DE3942728C1 - New immunologically active proteins derived from Borelia burgdorferiensity polyethylene vessel and a high density polyethylene sealing cap - useful as vaccine and for quick accurate diagnosis of Borelia infections - Google Patents

Abstract

New pure immunologically active proteins derived from Borrelia burgdorferei are claimed. The proteins are produced from DNA isolated from Borrelia burgdoreferei (DSM No.5662). They can have molecular weights of 41, 22, 17 or 100 kDa. The following partial sequences are specifically claimed for the 22 kDa protein: Lys-lle-Thr-Asp-Ser-Asn -Ala-Thr-Val-Leu-Ala-Val-Lys. and/or Asp-Leu-Phe-Glu-Ser-Val -Glu-Gly-Leu-Leu-Lys. The 100 kDa protein preferably has a partial sequence of formula. Glu-Leu-Asp-Lys-Glu-Lys-Leu-Lys -Asp-Phe-Val-Asn-Leu-Asp -Leu-Glu-Phe-Val-Asn-Thr. Also claimed are monoclonal antibodies produced from B. burgdorferi DSM No.5662. USE/ADVANTAGE - For the preparation of vaccines against Borrelia infections (claimed), e.g. early summer meningoencephalitis or Lyme borreliosis. Because of their purity, the proteins are also useful for quick, economical and accurate diagnosis of such infections, without the risk of confusion with similar diseases such as syphilis.

Description

The invention relates to those specified in claims 1 to 7 immunologically active proteins from Borrelia burgdorferi, according to claim 8 directed against monoclonal antibodies and according to Claims 9 to 17 the use of the above proteins.

Lyme disease is in the Federal Republic of Germany the most common tick-borne infectious disease of the human. In contrast to early summer meningoencephalitis (TBE), which is also transmitted by ticks, Lyme disease is not limited to just a few Endemic areas, but occurs in all countries of the FRG. The main vector in Europe, Ixodes ricinus, with the causative agent of Lyme disease, the spirochete Borrelia burgdorferi, is around 20% in southern Germany of adults, approx. 10% of the nymphs and approx. 1% of the  Larvae (Wilske, B .; Steinhuber, R .; Bermeister, H .; Fingerle, V .; Schierz, G .; Preac-Mursic, V .; Vanek, E .; Lorbeer, B. (1987): Lyme disease in southern Germany. German Med. Wschr. 112, 1730-1736). The main vector in USA, Ixodes dammini, can be up to 100% in high-end areas to be infected with Borrelia.

B. burgdorferi belongs to the spirochete family. Spirochetes are helical bacteria with a length of 8-30 µm. They consist of an outer shell, the endoflagella in the Periplasma and the protoplasmic cylinder. The protoplasmic cylinder is a complex of cytoplasm, inner cell membrane and peptidoglycan (Barbour, A.G .; Hayes, S.F. (1986): Biology of Borrelia species. Microbiol. Rev. 50, 381-400). To the Human pathogenic representatives of the spirochetes also belong B. burgdorferi relapsing fever borrelia (e.g. recurrentis), the causative agent of syphilis (Treponema (T.) pallidum) and the leptospires. Because of the close immunological Cross-reactivity is a problem in the serological detection of antibodies in syphilis and Lyme disease with tests available to date.

Infection with B. burgdorferi leads to a complex Disease that is similar to syphilis, in can divide into three different stages. The most important Manifestations are:

Early phase:

Stage I
Erythema migrans

Stage II
lymphocytic meningoradiculitis Bannwarth (LMR)
Borrelia lymphocytoma

Late phase:

Stage III
Lymphoarthritis
Acrodermatitis chronica atrophicans (ACA)
chronic borrelia encephalomyelitis

Less common clinical manifestations are: carditis, myosilitis, Iritis and panophthalmitis. The diaplacentare transmission of the pathogen is possible, but so far only a few are Cases of connatal Lyme disease documented. The different stages can occur individually or in combination. B. burgdorferi infection can also be subclinical run. Epidemiological studies on 375 clinically Proven cases showed some peculiarities in the Age and gender distribution among the various clinical manifestations. So patients found themselves with Erythema migrans is most common in the 30s up to 60 years. Neurological manifestations showed two Age peak: the first in children and adolescents up to 20 years, the second for 40 to 70 year olds. The Lyme Arthritis was most commonly seen in 30-60 year olds. Patients with ACA were never younger than 30 years. The ACA affects women far more often than women Men. The serological examination showed in the immunofluorescence test predominantly in patients with erythema migrans positive IgM results, with neurological manifestations predominantly positive IgG results. At the late manifestations The IgG titers were regular for ACA and Lyme arthritis increased and IgM antibodies can only be detected in exceptional cases (Wilske, B .; Schierz, G .; Preac-Mursic, V .; Weber, K .; Pfister, H.-W .; Einhäupl, K. (1984): Serological diagnosis of Erythema migrans disease and related disorders. Infection, 12, 331-337; Herzer, P .; Wilske, B. (1986) Lyme arthritis in Germany. Zbl. Bakt. Hyg. A 263, 268-274).

Professionally strongly exposed to ticks like forest workers show a significantly increasing prevalence with increasing age increased antibody titer against B. burgdorferi (Münchhoff, P .; Wilske, B .; Preac-Mursic, V .; Schierz, G. (1986): Antibodies against Borrelia burgdorferi in Bavarian forest workers. Zbl. Bakt. Hyg. A 263, 412-419). For diagnostics there is both the pathogen detection and the antibody detection to disposal. Pathogen detection from patient material (Skin biopsies, cerebrospinal fluid, punctures) is special in the early stages (erythema migrans), in which a  Antibody detection is often negative, recommended. Indeed is a complex one for growing B. burgdorferi Nutrient medium required (Preac-Mursic, V .; Wilske; B .; Schierz, G. (1986): European Borreliae burgdorferi isolated form humans and ticks - culture conditions and antibiotic susceptibility. Zbl. Bakt. Hyg. A 163, 112-118) and the cultivation is therefore reserved for special laboratories. For insulation the pathogen also has a time up to 5 Weeks needed. B. burgdorferi is isolated from skin samples in 50-70% of cases with skin manifestations and in 3-5% of cases with neuroborreliosis (Preac-Mursic, V .; unpublished. Results).

The antibody detection (IgM, IgG) is in the serum and at neurological manifestations also performed from cerebrospinal fluid. The serological findings depend on the stage of Illness, the duration of the symptoms and possibly already antibiotic therapy. So is the antibody detection with tests available so far for Erythema migrans successful only in 20-50% of cases, with neurological Manifestations in 50-90% and in ACA and arthritis 90-100%. Elevated IgG titers are not always diagnostic clearly interpretable: on the one hand it can be a clinically manifest infection, on the other hand by a titer of infection act. The main diagnostic parameter neuroborreliosis is intrathecal evidence formed antibody. For this purpose, the CSF / serum pair contains the Total IgG content and Borrelia-specific content IgG antibodies determined by ELISA and a special index calculated (Wilske, B .; Schierz, G .; Preac- Mursic, V .; v. Busch, K .; Kuehlbeck, R .; Pfister, H.-W .; Einhäupl. K. (1986): Intrathecal production of specific antibodies against Borrelia burgdorferi in patients with lymphocytic meningoradiculitis (Bannwarth’s syndrome). J. Infect. Dis. 153, 304-313).

The therapy for Lyme disease is predominantly with penicillin G, tetracyclines, erythromycin or cephalosporins carried out. Lyme disease heals in the early stages Stages are often spontaneous, but even then they are late manifestations  not excluded. Hence therapy indispensable in the early stages. It is also a clinical one Healing after antibiotic therapy for late manifestations can only be achieved in part of the cases (e.g. only approx. 50% in Lyme arthritis).

Therefore, Lyme disease should be started as early as possible be diagnosed. Since (as already explained) the pathogen isolation expensive, time consuming and also not always successful, better serodiagnostic Tests are developed. The procedures used so far (Immunofluorescence test (IFT)), indirect hemagglutination test (IHA), enzyme-linked immunosorbent assay (ELISA) fail often in the early stages. As antigens for these tests whole B. burgdorferi cells or whole cell Ultrasonics used. The use of different B. burgdorferi strains as antigen leads in ultrasonics ELISA on different test results, in particular it was found that the use of the isolate PKo (Preac-Mursic, V .; Wilske, B .; Schierz, G. (1986): European Borreliae burgdorferi isolated form humans and ticks culture conditions and antibiotic susceptibility. Zbl. Bakt. Hyg. A 163, 112-118) for significantly better results in IgM Led by ELISA. The cells are placed on slides or Ultrasonics antigen fixed on microtiter plates, with Incubated serum or cerebrospinal fluid and the Borrelia specific Antibodies labeled with a second fluorescent or peroxidase Antibodies of the corresponding immunoglobulin class detected. The reaction is then either in the fluorescence microscope (IFT) or after a color reaction in the photometer (ELISA) evaluated. A problem for specificity the tests are broad cross-reactions of the pathogen B. burgdorferi to other bacterial pathogens, especially T. pallidum, the causative agent of syphilis. Since the test antigens in generally consist of lysates of the entire pathogen, antibodies against so-called "common antigens" are also detected (Hansen, K .; Hindersson, P .; Pederson, N. S. (1988): Measurement of antibodies to the Borrelia burgdorferi flagellum  improves serodiagnosis in Lyme disease. J. Clin. Microbiol., 26, 338-346). "Common antigens" are widespread and Proteins highly conserved in their sequence, i.e. H. the "Common antigens" from Borrelia, Treponemen, but also numerous other bacteria have common epitopes. In addition, false positive reactions in the IgM-IFT or IgM ELISA occur in sera with rheumatoid factor activity. In order to make the tests more specific, it is used in the detection of IgG and IgM antibodies a pre-absorption of the Sera with a Treponema ultrasonics and additionally with Detection of IgM antibodies with an absorption Rheumatoid factor absorbent performed.

Von Luft et al., Infection and Immunity, Nov. 1989, p. 3637-3645 were cell lysates of the American Borrelia strain "B 31" using SDS gel electrophoresis separated and shown that some proteins a have immunological activity. This release however, it cannot be extracted from proteins like Borrelia strains can be produced in pure form. Nor does this work show how a sensitive and above all specific detection of Antibodies against Borrelia in test liquids can be performed.

The object of the present invention is therefore immunological active proteins from Borrelia burgdorferi are available to be used in a test kit, that does not have the disadvantages mentioned above. Farther this use is intended to provide quick and reliable evidence of antibodies directed against Borrelia burgdorferi enable.

Another object of the present invention is to provide monoclonal antibodies against certain immunologically active proteins from Borrelia burgdorferi are directed. They are also said to be immunological  active proteins are provided, which are known as Vaccines against infections caused by Borrelia strains own.

When examining patient sera from different Disease stages of Lyme disease in the Western Blot as well as examining non-Lyme disease Patients (especially syphilis patients) for cross reactivity with B. burgdorferi became immunologically active Proteins (B. burgdorferi antigens) found on the one hand produce a good antibody response after infection and on the other hand a low cross-reactivity with not  B. show Burgdorferi-positive sera (Example 1). It showed that a certain strain of B. burgdorferi with the PKo label inside the laboratory, the one used by Deutsche Collection for microorganisms (DSM) under the number 5662 was deposited, including an immunodominant protein in the molecular weight range around 22 kD (pC protein) owns. The determination of the molecular weight of the invention Proteins were made according to known Methods especially by SDS gel electrophoresis. It was found this protein to be immunodominant for the IgM response is. This protein is not found in all B. burgdorferi- Strains pronounced in the same way. According to the invention this immunologically active protein (pC protein) genetically manufactured (Ex. 3).

Other immunologically active proteins (antigens) that is particularly suitable for use in test kits are generally available and commercially available available Escherichia coli cells, such as the strains JM 105 or DH 5. For this purpose, the B. coding for these proteins were burgdorferi DNA fragments isolated and then in effective expression vectors used (Ex. 2 and 3).

The identification and isolation of the corresponding DNA Fragments were made using different methods. So it was an immunologically active protein with a molecular weight of approximately 41 kD, also referred to below as p41 protein by means of polymerase chain reaction (PCR) and specific primers whose sequences are made synthetically were shown (Ex. 2).

Furthermore, a gene bank of the B. burgdorferi genome was created created by means of monoclonal antibodies on the direct expression of immunologically active proteins was searched.  

Another method was to select certain ones immunologically active proteins (antigens) from B. burgdorferi- Purify lysates and amino acid sequences of these antigens to determine. Then the amino acid sequence corresponding oligodeoxynucleotides are synthesized and those clones of the gene bank identified by hybridization, the coding for the immunologically active proteins DNA sequences. The latter two Methods are explained in more detail in Example 3.

After characterization, sequencing and recloning of the Genes in corresponding expression vectors became the antigens expressed in E. coli cells and then purified. A preferred cleaning procedure is in Example 4 described.

The immunologically active produced according to the invention Proteins from Borrelia burgdorferi (DSM No. 5662) can be found in Test kits are used that are surprisingly sensitive Detection of antibodies against B. burgdorferi in different Provide test fluids. An advantage of the immunologically produced according to the invention active proteins is that the preparations only from the desired protein and possibly such proteins that insist on degradation and / or incomplete translation are. These preparations do not contain such B. burgdorferi proteins that are not recombinantly produced Correspond to protein because they are genetically engineered were.

Under the term "test kits" is a set of test reagents understood the detection of certain antibodies enables. The principles underlying the test kits were described in "Immunoassays for the 80s" (1981) by A. Voller et al., Published by MTP Press Ltd, Falcon House, Lancaster, England. The test reagents show the most important component is the antigen (s) and, if appropriate specific, preferably monoclonal antibodies on.  

The use according to the invention for the detection of antibodies against Borellia burgdorferi is characterized by that uses at least one immunologically active protein that is from the protein without contamination by other proteins Borrelia burgdorferi strain is available. This immunologically active protein acts as an antigen and reacts with the antibodies present in the test liquid. The ones used according to the invention preferably have Test kits two to four immunologically active proteins that are free from contamination by other proteins from B. burgdorferi are available. It also contains Test kit a display component that detects the presence of Allows complexes of antigen and antibody.

The test kits used according to the invention can be based on known principles. Basically the antigen can carry a label, the Label from a radioactive isotope or an enzyme can exist that catalyzes a color reaction. As well the antigen can be attached to a solid support (microtiter plates or beads) and the display component can be in an antibody directed against antibodies consist of who carries a mark, the mark consist of a radioactive isotope or an enzyme that catalyzes a color reaction.

In the context of the present invention, the use the so-called ELISA (enzyme-linked immunosorbent assay) prefers. An embodiment thereof is shown in Example 5 closer  described. The results of this example show that Surprisingly, by using only one of the invention immunologically active protein has a very high specificity of the test kit could be achieved. Furthermore surprisingly enable the test kits according to the invention a differentiation correlated with the disease stage. The combined use of several antigens in a test kit enables the detection of antibodies against Borrelia burgdorferi also in cases where there is the symptoms of the disease have not yet been clinically manifested to have. Infections with B. burgdorferi can also be diagnosed where the patient is only a subclinical Infection goes through. The statement made by the Test kits according to the invention can be obtained is in particular Significant in cases where a tick bite could be determined, but it is not clear whether a Infection with a Borrelia strain is present.

In the context of the present invention, the combined Use of several of the immunologically active proteins prefers. A combination is very particularly preferred the proteins p41, pC, p17 and / or p100. By using it of the ELISA test kits preferred according to the invention also a differentiation regarding the nature of the antibodies respectively. For example, IgM antibodies should be detected the so-called µ-capture assay can be used will; they are directed against IgM antibodies Antibody bound to the solid phase. After incubation of the Test plates with the liquid to be examined the IgM antibodies present in the test liquid bound to the solid phase. More unspecific after saturation Bindings can then be an immunologically active one Protein of the present invention can be added. This Antigen is then detected by a display molecule. The antigen can be biotinylated, followed by Avidin is added to the covalently bound Has peroxidase. The peroxidase catalyzes one Reaction that leads to color formation.  

Another possibility is that such monoclonal Antibody to the complex carrier / anti-IgM antibody / Antibody to be detected / antigen according to the invention given that are specific for the antigen and are biotinylated. The biotinylation is for example in Monoclonal Antibodies (1985) Springer Verlag, J. H. Peters et al. described. The complex is verified in it by adding avidin to which a color reaction catalyzing enzyme is coupled.

Another embodiment of the present invention is that the IgM detection by indirect ELISA to be led. The antigens according to the invention bound on microtiter plates with the one to be examined Incubated liquid and after washing the detection is done the immune complexes using anti-µ conjugate.

Another aspect of the present invention is in the production of monoclonal antibodies against the immunologically active proteins from Borrelia burgdorferi (DSM No. 5662). The manufacture of such monoclonal antibody is explained in more detail in Example 6. Such monoclonal antibodies can be used as reagents for direct pathogen detection. It can but also monoclonal antibodies to the solid phase of a Microplate are coupled. After adding the immunologically active proteins (antigens) are made by these Antibody-antigen binding to the microtiter plate fixed. Then the test liquid (which is, for example, serum or cerebrospinal fluid) can) added. The one in the test liquid existing antibodies then bind to the antigen and can be demonstrated with the help of a display component.  

In addition, the monoclonal antibodies can be very good for cleaning the immunologically active proteins (antigens) use. The advantage here is that the cleaning is particularly gentle. For this, the monoclonal Antibodies bound to a solid matrix. Preferably this solid matrix is in the form of a column. Subsequently the partially pre-purified antigens physiological conditions with those attached to a solid matrix coupled antibodies offset. After washing the matrix Antibody-antigen complex can elute the antigens will. Usually high salt concentrations are used or buffer with such a pH value that enables elution.

Furthermore, DNA sequences are made available that the amino acid sequence of the immunologically active proteins fully or partially. These DNA sequences can preferred for the detection of Borrelia strains in the test material can be used by hybridization. To an oligonucleotide is produced which corresponds to the DNA sequence partially corresponds. This oligonucleotide becomes radioactive marked. On the other hand, the DNA from the test material a suitable filter, preferably nitrocellulose bound and then with the radioactive labeled oligonucleotide hybridized. Likewise, the DNA sequences according to the invention for in-situ hybridization for the direct detection of B. burgdorferi in infected Tissues are used. Instead of the chemically synthesized Oligonucleotides can also have corresponding DNA Fragments are propagated in bacteria and then made the vectors using restriction endonucleases be cut out. After isolating these DNA fragments these can be radioactively marked and as above described can be used for hybridization.

Another aspect of the present invention is in that the immunologically active according to the invention Proteins (antigens) from Borrelia burgdorferi as vaccines can be used. For this purpose, the invention Antigens presented in pure form. Then be  them individually or in combination with or without a die Immune response stimulating agent of the person to be vaccinated applied. This will result in the formation of antibodies stimulated specifically against Borrelia burgdorferi strains are.

The proteins and monoclonal antibodies of the invention can be used in different areas. So the test kits can also be used to detect B. burgdorferi- Infections in animals are used and the proteins can also be used to vaccinate animals, especially valuable ones Animals.

Using the following tables, illustrations and examples become the preferred embodiments of the present Invention explained in more detail.

Example 1 Determination of the immune-relevant and gender-specific Borrelia proteins

It was based on specific, frequently occurring serum Antibodies searched for against certain individual B. burgdorferi antigens are directed as little as possible Show cross-reactivity with proteins of related pathogens and additionally a correlation with the individual Lyme Allow Lyme disease stages.

The search for frequently recognized antigens was carried out using Western blot. A bacterial extract from B. burgdorferi (PKo strain) (Preac-Mursic, V .; Wilske, B .; Schierz, G. (1986): European Borreliae burgdorferi isolated from humans and ticks - culture conditions and antibiotic susceptibility. Zbl. Bakt. Hyg. A 163, 112-118) after Pelletize, resuspend in PBS / NaCl and Ultrasound treatment in SDS polyacrylamide gel electrophoretically separated (Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685)

The gels consisted of a collection gel with pockets for the Samples and a separating gel. The composition of the separating gels was as follows: 15% acrylamide, 0.026% Diallyltartardiamide (DATD per percent acrylamide, 0.15% SDS, 375mM Tris-HCl pH 8.5, 0.14mM Ammonium peroxodisulphate (AMPER) and 0.035% N, N, N'-N'-tetramethylethylenediamine (TEMED). AMPER and TEMED served as radical initiators for the polymerization. 2-4 h after polymerization, the stacking gel (3.1% Acrylamide, 0.08% diallyltartardiamide, 0.1% SDS, 125 mM Tris HCl pH 7.0, 3 mM AMPER and 0.05% TEMED) via the separating gel poured and provided with a Teflon comb. The anode and The cathode chamber was filled with an identical buffer solution: 25mM Tris base, 192mM glycine and 0.1% SDS, pH 8.5.  

In each case 20 μl of sample in lysis buffer (3% sucrose, 2% SDS, 5% β-mercaptoethanol, 20 mM Tris-HCl pH 7.0, Bromophenol blue; 5 minutes at 100 ° C) per bag applied. The electrophoresis was over at room temperature Night with constant current of 6 mA for gels of size 20 × 15 cm. The gels were then opened Nitrocellulose (NC) transferred.

For the protein transfer, the gel was with the adjacent nitrocellulose between Whatman 3MM filter paper, conductive, 1 cm thick foam and two carbon plates, which conducted the current through platinum electrodes. Filter paper, Foam and nitrocellulose were good with blot buffer (192mM glycine, 25mM Tris base, 20% methanol, pH 8.5) soaked. The transfer took place at 2 mA / cm² for 2 h. Free Binding sites on the nitrocellulose were at 37 ° C for 1 h with Cohen buffer (1 mg / ml Ficoll 400, 1 mg / ml Polyvinylpyrrolidone, 16 mg / ml bovine serum albumin, 0.1% NP 40, 0.05% Bacto-Gelatin in sodium borate buffer pH 8.2); (Cohen G. H., Dietzschold, B., Ponce de Leon, M., Long, D., Golub, E., Varrichio, A., Pereira, L. and Eisenberg, R. J .: Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibodies. J. Virol. 49 (1984) 4183-4187) saturated. The blot was run overnight at room temperature the patient sera (dilution 1: 100 in 154 mM NaCl and 10 mM Tris-HCl pH 7.5) with shaking.

After the serum incubation, the blot was washed with TTBS (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.01% Tween 20) four times each Washed for 15 minutes. The blot was then coupled with peroxidase anti-human immunoglobulin (Dilution 1: 1000 in 154 mM NaCl and 10 mM Tris-HCl, pH 7.5) for Incubated for 2 h at room temperature. After washing several times TTBS was blotted with 0.5 mg / ml diaminobenzidine and 0.01% Stained hydrogen peroxide in 50 mM Tris-HCl pH 7.5. The Staining was then stopped with 1N sulfuric acid Blot washed acid-free with water and between filter paper dried.  

A selection of the reaction patterns of different sera with the Western blot strips is shown in Figures 1, 2a and b shown.

The following proteins proved to be immunodominant: p17 (17kDa), pC (22kDa) p41 (41kDa) and p100 (100kDa with Size variation in different B. burgdorferi isolates). To on p41 are the biological functions of these antigens unknown; p41 represents the flagellin protein (Barbour, A.G.S., Hayes, S.F., Heiland, R.A., Schrumpf, M.E. and Tessier, S. L .: A Borrelia genus-specific monoclonal antibody binds to a flagellar epitope. Infect. Immune. 52 (1986) 549- 554.

Because of these analyzes, which are carried out with a larger number of Patient sera from the various stages of the disease was carried out, there were indications that with a only antigen not always with B. burgdorferi Infected be detected. It turned out that especially at Sera with IgM antibodies (fresh infection) next to the Flagellin (p41) is another protein (pC) in the 22 kD range is recognized particularly frequently. The simultaneous occurrence however, both antibodies were not mandatory. It could both Sera that only have antibodies against p41 or only antibodies against possessed the pC protein, can be found (Fig. 1 and 2a, Western blots). In the case of neuroborreliosis, the evidence is the intrathecally formed antibodies in the cerebrospinal fluid of large Importance. IgG western blots in 12 CSF / serum pairs from Patients with lymphocytic meningoradiculitis Bannwarth showed a local intrathecal immune response in all cases against p41. In the late stage, in addition to IgG antibodies against the flagellin primarily antibodies against proteins in the 100 kD Range (p100) and in the 17 kD range (p17) found in the Early stages were not or only rarely detectable. Consequently are antibody reactivities against the p17 and p100 proteins good markers for reaching stage III (Fig. 2b, Western blot).

Using these four antigens can be a better one Standardization of the tests can be achieved.  

The proteins p41, pC and p17 also show only a small amount Cross-reactivity to other bacterial strains, the protein p100 proved to be a genus-specific protein with B. burgdorferi specific epitopes. Summarizing are in Tab. 2 (Reactivity of immune sera against various bacterial Pathogen with proteins from B. burgdorferi) cross-reactivity of sera against various related pathogens B. burgdorferi antigens listed by Western blot analysis. In experiments, the four proteins (p41, pC, p17, p100) from B. Cleaning burgdorferi extracts was found to be great Amounts of starting material are required. Special The cleaning of p100, which in the Total extract is underrepresented. Because the cultivation is complex and expensive, had to be genetically engineered Ways of producing these antigens are being sought. Analysis of patient sera using Western blot has shown that with a combination of genetic engineering p41, pC, p17 and p100 almost produced an antigen complete collection of all positive sera can take place, and continues to be correlated to the stage of the disease is.

Example 2 Genetic engineering production of p41 (flagellin) from B. Burgdorferi in Escherichia coli

The coding region of p41 was determined using DNA Amplification by a "polymerase chain reaction" (PCR) from a B. burgdorferi (DSM No. 5662 P / Ko2 / 85) total DNA Preparation won. The sequence thus obtained was then under the control of inducible promoters and after transfection in E. coli for expression brought (Maniatis, T .; Fritsch, E. F .; Sambrook, J. (1982) Molecular cloning. Cold Spring Harbor).

For this, the B. burgdorferi cells were in at 37 ° C for 2 weeks 2 l modified Barbour-Stoenner-Kelly- (BSK) medium (Preac- Mursic, V .; Wilske, B .; Schierz, G. (1986): European Borreliae burgdorferi isolated form humans and ticks - culture  conditions and antibiotic susceptibility. Zbl. Bakt. Hyg. A 163, 112-118) cultivated, pelleted at 6000 rpm, in TEN- Buffer (10 mM Tris-HCl pH 7.6; 1 mM EDTA; 10 mM NaCl) washed and in 20 ml lysozyme buffer (20% sucrose, 50 mM Tris-HCl pH 7.6, 50 mm EDTA, 5 mg / ml lysozyme) resuspended. After incubation for 30 min at 37 ° C, the by the Exposure of the lysozyme to the cell wall Protoplasts by adding 1 ml 25% SDS (Sodium dodecyl sulfate) lysed. After a further 10 min 4 ml of a 5 M NaCl solution were added. Protein was through Add an equal volume of TE-saturated (TE: 10 mM Tris / HCl, pH 7.8, 1 mM EDTA) phenol denatured. By Centrifugation at 4 ° C and 6500 rpm for 5 min Separation of the phases. The upper DNA-containing, aqueous phase was opened with a pipette with a wide opening (to avoid Shear forces) carefully transferred to a fresh tube and then again with the same volume Phenol / chloroform / isoamyl alcohol (1: 1: 0.04) extracted. To the separation again became the upper aqueous phase carefully transferred into a new tube and the DNA with the twice the volume of ethanol precipitated. After about 5 minutes the DNA that has failed as a long, threadlike structure Wind up on a glass rod and remove in a 70% Conveyed ethanol solution for washing. The through on the glass rod Adhesion-bound DNA was then at 2 h Stored at room temperature to allow the ethanol to evaporate effect, and then transferred to 4 ml of TEN buffer.

1 µl of the total B. burgdorferi DNA thus obtained was added to five 100 µl PCR batches amplified.

Catalyzed as a specific primer for the polymerase Amplification sequences were chosen that contain the information for the translational start and the 3′-end of p41 (Flagellin) contained. This was done in Fig. 3 DNA sequences shown used. The two Oligodeoxynucleotides were on a DNA Synthesizer 8700 synthesized in 1 µmol columns, according to the Roughly cleaned with ammonia by ethanol precipitation  and added in 400 µl H₂O. 1 µl each Oligodeoxynucleotide solution were used per PCR approach; the buffers, nucleotides and Taq polymerase were derived a commercially available test kit and were also after the test descriptions used. The temperature conditions for the individual cycles were:

2 min denaturation at 94 ° C
2 min annealing at 45 ° C
4 min DNA synthesis at 73 ° C

50 cycles were performed.

The batches from the PCRs were then combined, and the DNA after the addition of NaCl in a final concentration of 0.2 M with 2.5 times ethanol at -20 ° C for 5 h. After pelleting and washing in 70% ethanol, the DNA dissolved in 200 µl H₂O and after adding appropriate buffers with the restriction enzymes BAM HI and Pst I split according to the manufacturer's instructions.

After gel electrophoretic separation in a 1.5% The amplified DNA fragment (approx. 1000 bp) was agarose gel isolated and into a pUC8 cut with BamHI and Pst I Vector (Vierira, J .; Messing, J. (1982): The pUC plasmids, and M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259- 268) with 0.25 µg of the vector, 0.5 µg of p41 Fragments and 2U T4 DNA ligase with buffer according to the Manufacturer were used.

After transformation of the ligated DNA fragments in the E. coli Strain JM 109 (Yanisch-Perron, C .; Vieira, J .; Messing, J. (1985): Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119) and plating on agar plates with ampicillin (50 µg / ml) and X-Gal (30 µg / ml) turned white Colonies raised in 5 ml L-Broth medium, and the isolated plasmids by restriction enzyme cleavage their inserts examined.  

The DNA fragment coding for B. burgdorferi flagellin is located thus behind the inducible lacUV5 promoter of the vector im same reading frame as that started by this promoter lacZα-coding transcript. This creates a flagellin, which has some pUC8-encoded amino acids at its N-terminus contains. This area is listed below:

The vector of positive E. coli clones (e.g. pUC8 ly13) DNA insert in the expected length (1000 bp) were contained in turn, liquid cultures were created, and the transcription of the lac promoter of the plasmid by induction for 3 hours with 1 mM IPTG induced at 37 ° C and shaking. 1.5 ml of this Cultures were then briefly pelleted, the bacteria with "boiling mix" (3% sucrose, 2% SDS, 5% β-mercaptoethanol, 20 mM Tris-HCl pH 7.0, 2% bromophenol blue) at 100 ° C for Lysed for 10 min, and the proteins using 17.5% SDS-PAGE separated. After protein staining by Coomassie brilliant blue was seen in the cells with plasmid insert a new, additional band at approx. 41 kD that of the expected The size of the flag line corresponds. A specific reaction this recombinant antigen with a serum of a Lyme Lyme disease patients as well as with a monoclonal antibody against B. burgdorferi p41 Flagellin is shown in Fig. 4 demonstrated immunoblot.

Just like pUC 8, any other inducible plasmid is that starts a transcript in the same reading frame for which Production of p41 suitable. By splitting the p41- coding region at the translation start with BspHI (Tc ATG A) and PstI (at the 3′-end) and inserting the fragment into the NcoI Agency (CC ATG G) and PstI office of a so-called ATG Vector is also the expression of an authentic p41 possible, which has fused on no foreign amino acids.  

The clone was used for the methods shown below pUc8ly17 used.

Example 3 Production of pC and p100 in E. Coli from B. burgdorferi Gene banks

To provide B. burgdorferi specific DNA sequences a chromosomal gene bank was created in E. coli. With Using suitable methods such as immunoscreening or Hybridization with selected oligonucleotides was possible in of these gene bank E. coli clones are identified which corresponding B. burgdorferi specific DNA sequences contained. After restriction enzyme analysis, a Restriction enzyme map created. This could be used the targeted DNA sequences in expression Transfer vectors or perform their sequencing. The detailed procedure was as follows:

For isolation B. burgdorferi (DSM No. 5662) DNA (chromosomal DNA such as plasmid DNA), the cells were as in Example 2 described cultivated. After centrifugation at 12,000 rpm for The cells were washed for 20 minutes and in SET buffer (20% Sucrose, 50 mM Tris-HCl pH 7.6; 50 mM EDTA) resuspended. By adding 15 mg / 5 ml of lysozyme for 20 minutes Partially split cell wall. Then the protoplasted cells by adding SDS (n-dodecyl sulfate Sodium salt) final concentration 1% lysed. After 20 minutes at Proteinase K (final concentration 1 mg / ml) was at 37 ° C for two times Added 1 hour and the DNA containing solution to 100 mM NaCl with TEN buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 300 mM NaCl) set. There was a phenol extraction and two more phenol / chloroform / iso-amyl alcohol extractions (Phenol: chloroform in a ratio of 1: 1; chloroform: iso- Amyl alcohol in a ratio of 24: 1). The supernatant extracted in this way was with 2.5 vol. 95% ethanol added and the DNA at -20 ° C precipitated. By winding on a glass rod, the DNA that has failed is washed out and washed in 70% ethanol  will. After a short drying in the desiccator, the DNA was in TE buffer (10mM Tris-HCl pH 7.6, 1mM EDTA) from the RNAse (20 µg / ml) contained. The DNA prepared in this way was used for the next steps.

B. burgdorferi DNA was treated with the restriction enzyme Sau 3A according to the manufacturer's instructions incubated. By choosing suitable enzyme dilutions or The enzyme was exposed to partial cleavage same reached. Partially cleaved DNA was thus obtained with, BamH I-restricted and by treatment with alkaline phosphatase dephosphorylated vector DNA (pUC18 or other suitable vector DNA). For this T4 DNA Ligase according to the manufacturer used. For each transformation approach, 0.2-0.5 µg / µl Total DNA used. E. coli JM 109 (or other suitable E. coli strains) were made according to the Hanahan protocol (Hanahan, D. (1985): Techniques of Transformation of Escherichia coli, pp. 109-135. In: D. M. Glover (ed.) DNA cloning, vol. 1. A practical approach. IRL Press, Oxford) or according to Maniatis et. al. (Maniatis, T. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) with the ligated DNA. Recombinant E. coli clones were placed on LB medium (10 g trypton, 5 g yeast extract, 5 g NaCl, which 100 µg / ml ampicillin (or other suitable Antibiotic) contained, selected and cultivated. The Colony pattern was transferred to LB plates and identical Colony impressions created on nitrocellulose. The cells of this Colony impressions were made depending on the screening procedure used lysed differently on the filter. When using mono- or polyclonal sera (immunoscreening) for detection B. burgdorferi specific gene products by a recombined DNA, the cells were induced Lysed over saturated chloroform vapor for 15 min. To Saturation of the filter treated in this way with a skim milk Solution for 2 hours, the filters were left overnight various sera, incubated several times with TTBS buffer  washed (see above) and for 2 hours with the second peroxidase conjugated antibody. Again Washing with TTBS buffer was used for the reduction non-specifically bound peroxidase conjugated antibody. By enzymatic conversion of the substrates diaminobenzidine and H₂O₂ in an insoluble brown pigment could be positive, H. B. burgdorferi antigen-producing E. coli clones are recognized. The positive E. coli recognized in this way Clones were inoculated from the exit plate and analyzed. When using specific oligonucleotides for Hybridization and thus for the detection of specific B. burgdorferi antigen sequences (screening by hybridization) the cells were on the nitrocellulose filter alkaline lysed (by wetting the filter for 5 Minutes with 0.5 M NaOH, 1.5 M NaCl). After neutralization (by wetting the filter for 5 minutes in 1.5 M NaCl 0.5 M Tris-HCl pH 8.0), the filters with the denatured DNA with 2 × SSPE (20 × SSPE: 3.6 M NaCl, 200 mM NaH₂PO₄, pH 7.4, 20 mM EDTA, pH 7.4) wetted and dried. By baking the The DNA was fixed in a filter for 2 hours at 80 ° C. The so Treated filters were then used for hybridization used. Hybridization was done using radioactive (32 P) or non-radioactive (e.g. digoxigenin) Detection methods. Here was after known (Maniatis, T. (1982): Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor) or recommended by the manufacturer Labeling methods (32 P-labeling with 32 P-gamma-ATP and Kinase reaction or digoxygenin labeling with Dig-11-UTP and terminal transferase reaction). From positive E. a restriction enzyme analysis was performed and coli clones with this information an expression of the antigen coding DNA sequences in suitable vectors or their sequencing carried out.

Synthetic samples were initially used as hybridization samples Oligodeoxynucleotide used, based on their sequence  of p100 and pC amino acid sequences were selected.

The procedure was as follows:

The two proteins were formed from lysates from B. burgdorferi by extraction with n-octyl β-D-thioglucopyranoside partially cleaned and further by SDS polyacrylamide gel Electrophoresis separated. Then the antigens transferred to a glass fiber matrix by Western blotting and the corresponding pieces with the B. burgdorferi proteins cut out. p100 was then sequenced at the N-terminal and determined the first 22 amino acids of the amino terminus (this The method of "micro-sequencing" is described in: Eckerskorn, C., Mewes, W., Goretzki, H. and Lottsspeich, F .: A new siliconicide fiber as support for protein-chemical analysis of electroblotted proteins. Eur. J. Biochem. 176: 509-519 (1988). A direct sequencing was not possible with pC because the N-terminus of a sequencing is not directly accessible d. H. that there may be a myristilation or similar There are modifications. That is why this protein tryptically cleaved, the fragments separated by HPLC and then sequenced two of them. From the so obtained Amino acid sequences were then listed below Oligodeoxynucleotide sequences derived. Since usually several Codon possibilities for an amino acid had to exist the corresponding positions of the oligonucleotide Base variations are taken into account and during the Synthesis in equimolar ratios.

p100-p1 - p100 - amino acid sequence:
Glu Leu Asp Lys Glu Lys Leu Lys Asp Phe Val Asn Leu Asp Leu Glu Phe Val Asn Thr

p100 - oligodeoxynucleotide sequence given in parentheses and denoted by ";" separate bases were built in during the synthesis (on DNA synthesizer) in equimolar ratios:
GA (G; A) (C; T) T (G; T; A) GA (C; T) AA (G; A) GA (G; A) AA (G; A) (C; T) T ( G; T; A) AA (G; A) GA (C; T) TT (C; T) GT (T; A) AA (C; T) (C; T) T (G; T; A) GA (C; T) (C; T) A (G; T; A) GA (G; A) TT (C; T) GT (T; A) AA (C; T) TA (C; T) A

pC - amino acid sequences:
p1: Lys Ile Thr Asp Ser Asn Ala Thr Val Leu Ala Val Lys
p2: Asp Leu Phe Glu Ser Val Glu Gly Leu Leu Lys

pC-p1 oligodeoxynucleotide sequence:
AA (G; A) AT (T; A) AC (A; T) GA (T; C) (A; T) C (A; T) AA (T; C) GC (A; T) AC (A ; T) GT (A; T) (T; C) T (G; A; T) GC (A; T) GT (A; T) AA (A; G) A

pC-p2 oligodeoxynucleotide sequence:
GA (T; C) (C; T) T (G; A; T) TT (T; C) GA (G; A) (T; A) C (A; T) GT (A; T) GA ( G; A) GG (A; T; C) (T; C) T (G; A; T) (T; C) T (G; A; T) AA (A; G) A

Example 4 Purification of the recombinantly produced B. burgdorferi antigens using the example of the p41 (Flagellin)

A 50 ml overnight culture of that described in Example 2 Clones pUC8ly2 were placed in 1.5 ml of fresh L-Broth medium and incubated at 37 ° C with intensive shaking. When reached the optical density was 0.7 with IPTG in a final concentration of 1 mM and induced for a further 3 h incubated. The bacteria were pelleted (6000 rpm, 10 min), in 300 ml 20 mM Tris-HCl pH 8.0, 50 mM EDTA, 0.5 mg / ml lysozyme resuspended and in a 37 ° C for 45 min Given water bath. After adding NaCl in one Final concentration of 150 mM and Triton-X-100 in one Final concentration of 1% was continued for 45 min at 37 ° C incubated, and then the suspension three times for each Treated with ultrasound for 5 min. Insoluble components were pelleted at 9000 rpm in 30 min, in 20 mM Tris-HCl  pH 8.0, 10 mM dithiothreitol and 1% octyl-glucopyranoside resuspended and for 1 h at Room temperature stirred. After the subsequent pelleting insoluble components at 17,000 rpm in 30 min Decant the supernatant carefully.

The pellet was then dissolved in 150 ml of 20 mM Tris-HCl pH 8.0, 8 M urea, 1% β-mercaptoethanol by stirring for 2 h sesuspended. Insoluble constituents were again here separated by centrifugation at 17,000 rpm in 30 min, and the supernatant on a DEAE Sephacel column with a gel volume of 550 ml (3 cm diameter, 80 cm high). The p41 antigen was eluted in a NaCl gradient of 0-800 mM in a total volume of 600 ml. The recombinant p41 is at a NaCl concentration of approximately 0.25 M. The appropriate fractions were combined and further by HPLC with a Mono Q column (Anion exchanger) cleaned (Fig. 4). An elution profile with the purified p41 in a NaCl gradient of 0-800 mM is shown in Fig. 5. The p41-positive fractions here (after Western blot analysis) were against 20 mM Tris-HCl pH 8.0, 10 mM MgCl₂ and 0.1% β-mercaptoethanol dialyzed and then used for the tests shown in Example 5. From a purification of p41 based on 1 l bacterial culture can typically expect a yield of 5 to 10 mg will.

Example 5 Use of recombinantly produced B. burgdorferi antigens (Example p41) in the ELISA

Due to the high purity of the recombinant produced B. burgdorferi specific tests are possible antigens machine-readable and without large technical and human resources Effort is feasible.

Microtiter plates with 50 µl each of the purified p41 (Concentration 0.5 - 5 µg / ml) coated per bowl. The Plates were made using standard methods at 4 ° C overnight  incubated, washed and the binding sites still free Saturated 2% bovine serum albumin solution. Subsequently 50 µl of serum (dilution 1: 200) were pipetted in, incubated for 2 h at 37 ° C, washed unbound parts and the bound immune complexes with 50 µl peroxidase labeled anti-human IgG (dilution 1: 1000) was detected. To the dishes were washed again with 100 µl of ortho- Phenylenediamine (concentration 0.1% in 0.1 M phosphate buffer pH 6.0 with 0.03% H₂O₂) charged as a dye reagent and in Staining carried out in the dark after 10 min with 100 μl of 1 N Sulfuric acid stopped. The microtiter plate was in one Photometer evaluated at 486 nm (Fig. 6).

In the example shown here, 7 were positive and 8 negative anti-B. Burgdorferi sera tested. Of the clinically proven Lyme positive sera were three on Western blot strips with B. burgdorferi as antigen none Showed reaction with p41; H. Early - stage sera Infection. In the ELISA they reacted with the recombinant antigen also only borderline. Normal p41- positive sera, on the other hand, responded very well, while Lyme- negative sera remained below OD = 0.3.

Example 6 Production of B. burgdorferi specific monoclonal antibody

Female Balb / c mice were treated with B. burgdorferi (DSM no. 5662) immunized intraperitoneally. The 1st immunization was done with complete Freund's adjuvant, 2-5 more Immunizations with incomplete Freund's adjuvant followed every 2 weeks. 2 weeks later the antigen applied without adjuvant and the mice killed 3 days later and removed the spleen. The spleen lymphocytes were treated with mouse myeloma cells (Ag8-653) mixed in a ratio of 1: 1, sedimented and with a fusion solution (2.5 g polyethylene glycol (PEG), 2.5 ml RPMI medium, 250 µl DMSO) added: 1 min addition of the fusion solution, 90 sec.  Incubation at 37 ° C. The cells were sedimented again, the PEG removed and culture medium (HAT medium) added. Finally, the cell suspension was in microtiter plates, which Macrophages contained, seeded and incubated. Hybridoma supernatants were undiluted in the indirect Immunofluorescence (IFT) tested (Wilske, B .; Schierz, G .; Preac-Mursic, V .; Weber, K; Pfister, H.-W .; Einhäupl, K. (1984): Serological diagnosis of Erythema migrans disease and related disorders. Infection, 12, 331-337).

IFT-positive cell supernatants were determined using Western blot analyzed. Hybridomas reactive in the Western blot were 4 times subcloned using "limiting dilution" and with regard to their Immunoglobulin class and IgG subclass identified.

In this way the following monoclonal antibodies (MAB) receive:

• 1. MAB against p41:
  • (a) L41 1C11
    This antibody was reactive with all 30 B. burgdorferi strains tested and with relapsing fever borrelia (except B. hermsii), but not with treponemes.
  • (b) L41 1D3
    This antibody was reactive with the majority (21 out of 24) of the B. burgdorferi strains, but not with relapsing fever borrelia and treponemes.
• 2. MAB against p100 (L100 1D4):
  This antibody was reactive with all 30 B. burgdorferi strains tested, but not with relapsing fever borrelia or treponemes.
• 3. MAB against pC (L22 1F8):
  This MAB was reactive with pC proteins from skin and CSF strains, whereas the pC proteins of some but not all tick strains were negative.
• 4. MAB vs. OspA:
  OspA is a major protein (30 kD range) of the outer membrane of most B. burgdorferi strains. OspA proteins from European B. burgdorferi strains are antigenetically heterogeneous and differ antigenetically from the American strains. The few OspA-negative strains have pC proteins (Wilske, B; Preac-Mursic, V .; Schierz, G .; Kühbeck, R .; Barbour, AG; Kramer, M. (1988): Antigenic variability of Borrelia burgdorferi. Ann. NY Acad. Sci. 539, 126-143).
  • (a) L32 2E7:
    A total of 29 out of 32 strains were reactive. The negative strains had no OspA protein. The 3 negative strains were reactive with the pC-specific MAB L22 1F8.
  • (b) L32 1G3:
    This MAB was only reactive in 3 out of 25 strains tested.
• The combination of MAB L32 2E7 and MAB L22 1F8 as well as the Reaction with MAB L100 1D4 allows the identification of B. burgdorferi borrelia and treponemes. A safe one Identification and differentiation of B. burgdorferi was not with previously available monoclonal antibodies possible (Wilske, B .; Preac-Mursic, V .; Schierz, G .; Kühbeck, R .; Barbour, A.G .; Kramer, M. (1988): Antigenic variability of Borrelia burgdorferi. Ann. N. Y. Acad. Sci. 539, 126-143).

Description of the tables Table 1 Reactivity of Lyme disease sera from various Disease stages with B. burgdorferi antigens (p17, pC, p41, p100) in a Western blot with B. burgdorferi lysate as antigen

Table 1 gives an overview of the immunominants Proteins in various stages of lymeborreliosis.

1.1 sera from healthy individuals and to a greater extent from Syphilis patients showed antibodies against p60 ("common antigen "). Antibodies against p41 detected.

1.2 Prove to be immunodominant proteins Early manifestations (EM and LMR) of the flagella protein p41 and the pC protein. pC is the immunodominant protein for the early immune response. In particular IgM antibodies against pC can occur earlier than IgM antibodies against p41 (see also Fig.2a)

1.3 Sera from patients with late manifestations (ACA and Arthritis) reacted in all cases (n = 22) with p41 or p100 and in 21 cases with p100 or p17. p17 was in 17, p100 in 19 and p41 reactive in 20 cases.

1.4 The intrathecal IgG immune response was in all 12 investigated cases directed against p41. Antibodies against p41 were not detectable in 3 cases in the serum.

Table 2 Reactivity of immune sera (against various bacterial Pathogen) with proteins from B. burgdorferi (Western blot)

Western blot strips with electrophoretically separated B. Burgdorferi lysate was as described in Example 1 manufactured and with sera against different more or less related and therefore cross-reactive pathogens incubated. The  Sera were from rabbits with the respective pathogens had been immunized. The least cross reactivity has p100; only one (anti-B. hermsii) of the 15 tested Pathogen-specific sera reacts with this protein. p41 and pC react with three of the sera and shine therefore also suitable for diagnostic use. The presence of immune-conserved is clear To recognize antigens; for example 14 or 12 react of the sera tested with proteins of the size 40 or 60 kD (p40; p60). These "common antigens" are therefore unsuitable for diagnostic use.  

Tab. 1: Immunodominant proteins for the humoral immune response in Lyme borrellosis

1.1. Reactivity of human control sera (IgG Western Blot)

1.2. Immune response to pC and p41 in erythema migrans (EM) and lymphocytic meningoradiculitis (LMR) (Western blot)

1.3. Immune response to p100, p41 and p17 (IgG Western Blot)

1.4. Intrathecal immune response in lymphocytic meningoradiculitis (IgG Western Blot)

Description of the pictures Fig.1a and b Reactivity of B. burgdorferi-infected patients with Lysates from 5 different B. burgdorferi strains in the Western Blot

Sera from stages II and III (neuroborreliosis, Stage II (IgM and IgG); Acrodermatitis (IgG) and arthritis (IgG), stage III). The early immune response is independent of Test strain against a narrow spectrum of Borrelia proteins directed (pC and p41). The late immune response is against one broad panel of Borrelia proteins. Immune dominant Proteins are (regardless of the test strain) p100 (with variable Molecular weight) and p41.

Fig. 2 2a) Follow-up (IgM Western Blot) in Erythema migrans

The pC protein can be the immunodominant protein of the early Be immune response. Antibodies to p41 can later occur and are weak. With longer ones Disease duration can also be IgM antibodies against p17 occur.

2b) IgG western blot in late manifestations

IgG antibodies recognize a wide range of Borrelia proteins. Prove PKo when using the strain p17 and p100 as immunodominant proteins. p17 is from PKo strain very pronounced (in contrast to other strains; see Fig. 1). The flagellin p41 was used in 2 of these examples (Serum 1 and 2) not recognized.

Fig. 3 Scheme of DNA amplification of the p41 coding region

A; Section of the B. burgdorferi DNA with the p41-coding Region (black bar).  

B; Enlargement of the 5 'or 3' end of the p41 gene with the respective DNA sequences. The is also given Translation start (ATG) and the stop codon at the 3′-end (TAA). The primer sequences used for the PCR are additional under (primer 1) or over (primer 2) the DNA encoding p41 Double strand specified. Hybridization of the primers can only with the respective 3'-areas. The 5'-ends contain non-hybridizing parts that interface for Restriction enzymes represent: GGATCC - BamHI; TCATGA - BspHI, at the 5′-end; GACGTC - PstI at the 3′-end.

Fig. 4 Expression, reactivity and purification of recombinant p41

Left part: coomassie blue colored SDS polyacrylamide gel. The individual tracks were loaded as follows:

• 1, E. coli lysate, negative control;
• 2, E. coli lysate with pUC8ly17 according to IPTG Induction, the recombinantly produced p41 is additional Band in the range of approx. 45 kDa can be seen;
• 3, supernatant of Lysate from 2 after cell disruption as in Example 4 described;
• 4, pellet fraction of the lysed cells with the recombinant p41;
• 5, octyl-gluco-pyranoside supernatant;
• 6, like 5, but pellet fraction;
• 7-10, fractions after elution from p41 from a MonoQ column through a salt gradient;
• Lanes 9 and 10 contain recombinant p41,

due Degradation and incomplete translation there are small fragments in addition to the complete product on, which, however, is also reflected in authentic p41 material B. find burgdorferi.

right part: immunostained Western blot of an SDS gel with Samples of the Coomassie-stained gel. The immunostaining was done with a monoclonal antibody described in Example 6 carried out. Name of the tracks or samples like Coomassie stained gel; Track 0, empty track.  

Fig. 5 HPLC elution profile of p41 from an ion exchange column with a salt gradient

After the anion exchanger cleaning (MonoQ) from p41 the antigen against 4M urea was without Salt dialyzed back and again onto the MonoQ column given to check the purity. The elution profile shows only one protein adsorption peak. The smaller one Summit immediately before the main part corresponds to that in Fig. 4, lane 8 visible p41 fragment with a size of approx. 30 kD (tested by Western Blot).

Fig. 6 IgG ELISA with recombinant p41 as an antigen

The recombinant purified via anion exchanger (MonoQ) Antigen (see Fig. 5) was at a concentration of 0.5 µg / ml used. There were 7 sera from patients with one clinical defined Lyme disease and 8 sera tested by healthy individuals. Four sera from Lyme disease patients were in the Western blot strongly reactive with the recombinant p41 (= positive), 3 sera weakly reactive (= borderline), the serums of the healthy did not react (= negative). The IgG ELISA showed one comparable result. Y axis: Optical density at Wavelength 486 nm; borderline = borderline

Fig. 7 Reactivity of monoclonal antibodies against various B. burgdorferi antigens

Six monoclonal antibodies against B. burgdorferi were included 30 different B. burgdorferi strains, 4 Relapsing fever borrelia strains and 2 different treponemes tested. In the picture there are three different B. burgdorferi isolates (1 = B31, American strain; 2 = PKo, German Skin trunk; 3 = PBi, German CSF strain), one Relapsing fever borrelia (4 = B. Hermsii) and a Treponemen strain (5 = T. Phagedenis) shown as an example. It was the  monoclonal antibodies prepared according to Example 6 used.

Claims (17)

1. immunologically active protein from Borrelia burgdorferi, that in one from other Borrelia burgdorferi Protein free form is present and genetically engineered using from Borrelia burgdorferi (DSM No. 5662) isolated DNA. 2. Immunologically active protein according to claim 1, characterized characterized in that it has a molecular weight of about 41 has kDa. 3. Immunologically active protein according to claim 1, characterized characterized in that it has a molecular weight of about 22 has kDa. 4. Immunologically active protein according to claim 1 or 3, characterized in that it is the amino acid partial sequence Lys Ile Thr Asp Ser Asn Ala Thr Val Leu Ala Val Lys and / or Asp Leu Phe Glu Ser Val Glu Gly Leu Leu Lys has. 5. Immunologically active protein according to claim 1, characterized characterized in that it has a molecular weight of about 17 has kDa. 6. Immunologically active protein according to claim 1, characterized characterized in that it has a molecular weight of about 100 has kDa.   7. Immunologically active protein according to claim 1 or 6, characterized in that it is the amino acid partial sequence Glu Leu Asp Lys Glu Lys Leu Lys Asp Phe Val Asn Has Leu Asp Leu Glu Phe Val Asn Thr. 8. Monoclonal antibodies against one of the immunological active proteins according to any one of claims 1 to 7 are directed. 9. Use of immunologically active protein after one of claims 1-7 for the detection of antibodies against Borrelia strains, characterized in that at least an immunologically active protein according to one of the claims 1 to 7 is used, which with the in the Examination fluid react to existing antibodies can and that at least one display component is used, the detection of complexes enables immunologically active protein and antibodies. 10. Use according to claim 9, characterized in that 2 to 4 immunologically active proteins according to the claims 1 to 7 can be used. 11. Use according to one of claims 9 or 10, characterized characterized in that the display component against the Antibody to be detected is directed antibody that has a mark. 12. Use according to claim 11, characterized in that that the label is a radioactive isotope. 13. Use according to any one of claims 9 to 11, characterized characterized in that the label from an enzyme exists that can catalyze a color reaction.   14. Use according to claims 9 or 10, characterized in that that the immunologically active protein or a monoclonal antibody directed against it is biotinylated and the display component is avidin or Streptavidin with enzyme covalently attached to it, in particular Peroxidase is. 15. Use according to one of claims 9 to 11, characterized characterized that they are in the form of an ELISA test kit is carried out. 16. Use according to claim 15, characterized in that at least an immunologically active protein after one of claims 1 to 7 coupled to microtiter plates and the display component is made of anti-human Immunoglobulin, especially IgG and / or IgM antibodies exists to which a color reaction catalyzes Enzyme is coupled. 17. Use of immunologically active proteins after one of claims 1 to 7 for the production of vaccines to protect against infections of Borrelia Bacteria, preferably Borrelia burgdorferi strains.