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Title:  Immunologically active proteins from Borrelia burgdorferi

United States Patent:  6,509,019

Issued:  January 21, 2003

Inventors:  Fuchs; Renate (Deisenhofen, DE); Wilske; Bettina (Munchen, DE); Preac-Mursic; Vera (Munchen, DE); Motz; Manfred (Munchen, DE); Soutscheck; Erwin (Munchen, DE)

Assignee:  Mikrogen Molekularbiologische Entwicklungs-GmbH (Munich, DE)

Appl. No.:  711546

Filed:  November 13, 2000

Abstract

Various immunologically active proteins from Borrelia burgdorferi have been prepared by genetic manipulation in microorganisms. To do this, the specific DNA sequences were selected from a B. burgdorferi gene bank using suitable screening methods, or were prepared directly by DNA amplification using selected hybridization probes, and were placed under the control of inducible promoters such as, for example, the lac promoter. It has been possible, owing to description of efficient purification methods for the expressed antigens, to provide the proteins in a suitable way. These proteins can be used to produce specific and sensitive diagnostic assay kits. The specific combination of the immunologically active proteins makes precise diagnosis possible. Furthermore, monoclonal antibodies have been generated and are used as reagents for detecting pathogens directly in test samples or after cultivation. The Borrelia burgdorferi-specific DNA sequences can be employed for direct detection of the pathogen in patients' samples (for example by means of the PCR reaction).

Description of the Invention

Lyme borreliosis is the commonest infectious disease of humans transmitted by ticks in the Federal Republic of Germany. In contrast to Russian spring-summer encephalitis (RSSE) which is likewise transmitted by ticks, Lyme borreliosis is not confined to a few endemic areas but occurs in all the states of the FRG. Infestation of the main vector in Europe, Ixodes ricinus, with the pathogen of Lyme borreliosis, the spirochete Borrelia burgdorferi, in Southern Germany is about 20% of adults, about 10% of nymphs and about 1% of larvae. The main vector in the USA, Ixodes dammini, may be up to 100% infected by Borrelia in highly endemic areas.

B. burgdorferi belongs to the family of spirochetes. Spirochetes are spiral bacteria 8-30 .mu.m long. They consist of an outer coat, the endoflagella in the periplasm and the protoplasmic cylinder. The protoplasmic cylinder is a complex of cytoplasm, internal cell membrane and peptidoglycan. Representatives of the spirochetes which are pathogenic for humans include, beside B. burgdorferi, the Borrelia of relapsing fever (for example B. recurrentis), the pathogen of syphilis (Treponema (T.) pallidum) and the Leptospira. As a result of the close immunological relationship of the pathogens, cross-reactions are a problem in the serological detection of antibodies in cases of syphilis and Lyme borreliosis with assays currently available.

Infection with B. burgdorferi results in a complex clinical picture which can, similarly to syphilis, be divided into three different stages. The principal manifestations are:

        Early phase:    Stage I       Erythema migrans
                                      Bannwarth's lymphocytic
                                      meningoradiculitis (LMR)
                                      Borrelia lymphocytoma
        Late phase:     Stage III     Lyme arthritis
                                      Acrodermatitis chronica
                                      atrophicans (ACA)
                                      Chronic Borrelia
                                      encephalomyelitis



Less common clinical manifestations are: carditis, myositis, iritis and panophthalmitis. Transmission by the pathogen crossing the placenta is possible but to date only a few cases of congenital Lyme borreliosis have been recorded. The various stages may occur singly or in combination. B. burgdorferi infection may also have a sdbclinical course. Epidemiological studies on 375 clinically confirmed cases show some peculiarities in the age and sex distribution of the various clinical manifestations. Thus, patients with Erythema migrans were commonest in the 30 to 60 year age group. Neurological manifestations showed two peaks with age: the first in children and young people up to 20 years of age, and the second in 40 to 70 year-olds. Lyme arthritis was observed to be commonest in 30 to 60 year-olds. Patients with ACA were never below 30 years of age. ACA affects women distinctly more often than men. Serological testing showed predominantly positive IgM findings in patients with Erythema migrans, and predominantly positive IgG findings when there were neurological manifestations, in an immunofluorescence assay. With the late manifestations of ACA and Lyme arthritis, the IgG titers were regularly elevated, and IgM antibodies were now detectable only in exceptional cases.

Available for diagnosis are both pathogen detection and antibody detection. Pathogen detection in material from patients (skin biopsies, CSF, puncture fluids) is recommended especially in the early stage (Erythema migrans) when antibody detection is frequently negative. However, a complex nutrient medium is required for culturing B. burgdorferi (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) and cultivation is therefore restricted to special laboratories. In addition, a time of up to 5 weeks is required to isolate the pathogen. B. burgdorferi is isolated from skin samples in 50-70% of cases with cutaneous manifestations and in 3-5% of cases with neuroborreliosis (Preac-Mursic, V.; unpublished results).

Antibody detection (IgM, IgG) is carried out on serum and, when there are neurological manifestations, also from CSF. The serological finding depends on the stage of the disease, the duration of the symptoms and any antibiotic therapy which has already been applied. Thus, antibody detection with assays available to date is successful only in 20-50% of cases with Erythema migrans, in 50-90% of cases with neurological manifestations and in 90-100% in cases with ACA and arthritis.

Therapy of Lyme borreliosis is predominantly carried out with penicillin G, tetracyclines, erythromycin or cephalosporins. Although Lyme borreliosis frequently resolves spontaneously in the early stages, even then late manifestations are not ruled out. This is why therapy in the early stage is indispensable. In addition, clinical resolution after antibiotic therapy can be achieved when there are late manifestations only in some of the cases (for example only about 50% of cases with Lyme arthritis).

This is why Lyme borreliosis should be diagnosed as early as possible. Since (as already explained) pathogen isolation is costly, time-consuming and, moreover, not always successful, better serodiagnostic assays ought to be developed. The methods used to date (immunofluorescence assay (IFA), indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA)) frequently fail in the early stages. The antigens employed for these assays are all B. burgdorferi cells or whole-cell ultrasonicates. The use of different B. burgdorferi strains as antigen in the ultrasonicate ELISA leads to differing test results. Immobilization of cells on slides or ultrasonicate antigen on microtiter plates is followed by incubation with serum or CSF and detection of the Borrelia-specific antibodies with a second fluorescence- or peroxidase-labeled antibody of the appropriate immuno-globulin class. The reaction is then quantified either in a fluorescence microscope (IFA) or after a color reaction in a photometer (ELISA).

Broad cross-reactions of the pathogen B. burgdorferi with other bacterial pathogens, especially with T. pallidum, the syphilis pathogen, is a problem for the specificity of the assays. Since the assay antigens generally consist of lysates of the whole pathogen there is also detection of antibodies against so-called common antigens (Hansen, K.; Hindersson, P.; Pedersen, 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 widely distributed proteins with highly conserved sequences, that is to say the common antigens of Borrelia, Treponema as well as many other bacteria have common epitopes. Besides this, false-positive reactions may occur in the IgM-IFA or IgM-ELISA when the sera have rheumatoid factor activity. Therefore, in order to make the assays more specific, in the detection of IgG and IgM antibodies a preabsorption of the sera with a Treponema ultrasonicate, and additionally for the detection of IgM antibodies also absorption with rheumatoid factor absorbent, is carried out.

An object of the present invention is therefore to provide immunologically active proteins from Borrelia burgdorferi which are used in an assay kit which does not have the abovementioned disadvantages. An additional aim is that this assay kit makes it possible rapidly and reliably to detect antibodies directed against Borrelia burgdorferi.

Another object of the present invention is to provide monoclonal antibodies which are directed against particular immunologically active proteins from Borrelia burgdorferi. A further aim is to provide immunologically active proteins which are suitable as vaccines against infections caused by Borrelia strains.

Testing of patients' sera from different stages of the disease of Lyme borreliosis in a Western blot, and testing of non-Lyme borreliosis patients (especially syphilis patients) for cross-reactivity with B. burgdorferi resulted in the finding of immunologically active proteins (B. burgdorferi antigens) which, on the one hand, elicit a good antibody response after infection and, on the other hand, show a low cross-reactivity with sera which are not B. burgdorferi-positive (Example 1). It emerged that a particular strain of B. burgdorferi which has the internal laboratory identifier PKo and which was deposited at the Deutsch Sammlung fur Mikroorganismen (DSM) under No. 5662 possesses, inter alia, an immunodominant protein in the molecular-weight region about 22 kD (pC protein). Under the provisions of the Budapest Treaty, representative samples of the Borrelia burgdorferi strain (internal laboratory identifier PKo) were deposited at the DSM Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH, Mascheroder Weg 1 B, D-3300 Braunschweig, Germany, under accession number DSM 5662, on Nov. 30, 1989. The molecular weight of the proteins according to the invention was determined by methods known per se, in particular by SDS gel electrophoresis. It was found that this protein is immunodominant for the IgM response. This protein is not expressed in the same way in all B. burgdorferi strains. This immunologically active protein (pC protein) was prepared by genetic manipulation according to the invention (Example 3).

Other immunologically active proteins (antigens) which are particularly suitable for use in assay kits were also prepared in generally accessible and commercially available Escherichia coli cells such as, for example, strains JM 105 (Pharmacia) or DH 5 (Gibco-BRL). To do this, the B. burgdorferi DNA fragments coding for these proteins were isolated and subsequently inserted into efficient expression vectors (Examples 2 and 3).

The appropriate DNA fragments were identified and isolated by various methods. Thus, an immunologically active protein with a molecular weight of about 41 kD, which is also called p41 protein hereinafter, was prepared by means of the polymerase chain reaction (PCR) and specific primers whose sequences were prepared by synthesis (Example 2).

In addition, a gene bank of the B. burgdorferi genome was constructed and was screened using monoclonal antibodies for the direct expression of immunologically active proteins.

In a corresponding way, proteins with molecular weights of about 100 kD and 31 kD were also cloned and sequenced.

Another method comprised purifying particular selected immunologically active proteins (antigens) from B. burgdorferi lysates and determining the amino-acid sequences of these antigens. Subsequently, oligodeoxynucleotides corresponding to the amino-acid sequence were synthesized and, by hybridization, those clones in the gene bank which have DNA sequences coding for the immunologically active proteins were identified. The two latter methods are explained in detail in Example 3.

After characterization, sequencing and recloning of the genes into appropriate expression vectors, the antigens were expressed in E. coli cells and subsequently purified. A preferred purification method is described in Example 4.

The immunologically active proteins from Borrelia burgdorferi which have been prepared according to the invention can be used in assay kits which provide a surprisingly sensitive detection of antibodies against B. burgdorferi in various test fluids. One advantage of the immunologically active proteins prepared according to the invention is that the preparations consist only of the required protein and possibly those proteins which are attributable to degradation events and/or incomplete translation. These preparations contain no B. burgdorferi proteins which do not correspond to the protein produced by recombination because they have been prepared by genetic manipulation.

The term "assay kits" means a set of assay reagents which makes it possible to detect particular antibodies. The principles on which assay kits are based have been described in "Immunoassays for the 80s" (1981) by A. Voller et al., published by MTP Press Ltd., Falcon House, Lancaster, England. The assay reagents display as principal component the antigen(s) and, where appropriate, specific, preferably monoclonal, antibodies.

The assay kits according to the invention for detecting antibodies against Borrelia burgdorferi contain at least one immunologically active protein which is available without contamination by other proteins from the Borrelia burgdorferi strain. This immunologically active protein acts as antigen and reacts with the antibodies present in the test fluid. Assay kits according to the invention preferably have two to four immunologically active proteins which are available without contamination by other proteins from B. burgdorferi. The assay kit furthermore contains an indicator component which makes the detection of the presence of complexes of antigen and antibody possible.

The assay kits according to the invention can be based on a variety of principles known per se. In principle, the antigen can carry a label, and the label can consist of a radioactive isotope or an enzyme which catalyzes a color reaction. It is likewise possible for the antigen to be bound to a solid support (microtiter plates or beads), and the indicator component can comprise an antibody which is directed against antibodies and carries a label, and the label can comprise a radioactive isotope or an enzyme which catalyzes a color reaction.

The assay kit preferred for the purposes of the present invention is the so-called ELISA (enzyme-linked immunosorbent assay). One embodiment thereof is described in detail in Example 5. The results of this example show that it was surprisingly possible to achieve a very high specificity of the assay kit by using only one immunologically active protein according to the invention. Furthermore, the assay kits according to the invention surprisingly make possible a differentiation correlated with the stage of the disease. The combined use of a plurality of antigens in one assay kit makes it possible to detect antibodies against Borrelia burgdorferi even in cases in which the symptoms of the disease have not yet become clinically manifest. It is likewise possible to diagnose infections with B. burgdorferi in which the patient experiences only a subclinical infection. The information which can be obtained from the assay kits according to the invention is particularly important in cases in which it has been possible to find a tick bite but it is unclear whether an infection with a Borrelia strain is present.

Combined use of a plurality of the immunologically active proteins is preferred for the purpose of the present invention. A combination of the proteins p41, pC, p17 and/or p100 is very particularly preferred. The use of the ELISA assay kit preferred according to the invention also makes possible a differentiation with regard to the nature of the antibodies. If, for example, IgM antibodies are to be detected, the so-called .mu. capture assay can be employed, in which antibodies directed against IgM antibodies are bound to the solid phase. After the assay plates have been incubated with the fluid to be tested, the IgM antibodies present in the test fluid are bound to the solid phase. It is then possible, after saturation of non-specific bindings, to add an immunologically active protein of the present invention. This antigen is then detected by an indicator molecule. In this case the antigen can be biotinylated, and subsequently avidin which has covalently bonded peroxidase is added. The peroxidase then catalyzes a reaction which leads to color formation.

Another possibility comprises adding monoclonal antibodies, which are specific for the antigen and are biotinylated, to the complex of support/anti-IgM antibody/antibody to be detected/antigen according to the invention. Biotinylation is described, for example, in Monoklonale Antikorper [Monoclonal antibodies] (1985) Springer Verlag, J. H. Peters et al. Detection of the complex is effected therein by adding avidin to which an enzyme catalyzing a color reaction is coupled.

Another embodiment of the present invention comprises detecting IgM by indirect ELISA. This entails the antigens according to the invention being bound to microtiter plates, incubated with the fluid to be detected and, after washing, the immune complexes being detected by means of anti-.mu. conjugate.

Another aspect of the present invention comprises a generation of monoclonal antibodies which are directed against the immunologically active proteins of Borrelia burgdorferi. The preparation of monoclonal antibodies of this type is explained in detail in Example 6. It is possible to use monoclonal antibodies of this type as reagents for direct pathogen detection. However, monoclonal antibodies can also be coupled to the solid phase of a microtiter plate. The immunologically active proteins (antigens) are added and then immobilized by antibody-antigen binding to the microtiter plate. The test fluid (which can be, for example, serum or CSF) is subsequently added. The antibodies present in the test fluid then bind to the antigen and can be detected with the aid of an indicator component.

Furthermore, the monoclonal antibodies can be used very satisfactorily for purifying immunologically active proteins (antigens). The advantage in this case is that the purification is particularly gentle. To do this, the monoclonal antibodies are bound to a solid matrix. This solid matrix is preferably in the form of a column. The partially prepurified antigens are then mixed under physiological conditions with the antibodies coupled to a solid matrix. After the matrix-antibody-antigen complex has been washed it is possible to elute the antigens. It is normal to use for this high salt concentrations or buffers with a pH which makes the elution possible.

In another aspect of the present invention, DNA sequences which correspond in whole or in part to the amino-acid sequence of the immunologically active proteins are provided. These DNA sequences can preferably be used to detect Borrelia strains in test material by hybridization. To do this, an oligonucleotide which partly corresponds to the DNA sequence is prepared. This oligonucleotide is radioactively labeled. On the other hand, the DNA from the test material is bound to a suitable filter, preferably nitrocellulose, and subsequently hybridized with the radioactively labeled oglionucleotide. It is likewise possible to use the DNA sequences according to the invention for in situ hybridization for direct detection of B. burgdorferi in infected tissue. In place of the chemically synthesized oligonucleotides it is also possible for appropriate DNA fragments to be replicated in bacteria and subsequently cut out of the vectors with the aid of restriction endonucleases. After isolation of these DNA fragments they can be radioactively labeled and used as described above for the hybridization.

Another aspect of the present invention comprises the possibility of using the immunologically active proteins (antigens) according to the invention from Borrelia burgdorferi as vaccines. To do this, the antigens according to the invention are prepared in pure form. They are subsequently administered, singly of in combination with or without an agent stimulating the immune response, to the person to be immunized. This stimulates the formation of specific antibodies against Borrelia burgdorferi strains.

The proteins, DNA sequences and monoclonal antibodies according to the invention can be used in various areas. Thus, the assay kits according to the invention can also be used to detect B. burgdorferi infections in livestock, and the proteins can also be used for immunizing livestock, especially valuable livestock.

To the extent that the present invention relates to proteins from Borrelia burgdorferi, these can also be protein fragments which have only a partial sequence of the complete amino-acid sequence. Partial sequences of this type usually have at least 10 amino acids and preferably at least 15 amino acids.

However, the protein fragments are normally larger. Thus, for example, it has been found with the protein with a molecular weight of about 41 kD that deletion of about 20 to 25 amino acids at the N terminus of the protein leads to a protein which has an increased specificity. The reason for this might be that a so-called common epitope is deleted and specific epitopes remain. The use of proteins with deletions of this type is particularly preferred in this connection.

Proteins with a molecular weight of about 22 kD or 100 kD are particularly preferred for the purpose of the present invention. These proteins can also derive from other Borrelia burgdorferi strains.

Claim 1 of 5 Claims

What is claimed is:

1. A purified protein obtained from Borrelia burgdorferi wherein the protein is characterized in that it

a. elicits an immunological response against Borrelia burgdorferi from a mammal;

b. has been prepared by expression in a bacterium other than Borrelia burgdorferi;

c. is free of other proteins derived from Borrelia burgdorferi; and

d. is a protein comprising SEQ ID NO: 5 or is a protein consisting of at least 10 amino acids of SEQ ID NO: 5.
 


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