CN1159831A - Vaccines containing Borrelia brucei OSPG - Google Patents

Abstract

The present invention provides a novel antigen, OspG, from Borrelia burgdorferi which is useful in the prevention and diagnosis of Lyme disease. The invention discloses the cloning, sequence and expression of this antigen.

Description

Vaccines containing Borrelia brucei OspG

The present invention relates to neoantigens derived from Borrelia burgdorferi and derivatives thereof, processes for their preparation, their use in human and animal medicine and diagnosis and pharmaceutical compositions containing them.

Specifically, the present invention provides clonal expression of a novel polymorphic lipoprotein-OspG of B. burgdorferi. OspG was previously identified as 1p77. The deduced amino acid sequence of this protein showed no significant homology to other known Borrelia antigens, such as OspA, OspB, OspC, OspD, OspE, OspF and P27. These outer surface proteins share 41%-65% similarity to OspG (Table 1).

Lyme disease (Lyme disease) is the most common infectious disease transmitted by vector animals in warm climates. Borrelia brucei in its pathogenic spirochete causes diseases in multiple systems of the human body, affecting the skin, nervous system, and joints and heart (8, 44). Borrelia brucei isolates from different biological sources and geographic regions are heterogeneous (2, 19, 38, 45, 50), and disease manifestation patterns are thought to be influenced by antigenic differences among Borrelia strains. Although there have been recent reports of attempts to classify B. brucei using immunological or molecular criteria, the taxonomy of B. brucei remains a controversial but active area of research.

So far, various Borrelia bruceli antigens, such as outer surface protein A (OspA), OspB, PC, and p100, have been used in serological diagnosis and as options for future vaccine development (13, 14, 36 , 38, 40, 41). However, due to their apparent heterogeneity, clear scales are still lacking for the development of species-specific diagnostic criteria and for the development of a peptide vaccine that would confer protection against each subspecies.

The inventors of the present invention have further discovered a lipoprotein called OspG from Borrelia brucei. The characteristics of the molecule are: the apparent molecular weight is about 22KDa, the isoelectric point PI=5.2 and contains 196 amino acids. The mature protein is further characterized by a large hydrophobic domain of about 20 amino acids in the amino-terminal portion of OspG. This N-terminal peptide corresponds to the leader signal peptide of a typical prokaryotic lipoprotein precursor. The carboxy-terminus of the hydrophobic core is a cleavage site likely recognized by the Borrelia brucei signal peptidase. This possible cleavage site in OspG is located between serine at position 19 and cysteine at position 20, and the sequence near the cleavage site of OspG is L-l-l-S-C.

Unlike the genes of OspA, B, C, D, E, F and P27, the OspG gene is naturally located on the 55Kb plasmid of Borrelia brucei ZS7. Also note the weak cross-reactivity of the OspG probe with the 45Kb plasmid. The amino acid sequence of OspG is roughly shown in FIG. 1 . Its further characteristic is that it is only expressed during the infection period, and cannot be detected in Borrelia brucei cultured in vitro.

Therefore, the present invention provides an isolated protein obtained from Borrelia brucei, characterized in that: the molecular weight measured by two-dimensional SDS gel electrophoresis is between 20-22KDa, and the isoelectric point is between 4.9-5.4 .

The inventors of the present invention also provide a protein, or an immunologically or antibody equivalent fragment or derivative thereof, which has at least 80% homology with the amino acid sequence shown in FIG. 1 .

This protein and its corresponding DNA and RNA sequences have applications in both vaccines and diagnostics.

The invention preferably 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 invention may be a fusion protein, in which case it is characterized in that it contains part of its amino acid sequence or a fragment thereof. Preferably, this portion has at least 80% homology, more preferably 85%, more preferably 90%, and most preferably 95% homology with the protein sequence in Figure 1 .

The preferred protein purity is determined by SDS-polyacrylamide gel electrophoresis at least 70%, most preferably 80%, more preferably at least 90%.

The proteins of the invention may be lipoproteins or proteins prepared without lipids. When produced by recombinant techniques, lipoproteins are expressed together with a signal sequence. Excision of the signal sequence and removal of the N-terminal 19 amino acids resulted in a non-lipidated molecule.

Western blot analysis showed that OspG was recognized by sera from mice pre-infected with intact spirochetes. The results of this analysis indicate that the native protein is immunogenic.

The inventors of the present invention further identified and sequenced the OspG gene. Accordingly, the present invention provides DNA sequences encoding OspG proteins or fragments or derivatives thereof. A preferred DNA sequence is roughly as shown in FIG. 1 .

The word "approximately" used here means at least 70% identical to the sequence in Figure 1, preferably 80% identical, more preferably more than 85%, most preferably at least 95% identical. In particular, the present invention provides a DNA sequence approximately identical to the sequence of Figure 1, or a fragment thereof, or a DNA sequence capable of hybridizing to said sequence and encoding a protein exhibiting OspG antigenicity.

The DNA of the present invention can be prepared by enzymatic polymerization of DNA, which can be carried out in vitro with a DNA polymerase, such as DNA polymerase I (Klenow fragment), in a suitable buffer solution. The buffer solution contains the required nucleoside triphosphates dATP, dCTP, dGTP and dTTP, the temperature is 10°-37°C, and the volume is usually 50 μl or less. Enzymatic ligation of DNA fragments can use DNA ligase, such as T4 DNA ligase, in a suitable buffer, such as containing 0.05M Tris (pH7.4), 0.01M MgCl ₂ , 0.01M dithiothreitol, 1mM The reaction is carried out in a buffer solution of spermidine, 1 mM ATP and 0.1 mg/ml bovine serum albumin at an ambient temperature of 4°C, and the reaction volume is usually 50 μl or less. Chemical synthesis of DNA polymers or fragments can be performed by conventional phosphotriester, phosphite, or phosphoramidite chemistry using solid-phase techniques such as in "Chemical and Enzymatic Synthesis of Gene Fragments—A Laboratory Manual" (Chemical and Enzymatic Synthesis of GeneFragments—A Laboratory Manual) (eds. HG Gassen and A. Lang, Verlag Chemie, Weinheim, 1982), or in other scientific literature, such as MJ Gait, HWD Matthes, M. Singh, BSSproat and RCTitmas "Nucleic Acids Research" (Nucleic Acids Research), 1982, 10, 6243; "Tetrahedron Letters" by BSSproat and W.Bannwarth, 1983, 24, 5771; "Tetrahedron Letters" by MDMatteucci and MHCuthers, 1980 , 21, 719; MD Matteucci and MH Caruthers, "Journal of the American Chemical Society", 1981, 103, 3185; SPAdams et al., "Journal of the American Chemical Society", 1983, 105, 661; NDSinha, J. Biernat, J. McMannus and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and HWDMatthes et al., EMBO Journal, 1984 , 3, 801.

Alternatively, the coding sequence can be obtained from Borrelia brucei mRNA and commercially available cDNA kits using known techniques (eg, reverse transcription of mRNA to generate complementary cDNA strands).

The present invention is not limited to the specific sequences disclosed, but includes all molecules encoding the protein or its immunogenic derivatives described above.

A DNA polymer encoding a mutant protein of the present invention can be prepared by a conventional method of site-directed mutagenesis of a cDNA encoding the protein. This method is disclosed in "Nature" (Nature) 1982, 299, 756-758 by G. Winter et al. or "Nucl. Acids Res." (Nucl. Acids Res.) by Zoller and Smith 1982, 10, 6487-6500. Or prepare by deletion mutagenesis, as by Chan and Smith in "Nucleic Acids Research" (Nucl.Acids Res.), 1984,12,2407-2419 or Winter et al. in "Journal of Biochemical Association" (Biochem.Soc.Trans .), 1984, 12, 224-225 described.

In one aspect, the invention provides a method comprising the steps of:

i) Preparation of a replicable or integrated expression vector capable of expressing a DNA polymer in a host cell containing a nucleotide sequence encoding said OspG protein or an immunogenic derivative thereof ;

ii) transforming a host cell with said vector;

iii) cultivating said transformed host cell under conditions in which said DNA polymer is capable of expressing said protein; and

iv) recovering the protein.

The term "transformation" here refers to the introduction of foreign DNA into host cells by means of transformation, transfection or infection with an appropriate plasmid or virus vector. For example, with conventional methods, see the description of "Genetic Engineering" (S.M. Kingsman and A.J. Kingsman eds., Blackwell Scientific Publications, Oxford, UK, 1988). "Transformed" or "transformant" is used hereinafter to refer to a resulting host cell that contains and expresses a desired foreign gene.

The expression vector is novel and also forms part of the present invention.

A replicable expression vector can be prepared according to the method of the present invention. By cleaving a vector compatible with the host cell, a linear DNA fragment with an intact replicon is formed and combined with one or more DNA molecules. These DNA molecules are combined with said linear fragment encoding the desired product, such as a DNA polymer encoding OspG protein or a fragment thereof, under ligation conditions.

In this way, DNA polymers can be prepared or synthesized as desired in vector construction.

The choice of vector will depend in part on the host cells, which may be prokaryotic or eukaryotic. Suitable vectors include plasmids, phage, cosmids and recombinant viruses.

The replicable expression vector can be prepared by conventional methods, using appropriate enzymes to carry out restriction enzyme digestion of DNA, polymerization reaction and ligation. For example, as in Maniatis et al., cited above.

According to the present invention, the preparation of the recombinant host cell is realized by transforming the host cell with the replicable expression vector of the present invention under transformation conditions. Suitable transformation conditions are conventional conditions, such as the method described by Maniatis et al. cited above, or "DNA Cloning" (Vol. II, edited by D.M. Glover, IRL Publishing Co., 1985).

The choice of transformation conditions depends on the host cell. For bacterial hosts, such as Escherichia coli (E.coli) can be treated with CaCl ₂ solution (Cohen et al., "Proc. Nat. Acad. Sci.), 1973, 69, 2110) or with RbCl containing , MnCl ₂ , potassium acetate and glycerol, followed by 3-[N-morpholino]-propane-sulfonic acid, RbCl and glycerol. Cultured mammalian cells can be transformed into cells by the calcium co-precipitation method. The invention also extends to a host cell transformed with a replicable expression vector of the invention.

The transformed host cells are cultured under conditions in which the DNA polymer can be expressed, in a conventional manner, as described by Maniatis et al. or in "DNA Cloning" cited above. In this way, it is preferable to provide nutrients to the cells and to culture them below 45°C.

The recovery of the product is carried out according to the conventional method depending on the host cell. When the host cells are bacteria, such as E. coli, they can be lysed chemically or enzymatically and the protein product isolated from the resulting lysate or supernatant. When the host cells are mammalian cells, the product can usually be isolated from the nutrient medium or cell-free extract. Conventional protein separation techniques include: selective precipitation, absorption chromatography, and affinity chromatography including monoclonal antibody affinity columns.

Alternatively, expression can be performed in insect cells using a suitable vector, such as baculovirus. In a particular aspect of the invention, said protein is expressed in Lepidopteran insect cells in order to produce an immunogenic polypeptide. For expression of proteins in Lepidopteran cells, it is preferred to use the baculovirus expression system. In such systems, an expression cassette containing the protein coding sequence operably linked to a baculovirus promoter is typically located in a shuttle vector. Such vectors contain sufficient amounts of bacterial DNA to allow propagation of the shuttle vector in E. coli or other suitable prokaryotic hosts. The shuttle vector also contains sufficient amounts of baculovirus DNA flanking the desired protein-coding sequences to enable recombination between wild-type baculovirus and the heterologous gene. The recombinant vector is then co-transfected into Lepidopteran insect cells together with wild-type baculovirus DNA. Recombinant baculoviruses produced by homologous recombination are then selected and plaque purified according to standard methods. See Summers et al., "Texas Agricultural Experimental Station Bulletin (TAES Bull), NR1555, May 1987.

Methods for expressing circumsporozoite protein (CS) in insect cells are described in detail in USSN 287,934 to Smith Kline RIT (WO/US89/05550).

Production in insect cells can also be achieved by infecting insect larvae. For example, this protein can be obtained from the larvae of tobacco bud moth (Heliothis virescens), that is, feed the larvae with the recombinant baculovirus of the present invention and a small amount of wild-type baculovirus, and extract it from hemolymph after about 2 days the protein. See for example PCT/WO88/02030 to Miller et al.

The novel proteins of the present invention can also be expressed in yeast cells, like the expression of the CS protein in EP-A-0278941.

The present invention also relates to vaccine compositions comprising OspG or fragments or derivatives thereof.

The vaccine of the present invention can directly use the protein aqueous solution. Alternatively, the protein, lyophilized or not, is mixed with or absorbed with any known adjuvant. These adjuvants include, but are not limited to, aluminum hydroxide, muramyl dipeptides and saponins (such as Quil A). Particularly preferred adjuvants are monophosphoryl lipid A (MPL) and 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (3De-O-acylated monophosphoryl lipid A). The preferred adjuvant is also QS21. 3D-MPL can be obtained from Ribi Immunochemical Company or prepared according to the method disclosed in British Patent No.2220211; QS21 can be obtained from Cambridge Biotechnology Company or prepared according to the method disclosed in US Patent No.5,057,540 be made of. As another exemplary approach, the protein is encapsulated in microparticles, such as liposomes, or mixed with oil in an emulsion in water. Yet another illustrative approach is conjugation of the protein to an immunostimulatory macromolecule such as inactivated Bordetella or tetanus toxoid. In a preferred embodiment of the invention, the antigens of the invention comprise antigens of other Borrelia, in particular OspA.

The protein of the present invention can be expressed by live vectors, such as BCG, Listeria or Salmonella, and these vectors can be used to prepare live vaccines.

Vaccine preparation is generally described in "New Trends and Developments in Vaccines" (New Trends and Developments in Vaccines, edited by Voller et al., published by University Park Press, Baltimore, MD, 1978). Methods of encapsulating liposomes are described in US Patent 4,235,877 to Fullerton. Conjugation of proteins to macromolecules is described in US Patent 4,372,945 to Likhite and US Patent 4,474,757 to Armor et al.

The use of Quil A is described by Dalsgaard et al., Acta VetScand, 18:349 (1977).

The amount of protein of the present invention in each vaccine dose is selected based on the amount in a typical vaccine that induces a protective immune response without significant adverse side effects. This amount will vary depending on the particular immunogen employed and whether the vaccine is adjuvanted or not. Usually 1-1000 μg of protein is contained in each dose, preferably 1-200 μg. The optimal amount for a particular vaccine can be determined by standard methods, including observation of antibody titers and other responses in test subjects. Following the initial vaccination, the subject may be re-administered with the vaccine to enhance their immune response.

The invention also relates to antibodies, preferably monoclonal antibodies specific for OspG. This monoclonal antibody can be used in the diagnosis and prevention of Lyme disease.

In another embodiment, the present invention provides a diagnostic kit comprising OspG antigen. 1 Materials and methods 1.1 Borrelia strains

The B. Burgdorferi strains used in this study are described elsewhere (45). Borrelia were grown on modified Barbour-Stoenner-Kelly II (BSK II) medium at 33°C. Spirochetes were harvested by centrifugation at 10,000g for 20 minutes at 4°C, washed twice with phosphate buffered saline (PBS), and counted under a dark-field microscope. Preparation and Screening of Expression Libraries of Borrelia brucei

Genomic DNA of Borrelia brucei strain ZS7 was prepared by the lysozyme/SDS method, and DNA fragments were produced by ultrasonic treatment. The blunt-ended DNA was inserted into the pUEX1 vector by adapter cloning (7, 31). The ligated DNA was transformed into Escherichia coli MC1061 and then screened for expression with immune sera from DBA/2 mice that had been previously inoculated with 104 (or less) Borrelia brucei (ZS7) bacteria. 1.2 Southern blot hybridization

Total genomic DNA was extracted from Borrelia organisms as previously described (32). About 5 μg of DNA was digested with 100 U of restriction endonuclease (HindIII) according to the method recommended by the manufacturer (Boehringer, Mannheim). Samples were electrophoresed on a 0.7% agarose gel. The DNA fragments were transferred to Hybond ^™ -N nylon membrane (Amersham), followed by UV cross-linking and hybridization. Briefly, ³² P-labeled probes were hybridized overnight at 65°C in a solution containing 0.5M NaHPO ₄ /7% NaDodSO ₄ , pH 7.2. The membrane was washed in 40nM NaHPO ₄ /1%NaDodSO ₄ , pH 7.2 at room temperature for 30 minutes, and the dried membrane was autoradiographed with Kodak XAR-5 film at -80°C for 1-12 hours under the condition of using an intensifying screen. The hybridization probe was a 500bp DNA fragment containing the LA7 coding region. The desired gene fragment was recovered by low-melting point agarose gel, precipitated by ethanol treatment, and radiolabeled using the random primer reaction described above. 1.5 Gel electrophoresis

According to the method of Laemmli (21), perform electrophoresis on a one-dimensional SDS/PAGE slab gel, and mix 40 μl (equivalent to about 10 ⁸ organisms) from each lysate with 10 μl 5× reducing sample buffer.

Two-dimensional polyacrylamide gel electrophoresis was carried out according to the method described by O'Farrel (29), using isoelectric focusing (Pharmacia/LKB ampholyte: 1.45% pH3.5-10, 0.1% pH2.5- 4.0, 0.2% pH4-6, 0.2% pH9-11). Take the same amount of lysate as that used for one-dimensional gel electrophoresis for electrophoresis. Gels were stained with silver or subjected to Western blotting (15).

Surface proteolysis was treated with proteinase K (Boehringer, Mannheim, Germany) according to the method of Barbour et al. (20). The proteins were then separated by SDS-PAGE, and each antigen was identified by immunoblotting. 1.6 Western blotting

After two-dimensional SDS-PAGE electrophoresis, according to the manufacturer's recommendation, in a semi-dry electroblotting box (BIO-RAD, Munich, Germany), the protein was placed on a Hybond C nitrocellulose membrane (Amersham) at a constant current (60mA). Electroblot for 1 hr. After overnight incubation in blocking buffer (50 mM Tris-HCl, 150 mM NaCl, 5% non-fat dry milk), immunoblots were placed in 50 mM Tris- HCl, 150mM NaCl, 1% dry milk, 0.2% Tween20 solution or mouse monoclonal antibody (LA7) culture supernatant, incubated at room temperature for 2 hours. Nitrocellulose filters were washed 5 times in dilution buffer and incubated with alkaline phosphatase-conjugated goat anti-rabbit antiserum (Dianova, Hamburg, Germany, 1:400 v/v) for 1 hour. The blot was washed 4 times in the above buffer and 2 times in TBS, and then 20 ml of DEA buffer [0.1M diethanolamine (Sigma), 0.02% NaN ₃ , 5 mM MgCl ₂ , pH 9.0] was added, supplemented with 5 -Bromo-4-chloro-3-indole-phosphate (BCIP, Sigma; 165 μg/ml) and nitroblue tetrazolium (NBT, Sigma; 330 μg/ml) were used as substrates to develop immunoreactive bands. The reaction was terminated by washing the membrane in 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA. 1.7 DNA sequence

The Borrelia brucei genomic DNA fragment cloned into the pUEX1 plasmid (Amersham) was sequenced using the T7 sequencing kit (Pharmacia) according to the method recommended by the manufacturer. 1.8 Amino acid sequence analysis

HUSAR software was used to compare the protein sequences and construct the phylogenetic tree at the same time. 1.9 Immunofluorescence

Borrelia brucei was washed twice in PBS, transferred to an adhesive glass slide (BadMergentheim, Superior, Germany) (1× ¹⁰⁵ spirochetes/reaction area), fixed with absolute alcohol (2 minutes, -20°C) and air-dried . The fixed spirochetes were incubated with each diluted monoclonal antibody, and the spirochetes were then incubated for 30 minutes in a fluorescein isothiocyanate wet box. After washing three times in PBS, they were examined with a fluorescent microscope, and photographed and recorded with 400ASA black and white film (HP5; Illford, UK). 1.10 Immunofluorescence

Borrelia brucei was washed twice in PBS, transferred to an adhesive glass slide (BadMergentheim, Superior, Germany) (10 ⁵ spirochetes/each reaction area), fixed with absolute alcohol (2 minutes, -20°C), and air-dried. Fixed spirochetes were incubated with mAbs diluted in PBS for 30 min in a humid chamber. After washing three times with PBS, the spirochetes were incubated with fluorescein-isothiocyanate-labeled goat anti-mouse immunoglobulin antiserum (Medac, Hamburg, Germany) for 30 minutes in a dark humid chamber. After three washes in PBS, the preparations were examined under a fluorescence microscope and photographically documented with 400 ASA black and white film (HP5; Illford, UK).

ELISA Borrelia brucei-specific antibodies were determined using the B31 antigen in a solid-phase ELISA system as described previously (15). 2.1 PCR amplification of a part of ospG gene

In the absence of the ospG gene encoding a hydrophobic leader peptide, oligonucleotide primers were used: 5′-GTGGATCCAAGATTGATGCGAGTAGTG-3′ (corresponding to nucleotides 61 to 79) and 5′-GTGAATTCTATTTTTTATCTTCTATATTTTGAGGCTCTG-3′ (corresponding to nucleotides 560 from position to nucleotide 590) for PCR amplification. The pZS7 plasmid DNA was subjected to 30 cycles of PCR amplification on a DNA thermal cycler (Bio-Med60). Denaturation at 94°C for 60 seconds, annealing at 48°C for 90 seconds, and extension at 72°C for 90 seconds. The amplified fragment was digested with BamHI and EcoRI, connected to the frame of glutathione S-transferase (GST) gene and transferred into pGEX-2T vector, which was used to transform DH5α host cells. 2.2 Expression and purification of recombinant OspG

Expression of glutathione S-transferase-OspG fusion protein in E. coli DH5α strain. The methods of affinity purification and digestion of fusion protein with endoprotease thrombin were all carried out according to the method recommended by the manufacturer (Pharmacia). 2.3 [ ³ H]palmitic acid labeling and TritonX-100 phase assignment

Escherichia coli transformed with a plasmid (pZS77) with a truncated full-length ospG gene (pOspG) was cultured in a medium containing [9,10-(n) ^-3H ] palmitic acid (specific activity ≈ 50Ci/mmol, Amersham) culture medium. Radiolabeled lipoproteins were extracted by Triton X-114 phase partitioning as described previously (46). 2.4 Immune serum generation and serology

Immune sera were obtained from mice that had previously been inoculated in the tail with 10 ⁸ (CB-17; immune serum (IS) anti-10 ⁸ ) or 10 ³ (DBA/2; immune serum (IS) anti-10 ³ ) live Brucella Borrelia ZS7 strain, or first immunized with 5-10 μg lipidated OspA (lipOspA) (BALB/c; immune serum anti-lipOspA) or recombinant (rec) recOspG (BALB/c) containing adjuvant (ABMZ; Aidenbach Sebac, Germany) Serum anti-recOspG) subcutaneously (sc), followed by a booster dose 10-20 days later. All sera were analyzed for the amount of Borrelia-specific immunoglobulin (Ig) in total Borrelia lysate (Bb Ig) or recombinant OspA (OspA Ig) or recombinant OspG (OspG Ig) by ELISA as previously described. 2.5 Protection experiment

Severe combined immunodeficiency disease (SCID) mice were left untreated, or reconstituted with normal mouse serum (NMS) or pooled immune serum (IS). The amount of Borrelia-specific immunoglobulin (IG) transferred with individual IS (measured by ELISA in total Borrelia brucella cell lysate) was as follows: IS anti-10 ⁸ , 4.4 μg Ig/mouse; IS anti-10 ³ , 4.5 μg /mouse; IS anti-lipOspA, 5 μg/mouse; IS anti-recombinant OspG (recOspG), 72 ng/mouse. When tested against recOspG, the amount of specific Ig was about 10-20 times higher in IS anti-recOspG than in IS anti- ¹⁰³ . IS was injected intraperitoneally (ip) and mice receiving IS were infected one hour later with 10 ^{<5 >} Borrelia (Borrelia brucei strain ZS7) subcutaneously. Mice were observed for the development of clinical arthritis under sham conditions, and for the presence of spirochetes in ear biopsies cultured on BSK medium as previously described (37, 40). 2.6 Pathology

The onset of arthritis in the tibiotarsal joint was monitored under blinded conditions as previously described (37, 40). The grading criteria are as follows, ++severe; +serious; (+) moderate; +/-, mild swelling; (+/-) slightly red and swollen; - no clinical symptoms of arthritis. Results Cloning and recombinant structure analysis of OspG

The genomic DNA expression library of Borrelia brucei strain ZS7 was screened with immune serum from mice previously infected with Borrelia 10 ³ (IS anti-10 ³ ). This IS was previously shown to lack antibodies to OspA and OspB, but protects against subsequent infection in SCID mice (35). Moreover, this IS can recognize 4 different proteins with a relative molecular weight of 19-20KDa and 2 proteins with a molecular weight of about 40KDa in the western blot test. These proteins were obtained from whole cell lysates of the ZS7 strain and separated by two-dimensional gel electrophoresis. About 20 clones were identified, one of which, designated pZS77, was exceptionally reactive. This recombinant pZS77 plasmid was subjected to restriction analysis, subcloning and sequencing. The nucleotide sequence of ospG and the deduced amino acid sequence of OspG are shown in FIG. 1 . The consensus ribosome binding site (GGAG) is located 10 bp upstream of the ATG initiation codon of the ospG gene. Further upstream of this translation initiation sequence is the -10 region (TATATT) at positions -70 to -64 and the -35 region (TTGTTA) at positions -105 to -100. This upstream region at positions -118 to -49 contains 2 short inverted repeats ATATTT and TTACATTT. Following the ATG initiation codon of the ospG gene at position +1 is an open reading frame of 588 nucleotides, corresponding to a protein of 196 amino acids with a calculated molecular weight of 22,049 Da. It was determined that there may be a ρ-independent terminator between positions 620 and 656. Comparing the DNA sequence upstream of the ATG initiation codon of the ospG gene with the recently reported sequence of the promoter region of the ospE-ospF operon, the similarity calculated by the GAP formula is 94%. It was also found that the ospG promoter contains 2 highly conserved octameric DNA motifs: ATGTATTT (at -187 to -180) and AATTACAT (at -120 to -113), which were previously studied It has been shown to be involved in the protein binding site of a negative regulatory molecule called MATα2, and to regulate gene expression during yeast differentiation (5, 25). These two motifs have been identified in two genes of Saccharomyces cerevisiae, MFα2 and BAR1, respectively, and they also contain an incomplete inverted repeat motif similar to the immunoglobulin octamer motif ATTTGCAT. Most interestingly, between the two S. cerevisiae-like motifs, there is also the similar sequence ATTTGCAA of the immunoglobulin octamer (located between -154 and -147), which is similar to that of the immunoglobulin octamer The difference is only one base conversion substitution. OspG amino acid sequence analysis

The hydrophilicity profile of OspG suggests that this protein is predominantly hydrophilic, but has a hydrophobic region of about 20 amino acids in the amino-terminal portion. This N-terminal region exhibits similarity to the leader signal peptide in typical prokaryotic lipoproteins (51, 52). At the carboxy-terminus of the signal sequence is a putative signal peptidase II recognition motif, Leu-X-Y-Cys (Fig. 3a). The potential cleavage site in OspG is located between serine at position 19 and cysteine at position 20. The calculated isoelectric point PI=5.2. The amino acid sequence of OspG was compared with all known outer layer surface proteins OspA-OspF and P27 of Borrelia brucei, and the results showed that OspG had the highest homology (65%) with OspF (Table 1). Moreover, the basic N-terminal peptide motif M-N-K-K-M of OspG is consistent with that observed in OspE and OspF. OspG mapping

Plasmid and chromosomal DNA of several strains of Borrelia brucei were separated by pulsed field gel electrophoresis and hybridized with ospA and ospG specific probes. We estimated the size of the ospA-containing plasmid in isolate ZS7 of Borrelia brucei germania to be 53 Kb. Due to the small amount of DNA added to the gel, the ospA-containing plasmid in the ACA-1 strain was barely visible after electrophoresis, but could be seen after prolonged exposure (data not shown). An obvious band was seen with the ospG probe, a linear plasmid of about 48Kb, and a weaker band was a 45Kb plasmid of the ZS7 strain. In contrast, in the American B31 strain, two plasmids with a size of 45 Kb and about 35 Kb hybridized to the ospG gene probe. It was also found that when B. garinii ZQ1 strain, 20047 strain and B. japonica HO14 strain were used, no hybridization phenomenon was observed. In a control experiment, an assay was performed with the ospE-ospF probe from the ZS7 strain to see if different bands could be observed with these genomic DNAs. This experiment showed that ZS7, B31, 20047 and 21038 strains contained two plasmids of 45Kb and 35Kb. Borrelia from other species, such as B. coriaceae Co53, B. hermsii, B. turicatae, and B. pallidum from Treponema pallidum) did not hybridize to the ospG probe, indicating the specificity of ospG for Borrelia brucei. Restriction fragment length polymorphism (RFLP) of the ospG gene

Restriction fragment length polymorphism (RFLP) analysis of ospG with endonuclease HindIII showed that there were at least 7 different hybridization band types in the 20 isolates of Borrelia brucei tested: most Borrelia brucei isolates in the narrow sense are characterized by containing two hybridizing fragments of 1.8 Kb and 3.8 Kb (Fig. 6, lane 1); 3 of the 6 Borrelia isolates tested did not detect with ospG. 20047 and S90 strains of Borrelia garzii showed 1.8Kb fragments and 1.7 and 3Kb fragments respectively (data not shown); in the strains of Borrelia azeri, at least three different hybridization types were observed: ACA The -1 (electrophoresis lane 3) strain has a 2.4Kb band, the MMS strain (electrophoresis lane 4) has two bands of 1.9 and 2Kb, and the NE40 strain (electrophoresis lane 5) has a 2kb and 5kb band. Expression of OspG recombinant protein in Escherichia coli

For PCR amplification of OspG, primers were chosen such that the final recombinant product lacked the 20 amino acid residues that make up the leader peptide (46). The amplified OspG coding product was inserted into the carrier protein framework of the expression vector pGEX-2T (Pharmacia, Freiburg, Germany), and induced by isopropylthioβ-D-galactoside (IPTG) to obtain GST-OspG of about 44KDa fusion protein. GST-OspG fusion protein was enriched from E. coli lysate by glutathione-agarose beads followed by site-specific protease digestion of bound GST-OspG fusion protein. [ ³ H]palmitic acid labeling

To determine whether OspG is expressed as a lipoprotein in E. coli strain DH5α, cells were transformed with pZS77 or pOspG plasmids and labeled with [ ³ H]palmitic acid ( FIG. 8 ). Plasmid pZS77 encodes the full-length OspG precursor protein with normal N-terminal signal sequence, and the protein encoded by pOspG has the first 21 residues of OspG precursor replaced by Met-Lys sequence. After detergent-phase partition extraction and separation by SDS-PAGE, radioactive products were visualized by fluorescence visualization. E. coli cells containing the full length of the ospG gene (pZS77 plasmid) express a 20 kDa lipoprotein that partitions into the detergent phase. No lipoprotein was observed in DH5α cells containing truncated ospG gene. Subcellular localization of OspG

To determine the subcellular localization of OspG in intact organisms, spirochetes were treated with proteinase K. Subsequent analysis was performed by immunoblotting. The anti- ¹⁰³ serum used was the serum used to isolate ospG from the expression library. Four low-molecular-weight proteins were detected in anti- ¹⁰³ immune serum, which partially disappeared after proteolysis, while two structures with a molecular weight of approximately 40 kDa were not affected by protease treatment (17). Whether rOspG was recognized by anti-10 ³ serum was determined by Western blot. After absorbing anti-10 ³ serum, rOspG can recognize some major proteins of the same size (Fig. 7A). This result suggests that there may be no OspG in the low-molecular-weight proteins contained in the lysate of the ZS7 strain cultured in vitro, or OspG may have occurred proteolysis. To confirm the identity of OspG and whether it could be expressed in the ZS7 strain of Borrelia brucei in vitro, hyperimmunized mouse anti-rOspG serum could not detect OspG from 10 ⁹ of the ZS7 strain in vitro. In some experiments, an unexpectedly weak activity of the 40KDa polypeptide was observed, which may be a variant of OspG or a protein of Borrelia brucei that shows some common epitopes with OspG. To measure the expression of the ospG gene from in vitro cultured Borrelia brucei ZS7 strains, total RNA was isolated and analyzed for the presence of ospG transcripts by Northern blot hybridization. Nitrocellulose membranes were probed with the ospA gene as a control for in vitro expression and RNA degradation. In contrast to the ability of the ospA probe to bind a single -2 kb transcript, the ospG probe failed to detect the ospG transcript. These results suggest that the defective expression of OspG in vitro seems to be due to the mRNA stability of ospG at the transcriptional level. OspG is undetectable in lysates of Borrelia brucei strain ZS7 grown in vitro, and OspG expression may vary during growth in mammalian hosts. Western blot analysis of representative Lyme disease patient and experimentally infected mouse serum samples revealed the presence of antibodies specific for OspG. Anti-OspG antibodies were found in 6 of 13 serum samples from patients with confirmed Lyme borreliosis. In contrast, none of the sera of healthy blood donors (n=8) contained antibodies recognizing rOspG (data not shown). These findings suggest that OspG is only expressed during infection. SCID mice were partially protected with anti-OspG immune serum

To determine the protective capacity of anti-OspG immune sera, SCID mice were treated with the following amount of Borrelia brucei-specific immunoglobulin (Ig) from pooled immune sera (IS) preparations: CB-17IS anti-10 ⁸ (4.4 μg B.bIg/mouse), DBA/2 IS anti-10 ³ (4.5 μg B.bIg/mouse), BALB/c IS anti-lipOspA (5 μg B.bIg/mouse) and BALB/c IS anti-lipOspA (5 μg B.bIg/mouse) and BALB/ cIS anti-recOspG (72ng B.blg/mouse). However, compared with the anti-recOspG immune serum, the amount of anti-recOspG antibody contained in the anti-recOspG immune serum is more than ¹⁰ times that of the latter. SCID mice were injected intraperitoneally with any one of the ISs, followed by an infection test with 10 ⁵ amounts of Borrelia brucei. The onset of clinical arthritis and the appearance of spirochetes in ear tissue biopsies were observed. Inoculated with spirochetes without other treatment or NMS (normal mouse serum)-treated SCID mice, arthritis appeared on the 6th day after infection, and from the 13th day to the 24th day (terminal time), the tibia Severe swelling of the hock joints. As mentioned before, SCID mice passively immunized with anti-10 ⁸ or anti-10 ³ and anti-lipOspA immune sera did not appear or only mild symptoms of clinical arthritis. In contrast, SCID mice passively immunized with anti-recOspG only delayed the onset of arthritis based on their mild swelling from day 6 to day 18. At a later time, the mice developed more severe symptoms of arthritis, suggesting that this immune serum was less effective at controlling infection than other immune sera. Anti-recOspG immune sera contained more than 10 times more antibodies against recOspG than anti- ¹⁰³ immune sera, but were much less protective, suggesting that in anti- ¹⁰³ immune sera, another specific substance was controlling play a role in infection.

Table 1: Borrelia bruceli outer surface lipoproteins name source (strain) length (aa) Molecular weight (KDa) Isoelectricity (PI) Plasmid location Similarity to OspG (%) references OspAOspBOspCOspDOspEOspFOspGP27 ZS7B31B31B31N40N40ZS7B29 273296210257171230196248 29.331.822.328.419.226.12228.9 8.99.98.65.28.15.35.28.6 49kb linear 49kb linear 26kb circular 38kb linear 45kb 45kb 55kb 49kb linear 48.443.141.845.652.964.945.2 (47)(4)(30)(28)(22)(22)This study(32)

Table 2: Onset of clinical arthritis in SCID mice that were not injected with immune serum or pre-peritoneally injected with the indicated immune serum and then inoculated with 1×10 ⁵ Borrelia brucei ZS7 strain Number of mice transferred Degree of clinical arthritis after infection Recultured (ear) serum 6 13 18 20 27 None1 - +/+ ++/++ ++/++ ++/++ +2 (±)/(±) +/+ ++/++ ++/++ ++/++ +NMS 1 (±)/± +/+ ++/++ ++/++ ++/++ +[5 μg/mouse] 2 ±/(±) +/+ ++/++ ++/++ ++/++ +3 (±)/(±) +/+ ++/++ ++/++ ++/++ +IS Anti-10 ³ 1 - - - - - -(DBA/2) 2 - - - - - - [4, 5 μg sample 3 ±/± - - - ±/(±) -lg/mouse] 4 - - - (±)/- ±/- -IS anti-10 ⁸ 1 ± /- ±/- - - - -(CB-17) 2 - -/± - - - -[4, 4 μg sample 3 - - - - - -lg/mouse] 4 ±/± -/± - - ± /- -IS anti- 1 (±)/(±) - - - (±)/- -lipOspA 2 ±/± - - - - -(BALB/c)[5 μ 3 - - - - - -g sample 4 - - - -/(±) - -lg/mouse]IS anti-1 - (+)/(+) ±/- +/++ ++/++ +recOspG 2 - (+)/(+) ±/± (+)/(+) ++/++ +(BALB/c)[72 3 - (+)/(+) ±/± ++/++ ++/++ +ng sample 4 - (+)/(+) (+)/+ ++/++ ++/++ +lg/mouse] mAb LA 10 ^* 1 - - - (±)/± (+)/(+) +[ 5 μg sample 2 - (+)/(+) +/+ ++/++ ++/++ +lg/mouse] 3 - (+)/(+) +/+ ++/++ ++/ (+) +4 - (+)/(+) ++/++ ++/++ ++/++ +mAB LA2 ^* [5 1 - - - - - -μg sample 2 - ±/- (± )/- - - -lg/mouse] 3 - ±/- - - - -4 - - - - - - oEvaluation criteria: ++serious; +serious; (+) moderate; ±slightly swollen; (±) slightly red and swollen; ^* mAB is described earlier (20, 35, 39) references

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Sequence Listing (1) General Information (i) Applicant

(A) Name: Max Planck Company (Max Planck Gesellschaft zur Forderungder Wissenschaft eV)

(B) Street: Busenstrasse 10

(C) City: Gottingen

(E) Country: Germany

(F) Zip code (zip): D-3400

(A) Name: German Cancer Research Center (Deutsches Krebsforschungszentrum Stitting des offentilichen Rechts)

(B) Street: 280 Neuenheimer

(C) City: Heildelberg Heidelberg

(E) Country: Germany

(F) Zip code (zip): D-6900 (ii) Title of invention: Vaccine (iii) Number of sequences: 2 (iv) Computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible

(C) Operating system: PC-DOS/MS-DOS

(D) Software: PatentIn Release#10, #1.30 version (EPO) (2) Sequence 1 data: (i) Sequence features:

(A) Length: 196 amino acids

(B) Type: amino acid

(C) Chain type: single strand

(D) Topology: Linear (ii) Molecular Type: Protein (iii) Hypothetical Structure: None (iv) Antisense Strand: None (vi) Source

(A) Organism: B. burgdorferi

(C) Individual separation: OSPG (xi) sequence description: Sequence 1: Met Asn Lys Lys Met Lysn Leu Ile Cys Ala Val Phe Val Leu1 5 10 15ILE Serle ASP Ala Seru ASP Leu LYS Gln Asn

20 25 30Val Lys Glu Lys Val Glu Gly Phe Leu Asp Lys Glu Leu Met Gln Gly

35 40 45Asp Asp Pro Asn Asn Ser Leu Phe Asn Pro Pro Pro Val Leu Pro Ala

50 55 60r Serp asn Thr Pro Val Leu Lys Ala Val Gln Ala Lysp65 75 80Gly Gln Gln GLU GLU GLU GLU LYS Gln Gln Gln Gln Gln Glu

85 90 95Leu Lys Asp Lys Ile Asp Lys Arg Lys Lys Glu Leu Glu Glu Ala Arg

100 105 110Lys Lys Phe Gln Glu Phe Lys Glu Gln Val Glu Ser Ala Thr Gly Glu

115 120 125Ser Thr Glu Lys Val Lys Lys Gln Gly Asn Ile Gly Gln Lys Ala Leu

130 135 140lys Tyr Ala Lys Glu Leu Gly Val Asn Gly Ser Val Val ASP145 150 155 160GLY Thr Asn Asn ASP ALA Leu Ala Leu Ala Leu

165 170 175Lys Asn Ile Glu Glu Glu Leu Glu Lys Leu Ala Glu Pro Gln Asn Ile

180 185 190Glu Asp Lys Lys

  195(2) Sequence 2 data: (i) Sequence characteristics:

(A) Length: 591 base pairs

(B) type: nucleic acid

(C) Chain type: single strand

(D) Topological structure: linear (ii) Molecular type: DNA (genome) (iii) Hypothetical structure: None (iv) Antisense strand: None (vi) Source:

(A) Organism: Borrelia burgdorferi

(B) strain: ZS7 (vii) direct source:

(B)克隆：ospG(xi)序列描述：序列2：ATGAATAAGA AAATGAAAAA TTTAATTATT TGTGCAGTTT TTGTTTTGAT AATTTCTTGC60AAGATTGATG CGAGTAGTGA AGATTTAAAA CAAAATGTAA AAGAAAAAGT TGAAGGATTT120TTAGATAAAG AGTTAATGCA AGGTGACGAT CCTAATAACA GTCTGTTTAA TCCACCACCA180GTATTGCCGG CAAGTTCCCA CGATAACACA CCCGTATTAA AAGCGGTGCA AGCAAAAGAT240GGTGGTCAAC AAGAAGGAAA AGAAGAGAAA GAAAAAGAAA TTCAAGAATT AAAGGATAAA300ATAGATAAAA GAAAAAAAGA ATTAGAAGAG GCTAGAAAGA AATTTCAAGA ATTTAAAGAA360CAAGTTGAAT CTGCAACTGG AGAAAGTACT GAGAAAGTTA AAAAACAAGG AAATATTGGA420CAAAAAGCTT TAAAGTATGC TAAAGAATTG GGTGTAAATG GAAGTTATTC TGTTAATGAT480GGTACTAATA CTAATGATTT TGTAAAAAAG GTTATAGATG ATGCTCTTAA AAATATTGAG540GAAGAACTTG AAAGCTAGC AGAGCCTCAA AATATAGAAG ATAAAAAATA A5

Claims (18)

CLAIMS 1. An OspG purified from B. burgdorferi, or an immunologically functional derivative thereof. 2. An OspG purified from Borrelia brucei, characterized by an apparent molecular weight of 22KDa, an isoelectric point PI of about 5.2 and 196 amino acids, or its derivatives with immune function. 3. A purified OspG from Borrelia brucei, roughly as shown in Figure 1. 4. The purified OspG according to claim 3, which has at least 80% homology to the amino acid sequence shown in Figure 1 . 5. Purified OspG as claimed herein for use in medicine. 6. A vaccine composition comprising purified OspG as claimed in any one of claims 1-4. 7. The vaccine composition of claim 6, further comprising a Borrelia brucei antigen. 8. The vaccine composition of claim 6, further comprising an antigen from Borrelia. 9. The vaccine composition of any one of claims 6-8, further comprising QS21 or 3D-MPL. 10. The use of the OspG antigen in claims 1-4 to prepare a vaccine for immunoprophylaxis or immunotherapy of Lyme disease patients or those susceptible to the disease. 11. A method of treating a Lyme disease patient or a patient susceptible to the disease comprising administering an effective amount of a composition according to claims 6-9. 12. An isolated DNA sequence encoding the OspG protein as claimed in claims 1-4 or an immunologically functional derivative thereof. 13. An isolated DNA sequence encoding the OspG protein as claimed in claim 12, characterized in that it is substantially identical to the sequence shown in Figure 1 . 14. An expression vector comprising a DNA sequence as claimed in claims 12-13. 15. A host cell transformed with the DNA sequence of claim 12 or 13. 16. A method for producing purified OspG as claimed in claims 1-4, comprising transforming a cell with the expression vector of claim 14, isolating the cultured cells and isolating the resulting protein. 17. A method of producing a vaccine comprising OspG according to claims 1-4, comprising mixing said OspG or an immunologically functional derivative with a pharmaceutically acceptable excipient. 18. A diagnostic kit comprising OspG from Borrelia brucei.

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