WO1999012960A2 - ANTIGENES P13 PROVENANT DE $i(BORRELIA) - Google Patents

ANTIGENES P13 PROVENANT DE $i(BORRELIA) Download PDF

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WO1999012960A2
WO1999012960A2 PCT/IB1998/001424 IB9801424W WO9912960A2 WO 1999012960 A2 WO1999012960 A2 WO 1999012960A2 IB 9801424 W IB9801424 W IB 9801424W WO 9912960 A2 WO9912960 A2 WO 9912960A2
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borrelia
polypeptide
seq
nucleic acid
protein
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PCT/IB1998/001424
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English (en)
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WO1999012960A3 (fr
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Sven Bergström
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Symbicom Ab
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Priority to AU88811/98A priority Critical patent/AU8881198A/en
Priority to CA002300365A priority patent/CA2300365A1/fr
Priority to EP98940504A priority patent/EP1012269A2/fr
Publication of WO1999012960A2 publication Critical patent/WO1999012960A2/fr
Publication of WO1999012960A3 publication Critical patent/WO1999012960A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to nucleic acid sequences encoding antigenic proteins associated with Borrelia burgdorferi sensu lato (Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii; collectively designated Bb hereinafter), particularly polypeptides associated with virulence; vaccine formulations comprising these poly- peptides are also part of the invention.
  • the invention also relates to methods for pro- ducing Bb immunogenic polypeptides and corresponding antibodies.
  • Other embodiments of the invention relate to methods for detecting Lyme disease and transformed cells comprising Bb associated nucleic acids.
  • Bb is not a homogeneous group but has a variable genetic content, which may in turn affect its virulence, pattern of pathogenesis and immunogenicity.
  • Lyme borreliosis associated borreliae are so far taxonomically placed into three species, Borrelia burgdorferi senso stricto, Borrelia garinii, and Borrelia afzelii (Burgdorfer et al., 1 983, Baranton et al., 1992, Canica et al., 1993).
  • OspA In different animal models efficient protection is achieved by passive and active immunisation with OspA (Schaible et al., 1 990, Fikrig et al., 1992, Erdile et al., 1 993), and therefore, OspA remains one of the main candidates for Borrelia vaccine. It is unclear, however, whether inter- and intra-species heterogeneity of OspA, as well as other competitors for immunoprophylaxis, allow efficient cross-protection (Fikrig et al., 1 992, Norris et al., 1 992). Furthermore, it was recently suggested that certain protective antibodies produced early in the course of Borrelia infection are unrelated to OspA (Norton Hughes et al., 1 993, Barthold and Bockenstedt, 1993).
  • Serological assays for the diagnosis and detection of Lyme disease are thought to offer the best promise for sensitive and specific diagnosis.
  • serologic assays generally use whole Bb as antigen and suffer from a low "signal to noise" ratio, i.e., a low degree of reactivity in positive samples, particularly early in the disease, as compared to negative samples. This problem results in high numbers of false negatives and the potential for false positives. Background reactivity in negative controls may be due in part to conserved antigens such as the 41 K flagellin and the 60K "Common Antigen".
  • These Bb proteins possess a high degree of sequence homology with similar proteins found in other bacteria. Therefore normal individuals will often express anti-flagellar and anti-60K antibodies.
  • Unique, highly reactive Bb antigens for serological assays are therefore desirable but heretofore unavailable.
  • Diagnosis of Lyme disease remains a complex and uncertain endeavour, due to lack of any single diagnostic tool that is both sensitive and specific. Clinical manifestations and history are the most common bases for diagnosis. However, there is a pressing need for specific, sensitive, reproducible and readily available confirmatory tests.
  • Direct detection offers proof of infection but is hampered by the extremely low levels of Bb that are typically present during infection, as well as the inaccessibility of sites that tend to be consistently positive (e.g., heart and bladder). Culture, although sensitive, is cumbersome and requires 1 -3 weeks to obtain a positive result. PCR appears to offer promise in terms of direct detection (Lebech et al., 1 991 ) and indeed Goodman et al.
  • Lyme disease Another problem in detection of Lyme disease is the substantial number of humans exposed to Bb who develop inapparent or asymptomatic infections. This number has been estimated to be as high as 50% (Steere et al., 1 986).
  • Bb-specific antigens e.g., for the development of diagnostic tests for Lyme disease.
  • Adequate assays do not exist and should ideally meet several criteria, including ( 1 ) expression of an antigen by all pathogenic Bb strains, (2) elicitation of an immune response in all Lyme disease patients, (3) high immunogenicity with a detectable antibody response early in the infection stage, (4) antigens unique to Bb without cross-reactivity to other antigens, and (5) distinction between individuals exposed to non-pathogenic as opposed to pathogenic forms of Bb.
  • Sadziene et al. (1994) when analysing an Osp-less B. burgdorferi strain identified a 1 3 kDa surface exposed protein which was designated p1 3.
  • the inventors have surprisingly found that an antigen from Bb with an apparent molecular weight of 1 3 kDa (determined by SDS-PAGE, and subsequent visualization such as staining with Coomassie Blue) is highly conserved in the three strains B. burgdor- feri sensu stricto B31 , B. garinii IP90, and B. afzelii ACAI, whereas this antigen cannot be found in Borrelia species related to relapsing fever and avian borreliosis.
  • the disclosed antigens are therefore excellent candidates for vaccines and diagnostics relating to infections with Bb.
  • the antigens will be termed P1 3.
  • the present invention thus addresses one or more of the foregoing or other problems associated with the preparation and use of Bb specific antigens, particularly those antigens associated with virulence and which are useful for developing detection and diagnostic methods for Lyme disease.
  • the present invention involves the identification of such antigens, which herein are designated P13 as well as the identification and isolation of Bb nucleic acid sequences that encode P1 3 antigens or antigenic polypeptides derived therefrom. These sequences are useful for preparing expression vectors for transforming host cells to produce recombinant antigenic polypeptides. It is further proposed that these antigens will be useful as vaccines or immunodiag- nostic agents for Bb associated diseases such as Lyme disease in particular.
  • the DNA of the present invention was isolated from Bb.
  • the microorganism is a spiral-shaped organism approximately 0.2 micron in diameter and ranging in length from about 10 to 30 microns. Like other spirochaetes, it possesses an inner membrane, a thin peptidoglycan layer, an outer membrane, and periplasmic flagella which lie between the inner and outer membranes.
  • Bb is an obligate parasite found only in association with infected animals and arthropod vectors in endemic areas.
  • Bb-like organisms have also been identified in birds raising the possibility that birds could also serve as an animal reservoir. While some Bb isolates have been cloned, most isolates have not been cloned and most likely represent mixtures of different variants even at the time of culture origin.
  • Bb has similarities with other relapsing fever organisms such as B. hermsii.
  • Bb has a single chromosome with two unusual features, linear conformation and small size (approximately 900 kilobase pairs).
  • Fresh isolates of Bb contain up to four linear plasmids and six circular supercoiled plasmids.
  • the plasmid content of different Bb isolates is highly variable. For example, in one study only two of thirteen strains had similar plasmid profiles. Some plasmids are lost during in vitro passage which may correlate with loss of virulence.
  • Outer surface proteins OspA and OspB are encoded on the 49 kbp linear plasmid.
  • the P1 3 membrane-associated/outer surface proteins discovered by the inventors are encoded on the Bb chromosome.
  • the P13 protein gene being localised to the chromosome of borreliae shows a higher degree of conservation among Lyme disease associated borreliae contrary to the plasmid-encoded major outer surface proteins A, B, and C which exhibit a significant species and strain dependent genetic and antigenic polymorphism (Barbour 1 986, Jonsson et al., 1 992, Wilske et al., 1 993).
  • the level of similarity and identity between the deduced amino acid sequence of the P1 3 protein from different borrelia strains further shows that this protein can be useful as a vaccine against Lyme disease as well as a target for diagnostic use.
  • DNA libraries were prepared by restriction enzyme digestion of DNA prepared from the strains B. burgdorferi B31 , B. afzelii ACAI and B. garinii IP90.
  • Codons for the amino acid sequence obtained, SEQ ID NO: 1 were selected by reverse translation based on ( 1 ) conclusion that codons containing A or T were favoured and (2) knowledge of published DNA sequences for several Bb proteins. A choice favouring A or T containing codons was based on the observation that the G + C content of Bb is only 28-35% (Burman et al., 1 990). Two oligonucleotides were synthesized having the sequences shown in in SEQ ID NO: 2 and SEQ ID NO:3. These were used as primers in a PCR reaction with DNA prepared from B. burgdorferi B31 as template.
  • the amplified fragment was sequenced, SEQ ID NO: 4, and verified to code for the amino acid sequence, SEQ ID NO: 1 .
  • a DNA probe, designated Y7 (SEQ ID NO:7), was designed and used to screen the DNA library prepared from B. burgdorferi B31 in an attempt to identify DNA encoding the P1 3 protein from this Bb species. This attempt proved unsuccessful.
  • the identified sequence of the P1 3 gene from B. burgdorferi B31 was used to design PCR primers which were subsequently used to clone the P1 3 gene from B. afzelii ACAI and B. garinii IP90.
  • the P13 protein which has been cloned by the inventors of the present invention has been shown to have a molecular weight of about 1 9,000 as calculated from the deduced ammo acid sequence of the full-length protein but a molecular weight of about 14,000 as determined by MS but nevertheless to be identical to a protein from Bb which has an apparent molecular weight in SDS-PAGE of 1 3 kDa. This difference can be explained by post-translational modifications of the P1 3 protein. This is in accordance with the observation that it was not possible by standard methods to obtain an N-terminal ammo acid sequence of P1 3 protein prepared from Bb.
  • leader peptide sequences are typical of exported proteins with a basic residue followed by a hydrophobic and a potential leader peptidase I cleavage site according to the criteria established by von Heijne ( 1 986).
  • Ammo acid sequences resembling the signal sequences of bacterial lipoproteins can also be found in the N-terminal region of the deduced ammo acid sequences.
  • the N- terminal methionine is followed by a hydrophobic region and a signal peptidase II recognition sequence.
  • the signal sequences, Leu Ala Thr Phe Cys for B. burgdorferi B31 , Leu Leu Ala Phe Cys for B. afzelii ACAI and Leu Val lie Phe Cys for B.
  • garinii IP90 differed somewhat from the consensus signal peptidase II recognition sequence (Leu Xaa Xaa Cys) found in most bacteria, but resembled the cleavage sequence Leu Ser lie Ser Cys of the outer surface protein D (OspD) of Bb and Leu Met Me Gly Cys of the variable major proteins Vmp7 and Vmp21 of B. hermsii. These surface anti- gens have been shown to be lipoproteins (Norris et al., 1992; Burman et al., 1 990).
  • leader sequence may imply that mature P1 3 proteins are translocated across the cytoplasmic membrane and are anchored to the cytoplasmic membrane and/or outer membranes via fatty acids associated with an N-terminal cysteinyl residue.
  • Lipidated forms of the outer surface protein A (OspA) from Bb have been shown to be more immunogenic that non-iipidated forms of OspA (Erdile et al., 1 993).
  • Antigenicity of the P1 3 protein was verified by immunisation of a rabbit. Antiserum collected from rabbits injected with the P1 3 protein prepared from B. burgdorferi B31 3 was found to recognise the P13 protein of B. burgdorferi B31 , B. afzelii ACAI, and B. garinii IP90. There was no apparent reactivity of the antiserum with B. hermsii, B. crocidurae, B. anserina.
  • the nucleic acid segments of the present invention encode antigenic amino acid sequences associated with Bb. These sequences are important for their ability to selectively hybridise with complementary stretches of Bb gene segments. Varying conditions of hybridization may be desired, depending on the application envisioned and the selectivity of the probe toward the target sequence. Where a high degree of selectivity is desired, one may employ relatively stringent conditions to form the hybrids, such as relatively low salt and/or high temperature conditions. Under these conditions, little mismatch between the probe and template or target strand is tolerated. Less stringent conditions might be employed when, for example, one desires to prepare mutants or to detect mutants when significant divergence exists.
  • nucleic acid segments of the present invention may be used in combination with an appropriate means, such as a label, to determine hybridization with DNA of a pathogenic organism.
  • Typical methods of detection might utilise, for example, radioactive species, enzyme-active or other marker ligands such as avidin/biotin, which are detectable directly or indirectly.
  • an enzyme tag such as alkaline phos- phatase or peroxidase rather than radioactive or other reagents that may have undesirable environmental effects.
  • Enzyme tags for example, often utilise colorimetric indicator substrates that are readily detectable spectrophotometrically, many in the visible wavelength range. Luminescent substrates could also be used for increased sensitivity.
  • Hybridisable DNA segments may include any of a number of segments of the dis- closed DNA. For example, relatively short segments of at least 12 or so base pairs may be employed, or, more preferably when probes are desired, longer segments of at least 20, at least 30, and at least 40 base pairs, depending on the particular applications desired. Shorter segments are preferred as primers in molecular amplification techniques such as PCR, while some of the longer segments are generally preferable for blot hybridizations.
  • sequences disclosed for the DNA segments of the present invention are defined by SEQ ID NO: 1 8, SEQ ID NO: 20 and SEQ ID NO: 22, a certain amount of variation or base substitution would be expected, e.g., as may be found in mutants or strain variants, but which do not significantly affect hybridization characteristics. Such variations, including base modifications occurring naturally or otherwise, are intended to be included within the scope of the present invention.
  • the invention also relates to at least partially purified antigenic Bb proteins or polypeptides which are capable of producing an in vivo immunogenic response when challenged with Bb.
  • These proteins may comprise all or part of the amino acid sequence encoded by the DNA disclosed herein.
  • Particularly preferred antigenic proteins have the amino acid sequence shown in SEQ ID NO: 1 9, SEQ ID NO: 21 and SEQ ID NO: 23.
  • Post-translationally modified forms of these antigenic proteins are also the subject of this invention.
  • These proteins as well as their epitopes will be useful in connection with vaccine development, and as antigen(s) in immunoassays for detection of Bb antibodies in biological fluids such as serum, seminal or vaginal fluids, urine, saliva, body exudates and the like.
  • Expression vectors may be constructed to include any of the DNA segments herein- above disclosed. Such DNA might encode an antigenic protein specific for virulent strains of Bb or even hybridization probes for detecting Bb nucleic acids in samples. Longer or shorter DNA segments could be used, depending on the antigenic protein desired. Epitopic regions of the disclosed proteins of the present invention expressed or encoded by the disclosed DNA could be included as relatively short segments of DNA.
  • a wide variety of expression vectors are possible including, for example, DNA segments encoding reporter gene products useful for identification of heterologous gene products and/or resistance genes such as antibiotic resistance genes which may be useful in identifying transformed cells.
  • Recombinant vectors such as those described are particularly preferred for transforming bacterial host cells. Accordingly, a method is disclosed for preparing transformed bacterial host cells that generally includes the steps of selecting a suitable bacterial host cell, preparing a vector containing a desired DNA segment and transforming the selected bacterial host cell.
  • bacterial host cells include Bb, E. coli, B. subtilis, and the like as well as prokaryotic host cells.
  • Transformed cells may be selected using various techniques, including screening by differential hybridization, differential display techniques, identification of fused reporter gene products, resistance markers, anti-antigen antibodies and the like. After identification of an appropriate clone, it may be selected and cultivated under conditions appropriate to the circumstances, as for example, conditions favouring expression or, when DNA is desired, replication conditions.
  • antigens from segments of a known immunogenic protein or polypeptide.
  • Certain epitopic regions may be used to produce responses similar to those produced by the entire antigenic polypeptide.
  • Potential antigenic or immunogenic regions may be identified by any of a number of approaches, e.g., Jameson- Wolf or Kyte-Doolittle antigenicity analyses or Hopp and Woods ( 1 981 ) hydropho- bicity analysis (see, e.g., Kyte and Doolittle, 1 982, or U.S. Patent No. 4,554, 101 ).
  • Hydrophobicity analysis assigns average hydrophilicity values to each amino acid residue, and from these values average hydrophilicities can be calculated and regions of greatest hydrophilicity determined. Using one or more of these methods, regions of predicted antigenicity may be derived from the amino acid sequence of the disclosed P1 3 polypeptides.
  • Assays using the disclosed P1 3 proteins or antigenic polypeptides thereof are expected to give superior results both in terms of sensitivity and selectivity when compared to assays that use whole Bb or even purified flagella in either an indirect ELISA or an antibody capture ELISA format.
  • Western immuno- blots based on reactions with such antigens have been difficult to interpret due to the presence of antibodies in sera from unexposed individuals. These antibodies cross-react with Bb antigens, most particularly the 41 kDa flagellin and the 60 kDa common antigen protein.
  • assays which use whole organisms or purified flagella tend to contain antigens with epitopes that will cross-react with other bacterial antigens.
  • the N and C terminal regions of the Bb flagellin possess 52-55% sequence identity with the Salmonella typhimu- rium and Bacillus subtilis sequences (Wallich et al., 1 990), exemplifying the highly conserved nature of flagellin structure.
  • the 60 kDa Bb protein is likewise 58% homologous with the E. co/ protein (Shanafelt et al., 1 991 ).
  • Such cross-reactivity is not likely with the disclosed P1 3 antigens, which are apparently unique to Bb. It is further anticipated that recombinantly derived P13 Bb proteins will be particularly preferred for detecting Bb infections.
  • Unexposed individuals should have a low reactivity to one or more epitopes of the P1 3 proteins thereby making it possible to use lower dilutions of serum and increase sensitivity. Using a combination of more than one of these unique antigens may also enhance sensitivity without sacrificing specificity.
  • Yet another aspect of the invention is a method of detecting Bb nucleic acid in a sample.
  • the presence of Bb nucleic acid in the sample may be indicated by the presence of the polypeptide products which it encodes.
  • the method therefore includes detecting the presence of at least a portion of any of the polypeptides herein disclosed. Suitable detection methods include, for example, immunodetection reagents, PCR amplification, and hybridization.
  • Yet another aspect of the invention includes one or more primers capable of priming amplification of the disclosed DNA of SEQ ID NO: 1 8, SEQ ID NO: 20 and SEQ ID NO: 22.
  • primers are readily generated taking into account the base sequence of the DNA segment of SEQ ID NO: 1 8, SEQ ID NO: 20 and SEQ ID NO: 22, the dis- closed DNA, or deriving a base sequence from the amino acid sequence of a purified polypeptide encoded by the DNA.
  • Primers are analogous to hybridization probes, but are generally relatively short DNA segments, usually about 7-20 nucleotides.
  • antibody-based methods are contemplated for development; for example, an indirect ELISA using the P1 3 proteins or other Bb proteins as an antigen.
  • the P1 3 proteins may be produced in large quantities by recombinant DNA vectors already disclosed and purified. Optimum concentration of the antigen could be determined by checker board titration and diagnostic potential of the P1 3 proteins assay examined further by testing serum from mice at different stages of infection and infected with different strains of Bb. These results could indicate the relative time course for serum conversion for each of the assays and would also show whether infection with different strains causes variation in anti-P1 3 protein titers.
  • reactive epitopes of the P1 3 polypeptides are contemplated as useful either as antigens in an ELISA assay or to inhibit the reaction of antibodies toward intact P1 3 proteins bound to a well.
  • Epitopic peptides could be generated by recombinant DNA techniques previously disclosed or by synthesis of peptides from individual amino acids. In either case, reaction with a given peptide would indicate presence of antibodies directed against one or more epitopes. In addition to its diagnostic poten- tial, this method is seen as being particularly effective in characterising monoclonal antibodies against the P1 3 proteins and other virulence associated proteins.
  • the present invention concerns a kit for the detection of Bb antigens, the kit including a protein or peptide which includes an epitope thereof, to- gether with means for detecting a specific immunoreaction between an antibody and its corresponding antigen.
  • suitable means include labels attached directly to the antigen or antibody, a secondary antibody having specificity for human Ig, or protein A or protein G.
  • avidin-biotin mediated Staphylococcus aureus binding could be used.
  • the monoclonal antibody may be biotinylated so as to react with avidin complexed with an enzyme or a fluorescent compound.
  • a particular kit embodiment of the invention concerns detection of antibodies against the described Bb P1 3 antigens, epitopes thereof as represented by portions of the amino acid sequences, or closely related proteins or peptides, such as epitopes asso- ciated with other virulence-associated proteins detected by comparison of low-passage, virulent and high-passage, avirulent strains of Bb.
  • the antigen for the kit(s) consists of the Bb P1 3 proteins or portions thereof produced by a recombinant DNA vector in E. co/i or another bacterial or non-bacterial host. Alternatively, the antigen may be purified directly from Bb or manufactured as a synthetic peptide.
  • Samples for the assays may be body fluids or other tissue samples from humans or animals.
  • the presence of reactive antibodies in the samples may be demonstrated by antibody binding to antigen followed by detection of the antibody-antigen complex by any of a number of methods, including ELISA, TRIFMA (time-resolved immunofluorometric assay), RIA, fluorescence, agglutination or precipitation reactions, nephelometry, or any of these assays using avidin-biotin reactions.
  • the degree of reactivity may be assessed by comparison to control samples, and the degree of reactivity used as a measure of present or past infection with Bb.
  • the assay(s) could also be used to monitor reactivity during the course of Lyme disease, e.g., to determine the efficiency of therapy.
  • the invention contemplates a kit for the detection of Bb nucleic acids in the sample, wherein the kit includes one or more nucleic acid probes specific for the P1 3 genes, together with means for detecting a specific hybridization between such a probe and Bb nucleic acid, such as an associated label.
  • FIG. 1 A, 1 B and 1 C Analysis of Borrelia proteins.
  • FIG. 1A and 2B Demonstration of outer membrane association of the 1 3 kDa protein.
  • A Electron micrographs of immunogold-stained cells from B. burgdorferi B31 .
  • B Electron micrographs of immunogold-stained cells from B. burgdorferi B313.
  • Monoclonal antibody 15G6 was used as the primary antibody.
  • FIG. 3A and 3B Analysis of membrane Fraction B.
  • A Coomassie-blue stained 1 5% SDS-PAGE gel showing protein profiles of Fraction B (BF) prepared from cells from B. burgdorferi B31 , B. afzelii ACA1 and B. garinii IP90.
  • B The corresponding Western Blot probed with the monoclonal antibody 1 5G6. Arrows indicate the position of 13 kDa protein. Mw - molecular weight standard, kD - kilodalton.
  • FIG. 5A and 5B Gene localisation analysis of the P13 gene.
  • A Separation of total DNA prepared from B. burgdorferi B31 , B. burgdorferi B31 3, B. afzelii ACA1 and B. garinii IP90 by pulse-field agarose gel electrophoresis (AGE).
  • B The corresponding Southern blot using an ⁇ - 32 P labelled probe prepared by PCR amplification of a part of the P1 3 gene.
  • Figure 7A and 7B Expression of recombinant P1 3 in E. coli.
  • A Coomassie-blue stained 1 5% SDS-PAGE gel showing protein profiles of whole cell lysates of E. coli transfected with plasmid pLY313F and plasmid pLY313T.
  • B-fract.B31 B-fraction prepared from B. burgdorferi B313. Mw - molecular weight standard, kD - kilodalton.
  • FIG. 8A and 8B Constructs for DNA vaccination.
  • A Schematic representation of the insert of plasmid pLY-H used in DNA vaccination for the expression of recombinant P13.
  • B Schematic representation of the insert of plasmid pLY-HA used in DNA vaccination for the expression of fusion of recombinant P1 3 and recombinant OspA.
  • Figure 9. Electron mass spectrum of purified P1 3 protein.
  • the present invention relates to the utility of Bb associated nucleic acid fragments as diagnostic or preventive tools in Lyme disease as well as for the preparation of P1 3 and useful P1 3 analogues.
  • the present invention therefore relates to an isolated nucleic acid fragment encoding a polypeptide fragment which exhibits a substantial immunological reactivity with a rabbit polyclonal antibody raised against a polypeptide having an apparent molecular weight of 1 3 kDa as determined by SDS-PAGE and subsequent visualization, said polypeptide being derived from Borrelia burgdorferi B313 and consisting of the amino acid sequence of SEQ ID NO: 1 9 or a post-translationally modified form thereof, said rabbit polyclonal antibody exhibiting substantially no immuno- logical reactivity with proteins from at least 95% of spirochaetes randomly selected from the group consisting of Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica.
  • nucleic acid fragment as used herein is meant a fragment of DNA or RNA, but also of PNA (cf. Nielsen et al., 1 991 ), having a length of at least two joined nucleotides.
  • PNA cf. Nielsen et al., 1 991
  • RNA fragment in e.g. a viral vector, the genome of which is natively composed of RNA.
  • PNA fragments may prove useful, as these artificial nucleic acids have been demonstrated to exhibit very dynamic hybridization properties.
  • a substantial immunological reactivity is meant to designate a marked immunological binding between an antibody/antiserum on the one hand, and on the other hand an antigen, under well-defined conditions with respect to physicochemical parameters as well as concentrations of antigens and antibodies.
  • a substantial immunological reactivity should be clearly distinguishable from a non-specific interaction between an antibody/antiserum and an antigen. This distinction can for instance be made by reacting the antibody/antiserum with a known concentration of an antigen which has previously been shown not to react with the antibody/antiserum, and then using this reaction as a negative control.
  • a positive control could suitably be the reaction between the antibody/antiserum and the same concentration of the antigen used for the immunisation resulting in the production of the antibody/antiserum.
  • an antigen resulting in a relative signal of at least 10% (calculated as S m -(S P -S n )-100, where S m is the measured signal, S p the positive control signal, and S n the negative control signal) is regarded as having a substantial immunological reactivity.
  • An antigen exhibiting "substantially no immunological reactivity" is therefore defined as an antigen giving a signal of less than 10%.
  • the concentration of the amino acid sequence/polypeptide in a bacterium where it is “present” is at least 100 times higher than in a bacterium where it is substantially absent.
  • the ratio of the concentrations are at least 1000, and more preferred at least 10,000, 100,000 or even higher. It is especially preferred that no concentration of the amino acid sequence/polypeptide can be observed in the bacterium from where it is substantially absent.
  • the cross-reactivity will be less than 5% (since there is no reactivity with at least 95% of randomly chosen Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica), and according to the invention this cross- reactivity may be even lower, such as at the most 4% and 3%, preferably at the most 2%, such as 1 %. According to the invention the cross-reactivity is most preferred at most 1 /2%, such as 0%.
  • cross-reactivity is herein meant the phenomenon that two species exhibit a common feature which is detected in a reaction.
  • cross-reactivity is used for similar reactions in antigen-antibody interactions as well as in hybridization interactions.
  • the above-cited considerations concerning cross-reactivity of polypeptides apply for ail cross-reactions between on the one hand the polypeptides/DNA fragments of the invention and on the other hand material from Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica; this is also true for the quantitative assessment of whether cross-reactivity is present or not.
  • Nucleic acid fragments of the invention useful as hybridization probes and/or primers are not necessarily those fragments encoding immunologically useful polypeptides. Therefore the invention also relates to nucleic acid fragments which hybridise readily under highly stringent hybridization conditions with a DNA fragment having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 8, SEQ ID NO: 20, and SEQ ID NO: 22, or with a DNA fragment complementary thereto, but exhibit no substantial hybridization when the hybridization conditions are highly stringent with genomic DNA from at least 95% of spirochaetes randomly selected from the group consisting of Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica.
  • highly stringent when used in conjunction with hybridization conditions is as defined in the art, i.e. 5-10°C under the melting point T m , cf. Sambrook et al, 1989, pages 1 1 .45-1 1 .49.
  • nucleic acid fragments of the invention encode a polypeptide fragment comprising an amino acid sequence comprised in a polypeptide, said polypeptide being present in whole cell preparations of Borrelia burgdorferi B31 , Borrelia burgdorferi B31 3, Borrelia garinii IP90, and/or Borrelia afzelii ACAI but being substantially absent from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica.
  • said polypeptide is a protein having an apparent molecular weight of 1 3 kDa
  • the encoded polypeptide fragment comprises at least one epitope (such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 1 2, 1 3, 14, 1 5, 1 6, 1 7, 1 8, 1 9, 20, or at least 25 epitopes) being present in whole cell preparations of Borrelia burgdorferi B31 , Borrelia burgdorferi B313, Borrelia garinii IP90, or Borrelia afzelii ACAI but being substantially absent from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica.
  • This at least one epitope is preferably one from a protein having an apparent molecular weight of 1 3 kDa.
  • epitopes and “epitopic region” is meant the spatial part of an antigen responsible for the specific binding to the antigen-binding part of an antibody. It goes without saying that the identification of epitopes of the disclosed antigens will facilitate the production of polypeptides which exhibit marked antigenicity thus making them interesting with respect to diagnosis of Borreliosis and vaccination against infections with Bb; identification of epitopes has been discussed above.
  • Preferred nucleic acid fragments of the invention are DNA fragments, especially those which have nucleotide sequences with a sequence identity of at least 70% with SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 or with subsequences thereof of at least 12 nucleotides.
  • the degree of sequence identity may be even higher such as at least 75%, 80%, 85%, 87%, and 89%. It is preferred that the degree of sequence identity is at least 90%, such as 92%, 94% or 95%, and especially preferred are DNA fragments with a sequence identity of at least 96% with SEQ ID NO: 1 8, SEQ ID NO: 20, or SEQ ID NO: 22. Especially for high accuracy hybridization assays, a total sequence identity is necessary, and therefore preferred.
  • Other preferred nucleotide acid fragments of the invention are those which encode a polypeptide of the invention (cf.
  • sequence identity of the encoded polypeptide fragment is preferably higher, such as at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, and at least 96%.
  • identity is, with respect to nucleotide fragments such as DNA fragments, intended to indicate the identity between the nucleotides in question between which the identity is to be established, in the match with respect to nucleotide composition and position in the DNA fragments.
  • polypeptides and fragments thereof described herein the term means an identity between the amino acids in question between which the homology is to be established, in the match with respect to amino acid composition and their position in the polypeptides.
  • sequence identity thus indicates a quantitative measure of the degree of homology between two amino acid sequences or between two nucleotide sequences of equal length:
  • sequence identity can be calculated as Nref 1 , wherein N dit is the total number of non-identical residues in the two sequences when aligned and wherein N r ⁇ f is the number of residues in one of the sequences.
  • nucleic acid fragments of the invention are those comprising a sequence encoding a polypeptide which comprises at least one amino acid sequence selected from the group consisting of amino acid residues 1 9-27, 33-36, 41 -47, 95-104, 1 38-147 and 174-1 79 in SEQ ID NO: 1 9; amino acid residues 1 9-26, 32-35, 40-47, 94-101 , 1 37-146, and 174-1 78 in SEQ ID NO: 21 ; and amino acid residues 18-26, 30-33, 39-46, 91 -104, 137-145 and 173- 1 77 in SEQ ID NO: 23, all of which have been identified as putative epitopic regions.
  • nucleic acid fragments are those which encode mature P1 3, i.e. nucleic acid fragments which encode a protein having an apparent molecular weight of 1 3 kDa which is present in whole cell preparations of Borrelia burgdorferi B31 , Borrelia burgdorferi B31 3, Borrelia garinii IP90, or Borrelia afzelii ACAI but which is substantially absent from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica.
  • this encoded protein is present in fraction B from Borrelia burgdorferi B31 , Borrelia burgdorferi B313, Borrelia garinii IP90, or Borrelia afzelii ACAI.
  • P1 3 is a surface exposed polypeptide in Bb, and therefore preferred nucleic acid fragments are those which encode a surface exposed protein of Borrelia burgdorferi B31 , Borrelia burgdorferi B31 3, Borrelia garinii IP90, or Borrelia afzelii AC Al.
  • nucleic acid fragments of the invention comprise a nucleotide sequence encoding a polypeptide fragment which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 9, 21 , and 23, and of these, those which comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 8, 20, and 22 are especially preferred.
  • Most preferred nucleic acid fragments of the invention are those which consist of a nucleotide sequence encoding a polypeptide fragment consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 9, 21 , and 23, and the three most preferred are those which consist of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 8, 20, and 22. 5
  • Analogues of the disclosed nucleic acid fragments which also form part of the invention are nucleic acid fragments which are fused to at least one other nucleic acid fragment which encodes a fusion partner.
  • This fusion partner can be a protein enhancing the immunogenicity of the fused protein relative to a protein without the 0 encoded fusion partner.
  • encoded proteins may e.g. be lipoproteins, e.g. the outer membrane lipoprotein from E. coli and OspA from Borrelia burgdorferi sensu lato; viral proteins, e.g. from Hepatitis B surface antigen, Hepatitis B core antigen, and the influenza virus non-structural protein NS1 (cf.
  • EP-A-0 366 238 and WO 88/01875 immunoglobulin binding proteins, e.g. protein A, protein G, and the syn- 5 thetic ZZ-peptide (cf. EP-A-0 243 333 and US 5,41 1 ,732); T-cell epitopes; or B-cell epitopes.
  • nucleic acid fragments to form part of a nucleic acid fragment of the invention encoding a fusion polypeptide are those encoding polypeptides which facilitate 0 expression and/or purification of the fused peptide.
  • Such encoded polypeptides could, according to the invention, be bacterial fimbrial proteins, e.g. the pilus components piiin and papA; protein A; the ZZ-peptide; the maltose binding protein; glutathione S- transferase; ⁇ -galactosidase; calmodulin binding protein; or poly-histidine.
  • nucleic acid fragments to form part of a nucleic acid fragment of the invention encoding a fusion polypeptide are those encoding polypeptides which facilitate modulation in drug resistance of the host thereby facilitating selection of the host harbouring the plasmid.
  • Such hosts could, according to the invention, be a bacterium, a yeast, a protozoan, or a cell derived from a multicellular organism selected from a
  • fungus an insect cell, a plant cell, and a mammalian cell.
  • fusion partners derived from P13 are interesting and these could be those which have the same amino acid sequence as at least one amino acid sequence selected from the group consisting of amino acid residues 19-27, 33-36, 41 -47, 95- 35 104, 138-147 and 1 74-179 in SEQ ID NO: 19; amino acid residues 1 9-26, 32-35, 40-47, 94-101 , 1 37-146, and 1 74-1 78 in SEQ ID NO: 21 ; and amino acid residues 1 8-26, 30-33, 39-46, 91 -104, 1 37-145 and 173-177 in SEQ ID NO: 23
  • various analogues and subsequences of the nucleic acids disclosed in the sequence listing herein are interesting aspects of the invention, as are nucleic acid fragments encoding fused polypeptides including polypeptides encoded by nucleic acid fragments of the invention.
  • nucleic acid fragments of the invention is intended to indicate a nucleotide sequence which encodes a polypeptide identical or substantially identical to a polypeptide encoded by a nucleic acid fragment of the invention (SEQ ID NOs: 18, 20, and 22).
  • nucleic acid fragment of the invention may be exchanged by others which, when expressed, result in a polypeptide identical or substantially identical to the polypeptide encoded by the nucleic acid fragment in question.
  • analogue is used in the present context to indicate a nucleic acid fragment or a nucleic acid sequence of a similar nucleotide composition or sequence as the nucleic acid sequence encoding the amino acid sequence having the immunological properties discussed above, allowing for minor variations which do not have an adverse effect on the biological function and/or immunogenicity as compared to the disclosed polypeptides, or which give interesting and useful novel binding properties or biological functions and immunogenicities etc. of the analogue.
  • the analogous nucleic acid fragment or nucleic acid sequence may be derived from an animal or a human or may be partially or completely of synthetic origin as described herein.
  • the analogue may also be derived through the use of recombinant nucleic acid techniques.
  • analogue and “subsequence” are intended to allow for variations in the sequence such as substitution, insertion (including introns), addition, deletion and rearrangement of one or more nucleotides, which variations do not have any substantial effect on the polypeptide encoded by a nucleic acid fragment or a subsequence thereof.
  • substitution is intended to mean the replacement of one or more nucleotides in the full nucleotide sequence with one or more different nucleotides
  • addition is understood to mean the addition of one or more nucleotides at either end of the full nucleotide sequence
  • insertion is intended to mean the introduction of one or more nucleotides within the full nucleotide sequence
  • deletion is intended to indicate that one or more nucleotides have been deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it
  • “rearrangement” is intended to mean that two or more nucleotide residues have been exchanged with each other.
  • the present invention relates to the utility of Bb associated antigenic proteins as diagnostic or preventive tools in Lyme disease. Proteins have been indentified as associated only (or predominantly) with virulent isolates of Bb, providing a basis for several types of diagnostic tests for Lyme disease, including immunodiagnostic and nucleic acid identification, such as those based on amplification procedures. All these embodiments rely on the availability of the P1 3 proteins and their analogues.
  • Another part of the invention therefore pertains to a polypeptide fragment which exhibits a substantial immunological reactivity with a polyclonal rabbit antibody raised against a polypeptide having an apparent molecular weight of 1 3 kDa as determined by SDS PAGE, followed by visualization, and being derived from Borrelia burgdorferi B31 3, said polypeptide comprising the amino acid sequence 1 -1 67 of SEQ ID NO: 1 9, said polyclonal rabbit antibody exhibiting substantially no immunological reactivity with whole cell preparations from at least 95% of randomly selected B. hermsii, B. crocidurae, B. anserina, or B.
  • polypeptide is essentially free from other Borrelia-de ved antigens when it is identical in amino acid sequence to a 1 3 kDa surface exposed polypeptide which can be extracted from Borrelia burgdorferi sensu la to.
  • the 1 3 kDa polypeptide has previously been identified in SDS gels. However, the 13 kDa polypeptide has never been purified to homogeneity, let alone been cloned and sequenced.
  • the present invention is therefore the first to provide the 1 3 kDa polypeptide in a form which is totally free from contaminating and potentially harmful (for e.g. vaccine purposes) Borrelia antigens, i.a. the 1 3 kDa polypeptide in a substantially pure form.
  • the present invention is also the first to provide useful variants of the 1 3 kDa polypeptide (such variants including subsequences of the polypeptide as well as analogues wherein changes have been made to the native amino acid sequence).
  • polypeptide fragment of the invention is otherwise precisely as described above when discussing the nucleic acid fragments of the invention and all discussions pertaining to polypeptide fragments encoded by the nucleic acid fragments of the invention apply mutatis mutandis to the polypeptide fragments of the invention. Hence, all considerations regarding the presence or absence of the polypeptide fragments and their epitopes in various borrelial species as well as other considerations, apply to the polypeptide fragments of the invention.
  • analogues of the P1 3 polypeptides of the invention are embraced by the present invention.
  • analogue and “subsequence” in connection with polypeptides is meant any polypeptide having the same immuno- logical characteristics as the polypeptides of the invention described above with respect to the ability to confer an equivalent and increased resistance to infections with Borrelia burgdorferi sensu lato through immune responses against P1 3.
  • a polypeptide from different sources such as other bacteria or even from eukaryotic cells.
  • analogue and “subsequence” with regard to a polypeptide of the invention are also used in the present context to indicate a protein or polypeptide of a similar amino acid composition or sequence as the characteristic amino acid sequences shown in SEQ ID NOs: 19, 21 and 23, allowing for minor variations which do not have an adverse effect on the ligand binding properties and/or biological function and/or immunogenicity, or which may give interesting and useful novel binding properties or biological functions and immunogenicities etc.
  • the analogous polypeptide or protein may be derived from other microorganisms, cells, or animals and the analogue may also be derived through the use of recombinant DNA techniques as described herein.
  • the invention also comprises polypeptides which can be the product of post-translational modifications of the polypeptides of the invention described above.
  • post-translational modification with regard to a polypeptide of the invention is meant any modification or processing of the full-length polypeptide that can occur during the production of the peptides in Bb, or in the case of recombinant polypeptides in the production of the polypeptides in a host cell.
  • modifications include, but are not limited to, the processing by various peptidases, such as signal pepti- dases, and modifications such as glycosylations, phosphorylations, acetylations, formylations, acylations, palmitylations, sulphations and lipidations.
  • the term "immunologically equivalent” means that the analogue or subsequence of the polypeptide is functionally equivalent to the polypeptide with respect to the ability of evoking a protective immune response against Borrelia burgdorferi sensu lato infections.
  • fusion polypeptides as described above are part of the invention and this is also true for all considerations relating to fusion partners etc. which are discussed above when dealing with the nucleic acid fragments of the invention.
  • the invention also allows for the preparation of P1 3 and variants thereof by means of genetic engineering.
  • Useful tools in this connection are cloning and expression vectors and therefore another important part of the invention is a non-borreliai vector carrying the nucleic acid fragment according to the invention and described in detail above.
  • a vector of the invention is preferably capable of autonomous replication.
  • Preferred vectors are selected from the group consisting of a plasmid, a phage, a cosmid, a mini-chromosome, and a virus.
  • vectors which, when introduced in a host cell, are integrated in the host cell genome are especially preferred due to the increased stability of the obtained transformed cells.
  • a preferred vector of the invention comprises, in the 5' ⁇ 3' direction and in operable linkage, a promoter for driving expression of the nucleic acid fragment of the invention, a nucleic acid sequence encoding a leader peptide enabling secretion of or integration into the membrane of the polypeptide, the nucleic acid fragment according to the invention, and a nucleic acid sequence encoding a terminator.
  • the promoter drives expression in a euka- ryotic cell and also that the leader peptide enables secretion from or integration into the membrane of a mammalian cell.
  • a stable cell line is preferred and therefore the invention also relates to a stable cell line producing the polypeptide of the invention, which carries a vector of the invention, and which expresses the nucleic acid fragment of the invention.
  • prokaryotes are preferred for the initial cloning of DNA sequences and constructing the vectors useful in the invention.
  • prokaryotes are preferred for the initial cloning of DNA sequences and constructing the vectors useful in the invention.
  • strains such as E. coli K1 2 strain 294 (ATCC No. 31446), E. coli B, and E. coli X 1 776 (ATCC No. 31 537).
  • E. coli K1 2 strain 294 ATCC No. 31446
  • E. coli B E. coli B
  • E. coli X 1 776 ATCC No. 31 537
  • 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 ⁇ s typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., 1977).
  • the pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid or another microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.
  • the promoters most commonly used in recombinant DNA construction include the ⁇ - lactamase (penicillinase) and lactose promoter systems (Chang et al., 1 978; Itakura et al., 1 977; Goeddel et al., 1 979) and a tryptophan (trp) promoter system (EP-B- 0 036 776). While these are the most commonly used, other microbial promoters have been discovered and utilised, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plas- mid vectors (Siebeniist et al., 1 980). Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, thus avoiding the need for addition of another promoter by artificial means.
  • eukaryotic microbes such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • the plasmid YRp7 for example, is commonly used (Stinchcomb et al., 1 979; Kingsman et al., 1979; Tschumper et al., 1 980).
  • 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, 1 977).
  • the use of the trpl lesion as a characteristic of the yeast host cell
  • 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 tran- scriptional terminator sequences.
  • An 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 viruses (e.g., Polyoma, Adeno, VSV, BPV) 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 viruses (e.g., Polyoma, Adeno, VSV, BPV) 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.
  • post-translational modifications preferably involve iipidation or glycosylation when these modifications have not been accomplished by means of the preparative procedure itself. Applicable methods for accomplishing Iipidation and/or glycosylation are well-known to the skilled person.
  • Other post-translational modifications include cleavage or elongation of the obtained product.
  • the host cell or cell line also processes the translation product so as to obtain a processed polypeptide.
  • the present invention has addressed the cloning of nucleic acids encoding certain antigenic polypeptides related to the P1 3 proteins.
  • Site-specific mutagenesis allows the production of mutants through the use of specific o gonucleotide 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 1 7 to 25 nucleotides in length is preferred, with about 5 to 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 publications (Adelman et al., 1 983).
  • 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 M 1 3 phage (Messing et al , 1 981 ). These phages are readily commercially available and their use is generally well known to those skilled in the art.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector which includes within its sequence a DNA sequence which encodes the P1 3 antigens.
  • An ohgonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Crea et al. (1 978).
  • This primer is then annealed with the single stranded vector, and subjected to DNA polymerising enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA polymerising enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • 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 P13 genes using site-directed mutagenesis is provided as a means of producing potentially useful species of the P1 3 genes and is not meant to be limiting as there are other ways in which sequence variants of the P1 3 genes may be obtained.
  • recombinant vectors comprising the desired P1 3 genes may be treated with mutagenic agents to obtain se- quence variants (see, e.g., a method described by Eichenlaub, 1979) for the mutagenesis of plasmid DNA using hydroxylamine.
  • another embodiment of the invention is a nucleic acid fragment substantially identical to a nucleic acid fragment of the invention which can be provided e.g. by dual hybridisation.
  • This method employs a nucleic acid fragment which specifically hybridizes under stringency hybridization conditions to a complementary nucleic acid fragment which in turn specifically hybridizes under stringency hybridization conditions to a third nucleic acid fragment encoding a polypeptide comprising the amino acid sequences of the invention.
  • This third nucleic acid fragment will thus be substantially identical to initial nucleic acid fragment.
  • a vaccine of the invention comprises an amount of a polypeptide of the invention or produced according to the invention, said amount being effective to confer substantially increased resistance to infections with Borrelia burgdorferi sensu lato in an animal, including a human being, the polypeptide being formulated in combination with a pharmaceutically acceptable carrier, diluent or vehicle and the vaccine optionally further comprising an adjuvant.
  • the vaccine is generally used in a method of immunizing an animal, including a human being, against infections with Borrelia burgdorferi sensu lato, the method comprising administering an immunogenically effective amount of the vaccine to the animal.
  • a pharmaceutically acceptable carrier vehicle, or diluent, and optionally
  • 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.
  • Suitable carriers are selected from the group consisting of a polymer to which the polypeptide(s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the polypeptide(s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin.
  • Suitable vehicles are selected from the group consisting of a diluent and a suspending agent.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional 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, phar- maceutical 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 acid addition salts (formed with the free amino groups of the peptide) 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 may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyiamine, 2- ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such an 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 synthesise antibodies, and the degree of protection desired.
  • Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination.
  • suitable dosages can be in the range of 1 -1000 ⁇ g, such as between 2 and 750 ⁇ g, between 5 and 500 ⁇ g, between 7.5 and 250 ⁇ g, between 10 and 1 50 ⁇ g, between 10 and 100 ⁇ g, between 10 and 75 ⁇ g, and between 10 and 50 ⁇ g.
  • 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 10 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 include use of agents such as aluminium 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 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70°C and 101 °C for
  • the adjuvant is preferably selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), Quil A, poly l:C, Freund's incomplete adjuvant, IFN- ⁇ , IL-2, IL-1 2, monophosphoryl lipid A (MPL), and muramyl dipeptide (MDP).
  • DDA dimethyldioctadecylammonium bromide
  • Quil A Quil A
  • poly l:C poly l:C
  • Freund's incomplete adjuvant IFN- ⁇
  • IL-2 interleukin-1 2
  • MPL monophosphoryl lipid A
  • MDP muramyl dipeptide
  • 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,
  • the course of the immunisation may be followed by assays for antibodies for the supernatant antigens.
  • the assays may be performed by labelling 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, which are illustrative of these types of assays.
  • polypeptide of the invention can also be part of a multi-component or combina- tion vaccine, which is also an important part of the invention.
  • a vaccine contains
  • an amount of the polypeptide fragment of the invention or of a polypeptide fragment prepared according to the invention the amount of the polypeptide fragment being effective to confer substantially increased resistance to infections with Borrelia burgdorferi sensu lato in an animal, including a human being;
  • polypeptide fragment and the antigen being formulated in combination with a pharmaceutically acceptable carrier, vehicle, or diluent and the vaccine optionally further comprising an adjuvant. All components of such a vaccine apart from the at least one further Borrelia antigen are as described in detail herein.
  • the at least one further Borrelia antigen it is preferred that it is selected from the group consisting of OspA, OspB, OspC, OspD, OspE, OspF, OspG, PC, Oms28, Oms45, Oms 66, decorin binding protein (dbp), LpLA7, EppA, T5, S1 , 26 kDa, 39 kDa, 66 kDa, 79 kDa, 85 kDa, and 1 1 0 kDa antigen.
  • dbp decorin binding protein
  • a combination vaccine of the invention comprises at least two non-identical polypeptide fragments of the present invention or at least two non- identical polypeptide fragments prepared by the method of the invention, the vaccine comprising an amount of the polypeptides effective to confer substantially increased resistance to infections with Borrelia burgdorferi sensu lato in an animal, including a human being, in combination with a pharmaceutically acceptable carrier, vehicle, or diluent, the vaccine optionally further comprising an adjuvant. Also in this case, all components of the vaccine have been described elsewhere herein.
  • the invention also contemplates the use of disclosed nucleic acid segments in the construction of expression vectors or plasmids and use in host cells with a view to vaccination of the individual housing the host cells.
  • the invention also pertains to a vaccine comprising a nucleic acid fragment or a vector of the invention, the vaccine effecting in vivo expression of antigens by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigens being effective to confer substantially increased resistance to infections with Borrelia burg- dorferi sensu lato in an animal, including a human being.
  • the related vaccination method consists of administering an amount of this vaccine which is effective to confer an increased resistance to such infections upon the mammal to which it has been administered.
  • the present invention also provides for a DNA-based vaccine or immunological composition against Lyme disease (e.g., Borrelia burgdorferi, afzelii, or garinii) which can elicit an immunological response, which can confer protection, even up to 100%, in mice against challenge with an infectious strain of Borrelia burgdorferi.
  • Lyme disease e.g., Borrelia burgdorferi, afzelii, or garinii
  • An exemplary plasmid of the invention contains the human cytomegalovirus immediate early promoter driving expression of the P1 3 protein. To facilitate expression in eukaryotic cells, the natural leader sequence of the gene encoding P13 has been replaced with the human tissue plasminogen activator leader sequence.
  • mice by injecting, intramuscularly, naked plasmid DNA and subsequently challenging with Bb spirochaetes. Following vaccination sera will contain high titers of antibody to P13 which will inhibit spirochaete growth in vitro.
  • a DNA vaccine or immunological composition expressing a P13 antigen, from Borrelia burgdorferi, Borrelia afzelii or Borrelia garinii or any combination thereof, can protect mice against infection by a Borrelia genospecies, the etiologic agent of Lyme disease.
  • the composition is thus useful for eliciting a protective response in a host susceptible to Lyme Disease, as well as for eliciting antigens and antibodies, which are also useful in and of themselves.
  • mice From present dog and human trials based on efficiency studies with mice (Erdile et al., 1 993; USSN 08/373,455), it is clear that mice are now a suitable animal model with respect to Borrelia and Lyme disease for extrapolation to domestic animals, humans, and other animals susceptible to Lyme disease or Borrelia infection (e.g., wild animals such as deer).
  • the DNA encoding P1 3 or an immunologically active fragment thereof can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts taking into consideration such factors as the age, sex, weight, species and condition of the particular patient, and the route of administration.
  • DNA encoding P13 or an immunologically active fragment thereof can be administered alone, or can be co-administered or sequentially administered with other Bb antigens, or with DNA encoding other Bb antigens; and the DNA encoding P1 3 or an immunologically active fragment thereof can be sequentially administered, e.g., each spring as the "Lyme Disease season" is about to begin.
  • the invention also pertains to plasmids comprising DNA including P1 3 encoding DNA for expression by eukaryotic cells.
  • the DNA from upstream to downstream (5' to 3'), can comprise: DNA encoding a promoter for driving expression in eukaryotic cells, DNA encoding a leader peptide which enables secretion of a prokaryotic protein sequence from a mammalian cell, DNA encoding a P1 3 antigen (or antigens) or an immunologically active fragment thereof, DNA encoding other Bb antigens such as OspA, OspB, OspC or OspD or an immunologically active fragment thereof, and DNA encoding a terminator.
  • the promoter can be a eukaryotic viral promoter such as a herpes vviirus promoter, e.g., human cytomegalovirus promoter DNA.
  • a herpes vviirus promoter e.g., human cytomegalovirus promoter DNA.
  • the DNA encoding a leader peptide which enables secretion of a prokaryotic protein sequence from a mammalian cell is any DNA encoding any suitable leader for this purpose such as DNA encoding a eukaryotic, preferably mammalian, leader sequence; for instance, DNA encoding a leader peptide of a peptide hormone, or, for example, of insulin, renin, Factor VIII, TPA, and the like, with DNA encoding human tissue plasminogen activator (TPA) leader peptide being presently preferred.
  • TPA tissue plasminogen activator
  • the transcriptional terminator sequence can be any suitable terminator, such as a eukaryotic terminator, for instance, DNA encoding a terminator for a mammalian peptide, with the BGH terminator being presently preferred.
  • a eukaryotic terminator for instance, DNA encoding a terminator for a mammalian peptide, with the BGH terminator being presently preferred.
  • the plasmid can be in admixture with any suitable carrier, diluent or excipient such as sterile water, physiological saline, and the like.
  • a suitable carrier diluent or excipient
  • the carrier, diluent or excipient should not disrupt or damage the plasmid DNA.
  • the plasmid can be administered in any suitable manner.
  • the plasmid can be in a composition suitable for the manner of administration.
  • the compositions can include: liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric administration and the like, such as solutions, suspensions, syrups, elixirs; and liquid preparations for parenteral, subcutaneous, intradermal, intramuscular, intravenous administration, and the like, such as sterile solutions, suspensions or emulsions, e.g., for administration by injection. Intramuscular administration and compositions therefor are presently preferred.
  • the plasmids of the invention can be used for in vitro expression of antigens by eukaryotic cells. Recovery of such antigens can be by any suitable techniques; for instance, techniques analogous to the recovery techniques employed in the documents cited herein (such as the applications cited under Related Applications and the documents cited therein).
  • the thus expressed antigens can be used in immunological, antigenic or vaccine compositions, with or without an immunogenicity-enhancing adjuvant ("expressed antigen compositions").
  • Such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts taking into consideration such factors as age, sex, weight, species, condition of the particular patient, and the route of administration.
  • These compositions can be administered alone or with other compositions, and can be sequentially administered, e.g., each spring as the "Lyme Disease season" is about to begin.
  • the expressed antigen compositions can be solutions, suspensions, emulsions, syrups, elixirs, capsules (including "gelcaps" - a gelatin capsule containing a liquid antigen or fragment thereof - preparations), tablets, hard-candy-like preparations, and the like.
  • the expressed antigen compositions may contain a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • the compositions can also be lyophiiised.
  • compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavouring agents, colours, and the like, depending upon the route of administration and the preparation desired.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavouring agents, colours, and the like, depending upon the route of administration and the preparation desired.
  • Standard texts such as Remington: The Science and Practice of Pharmacy, 19 th ed., Gennaro, AR, 1995, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
  • Suitable dosages for plasmid compositions and for expressed antigen compositions can also be based upon the examples below, and upon the documents cited herein.
  • suitable dosages can be in the range of 1 -1000 ⁇ g, such as between 2 and 750 ⁇ g, between 5 and 500 ⁇ g, between 7.5 and 250 ⁇ g, between 10 and 1 50 ⁇ g, between 10 and 100 ⁇ g, between 10 and 75 ⁇ g, and between 10 and 50 ⁇ g, in expressed antigen compositions.
  • the dosage should be a sufficient amount of plasmid to elicit a response analogous to the expressed antigen compositions; or expression analogous to dosages in expressed antigen compositions.
  • suitable quantities of plasmid DNA in plasmid compositions can be 0.1 to 2 mg, preferably 1 -10 ⁇ g.
  • the invention further provides a method comprising administering a composition containing plasmid DNA including DNA encoding a P1 3 antigen or antigens, for expression of the antigen or antigens in vivo for eliciting an immunological, antigenic or vaccine (protective) response by a eukaryotic cell; or for ex vivo or in vitro expression (that is, the cell can be a cell of a host susceptible to Lyme Disease, i.e., the administration can be to a host susceptible to Lyme Disease such as a mammal, e.g., a human; or the cell can be an ex vivo or in vitro cell).
  • the invention further provides a composition containing a P13 antigen or antigens from expression of the plasmid DNA by a eukaryotic cell, in vitro or ex vivo, and methods for administering such compositions to a host mammal susceptible to Lyme disease to elicit a response.
  • the inventive methods can be used for merely stimulating an immune response (as opposed to also being a protective response) because the resulting antibodies (without protection) are nonetheless useful.
  • monoclonal antibodies can be prepared and the monoclonal antibodies can be em- ployed in well known antibody binding assays, diagnostic kits or tests to determine the presence or absence of a P1 3 antigen or to determine whether an immune response to the bacteria has simply been stimulated.
  • the monoclonal antibodies can also be employed in recovery or testing procedures, for instance, in immunoadsorp- tion chromatography to recover or isolate a P13 antigen.
  • the DNA therein is preferably ligated together to form a plasmid.
  • the promoter, leader sequence, antigen and terminator DNA is preferably isolated, purified and ligated together in a 5' to 3' upstream to downstream orientation.
  • a three-way ligation as exemplified below, is presently preferred.
  • Also contemplated within the scope of the present invention is the use of the disclosed DNA as a hybridization probe. While particular examples are provided to illustrate such use, the following provides a general background for hybridization applications taking advantage of the disclosed nucleic acid sequences of the invention.
  • the DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridise to Bb gene sequences.
  • nucleic acid probes of an appropriate length are prepared based on a consideration of the sequence, e.g., SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22 or derived from flanking regions of these genes.
  • the ability of such nucleic acid probes to speci- fically hybridise to the Bb gene sequences lend them particular utility in a variety of embodiments.
  • the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample. However, either uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructs.
  • the preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 10 to 40, or so, nucleotide stretch of the selected sequence, such as that shown in SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22.
  • a size of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over stretches more than 10 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.
  • nucleotide sequences of the invention are important for their ability to selectively form duplex molecules with complementary stretches of Bb gene segments.
  • relatively stringent conditions for applications requiring a high degree of selectivity, one will typically desire to employ relatively low salt and/or high temperature conditions, such as provided by 0.02M-0.1 5M NaCI at temperatures of 50°C to 70°C. These conditions are particularly selective and tolerate little, if any, mismatch between the probe and the template or target strand.
  • nucleic acid sequences of the present invention are used in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • an enzyme tag such as alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents.
  • colorimetric indicator substrates are known which are employed to provide a means visible to the human eye or spectrophotometrically to identify specific hybridization with pathogenic nucleic acid-containing samples. Luminescent substrates, which give off light upon enzymatic degradation, could also be employed and may provide increased sensitivity.
  • 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) from suspected clinical samples such as exudates, body fluids (e.g., amniotic fluid, cere- brospinal fluid) or even tissues, 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.). Following washing of the hybridised surface so as to remove non-specifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
  • synthetic single stranded nucleotides can be pro- prised (by a series of photolitograpic and chemical steps) on a solid phase based on nucleic acid sequences or the complementary sequence of the invention and sequences comprised thereof.
  • Single-stranded nucleic acid fragments from suspected clinical samples such as exudates, body fluids (e.g., amniotic fluid, cerebrospinal fluid) or even tissues are then subjected to specific hybridisation under desired conditions. These single-stranded nucleic acid fragments are labeled for detection prior to hybridisation.
  • the selected conditions for hybridisation 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 quantified, by means of the label.
  • the invention discloses a DNA segment encoding an antigenic Bb protein. Detection of that DNA or various parts thereof is expected to provide the basis for a useful amplification assay.
  • One method of detecting the P13 antigen genes is based on selective amplification of known portions of the gene.
  • a particular method utilises PCR amplification, using any one of a number of primers that could be prepared from knowledge of the nucleic acid sequence of SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22.
  • primers are relatively short, e.g., 7-28 base pairs in length, and may be derived from the respective sense or anti-sense strands of the disclosed DNA segment. Synthesis of these primers may utilise standard phosphor- amidite chemistry (Beaucage et al., 1981 ).
  • this part of the invention relates to a diagnostic composition adapted for the determination of Borrelia burgdorferi sensu lato in a sample, the composition comprising an amount of the nucleic acid fragment of the invention which is effective to detectably bind to a nucleic acid fragment from Borrelia burgdorferi sensu lato present in the sample, the composition optionally comprising a detectable label.
  • another embodiment of the invention is a method of determining the presence of Borrelia burgdorferi sensu lato nucleic acids in a sample, comprising incubating the sample with the nucleic acid fragment of the invention, and detecting the presence of hybridized nucleic acids resulting from the incubation.
  • a method comprises subjecting the nucleic acid fragment of the invention to a molecular amplification reaction, such as PCR, and detecting the presence of amplified nucleic acid which is specific for Borrelia burgdorferi sensu lato.
  • the invention also provides a diagnostic kit comprising
  • nucleic acid fragment of the invention and a means for detecting the binding between the nucleic acid fragment and nucleic acid bound thereto, or
  • nucleic acid primers which, when used in a molecular amplification procedure together with the nucleic acid fragment of the invention, will result in specific amplification of said nucleic acid fragment, and a means for detecting the amplified nucleic acid fragment.
  • Antibodies could be produced and used for screening strains for protein expression, for determining structural location and for examining bactericidal activity of antibodies against these proteins. Means and measures for producing both monoclonal and polyclonal antibodies against P13 are easily applied by the skilled person on the basis of the teachings herein.
  • an embodiment of this part of the invention is a diagnostic composition adapted for the determination of Borrelia burgdorferi sensu lato in a sample, the com- position comprising the polypeptide of the invention or prepared thereby, the amount of the polypeptide being effective to detectably react with antibodies present in the sample, the antibodies being directed against Borrelia burgdorferi sensu lato, the composition optionally comprising a detectable label, e.g. as described above.
  • a detectable label e.g. as described above.
  • another embodiment of the invention i.e.
  • a method of determining the pre- sence of antibodies directed against Borrelia burgdorferi sensu lato in a sample comprising incubating the sample with the polypeptide of the invention or prepared by the method of the invention, and detecting the presence of bound antibody resulting from the administration or incubation.
  • this part of the invention also pertains to a diagnostic kit comprising a polypeptide of the invention and a means for detecting the polypeptide with antibody bound thereto.
  • B. hermsii B. crocidurae
  • B. hispanica B. anserina
  • B. anserina the causative agent of avian borreliosis.
  • BSK II medium Barbour, 1984
  • the cells were harvested in late-log phase by centrifugation at 5,000 rpm for 20 min.
  • E. coli strains DH5 ⁇ and BL21 were used for transformation with the recombinant plasmids in DNA cloning and gene expression experiments, respectively.
  • E. coli strains were grown in Luria broth medium (Gibco BRL, Gaithersburg, MD) supplemented, when required, with carbenicillin (Sigma, St. Louis, MO) at 50 ⁇ g/ml.
  • Monoclonal antibodies 1 5G6 and 7D4 were obtained from Dr. Alan G. Barbour (Sadziene et al., 1 994)
  • DNA fragments were sequenced by the dideoxy chain termination method, with ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit, with AmpliTaq ® DNA Polymerase, FS.
  • the sequence fragments were assembled using the GCG software for UNIX computer.
  • Bb proteins For the whole-cell protein preparations, bacteria harvested from 250 ml of BSK II medium were washed twice with phosphate-buffered saline-5mM MgCI 2 (PBS-Mg). The pellet was suspended in 2 ml of PBS, sonicated and the supernatant was collected after centrifugation at 10,000 rpm for 30 min.
  • PBS-Mg phosphate-buffered saline-5mM MgCI 2
  • Fraction B The subcellular fraction of borreliae outer membrane components (designated Fraction B) was prepared as described elsewhere (WO 90/0441 1 ). Briefly, cells harvested from 1 .5 I of the culture were washed three times with 10 mM Tris-HCI (pH 7.4), 1 50 mM NaCI and 5 mM MgCI 2 (TSM buffer). Octyl- ⁇ -D-glucopyranoside (OGP) (Sigma St. Louis, MO) was added to a final concentration of 2% in 10 ml TSM buffer and the suspension was incubated at 37°C for 60 min. The cell lysate was centri- fuged and the supernatant was incubated at 56°C for 30 min.
  • Tris-HCI pH 7.4
  • OGP Octyl- ⁇ -D-glucopyranoside
  • Fraction B The precipitate was removed by centrifugation at 20,000 rpm for 30 min at 37°C, and the supernatant was dialysed against water at 4°C for 2 days.
  • the precipitate (Fraction B) formed in the dialysis bag was recovered by centrifugation at 20,000 rpm for 30 min at 25°C.
  • the proteins were transferred to a PVDF-membrane (BioRad, Hercules, CA) by elec- troblotting at 0.8 mA/cm 2 for 1 h.
  • the non-specific binding was blocked by immersing the filter for 2 h into 5% non-fat milk powder (Semper, Sweden) in PBS containing 0.05% Tween-20 (PBS-T).
  • Primary or secondary antibodies were diluted with 2.5 % milk-powder in PBS-T, and both incubations of the filter for 1 h were followed by washing in PBS-T.
  • the substrate for the alkaline phosphatase conjugate was 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma, St. Louis, MO).
  • a 13 kDa protein was purified by 1 5% SDS-PAGE of Fraction B obtained from the B. burgdorferi B313 spirochaetes. The appropriate band was visualised by staining the gel with 250 mM KCI in ice-cold water without fixation in MeOH and acetic acid. Elution of protein from the gel was performed in a Schleicher and Schuell Biotrap in 1 5 mM NH 4 HCO 3 (200V for 8 hr).
  • the proteolytic treatment was stopped by the addition of 10 ⁇ l from a solution of the peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, MO) (50 mg of PMSF per 1 ml of isopropanoi), and the cells were centrifuged and washed twice with PBS-Mg. The pellets were resuspended in TSM buffer. One-third of the cell suspension of each preparation was subjected to the whole cell protein extraction by boiling in SDS-PAGE sample buffer. The remaining part of the suspensions was used to prepare the subcellular fraction of the borrelial outer membrane components, Fraction B, as described above.
  • PMSF peptidase inhibitor phenylmethylsulfonyl fluoride
  • the monoclonal antibody 1 5G6 raised against the 13 kDa protein was used as the primary antibody for immunogold staining of intact B. burgdorferi B31 and B. burgdorferi B31 3.
  • Cells from strain B31 ( Figure 2A) were labelled to a minor extent than cells from the strain B313 ( Figure 2B). This was probably due to the presence of outer surface proteins, i.e. OspA, OspB, OspC, OspD, on the surface of the B31 cells.
  • the labelling was confined to the outer surface membrane for both strains indicating that the 13 kDa protein is an outer surface protein.
  • the CB stained SDS-PAGE of the whole-cell protein preparations of Lyme disease borreliae is shown in Figure 1 A.
  • the 1 3 kDa protein was present in the whole-cell preparations (WC) and enriched in the membrane fraction (BF) of B. burgdorferi B31 , B. afzelii ACAI, and B. garinii IP90.
  • the PAGE revealed no major differences among the borrelial strains in respect of either apparent molecular weight or expression level of the 13 kDa protein.
  • the analogous preparations from B. hermsii, B. crocidurae and B. anserina no visible band corresponding to the 1 3 kDa protein was detectable.
  • polyclonal rabbit antiserum (described in Example 2.2) was able to recognise the 13 kDa protein from all three Lyme Disease species, i.e. B. burgdorferi B31 , B. afzelii ACAI and B. garinii IP90 ( Figure 1 B).
  • the 1 3 kDa protein band was isolated and cut from a SDS-PAGE gel and eluted in a Biotrap as described above, Example 2.1 .
  • N-terminal amino acid sequencing of the purified 1 3 kDa protein was attempted but no sequence was obtained. It was concluded that the N-terminus of the 1 3 kDa protein was blocked. Therefore, the purified protein was digested with Staphylococcus aureus V8 protease. The protein cleavage resulted in two fragments of about equal size. As one of the fragments is blocked, only one can be sequenced. After cleavage the fragments were transferred to a PVDF membrane (Biorad, Hercules, CA) by soaking the membrane in the protein solution over night. N-terminal amino acid sequence analysis was performed on a 477A sequenator (Applied Biosystems, Foster City, CA) at Umea University.
  • the sequence of the 25 amino acid fragment was used to design two oligonucleo- tides, one designated Y5.2 (SEQ ID NO: 2), and one designated Y6.2 (SEQ ID NO: 3). Codons for the amino acid sequence obtained, SEQ ID NO: 1 , were selected by reverse translation based on (1 ) conclusion that codons containing A or T were favoured and (2) knowledge of published DNA sequences for several Bb proteins. A choice favouring A or T containing codons was based on the observation that the G + C content of Bb is only 28-35% (Burman et al., 1990). These oligonucleotides were used in a PCR reaction with DNA prepared from B.
  • RNA sequence was cloned into the T-vector (Novagen, Madison, Wl) and sequenced, SEQ ID NO: 4. It was verified that the obtained PCR fragment coded for the N-terminal amino acid sequence of the peptide fragment obtained after protease cleavage of the 1 3 kDa protein, SEQ ID NO: 1 . Based on the sequence of the PCR fragment an oligonucleotide designated Y7 (SEQ ID NO: 5) was designed. This oligonucleotide was to be used as a probe.
  • the recombinant plasmids were transformed into competent E. coli DH5 ⁇ cells. Initially, a B. burgdorferi B31 and B313 EcoR digested DNA library was screened with the DNA probe Y7 (SEQ ID NO: 5). This screening did not result in any positive clones. An Rsal restriction site was identified in the sequence of the PCR fragment, SEQ ID NO: 4. This site was used in a further attempt to clone the gene encoding the 1 3 kDa protein as described below. DNA prepared from B. burgdorferi B31 was cut with Rsa ⁇ and the fragment ligated into HincW digested pUC1 8 (Pharmacia, Uppsala, Sweden) plasmid.
  • the obtained total sequence coded for a protein with a calculated molecular weight of 7 kDa This did not correspond to the expected molecular weight of the full-length DNA fragment coding for the 1 3 kDa protein.
  • a new oligonucleotide designated Y9, SEQ ID NO: 7, was designed and used together with the oligonucleotide Y7R, SEQ ID NO: 6, to generate a new PCR fragment.
  • This PCR fragment was used as a probe to screen a library of Xba ⁇ digested DNA prepared from B. burgdorferi B31 in an attempt to isolate a full-length DNA fragment encoding the 1 3 kDa protein.
  • a recombinant plasmid designated pLY-100 recovered from one positive E. coli DH5 ⁇ clone was isolated.
  • the DNA insert of this plasmid was sequenced and found to comprise a gene fragment containing 537 bp including an ATG start codon followed by an open reading frame (ORF), SEQ ID NO: 18.
  • the full-length 13 kDa protein gene was retrieved by PCR amplification followed by ligation into a pT7Blue vector (Novagen, Madison, Wl). Multiple amplifications were used to ensure that the DNA Taq polymerase did not introduce any errors in the sequence.
  • the nucleotide sequences of the gene encoding the 1 3 kDa protein of B. burgdorferi B31 , B. afzelii AC Al, B. garinii IP90, are shown in SEQ ID NO: 1 8, SEQ ID NO: 20, and SEQ ID NO: 22, respectively.
  • the ATG start codon was followed by an ORF of 1 5 534 and 531 nucleotides for strains ACAI and IP90, respectively.
  • the nucleotide sequence of the gene encoding the 13 kDa protein of B. burgdorferi B313 was identical to the nucleotide sequence of the gene encoding the 1 3 kDa protein of B. burgdorferi B31 .
  • the deduced amino acid sequences of the 1 3 kDa protein of B. burgdorferi B31 , B. afzelii ACAI and B. garinii IP90 are presented in SEQ ID NO: 1 9, SEQ ID NO: 21 and SEQ ID NO: 23.
  • the deduced amino acid sequences of the full-length protein consist of 179, 178 and 177 amino acids for the respective strain.
  • the amino acid sequence of the P13 protein from B. burgdorferi B31 was 87.9% and 87.5 % identical to the sequences from B. afzelii ACAI and B. garinii IP90, respectively. When compared with each other, the two latter strains showed 90.5% identity.
  • the level of similarity and identity between the deduced amino acid sequence of the P1 3 protein from different Borrelia strains further shows that this protein can be useful as a vaccine against Lyme disease as well as a target for diagnostic use.
  • the P13 proteins were examined for the sequence similarity to other known proteins in database libraries. There were no other sequences related significantly to the P13 proteins.
  • the best hit in an EMBL database search was a 95 residues long amino acid sequence encoded by the p1 1 gene on the Bb 49 kb linear plasmid. The encoded amino acid sequence was 41 .5% identical in sequence in a 82 amino acid overlap (corresponding to amino acid residues -1 2 to 70 in SEQ ID NO: 1 9). For the purposes of reference, this best hits are included herein as SEQ ID NOs: 30 and 31 .
  • the P13 gene sequence was compared to the recently completed B.
  • the DNA prepared from B. burgdorferi B31 , B. burgdorferi B31 3, B. afzelii ACAI and B. garinii IP90 was recovered in 1 % agarose blocks as previously described (Ferdows and Barbour, 1 989).
  • One-dimensional AGE and pulse- field AGE were performed in 1 % agarose in TBE buffer.
  • For the pulse-field AGE pulse times were 0.5 s for 30 min, 8 s for 30 min, 1 s for 3 h, 2 s for 3 h, 4 s for 6 h, 8 s
  • the DNA was transferred to a Hybond-N membrane (Amersham, Buckinghamshire, UK) by the
  • the temperature was 55°C for probing with a PCR fragment obtained by amplification using the primers Y9 (SEQ ID NO: 7) and Y7R (SEQ ID NO: 6) (see above) [ ⁇ - 32 P]dATP (Amersham, Buckinghamshire, UK), radio- labeled by random primer technique.
  • the P1 3 protein gene being localised to the chromosome of borreliae shows a higher degree of conservation among Lyme disease associated borreliae contrary to the plasmid-encoded major outer surface proteins A, B, and C which exhibit a significant species and strain dependent genetic and antigenic polymorphism (Barbour 1 986, Jonsson et al., 1 992, Wilske et al., 1 993).
  • oligonucleotide primers Y14 (SEQ ID NO: 1 2) and Y33 (SEQ ID NO: 1 5), were designed to anneal to the 5 ' end containing the Iipidation signal and the 3 ' end of the P1 3 gene from B. burgdorferi B31 .
  • the primers contained Bam ⁇ and EcoRl restriction sites, respectively, and were used to amplify the P13 gene in the PCR.
  • PCR amplification was performed using Ampli-Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT).
  • the DNA encoding TPA 5' UTR and leader peptide, P13 and OspA were isolated from previous plasmid constructs or amplified.
  • the TPA signal was isolated from VR2210 (Luke et al., 1997) by digestion with Pst ⁇ /Kpn ⁇ .
  • the P13 gene was PCR amplified from pLY100 using the primers L1 (SEQ ID NO: 24) and L2 (SEQ ID NO: 25).
  • the P13 containing fragment was digested with Kpn ⁇ lXba ⁇ and introduced together with the Pst ⁇ IKpn ⁇ isolated TPA signal into VR1020 digested with Pst ⁇ /Xba ⁇ .
  • the TPA signal was PCR amplified with the primers L5 (SEQ ID NO: 28) and L6 (SEQ ID NO: 29).
  • the P13 gene was PCR amplified from pLY100 using the primers L3 (SEQ ID NO: 26) and L4 (SEQ ID NO: 27)
  • the PCR fragments were digested with the appropriate restriction enzymes.
  • the OspA gene was isolated from VR2210 by digestion with Kpn ⁇ /Xba ⁇ . All three fragments were combined in a three fragment ligation into the Pst ⁇ IXba ⁇ digested VR1020 to yield pLY-HA.
  • mice will be injected with the plasmids pLY-h and pLY-HA as well as a negative control plasmid not containing a coding sequence for a Borrelia antigen.
  • the plasmid and control DNA are diluted in standard saline.
  • Three bilateral injections of DNA will be given at two week intervals at a dosage of 50 ⁇ g/leg into the rectus femoris muscle.
  • Sera will be collected after each injection and analyzed by 1 ) antibody ELISA and 2) growth inhibition of spirochaetes.
  • mice After the last injection, mice will be challenged with B. burgdorferi sensu stricto N40 spirochaetes (same OspA serogroup as B31 ). Spirochetes will be either injected intradermally in the tail or by the tick challenge model (Telford et al., 1 993). Mice will be sacrificed following the challenge. Bladder, heart, plasma, and cross-cuttings of the tibiotarsal joints will be cultured in growth medium. Cultures will be examined for the presence of spirochaetes by phase-contrast microscopy and scored as negative if no spirochaetes are seen in 50 high-power fields.
  • the clone pMG2 was obtained from Dr. Michael Norgard's laboratory. This clone had been isolated from a library prepared from partial Sau3A ⁇ digested DNA of Borrelia burgdorferi 297 cloned into the BamH ⁇ site of the plasmid pGEX-1 (Pharmacia). The library had been screened with the monoclonal antibody 1 5G6. After IPTG induction of E. coli DH5 cells transfected with the plasmid pMG2, a glutathione-S-transferase (GST) fusion protein reacting with the monoclonal antibody 1 5G6 could be seen in an immunoblot experiment. The fusion protein had a molecular weight of about 36 kDa, 26 kDa for the fusion partner GST plus 10 kDa for the Bb protein fragment.
  • GST glutathione-S-transferase
  • this clone contained an approximately 300 base pair insert.
  • DNA sequencing showed an open reading frame of 251 bases.
  • the insert was PCR amplified, oligolabelled, and used as a probe to screen libraries prepared from partial Hind ⁇ digested DNA from B. burgdorferi B31 , B. afzelii AC Al, and B. garinii IP90, respectively.
  • P1 3 Molecular weight determinations of P1 3 were performed on a VG Platform II mass spectrometer with a range of 2000 m/z equipped with an electrospray source (Micromass, Altrincham, UK). Prior to injection, the purified P1 3 preparation was precipitated with methanohchloroform to remove any trace of SDS contamination. A P1 3 solution of 20 pmol/ml in water-acetonitrile (50:50 [voi/vol]) was mixed with 5 % formic acid and introduced directly into the electrospray source at a flow rate of 5 ml/min. Calibration was performed by a separate introduction using horse heart myoglobin (1 6,951 .5 Da). The MassLynx software was used to calculate the mole- cular weight.
  • variable antigens Vmp7 and Vmp21 of the relapsing fever bacterium Borrelia hermsii are structurally analogous to the VSG proteins of the African trypanosome. Molecular Microbiology, 4: 1 71 5-1 726. 5
  • Fiers W Contreras R, Haegemann G, Rogiers R, Van de Voorde A, Van Heuverswyn H, Van Herreweghe J, Volckaert G, Ysebaert M. 1978. Complete nucleotide 35 sequence of SV40 DNA. Nature. 273(5658): 1 1 3-20.
  • Fikrig E Barthold SW, Marcantonio N, DePonte K, Kantor FS, Flaveli RA. 1992. Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in laboratory mice. Infection and Immunity, 60: 657-661 .
  • Porcella SF Popova TG, Akins DR, Li M, Radolf JD, Norgard MV. 1996. Borrelia burgdorferi supercoiled plasmids encode multicopy tandem reading frames and a lipoprotein gene family. Journal of Bacteriology. 1 78: 3293-3307.
  • Zingg BC Zingg BC, Anderson JF, Johnson RC, LeFebvre RB. 1993. Comparative analysis of genetic variability among Borrelia burgdorferi isolates from Europe and the United States by restriction enzyme analysis, gene restriction fragment length polymorphism, and pulse-field gel electrophoresis. Journal of Clinical Microbiology, 31 : 31 1 5-31 22.

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  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un fragment d'acide nucléique isolé qui: - code un fragment de polypeptide présentant une réactivité immunologique importante avec un anticorps polyclonal de lapin produit contre un polypeptide possédant un poids moléculaire apparent de 13 kDa déterminé par une électrophorèse de polyacrylamide en présence de sulfate de sodium dodécylique suivie par une visualisation, ce polypeptide dérivant de Borrelia burgdorferi B313 et étant codé par la séquence nucléotidique SEQ ID NO 18; l'anticorps polyclonal de lapin ne présente sensiblement aucune réactivité immunologique avec des protéines provenant à au moins 95 % de spirochètes sélectionnés au hasard dans le groupe formé par Borrelia hermsii, Borrelia crocidurae, Borrelia anserina et Borrelia hispanica; et/ou s'hybride facilement dans des conditions d'hybridation hautement rigoureuses avec un fragment d'ADN possédant une séquence nucléotidique sélectionnée dans le groupe formé par SEQ ID NO 18, SEQ ID NO 20 et SEQ ID NO 22, ou avec un fragment d'ADN complémentaire de ce dernier; mais le fragment d'acide nucléique ne présente aucune hybridation sensible lorsque les conditions d'hybridation sont hautement rigoureuses avec un ADN génomique provenant à au moins 95 % de spirochètes sélectionnés dans le groupe formé par Borrelia hermsii, Borrelia crocidurae, Borrelia anserina et Borrelia hispanica. L'invention concerne en outre des fragments de polypeptides, des vecteurs, des cellules et lignes cellulaires transformées, une méthode de préparation d'un fragment polypeptidique et de vaccins, ainsi que des compositions et trousses de diagnostic.
PCT/IB1998/001424 1997-09-10 1998-09-04 ANTIGENES P13 PROVENANT DE $i(BORRELIA) WO1999012960A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU88811/98A AU8881198A (en) 1997-09-10 1998-09-04 P13 antigens from (borrelia)
CA002300365A CA2300365A1 (fr) 1997-09-10 1998-09-04 Antigenes p13 provenant de borrelia
EP98940504A EP1012269A2 (fr) 1997-09-10 1998-09-04 Antigenes p13 provenant de borrelia

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DK104197 1997-09-10
DK1041/97 1997-09-10
US5903697P 1997-09-16 1997-09-16
US60/059,036 1997-09-16

Publications (2)

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WO1999012960A2 true WO1999012960A2 (fr) 1999-03-18
WO1999012960A3 WO1999012960A3 (fr) 1999-05-27

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PCT/IB1998/001424 WO1999012960A2 (fr) 1997-09-10 1998-09-04 ANTIGENES P13 PROVENANT DE $i(BORRELIA)

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EP (1) EP1012269A2 (fr)
AU (1) AU8881198A (fr)
CA (1) CA2300365A1 (fr)
WO (1) WO1999012960A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078800A2 (fr) * 1999-06-18 2000-12-28 Medimmune, Inc. Compositions combinant la proteine de liaison de decorine et la proteine de surface externe et procedes correspondants
CN108484745A (zh) * 2018-05-17 2018-09-04 青岛大学 一种小麦白粉病抗性相关蛋白TaSARD1及其编码基因与应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990004411A1 (fr) * 1988-10-24 1990-05-03 Symbicom Aktiebolag Fractions de borrelia burgdorferi avec action immunogene
EP0540457A1 (fr) * 1991-10-22 1993-05-05 Symbicom Ab Améliorations dans le diagnostic et dans le prophylaxie de Borrelia burgdorferi
WO1995012676A1 (fr) * 1993-11-01 1995-05-11 Associated Universities, Inc. Proteines chimeres comprenant des polypeptides de borrelia et leurs utilisations
WO1995035119A1 (fr) * 1994-06-22 1995-12-28 Board Of Regents, The University Of Texas System PROCEDES ET COMPOSITIONS COMPRENANT UNE PROTEINE DE B. BURGDORFERI DE 13 kD

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990004411A1 (fr) * 1988-10-24 1990-05-03 Symbicom Aktiebolag Fractions de borrelia burgdorferi avec action immunogene
EP0540457A1 (fr) * 1991-10-22 1993-05-05 Symbicom Ab Améliorations dans le diagnostic et dans le prophylaxie de Borrelia burgdorferi
WO1995012676A1 (fr) * 1993-11-01 1995-05-11 Associated Universities, Inc. Proteines chimeres comprenant des polypeptides de borrelia et leurs utilisations
WO1995035119A1 (fr) * 1994-06-22 1995-12-28 Board Of Regents, The University Of Texas System PROCEDES ET COMPOSITIONS COMPRENANT UNE PROTEINE DE B. BURGDORFERI DE 13 kD

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EMBL Data Base Empro:AE001117 Ac. No. AE001117, 16 December 1997 Fraser et al.: 'Genomic sequence of a Lyme disease spirochete, Borrelia burgdorferi' XP002096131 -& FRASER ET AL.: NATURE , vol. 390, 11 December 1997, pages 580-586, XP002096130 *
EMBL Data Base Pir2:B70104 Ac. No. B70104, 13 February 1998 Fraser et al.: 'Genomic sequence of a Lyme disease spirochete, Borrelia burgdorferi' XP002096132 & FRASER ET AL.: NATURE, vol. 390, 11 December 1997, pages 580-586, *
FENG S ET AL: "CHARACTERIZATION OF TWO GENES, P11 AND P5, ON THE BORRELIA BURGDORFERI 49-KILO BASE LINEAR PLASMID" BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1307, no. 3, 17 July 1996, pages 270-272, XP000613914 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078800A2 (fr) * 1999-06-18 2000-12-28 Medimmune, Inc. Compositions combinant la proteine de liaison de decorine et la proteine de surface externe et procedes correspondants
WO2000078800A3 (fr) * 1999-06-18 2001-07-19 Medimmune Inc Compositions combinant la proteine de liaison de decorine et la proteine de surface externe et procedes correspondants
CN108484745A (zh) * 2018-05-17 2018-09-04 青岛大学 一种小麦白粉病抗性相关蛋白TaSARD1及其编码基因与应用
CN108484745B (zh) * 2018-05-17 2021-06-25 青岛大学 一种小麦白粉病抗性相关蛋白TaSARD1及其编码基因与应用

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AU8881198A (en) 1999-03-29
CA2300365A1 (fr) 1999-03-18
EP1012269A2 (fr) 2000-06-28
WO1999012960A3 (fr) 1999-05-27

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