Title: Immunologically active proteins from Borrelia
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
Appl. No.: 711546
Filed: November 13, 2000
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
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
Late phase: Stage III Lyme arthritis
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
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
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
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
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
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.
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
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
b. has been prepared by expression in a bacterium other than Borrelia
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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