WO1995009919A1 - Vaccines and diagnostics for borrelia burgdorferi - Google Patents

Abstract

The present invention provides a novel protein from Borrelia burgdorferi. The protein, known as LpLA7, is useful in the diagnosis of Lyme disease. The protein may also be utilised in vaccine compositions against Lyme disease.

Description

VACCINES AND DIAGNOSTICS FOR BORRELIA BURGDORFERI

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, LpLA7. The deduced amino acid sequence of this protein exhibits no significant homologies to other known Borrelial antigens such as OspA, OspB, pC or OspD (4,10,16,20). The present invention further provides LpLA7 in a purified form.

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 (29). B. burgdorferi strains isolated from different biological sources and geographic areas are heterogeneous (1,33,34) 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 (35).

To date, a variety of B.burgdorferi antigens such as outer surface protein A (OspA), OspB, pC and p100, are employed for serological diagnosis and as putative candidates for vaccine development (25). 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 LpLA7. This molecule is characterised by having a in apparent molecular weight of approximately 21.9 kDa, and an isoelectric point of PI =5.7 when obtain from certain isolates it is reactive with the monoclonal antibody LA7. The hybridoma secreting LA-7 monoclonal has been deposited at the Public Health Laboratory Services PHLS Centre for applied Microbiology and Research European Collection of Animal Cell Cultures, Division of Biologies, Salisbury Wiltshire SP4 0J9, United Kingdom, on 21 September 1993 under accession No. 93092118. The mature protein is further characterised as having a sequence of 173 amino acids. Unlike the gene for OspA, B or C, the gene is located naturally on the bacterial chromosome. LpLA7 has the amino acid sequence substantially as set forth in sequence ID 1. This sequence includes the signal sequence. Accordingly, the present invention provides an isolated protein derived from B.burgdorferi characterised in that it has a molecular weight of between 21.5-22.5 kDa as determined by SDS gel electrophoresis and an isoelectric point of between 5.4 and 5.9 and is recognised by the monoclonal antibody LA7 or an

immunologically or antigenically equivalent fragment or derivative thereof.

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 sequence ID 1.

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 sequence ID 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 sequence ID 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 in E.coli, the LpLA7 lipoprotein will be expressed without the signal sequence.

Cleavage of the signal sequence, to remove the N-teπninal 21 amino acids will result in a molecule being produced in E.coli which is lipidated at amino acid # 22 (cysteine).

Immunoblot analysis of individual B.burgdorferi isolates with mAb LA7 revealed differential expression of the epitope: all strains of the OspA genotypes I, III and V strained positive, whereas all strains of the genotypes II, IV and VI strained negative. The fact that DNA from all B.burgdorferi isolates exhibited characteristic RFLP signals with the radiolabeled insert from plasmid pLA7-2 indicates that all tested strains contain the respective gene but not necessarily the LA7 epitope. Surface proteolysis of intact B.burgdorferi which led to nearly complete degradation of OspA and OspB did not significantly affect the LpLA7. The finding indicates that the majority of the LpLA7 lipoprotein is either not surface exposed or resistant to proteinase K digestion.

Immunoblot analysis demonstrated that LpLA7 is recognised by sera from mice previously infected with intact spirochetes that suggests that the native protein is immunogenic in this species. In preliminary experiments using human sera from forest workers four out of eight sera tested contained antibodies active with LpLA7 (see table 1) indicating the LpLA7 is also immunogenic in humans and may be a useful additional marker for diagnosis of Lyme disease. In such cases, either the antigen or the monoclonal antibody may be utilised in suitable diagnostic kits. This finding is even more significant since those workers with a strong LpLA7 antibody response were asymptomatic indicating that this protein has a role in the protection from Lyme disease. Finally, LpLA7 may contribute to the diverse cellular and humoral immune phenomena in mice and humans. The fact that lipoproteins have been shown before to be potent activators of macrophages and lymphocytes suggests that it is possible that spirochetal lipoproteins including LpLA7 may contribute to various host responses associated with either protection and/or pathogenesis

(5,9,12).

The present inventors have further indentified and sequenced the gene for LpLA7. Accordingly, the invention provides a DNA molecule having the sequence encoding for a protein LpLA7 or fragment or derivative thereof. Preferably the DNA sequence is substantially as set forth in sequence ID 2. In an analogous manner, the present invention also provided the corresponding RNA molecule.

The term substantially as used herein means at least 70% identity to the sequence set forth in sequence ID 2, 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 ID 2, or a fragment thereof, or a DNA sequence which hybridises to said sequence and which codes for a protein which exhibits LpLA7 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 50ml 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 MgCl- , 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 50ml 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 LpLA7 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 LpLA7 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, Maniatis 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 physically, chemically or enzymatically and the protein product isolated from the resulting lysate. 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 baculo virus 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 LpLA7 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 me 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.

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 mg of protein, preferably 1-200 mg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody litres and other responses in subjects. Following an initial vaccination, subjects may receive an additional administration to enhance their immune response.

The DNA or RNA of the present invention may also be utilised in direct immunisation, by administering the DNA or RNA directly to a patent.

The use of nucleic acid for vaccination purposes is known in the art, for example from International patent application No. WO90/11092 (Vical), and International Patent application No.17681/92 (Pasteur Merieux).

Accordingly in one aspect of the present invention there is provided a pharmaceutical composition comprising a nucleic acid molecular encoding LpLA7 or an immunologically equivalent derivative thereof and a pharmaceutically acceptable excipient.

The vaccines of the present invention may additionally include other antigens, or nucleic acids encoding therefor.

In particular, the vaccines of the present invention may additionally include one or more of the outer surface proteins of B.burgdorferi, such as OspA or OspB.

The present invention also relates to antibodies, preferably monoclonal antibodies which are specific for LpLA7. Such antibodies find utility in the diagnosis and also in the prevention of Lyme disease. Examples

I. Materials and Meth ods

1.1 Borrelia strains

B.Burgdorferi strains used in this study (Table 2) were described elsewhere (26,27). Borrelia were grown in modified Barbour-Stoenner-Kelly II (BSK II) medium (2) at 33°C. Spirochetes were harvested by centifugation at 10,000 × 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 MCI 061 followed by expression screening using a mAb LA7 (32).

1.2 South ern blot hybridization

Total genomic DNA was extracted from Borrelia organisms as described previously (32). Approximately 5 mg 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 as described (33). Briefly, using ³²p-labeled probes hybridization was done over night at 65°C in 0.5 M NaHPO₄/7% NaDodSO_4, pH7.2. After washing in 40 mM NaHPO₄/1 % NaDodSO_4, 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 (8).

1.3 Triton X-114 phase partitioning. B.burgdorferi was grown in BSKII medium, and borrelial lipoprotein-enriched preparations where obtained by extraction and phase separation with the nonionic detergent Triton X-114. In brief, washed bacteria were suspended in ice-cold 2% (vol/vol) Triton X-114 (Fluka Chemie, Buchs, Switzerland) in PBS (pH 7.4) at 5 × 10⁹ organisms per ml of detergent. After incubation overnight at 4°C on an end-over-end rotating wheel, insoluble material was removed by centrifugation at 20,000 x g for 30 min. at 4°C. The supernatant was phase separated by being warmed to 37°C for 15 min in a water bath and then was centrifuged for 15 min at room temperature in a

microcentrifuge. The separated detergent and aqueous phases were then washed four times in the following manner. The detergent phase was suspended to its original volume in ice-cold PBS, while the aqueous phase was brought to a final concentration of 2% detergent by the addition of cold 10% Triton X-114 and then was phase separated as described above. Protein concentrations were determined by the bicinchoninic acid method (Pierce Chemical Co. , Rockford III.).

1.4 Intrinsic radiolabeling of B.burgdorferi proteins

B.burgdorferi was grown in BSKII medium at 33 °C to a density of 10⁸ organisms per ml. Radiolabeled palmitate was then added to a final concentration of 0.25mCi/ml, and incubation was continued for 2 days.

1.5 Gel electrophoresis

For electrophoresis on one-dimensional SDS/PAGE slab gels according to Laemmli (17), 40 ml of each lysate (equivalent to≈10⁸ organisms) were mixed with 10ml 5 x reducing sample buffer. Two-dimensional polyacrylamide gel electrophoresis was carried out as described by O'Farrel (21), 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 Norris et al. (20). The proteins were then separated by SDS-PAGE and individual antigens were identified by immunoblotting.

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 mg/ml) and nitro blue tetrazolium (NBT, Sigma; 330 mg/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 T⁷Sequencing kit (Pharmacia) according to the manufacturer's recommendations (32).

1.8 Amino acid sequence analyses

[[Simultaneous alignment for protein sequences and phylogenetic tree construction were performed by using the HUSAR software (30).

1.9 Immunofluorescence

B.burgdorferi organisms were washed twice in PBS, transferred to adhesion slides (Superior, Bad Mergentheim, Germany) 10⁵ 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 fiuorescein isothiocyanatelabeled 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 B331 antigens as described previously (15). II. Results

Analysis of LpLA7 by phase partitioning with Triton X-114. B.Burgdorferi ZS7 was labeled with [³H]palmitate and subsequently extracted with Triton X-114. Radiolabeled proteins were separated by one-dimensional SDS-PAGE and visualized by fluorography. From the unpartitioned preparations of strain ZS7, at least seven lipoproteins with apparent molecular masses of about 55, 34, 31, 24, 21, 19, and 14: kDa could be identified. The most prominent polypeptides, with molecular masses of 31 and 34 kDa, are likely to represents the OspA and OspB antigens, respectively. All of the [³H]palmitate-labeled polypeptides partitioned exclusively into the detergent phase. One of the low-molecular-mass proteins (about 20 kDa) presumably represents the LpLA7 antigen. For confirmation of this assumption, two-dimensional gel electrophoresis was performed with [³H]palmitate-labeled B.burgdorferi ZS7. The proteins identified by two-dimensional SDS-PAGE of whole-cell lysates included LpLA7, OspA. Because of the limited pi range (4 to 8), the OspB molecule was not seen; it has a calculated pi of 9.7.

II.l Cloning and sequence analysis of th e LA7 gene

A pUEXl expression library of B.Burgdorferi ZS7 genomic DNA was screened using mAb LA7. Three independent recombinant clones were identified. All three clones overlapped and comprised a colinear sequence encompassing the entire coding region of the putative LA7 gene. The protein encoding sequence of the LA-7 gene is dupicted in ID No:2. Six bases upstream of the ATG start codon a consensus ribosomal binding site (-AAGGGAGA-) is located. Further upstream from this translational start point at position -34 to -28, a putative RNA polymerase start site, also known as '-10' box (-TAATATG-), is located, which is preceeded by a '-35' region (-TGTGTACAAAA-) at position -77 to 67 (24). Downstream of the protein encoding region, a perfect 12-mer palindromic sequence (- ATAGGCTTTAAT-) starting at nucleotide 603 is found. The relatively high expression of IpLA7 in E. Coli suggests that similar regulatory sequences are functioning in both species. The molecular weight of the protein predicted from the amino acid sequence of 194 residues is 21.865 kDa. Molecular analysis and sequence comparison of LpLA7 with other proteins reveals sequence similarity to the signal peptides of prokaryotic lipoproteins (36,37). LpLA7 resembles other outer membrane proteins comprising a large portion of charged amino acid residues ( > 30%) and one single segment of hydrophobicity in the N-terminal region (19). Beyond the leader sequence, the deduced LpLA7 sequence is largely hydrophilic and predominantly alpha-helical as predicted by secondary structure analyses (data not shown). The isolation of two additional low-molecular- weight proteins from B.burgdorferi (pC and OspD) was reported recently (10,20). All three sequences indicates the presence of Leu-X- Y-Z-Cys at the COOH terminal end of the signal sequence which represents a common feature of lipoprotein precursors in bacteria (36). Cleavage at the cysteine residue by signal peptidase II would yield a polypeptide with a predicted mass of 19.341 Da. The first fifty amino acids of LpLA7 show a homology of 30% with the N-teπήinal sequence of pC and of 20% with that of OspD (Fig.2). No significant homology was found to the N-terminal amino acid sequence of a low-molecular-weight (22 kDa) protein recently described by Luft et al. (18).

II.2 Restriction fragment length polymorphism of th e LA-7 gene

The RFLP analysis for LA7 using endonuclease HindIII revealed at least six distinct hybridization patterns among the 40 B.burgdorferi isolates tested. All strains previously shown to express OspA genotype I antigens (e.g. B31, ZS7; 33) are characterized by one hybridization fragment of 2 kb, those expressing OspA genotype II (e.g. ZQ1) by one fragment of 0.8 kb. B.burgdorferi strains expressing OspA genotype III (19857 and 21038) exhibit one hybridizing fragment of 0.75 kb and all isolates of OspA genotype FV (e.g. ACA-1) showed one fragment of 1.9 kb. B.burgdorferi strains with OspA genotypes V (20047) and VI (S90) exhibited either a fragment of 0.6 kb or of > 0.8 kb, respectively.

The distinct patterns of the LA7-specific RFLP correlated with the differential expression of the epitope defined by mAb LA7. Only B.burgdorferi strains of the OspA genotypes I, III and V (e.g. ZS7, 19857, 20047) but not those of OspA genotype II, IV and VI (e.g. ZQ1, ACA-1, S90 reacted with mAb LA7 (Table 2).

In contrast to the OspA gene which is located on the 49-kilobase

extrachromosomal linear plasmid, LpLA7 is encoded by a chromosomal gene as elucidated by pulse field gel electrophoresis (data not shown). DNAs isolated from members of other species of Borrelia such as Borrelia coriaceae Co53, B.hermsii, B.turicatae, and T. pallidum (data not shown) did not hybridize to the LA7-specific probe indicating specificity for B.burgdorferi. II.3 2D immunoblot analysis of LpLA7

To analyse the expression of lpLA7 in B.burgdorferi organisms, whole-cell lysates were separated on 2D gels and subsequently either silver stained or immuno-blotted using mAbs directed against OspA, OspB and flagellin.LpLA7 is clearly separated from other low molecular weight proteins. Another 20-kDa protein migrated at a charge position similar to that of OspB and reacted with OspB mAb LA32 (15). Whether mAb LA32 recognizes a truncated version of OspB or whether it recognizes an antigenic epitope shared by two independent proteins needs further investigations. Yet, the finding is reminiscent of recently published data by Norris et al. showing that mAb H6831 recognizes a crossreactive epitope expressed on both OspB and a 20 kDa polypeptide (20).

II.4 Expression of LpLA7 in E.coli

We examined the expression of IpLA7 in E.coli transformed with plasmids carrying full length (pLA7-2) or truncated versions which is devoid of the 21 N- terminal amino acids containing the signal sequence (ρLA7-A2) of the LA7 gene. As controls the full length (pOspA) and truncated versions (pOspA-17) of the OspA gene were used in similar experiments. The respective E.coli cells were extracted by detergent phase partioning and analysed by gel electrophoresis. E.coli cells carrying plasmid pLA7-2 expressed one band widi an apparent molecular mass of 21 kDa, E. coli cells harbouring pOspA expressed a band of 31 kDa in size. In contrast, no lipoproteins of the expected molecular masses were formed from the trucated genes of LA7 (pLA7-A2) and OspA (pOspA-17). However, expression of non-lipidated forms of OspA and IpLA7 by the two truncated clones, pOspA-17 and pLA7-A2, was revealed by immuno-blotting. II.5 Subcellular localization of LpLA7

To explore the localization of the LA7 epitope in intact spirochetes, mAb LA7 as well as a selected range of mAbs directed against different B.burgdorferi structures were tested for staining the bacteria by immunofluorescence. As shown previously, two mAb specific for OspA (LA2 and LA26) and one mAb specific for OspB (LA25) gave bright fluorescence staining, whereas another mAb specific for OspA (LA5) as well as the flagellin mAb LA21 stained much weaker. A patchy and weak staining pattern was also observed with mAb LA7 suggesting that the relevant epitope is not readily accessible to this mAb.

To further elucidate the localization of LpLA7 protein, intact B. Burgdorferi

ZS7 organisms were treated with proteinase K and subsequently analysed by SDS-PAGE. The bands corresponding to OspA, OspB and a 24-kDa protein showed a decreased intensity compared to the untreated control. In contrast, LpLA7 was resistent to degradation under these conditions, as revealed by Western blot analysis using mAb LA7. The fact, that other internally localized proteins such as flagellin were also not affected under similar conditions suggests that LpLA7 is also not accessible for exogenous proteolytic enzymes. II.C Immunoreactivity of LpLA7

Sera derived from mice previously inoculated with 10- B. burgdorferi (ZS7) reacted with native LpLA7 antigen as well as with the recombinant proteins encoded by pLA7-2 or pLA7-8 but not with lysates of E. coli carrying only the cloning vector. In the affinity purified preparation of recombinant LpLA7 two additional larger bands were stained with mAb LA7; these bands may represent artefacts generated during enrichment.

In further experiments, human sera from (i) healthy donors, (ii) patients with ECM and A.C. A, and (iii) members of a high-risk group (forest workers) were similarly analyzed for the presence of LpLA7-reactive antibodies. As shown in Table 3, all serum specimens from healthy individuals (m = 5) were negative when tested in an ELISA (on B31 sonicate) and by Western blot analysis with a total B. burgdorferi lysate as well as recombinant LpLA7. All serum specimens from patients with ECM and ACA (m = 12) were positive when tested in an ELISA (on B331 sonicate) and by Western blot analysis with a total lysate but negative when tested by Western blot analysis with recombinant LpLA7. In contrast, three serum specimens from forest workers who were healthy but were found seropositive in an

ELISA (on B31 sonicate) and by Western blot analysis with a total lysate were also reactive with recombinant LpLA7 in Western blotting (Tables 1 and 3).

The data indicates that LpLA7 is immunogenic in humans and, moreover, that this lipoprotein maybe a useful marker for the diagnosis of Lyme disease.

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Claims

C l a i m s

1. A purified B.burgdorferi protein characterised in that it: a) reacts with monoclonal antibody LA7.

b) has a molecular weight of between 21.5-22.5 as determined by SDS gel electrophorosis and an isoelectric point of between 5.4 and 5.9 or an

immunologically or antigenically equivalent fragment or dervative thereof.

2. A B.burgdorferi protein or an immunologically or antigenically equivalent fragment or derivative thereof having at least 80% homology to the amino acid sequence depicted in sequence ID No.1.

3. A nucleic acid molecule encoding a protein or fragment or derivative thereof as claimed in claim 1 or 2.

4. A protein, fragment or derivative thereof as claimed in claim 1 or 2 for use in medicine or diagnosis.

5. A pharmaceutical or diagnostic composition comprising a protein, fragment or derivative as claimed in claim 1 or 2 and a pharmaceutically acceptable excipient, diluent or adjuvant.

6. A nucleic acid molecule as claimed in claim 3 for use in medicine.

7. A pharmaceutical compositions comprising a nucleic acid molecule of claim 3, and a pharmaceutical acceptable excipient.

8. A monoclonal antibody secreted by the hybridoma LA7.

9. A monoclonal antibody specific for LpLA7.

10. A vector containing a nucleic acid molecule according to claim 3.

11. A host cell transformed with a nucleic acid molecule according to claim 3.

12. A method of diagnosing a patient suffereing from Borrelia infection, the method comprising contacting a sample from a patient with an antibody or antigen as claimed in any of claims 1, 2 8 or 9.

13. A method of treatment of a patient susceptible to Borrelia infection, me method comprising administering an effective, non-toxic amount of a nucleic acid, protein or antibody as claimed in any of claims 1, 2, 3, 8 or 9.