VACCINES CONTAINING BORRELIA BURGDORFERI OspG

Abstract: The present invention provides a new antigen, OspG, from B. burgdorferi which is useful in the prevention and diagnosis of Lyme disease. The cloning and sequence and expression of the antigen are disclosed.

Description: Vacci nes contai n ing Borrel l a burgdorferi OspG The present invention relates to novel antigens and derivatives thereof derived from Borrelia burgdorferi, to methods of their production, to their use in human and animal medicine and diagnosis and to pharmaceutical compositions containing them. In particular, the present invention provides the cloning expression of a novel polymorphic B. burgdorferi lipoprotein, OspG. This has previously been known as lp77. The deduced amino acid sequence of this protein exhibits no significant homologies to other known Borrelial antigens such as OspA, OspB, OspC, OspD, OspE, OspF and P27. These outer surface proteins exhibit between 41 % and 65% similarity with OspG (Table 1). Lyme disease is the most common vector borne infectious disease of the temperate climate. The etiological agent, the spirochete Borrelia burgdorferi, causes a multisystemic illness in humans which may affect skin, nervous system, joints and heart (8, 44). B. burgdorferi strains isolated from different biological sources and geographic areas are heterogeneous (2, 19, 38, 45, 50) and it is assumed, that the patterns of disease manifestations are influenced by antigenic differences of the spirochetal strains. Although several attempts to classify B. burgdorferi by immunological or molecular criteria have recently been reported, the taxonomy of B. burgdorferi is still a matter of controversy and active research. To date, a variety of B. burgdorferi antigens such as outer surface protein A (OspA), OspB, pC and plOO, are employed for serological diagnosis and as putative candidates for vaccine development (13, 14, 36, 38, 40, 41). However, because of their obvious heterogeneity unambiguous criteria for the generation of a species- specific diagnostic standard and for the development of a polypeptide vaccine that would guarantee protection against any subspecies are still lacking. The present inventors have discovered a further lipoprotein from B. burgdorferi which is designated OspG. This molecule is characterised by having an apparent molecular weight of approximately 22kDa, and an isoelectric point of PI =5.2 and comprises 196 amino acids. The mature protein is further characterised as having one large hydrophobic domain of about 20 amino acids at the amino- terminal portion of OspG. This N-terminal peptide corresponds to the leader signal peptide found in typical prokaryotic lipoprotein precursors. At the carboxy terminus of the hydrophobic core is a cleavage site presumably recognized by a B. burgdorferi signal peptidase. The potential cleavage site in OspG is found between serine at position 19 and cystein at position 20. The sequence around the cleavage site of OspG is L-1-l-S-C. Unlike the gene for OspA, B, C, D, E, F and P27 the OspG gene is located naturally on the 55kb plasmid in B. burgdorferi ZS7. Weak cross reactivity of the OspG probe with the 45-kb plasmid was noted. OspG has the amino acid sequence substantially as set forth in figure 1. It is further characterised by being expressed only during infection and is not detectable in B. burgdorferi cultivated in vitro. Accordingly, the present invention provides an isolated protein derived from B. burgdorferi characterised in that it has a molecular weight of between 20-22kDa as determined by two-dimensional SDS gel electrophoresis and an isoelectric point of between 4.9 and 5.4. The present inventors further provide a protein or an immunologically or antigenically equivalent fragment or derivative thereof having at least 80% homology to the amino acid sequence depicted in figure 1. The protein and its corresponding DNA and RNA sequences find utility in both the vaccine and diagnostic field. Preferably the present invention provides a protein having at least 85% homology, more preferably 90% homology and most preferably at least 95% homology to the protein depicted in figure 1. The protein of the present invention may be a fusion protein, in which case the fusion protein is characterised by having a portion of its amino acid sequence or a fragment thereof. Preferably the portion is at least 80%, preferably 85%, more preferably 90%, and most preferably 95% homologous to the protein sequence of figure 1. Preferably the protein is at least 70% pure as determined by SDS polyacrylamide gel electrophoresis, and most preferably 80% pure, and more preferably at least 90% pure. The protein of the present invention maybe a lipoprotein or produced as a protein without any associated lipids. When produced by recombinant techniques, the lipoprotein will be expressed with the signal sequence. Cleavage of the signal sequence, to remove the N-terminal 19 amino acids will result in a non-lipidated molecule being produced. Immunoblot analysis shows that OspG is recognised by sera from mice previously infected with intact spirochetes that suggests that the native protein is immunogenic in this species. The present inventors have further indentified and sequenced the gene for OspG. Accordingly, the invention provides a DNA sequence encoding for a protein OspG or fragment or derivative thereof. Preferably the DNA sequence is substantially as set forth in Figure 1. The term substantially as used herein means at least 70% identity to the sequence set forth in Figure 1, preferably 80% identity and more preferably at least 85% and most preferably at least 95% identify. In particular, the present invention provides a DNA sequence having substantially the sequence depicted in Figure 1 , or a fragment thereof, or a DNA sequence which hybridises to said sequence and which codes for a protein which exhibits OspG antigenicity. The DNA of the present invention may be prepared by enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50μl or less. Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl2, 0.01M dithiothreitol, ImM spermidine, ImM ATP and 0. lmg/ml bovine serum albumin, at a temperature of 4°C to ambient, generally in a volume of 50μl or less. The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982),or in other scientific publications, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D. Matteucci and M.H Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D. Matteucci and M.H. Caruthers, Journal of the American Chemical Society, 1981 , 103, 3185; S.P. Adams et al., Journal of the American Chemical Society, 1983, 105, 661; N.D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W.D. Matthes et al., EMBO Journal, 1984, 3, 801. Alternatively, the coding sequence can be derived from B. burgdorferi mRNA, using known techniques (e.g. reverse transcription of mRNA to generate a complementary cDNA strand), and commercially available cDNA kits. The invention is not limited to the specifically disclosed sequence, but includes all molecules coding for the protein or an immunogenic derivative thereof, as described above. DNA polymers which encodes mutants of the protein of the invention may be prepared by site-directed mutagenesis of the cDNA which codes for the protein by conventional methods such as those described by G. Winter et al in Nature 1982, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6500, or deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter et al in Biochem. Soc. Trans., 1984, 12, 224-225. In one aspect, the present invention provides a process comprising the steps of: i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes said OspG protein or an immunogenic derivative thereof; ii) transforming a host cell with said vector; iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein; and iv) recovering said protein. The term 'transforming' is used herein to mean the introduction of foreign DNA into a host cell by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kingsman and A.J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term 'transformed' or 'transformant' will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest. The expression vector is novel and also forms part of the invention. The replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the OspG protein, or fragments thereof, under ligating conditions. Thus, the DNA polymer may be preformed or formed during the construction of the vector, as desired. The choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses. The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al cited above. The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Mamatis et al cited above, or "DNA Cloning" Vol. II, D.M. Glover ed., IRL Press Ltd, 1985. The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such as E. coli may be treated with a solution of CaCl2 (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol. Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells. The invention also extends to a host cell transformed with a replicable expression vector of the invention. Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al and "DNA Cloning" cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 45°C. The product is recovered by conventional methods according to the host cell. Thus, where the host cell is bacterial, such as E. coli it may be lysed, chemically or enzymatically and the protein product isolated from the resulting lysate or super variant. Where the host cell is mammalian, the product may generally be isolated from the nutrient medium or from cell free extracts. Conventional protein isolation techniques include selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column. Alternatively, the expression may be carried out in insect cells using a suitable vector such as the Baculovirus. In a particular aspect of this invention, the protein is expressed in Lepidoptera cells to produce immunogenic polypeptides. For expression of the protein in Lepidoptera cells, use of a baculovirus expression system is preferred. In such system, an expression cassette comprising the protein coding sequence, operatively linked to a baculovirus promoter, typically is placed into a shuttle vector. Such vector contains a sufficient amount of bacterial DNA to propagate the shuttle vector in E. coli or some other suitable prokaryotic host. Such shuttle vector also contains a sufficient amount of baculovirus DNA flanking the desired protein coding sequence so as to permit recombination between a wild-type baculovirus and the heterologous gene. The recombinant vector is then cotransfected into Lepidoptera cells with DNA from a wild-type baculovirus. The recombinant baculoviruses arising from homologous recombination are then selected and plaque purified by standard techniques. See Summers et al., TAES Bull (Texas Agricultural Experimental Station Bulletin) NR 1555, May, 1987. A process for expressing the CS protein in insect cells is described in detail in USSN 287,934 of SmithKline RIT (WO/US 89/05550). Production in insect cells can also be accomplished by infecting insect larvae. For example, the protein can be produced in Heliothis virescens caterpillars by feeding the recombinant baculovirus of the invention along with traces of wild type baculovirus and then extracting the protein from the hemolymph after about two days. See, for example, Miller et al., PCT/WO88/02030. The novel protein of the invention may also be expressed in yeast cells as described for the CS protein in EP-A-0 278 941. The present invention also relates to vaccine composition comprising OspG or fragment or derivative thereof. In the vaccine of the invention, an aqueous solution of the protein(s) can be used directly. Alternatively, the protein, with or without prior lyophilization, can be mixed or absorbed with any of the various known adjuvants. Such adjuvants include, but are not limited to, aluminium hydroxide, muramyl dipeptide and saponins such as Quil A. Particularly preferred adjuvants are, MPL (monophosphoryl lipid A) and 3D-MPL (3 De-O-acylated monophosphoryl lipid A). A further preferred adjuvant is known as QS21. 3 D-MPL can be obtained from Ribi Immunochem or by the methods disclosed in UK patent No. 2220211, whereas QS21 can be obtained from Cambridge Biotech or by the method disclosed in US patent No. 5,057,540. As a further exemplary alternative, the proteins can be encapsulated within microparticles such as liposomes or associated with oil in water emulsions. In yet another exemplary alternative, the proteins can be conjugated to an immuostimulating macromolecule, such as killed Bordetella or a tetanus toxoid. In a preferred embodiment of the invention, the antigen of the present invention will contain other Borrelia antigens, in particular OspA. The proteins of the present invention may be expressed by live vectors such as BCG, Listeria or Salmonella and formulated as live vaccines using such vectors. Vaccine preparation is generally described in New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Maryland, 1978. Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, US Patent 4,372,945 and Armor et al., US Patent 4,474,757. Use of Quil A is disclosed by Dalsgaard et al., Acta Vet Scand, 18:349 (1977). The amount of the protein of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 1-200 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects may receive an additional administration to enhance their immune response. The present invention also relates to antibodies, preferably monoclonal antibodies which are specific for OspG. Such antibodies fmd utility in the diagnosis and also in the prevention of Lyme disease. In an alternative embodiment the invention provides a diagnostic kit comprising an OspG antigen. I. Materials and Methods 1.1 Borrelia strains B. Burgdorferi strains used in this study were described elsewhere (45). Borrelia were grown in modified Barbour-Stoenner-Kelly II (BSK II) medium (2) at 33°C. Spirochetes were harvested by centifugation at 10,000 x g at 4°C for 20 min, washed two times in PBS, and enumerated by dark-field microscopy. Preparation and screening a B. burgdorferi expression library Genomic DNA was prepared from B. burgdorferi strain ZS7 by the lysozyme/SDS method, and DNA fragments were generated by sonication. Blunt- ended DNA was inserted into the pUEXl vector using an adaptor cloning strategy (7,31). The ligated DNA was transformed into E.coli MC1061 followed by expression screening using an immune sera taken from DBA/2 mice inoculated with 104 (and fewer) B.burgdorferi (ZS7) organisms. 1.2 Southern blot hybridization Total genomic DNA was extracted from Borrelia organisms as described previously (32). Approximately 5 μg of DNA was digested with 100 U of restriction nuclease (Hindlll) according to the manufacturer's recommendations (Boehringer, Mannheim). Samples were subjected to electrophoresis using a 0.7% agarose gel. DNA fragments were transferred to Hybond ™-N nylon membrane (Amersham) followed by UV-cross-linking and hybridization. Briefly, using 32p. labeled probes hybridization was done over night at 65°C in 0.5 M NaHPO4/7% NaDodSO^ pH7.2. After washing in 40 nM NaHPO^l % NaDodSO^ pH7.2 at room tempeature for 30 min the dry membrane was autoradiographed on Kodak XAR-5 film with intensifying screens at -80°C for 1 to 12 h. As hybridization probe, a 500-bp DNA fragment encompassing the LA7 coding region was used. The gene fragment of interest was recovered for low melt agarose gel, precipitated by ethanol treatment, and radiolabeled by random primed reaction as described. 1.5 Gel electrophoresis For electrophoresis on one-dimensional SDS/PAGE slab gels according to Laemmli (21), 40 μl of each lysate (equivalent to *10^ organisms) were mixed with lOμl 5 x reducing sample buffer. Two-dimensional polyacrylamide gel electrophoresis was carried out as described by O'Farrel (29), using IEF (Pharmacia/LKB ampholytes: 1.45% pH 3.5- 10, 0.1 % pH 2.5-4.0, 0.2% pH 4-6, 0.2% pH 9-11) in the first dimension. The same amount of lysate was applied as in the case of one-dimensional gel electrophoresis. Gels were either silver strained or processed for Western blotting (15). Surface proteolysis using proteinase K (Boehringer-Mannheim, Germany) was carried out according to the method of Barbour et al. (20). The proteins were then separated by SDS-PAGE and individual antigens were identified by immuno- blotting. 1.6 Western blotting Following a two dimensional SDS-PAGE, proteins were electroblotted for 1 hr at constant current (60 mA) onto Hybond C nitrocellulose sheets (Amersham) employing a semi-dry electroblotting chamber (BIO-RAD, Munich, Germany) according to the manufacturers' recommendations. Following an overnight incubation in blocking buffer (50 mM Tris-HCl, 150 mM NaCl, 5% non-fat dried milk), immunoblots were incubated for 2h at room temperature with a 1: 100 (v/v) dilution of mouse and human antisera in 50 mM Tris-HCl, 150 mM NaCl, 1 % dried milk, 0.2% Tween 20 or with culture supernatant of mouse mAbs (LA7). Nitrocellulose filters were washed five times in dilution buffer and incubated for an additional hour with an alkaline phosphatase-conjugated goat anti-rabbit antiserum (Dianova, Hamburg, Germany, 1:400 v/v). Blots were washed four times in the above mentioned buffer and twice in TBS and immunoreactive bands were then visualized by addition of 20 ml DEA-buffer (0.1M diethanolamine (Sigma), 0.02% NaN3, 5mM MgCl2, pH9.0] supplemented with 5-bromo-4-chloro-3- indolyphosphate (BCIP, Sigma; 165 μg/ml) and nitro blue tetrazolium (NBT, Sigma; 330 μg/ml) as substrate. The reaction was stopped by washing the membrane in 50mM Tris-HCl, 150 mM NaCl, 5 mM EDTA. 1.7 DNA sequence B. burgdorferi genomic DNA fragments cloned in pUEXl plasmids (Amersham) were sequenced by using a ^Sequencing kit (Pharmacia) according to the manufacturer's recommendations. 1.8 Amino acid sequence analyses Simultaneous alignment for protein sequences and phylogenetic tree construction were performed by using the HUSAR software. 1.9 Immunofluorescence B. burgdorferi organisms were washed twice in PBS, transferred onto adhesion slides (Superior, Bad Mergentheim, FRG) (1 x 10-5 spirochetes/reaction field), fixed in absolute ethanol (2 min., -20°C) and air dried. The fixed spirochetes were incubated with the respective mAbs diluted spirochetes were incubated with a fluoresceine isothiocyanate moist chamber for 30 min. After three washings in PBS, the preparations were examined using a fluorescence microscope, and documented using a 400 ASA black and white film (HP5; Illford, UK). 1.10 Immunofluorescence B. burgdorferi organisms were washed twice in PBS, transferred to adhesion slides (Superior, Bad Mergentheim, Germany) 10^ spirochetes per reaction field), fixed in absolute ethanol (2min, -20°C), and air dried. The fixed spirochetes were incubated with the individual MAb diluted in PBS in a moist chamber for 30 min. After three washings in PBS, the spirochetes were incubated with a fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin antiserum (Medac, Hamburg, Germany) in a dark moist chamber for 30 min. After three washings in PBS, the preparations were examined with a fluorescence microscope and documented with 400- ASA black-and-white film (HP5; •••, Illford, United Kingdom). ELISA B.burgdorferi-specific antibodies were measured in a solid-phase ELISA system with B. burgdorferi B31 antigens as described previously (15). 2.1 PCR amplification of a portion of the ospG gene The ospG gene lacking the sequence encoding the hydrophobic leader peptide was PCR amplified with oligonucleotide primers 5'- GTGGATCCAAGATTGATGCGAGTAGTG-3' (corresponds to nucleotides 61 to 79) and 5'-GTGAATTCTATTTTTTATCTTCTATATTTTGAGGCTCTG-3' (corresponds to nucleotides 560 to 590). Plasmid pZS7 DNA was subjected to 30 cycles of PCR in a DNA Thermal Cycler (Bio-Med60). Denaturation was carried out at 94°C for 60 s, annealing at 48°C for 90 s and extension at 72°C for 90 s. The amplified fragment was ligated in frame with the glutathione S-transferase gene into the pGEX-2T vector after digestion with BamHI and EcoRI and used for transformation of DH5α host cells. 2.2 Expression and purification of the recombinant OspG Expression of the glutathione S-transferase-OspG fusion protein in E. coli DH5α, affinity purification and endoproteinase thrombin cleavage of fusion protein was performed as recommended by the manufacturer (Pharmacia). 2.3 [^H]palmitate labelling and Triton X-100 phase partitioning E. coli organisms transformed with plasmids carrying full-length (pZS77) of truncated (pOspG) versions of the ospG gene were grown in the presence of [9, 10- (n)- 3 H|palmitic acid (specific activity, «50Ci/mmol, Amersham), and radiolabelled lipoproteins were extracted by Triton X-114 phase partitioning as described previously (46). 2.4 Generation of immune sera and serology Immune sera were taken from mice previously either inoculated in the tail with 10 8 (C.B-17; IS anti-10 8 ) or 10 3 (DBA/2; IS anti-10 3 ) viable B. burgdorferi spirochetes of strain ZS7 or primed with 5-10 / xg of lipidated (lip) HpOspA (BALB/c; IS anti- lipOspA) or of recombinant (rec) recOspG (BALB/c; IS anti-recOspG) s.c. in adjuvant (ABM2; Sebac, Aidenbach, Germany) and boosted after 10 and 20 days. All sera were analysed by ELISA for the amount of spirochete-specific immunoglobulin (lg) on either whole spirochetal lysate (B.b. lg) or on recOspA (OspA lg) or recOspG (OspG lg) as described previously (39). 2.5 Protection experiments SCID mice were either left untreated or reconstituted with either normal mouse serum (NMS) or pooled Immune Sera (IS). The amount of spirochete-specific IG (ELISA on whole B. burgdorferi cell lysates) transferred with the individual IS was as follows: IS anti-10 8 , 4,4μg lg/mouse; IS anti-10 3 , 4,5μg/mouse; IS anti- lipOspA, 5 ig/mouse; IS anti-recOspG, 72ng/mouse. When tested on recOspG, the amount of specific lg was around 10-20 fold higher in IS anti-recOspG as compared to IS anti-10 3 . IS were given i.p. and one hour later the recipients were challenged s.c. with 10-5 spirochetes (B. burgdorferi ZS7). Mice were investigated for the development of clinical arthritis under blinded conditions and for the presence of spirochetes by cultivating ear biopsies in BSK medium as described previously (37, 40). 2.6 Pathology The development of clinical arthritis in the tibiotarsal joints was monitored under blinded conditions as described earlier (37, 40). The scoring used was as follows: + +, severe; +, less severe; (+), moderate; +/-, mild swelling; (+/-), marginal swelling, reddening; and -, no clinical signs of arthritis. Results Cloning of OspG and analysis of the recombinant constructs A B. burgdorferi ZS7 genomic DNA expression library was screened with an immune serum from mice previously infected with 10 3 spirochetes (IS anti-10 3 ). This IS was shown before to lack antibodies to OspA and OspB but convey protection in SCID mice against subsequent infection (35). Furthermore, this IS recognised four individual proteins with relative molecular masses of 19-20-kDa and two proteins of * 40-kDa when tested on immuno-blots from whole-cell lysates of strain ZS7 separated by two dimensional gel electrophoresis. Approximately 20 clones were identified with one clone, designated pZS77, being particular reactive. The recombinant plasmid pZS77 was subjected to restriction analysis, subcloning and sequencing. The nucleotide sequence of ospG together with the deduced amino acid sequence of OspG is shown in Figure 1. A consensus ribosome binding site (GGAG) is located 10 bp upstream of the ATG start codon of the ospG gene. Further upstream of this translational initiation sequence are the -10 region (TATATT), at positions -70 to -64, and the -35 region (TTGTTA), at positions - 105 to -100. Two short inverted repeats with sequences ATATTT and TTACATTT were contained in this upstream region between positions -118 to -49. The ATG start codon of the ospG gene at position + 1 is followed by an open reading frame of 588 nucleotides, corresponding to a 196-amino-acid protein with a calculated molecular mass of 22.049 Da. A possible rho-independent terminator was identified between position 620 and 656. Alignment of the DNA sequence upstream of the ATG start codon of d e ospG gene with the recently reported promoter region of the ospE-ospF operon reveals identity of 94% as determined by the GAP algorithm. Note that the ospG promoter contains two highly conserved octamer DNA motifs, ATGTATTT (at position -187 to -180) and AATTACAT (at position - 120 to -113), which have been shown before to be associated with protein binding sites for a negative regulator molecule, named MATα2 and to regulate gene expression during yeast differentiation (5, 25). These DNA motifs have been identified on two Saccharomyces cerevisiae genes MFα2 and BAR1, respectively, and contain imperfect inverted repeats of a motif that resembles the immunoglobulin octamer motif ATTTGCAT. Most interestingly, a further immunoglobulin (lg) octamer-like sequence ATTTGCAA (at position - 154 to -147) is located between both S. cerevisiae-like motifs that differs from the lg-octamer motif by only one transition substitution. Amino acid sequence analysis of OspG The hydropathy profile of OspG suggest that the protein is largely hydrophilic with one hydrophobic domain of about 20 amino acids at the amino-terminal portion. This N-terminal region reveals similarities to leader signal peptides present in typical prokaryotic lipoproteins (51, 52). At the COOH-terminal end of the signal sequence is a putative signal peptidase 11 recognition motif Leu-X-Y-Z-Cys (Figure 3a). The potential cleavage site in OspG is located between serine at position 19 and cystein at position 20. The calculated isolelectric point is at pi 5.2. Comparison of the amino acid sequence of OspG with the sequences of all known B. burgdorferi outer surface proteins, OspA - OspF and P27, reveals that OspG exhibits the highest homology with OspF (65%) (Table 1). Furthermore, the basic N-terminal peptide motif M-N-K-K-M of OspG is identical to that observed for OspE and OspF. Mapping of OspG Plasmid and chromosomal DNA of several B burgdorferi strains were separated by pulse-field gel electrophoresis and hybridized to ospA and ospG specific probes. We estimated the size of the plasmid containing ospA to be 53 kb for the German B. burgdorferi isolate ZS7. The ospA containing plasmid of strain ACA-1 was barely visible because of low amounts of DNA loaded onto the gel, but was seen after prolonged exposure (data not shown). Using the ospG probe a prominent band was seen with a linear plasmid of approximately 48 kb and a weaker one with a 45 kb plasmid of strain ZS7. In contrast, in the American strain B31 two plasmids of 45 kb and about 35 kb in size hybridized with the ospG gene probe. Note that hybridization was not observed when genomic DNA were applied from B. garinii strains ZQ1, and 20047 as well as the B. japonica strain HO14. In a control experiment, the ospE-ospF probe derived from strain ZS7 was used to determine whether distinct bands could be observed with these genomic DNAs. This experiment revealed two plasmids of 45 kb and 35 kb for strains ZS7, B31, 20047 and 21038. DNAs isolated from other Borrelia species, such as B. coriaceae Co53, B. hermsii, and B. turicatae, and from Treponema pallidum did not hybridize to the ospG probe, indicating specificity for the species B. burgdorferi. RFLP of the ospG gene Restriction fragment length polymorphism (RFLP) analysis of ospG with endonuclease Hindlll revealed at least seven distinct hybridization patterns among the 20 B. burgdorferi isolates tested: the majority of B. burgdorferi sensu stricto isolates are characterized by two hybridization fragments of 1.8 and 3.8 kb (Figure 6, lane 1); 3 out of 6 B. garinii isolates tested did not hybridize with the ospG probe and B garinii strains 20047 and S90 exhibited fragments of 1.8 kb and 1.7 and 3 kb, respectively (data not shown); among strains of the species B. afzelii at least three different hybridization patterns could be observed: one band of 2.4 kb for strain ACA-1 (lane 3) and two bands of either 1.9 and 2 kb for strain MMS (lane 4) or 2 and 5 kb for strain NE40 (lane 5). Expression of the OspG recombinant protein in E. coli To amplify OspG by PCR, primers were selected in such a way that the final recombinant product lack the 20 amino acid residues composing the leader peptide (46). The amplified OspG-encoding product was inserted in frame with the carrier protein of the expression vector pGEX-2T (Pharmacia, Freiburg, Germany) and after induction with IPTG, and approximately 44-kDA GST-OspG fusion protein was obtained. The GST-OspG fusion protein was enriched from E. coli lysate by use of glutathione-agarose beads and subsequent digestion of the bound GST-OspG fusion proteins with a site-specific protease. [ H]palmitate labelling To determine whether OspG is expressed as a lipoprotein in E coli DH5α cells were transformed with either plasmid pZS77 or pOspG and labelled with [ H]palmitate (Figure 8). Plasmid pZS77 encodes the full-length OspG precursor protein with its normal N-terminal signal sequence, whereas pOspG specifies a protein that has the first 21 residues of the OspG precursors replaced with the sequence Met-Lys. After extraction by detergent-phase partitioning and separation by SDS-PAGE radioactive products were visualised by fluorography. E coli cells containing the full-length ospG gene (plasmid pZS77) expressed a 20 kDa lipoprotein that partitioned into the detergent phase whereas lipoproteins could not be observed in DH5α cells containing the truncated ospG gene. Subcellular localization of OspG To determine the subcullular localization of OspG in intact organisms, spirochetes were treated with proteinase K and subsequently analysed by immuno-blotting using the anti-10 3 serum applied to isolate ospG from the expression library. Anti-10 3 immunserum detected four low-molecular-weight proteins that partially disappeared after proteolysis whereas two structures in the molecular weight range of * 40 kDa were not affected by the proteinase treatment (17). To determine whether rOspG is recognized by anti-10 3 serum Western blotting was performed. Following absorption of anti-10 3 serum with rOspG the absorbed anti-10 3 serum recognised the same major proteins identical in size (Fig. 7 A) suggesting that OspG may not be present among the low-molecular- weight proteins contained in lysates from in vitro cultured ZS7 or alternatively, proteolysis of OspG may have occured. In order to confirm the identity of OspG and to see whether OspG is capable of being expressed in in vitro cultivated B. burgdorferi ZS7, hyperimmune murine anti-rOspG sera failed to detect OspG from 10^ in vitro-cultivated ZS7 organisms. In some experiments a weak unexpected reactivity could be observed with a 40-kDa polypeptide that may either be a variant of OspG or represent a B. burgdorferi protein that shares some epitopes with OspG. In order to assess expression of the ospG gene from in vitro cultured B. burgdorferi ZS7 total RNA was isolated and analysed for the presence of an opgG transcript by Northern blot hybridization. As a control for in vitro expression and RNA degradation, the NC filter was probed with an ospA gene probe. In contrast to the ospA probe that bound to a single approximately 2-kb transcript the ospG probe failed to detect an ospG transcript. These results suggest that deficiency in OspG expression during in vitro cultivation appears to be at the level of ospG transcription of mRNA stability. While OspG was undetectable in lysates from in vitro cultured B. burgdorferi ZS7, expression of OspG may be different during growth in the mammalian host. Immunoblot analysis using representative serum samples from both human Lyme disease patients and from experimentally infected mice revealed that antibodies with specificity for OspG were present. 6 out of 13 serum specimens from patients with documented Lyme borreliosis were shown to contain anti-OspG antibodies. In contrast, all sera from healthy donors (n = 8) did not contain antibodies that recognised rOspG (data not shown). These findings suggest that OspG is expressed only during the course of infection. Partial protection of SCID mice by anti-OspG immune serum In order to test the protective capacity of anti-OspG immune serum, SCID mice were treated with the following amounts of B. burgdorferi specific lg from pooled (Immune Sera) IS preparations: C.B-17 IS anti-10 8 (4,4μg B.b. lg/mouse), DBA/2 IS anti-10 3 (4.5μg B.b. lg/mouse), BALB/c IS anti-lipOspA (5μg B.b. lg/mouse), and BALB/c IS anti-recOspG (72 ng B.b. lg/mouse). However, IS anti-recOspG contained > 10-fold more antibodies to recOspG compared to IS anti-10 3 . SCID mice were injected i.p. with either of the indicated IS and subsequently challenged with 10-5 B. burgdorferi organisms. The development of clinical arthritis and the presence of spirochetes in ear biopsies were monitored. Inoculated but otherwise untreated or (Normal Mouse Sera) NMS-treated SCID mice developed clinical arthritis starting from day 6 p.i. on with severe swellings of the tibiotarsal joints developing between days 13 to 24 (end point). As described before, SCID mice passively immunised with IS anti-10 8 or IS anti-10 3 and IS anti-lipOspA showed none or only marginal signs of clinical arthritis. In contrast, in SCID mice passively immunized with IS anti-recOspG development of arthritis was only retarded as indicated by the development of mild swelling between days 6 and 18. At later time points, these mice developed more severe forms of arthritis indicating that this IS less efficient than the other IS to control infection. The fact that the IS anti-recOspG contained > 10-fold more antibodies to recOspG than the IS anti-10 3 but was much less protective suggest that in the IS anti-10 3 additional specificity's contribute to the control of infection. References 1. Aron, L., M. Alekshun, L. Perlee, I. Schwartz, H.P. Godfrey and F.C. Cabello. 1994. Cloning and DNA sequence analysis of bmpC, a gene encoding a potential membrane liproprotein of Borrelia burgdorferi. FEMS Microbiol. Lett. 123: 75-82. 2. Barbour, A.G., S.L. Tessier, and S.F. Hayes. 1984. Variation in a major surface protein of Lyme disease spirochetes. Infect. Immun. 45:94-100. 3. Barbour, A.G., and H.G. Stoenner. 1985. Antigenic variation of Borrelia hermsii. UCLA Symp.MolCell.Biol.New Ser. 20:123-125. 4. Bergstrόm, S., V.G. Bundoc, and A.G. Barbour. 1989. Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mol. Microbiol. 3:479-486. 5. Borst, P. and D.R. Greaves. 1987. Programmed gene rearrangements altering gene expression. Science 235:658-667. 6. Brandt, M.E., B.S. Riley, J.D. Radolf, and M.V. Norgard. 1990. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins. Infect. Immun 58:983-991. 7. Bressan, G.M. and K.K. Stanley. 1987. pUEX, a bacterial expression vector related to pEX with universal host specificity. Nucl. Acids Res. 15: 10056. 8. Burgdorfer, W., A.G. Barbour, S.F. Hayes, J.L. Benach, E. Grunwaldt, and J.P. Davis. 1982. Lyme disease - a tick-borne spirochestosis? Science 216: 1317-1319. 9. Champion, C.I., D.R. Blanco, J.T. Skare, D.A. Haake, M.Giladi, D.Foley, J.N. Miller, and M.A. Lovett. 1994. 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Immun. 60:657-661. 15. Fikrig, E., H. Tao, F.S. Kantor, S.W. Barthold, and R.A. Flavell. 1993. Evasion of protective immunity by Borrelia burgdorferi by truncation of outer surface protein B. Proc. Natl. Acad. Sci. USA 90:4092-4096. 16. Fuchs, H., R. Wallich, M.D. Kramer, and M.M. Simon. 1994. The outer surface protein A of Borrelia burgdorferi is a plasmin (ogen) binding protein. Proc. Natl. Acad. Sci. USA, 91: 12594-12598. 17. Gern, L., U.E. Schaible, and M.M. Simon. 1993. Mode of inoculation of the Lyme disease agent Borrelia burgdorferi influences infection and immune responses in inbred strains of mice. J. Infect. Dis. 167:971-975. 18. Hughes, C.A.N., S.M. Engstrom, L.A. Coleman, C.B. Kodner, and R.C. Johnson. 1993. Protective immunity is induced by a Borrelia burgdorferi mutant that lacks OspA and OspB. Infect. Immun. 61 :5115-5122. 19. Jonsson, M. , L. Noppa, A.G. Barbour, and S. Bergstrόm. 1992. Heterogeneity of outer membrane proteins in Borrelia burgdorferi: Comparison of osp operons of three isolates of different geographic origins. Infect. Immun. 60: 1845-1853. 20. Kurashige, S., M. Bissett, and L. Oshiro. 1990. Characterisation of a tick isolate of Borrelia burgdorferi that posseses a major low-molecular-weight surface protein. J. Clin. Microbiol. 28: 1362-1366. 21. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. 22. Lam, T.T., T.-P.K. Nguyen, R.R. Montgomery, F.S. Kantor, E. Fikrig, and R.A. Flavell. 1994. Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease. Infect. Immun. 62:290-298. 23. Margolis, N., and P. A. Rosa. 1993. Regulation of expression of major outer surface proteins in Borrelia burgdorferi. Infect. Immun. 61:2207-2210. 24. Masuzawa, T., H. Kawabata, Y. Beppu, K. Miyamoto, M. Nakao, N. Sato, K. Muramatsu, R.C. Johnson, and Y. Yanagihara. 1994. Characterisation of monoclonal antibodies for identification of Borrelia japonica, isolates from Ixodes ovatus. Microbiol. Immunol. 38:393-398. 25. Miller, A.M., V.L. MacKay, and K. A. Nasmyth. 1985. Identification and comparison of two sequence elements that confer cell-type specific transcription in yeast. Nature 314: 598-603. 25a. Miller, J.F., J.J. Mekalanos, and S. Falkow. 1989. Co-ordinate regulation and sensory transduction in the control of bacterial virulence. Science 243:916-922. 26. Needleman, S.B., and CD. Wunsch. 1970. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48:443-453. 27. Nguyen, T.-P.K., T.T. Lam, S.W. Barthold, S.R. Telford, in, R.A. Flavell, and E. Fikrig. 1994. Partial destruction of Borrelia burgdorferi within ticks that engorged on OspE- or OspF-immunised mice. Infect. Immun. 62:2079- 2084. 28. Norris, S.J., C.J. Carter, J.K. Howell, and A.G. Barbour. 1992. Low- passage-associated proteins of Borrelia burgdorferi B31: Characterisation and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein. Infect. Immun. 60:4662-4672. 29. O'Farrel, P.H. 1975. High resolution two-dimensional poly-acrylamide gel electrophoresis. J. Biol. Chem. 250:4007-4021. 30. Padula, S.J., A Sampieri. F. Dias, A. Szczepanski, and R.W. Ryan. 1993. Molecular characterisation and expression of p23 (OspC) from a North American strain of Borrelia burgdorferi. Infect. Immun. 61:5097-5105. 31. Preac-Mursic, V., B.Wilske, E.Patsouris, S. Jauris, G. Will, E. Soutschek, S. Rainhardt, G. Lehnert, U. Klockmann, and P. Mehraein. 1992. Active immunisation with pC protein of Borrelia burgdorferi protects gerbils against B. burgdorferi infection. Infection 20: 342-349. 32. Reindl, M., B. Redl, and G. Stόffler. 1993. Isolation and analysis of a linear plasmid-located gene of Borrelia burgdorferi B29 encoding a 27 kDa surface lipoprotein (P27) and its overexpression in Escherichia coli. Mol. Microbiol. 8: 1115-1124. 33. Reindl, M. , B. Redl, and G. Stόffler. 1994. A restriction map of the ospAB operon containing linear plasmid of Borrelia garinii B29, p.57-60. In: R. Cevenini, V. Sambri, M. Placa, (eds.) Advances in Lyme Borreliosis research. Proc. of the VI. Int. Conf. on Lyme Borreliosis. Bologna, Italy. 34. Sadziene, A., B. Wilske, M.S. Ferdows, and A.G. Barbour. 1993. The cryptic ospC gene of Borrelia burgdorferi B31 is located on a circular plasmid. Infect. Immun. 61:2192-2195. 35. Schaible, U.E., L. Gern, R. Wallich, M.D. Kramer, M.Prester, and M.M. Simon. 1993. Distinct patterns of protective antibodies are generated against Borrelia burgdorferi in mice experimentally inoculated with high and low doses of antigen. Immunol. Lett. 36:219-226. 36. Schaible, U.E., M.D. Kramer, K. Eichmann, M. Modolell, C. Museteanu, and M.M. Simon. 1990. Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc. Natl. Acad. Sci USA 87: 3768- 3772. 37. Schaible, U.E., M.D. Kramer, C. Museteanu, G. Zimmer, H. Mossmann, and M.M. Simon. 1989. The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis. J. Exp. Med. 170:1427-1432. 38. Schaible, U.E., R. Wallich, M.D. Kramer, L. Gern, J.F. Anderson, C. Museteanu, and M.M. Simon. 1993. Immune sera to individual Borrelia burgdorferi isolates or recombinant OspA thereof protect SCID mice against infection with homologous strains but only partially or nt at all agaisnt those of different OspA/OspB genotype. Vaccine 11 : 1049-1054. 39. Schouls, L.M., H.G.J. van der Heide, and J.D.A. van Embden. 1991. Characterisation of the 35-kilodalton Treponema pallidum subsp. pallidum recombinant lipoprotein TmpC and antibody response to lipidated and nonlipidated T. pallidum antigens. Infect. Immun. 59:3536-3546. 40. Simon, M.M., U.E. Schaible, M.D. Kramer, C.Eckerskorn, C. Museteanu, H.K. Mϋller-Hermelink, and R. Wallich. 1991. Recombinant outer surface protein A from Borrelia burgdorferi induces antibodies protective against spirochetal infection in mice. J. Infect. Dis. 164: 123-132. 41. Simon, M.M., U.E. Schaible, R. Wallich, and M.D. Kramer. 1991. A mouse model for Borrelia burgdorferi infection: Approach to a vaccine against Lyme disease. Immunol. Today 12: 11-16. 42. Simpson, W.J., W. Burgdorfer, M.E. Schrumpf, R.H. Karstens, and T.G. Schwan. 1991. Antibody to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals. J. Clin. Microbiol. 29:236-243. 43. Simpson, W.J., W. Cieplak, M.E. Schrumpf, A. G. Barbour, and T.G. Schwan. 1994. Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen. FEMS Microbiol. Lett. 119:381-388. 44. Steere, A.C. 1989. Lyme disease. N. Engl. J. Med. 321:586-596. 45. Wallich, R., C. Helmes, U.E. Schaible, Y. Lobet, S.E. Moter, M.D. Kramer, and M.M. Simon. 1992. Evaluation of genetic divergence among Borrelia burgdorferi isolates by use of OspA, fla, HSP60, and HSP70 gene probes. Infect. Immun. 60:4856-4866. 46. Wallich, R., S.E. Moter, M.M. Simon, K. Ebnet, A. Heiberger, and M.D. Kramer. 1990. The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): Molecular cloning, expression, and amplification of the gene. Infect. Immun. 58: 1711-1719. 47. Wallich, R., U.E. Schaible, M.M.Simon, A. Heiberger, and M.D. Kramer. 1989. Cloning and sequencing of the gene encoding the outer surface protein A (OspA) of a European Borrelia burgdorferi isolate. Nucleic Acids Res. 17:8864. 48. Wallich, R., M.M. Simon, H. Hofmann, S.E. Moter, U.E. Schaible, and M.D. Kramer. 1993. Molecular and immunological characterisation of novel polymorphic lipoprotein of Borrelia burgdorferi. Infect. Immun. 61 :4158-4166. 49. Wallich, R., et al. Unpublished data. 50. Wilske, B. , A.G. Barbour, S.Bergstrόm, N. Burman, B.I. Restrepo, P. A. Rosa, T. Schwan, E. Soutschek, and R. Wallich. 1992. Antigenic variation and strain heterogeneity in Borrelia spp. Res. Microbiol. 143:583-596. 51. Wu, H.C. 1987 Posttranslational modification and processing of memberane proteins in bacteria, p. 37-74; In: M. Inouye (ed.) Bacterial outer membranes as model systems. John Wiley & Sons, Inc., New York. 52. Wu, H.C. and M. Tokunaga. 1986. Biogenesis of lipoproteins in bacteria. Curr. Top. Microbiol. Immunol. 125:127-157. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Max Planck Gesellschaft zur Forderung der Wissenschaft e v (B) STREET: Busenstrasse 10 (C) CITY: Gottingen (E) COUNTRY: Germany (F) POSTAL CODE (ZIP) : D-3400 (A) NAME: Deutsches Krebsforschungszentrutn Stitung des offentilichen Rechts (B) STREET: Im Neuenheimer Feld 2Θ0 (C) CITY: Heildelberg (E) COUNTRY: Germany (F) POSTAL CODE (ZIP) : D-6900 (ii) TITLE OF INVENTION: Vaccines (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 196 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO ( iv) ANTI- SENSE : NO (vi ) ORIGINAL SOURCE : (A) ORGANISM : B Burgdorferi (C) INDIVIDUAL ISOLATE : Osp G (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 1 : Met Asn Lys Lys Met Lys Asn Leu lie lie Cys Ala Val Phe Val Leu 1 5 10 15 lie lie Ser Cys Lys lie Asp Ala Ser Ser Glu Asp Leu Lys Gin Asn 20 25 30 Val Lys Glu Lys Val Glu Gly Phe Leu Asp Lys Glu Leu Met Gin Gly 35 40 45 Asp Asp Pro Asn Asn Ser Leu Phe Asn Pro Pro Pro Val Leu Pro Ala 50 55 60 Ser Ser His Asp Asn Thr Pro Val Leu Lys Ala Val Gin Ala Lys Asp 65 70 75 80 Gly Gly Gin Gin Glu Gly Lys Glu Glu Lys Glu Lys Glu lie Gin Glu 85 90 95 Leu Lys Asp Lys lie Asp Lys Arg Lys Lys Glu Leu Glu Glu Ala Arg 100 105 110 Lys Lys Phe Gin Glu Phe Lys Glu Gin Val Glu Ser Ala Thr Gly Glu 115 120 125 Ser Thr Glu Lys Val Lys Lys Gin Gly Asn lie Gly Gin Lys Ala Leu 130 135 140 Lys Tyr Ala Lys Glu Leu Gly Val Asn Gly Ser Tyr Ser Val Asn Asp 145 150 155 160 Gly Thr Asn Thr Asn Asp Phe Val Lys Lys Val lie Asp Asp Ala Leu 165 170 175 Lys Asn lie Glu Glu Glu Leu Glu Lys Leu Ala Glu Pro Gin Asn lie 180 185 190 Glu Asp Lys Lys 195 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 591 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Borrelia burgdorferi (B) STRAIN: zs7 (vii) IMMEDIATE SOURCE: (B) CLONE: ospG (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATGAATAAGA AAATGAAAAA TTTAATTATT TGTGCAGTTT TTGTTTTGAT AATTTCTTGC 60 AAGATTGATG CGAGTAGTGA AGATTTAAAA CAAAATGTAA AAGAAAAAGT TGAAGGATTT 120 TTAGATAAAG AGTTAATGCA AGGTGACGAT CCTAATAACA GTCTGTTTAA TCCACCACCA 180 GTATTGCCGG CAAGTTCCCA CGATAACACA CCCGTATTAA AAGCGGTGCA AGCAAAAGAT 240 GGTGGTCAAC AAGAAGGAAA AGAAGAGAAA GAAAAAGAAA TTCAAGAATT AAAGGATAAA 300 ATAGATAAAA GAAAAAAAGA ATTAGAAGAG GCTAGAAAGA AATTTCAAGA ATTTAAAGAA 360 CAAGTTGAAT CTGCAACTGG AGAAAGTACT GAGAAAGTTA AAAAACAAGG AAATATTGGA 420 CAAAAAGCTT TAAAGTATGC TAAAGAATTG GGTGTAAATG GAAGTTATTC TGTTAATGAT 480 GGTACTAATA CTAATGATTT TGTAAAAAAG GTTATAGATG ATGCTCTTAA AAATATTGAG 540 GAAGAACTTG AAAAGCTAGC AGAGCCTCAA AATATAGAAG ATAAAAAATA A 591 Table 1 : Outer Surface Lipoproteins of Borrelia burgdorferi Table 2: Development of clinical arthritis in individual SCID mice nontreated or pretreated i.p. with indicated immunsera and subsequently inoculated with 1 xlO^ Borrelia burgdorferi ZS7 Serum Mouse clinical arthritis at price p.i.° recultivation transferred no. 6 13 18 20 27 (ear) None 1 - +/ + + +/ + + + +/+ + + +/+ + + 2 (±)/(±) +/+ + +/ + + + +/ + + + +/ + + + NMS 1 (±)/± +/ + + +/+ + + +/ + + + +/ + + + [5 μg/mouse] 2 ± (±) +/ + + +/+ + + +/ + + + +/ + + + 3 (±)/(±) +/ + + +/+ + + +/+ + + +/+ + + IS anti-10 3 1 _ _ . _ _ _ (DBA/2) 2 - - - - - - [4,5 μg spec. 3 ±/± - - - ±/(±) - lg/mouse] 4 - - - ( ± ) /- ±1- - IS anti-10 8 1 ±1- ±1- - . _ _ (C.B-17) 2 - -l± - - - - [4,4 μg spec. 3 - - - - - - lg/mouse] 4 ±/± -I± - - ±1- - IS anti- 1 (±)/(±) - - . (±)/- _ lipOspA 2 ±/± - - - - - (BALB/c) [5 μ 3 - - - - - - g spec. 4 - - - -/(±) - - lg/mouse] IS anti- 1 - (+)/(+) ±J- +/ + + + +/+ + + recOspG 2 - (+)/(+) ±J± (+)/(+) + +/+ + + (BALB/c) [72 3 - (+)/(+) ±/± + +/+ + + +/+ + + ng spec. 4 - (+)/(+) (+)/ + + +/+ + + +/+ + + lg/mouse] mAb LA 10* 1 - - - (±)/± (+)/(+) + [5 μg spec. 2 - (+)/(+) +/ + + +/ + + + +/+ + + lg/mouse] 3 - (+)/(+) +/ + + +/+ + + +/(+) + 4 - (+)/(+) + +/+ + + +/+ + + +/ + -I- + mAB LA2* [5 1 . - _ _. . . μg spec. 2 - ±1- (±)/- - - - lg/mouse] 3 - ±1- - - - - 4 - - - - - - "Scoring: + + severe; -I- less severe; (+) moderate; ± mild swelling; (±) marginal swelling, reddening; - no clinical signs in the left or right tibiotarsal joint. *mAB were described previously (20,35,39)

Claims: Claims 1. A purified OspG from B. burgdorferi, or an immunologically functional derivative thereof. 2. A purified OspG from B. burgdorferi characterised in that it has an apparent molecular weight of 22kDa, an isoelectric point of PI about 5.2, and has 196 amino-acid, or an immunologically functional derivative thereof. 3. A purified OspG from B. burgdorferi substantial as set out in figure 1. 4. A purified OspG as claimed in claim 3 having at least 80% homology with the amino acid sequence set out in figure 1. 5. A purified OspG as claimed herein, for use in Medicine. 6. A vaccine composition comprising a purified OspG as claimed in any of claims 1 to 4. 7. A vaccine composition as claimed in claim 6 additionally comprising a further B.burgdorferi antigen. 8. A vaccine composition as claimed in claim 6 additionally comprising an OspA antigen from Borrelia. 9. A vaccine composition as claimed in any of claims 6-8 additionally comprising QS21 or 3D-MPL. 10. Use of an OspG antigen as claimed in claims 1 to 4 for the preparation of a vaccine for the immunoprophylatic or immuno therapeutic treatment of a patient suffering from or susceptible to Lyme disease. 11. A method of treating a patient suffering from or susceptible to lyme disease comprising administering an effective amount of a composition according to claims 6-9. 12. An isolated DNA sequence encoding an OspG protein or immunologically functional derivative as claimed in claims 1 to 4. 13. An isolated DNA sequence encoding an OspG protein as claimed in claim 12 further characterised by having the sequence substantially as set forth in figure 1. 14. An expression vector containing a DNA sequence as claimed in claim 12 to 13. 15. A host cell transformed with a DNA sequence of claim 12 or 13. 16. A method of producing a purified OspG as claimed in claims 1 to 4, comprising transforming a cell with an expression vector of claim 14, and isolating culturing the cells, and isolating the resulting protein. 17. A method of producing a vaccine comprising OspG of claims 1 to 4, comprising admixing said OspG or immunologically functional derivative with a pharmaceutically acceptable excipient. 18. A diagnostic kit comprising an OspG from B.burgdorferi.

Inventors: KRAMER MICHAEL D, SIMON MARKUS M, WALLICH REINHARD, KRAMER, MICHAEL, D., SIMON, MARKUS, M., WALLICH, REINHARD
Applicants: DEUTSCHES KREBSFORSCH, KRAMER MICHAEL D, MAX PLANCK GESELLSCHAFT, SIMON MARKUS M, WALLICH REINHARD, DEUTSCHES KREBSFORSCHUNGSZENTRUM, KRAMER, MICHAEL, D., MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSC, SIMON, MARKUS, M., WALLICH, REINHARD
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, A61K39, A61P33, C07K14, C12N5, C12N15, C12P21, G01N33