attaches itself to the host and, upon feeding, deposits the spirochetes into the dermis of the skin.
  • B. burgdorferi replicates before endovascular dissemination to organs.
  • an annular spreading skin lesion, erythema migrans forms from the site of the tick bite.
  • Early symptoms of Lyme disease are flu-like and may include fatigue and lethargy. Left untreated, Lyme disease can develop into a chronic, multisystemic disorder involving the skin, joints, heart, and central nervous system.
  • the spirochetes become associated with and appear to colonize the collagen fibers.
  • Skin is the most consistent site of spirochete- positive culture. In persistent infection, the skin may provide a protective niche for replication, thereby acting as a reservoir of spirochetes for subsequent distribution to other tissues.
  • B. burgdorferi disseminates to other organs, the organisms appear to localize to the extracellular spaces of these tissues as well.
  • tendon Barthold et al , 1993; 1991
  • ligament Haupl et al , 1993
  • heart Zimmer et al , 1990
  • muscle Barthold et al , 1992; Duray, 1992
  • B. burgdorferi spirochetes are found primarily in close association with collagen fibers, suggesting that this association is an important mechanism of tissue adherence in different stages of infection.
  • Lyme disease is typically treated with antibiotics, which are generally effective in the early stages of the disease. Later stages involving cardiac, arthritic, and nervous system disorders are often non- responsive.
  • OspA-based vaccines may prove to have limited efficacy in treating Lyme disease in humans: a) Modulation of OspA expression by B. burgdorferi may limit the site of action of OspA-specific antibodies to spirochetes residing in the tick midgut as these antibodies are ineffective shortly after infection; b) Human immune responses to OspA subunit vaccines have not matched those of rodents in level or duration; and c) OspA is serologically diverse, particularly among European and Asian B. garinii and B. afzelii isolates. Reactivity with panels of OspA monoclonal antibodies (mAbs), and DNA sequence analysis has shown that as many as seven different OspA subgroups can be distinguished (Wilske et al. ,
  • cross-protection was seen by one group using an immunocompetent mouse model (Fikrig et al. , 1995), but cross-protection was weak or absent in SCID mouse or hamster models used by other (Schaible et al , 1993;
  • OspA protein is either poorly i munogenic in humans, or not expressed by B. burgdorferi in vivo until late in infection. Lyme disease patients, mice, hamsters, and dogs infected by tick bite or low-doses of cultured B. burgdorferi fail to mount substantial anti-OspA immune responses for many months following infection although they do mount early responses to other B. burgdorferi antigens (flagellin, OspC, etc.) (Steere, 1989;
  • OspA is expressed by B. burgdorferi within ticks (Barbour et al , 1983), but detection of OspA on borrelias in tissue early after infection is difficult. Passive immunization of mice with OspA antibody (Schaible et al , 1990), or immunization with recombinant OspA, after challenge does not eliminate infection and only partially alters disease. Unfortunately, OspA-immunized mice are not protected from a challenge with host-adapted spirochetes delivered in the form of skin biopsy transplants from infected mice (Barthold et al , 1995).
  • the bacteria appear to express OspA in vivo only at later stages when the infection becomes disseminated. This would be explained by down-regulation of OspA expression by borrelia shortly after initiation of feeding by the tick.
  • de Silva et al. (1996) demonstrated that when OspA-specific antibodies were administered to mice before or at the time of attachment of borrelia-infected ticks these mice were protected from spirochetal infection. However, when OspA-specific antibody was administered 48-hr after tick attachment no protection was observed. Modulation of borrelia antigen expression within feeding ticks has recently been reported for OspC; initially low in resting ticks, OspC levels increase on B.
  • OspC might appear to be a promising in vivo target, but its high level of antigenic variation complicates its development as a vaccine (Probert and LeFebvre, 1995).
  • In vitro cultivation of B. burgdorferi suggests that the genes for OspA and
  • OspC are inversely regulated. Preliminary findings of some researchers do suggest that OspA levels similarly decrease after initiation of tick feeding. If these findings are confirmed, OspA antibodies will need to pre-exist at high levels in human or animal hosts prior to the tick bite to be effective against OspA-expressing borrelias in the tick, and may receive little or no boosting upon delivery of the spirochetes into the skin within the milieu of immunosuppressive components of the tick saliva (Urioste et al , 1994).
  • the present invention overcomes one or more of these and other drawbacks inherent in the prior art by providing novel compositions and methods for their use in the treatment of Lyme disease using non-antibiotic strategies.
  • Disclosed are methods for the use of novel peptides and antibody compositions in the treatment of Lyme disease mediated by the inhibition of B. burgdorferi binding to the host cell ECM component, Den.
  • Also disclosed are methods for active and passive immunization against B. burgdorferi and related borrelias including B. afzelii, B. andersonii, B. japonica, and B. garinii using novel native and site-specifically-altered DBP compositions and DBP-derived epitopic peptides from B. burgdorferi, B.
  • dbp gene means a nucleic acid sequence encoding a DBP.
  • Preferred dbp genes include the dbp A and dbpB genes, in particular those from B. burgdorferi, B. japonica, B. andersonii, B. afzelii, and B. garinii.
  • a preferred nucleic acid sequence encoding a dbpA gene is the nucleotide sequence of SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:52, and more preferably, the nucleotide sequence of the approximately 0.6-kb open reading frame (ORF) of the B. burgdorferi dbp A gene (from position 1471 to 2031 of SEQ ID NO:7), or strain variants or active fragments thereof.
  • a preferred nucleic acid sequence encoding a dbpB gene is the nucleotide sequence of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO: 61 , SEQ ID NO: 63, and SEQ ID NO: 65, and more preferably, the nucleotide sequence of the approximately 0.6-kb open reading frame (ORF) of the B. burgdorferi dbpB gene (from position 791 to 1351 of SEQ ID NO:7) or strain variants or active fragments thereof.
  • ORF open reading frame
  • genes encoding DBPs will vary in nucleic acid sequence from strain to strain, but that the variation in nucleic acid sequence will not preclude hybridization between sequences encoding the DBPs of each strain under moderate to strict hybridization conditions. It is also contemplated that the genes encoding DbpAs from various strains may vary in nucleic acid sequences, but that the variation will not preclude hybridization between sequences encoding a DbpA from each strain under moderate to strict hybridization conditions. Likewise, it is contemplated that the genes encoding DbpBs from various strains may vary in nucleic acid sequences, but that the variation will not preclude hybridization between sequences encoding a DbpB from each strain under moderate to strict hybridization conditions.
  • a strain variant of a DBP means any polypeptide encoded, in whole or in part, by a nucleic acid sequence which hybridizes under moderate to strict hybridization conditions to the nucleic acid sequence of SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:ll , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26 encoding the DbpA protein of strains 297, B31 , Sh.2.82, HB-19, PGau, IP90, LP4, LP7, and JD1, respectively, or to the nucleic acid sequence of SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, S
  • a strain variant of a DBP means any polypeptide encoded, in whole or in part, by a nucleic acid sequence which hybridizes under moderate to strict hybridization conditions to the nucleic acid sequence of SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11, or to the nucleic acid sequence of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO: 63, or SEQ ID NO: 65, encoding the DbpB protein of strains CA287 (DbpB protein: SEQ ID NO:54), IPS (DbpB protein: SEQ ID NO:56), JD1 (DbpB protein: SEQ ID NO:58), 297, SH2, and LP4 (Dbp
  • strain variants of DBPs include those proteins encoded by nucleic acid sequences which may be amplified using one or more of the dbpA-encoding nucleic acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, or SEQ ID NO:51, and more preferably from the nucleic acid sequence of SEQ ID NO:7, and most preferably, the nucleic acid sequence of the 0.6-kb ORF (from position 1471 to 2031 of
  • strain variants of DBPs include those proteins encoded by nucleic acid sequences which may be amplified using one or more of the dbpB-encoding nucleic acid sequences of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:63, or SEQ ID NO: 65, and more preferably from the nucleic acid sequence of SEQ ID NO: 7, and most preferably, the nucleic acid sequence of the 0.6-kb ORF (from position 791 to 1351 of SEQ ID NO: 7) encoding the B. burgdorferi 297 DbpB protein.
  • SEQ ID NO: 7 lists the nucleotide sequence of a 2.5-kb insert of borrelia genomic DNA cloned in pBlueScriptTM which comprises the dbpA and dbpB genes of B. burgdorferi 291.
  • This recombinant clone designated BG26:pB/2.5(5), has been deposited with the American Type Culture collection in E. coli strain JM101, and has been given the ATCC accession number ATCC69791.
  • Contained within the 2.5-kb DNA insert from position 1471 to 2031 is the approximately 0.6-kb ORF encoding the B. burgdorferi DbpA protein (SEQ ID NO: 8) and from position 791 to position 1351, the approximately 0.6-kb ORF encoding the B. burgdorferi DbpB protein (SEQ ID NO: 28).
  • the invention also comprises strain variants of DBPs and nucleic acid segments encoding DBPs, in particular, the dbpA and dbpB genes which encode the DbpA and DbpB proteins, respectively.
  • Strain variants are those nucleic acid compositions and polypeptide compositions expressed by various strains of B. burgdorferi and related borrelias including B. afzelii, B. andersonii, B. japonica, and B. garinii which specifically encode DBPs.
  • DBPs also bind Den and related proteoglycans and share similarity of structure and function with the DbpA and DbpB proteins of B. burgdorferi strain 297.
  • DbpA amino acid sequences include those DbpA sequences of related borrelia strains B31, Sh.2.82, HB-19, LP4, LP7, and JD1 of B. burgdorferi (SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26, respectively); strain pGau (SEQ ID NO: 18) of B. afzelii; or strain IP90 (SEQ ID NO:20) of B. garinii.
  • DbpA amino acid sequences are also disclosed in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:52.
  • DbpB amino acid sequences are disclosed in SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, and SEQ ID NO:56.
  • aspects of the invention concern the identification of such strain variants using diagnostic methods and kits described herein.
  • methods utilizing dbpA and dbpB gene sequences as nucleic acid hybridization probes and/or anti-DbpA and anti-DbpB antibodies in western blots or related analyses are useful for the identification of such strain variants.
  • the identity of potential strain variants of DBPs may be confirmed by Den binding assays, e.g. , by blot analysis with labeled Den, or alternatively by the demonstrating the ability of the strain-variant DBPs to lessen or prevent adherence of B. burgdorferi and related borrelias including B. afzelii, B. andersonii, B. japonica, and B. garinii to Den.
  • a DBP means a purified and isolated protein including a strain variant or an active fragment thereof, derived from B. burgdorferi or a similar species which induces Lyme disease in a similar way, and having the ability to bind Den.
  • a DBP is a DbpA or DbpB protein encoded by a nucleic acid sequence contained within the B. burgdorferi DNA insert shown in SEQ ID NO: 7, and more preferably a DbpA or DbpB protein which comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:28, which encode DbpA and DbpB from 297, respectively.
  • Preferred DbpA-encoding DNA sequences are those shown in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26, as well as those DbpA-encoding DNA sequences disclosed in SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, and SEQ ID NO:51, and all strain variants or active fragments thereof encoding all or portions of a borrelial DbpA protein.
  • Preferred DbpB-encoding DNA sequences are those shown in FIG. 3 (SEQ ID NO.
  • FIG. 6 SEQ ID NO:64
  • FIG. 7 SEQ ID NO:62
  • FIG. 8 SEQ ID NO:66
  • FIG. 9 SEQ ID NO:58
  • FIG. 10 SEQ ID NO:56
  • FIG. 11 SEQ ID NO:
  • a DBP composition is also understood to comprise one or more polypeptides that are immunologically reactive with antibodies generated against B. burgdorferi, B. garinii, B. afzelii, B. andersonii, B. japonica, or related
  • SEQ ID NO:59 SEQ ID NO:61, SEQ ID NO:63, or SEQ ID NO:65, or to active fragments, or to strain variants thereof.
  • a DBP composition of the present invention is understood to comprise one or more polypeptides that are capable of eliciting antibodies that are immunologically reactive with one or more DbpA or DbpB proteins encoded by one or more dbpA nucleic acid sequences contained in SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: l l , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, or SEQ ID NO:51, or by one or more dbpB nucleic acid sequences contained in SEQ ID NO:7,
  • DbpA proteins include one or more of the DbpA amino acid sequences disclosed in any of SEQ ID NO:8, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:52.
  • DbpB proteins include one or more of the DbpB amino acid sequences disclosed in any of SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, and SEQ ID NO:66.
  • a DBP composition of the present invention is also understood to comprise one or more polypeptides that elicit an immune response in a treated animal, this immune response being effective to lessen or prevent symptomatic disorders associated with Lyme disease or related borrelioses, or which polypeptides are capable of eliciting antibodies that are immunologically reactive with a DbpA encoded by a nucleic acid sequence of any of SEQ ID NO:7, SEQ ID NO.10, SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49
  • an active fragment of a DBP includes a whole or a portion of a DBP which is modified by conventional techniques, e.g. , mutagenesis, or by addition, deletion, or substitution, but which active fragment exhibits substantially the same structure and function as a native DBP as described herein.
  • portions of the protein not required to block adherence of B. burgdorferi to Den may be deleted or altered; additions to the protein may be made to enhance the protein's antigenicity according to conventional methods.
  • a DBP composition which confers protection against Lyme disease means a DBP composition which prevents or lessens adhesion of B.
  • burgdorferi to Den, or prevents or lessens adhesion the severity of any of the disorders associated with B. burgdorferi infection, including erythema migrans, arthritis, carditis, neurological disorders, or any other Lyme disease-related disorder.
  • Other aspects of the present invention concern isolated DNA segments and recombinant vectors encoding one or more DBPs, in particular, the DbpA and DbpB proteins from Borrelia such as B. burgdorferi, B. afzelii, B. andersonii, B. garinii, and B.
  • japonica and the creation and use of recombinant host cells through the application of DNA technology, that express one or more dbp gene products, and in particular, the dbpA and dbpB genes from Borrelia such as B. burgdorferi, B. afzelii, B. andersonii, B. garinii, and B. japonica, .
  • the invention concerns DNA segments comprising an isolated gene that encodes a DbpA protein or peptide that includes an amino acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:52.
  • DNA segments are represented by those that include a dbpA nucleic acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO.37, SEQ ID NO:39, SEQ ID NO.41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, or SEQ ID NO:53.
  • compositions that include a purified DbpA protein that has an amino acid sequence essentially as set forth by the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO.T9, SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO.27, SEQ ID NO:30, SEQ ID NO.32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:52 are also encompassed by the invention.
  • the invention also concerns DNA segments comprising an isolated gene that encodes a DbpB protein or peptide that includes an amino acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:66.
  • DNA segments are represented by those that include a dbpB nucleic acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO.53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO.61 , SEQ ID NO:63, or SEQ ID NO:65.
  • compositions that include a purified DbpB protein that has an amino acid sequence essentially as set forth by the amino acid sequence of SEQ ID NO: 28, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:66 are also encompassed by the invention.
  • the present invention concerns DNA segments, that can be isolated from virtually any bacterial source, that are free from total genomic DNA and that encode proteins having DBP-like activity. DNA segments encoding one or more DBP-like species may prove to encode proteins, polypeptides, subunits, functional domains, and the like.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a DBP refers to a DNA segment that contains one or more DBP coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term “DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
  • a DNA segment comprising an isolated or purified dbp gene refers to a DNA segment including DBP coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences.
  • the sequence encodes a DbpA or DbpB protein, and more preferably, comprises a dbpA or dbpB gene, in particular, a dbp A or dbpB gene from Borrelia such as B. burgdorferi, B. afzelii, B. andersonii, B. garinii, or B. japonica.
  • the term "gene” is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit.
  • this functional term includes both genomic sequences, extra- genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. Such segments may be naturally isolated, or modified synthetically by the hand of man.
  • isolated substantially away from other coding sequences means that the gene of interest, in this case, a gene encoding a DBP, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a DBP species that comprises an amino acid sequence essentially as set forth in any of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17.
  • SEQ ID NO: 19 SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:66, or biologically-functional equivalents thereof.
  • the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that comprises a sequence essentially as set forth in SEQ ID NO:7, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, or SEQ ID NO:65, or biologically-functional equivalent
  • nucleotide sequences are also considered to be essentially as those disclosed herein when they encode essentially the same amino acid sequences as disclosed, or that they encode biologically functional equivalent amino acids tot hose as disclosed herein.
  • preferred nucleotide sequences are those which encode the amino acid sequences of any of SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ
  • sequences that have between about 70% and about 80% ; or more preferably, between about 81 % and about 90%; or even more preferably, between about 91 % and about 99%; of amino acids that are identical or functionally equivalent to the amino acids disclosed herein, will be sequences that are "essentially as set forth in SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ
  • the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: l l , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:63 or SEQ ID NO:65.
  • SEQ ID NO:7 SEQ ID NO: 10
  • SEQ ID NO: 11 SEQ ID NO: 12
  • SEQ ID NO: 14 SEQ ID NO: 16
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or
  • nucleic acid sequences may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various upstream or downstream regulatory or structural genes.
  • the present invention also encompasses DNA segments that are complementary, or essentially complementary, to one or more of the sequences set forth in SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63 or SEQ ID NO: 65.
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules.
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to one or more of the nucleic acid segments of SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: ll, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:
  • nucleic acid segments of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to one or more of SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO: 63 or SEQ ID NO: 65, such as about 14 nucleotides, and that are up to about 10,000 or
  • intermediate lengths means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc. ; 21, 22, 23, etc. ; 30, 31, 32, etc. ; 50, 51, 52, 53, etc. ; 100, 101, 102, 103, etc. ; 150, 151, 152, 153, etc. ; including all integers through the 200-500; 500-1 ,000; 1 ,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 12,001 , 12,002, 13,001 , 13,002 and the like.
  • this invention is not limited to the particular nucleic acid sequences as disclosed in SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: l l , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO.31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO.43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63 or SEQ ID NO:65 or to the amino acid sequences as disclosed in SEQ ID NO:8, SEQ ID NO:
  • Recombinant vectors and isolated DNA segments may therefore variously include the DbpA and/or DbpB coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include a DBP coding region or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
  • DNA segments of the present invention encompass biologically functional equivalent DBPs and DBP peptides, in particular those DBPs isolated from prokaryotic sources such as bacteria.
  • DNA segments isolated from species of Borrelia and related bacteria which are shown to bind Den are particularly preferred for use in the methods disclosed herein. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g. , to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.
  • fusion proteins and peptides e.g. , where the DBP coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e. g. , proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
  • Recombinant vectors form further aspects of the present invention.
  • Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter.
  • the promoter may be in the form of the promoter that is naturally associated with a DBP gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DBP gene in its natural environment.
  • Such promoters may include dbpA or dbpB promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell.
  • promoters may include dbpA or dbpB promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell.
  • promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989).
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
  • Prokaryotic expression of nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promotor sequences such as those provided by tac, trp, lac, lacUV5 or T7.
  • expression vectors and promotor sequences such as those provided by tac, trp, lac, lacUV5 or T7.
  • An exemplary eukaryotic promoter system contemplated for use in high-level expression is the Pichia expression vector system (Pharmacia LKB Biotechnology).
  • DNA segments that encode peptide antigens from about 15 to about 100 amino acids in length, or more preferably, from about 15 to about 50 amino acids in length are contemplated to be particularly useful.
  • dbpA and dbpB genes and DNA segments derifed therefrom may also be used in connection with somatic expression in an animal or in the creation of a transgenic animal.
  • a recombinant vector that directs the expression of the full length or active DBP is particularly contemplated.
  • Expression of dbpA and or dbpB transgene in animals is particularly contemplated to be useful in the production of anti-DbpA or anti-DbpB antibodies for use in passive immunization methods for prevention of borrelial adhesion to Den, and treatment of infections due to Borrelia invasion, and particularly invasion by B. burgdorferi, B. afzelii, B. andersonii, B.
  • anti-DbpA or anti-DbpB antibodies are also contemplated for use in passive immunization methods for prevention of bacterial adhesion to Den, and treatment of infections caused by any bacterial species which binds to Den upon invasion.
  • nucleic acid sequences encoding DbpA proteins which do not share significant sequence homology with nucleic acid sequences encoding DbpB proteins, and vice versa.
  • Methods are provided for determining such sequences based on nucleic acid hybridization.
  • the invention discloses and claims a nucleic acid segment which comprises the nucleic acid sequence from between position 791 and position 1351 of SEQ ID NO:7, or the complement thereof, or a sequence that hybridizes to the sequence from between position 791 and position 1351 of SEQ ID NO: 7, but not to the sequence from between position 1471 and position 2031 of SEQ ID NO: 7 under the same hybridization conditions.
  • Such methods permit the identification of dbpB genes which do not cross-hybridize with dbpA gene sequences and vice versa.
  • the present invention also concerns recombinant host cells for expression of one or more isolated dbpA or dbpB genes. It is contemplated that virtually any host cell may be employed for this purpose, but certain advantages may be found in using a bacterial host cell such as E. coli, S. typhimurium, B. subtilis, or others. Expression in eukaryotic cells is also contemplated such as those derived from yeast, insect, or mammalian cell lines. These recombinant host cells may be employed in connection with "overexpressing" DBPs, that is, increasing the level of expression over that found naturally in Borrelia, in particular, B. burgdorferi, B. afzelii, B. andersonii, B. garinii, B. japonica, or related spirochete.
  • Proteins having amino acid sequences derived from, or similar to the DbpA or DbpB proteins of the present invention are contemplated to have affinity for Den and may be purified from other constituents oi Borrelia, in particular, B. burgdorferi, B. afzelii, B. andersonii, B. garinii, or B. japonica, or recombinant host cells by chromatography on matrices containing Den, so-called "affinity chromatography. " DBPs may also be purified by methodologies not relying on affinity for Den such as ion exchange chromatography, size exclusion chromatography, metal chelation chromatography, or the like.
  • Buffer, detergent, and other conditions may be dissimilar from those optimal for "affinity chromatography. "
  • an affinity matrix comprising Den or a related proteoglycan may be used for the isolation of DBPs from solution, or alternatively, isolation of intact bacteria expressing DBPs, or even membrane fragments of bacteria expressing DBPs.
  • a particular aspect of this invention provides novel ways in which to utilize recombinant DBPs or DBP-derived peptides, nucleic acid segments encoding these peptides, recombinant vectors and transfo ⁇ ned host cells comprising one or more dbp genes or dbp- ⁇ efwt ⁇ nucleic acid segments, recombinant vectors and transformed host cells comprising one or more dbp genes or dbp- ⁇ exw ' &d DNA segments, and recombinant vectors and transformed host cells comprising one or more Borrelia dbp- derived nucleic acid segments, in particular one or more dbpA or dbpB nucleic acid segments from B.
  • a suitable vector for expression in mammalian cells is that described in U. S. Patent 5,168,050, inco ⁇ orated herein by reference.
  • a highly purified vector be used, so long as the coding segment employed encodes a protein or peptide of interest (e.g. , a DbpA or DbpB protein from Borrelia, and particularly a DbpA or DbpB protein from B. burgdorferi, B.
  • useful nucleic acid sequences may include additional residues, such as additional non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various regulatory sequences.
  • an appropriate epitope-encoding nucleic acid molecule After identifying an appropriate epitope-encoding nucleic acid molecule, it may be inserted into any one of the many vectors currently known in the art, so that it will direct the expression and production of the protein or peptide epitope of interest (e.g. , a DbpA or DbpB protein from Borrelia and in particular, from B. burgdorferi, B. afzelii, B. andersonii, B. garinii, or B. japonica) when inco ⁇ orated into a host cell.
  • the coding portion of the DNA segment In a recombinant expression vector, the coding portion of the DNA segment is positioned under the control of a promoter.
  • the promoter may be in the form of the promoter which is naturally associated with a DBP-encoding nucleic acid segment, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.
  • Direct amplification of nucleic acids using the PCRTM technology of U.S. Patents 4,683,195 and 4,683,202 (herein inco ⁇ orated by reference) are particularly contemplated to be useful in such methodologies.
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a dbpA, dbpB, or gene segment in its natural environment.
  • a "dbp- ⁇ ike gene segment” is intended to mean any nucleic acid segment which hybridizes to a dbpA and/or dbpB gene under conditions of moderate to high stringency.
  • Such promoters may include those normally associated with other MSCRAMM-encoding genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the particular cell containing the vector comprising one or more DBP-encoding nucleic acid segments.
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level or regulated expression of the introduced DNA segment.
  • the currently preferred promoters are those such as CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter in combination with the SV40 enhancer.
  • the expression of recombinant DBPs may be carried out using prokaryotic expression systems, and in particular bacterial systems such as E. coli, B burgdorferi.
  • nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promotor sequences such as those provided by Ipp, tac, trp, lac, lacUV5 or T7 promotors.
  • expression vectors and promotor sequences such as those provided by Ipp, tac, trp, lac, lacUV5 or T7 promotors.
  • DbpA, DbpB, and DbpA- or DbpB-derived epitopes once a suitable clone or clones have been obtained, whether they be native sequences or genetically-modified, one may proceed to prepare an expression system for the recombinant preparation of one or more DBP or DBP-derived peptides.
  • the engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of one or more DBPs or DBP-derived epitopes.
  • DBPs or DBP-derived epitopes may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with /3-galactosidase, ubiquitin, Schistosoma japonicum glutathione S -transferase, S. aureus Protein A, maltose binding protein, and the like. It is believed that bacterial expression will ultimately have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.
  • Genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.
  • eukaryotic expression system may be utilized for the expression of one or more DBPs and DBP-derived epitopes, e.g. , baculo virus-based, glutamine synthase-based or dihydrofolate reductase-based systems may be employed.
  • plasmid vectors inco ⁇ orating an origin of replication and an efficient eukaryotic promoter as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use.
  • coding sequences For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides "downstream" of (i.e. , 3' of) the chosen promoter. Where eukaryotic expression is contemplated, one will also typically desire to inco ⁇ orate into the transcriptional unit which includes nucleic acid sequences encoding one or more DBPs or DBP-derived peptides, an appropriate polyadenylation site (e.g.
  • the poly-A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.
  • DBPs and DBP-derived epitopes can be used in connection with the expression of one or more DBPs and DBP-derived epitopes in accordance herewith.
  • Examples include cell lines typically employed for eukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, Wl 38, BHK, COS-7, RIN and MDCK cell lines.
  • a DbpA, DbpB, or epitopic peptides derived from one or more native or recombinant DBPs may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in human cells, or even relative to the expression of other proteins in a recombinant host cell containing a DBP-encoding DNA segment.
  • overexpression may be assessed by a variety of methods, including radiolabeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot.
  • a specific increase in the level of the recombinant protein or peptide in comparison to the level in natural DBP-producing cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, e.g., visible on a gel.
  • the term "engineered” or "recombinant” cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a DBP has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man.
  • Recombinantly introduced genes will either be in the form of a single structural gene, an entire genomic clone comprising a structural gene and flanking DNA, or an operon or other functional nucleic acid segment which may also include genes positioned either upstream and/or downstream of the promotor, regulatory elements, or structural gene itself, or even genes not naturally associated with the particular structural gene of interest.
  • constitutive eukaryotic promoters include viral promotors such as the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, or the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes.
  • CMV cytomegalovirus
  • LTR Rous sarcoma long-terminal repeat
  • the level of expression from the introduced genes of interest can vary in different clones, or genes isolated from different strains or bacteria.
  • the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each transfection experiment; once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in anterior pituitary cell lines.
  • nucleic acid compositions for diagnosing Lyme disease, and the detection of B. burgdorferi in clinical samples using nucleic acid compositions are also provided by the invention.
  • the nucleic acid sequences encoding one or more DBPs, and in particular DbpA or DbpB are useful as diagnostic probes to detect the presence of B. burgdorferi, and related borrelias including B. afzelii, B. andersonii, B. japonica, and B. garinii in a test sample, using conventional techniques.
  • dbpA and/or dbpB nucleic acid segments may be used in Southern hybridization analyses or Northern hybridization analyses to detect the presence of a dbpA- or dbpB-encoding nucleic acid segment within a clinical sample from a patient suspected of having such an infection.
  • a further aspect of the invention is the preparation of immunological compositions, and in particular anti-DbpA and anti-DbpB antibodies for diagnostic and therapeutic methods relating to the detection and treatment of infections caused by B. burgdorferi and related borrelias including B. afzelii, B. andersonii, B. garinii, and B. japonica.
  • the inventors contemplate an important aspect of the invention is the identification of DbpA amino acid sequences which do cross-react with antibodies specific for DbpB, and the identification of DbpB amino acid sequences which do not cross-react with antibodies specific for DbpA. Methods are provided for identifying such proteins based on immunological methods such as ELISA, Western blots, and the like.
  • the invention discloses a DbpB amino acid composition which does not cross-react with DbpA antibody compositions, and a DbpA amino acid composition which does not cross- react with DbpB antibody compositions.
  • a method of generating an immune response in an animal generally involves administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a peptide composition disclosed herein.
  • Preferred peptide compositions include the DbpA peptides disclosed in SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO.21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34.
  • SEQ ID NO:48 SEQ ID NO:50, and SEQ ID NO:52; as well as the DbpB peptides disclosed in SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, and SEQ ID NO:66.
  • the invention also encompasses DBP and DBP-derived peptide antigen compositions together with pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and other components, such as additional peptides, antigens, or outer membrane preparations, as may be employed in the formulation of particular vaccines.
  • the nucleic acid sequences of the present invention encode DBP and are useful to generate pure recombinant DBP for administration to a host. Such administration is useful to prevent adherence of Borrelia spp. , and in particular, B. burgdorferi, B. afzelii, B. andersonii, B. garinii, and B. japonica, to the host's tissues or as a vaccine to induce therapeutic antibodies.
  • antisera raised against and reactive with one or more DBPs is inhibitory to in vitro and in vivo growth of various Borrelia strains.
  • administration of antibodies reactive with one or more DBPs to at-risk subjects will be effective for prophylaxis of, and in the case of infected subjects for therapy of, Lyme disease.
  • Antibodies may be of several types including those raised in heterologous donor animals or human volunteers immunized with DBPs, monoclonal antibodies (mAbs) resulting from hybridomas derived from fusions of B cells from DBP- immunized animals or humans with compatible myeloma cell lines, so-called "humanized” mAbs resulting from expression of gene fusions of combinatorial determining regions of mAb-encoding genes from heterologous species with genes encoding human antibodies, or DBP-reactive antibody-containing fractions of plasma from human donors residing in Lyme disease-endemic areas. It is contemplated that any of the techniques described above might be used for the vaccination of subjects for the pu ⁇ ose of antibody production.
  • Optimal dosing of such antibodies is highly dependent upon the pharmacokinetics of the specific antibody population in the particular species to be treated, but it is anticipated that it will be necessary to maintain in these subjects a serum concentration of DBP-reactive antibodies that is at least twice that required for inhibition of in vitro growth of endemic borrelia strains. It is contemplated that the duration of dosing maintaining anti-DbpA and or anti-DbpB levels at these inhibitory antibody concentrations would be for at least four to eight weeks following presumptive exposure to a Borrelia, and in particular, B. burgdorferi, or throughout the duration of symptoms of Lyme disease and for at least four to eight weeks after cessation of these symptoms.
  • the present invention also provides methods of generating an immune response, which methods generally comprise administering to an animal, a pharmaceutically-acceptable composition comprising an immunologically effective amount of one or more DBP peptide compositions.
  • Preferred animals include mammals, and particularly humans. Other preferred animals include murines, bovines, equines, porcines, canines, and felines.
  • the composition may include partially or significantly purified DBP epitopes, obtained from natural or recombinant sources, which proteins or peptides may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such epitopes.
  • Smaller peptides that include reactive epitopes such as those between about 10 and about 50, or even between about 50 and about 100 amino acids in length will often be preferred.
  • the antigenic proteins or peptides may also be combined with other agents, such as other borrelial peptide or nucleic acid compositions, if desired.
  • immunologically effective amount an amount of a peptide composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B cell response), and/or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response will have utility in both the production of useful bioreagents, e.g., CTLs and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various prophylactic or therapeutic embodiments.
  • useful bioreagents e.g., CTLs and, more particularly, reactive antibodies
  • these methods for the stimulation of an immune response include vaccination regimens designed to prevent or lessen significant infections caused by borrelias or other bacteria expressing a DBP, and treatment regimens that may lessen the severity or duration of any infection, it will be understood that achieving either of these end results is not necessary for practicing these aspects of the invention.
  • Such treatment methods may be used particularly for the treatment of infections caused by pathogens such as B. burgdorferi, B. afzelii, B. andersonii, B. garinii, B. japonica, related borrelial species, and other bacteria which express one or more DBPs and in particular DbpA and/or DbpB and adhere to Den.
  • an immune response in an animal includes administering to the animal, or human subject, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding a DBP epitope, or an immunologically effective amount of an attenuated live organism that includes and expresses such a nucleic acid composition.
  • a pharmaceutically-acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding a DBP epitope, or an immunologically effective amount of an attenuated live organism that includes and expresses such a nucleic acid composition.
  • the "immunologically effective amounts” are those amounts capable of stimulating a B-cell and/or T-cell response.
  • Immunoformulations of this invention may comprise native, or synthetically-derived antigenic peptide fragments from these proteins.
  • antigenic functional equivalents of the proteins and peptides described herein also fall within the scope of the present invention.
  • An "antigenically functional equivalent" protein or peptide is one that inco ⁇ orates an epitope that is immunologically cross-reactive with one or more epitopes derived from any of the particular MSCRAMM proteins disclosed (e.g. , DBPs), and particularly the DBP of B. burgdorferi.
  • Antigenically functional equivalents, or epitopic sequences may be first designed or predicted and then tested, or may simply be directly tested for cross- reactivity.
  • DBP epitopes and/or their functional equivalents, suitable for use in immunoformulations, vaccines, or simply as antigens (e.g., for use in detection protocols), is a relatively straightforward matter.
  • Hopp as enabled in U.S. Patent 4,554, 101 , inco ⁇ orated herein by reference, that teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity.
  • the methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences.
  • the use of shorter antigenic peptides e.g. , about 25 to about 50, or even about 15 to 25 amino acids in length, that inco ⁇ orate epitopes of one or more DBPs will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
  • the present invention concerns immunodetection methods and associated kits.
  • the proteins or peptides of the invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect DBPs or DBP-derived peptides.
  • Either type of kit may be used in the immunodetection of compounds, present within clinical samples, that are indicative of Lyme disease or related infections caused by borrelias, and in particular B. burgdorferi.
  • the kits may also be used in antigen or antibody purification, as appropriate.
  • the preferred immunodetection methods will include first obtaining a sample suspected of containing a DBP-reactive antibody, such as a biological sample from a patient, and contacting the sample with a first DBP or peptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexes that are formed.
  • a DBP-reactive antibody such as a biological sample from a patient
  • Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable.
  • a label or marker such as a radioactive, fluorescent, biological or enzymatic label
  • enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable.
  • the particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined.
  • the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first protein or peptide.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labelled secondary antibodies and the remaining bound label is then detected.
  • diagnostic pu ⁇ oses it is proposed that virtually any sample suspected of containing the antibodies of interest may be employed.
  • Exemplary samples include clinical samples obtained from a patient such as blood or serum samples, cerebrospinal, synovial, or bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even perhaps urine samples may be employed. This allows for the diagnosis of Lyme disease and related infections caused by borrelias, and in particular, B. burgdorferi. Furthermore, it is contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like. Alternatively, the clinical samples may be from veterinary sources and may include such domestic animals as cattle, sheep, and goats. Samples from feline, canine, and equine sources may also be used in accordance with the methods described herein.
  • kits in accordance with the present invention contemplates the preparation of kits that may be employed to detect the presence of DBP-specific antibodies in a sample.
  • kits in accordance with the present invention will include a suitable protein or peptide together with an immunodetection reagent, and a means for containing the protein or peptide and reagent.
  • the immunodetection reagent will typically comprise a label associated with a DBP or peptide, or associated with a secondary binding ligand.
  • exemplary ligands might include a secondary antibody directed against the first DBP or peptide or antibody, or a biotin or avidin (or streptavidin) ligand having an associated label.
  • Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, e.g. , for protocols where the first reagent is a DBP peptide that is used to bind to a reactive antibody from a human sample.
  • a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.
  • kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen may be placed, and preferably suitably allocated. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed.
  • the kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • the DBP is useful as an agent to block B. burgdorferi adherence to Den, and proteoglycans which are structurally similar to Den such as Lmn, Fmn, Epn, and Bgn.
  • a therapeutically effective dose of one or more DBPs, and in particular one or more DbpA or DbpB proteins is administered to a subject to prevent or block adhesion of B. burgdorferi to the host's tissues by conventional methods.
  • the composition is preferably systemically administered, but may be applied topically, e.g. , to a localized lesion.
  • therapeutically effective dose means that amount of a DBP composition which is sufficient to lessen or prevent adherence of B.
  • compositions of the present invention is useful as a therapeutic agent to prevent adhesion of B. burgdorferi and thereby lessen or prevent disease induced by this microorganism.
  • nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments.
  • nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous sequence of SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO NO:
  • nucleic acid probes to specifically hybridize to DBP- encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample.
  • sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • the total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 14 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
  • hybridization probe of about 14-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequence set forth in SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63 or SEQ ID NO:65, or to any continuous portion of the sequence, from about 14-25 nu
  • the process of selecting and preparing a nucleic acid segment that includes a contiguous sequence from within SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:63 or SEQ ID NO: 65 may alternatively be described as preparing a nucleic acid fragment.
  • fragments may also be obtained by other techniques such as, e.g. , by mechanical shearing or by restriction enzyme digestion.
  • Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Patent 4,683,202 (inco ⁇ orated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire dbp gene or gene fragments.
  • relatively stringent conditions e.g. , one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of 50°C to 70°C.
  • Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating DBP genes.
  • nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
  • enzyme tags colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
  • specific hybridization is detected, or even quantitated, by means of the label.
  • administering induces in the subject antibodies which bind and neutralize a Borrelia bacterium (and particularly B. burgdorferi, B. garinii, B. afzelii, B. andersonii, B. japonica and related Borrelia spp.), present in the subject, thereby preventing the deleterious effects of this microorganism.
  • a Borrelia bacterium and particularly B. burgdorferi, B. garinii, B. afzelii, B. andersonii, B. japonica and related Borrelia spp.
  • antibodies generated in a first host animal provide antibodies which can be administered to a second subject for passive immunization or treatment against B. burgdorferi, B. garinii, B. afzelii, B. andersonii, or B. japonica infection.
  • Such an ⁇ -Borrelia antibodies are also useful as a diagnostic screen for the presence of Borrelias, and in particular B. burgdorferi, B. garinii, B. afzelii, B. andersonii, B. japonica or related Borrelia spp. in a test sample, using conventional immunoassay techniques.
  • novel nucleic acid sequences encode novel DBPs of B.
  • Strain variants are prepared and screened by amplification of nucleic acid sequences of other strains of B. burgdorferi or similar Lyme-disease inducing bacteria using oligonucleotide probes derived from the 2.5 kb insert of B. burgdorferi strain 297 (SEQ ID NO: 7) and preferably from the approximate 0.6 kb open reading frame. Clones obtained from the amplification procedures are then used as hybridization probes to isolate the full length nucleic acid encoding strain variants. Alternatively, DNA libraries for each strain are constructed and screened for clones expressing a DBP, e.g. , by their affinity for Den.
  • the present invention concerns novel antibody compositions which inhibit Den binding to bacteria.
  • antibodies to native and synthetically-modified epitopes from DBPs have been developed which inhibit Den binding to DBPs both in vitro and in vivo.
  • proteins, peptides and peptide epitopes have been produced to provide vaccine compositions useful in the prevention of Lyme disease and antibody compositions useful in the prevention of Den binding to Borrelias.
  • the present invention encompasses antibody compositions which enhance Den binding to bacterial cells.
  • the invention discloses an antibody that interacts with a DBP domain of a bacteria dbp gene product, and particularly, a DBP domain of a B. burgdorferi dbp gene product.
  • Such antibody may be monoclonal, or preferably polyclonal.
  • the invention discloses an antibody which inhibits bacterial adhesion, and the binding of the gene product to Den.
  • a method for detecting a bacterium expressing a DBP in a sample generally involves obtaining a sample suspected of containing a bacterium expressing such a protein, then contacting the sample with an antibody composition disclosed herein, and detecting the formation of immune complexes.
  • the bacterium is a borrelia, and most preferably, a B. burgdorferi, B. afzelii, B. andersonii, or B. garinii strain.
  • kits containing antibodies of the present invention and suitable immunodetection reagents such as a detectable label linked to a protein, peptide or the antibody itself. Alternatively, the detectable label may be linked to a second antibody which binds to an antibody of the invention.
  • diagnostic and therapeutic kits which include pharmaceutical ly-acceptable formulations of either the antibodies or peptide antigens disclosed herein. Such kits are useful in the detection of borrelias in clinical samples, and also useful for inhibiting or promoting the binding of borrelias to the ECM component, Den.
  • the bacteria detected using such kits include borrelias, and in particular, B. burgdorferi, B. afzelii, B. andersonii, B. garinii, B. japonica, and related species.
  • aspects of the invention include methods of inhibiting bacterial colonization, and particularly colonization by borrelias, in an animal by administering to the animal an antibody of the present invention which prevents or significantly reduces the binding of Den to the DBP expressed by the bacteria.
  • Administration of the antibody composition may be prophylactically prior to and/or following diagnosis of Lyme disease or other multisystemic disorders caused by Borrelioses which may involve the skin, joints, heart, and central nervous system.
  • the administration may also be made in passive immunization protocols designed to prevent and/or ameliorate systemic infections by susceptible pathogens, and in particular, to ameliorate the effects of infections by pathogenic B. burgdorferi.
  • the present invention includes proteins expressed from genes encoding a DBP such as that protein expressed from the DNA insert of recombinant clone BG26:pB/2.5(5). Also included are strain variants of the gene derived from B. burgdorferi strain 297 present in the recombinant clone BG26:pB/2.5(5) which also encode proteins capable of binding Den, which may hybridize to DNA derived from BG26:pB/2.5(5) under conditions of moderate or high stringency, or which may serve as templates for gene amplification by PCRTM using oligonucleotide primers derived from BG26:pB/2.5(5).
  • these variants may include genes containing codons not identical in nucleotide sequence to those of the dbp gene of strain 297, but encoding the same, or functionally equivalent amino acid, as is anticipated by those practiced in the art who understand the degeneracy of the genetic code. These variants may also include those genes similar to the dbp gene from strain 297, but having codons specifying relatively few amino acids that are different from those of the protein(s) encoded by BG26:pB/2.5(5), or having somewhat fewer or greater numbers of these codons.
  • sequences include those that have between about 60% and about 80%; or more preferably, between about 81 % and about 90%; or even more preferably, between about 91 % and about 99%; of amino acids that are identical or functionally equivalent to those of protein(s) encoded by BG26:pB/2.5(5).
  • amino acid sequences and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' nucleic acid sequences, and yet still be as set forth herein, so long as the sequence meets the criteria set forth above including the expression of a DBP protein.
  • additional sequences may, for example, include various transcriptional promoters, enhancers, or terminators, various secretion-directing leader peptides, various amino acid sequences directing posttranslational modifications, amino acids or peptides which may facilitate isolation and purification of DBP(s), and the like.
  • additional sequences may, for example, include various transcriptional promoters, enhancers, or terminators, various secretion-directing leader peptides, various amino acid sequences directing posttranslational modifications, amino acids or peptides which may facilitate isolation and purification of DBP(s), and the like.
  • DBPs may be desirable to administer DBPs to the human or animal subject in a pharmaceutically acceptable composition
  • a pharmaceutically acceptable composition comprising an immunologically effective amount of DBPs mixed with other excipients, carriers, or diluents which may improve or otherwise alter stimulation of B cell and/or T cell responses, or immunologically inert salts, organic acids and bases, carbohydrates, and the like, which promote stability of such mixtures.
  • Immunostimulatory excipients may include salts of aluminum (often referred to as Alums), simple or complex fatty acids and sterol compounds, physiologically acceptable oils, polymeric carbohydrates, chemically or genetically modified protein toxins, and various particulate or emulsified combinations thereof.
  • Alums aluminum
  • simple or complex fatty acids and sterol compounds simple or complex fatty acids and
  • Attenuated organisms may be engineered to express recombinant DBP gene products and themselves be delivery vehicles for the invention.
  • Particularly preferred are attenuated bacterial species such as Mycobacterium, and in particular M. bovis, M. smegmatis, or BCG.
  • pox-, polio-, adeno-, or other viruses, and bacteria such as Salmonella, Shigella, Listeria, Streptococcus species may also be used in conjunction with the methods and compositions disclosed herein.
  • the naked DNA technology has been shown to be suitable for protection against infectious organisms.
  • DNA segments could be used in a variety of forms including naked DNA and plasmid DNA, and may administered to the subject in a variety of ways including parenteral, mucosal, and so-called microprojectile-based "gene-gun" inoculations.
  • the use of dbp nucleic acid compositions of the present invention in such immunization techniques is thus proposed to be useful as a vaccination strategy against Lyme disease.
  • an optimal dosing schedule of a vaccination regimen may include as many as five to six, but preferably three to five, or even more preferably one to three administrations of the immunizing entity given at intervals of as few as two to four weeks, to as long as five to ten years, or occasionally at even longer intervals.
  • Particular aspects of the invention concern the use of plasmid vectors for the cloning and expression of recombinant peptides, and particular peptide epitopes comprising either native, or site-specifically mutated DBP epitopes.
  • the generation of recombinant vectors, transformation of host cells, and expression of recombinant proteins is well-known to those of skill in the art.
  • Prokaryotic hosts are preferred for expression of the peptide compositions of the present invention.
  • An example of a preferred prokaryotic host is E. coli, and in particular, E. coli strains ATCC69791 , BL21(DE3), JM101 , XLl-BlueTM, RR1, LE392, B, ⁇ 1776 (ATCC No.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli may be typically transformed using vectors such as pBR322, or any of its derivatives (Bolivar et al , 1977).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • pBR322 its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.
  • a preferred vector for cloning the dbp constructs is pBlueScriptTM, and in particular the construct BG26:pB/2.5(5), or alternatively, vectors based on the pET vector series (Novagen, Inc., Madison, Wl).
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • bacteriophage such as ⁇ GEMTM-l l may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • promoters most commonly used in recombinant DNA construction include the -lactamase (penicillinase) and lactose promoter systems (Chang et al. , 1978; Itakura et al , 1977; Goeddel et al , 1979) or the tryptophan (trp) promoter system (Goeddel et al , 1980).
  • lactose promoter systems Chang et al. , 1978; Itakura et al , 1977; Goeddel et al , 1979
  • tryptophan (trp) promoter system Goeddel et al , 1980.
  • the use of recombinant and native microbial promoters is well-known to those of skill in the art, and details concerning their nucleotide sequences and specific methodologies are in the public domain, enabling a skilled worker to construct particular recombinant vectors and expression systems for the pu ⁇ ose of producing compositions of
  • eukaryotic microbes such as yeast cultures may also be used in conjunction with the methods disclosed herein.
  • Saccharomyces cerevisiae, or common bakers' yeast is the most commonly used among eukaryotic microorganisms, although a number of other species may also be employed for such eukaryotic expression systems.
  • the plasmid YRp7 for example, is commonly used (Stinchcomb et al, 1979; Kingsman et al, 1979; Tschemper et al , 1980).
  • This plasmid already contains the trpL gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977).
  • the presence of the trpL lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Suitable promoting sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (Hitzeman et al , 1980) or other glycolytic enzymes (Hess et al , 1968; Holland et al , 1978), such as enolase, glyceraldehyde-3-phos ⁇ hate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • Other promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Any plasmid vector containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable.
  • cultures of cells derived from multicellular organisms may also be used as hosts in the routine practice of the disclosed methods.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years.
  • useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • control functions on the expression vectors are often provided by viral material.
  • promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40).
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al , 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the H dIII site toward the Bgtt. site located in the viral origin of replication.
  • promoter or control sequences normally associated with the desired gene sequence provided such control sequences are compatible with the host cell systems.
  • the origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g. , Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • an exogenous origin such as may be derived from SV40 or other viral (e.g. , Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • polypeptides may be present in quantities below the detection limits of the Coomassie brilliant blue staining procedure usually employed in the analysis of SDS/PAGE gels, or that their presence may be masked by an inactive polypeptide of similar M r .
  • detection techniques may be employed advantageously in the visualization of particular polypeptides of interest. Immunologically-based techniques such as Western blotting using enzymatically-, radiolabel-, or fluorescently-tagged antibodies described herein are considered to be of particular use in this regard.
  • the peptides of the present invention may be detected by using antibodies of the present invention in combination with secondary antibodies having affinity for such primary antibodies. This secondary antibody may be enzymatically- or radiolabeled, or alternatively, fluorescently-, or colloidal gold-tagged. Means for the labeling and detection of such two-step secondary antibody techniques are well-known to those of skill in the art.
  • FIG. 1A Identification of DBPs of B. burgdorferi N40. B. burgdorferi whole-cell lysate was subjected to SDS-PAGE (5 to 15%) under reducing conditions and stained with Coomassie brilliant blue. The same gel was transferred to a nitrocellulose membrane (FIG. IB).
  • FIG. IB Identification of DBPs of B. burgdorferi N40.
  • B. burgdorferi whole-cell lysate was subjected to SDS-PAGE (5 to 15%) under reducing conditions (FIG. 1A), then transferred to a nitrocellulose membrane. After blocking additional protein- binding sites, proteins on the membrane were probed with biotin-labeled Den and visualized by chemiluminescence. The migration of standard proteins with known molecular masses (in kDa) is shown on the left and right.
  • FIG. 2A DNA sequence of a 2.5 kb insert of B. burgdorferi strain 297 DNA contained in the plasmid BG26:pB/2.5(5), which comprises a nucleic acid sequence which encodes DbpA and DbpB.
  • the deduced amino acid sequences of DbpA and DbpB are shown in FIG. 3.
  • Depicted in FIG. 2A are nucleotides 1-1400 (SEQ ID NO: 10).
  • SEQ ID NO: 11 The DNA sequence from position 1401 to 2653 (SEQ ID NO: 11) is continued in FIG. 2B.
  • FIG. 2B Continuation of the DNA sequence of a 2.5 kb insert of B. burgdorferi strain 297 DNA contained in the plasmid BG26:pB/2.5(5), which comprises a nucleic acid sequence which encodes DbpA and DbpB. Depicted in FIG. 2B are nucleotides 1401 to 2653 (SEQ ID NO: 11). Nucleotides 1-1400 are shown in FIG. 2A.
  • FIG. 3 Nucleotide sequence of the dbpB gene from B. burgdorferi strains 297, SH2 and LP4 (SEQ ID NO:59), and amino acid sequence of the 187 amino-acid DbpB protein from B. burgdorferi strains 297, SH2 and LP4 (SEQ ID NO: 60).
  • FIG. 4 Nucleotide sequence of the dbp A gene from B. burgdorferi strains 297 and LP7 (SEQ ID NO:29), and amino acid sequence of the 187 amino-acid DbpA from B. burgdorferi strains 297 and LP7 (SEQ ID NO: 30).
  • FIG. 5. Nucleotide sequence of B. burgdorferi strains B31 , BR4, and 3028 DNA encoding DbpA (SEQ ID NO: 39). The translated amino acid sequence of the identical DbpAs from strains B31 , BR4, and 3028 is given in SEQ ID NO:40.
  • FIG. 6 Nucleotide sequence of B. burgdorferi dbpB gene from strains HB19, G3940, LP5, ZS7, NCH01 , FRED, and B. garinii dbpB gene from strain 20047. The sequence is identical for all seven strains (SEQ ID NO:63).
  • FIG. 7 Nucleotide sequence of B. burgdorferi dbpB gene from strains N40, LP7, and B. afzelii strain PKo. The sequence is identical for all three strains (SEQ ID NO:61). The amino acid sequence of B. burgdorferi DbpB protein from strains N40, LP7, and B. andersonii strain PKo is also identical for all three strains (SEQ ID NO: 62).
  • FIG. 8 Partial dbpB gene sequence from B. garinii strain IP90 (SEQ ID NO:65) and corresponding amino acid sequence of DbpB protein (SEQ ID NO:66).
  • FIG. 9 dbpB gene sequence from B. burgdorferi strain JD1 (SEQ ID NO:57) and corresponding amino acid sequence of DbpB protein (SEQ ID NO:58).
  • FIG. 10 dbpB gene sequence from B. burgdorferi strain IPS (SEQ ID NO:55) and corresponding amino acid sequence of DbpB protein (SEQ ID NO: 56).
  • FIG. 11 dbpB gene sequence from B. burgdorferi strain CA287 (SEQ ID NO:53) and corresponding amino acid sequence of DbpB protein (SEQ ID NO: 54).
  • FIG. 12 Nucleotide and deduced amino acid sequence of B. garinii strain IP90 dbp A gene (SEQ ID NO:51). The translated amino acid sequence of DbpA is given in SEQ ID NO:52.
  • FIG. 13 Nucleotide and deduced amino acid sequence of B. afzelii strain B023 dbpA gene (SEQ ID NO:49). The translated amino acid sequence of DbpA is given in SEQ ID NO:50.
  • FIG. 14 Nucleotide and deduced amino acid sequence of B. afzelii strain PGau dbpA gene (SEQ ID NO:47). The translated amino acid sequence of DbpA is given in SEQ ID NO:48.
  • FIG. 15 Nucleotide and deduced amino acid sequence of B. burgdorferi strain ZS7 dbpA gene (SEQ ID NO:45). The translated amino acid sequence of DbpA is given in SEQ ID NO:46.
  • FIG. 16 Nucleotide and deduced amino acid sequence of B. burgdorferi strain LP4 dbpA gene (SEQ ID NO:43). The translated amino acid sequence of DbpA is given in SEQ ID NO:44.
  • FIG. 17 Nucleotide and deduced amino acid sequence of B. burgdorferi strain G3940 dbpA gene (SEQ ID NO:41). The translated amino acid sequence of DbpA is given in SEQ ID NO:42.
  • FIG. 18 Nucleotide and deduced amino acid sequence of B. burgdorferi strain HB19 dbpA gene (SEQ ID NO: 37). The translated amino acid sequence of DbpA is given in SEQ ID NO:38.
  • FIG. 19 Nucleotide and deduced amino acid sequence of B. burgdorferi strain JD1 dbp A gene (SEQ ID NO: 35). The translated amino acid sequence of DbpA is given in SEQ ID NO:36.
  • FIG. 20 Nucleotide and deduced amino acid sequence of B. burgdorferi strain N40 dbpA gene (SEQ ID NO:33). The translated amino acid sequence of DbpA is given in SEQ ID NO: 34.
  • FIG. 21 Nucleotide and deduced amino acid sequence of B. burgdorferi strain SH2 dbp A gene (SEQ ID NO:31). The translated amino acid sequence of DbpA is given in SEQ ID NO:32.
  • FIG. 22 Comparison of amino acid sequence identities for the Dbp As from related borrelias.
  • the predicted DbpA amino acid sequences disclosed herein were compared in a pairwise fashion as to % identity using the BestFit algorithm of the University of Wisconsin Genetics Computer Group DNA analysis software package. Default parameters for gap weight and gap length weight were used.
  • FIG. 23A B. burgdorferi DBP expression, B. burgdorferi strain 297 (lane 1) and HP B31 (lane 2) whole-cell lysates were subjected to SDS-PAGE (5-15% gradient) under reducing conditions and stained with Coomassie.
  • FIG. 23B Gel from FIG. 23A transferred to a nitrocellulose membrane (B). After blocking additional protein-binding sites, proteins on the membrane were probed with digoxigenin-labeled Den and visualized by alkaline phosphatase reactivity. The migration of standard proteins with known molecular masses (in kilodaltons) are shown on the left and right.
  • FIG. 23C Attachment of 5. burgdorferi to Den substrata. Dcn-coated microtiter wells were incubated with B. burgdorferi 297 or HP B31. Attachment to the substrate was quantitated by an ELISA.
  • FIG. 24 Diagram of the dbpA and dbpB clones isolated from a ⁇ ZAPII expression library and construction of recombinant polyhistidine fusion subclones (DbpB:500, DbpA: 549, DbpA: 504) and site-directed mutant (DbpA:C25A).
  • the polyhistidine fusion (black), leader peptides (dotted, striped) and cysteine to alanine mutation are indicated.
  • FIG. 25 Activity of recombinant Dcn-binding proteins. Microtiter wells were coated with DbpA:549, DbpB:500, DbpA:C25A, or OspC. Biotin-Iabeled Den was allowed to bind, followed by alkaline phosphatase conjugated streptavidin. Den binding was quantitated by measuring absorbance at 405 nm in the presence of phosphatase substrate.
  • FIG. 26A Inhibition of the attachment of B. burgdorferi 297 to Den substrata. Dcn-coated microtiter wells were preincubated with DbpA:549, DbpB:500, or DbpA:C25A before B. burgdorferi were allowed to attach. Attachment to the substrate was quantitated by an ELISA.
  • FIG. 26B Inhibition of the attachment of B. burgdorferi N40 to Den substrata. Dcn-coated microtiter wells were preincubated with DbpA: 549, DbpB: 500, or DbpA:C25A before B. burgdorferi were allowed to attach. Attachment to the substrate was quantitated by an ELISA.
  • FIG. 27 Competition of DBPs for Den binding.
  • Microtiter wells were coated with DbpA:549, DbpB:500, or DbpA:C25A.
  • Biotin-labeled Den was preincubated with Dbps before being allowed to bind to Dbp coated wells. After incubating with alkaline phosphatase conjugated streptavidin, Den binding was quantitated by measuring absorbance at 405 nm in the presence of phosphatase substrate. Readings from BSA coated wells were subtracted background.
  • FIG. 28A DbpB Active protection study in BALB/c mice.
  • FIG. 28B DbpB Active protection study in BALB/c mice.
  • the technology described herein is used to develop methods and compositions that specifically interfere with bacterial adhesion and the subsequent colonization host tissues, thus resulting in the prevention of infection.
  • the technology is broadly applicable, has the potential to increase the effectiveness of antibiotic therapy in many situations, and replace antibiotic therapy in a number of other applications.
  • the technology is anticipated to be especially effective in treatment regimens for Lyme disease, and as a cost-effective prophylaxis for prevention of borrelial infections.
  • Serological variation of DbpA is less than that of OspA as antibodies reactive with DbpA-derived from B. burgdorferi sensu stricto are also growth- inhibitory to many strains of B. garinii and B. afzelii;
  • mice with either full-length DbpA or a recombinant truncated chimeric DBP protects against challenge with a heterologous B. burgdorferi strain
  • Antiserum against DbpA 297 provides either complete or partial protection against several additional heterologous B. burgdorferi, B. afzelii. and B. garinii strains;
  • Antibodies (FAb fragments) against DBPs block the binding of recombinant DBPs to Den;
  • the dbpA gene from B. burgdorferi 297 has been used to identify and isolate molecular clones of alleles of this gene present in different phylogenetic groups of Lyme disease spirochetes including B. garinii, B. japonica, B. afzelii, B. andersonii, and related Borrelia spp. by utilization of PCRTM techniques. These new dbp alleles have been shown to have a high level of sequence identity to the dbpA gene from strain 297. Indication was seen of important serologic differences among the various DBPs suggesting that it may be necessary to formulate a multiple-DBP antigen cocktail to achieve complete vaccine coverage of all clinically relevant B. burgdorferi strains.
  • DbpA and/or DbpB do not suffer the limitations of the prior art, particularly with respect to OspA and other antigens previously investigated as antigens for development of borrelia vaccines. Indeed, vaccine compositions comprising one or more DBPs, and in particular, DbpA and/or DbpB, are likely to be superior to those previously available containing OspA alone.
  • DbpA and DbpB appear to be less than that of OspA as antibodies reactive with DbpA and DbpB derived from B. burgdorferi sensu stricto are also growth-inhibitory to strains of B. garinii and B afzelii.
  • mice with recombinant DbpA or DbpB protects against challenge from heterologous strains of B. burgdorferi.
  • DbpA and DbpB remain a target for immune intervention for several days after initiation of infection, and is the only antigen shown to date to possess this desirable property with respect to potential vaccine efficacy.
  • DBP-targeted antibodies administered several days after initiation of infection can eliminate viable spirochetes from infected mice, suggesting that this antigen is also a potential target for immunotherapy of Lyme disease as well as for immunoprophylaxis.
  • Biochemical data is provided showing that DbpA and DbpB are lipoproteins, and are exposed at the surface of the spirochetal outer membrane. 4.2.6 dbp Nucleic Acid Segments Are Useful in Identifying Borrelial Isolates
  • novel dbpA and dbpB genes disclosed herein may be used to identify and isolate molecular clones of alleles of this gene present in different phylogenetic groups of Lyme disease spirochetes including B. burgdorferi, B. garinii, B. afzelii, B. andersonii, and B. japonica by utilization of such techniques as PCRTM.
  • MSCRAMMs MSCRAMMs
  • MSCRAMMs microbial surface components recognizing adhesive matrix molecules, Patti et al, 1994; Parti and Hook, 1994
  • MSCRAMMs microbial surface components recognizing adhesive matrix molecules, Patti et al, 1994; Parti and Hook, 1994
  • pathogenic bacteria have been shown to specifically recognize and bind to various components of the extracellular matrix in an interaction which appears to represent a host tissue colonization mechanism.
  • MSCRAMMs on the bacterial cell surface
  • ligands within the host tissue
  • MSCRAMMs on the bacterial cell surface
  • ligands within the host tissue
  • Complete blockage of microbial adhesion is not required to prevent infection. It is only necessary to keep the bacterial inoculum below a critical mass.
  • Several strategies have been developed which are particularly useful in combatting bacterial infections, such as infection by B. burgdorferi, by preventing bacterial adhesion to Den substrata including the extracellular matrix (ECM) of susceptible host cells. Such strategies are contemplated to be useful in the diagnosis, treatment, and prophylaxis of Lyme disease.
  • ECM extracellular matrix
  • the ECM contains numerous glycoproteins and proteoglycans which, through inter- and intramolecular interactions, form insoluble matrices.
  • the ECM has a structural function in the tissues but also affects the cellular physiology of the organism. Perhaps the best characterized biological functions of the ECM are related to its ability to serve as a substrata for the adhesion of host tissue cells. This process involves the integrins, a family of heterodimeric ( ⁇ / ⁇ ) cell surface receptors which recognize specific structures in many of the ECM proteins. It is clear that many bacteria also have taken advantage of the ECM as a substrate for adhesion. Like most eukaryotic tissue cells, many bacteria have developed several parallel adhesion mechanisms and this apparent redundancy may reflect the importance of host tissue adherence for the prosperity of the bacteria.
  • the adherence of microbes to various cell-surface and extracellular matrix components has been widely reported (Abraham et al , 1983; Coburn et ai, 1993; Fr ⁇ man et al, 1984; Isaacs, 1994; Maxe et al, 1986; Van Nhieu and Isber, 1993).
  • the present invention has identified a new class of bacterial MSCRAMMs which promotes bacterial adhesion to Den and other proteoglycans which are structurally similar to Den, which are found in conjunction with ECM components such as collagen.
  • the identification and characterization of DbpA and DbpB and the genes which encode them represents the first known bacterial proteins with affinity for Den.
  • Collagenous proteins are the major constituents of the ECM (Bornstein and Sage, 1980). Most collagens are synthesized intracellularly as precursor molecules and undergo extensive posttranslational processing prior to secretion and inco ⁇ oration into the ECM or other collagen-rich tissues such as cartilage (Ramachandran and Reddi. 1976). To date over 18 different type of collagens have been defined, and they are loosely categorized into five groups (Vanderrest and Garrone, 1991). These groups are:
  • collagen types XII, XIV, and IX which are fibril-associated with interrupted triple helices; 3) collagen types IV, VIII, and X which form sheets;
  • collagen type VII which forms anchoring fibrils.
  • the collagen network in skin is composed predominantly of collagens type I and type III. Den can inhibit transforming growth factor beta activity (TGF ⁇ ) (Yamaguchi et al, 1990) and inactivate the complement component Clq (Krumdieck et al. , 1992) and has been proposed to act as an anti-inflammatory agent through these interactions.
  • TGF ⁇ transforming growth factor beta activity
  • Clq complement component
  • Den also known as PG-40, PG-II, PG-S2 and CSIDS-PGII, is a small proteoglycan with a single chondroitin or dermatan sulfate chain attached to the fourth amino acid of the secreted 36-38 kDa protein (Chopra et al, 1985). Den has been found associated with collagen fibrils in virtually all connective tissues (Bianco et al. , 1990), perhaps near the d and e bands in the D period (Pringle and Dodd, 1990).
  • Den so named because it "decorates" collagen fibers in the intracellular matrix, has been shown to bind different collagen types and is believed to act as a regulator of collagen fiber formation.
  • the proteoglycan can be isolated from many different tissues, including skin, cartilage, and tendon.
  • Den consists of a 36 kDa core protein, a single, serine-linked glycosaminoglycan (GAG) chain of the chondroitin/dermatan sulfate type, and up to three N-linked oligosaccharide.
  • GAGs are unbranched polysaccharides consisting of repeating disaccharide units, highly sulfated and therefore highly negatively charged.
  • Den containing a chondroitin-4-sulfate chain is isolated from developing bone (Fisher et al, 1987) while Den containing a dermatan sulfate chain is generally isolated from articular cartilage (Choi et al. , 1989) or tendon (Vogel and Heinegard, 1985).
  • the Den is heterogenous with respect to glycosaminoglycan chain size and the average size of the chains differ with tissue and developmental age, however, the Alcian blue or StainsAll band generally are centered from 100 to 250 kDa. While Den size may differ in different tissues, it is almost always smaller than the biglycan (Bgn) proteoglycan in the same tissue.
  • Den has been localized to human chromosome 12 (McBride et al, 1990), and Den itself shows obvious homology to other small proteoglycans, including Bgn (Fisher et al, 1989), and Fmn (Oldberg et al, 1989), Epn, and Lmn. These proteoglycans are predominantly composed of 10-12 tandem repeats with each nominal 24 amino acid repeat having a pattern of hydrophobic amino acids (Fisher et al, 1989; Oldberg et al, 1989). These repeat sequences have been used many times in evolution when protein-protein, protein-cell or cell-cell interactions are required. While Den, like Fmn, is found associated with collagen fibrils (Oldberg et al, 1989), Bgn appears to be associated on or very near cell surfaces and not collagen bundles (Bianco et al, 1990).
  • Bgn (Fisher et al , 1989) is a small proteoglycan whose primary gene product is found associated with the cell surface or pericellular matrix of a variety of cells including specific subsets of developing mesenchymal (skeletal muscle, bone and cartilage), endothelial (blood vessels), and epithelial (keratinocytes cells) (Bianco et al. 1990). Other names for this proteoglycan includes; PG-1 , PG-I, DS-PGI, PG-S1 and DS-I.
  • Bgn is composed of two chondroitin (CS) or dermatan sulfate (DS) chains on a 38 kDa core protein that is predominantly made of 12 tandem 24 amino acid repeat structures, each characterized by ordered hydrophobic residues. Similar tandem repeat structures have been used throughout evolution when a protein is destined to bind another protein or perhaps a cell surface (Fisher et al , 1989; Patthy, 1987). The function of Bgn is unknown, but. like the homologous proteoglycan. Den, it may also bind to TGF- ⁇ .
  • the chondroitin sulfate containing Bgn is most commonly isolated from fetal or young bone (Fisher et al, 1987; 1983), while the dermatan sulfate containing form is isolated from articular cartilage (Choi et ai . 1989).
  • the Bgn is heterogeneous with respect to the size of the glycosaminoglycan chains which results in a broad band on SDS-PAGE centered anywhere from 200-350 kDa.
  • Bgn may differ in size between tissues and developmental stage, it is almost always larger than the other small proteoglycan, Den, when Den is also present. Bgn may occasionally be present with a single CS/DS chain, thus making it the same size as Den.
  • the gene for Bgn is on the human X chromosome (Xq27ter) (Fisher et al. , 1989) and its mRNA encodes a 42.5 kDa preproprotein.
  • the human (cDNA) (Fisher et al, 1989), bovine protein (Neame et al, 1989) and rat (Dreher et al, 1990) sequences have been reported. Bgn probably contains three disulfide bonds.
  • Bgn Unlike its close relatives, Den and Fmn, purified Bgn does not bind to collagen fibrils in vitro, nor is it found associated with classic collagen bundles in tissues. Bgn (both protein and mRNA) is expressed in a range of specialized cell types in developing human tissues including bone, cartilage, blood vessel endothelial cells, skeletal myofibrils, renal tubular epithelia, and differentiating keratinocytes (Bianco et al , 1990). Generally, the Bgn is immunolocalized to the cell surface or pericellular matrices, but in a tissue such as bone, the protein is detected in the matrix proper.
  • This localization in the extracellular matrix may be due to the adso ⁇ tion of the Bgn to hydroxylapatite crystals after having been shed from the osteoblasts. Localization of Bgn by immunoelectron microscopy has not yet been performed.
  • the human Bgn gene has been cloned and partially sequenced.
  • Fmn (or 59-kDa cartilage protein) is a keratan sulfate proteoglycan present in many types of connective tissues, e.g. cartilage, tendon and skin. Fmn is structurally related to the dermatan sulfate/chondroitin sulfate proteoglycans Den and Bgn. Fmn binds to collagen and affects the collagen fibrillogenesis in vitro (Heinegard and Oldberg, 1989).
  • the Fmn protein backbone consists of 357 amino acid residues (42 kDa) which can be divided into three structural domains (Oldberg et al, 1989).
  • the N-terminal domain has four cysteine residues of which two are involved in an intrachain disulfide bond. This region of the protein also contains five to seven closely spaced tyrosine sulfate residues.
  • the centra] domain which constitutes 60% of the protein, consists of ten repeats of 25 amino acid residues. This central repeat domain, with preferentially leucine residues in conserved positions, is homologous to similar repeats in a number of proteins including the interstitial proteoglycans Den and Bgn (Oldberg et al, 1989).
  • the C-terminal domain contains two cysteine residues which form an intrachain disulfide bond.
  • Fmn from cartilage, tendon and sclera contains asparagine-linked keratan sulfate chains (Oldberg et al , 1989; Plaas et al, 1990).
  • Four of the five potential N-glycosylation sites in Fmn from bovine articular cartilage is substituted with keratan sulfate chains (Plaas et al, 1990).
  • Fmn binds to type I and II collagen with a K d of 35 nM.
  • the protein also delays the collagen fibrillation in vitro and causes the formation of thinner fibrils. This collagen binding property is shared by Den but not by the structurally related Bgn (Hedbom and Heinegard, 1989; Brown and Vogel, 1989).
  • Epn epiphycan A small dermatan sulfate proteoglycan containing leucine-rich repeats has been isolated from fetal bovine epiphyseal cartilage. This proteoglycan is referred to as Epn based on its preparation from that tissue. This proteoglycan which seems to have restricted expression is closely similar to other leucine-rich repeat containing proteoglycans, Den and Bgn (Krusius and Ruoslahti, 1986; Day et al.
  • the molecular weight of the intact proteoglycan was approximately 133 kDa while the core protein was ⁇ 46 kDa and the GAG chains were -23-34 kDa. This analysis was determined in comparison with the fetal bovine epiphyseal Den and Bgn produced during the same preparation of Epn. Iodinated proteoglycan was analyzed for its ability to interact with collagens. Furthermore, tryptic peptides from this preparation of Epn were used to determine residues that have undergone posttranslational modification. 4.6.5 Lumican (Lmn)
  • a recent publication has identified a 1.9-kb cDNA clone encoding the chick Lmn (corneal keratan sulfate proteoglycan) (Blochberger et al, 1992).
  • the deduced sequence shows five potential N-linked glycosylation sites, four of which are in the leucine-rich region. These sites are also potential keratan sulfate attachment sites.
  • the cDNA clone to Lmn hybridized to a 2.0-kb mRNA found in tissues other than cornea, predominantly muscle and intestine.
  • the primary structure of lumican is similar to Fmn, Den, and Bgn.
  • B. burgdorferi has now been found to adhere to Den, but does not directly adhere to collagen types I or III. High-affinity binding of B. burgdorferi to Den is specific and appears to require the intact proteoglycan, rather than the isolated core protein or GAG chain. Partial purification of the membrane constituents of B. burgdorferi and affinity chromatography using Den bound to a solid support has permitted visualization of at least two DBPs, designated DbpA and DbpB, having apparent molecular weights of about 18-20 kDa.
  • dbp gene is used to refer to a gene or DNA coding region that encodes a protein, polypeptide or peptide that is capable of binding Den, Fmn, Bgn, Epn. or Lmn.
  • exemplary and preferred dbp genes include the dbpA and dbpB genes isolated from Borrelia, and in particular, from B. burgdorferi, B. afzelii, B. andersonii, B. garinii, or B. japonica.
  • dbp gene is a gene that hybridizes, under relatively stringent hybridization conditions (see, e.g.. Maniatis et al, 1982), to DNA sequences presently known to include a dbp gene sequence.
  • the definition of a dbp gene is a gene that hybridizes, under relatively stringent hybridization conditions (see, e.g.. Maniatis et al, 1982), to DNA sequences presently known to include a dbp gene sequence.
  • dbpA gene is a gene that hybridizes, under relatively stringent hybridization conditions (see, e.g., Maniatis et al, 1982), to DNA sequences presently known to include a dbpA gene sequence.
  • dbpB gene is a gene that hybridizes, under relatively stringent hybridization conditions (see, e.g., Maniatis et al, 1982), to DNA sequences presently known to include a dbpB gene sequence.
  • one or more than one genes encoding DBPs or peptides may be used in the methods and compositions of the invention.
  • the nucleic acid compositions and methods disclosed herein may entail the administration of one, two, three, or more, genes or gene segments.
  • the maximum number of genes that may be used is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting a significant adverse cytotoxic effect.
  • multiple dbp genes they may be combined on a single genetic construct under control of one or more promoters, or they may be prepared as separate constructs of the same or different types. Thus, an almost endless combination of different dbp genes and genetic constructs may be employed.
  • dbp A genes in combination with one or more dbpB genes either on the same, or different DNA segments in order to produce the polypeptides of the invention.
  • genes may either be on a single plasmid or vector, or, alternatively, present on more than one separate plasmids or vectors.
  • the genes may also be present as genomic or extragenomic sequences under the control or a single promoter or multiple independent promoters.
  • Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on formation of an immune response, or the development of antibodies to the dbpA and/or dbpB gene products encoded by such nucleic acid segments, or in the production of diagnostic and treatment protocols for borrelia infection, and in particular, infection with B. burgdorferi, B. afzelii, B. andersonii, B. japonica, or B. garinii, and those infections leading to Lyme disease. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene-protein combinations.
  • nucleic segment or gene could be administered in combination with further agents, such as, e.g. , proteins or polypeptides or various pharmaceutically active agents. So long as genetic material forms part of the composition, there is virtually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or tissues.
  • a therapeutic kit comprising, in suitable container means, one or more DBP composition s) of the present invention in a pharmaceutically acceptable formulation represent another aspect of the invention.
  • the DBP composition(s) may comprise: 1) one or more DBP proteins or peptides, and in particular DbpA and/or
  • DbpB proteins or peptides DbpB proteins or peptides
  • DBP-encoded peptide epitopes one or more DBP-encoded peptide epitopes, domains or motifs, and in particular DbpA- and/or DbpB-derived epitopes, domains or motifs;
  • one or more antibodies which bind to native, truncated, site-specifically or randomly mutated DBPs, or DBP-encoded peptide epitopes, domains or motifs, and in particular anti-DbpA and/or anti-DbpB;
  • nucleic acid segments encoding all or a portion of one or more dbp genes, and in particular one or more dbpA and or dbpB genes.
  • These nucleic acid segments may encode native DBPs, truncated DBPs, site-specifically or randomly mutated DBPs, or DBP-derived peptide epitopes, domains or motifs, and may be either native, recombinant, or mutagenized DNA or RNA segments; or, alternatively,
  • compositions 1) through 6 7) a combination of one or more of the compositions 1) through 6).
  • the kit may comprise a single container means that contains the DBP composition(s).
  • the container means may, if desired, contain a pharmaceutically acceptable sterile excipient, having associated with it, the DBP composition(s) and, optionally, a detectable label or imaging agent.
  • the formulation may be in the form of a gelatinous composition (e.g., a collagenous composition), a powder, solution, matrix, lyophilized reagent, or any other such suitable means.
  • the container means may itself be a syringe, pipette, or other such like apparatus, from which the DBP composition s) may be applied to a tissue site, skin lesion, wound area, or other site of borrelial infection.
  • the single container means may contain a dry, or lyophilized, mixture of one or more DBP composition(s), which may or may not require pre- wetting before use.
  • kits of the invention may comprise distinct container means for each component.
  • one or more containers would contain each of the DBP composition(s), either as sterile solutions, powders, lyophilized forms, etc.
  • the other container(s) would include a matrix, solution, or other suitable delivery device for applying the DBP composition to the body, bloodstream, or to a tissue site, skin lesion, wound area, or other site of borrelial infection.
  • a delivery device may or may not itself contain a sterile solution, diluent, gelatinous matrix, carrier or other pharmaceutical ly-acceptable components.
  • kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent.
  • a sterile, pharmaceutically acceptable buffer, diluent or solvent Such a solution may be required to formulate the DBP component into a more suitable form for application to the body, e.g. , as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms.
  • all components of a kit could be supplied in a dry form (lyophilized), which would allow for "wetting" upon contact with body fluids.
  • the kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition.
  • the container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, into which the components of the kit may placed.
  • the matrix and gene components may also be aliquoted into smaller containers, should this be desired.
  • the kits of the present invention may also include a means for containing the individual containers in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials or syringes are retained. Irrespective of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the placement of the ultimate matrix-gene composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.
  • Affinity chromatography is generally based on the recognition of a protein by a substance such as a ligand or an antibody.
  • the column material may be synthesized by covalently coupling a binding molecule, such as an activated dye, for example to an insoluble matrix.
  • the column material is then allowed to adsorb the desired substance from solution. Next, the conditions are changed to those under which binding does not occur and the substrate is eluted.
  • the requirements for successful affinity chromatography are:
  • a preferred embodiment of the present invention is an affinity chromatography method for purification of antibodies from solution wherein the matrix contains DBPs or peptide epitopes derived from DBPs such as those derived from the DBP of B. burgdorferi, covalently-coupled to a Sepharose CL6B or CL4B.
  • This matrix binds the antibodies of the present invention directly and allows their separation by elution with an appropriate gradient such as salt, GuHCl, pH, or urea.
  • Another preferred embodiment of the present invention is an affinity chromatography method for the purification of DBPs and peptide epitopes from solution. The matrix binds the amino acid compositions of the present invention directly, and allows their separation by elution with a suitable buffer as described above. 4.11 Methods of Nucleic Acid Delivery and DNA Transfection
  • nucleic acid segments disclosed herein will be used to transfect appropriate host cells.
  • Technology for introduction of DNA into cells is well-known to those of skill in the art.
  • Four general methods for delivering a nucleic segment into cells have been described:
  • the inventors contemplate the use of liposomes and/or nanocapsules for the introduction of particular peptides or nucleic acid segments into host cells. Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the nucleic acids, peptides, and/or antibodies disclosed herein.
  • the formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al, 1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).
  • Nanocapsules c m generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al, 1977; 1988).
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multiiamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • the following information may be utilized in generating liposomal formulations.
  • Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less- ordered structure, known as the fluid state. This occurs at a characteristic phase- transition temperature and results in an increase in permeability to ions, sugars and drugs.
  • Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendotheiial system such as macrophages and neutrophils; adso ⁇ tion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of iiposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
  • the present invention contemplates an antibody that is immunoreactive with a polypeptide of the invention.
  • one of the uses for DBPs and DBP-derived epitopic peptides according to the present invention is to generate antibodies.
  • Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies (mAbs), and parts thereof, either alone or conjugated with other moieties.
  • Antibody parts include Fab and F(ab) 2 fragments and single chain antibodies.
  • the antibodies may be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques.
  • an antibody is a polyclonal antibody.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
  • an immunogen comprising a polypeptide of the present invention
  • a wide range of animal species can be used for the production of antisera.
  • an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • Antibodies both polyclonal and monoclonal, specific for DBP and DBP-derived epitopes may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of particular DBPs can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against DBP peptide.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs (below).
  • polyclonal sera that is relatively homogenous with respect to the specificity of the antibodies therein.
  • polyclonal antisera is derived from a variety of different "clones,” i.e., B-cells of different lineage.
  • mAbs by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality.
  • peptides are used as antigens to raise polyclonal sera, one would expect considerably less variation in the clonal nature of the sera than if a whole antigen were employed.
  • Polyclonal antisera according to present invention is produced against peptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore, more stable in an immunologic sense and thus express a more consistent immunologic target for the immune system. Under this model, the number of potential B-cell clones that will respond to this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be higher.
  • the present invention provides for polyclonal antisera where the clonality, i.e., the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonality - 90%, 95% or greater - is contemplated.
  • mAbs To obtain mAbs, one would also initially immunize an experimental animal, often preferably a mouse, with a DBP-containing composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired peptide.
  • spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting mAbs against DBP.
  • Hybridomas which produce mAbs to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the DBP-specific mAbs. It is proposed that the mAbs of the present invention will also find useful application in immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures such as immunoprecipitation, immunocytological methods, etc. which may utilize antibodies specific to DBPs.
  • DBP antibodies may be used in immunoabsorbent protocols to purify native or recombinant DBPs or DBP-derived peptide species or synthetic or natural variants thereof.
  • the antibodies disclosed herein may be employed in antibody cloning protocols to obtain cDNAs or genes encoding DBPs from other species or organisms, or to identify proteins having significant homology to DBP. They may also be used in inhibition studies to analyze the effects of DBP in cells, tissues, or whole animals. Anti-DBP antibodies will also be useful in immuno localization studies to analyze the distribution of bacteria expressing DBPs during cellular infection, for example, to determine the cellular or tissue-specific distribution of borrelias under different physiological conditions. A particularly useful application of such antibodies is in purifying native or recombinant DBPs, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
  • DBP-specific antibody compositions are used to bind to the bacteria and prevent adhesion to host tissues. Moreover, antibody coated bacteria are more easily recognized by the immune system resulting in more efficient killing of the bacteria. Den, Lmn, Epn, Fmn, Bgn, etc., or other molecules mimicking specific structure(s) within host tissues that pathogenic bacteria attach, may be used to saturate the DBPs on the surface of the bacteria, thereby preventing adhesion. Alternatively, DBP derivatives, or other molecules mimicking DBP may be used to saturate the binding site within host tissues, resulting in the inhibition of bacterial adhesion.
  • a third alternative provided by the invention is anti-DBP antibody compositions which bind to DBP both in vitro and in vivo. Such antibodies make the DBPs unavailable for attachment to Den, and therefore, the bacteria expressing cell surface DBPs are unable to adhere to host tissues via Den binding. 4.15 Recombinant Expression of DBP
  • Recombinant clones expressing the dbp nucleic acid segments may be used to prepare purified recombinant DBP (rDBP), purified rDBP-derived peptide antigens as well as mutant or variant recombinant protein species in significant quantities.
  • the selected antigens, and variants thereof, are proposed to have significant utility in diagnosing and treating infections cause by borrelias and in particular, B. burgdorferi, B. garinii, B. andersonii, B. japonica and B. afzelii.
  • rDBPs may also be used in immunoassays to detect borrelias or as vaccines or immunotherapeutics to treat borrelia infections, and to prevent bacterial adhesion to ECM components such as Den in the same manner as native DBP compositions.
  • the present invention allows the ready preparation of so-called "second generation" molecules having modified or simplified protein structures. Second generation proteins will typically share one or more properties in common with the full-length antigen, such as a particular antigenic/immunogenic epitopic core sequence.
  • Epitopic sequences can be provided on relatively short molecules prepared from knowledge of the peptide, or encoding DNA sequence information.
  • Such variant molecules may not only be derived from selected immunogenic/ antigenic regions of the protein structure, but may additionally, or alternatively, include one or more functionally equivalent amino acids selected on the basis of similarities or even differences with respect to the natural sequence. This is particularly desirable in the preparation of blocking antibodies which prevent bacterial adhesion to Den, as outlined herein.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal.
  • an immunogenic composition in accordance with the present invention
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, w-maleimidobenzoyl-/V-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, inco ⁇ orated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide or peptide.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible.
  • the use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol.
  • B-cells B-lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately about 5 x 10 7 to about 2 x 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
  • NS-1 myeloma cell line also termed P3-NS-l -Ag4-l
  • Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described (Kohler and Milstein. 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding, 1986). Fusion procedures usually produce viable hybrids at low frequencies, about
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • HPRT hypoxanthine phosphoribosyl transferase
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
  • the cell lines may be exploited for mAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific mAb produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • native and synthetically-derived peptides and peptide epitopes of the invention will find utility as immunogens, e.g., in connection with vaccine development, or as antigens in immunoassays for the detection of reactive antibodies.
  • preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs), as are known to those of skill in the art.
  • ELISAs enzyme linked immunosorbent assays
  • DBP-derived proteins and peptides is not limited to such assays, and that other useful embodiments include RIAs and other non-enzyme linked antibody binding assays and procedures.
  • proteins or peptides inco ⁇ orating DBP, rDBP, or DBP-derived protein antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate.
  • a selected surface preferably a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate.
  • a nonspecific protein that is known to be antigenically neutral with regard to the test antisera, such as bovine serum albumin (BSA) or casein, onto the well. This allows for blocking of nonspecific adso ⁇ tion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • BSA bovine serum albumin
  • the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween®. These added agents also tend to assist in the reduction of nonspecific background.
  • the layered antisera is then allowed to incubate for, e.g., from 2 to 4 hours, at temperatures preferably on the order of about 25° to about 27°C.
  • the antisera-contacted surface is washed so as to remove non- immunocomplexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween®, or borate buffer.
  • the occurrence and the amount of immunocomplex formation may be determined by subjecting the complex to a second antibody having specificity for the first.
  • the second antibody will preferably be an antibody having specificity for human antibodies.
  • the second antibody will preferably have an associated detectable label, such as an enzyme label, that will generate a signal, such as color development upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions that favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS- containing solution such as PBS-Tween®).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 . in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 .
  • ELISAs may be used in conjunction with the invention.
  • proteins or peptides incorporating antigenic sequences of the present invention are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate.
  • a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk.
  • BSA bovine serum albumin
  • casein casein
  • the anti-DBP antibodies of the present invention are particularly useful for the isolation of DBP antigens by immunoprecipitation.
  • Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.
  • peptides For the isolation of cell-surface localized proteins such as DBP, peptides must be solubilized from the bacterial cell wall by treatment with enzymes such as lysozyme, lysostaphin or mutanolysin, or alternatively, into detergent micelles.
  • Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
  • the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g., enzyme-substrate pairs.
  • antibodies of the present invention are useful for promoting the binding of Den to dbp gene products. Such binding is readily measured by monitoring ligand binding using well-known procedures. Detection of the binding may be accomplished by using radioactively labeled antibodies or alternatively, radioactively-labeled Den. Alternatively, assays employing biotin-labeled antibodies are also well-known in the art as described (Bayer and Wilchek, 1980).
  • compositions of the present invention will find great use in immunoblot or western blot analysis.
  • the anti-DBP antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
  • a solid support matrix such as nitrocellulose, nylon or combinations thereof.
  • immunoprecipitation followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
  • the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross- reacting signal.
  • Immunologically-based detection methods in conjunction with Western blotting are considered to be of particular use in this regard.
  • the present invention contemplates vaccines for use in both active and passive immunization embodiments.
  • Immunogenic compositions proposed to be suitable for use as a vaccine may be prepared most readily directly from the novel immunogenic proteins and/or peptide epitopes described herein.
  • the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.
  • vaccines that contain peptide sequences as active ingredients
  • U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770 all incorporated herein by reference.
  • such vaccines are prepared as injectables, either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines.
  • a composition comprising DBP or DBP-derived proteins and/or native or modified epitopic peptides therefrom could also be the basis for human vaccines.
  • the preparation of such compositions that are essentially free from endotoxin can be achieved by following the published methodology, for example, U.S. Patent 4,271,147 (incorporated herein by reference) discloses methods for the preparation of Neisseria meningitidis membrane proteins for use in vaccines.
  • DBP and DBP-derived epitope-based vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • the proteins may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines may be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g. , the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered will be readily determinable by the skilled practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
  • Various methods of achieving adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol®) used as 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° and about 101 °C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated F(ab) antibodies to albumin, mixture with bacterial cells such as C.
  • agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol®) used as 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° and about 101 °C for 30 second to 2 minute periods respectively. Aggregation by reactiv
  • parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria emulsion in physiologically acceptable oil vehicles such as mannide monooleate (Aracel-ATM) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol- DATM) used as a block substitute may also be employed.
  • physiologically acceptable oil vehicles such as mannide monooleate (Aracel-ATM) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol- DATM) used as a block substitute may also be employed.
  • the vaccine will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations.
  • the vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1 -5 years, usually three years, will be desirable to maintain protective levels of the antibodies.
  • the course of the immunization may be followed by assays for antibodies for the supernatant antigens.
  • the assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791 ,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.
  • Immunogenic compositions proposed to be suitable for use as a vaccine, may be prepared most readily directly from immunogenic peptides prepared in a manner disclosed herein.
  • the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.
  • the preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S.
  • such vaccines are prepared as injectables. Either as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
  • compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1 % of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit.
  • the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained- release preparation and formulations.
  • the active compounds may also be administered parenterally or intraperitoneal ly.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol. liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the polypeptide may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • composition can be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035- 1038 and 1570-1580).
  • Host cells that have been transformed could be used in the screening of natural and artificially derived compounds or mixtures to select those that are capable of complexing with the DBP and DBP-derived proteins of the present invention. This could be useful in the search for compounds that inhibit or otherwise disrupt, or even enhance the ability of the microorganism to bind Den. It is contemplated that effective pharmaceutical agents could be developed by identifying compounds that complex with the particular DBP epitopes, including, for example, compounds isolated from natural sources, such as plant, animal and marine sources, and various synthetic compounds. Natural or man-made compounds that may be tested in this manner could also include various minerals and proteins, peptides or antibodies.
  • the present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more of the antibodies of the present invention.
  • the term "incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-DBP antibodies” is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a DBP polypeptide.
  • the level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the DBP polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen.
  • Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.
  • DBP epitopes such as those derived from dbp or dbp-Wk gene products and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter.
  • the methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf e/ al, 1988; U.S. Patent Number 4,554,101).
  • the amino acid sequence of these "epitopic core sequences" may then be readily inco ⁇ orated into peptides, either through the application of peptide synthesis or recombinant technology.
  • Preferred peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more preferably about 8 to about 20 amino acids in length. It is proposed that shorter antigenic peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
  • An epitopic core sequence is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on DBP epitope-specific antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term “complementary” refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
  • the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences.
  • the smallest useful core sequence expected by the present disclosure would generally be on the order of about 5 amino acids in length, with sequences on the order of 8 or 25 being more preferred.
  • this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.
  • the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
  • the peptides provided by this invention are ideal targets for use as vaccines or immunoreagents for the treatment of various borrelia-related diseases, and in particular, those caused by species which contain DBP and DBP-encoding genes, and hence those which express either dbp or dbp-like gene product(s) on the cell surface and in turn interact with ECM components such as Den to promote bacterial adhesion to host cells.
  • ECM components such as Den to promote bacterial adhesion to host cells.
  • particular advantages may be realized through the preparation of synthetic peptides that include epitopic/immunogenic core sequences. These epitopic core sequences may be identified as hydrophilic and/or mobile regions of the polypeptides or those that include a T cell motif.
  • the screening assays may be employed to identify either equivalent antigens or cross-reactive antibodies. In any event, the principle is the same, i.e., based upon competition for binding sites between antibodies and antigens.
  • Suitable competition assays include protocols based upon immunohistochemical assays, ELISAs, RIAs, Western or dot blotting and the like.
  • one of the binding components generally the known element, such as the DBP-derived peptide, or a known antibody, will be labeled with a detectable label and the test components, that generally remain unlabeled, will be tested for their ability to reduce the amount of label that is bound to the corresponding reactive antibody or antigen.
  • DBP DBP
  • a detectable label such as, e.g., biotin or an enzymatic, radioactive or fluorogenic label
  • the known antibody would be immobilized, e.g., by attaching to an ELISA plate.
  • the ability of the mixture to bind to the antibody would be determined by detecting the presence of the specifically bound label. This value would then be compared to a control value in which no potentially competing (test) antigen was included in the incubation.
  • the assay may be any one of a range of immunological assays based upon hybridization, and the reactive antigens would be detected by means of detecting their label, e.g., using streptavidin in the case of biotinylated antigens or by using a chromogenic substrate in connection with an enzymatic label or by simply detecting a radioactive or fluorescent label.
  • An antigen that binds to the same antibody as DBP, for example, will be able to effectively compete for binding to and thus will significantly reduce DBP binding, as evidenced by a reduction in the amount of label detected.
  • the reactivity of the labeled antigen, e.g., a DBP composition, in the absence of any test antigen would be the control high value.
  • the control low value would be obtained by incubating the labeled antigen with an excess of unlabeled DBP antigen, when competition would occur and reduce binding.
  • a significant reduction in labeled antigen reactivity in the presence of a test antigen is indicative of a test antigen that is "cross-reactive", i.e., that has binding affinity for the same antibody.
  • a significant reduction in terms of the present application, may be defined as a reproducible (i.e., consistently observed) reduction in binding.
  • peptidyl compounds described herein may be formulated to mimic the key portions of the peptide structure.
  • Such compounds which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents.
  • the generation of a structural functional equivalent may be achieved by the techniques of modelling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of a commercially-available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
  • peptides may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity.
  • agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5.
  • agents which will inhibit microbial growth such as sodium azide or Merthiolate.
  • the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 14 to about 25 nucleotides in length is preferred, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically.
  • This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation- bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • mutagenic agents such as hydroxylamine
  • PCRTM-based strand overlap extension (SOE) Ho et al, 1989
  • SOE strand overlap extension
  • the techniques of PCRTM are well-known to those of skill in the art, as described hereinabove.
  • the SOE procedure involves a two-step PCRTM protocol, in which a complementary pair of internal primers (B and C) are used to introduce the appropriate nucleotide changes into the wild-type sequence.
  • flanking PCRTM primer A (restriction site incorporated into the oligo) and primer D (restriction site inco ⁇ orated into the oligo) are used in conjunction with primers B and C, respectively to generate PCRTM products AB and CD.
  • the PCRTM products are purified by agarose gel electrophoresis and the two overlapping PCRTM fragments AB and CD are combined with flanking primers A and D and used in a second PCRTM reaction.
  • the amplified PCRTM product is agarose gel purified, digested with the appropriate enzymes, ligated into an expression vector, and transformed into E.
  • Clones are isolated and the mutations are confirmed by sequencing of the isolated plasmids. Beginning with the native dbp gene sequence, suitable clones and subclones may be made in BG26:pB/2.5(5), from which site-specific mutagenesis may be performed. Alternatively, the use of pET vectors (Novagen, Inc., Madison, Wl; U.S. Patent 4, 952,496, disclosed herein by reference) is contemplated in the recombinant production of DBP and DBP-derived polypeptides.
  • Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics.
  • the following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to Table 1.
  • certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. 5.
  • B. burgdorferi N40 Low-passage (fewer than 10 in vitro passages) B. burgdorferi N40 was used for all studies unless specified otherwise.
  • High-passage B. burgdorferi B31 (ATCC 35210) has undergone numerous in vitro passages.
  • B. burgdorferi was cultured in BSKII medium at 34°C (Barbour, 1984). Cultures were incubated in a GasPak chamber (BBL, Baltimore, MD) with 3 to 6% O 2 until the cells reached the mid-to late-log phase.
  • Cells were harvested by centrifugation at 14, 500 x g for 30 min and gently washed in sterile, filtered phosphate-buffered saline (PBS; pH 7.4; 0.137 M NaCl, 3 mM KC1, 4 mM Na 2 HPO 4 , 1 M KH 2 PO 4 ) three times.
  • PBS filtered phosphate-buffered saline
  • the spirochetes were resuspended in PBS, and the cell density was adjusted to 10 9 organisms per ml by use of a reference standard curve relating the A 600 to the organism number as determined by dark-field microscopy.
  • the spirochetes were stored at 4°C and retained Dcn-binding activity for up to 1 month.
  • Staphylococcus aureus Phillips (clinical osteomyelitis isolate) and PHI 00 (collagen adhesion-negative isogenic mutant of strain Phillips) were grown in brain heart fusion broth (Difco Laboratories, Detroit, MI) overnight at 37°C without antibiotics (Patti et al, 1994). The cells were washed and resuspended in PBS. 5.1.1.2 Labeling of Density
  • Bovine Den from fetal skin was purified as described previously (Choi et al. , 1989). Den was labeled with NHS-LC-Biotin (Pierce, Rockford, IL) as described in the manufacturer's directions and stored at -20°C. Den was iodinated by the chloramine T method as described by Hunter
  • Immulon-1® microtiter plate wells (Dynatech Labs, Chantilly, VA) were coated with Den, collagen type I from rat tail (Collaborative Biomedical Products, Bedford, MA), or collagen type III from calf skin (Sigma Chemical Co., St. Louis, MO). Den was dissolved in PBS, and collagens type I and III were dissolved in 20 mM acetic acid and adjusted to a concentration of 1 mg/ml. Two micrograms of each protein in a total volume of 50 ⁇ l was incubated in the microtiter plate wells at 4°C overnight. The wells were decanted and washed with 200 ⁇ l of PBS containing 0.1% bovine serum albumin (BSA) three times for 5 min each.
  • BSA bovine serum albumin
  • Additional protein-binding sites were blocked by incubating the microtiter wells with 100 ⁇ l of a 1 -mg/ml concentration of BSA in PBS for 2 h. The wells were washed and incubated for 1 h with 25 ⁇ l of a suspension containing 10 9 organisms per ml of PBS-0.1% bovine serum albumin (BSA) three times for 5 min each. Additional protein-binding sites were blocked by incubating the microtiter wells with 100 ⁇ l of a 1 mg/ml concentration of BSA in PBS for 2 h. The wells were washed and incubated for 1 h with 25 ⁇ l of a suspension containing 10 9 organisms per ml of PBS-0.1% BSA.
  • BSA bovine serum albumin
  • the wells were washed and subsequently incubated with 100 ⁇ l of a 1 : 1.000 dilution of goat anti-rabbit alkaline phosphatase conjugate (Bio-Rad, Hercules, CA) in PBS-0.1% BSA for 1 h and then washed and subsequently incubated with 100 ⁇ l of a 1 -mg/ml concentration of Sigma 104 phosphatase substrate dissolved in 1 M diethanolamine-0.5 mM MgCl 2 (pH 9.8) at 37°C for 30 to 45 min.
  • the A A05 was determined in a microplate reader (Molecular Devices, Menlo Park, CA). To assay inhibition of attachment, 100- ⁇ l suspensions containing 10 9 organisms of B.
  • burgdorferi N40 per ml were preincubated with 2 ⁇ g of the potential competitor (or as otherwise stated) for 1 h at room temperature.
  • the potential competitors included Den, BSA (The Binding Site, San Diego, CA, or ICN, Costa Mesa, CA), fetuin type IV (Sigma), thyroglobulin type II (Sigma), fibrinogen (KabiVitrium, Swiss, Sweden), aggrecan (Isolated from bovine cartilage), heparin (Sigma), and chondroitin sulfate type A (from whale and shark cartilage; Sigma).
  • One microliter of 10% BSA was added to obtain a final concentration of 1% BSA.
  • the suspensions were added to protein-coated microtiter wells, and the assay was continued as described above.
  • B. burgdorferi N40 cells (1.5 x 10 s ) were incubated with approximately 50,000 cpm of 125 l-labeled Den in a final volume of 0.5 ml of PBS containing 1% BSA for 1 h at room temperature. The reaction was stopped by the addition of 3 ml of PBS containing 1% BSA; centrifugation at 6,000 x g for 30 min followed. Radiolabeled Den associated with the bacterial pellet was quantitated in a Cobra II Auto-Gamma Counter (Packard Instruments, Meriden, CN). Radioactivity recovered in tubes incubated as described above, but without bacteria, was regarded as background and subtracted from the values obtained with bacteria. Time dependence of binding was assayed by incubating B. burgdorferi with I25 I-labeled Den as described above and stopping the reaction at the specified times.
  • Proteins from B. burgdorferi whole-cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) and probed with a Western blot-type assay.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • 2 lO 7 B. burgdorferi cells were lysed by boiling SDS under reducing conditions and subjected to electrophoresis through a 5 to 15% gradient acrylamide slab gel at 175 V for 30 min, or through a 12.5% acrylamide resolving gel at 200V for 35 min.
  • the proteins were transferred from the polyacrylamide gel to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH) by electroblot for 1.5 h at 4°C. Additional protein-binding sites on the membrane were blocked by incubating in 3% nonfat dry milk in TBST (0.15 M NaCl, 20 mM Tris-HCl, 0.05% Tween-20® [pH 7.4]) for 2 h at room temperature or overnight at 4°C.
  • TBST 0.15 M NaCl, 20 mM Tris-HCl, 0.05% Tween-20® [pH 7.4]
  • the membrane was incubated at room temperature with 0.1 ⁇ g of biotin-labeled Den per ml of TBST for 1 h, washed, and incubated with a 1 :3,000 dilution of avidin D horseradish peroxidase conjugate (Vector Laboratories, Burlingame, CA) in TBST for 1 h.
  • the membrane was washed and incubated in 1 ml of Enhanced Chemi-Luminescence detection reagents 1 and 2 (Amersham Life Science) for 1 min and exposed to X-ray film for 1 to 5 s.
  • the inventors used an in vitro attachment assay. Microtiter wells were coated with Den or collagens, and a suspension of spirochetes was incubated for 1 h in the protein-coated wells. Adherent spirochetes were detected by an immunological method after nonadherent organisms were removed by washing. B. burgdorferi N40 adhered to wells coated with Den, whereas spirochete adherence to collagen-coated wells was only marginally greater than adherence to BSA-coated wells.
  • Adherent spirochetes were detected by an immunological method after nonadherent organisms were removed by washing.
  • B. burgdorferi N40 adhered to wells coated with Den, whereas spirochete adherence to collagen-coated wells was only marginally greater than adherence to BSA-coated controls wells.
  • ELISA enzyme-linked immunosorbent assay
  • a modified in vitro binding assay was used to determine whether B. burgdorferi binds to soluble l25 I-labeled Den. Spirochetes were incubated in a suspension containing PBS, 1% BSA, and 125 I-labeled Den. At the end of the incubation, the bacteria were collected by centrifugation and the amount of Den bound was assayed by measuring radioactivity in the pellet. When binding was assayed as a function of time from 0 to 120 min, maximum binding was achieved in 15 min and remained constant for up to 2 h. Prolonged incubation for 3 to 4 h often resulted in a decrease of binding. A high-passage isolate of B. burgdorferi B31 showed no binding at any time points.
  • k d was calculated at 3 x 10 "7 M "1 , indicating a moderate affinity
  • n was calculated to be approximately 5 x 10 4 Dcn-binding sites per organism, indicating a small copy number. Although the standard deviation for Den bound is large, these values are semi-quantitative estimates
  • This assay revealed the presence of two DBPs with apparent molecular masses of 19 and 20 kDa in the mixture of proteins from B. burgdorferi N40. SDS-PAGE followed by staining with Coomassie brilliant blue indicated that these proteins constitute a small portion of the total proteins of B. burgdorferi.
  • the two DBPs run directly beneath OspC (the prominent band at -21 kDa) but are hardly visible, if at all, by Coomassie blue stain.
  • OspC the prominent band at -21 kDa
  • B. burgdorferi is predominantly an extracellular pathogen and that the spirochetes are often found in intimate association with collagen fibers (Barthold et ⁇ l, 1991 ; 1992; 1993; Duray, 1992).
  • the collagen network is composed mainly of collagen types I and III and the proteoglycan Den, which is associated with the collagen fibers.
  • B. burgdorferi adhered to substrata composed of Den but did not adhere to collagens type I or III. These data suggest that Den is a possible target for B. burgdorferi adherence in the skin. Previously, B. burgdorferi adherence to heparin Isaacs, 1994) and ⁇ Ilb ⁇ 3 integrin (Coburn et al, 1993; 1994) has been reported. Furthermore, heparin inhibits the adherence of B. burgdorferi to cultured HeLa cells (Isaacs, 1994) but, as shown in this study, does not affect the binding of Den to the spirochete. B. burgdorferi most likely possesses several mechanisms of host tissue adherence; however, it is unlikely that any of the previously described adherence mechanisms are responsible for the observed colonization of collagen fibers in the dermis.
  • B. burgdorferi also binds to soluble Den in a process that exhibits saturation kinetics and occurs in a time- and concentration-dependent manner. Maximal binding was achieved more quickly when Den was in solution than when it was immobilized onto microtiter wells. The reason for this discrepancy is unknown. Both the binding of soluble 125 I-labeled Den and the attachment of spirochetes to a Den substrate were effectively inhibited by Den. Other extracellular matrix proteins had only marginal effects, suggesting a high degree of specificity. Furthermore, neither isolated core protein nor GAG chain alone or in combination could inhibit binding.
  • Den The Borrelia binding site on the Den molecule has not been identified. Presumably, Den binds both collagen and borrelias at once, with the two interactions involving different sites on the proteoglycan. The requirement of intact Den adhesin on the spirochete may recognize a conformational motif that is destroyed upon separation of the core protein and the GAG chain.
  • the K t for the binding of B burgdorferi to Den was estimated to be approximately 3 x 10' 7 M " ', indicating moderate affinity.
  • the number of binding sites, n was calculated to be approximately 5 x 10 4 copies per organism, which is a low copy number. SDS-PAGE analysis also seems to indicate that the DBPs are not abundant B. burgdorferi proteins.
  • the 19-kDa protein may be a truncated product derived from the same gene as the 20-kDa protein; alternatively, the two proteins may be genetically distinct.
  • N-40 To approximately 1 x 10 9 organisms/ml phosphate-buffered saline (PBS) was added N-octyl-glucopyranoside to a concentration of 1.5%. The mixture was incubated twenty min at room temperature, rotating end over end. The incubated mixture was spun down at 30,000 ⁇ m for 5 min, and the supernatant containing the extracted membrane constituents was removed. The supernatant was dialyzed against PBS
  • the Den affinity column was prepared by first purifying Den from fetal bovine skin according to the method described by Choi. Purified Den was then covalently linked to CNBr-activated Sepharose® 4B (Pharmacia, Uppsala, SWEDEN), according to the manufacturer's instructions. The 2.0 ml Den column was equilibrated with PBS at room temperature, and the membrane preparation was poured through the column, followed by a 10 column volume wash with PBS. Protein bound to the Den column was eluted with 1 M NaCl, and eight 0.5 ml fractions were collected. Twenty microliters of each collected fraction was electrophoresed in duplicate
  • a portion of the lower gel band e.g., the 18-19 kDa band, was cut from the Coomassie Blue stained gel and transferred to a 5-20% gradient polyacrylamide gel together with endopeptidase. The separated proteins were transferred to PVDF
  • DBPs The adhesive function of DBPs, and their role as targets for growth-inhibitory antibodies, imply that the DBPs are localized to the borrelia outer membrane.
  • IM inner and outer membranes
  • OM inner and outer membranes
  • DbpA detergent phase portioning DbpA appears to be amphiphilic as are OspA and other borrelia membrane lipoproteins (Brandt et al, 1990). To confirm the presence of lipid on these proteins B.
  • DbpA is a lipoprotein as predicted by its sequence and membrane fractionation properties.
  • the ⁇ ZAPTM expression library was obtained from Dr. Robin Isaacs of the V.A. Medical Center in Jackson, MS.
  • the library had been constructed using B. burgdorferi strain 297 DNA according to the manufacturer's instructions (Stratagene, La Jolla, CA).
  • the library was created from DNA recovered from a B. burgdorferi 297 (p3) culture and included genomic and plasmid elements.
  • the DNA was partially digested with Sau3A and partially end-filled to enable ligation into the vector digested with Xho ⁇ and partially filled in.
  • the initial library contained about 2.1 x 10 5 clones with greater than 95% recombinants.
  • the average insert size was in the range of 2-4 kb.
  • the library was plated in six 90-mm plates and plaques were lifted onto HAFT nitrocellulose filters (Millipore, Bedford, MA) according to the protocol described by Sambrook et al, (1989).
  • the filters were incubated in TBST (0.15 M NaCl, 0.02 M Tris HCI, 0.05% Tween-20®, pH 7.4) containing 3% (w/v) bovine serum albumin (BSA) for 2 hours at room temperature.
  • the filters were washed with TBST three times for 5 min at room temperature.
  • the washed filters were incubated with 1 ⁇ g of digoxigenin-labeled Den (prepared as described above) per ml of TBST for one h at room temperature, then washed as described above and incubated with 1 :1000 anti-digoxigenin-POD Fab fragments in TBST for one h at room temperature.
  • the antibody marker was developed by washing and incubating in chloronapthol solution (30 mg of 4-chloro-l-napthol [Bio-Rad, Hercules, CA] in 10 ml of methanol, chilled at -20°C for ten min then 50 ml TBS [0.15 M NaCl, 0.02 M Tris HCI, pH 7.5] and 100 ⁇ l of 30% H 2 O 2 was added) at room temperature for 5-20 min until color development was complete.
  • chloronapthol solution (30 mg of 4-chloro-l-napthol [Bio-Rad, Hercules, CA] in 10 ml of methanol, chilled at -20°C for ten min then 50 ml TBS [0.15 M NaCl, 0.02 M Tris HCI, pH 7.5] and 100 ⁇ l of 30% H 2 O 2 was added
  • pBG26 contains an additional 500-bp of upstream sequence and pBG27 contains an additional 500-bp of downstream sequence (FIG. 24).
  • Analysis of the DNA sequence revealed two ORFs each of exactly 561 bp, within the 1500-kb overlapping sequence. The ORFs are not arranged in an operon, but have separate promoters and are separated by a 177-bp DNA sequence. These ORFs were subsequently shown to be genes which encode for DBPs, therefore, they were designated dbpA and dbpB.
  • Analysis of the deduced amino acid sequences revealed putative lipid attachment sites LISC (DbpA) and LVAC (DbpB). The nucleotide sequences for dbpA and dbpB were aligned using the MacVector program and were revealed to share 50% identity. The deduced amino acid sequences were aligned and shown to share only a 40% identity.
  • the sequence of the 2.5 kb (SEQ ID NO:7) insert is shown in FIG. 2A and FIG. 2B, with the approximate 0.6-kb open reading frame of dbpB beginning with the ATG sequence at nucleotide 791 and ending at nucleotide 1351 ; the approximate 0.6- kb open reading frame of dbp A begins with the ATG sequence at nucleotide 1471 and ends with the TCG at nucleotide 2031.
  • the amino acid sequences of DbpA and DbpB are shown in SEQ ID NOs:8 and 28.
  • the dbpB gene was not expressed at high levels by this clone, but its ORF was identical in length to dbpA. Su ⁇ risingly, the products of these two genes, however, share only about 40% amino acid sequence identity.
  • DBPs are believed to be lipoproteins based on their partitioning into the Triton X-1 14 detergent phase as determined by Western blot analysis and inco ⁇ oration of 3 H palmitate (DbpA).
  • DbpA Western blot analysis and inco ⁇ oration of 3 H palmitate
  • studies of other lipoproteins from B. burgdorferi have shown that signal peptidase II cleaves at the N-terminal side of cysteine after lipid attachment.
  • PCR was used to construct recombinant DBPs with the corresponding amino acid truncations; these constructs were designated DbpA:504 and DbpB:500 (FIG. 24). These proteins were expressed in E coli Ml 5 cells and polyhistidine (N-terminal) fusions.
  • DBPs were purified by immobilized nickel chelating chromatography. When necessary, the proteins were also subsequently purified by cation exchange chromatography (Mono-S, Pharmacia, Uppsala, Sweden). DbpB:500 appeared as a single band at approximately 19kDa by SDS-PAGE. DbpA:504 was highly insoluble so we created a construct lacking only nine amino terminal residues (designated DbpA:549, FIG. 23A and FIG. 23B). Purified DbpA:549 did appear to be soluble, however, when analyzed by SDS-PAGE, the protein exhibited two bands with apparent molecular masses of 20 and 40 kDa, indicating the formation of dimers.
  • the 40 kDa protein was confirmed to be DbpA:549 by Western blot. Although the dimers could be detected even when SDS-PAGE was performed under reducing conditions. However, they were almost completely eliminated by adding excess (0.1M) ⁇ - mercaptoethanol to both the sample and running buffers, suggesting that the aggregation was due to intermolecular disulfide bonding.
  • a site-directed mutant of DbpA:549 was created in which the sole cyestein at position 25 was mutated into an alanine residue (designated DbpA:C25A, FIG. 24). The resulting mutant did not appear to dimerize since it runs as a single band.
  • the activity of the recombinant Dbps was determined by Western blot-type assay and by a microtiter well binding assay.
  • the Western blot-type assay is based on a traditional Western blot with the modification of using a labeled ligand, in this case digoxigenin-labeled Den, as the probe. Purified proteins were separated by SDS- PAGE under reducing conditions and transferred to a nitrocellulose membrane.
  • digoxigenin-labeled Den was allowed to bind to proteins on the membrane, followed by binding of alkaline phosphates-conjugated anti-digoxigenin F ab fragments to the digoxigenin-labeled Den.
  • Dcn-binding proteins were visualized by phosphatase substrate reactivity. All three proteins, DbpA:549, DbpA:C25A and DbpB:500, showed roughly similar Dcn-binding activity as judged by relative band intensity.
  • Dcn-binding activity by binding assay To determine Dcn-binding activity by binding assay, separate microtiter wells were coated with increasing amounts of Dbps then biotin-labeled Den was allowed to bind for 1 hour. Using this assay, DbpA:549, DbpA:C25A, had at most approximately 80% activity compared with DbpA:549. It is possible that this discrepancy is due to differences in the proteins' affinity for the microtiter wells. It is significant to note that recombinant Osp used as a negative control (produced as a polyhistidine fusion protein from strain N40) did not exhibit any detectable Dcn-binding activity even at the highest concentration of 1000 ng per well.
  • burgdorferi N40 is inhibited 93% when the wells were preincubated with 200 ng of either DbpA protein and completely blocked when preincubated with 400 ng (FIG. 26B).
  • DbpB appeared to inhibit attachment weakly. This data suggested that different Borrelia strains may vary in their expression of DBPs. Alternatively, the two proteins could bind to different binding sites on Den.
  • a Western blot was used to address whether different Borrelia strains express different relative amounts of the two DBPs.
  • Rabbit anti-sera were generated against DbpA:549 and DbpB:500. Surprisingly, these anti-sera showed minimal cross- reactivity as determined by ELISA.
  • the few cross-reactive antibodies were adsorbed out using Dbps coupled to sepharose beads.
  • the proteins in whole cell lysate of B. burgdorferi 297 and N40 were separated by SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. The adsorbed anti-sera was used to probe for DbpA and DbpB in a Western blot.
  • DBPs were individually preincubated in approximately 1000 fold excess of biotin-labeled Den to saturate all of the binding sites on Den.
  • the DBPs were coated onto microtiter wells before the Dcn-DBP mixture was allowed to bind.
  • DbpA successfully competed the interaction between DbpB with Den and vice versa (FIG. 27).
  • OspC did not inhibit binding of either DBP to Den and each DBP was able lo inhibit Den binding with itself. Therefore, both proteins appear to bind to the same site on Den.
  • Labeled Den was used as a probe to clone Dbp genes from a ⁇ ZAPII genomic library constructed from B. burgdorferi 297 DNA. Two genes of exactly 561 bp were isolated. E. coli was able to recognize one or both of the DBP gene promoters since neither protein was being expressed as a fusion. The genes were localized by Southern hybridization to a 56-kb plasmid, the same plasmid on which the ospAB operon is located. The deduced amino acid sequences of DbpA and DbpB are 40% identical and their "normalized alignment score" was calculated to be 350, which indicates that the two proteins are evolutionarily related.
  • DBPs Both DBPs were produced in E coli as polyhistidine fusions proteins lacking their leader peptides and purified by nickel chelating chromatography.
  • the recombinant DBPs were active, but appeared to have different binding affinities for Den based on the microtiter well binding assay. However, these discrepancies could be due to incomplete coating on the wells. No differences in binding as judged by band intensity were detectable by Western blot-type assay.
  • DbpA is a more effective inhibitor of spirochete attachment to Den regardless of the relative expression of the DBPs.
  • DbpA and DbpB bind to the same site on Den.
  • DbpA:C25A was a slightly less effective inhibitor compared to DbpA:549 and DbpB:500, which may be a result of the mutation.
  • DbpB is just as efficient as DbpA at competing for Den binding (FIG. 27), despite being unable to compete with intact spirochetes (FIG. 26A and FIG. 26B). This difference may be an artifact resulting from using purified proteins in an in vitro assay since the DBPs present in Borrelia membranes may not be fully accessible.
  • B burgdorferi has two genes, designated dbpA and dbpB, encoding distinct Dcn-binding proteins, DbpA and DbpB, respectively.
  • the proteins are similar in size, sequence and function, yet are expressed under the control of separate promoters and do not elicit cross-reactive antibodies.
  • EXAMPLE 5 Identification of DBPs in Borrelial Isolates
  • One aspect of the present invention is the identification of borrelias using the DBP compositions disclosed herein as diagnostic indicators of borrelial infection.
  • Table 2 an assay of DBPs in borrelias using Western hybridization analyses, it was possible to identify the presence of DBPs in at least 13 strains of B. burgdorferi, 5 strains of B. garinii, and at least three strains of B. afzelii. These methods represent important diagnostic tools for the identification of bacteria in clinical isolates.
  • Strains were derived from Lyme patient material or Ixodes sp. ticks. ttwo bands with activity at 18-20 kDa.
  • CSF cerebrospinal fluid
  • EM reythema migrans
  • ACA acrodermatitis chronicum atrophicans.
  • the oligonucleotide primers were used in PCRTM to amplify portions of DbpA from the following strains of B. burgdorferi: N40, SH2, HB19, B31 (low passage); HPB31 (high passage), and 297. Amplification was carried out for 30 cycles of 94°C for one min; 50°C for 2 min; and 72°C for three min using standard PCRTM conditions. (100 microliters total volume: 71.5 microliters water; 10 microliters 1 OX PCRTM buffer; 5 microliters of 5 mM MgCl 2 ; 2 microliters DNA; 2 ⁇ l each dNTP; 1.5 microliters of each primers; and 0.5 microliters of Taq polymerase (GIBCO). Each mixture was covered with 100 microliters of mineral oil and PCRTM cycles begun).
  • the amplification primers derived from the nucleotide sequence of dbpA of B. burgdorferi strain 297 produced products for each of the variant strains tested, although the ability of specific primers to amplify a product in each strain varied considerably. This implies variation between strains of the bacteria in the nucleic acid sequence encoding DbpA.
  • Oligonucleotides derived from the DbpA DNA sequence of strain 297 were used as primers for PCRTM amplifications of dbp gene fragments from various borrelia strains. Using a western blot-like assay with tagged Den for assessment of Den binding activity, almost all strains were found to express a DBP in vitro of approximately the same size, 20 kDa + 2 kDa. Thus these strains were predicted to contain alleles of the dbpA and dbpB genes of strain 297 as confirmed by PCRTM.
  • PCRTM products may be used to identify DNA fragments containing dbpA and dbpB strain variants by
  • the sequence divergence of DbpA from some B. afzelii and B. garinii strains PGau with respect to the B. burgdorferi strains is consistent with the resistance of these strains to in vitro growth inhibition by anti-DbpA 297 serum.
  • Availability of the dbpA sequences from B. afzelii and B. garinii provides a the basis for obtaining clones of additional B. afzelii and B. garinii dbpA gene sequences, elucidation of the common epitope motifs which may differ from B. burgdorferi and facilitate design of broad coverage multi-DbpA or multi DbpA/DbpB cocktail vaccines.
  • Microtiter test wells were coated with purified Den prepared as described for Example 1 and shaken at 4°C, covered, overnight. (One microgram of Den in 50 ⁇ l PBS, 50 ⁇ l/well.) Control wells were coated with 1% BSA in PBS. Den coated wells were also incubated with 1% BSA in PBS for 1 hr at room temperature to block uncovered sites.
  • Recombinant DBP was obtained by growing BG26:pB/2.5(5) in LB medium containing 100 ⁇ g/ml ampicillin overnight at 37°C with shaking. 20 ml of the grown bacteria was placed in 1 1 of LB medium containing 100 ⁇ g/ml ampicillin and shaken at 37°C overnight till reaching an optical density of about 0.6-0.8 at A 600 . IPTG was added to a concentration of 0.2 mM, and the mixture was shaken at 37°C for about 2-3 hr. The cells were broken in a French press and the debris was pelleted at 40,000 ⁇ m for about 15 min.
  • the supernatant was filtered through a 0.45 ⁇ m filter and 5 ⁇ l of the filtered supernatant was added to a 2-ml Den affinity column previously equilibrated in PBS.
  • the column was washed with 10 volumes of PBS and the bound protein was eluted with 1 M NaCl.
  • a 5-15% acrylamide gradient gel was prepared, and 20 ⁇ l of each fraction was added and the gel run to determine those fractions having the DBP. Those fractions containing the protein were pooled, and the pool dialyzed against PBS overnight at 4°C, changing the buffer at least once.
  • the protein concentration of the dialyzed sample was determined by Lowry assay.
  • the coated microtiter plate was washed 3 times with 200 ⁇ l PBS containing 0.1% BSA, incubating about five min at room temperature on a shaker. DBP was diluted in PBS containing 0.1% BSA. 50 ⁇ l of the appropriate dilution was added to the well, and the plate was incubated for about 45 min at room temperature and on a shaker. The liquid was decanted and drained.
  • B. burgdorferi strain N40 was then added to the wells. Twenty five (25) microliters of bacteria (1 x 10 9 organisms/ml) in PBS containing 0.1% BSA was added to each well and incubated 45 min at room temperature. The solution was decanted and drained, and the wells washed three times with PBS containing 0.1% BSA to remove unattached bacteria.
  • Anti-5. burgdorferi rabbit serum (1 :1000, 100 ⁇ l/well) was added and incubated for 1 hr. (Rabbits were inoculated with 1 x 10 s organisms per ml of washed B. burgdorferi strain B31 , and serum was collected 3 weeks post-infection). After washing 3 times in PBS containing 0.1% BSA, the second antibody, 100 ⁇ l of 1 :1000 goat anti-rabbit alkaline phosphatase conjugate (BioRad, Hercules, CA) diluted in PBS containing 0.1% BSA was added and incubated for 1 hr at room temperature.
  • OspA and OspB Two other borrelial proteins, OspA and OspB, believed to be surface-exposed have been shown to be targets for bacterial killing by specific antibodies in the absence of complement (Sadziene et al , 1993). This prompted the inventors to examine antiserum against DbpA for a similar activity. Accessibility to bacteriostatic or bactericidal antibodies is one measure of a protein's exposure at the surface of a cell.
  • Table 6 shows the growth inhibitory activity of anti-rDbpA serum for diverse borrelia strains.
  • Rabbit antisera were serially diluted in 96-well plates in 0.1 ml BSKII medium, 10 5 borrelia in the mid-log phase of growth in 0.1 ml BSKII medium were added per well, the mixture was incubated for three days and cell viability (motility) was assessed by microscopy.
  • the results shown in Table 6 were those using rabbit serum raised against rDbpA derived from B. burgdorferi sensu stricto strain 297.
  • This serum was strongly inhibitory to growth of twelve of seventeen B. burgdorferi sensu stricto strains tested expressing DbpA (HB-19, CA-3- 87, and FRED express little or no DbpA in vitro), as well as several of the B. garinii and B. afzelii strains. Three B. afzelii strains and one B. garinii strain were slightly inhibited at a 1 :50 serum dilution. Strain 25015 was also inhibited by 1 :10 anti-DBP serum. B. andersonii strain 21038 was strongly sensitive. The strains inhibited by anti-DBP serum fall into at least four OspA serogroups, and have diverse geographic origins.
  • Serum against an irrelevant antigen, OspA was not inhibitory.
  • the 36 5. burgdorferi sensu lato strains were also tested for their sensitivy to growth inhibition by rabbit serum against rOspA from strain B31. Of the 36 strains, 17 were sensitive to anti-OspA serum, and all but three of these were B. burgdorferi sensu stricto isolates.
  • antibodies against DbpA from strain 297 inhibit the growth of more strains than anti-OspA serum, anti-DbpA antibodies inhibit more 5. afzelii and B. garinii strains than OspA.
  • Strains were derived from Lyme patient material or Ixodes sp. ticks. Endpoint titer for GI was for >90% reduction in cell nu 15 and motility; titer ⁇ 100 defined as positive inhibition.
  • CSF cerebrospinal fluid
  • ACA acrodermatitis chronicum atrophicans.
  • DNA fragments encoding either one or more native or genetically-modified dbp genes of B. burgdorferi or dbp-de ⁇ ved nucleic acid segments encoding either DBP epitopes, truncated DBPs or nucleic acid segments encoding mutated DBPs or DBP-derived epitopes are cloned into appropriate expression vectors.
  • Such vectors may contain a multiple restriction enzyme cloning site that situates the nucleic acid segment of interest such that its expression is controlled from an inducible promoter.
  • a particular aspect of the present invention is the production of recombinant proteins in large quantity. Such methods are well-known to those of skill in the art, and have been described in detail hereinabove. In an overall and general sense, the production of a large number of recombinant proteins may be produced in either prokaryotic or eukaryotic cells using various expression systems depending upon the particular construct, and the particular advantages of the various expression systems available for such protein production. Particular aspects of the present invention include the use of the following expression systems:
  • Recombinant proteins so prepared find utility in the present invention in a variety of embodiments, including compositions for immunoassay reagents, antigen preparation for generation of immune responses in susceptible animals, vaccine formulations, and substrates for antibody production for use in passive and active immunization methods.
  • Isopropyl-1-thio- ⁇ -D-galactopyranoside (IPTG; Gibco BRL, Grand Island, NY) (final concentration 0.2 mM) is added to the cells and growth continued for another 2.5 - 5 hr at 37°C.
  • the bacteria are collected by centrifugation and the bacterial pellets are resuspended in phosphate buffered saline (PBS; 10 mM phosphate, 0.14 M NaCl, pH 7.4).
  • the cells are lysed by passage through a French press (SLM Instrument Inc., Urbana, IL) twice at 20,000 lb./in 2 .
  • the bacterial lysate is centrifuged at 102,000 x g for 10 minutes to remove bacterial debris.
  • the supernatant containing the soluble proteins is filtered through a 0.45 ⁇ M membrane (Nalgene, Rochester, NY) and retained for further purification.
  • the present invention also concerns preparation of truncated DBPs expressed in recombinant systems.
  • One such example is the construction of truncated DBPs which still retain the ability to bind Den.
  • a vector was constructed to express proteins as fusions to the leader peptide and posttranslational modification sequence of the major E. coli lipoprotein, Lpp.
  • the Ndel-EcoRl restriction fragment containing the beginning of the translational region expressed from the T7 promoter was replaced with a synthetic sequence expressing the leader peptide of the Lpp protein and a peptide specifying lipoprotein posttranslational modifications generating the new vector pT7Lppl .
  • a fragment of the dbp A gene spanning amino acids Gly26 through Serl 87 was amplified by PCR® with primer-added BamHl and 7/ dIII sites. This fragment encoding a truncated DbpA protein was cloned into pT7Lppl to generate pMSH24.
  • the antibody compositions disclosed herein find particular utility in the passive immunization of animals to prevent bacterial adhesion to the ECM component Den, and related proteoglycans.
  • Native DBPs, truncated DBPs, and site-specifically mutagenized DBPs, in particular, DbpA and DbpB, are contemplated to be particularly useful as antigens to immunize animal donors, and particularly mammalian donors such as humans.
  • the immunoglobulin fraction (Ig) from the blood plasma of the donors can be purified and systemically administered to a target population.
  • Those individuals at high risk for developing borrelia infections include, but are limited to, patients in intensive care units, immunocompromised patients, surgery patients, children, and persons in areas having high incidence of ixodid tick infestations such as the northeastern, midwestern, and western pacific United States.
  • Two particular references which describe those at risk from borrelioses include Steere, 1994 and a report by the Centers for Disease Control, 1994.
  • the anti-DbpA and anti-DbpB Ig produced by immunization with peptides of the present invention find particular utility in several areas:
  • C3H/HeJ mice a widely-used strain susceptible to both infection and disease were challenged with an intradermal inoculum of 10 4 spirochetes and passively immunized just before challenge with rabbit sera against rDbpA, rOspA, or no serum.
  • tissue samples bladedder, heart, synovial fluid
  • evidence of borrelial outgrowth from these tissues were assessed microscopically after 2 and 3 weeks of in vitro culture. Protection was judged to be complete when no spirochetes are seen upon examination of 10-20 high power fields of samples from all three cultures.
  • mice Groups of five mice (BALB/c and C3H/HeJ) were immunized with 20 ⁇ g rDbpA or soluble protein extract of E. coli JMlOl/pBSII, or 5 ⁇ g rOspA lipoprotein in complete Freund's adjuvant (CFA), and boosted with protein in incomplete Freund's adjuvant (IFA) at 4 wk.
  • Groups of five BALB/c mice were also immunized with 20 ⁇ g rDbpB, or 5 ⁇ g rOspA or E. coli extract in CFA and boosted with protein in IFA at 3 weeks. At two week intervals sera were collected and analyzed for reactivity with the immunizing antigen by ELISA. At the time of challenge these serum ELISA titers were in excess of 1 :100,000 indicating all antigen preparations were highly immunogenic by this immunization regimen.
  • rDbpA antigen preparation was able to confer complete protection for all BALB/c mice. Protection was also seen with rOspA, but not with the irrelevant E. coli antigen extract. Interestingly, spirochetes were absent from skin and joint tissue of rDbpA-immunized C3H (H-2 haplotype) and BALB/c (H-2 d haplotype) mice.
  • the rDbpB antigen preparation was able to confer complete protection for three of five BALB/c mice, while the two infected mice had reduced levels of spirochetes in their tissues (FIG. 28A and FIG. 28B).
  • mice Groups of five mice (C3H/HeJ) were immunized. At two week intervals sera were collected and analyzed for reactivity with the immunizing antigen by ELISA. At the time of challenge these serum ELISA titers were in excess of 1 :100,000 indicating all antigen preparations were highly immunogenic by this immunization regimen.
  • the rDbpA antigen preparation was similar to the chimeric lipoprotein DbpA truncate in its ability to confer protection for C3H/HeJ mice. Protection was also seen with rOspA, but not with the irrelevant E. coli antigen extracts. Serum against rDbpA raised in rabbits completely protected C3H mice.
  • OspA-based vaccines the leading candidate antigen for a Lyme disease vaccine
  • antibodies against this protein are effective only if present at high levels prior to infection. This suggests that an infection-induced memory response to OspA will be of little or no benefit.
  • other borrelia surface proteins required for growth and persistence in vivo may not suffer this limitation as vaccine immunogens.
  • Many bacterial pathogens including borrelias initiate infection following adhesion to specific macromolecules of the host target tissue. These adhesins are exposed at the bacterial surface. Adhesins are effective vaccines against human and animal diseases, as demonstrated in several animal models.
  • Several of the recently developed acellular pertussis vaccines include the B.
  • OspA serum was ineffective after day 0. This shows that DbpA, even after a period of four days to grow and adapt to in vivo conditions present in the host remains a target for antibody-mediated elimination. This was not the case for OspA. It is possible this post-infection therapeutic effects can be extended to later in the infection process if multiple doses of antibody, or antibody from the homologous species, are administered to gain more favorable pharmacokinetics. The studies measured only infection rather than disease, however, antibody levels which are not sufficient to eliminate all borrelia may in fact be sufficient to prevent disease pathologies.
  • Oligonucleotides were used as primers for PCRTM amplifications of dbpA gene fragments from borrelia strains representing the three major phylogenetic groups of Lyme disease spirochetes.
  • Primers derived from the dbpA gene of strain 297 are able to function in the PCRTM to amplify candidate dbpA alleles of the expected size from seven of eight strains tested under a limited set of different amplification conditions.
  • Strains having variant dbpA alleles not yet amplified by the conditions described, such as PBi may yield detectable PCRTM products under less stringent annealing conditions, or with other combinations of these primers, or both.
  • nucleotide sequences of these alleles will allow elucidation of the most highly conserved regions which presumably would be common among all genes expressing DbpA. These common sequences would then be used to facilitate design of new primers to allow amplification of these strain variants. Alternately, these PCRTM products could be used to identify DNA fragments containing dbpA strain variants by Southern hybridization, and ultimately derive molecular clones of these genes.
  • Identification of candidate dbpA alleles from B. burgdorferi, B. afzelii, and B. garinii was accomplished using oligonucleotides as primers for PCRTM amplifications of dbpA gene fragments from borrelia strains representing the three major phylogenetic groups of Lyme disease spirochetes. Portions of the PCRTM amplification reactions were electrophoresed on 1 % agarose gels and the approximate sizes of the DNA products were estimated relative to size standards. Where a given primer pair yielded an amplification product of the size expected from the strain 297 sequence this is indicated with a check mark. In some cases additional amplification products were also obtained.
  • Table 9 shows a summary of the heterologous borrelia strain passive protection results discussed in Example 5.10. Data were compiled in tabular form and expressed as % of mice protected by anti-DBP 297 serum and anti-OspA B31 serum. Under these challenge conditions, broader protection was achieved with anti-DBP serum.
  • compositions comprising proteoglycans and/or their derivatives which recognize the native DBPs and synthetically-modified DBP-derived epitopes disclosed herein.
  • Such compositions are useful in the preparation of therapeutic formulations for preventing bacterial adhesion in an animal, and particularly, in humans.
  • Novel ligands mimicking native Den present in the ECM serve as chemopreventatives in bacterial infection by effectively saturating the MSCRAMM components present on the bacterial cell surface.
  • SEQ ID NO:7 DNA sequence of 2.5-kb dbpA/dbpB from B. burgdorferi 297.
  • SEQ ID NO:8 DbpA sequence of 0.6-kb ORF from B. burgdorferi 297.
  • SEQ ID NO:9 DbpA sequence of 0.6-kb ORF from B. burgdorferi 297.
  • SEQ ID NO:10 Nucleotides 1-1400 of 2.5-kb insert DNA of BG26:pB/2.5(5).
  • BG26:pB/2.5(5) BG26:pB/2.5(5).
  • SEQ ID NO: 12 dbp A gene of B. burgdorferi strain B31.
  • SEQ ID NO: 17 DbpA protein from B. burgdorferi strain HB-19.
  • SEQ ID NO: 18 dbpA gene from B. afzelii strain PGau.
  • SEQ ID NO:20 dbpA gene from B. garinii strain IP90.
  • SEQ ID NO:21 DbpA protein from B. garinii strain IP90.
  • SEQ ID NO:22 dbpA gene from B. burgdorferi strain LP4.
  • SEQ ID NO:23 DbpA protein from B. burgdorferi strain LP4.
  • SEQ ID NO:24 dbpA gene from B. burgdorferi strain LP7.
  • SEQ ID NO:25 DbpA protein from B. burgdorferi strain LP7.
  • SEQ ID NO:26 dbpA gene from B. burgdorferi strain JD1.
  • SEQ ID NO:27 DbpA protein from B. burgdorferi strain JD1.
  • SEQ ID NO:28 DbpA protein from B. burgdorferi 291.
  • SEQ ID NO: 30 DbpA protein from B. burgdorferi 297, LP4.
  • SEQ ID NO:32 DbpA protein from B. burgdorferi SH2.
  • SEQ ID NO:33 dbpA gene from B. burgdorferi N40.
  • SEQ ID NO:34 DbpA protein from B. burgdorferi N40.
  • SEQ ID NO:35 dbpA gene from B. burgdorferi JD1.
  • SEQ ID NO:36 DbpA protein from B. burgdorferi JD1.
  • SEQ ID NO:40 DbpA protein from B. burgdorferi B31, BR4, 3028.
  • SEQ ID NO:41 dbpA gene from B. burgdorferi G3940.
  • SEQ ID NO:42 DbpA protein from B. burgdorferi G3940.
  • SEQ ID NO:43 dbpA gene from B. burgdorferi IP90.
  • SEQ ID NO:44 DbpA protein from B. burgdorferi LP4.
  • SEQ ID NO:45 dbpA gene from B. burgdorferi ZS7.
  • SEQ ID NO:46 DbpA protein from B. burgdorferi ZS7.
  • SEQ ID NO:47 dbpA gene from B. afzelii PGau.
  • SEQ ID NO:48 DbpA protein from B. afzelii PGau.
  • SEQ ID NO:50 DbpA protein from B. afzelii B023.
  • SEQ ID NO:52 DbpA protein from B. garinii IP90.
  • SEQ ID NO:53 dbpB gene from B. burgdorferi CA287.
  • SEQ ID NO:54 DbpB protein from B. burgdorferi CA287.
  • SEQ ID NO:55 dbpB gene from B. burgdorferi IPS.
  • SEQ ID NO:56 DbpB protein from B. burgdorferi IPS.
  • SEQ ID NO:57 dbpB gene from B. burgdorferi JD1.
  • SEQ ID NO:58 DbpB protein from B. burgdorferi JD1.
  • SEQ ID NO:60 DbpB protein from B. burgdorferi 297. SH2 and LP4.
  • SEQ ID NO:61 SEQ ID NO:61. dbpB gene from B. burgdorferi N40, LP7, and B. afzelii PKo.
  • SEQ ID NO:62 DbpB from B. burgdorferi N40, LP7, and B. afzelii PKo.
  • SEQ ID NO:63 dbpB gene from B. burgdorferi HB19, G3940, LP5, ZS7,
  • NCH-1 NCH-1 , FRED, and B. garinii 20047.
  • NCH-1 NCH-1 , FRED, and B. garinii 20047.
  • SEQ ID NO:65 Partial dbpB gene from B. garinii IP90.
  • SEQ ID NO:66 Partial DbpB protein from B. garinii IP90.
  • AAAGACAAAA AATAACATCG GACTTTTGAA TGTCTTTAAA CA 2656
  • MOLECULE TYPE protein 10
  • SEQUENCE DESCRIPTION SEQ ID NO: 8:

Abstract

L'invention concerne le gène dbp et des segments d'acide nucléique dérivés de la dbp provenant de Borrelia burgdorferi, agent étiologique de la maladie de Lyme, ainsi que des segments d'ADN codant pour la dbp provenant de borrelias voisines. Elle concerne également des compositions de protéines de liaison de la décorine et leurs modes d'utilisation. Il est proposé d'utiliser la protéine DBP, ainsi que les épitopes antigéniques qui en sont dérivés, pour le traitement des infections pathologiques à Borrelia et notamment pour la prévention de l'adhésion bactérienne à la décorine. Les segments d'ADN codant pour ces protéines et les anticorps anti-(protéine de liaison de la décorine) peuvent également être utilisés pour diverses applications de criblage, de diagnostic et de thérapeutique, parmi lesquelles l'immunisation passive et active, ainsi que des méthodes de prévention de la colonisation par Borrelia chez l'animal. Il est proposé d'utiliser ces segments d'ADN et les peptides qui en sont dérivés pour la préparation de vaccins et comme protéines porteuses dans des formulations de vaccins, ainsi que dans la formulation de compositions destinées à la prévention de la maladie de Lyme.
PCT/US1996/017081 1995-04-24 1996-10-22 Compositions de proteines de liaison de la decorine et modes d'utilisation WO1997027301A1 (fr)

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JP9526823A JP2000503842A (ja) 1996-01-22 1996-10-22 デコリン結合タンパク質組成物および使用方法
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US6248517B1 (en) 1995-04-24 2001-06-19 The Texas A & M University System Decorin binding protein compositions and methods of use
US6312907B1 (en) 1995-04-24 2001-11-06 The Texas A & M University System DbpA compositions and methods of use
US5853987A (en) 1995-04-24 1998-12-29 The Texas A & M University System Decorin binding protein compositions and methods of use
US6214355B1 (en) 1995-04-24 2001-04-10 Texas A & M University System DbpA compositions
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
EP0913474A3 (fr) * 1997-10-28 1999-12-29 Smithkline Beecham Corporation Dbpa, une hélicase de Staphylococcus aureus
WO2000021989A1 (fr) * 1998-10-09 2000-04-20 Medimmune, Inc. Proteines dbp a et b liant la decorine et gene codant pour elles
AU5495500A (en) * 1999-06-16 2001-01-02 The Texas A & M University System Decorin binding protein essential peptides and methods of use
US6517838B1 (en) 2000-06-16 2003-02-11 The Texas A&M University System Decorin binding protein essential peptides and methods of use
US20120142023A1 (en) * 2010-12-02 2012-06-07 Ascoli Carl A Proteins and method for detection of lyme disease
CA2863661C (fr) * 2012-02-01 2021-06-01 Biopeptides Corp Peptides diagnostiques pour la maladie de lyme
WO2015085323A1 (fr) * 2013-12-06 2015-06-11 Biopeptides Corp. Peptides destinés au diagnostic de la maladie de lyme
FI3353551T3 (fi) 2015-09-25 2023-01-31 Koostumuksia ja menetelmiä lymen taudin diagnosoimiseksi ja lymen tautia aiheuttavan spirokeetan eliminaation ennustamiseksi hoidon jälkeen

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* Cited by examiner, † Cited by third party
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