Note: Descriptions are shown in the official language in which they were submitted.
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1
P13 antigens from Borrelia
FIELD OF THE INVENTION
The present invention relates to nucleic acid sequences encoding antigenic
proteins
associated with Borrelia burgdorferi sensu lato (Borreiia burgdorferi sensu
stricto,
Borrelia garinii, and Borrelia afzelii; collectively designated Bb
hereinafter), particularly
polypeptides associated with virulence; vaccine formulations comprising these
poly-
peptides are also part of the invention. The invention also relates to methods
for pro-
ducing Bb immunogenic polypeptides and corresponding antibodies. Other embodi-
ments of the invention relate to methods for detecting Lyme disease and
transformed
cells comprising Bb associated nucleic acids.
BACKGROUND OF THE INVENTION
Lyme disease is a multisystem disease resulting from tick transmission of the
infectious agent, Bb (Rahn and Maiawista, 1991 ). Although recognised as a
clinical
entity within the last few decades (Steere et al., 1977), case reports
resembling
Lyme disease date back to the early part of the 20th century. Cases of the
disease
have been reported in Europe, Asia and North America (Schmid, 19851. Despite a
relatively low total incidence compared to other infectious diseases, Lyme
disease
represents a significant health problem because of its potentially severe
cardio-
vascular, neurologic and arthritic complications, difficulty in diagnosis and
treatment
and high prevalence in some geographic regions.
Bb is not a homogeneous group but has a variable genetic content, which may in
turn
affect its virulence, pattern of pathogenesis and immunogenicity. Lyme
borreliosis
associated borreliae are so far taxonomically placed into three species,
Borrelia
burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii (Burgdorfer
et al., 1983,
Baranton et al., 1992, Canica et al., 1993). It is well documented that
considerable
genetic, antigenic and immunogenic heterogeneity occurs among them, as well as
among the strains within the separate species fBaranton et al., 1992, Canica
et al.,
1993, Zingg et al., 1993, Wilske et al., 1993, Adam et al., 1991, Marconi and
Garon
1992). The major evidence of this phenomenon is provided by the molecular
studies
of the plasmid-encoded outer surface protein A (OspA), OspB and OspC (Barbour
et
al., 1984, Jonsson et al., 1992, Wllske et al., 1993, Marconi et al., 19931.
In diffe-
rent animal models efficient protection is achieved by passive and active
immunisa-
tion with OspA (Schaible et al., 1990, Fikrig et al., 1992, Erdile et al.,
1993), and
therefore, OspA remains one of the main candidates for Borrelia vaccine. It is
unclear,
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however, whether inter- and intra-species heterogeneity of OspA, as well as
other
competitors for immunoprophylaxis, allow efficient cross-protection (Fikrig et
al.,
1992, Norris et al., 1992). Furthermore, it was recently suggested that
certain pro-
tective antibodies produced early in the course of Borrelia infection are
unrelated to
OspA (Norton Hughes et al., i 993, Barthold and Bockenstedt, 1993).
Its virulence factors, pathogenetic mechanisms and means of immune evasion are
unknown. At the level of patient care, diagnosis of the disease is complicated
by its
varied clinical presentation and the lack of practical, standardised
diagnostic tests of
high sensitivity and specificity. Antimicrobial therapy is not always
effective, parti-
cularly in the later stages of the disease.
Variation among Bb strains and species and the changes resulting from in vitro
pas-
sage add to the problems of developing vaccines or immunodiagnostics from
either
the whole organism or specifically associated proteins. Using a PCR assay, it
was
found that one set of oligonucleotide primers was specific for North American
Bb
isolates, another for most European isolates and a third set recognised all Bb
strains
(Rosa et al., 1989).
Serological assays for the diagnosis and detection of Lyme disease are thought
to
offer the best promise for sensitive and specific diagnosis. However,
serologic assays
generally use whole Bb as antigen and suffer from a low "signal to noise"
ratio, i.e., a
low degree of reactivity in positive samples, particularly early in the
disease, as com-
pared to negative samples. This problem results in high numbers of false
negatives
and the potential for false positives. Background reactivity in negative
controls may
be due in part to conserved antigens such as the 41 K flagellin and the 60K
"Common
Antigen". These Bb proteins possess a high degree of sequence homology with
similar proteins found in other bacteria. Therefore normal individuals will
often
express anti-flagellar and anti-60K antibodies. Unique, highly reactive Bb
antigens for
serological assays are therefore desirable but heretofore unavailable.
Diagnosis of Lyme disease remains a complex and uncertain endeavour, due to
lack
of any single diagnostic tool that is both sensitive and specific. Clinical
manifesta-
tions and history are the most common bases for diagnosis. However, there is a
pressing need for specific, sensitive, reproducible and readily available
confirmatory
tests. Direct detection offers proof of infection but is hampered by the
extremely low
levels of Bb that are typically present during infection, as well as the
inaccessibility of
sites that tend to be consistently positive (e.g., heart and bladder).
Culture, although
sensitive, is cumbersome and requires 1-3 weeks to obtain a positive result.
PCR
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appears to offer promise in terms of direct detection (Lebech et al., 19911
and indeed
Goodman et ai. (19911 have reported detection of Bb DNA in the urine of
patients
with active Lyme disease using a PCR method. However, it is unlikely that PCR
assays will become commonly used in clinical laboratories because of the
degree of
skill required for its use and the high risk of DNA contamination.
Another problem in detection of Lyme disease is the substantial number of
hurnans
exposed to Bb who develop inapparent or asymptomatic infections. This number
has
been estimated to be as high as 5096 (Steere et al., 1986).
There is clearly a need for means of preparing Bb-specific antigens, e.g., for
the deve-
lopment of diagnostic tests for Lyme disease. Adequate assays do not exist and
should ideally meet several criteria. including 11 ) expression of an antigen
by all
pathogenic Bb strains, (2) elicitation of an immune response in all Lyme
disease
patients, (3) high immunogenicity with a detectable antibody response early in
the
infection stage, (4) antigens unique to Bb without cross-reactivity to other
antigens,
and (5) distinction between individuals exposed to non-pathogenic as opposed
to
pathogenic forms of Bb.
There have been several studies describing low molecular weight proteins from
Bb.
Katona et al. showed the presence of a major low-molecular weight lipoprotein
specific for B. burgdorferi and raised the possibility that it was a borrelial
equivalent
of Braun's lipoprotein (Katana et al., 19921. Another study reported an
immunogenic
14 kDa surface protein of B, burgdorferi recognised by sera from Lyme disease
patients (Sambri et al., 19911. A 14 kDa mitogenic lipoprotein of B.
burgdorferi was
reported by Honarvar et al. ( 1994).
Sadziene et al. (1994) when analysing an Osp-less B, burgdorferi strain
identified a
13 kDa surface exposed protein which was designated p13.
OBJECT OF THE INVENTION
It is an object of the invention to provide novel nucleic acid fragments and
poly-
peptide fragments which are useful in the preparation of diagnostics and
prophylactic
means and compositions relating to infections with Bb. It is a further object
to pro-
vide novel vaccines and diagnostic means as well as methods for the
preparation and
use of such vaccines and diagnostic means. Finally, it is an object of the
invention to
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provide tools such as vectors and transformed cells which facilitate the
preparation of
the polypeptide fragments and the vaccines.
SUMMARY OF THE INVENTION
The inventors have surprisingly found that an antigen from Bb with an apparent
mole-
cular weight of 13 kDa (determined by SDS-PAGE, and subsequent visualization
such
as staining with Coomassie Blue) is highly conserved in the three strains B.
burgdor-
feri sense stricto B31, B. garinii IP90, and B. afzeiii ACAI, whereas this
antigen
cannot be found in Borrelia species related to relapsing fever and avian
borreliosis.
The disclosed antigens are therefore excellent candidates for vaccines and
diag-
nostics relating to infections with Bb. The antigens will be termed P13.
The present invention thus addresses one or more of the foregoing or other
problems
associated with the preparation and use of Bb specific antigens, particularly
those
antigens associated with virulence and which are useful for developing
detection and
diagnostic methods for Lyme disease. The present invention involves the
identifica-
tion of such antigens, which herein are designated P13 as well as the
identification
and isolation of Bb nucleic acid sequences that encode P13 antigens or
antigenic
polypeptides derived therefrom. These sequences are useful for preparing
expression
vectors far transforming host cells to produce recombinant antigenic
polypeptides. It
is further proposed that these antigens will be useful as vaccines or
immunodiag-
nostic agents for Bb associated diseases such as Lyme disease in particular.
The DNA of the present invention was isolated from Bb. The microorganism is a
spiral-shaped organism approximately 0.2 micron in diameter and ranging in
length
from about 10 to 30 microns. Like other spirachaetes, it possesses an inner
mem-
brane, a thin peptidoglycan layer, an outer membrane, and periplasmic flagella
which
lie between the inner and outer membranes. Bb is an obligate parasite found
only in
association with infected animals and arthropod vectors in endemic areas. Bb-
like
organisms have also been identified in birds raising the possibility that
birds could
also serve as an animal reservoir. While some Bb isolates have been cloned,
most
isolates have not been cloned and most likely represent mixtures of different
variants
even at the time of culture origin.
Bb has similarities with other relapsing fever organisms such as B. hermsii.
Bb has a
single chromosome with two unusual features, linear conformation and small
size
(approximately 900 kilobase pairs). Fresh isolates of Bb contain up to four
linear
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plasmids and six circular supercoiled plasmids. The plasmid content of
different Bb
isolates is highly variable. For example, in one study only two of thirteen
strains had
similar plasmid profiles. Some plasmids are lost during in vitro passage which
may
correlate with loss of virulence. Outer surface proteins OspA and OspB are
encoded
5 on the 49 kbp linear plasmid. The P13 membrane-associatediouter surface
proteins
discovered by the inventors are encoded on the Bb chromosome. The P13 protein
gene being localised to the chromosome of borreliae shows a higher degree of
con-
servation among Lyme disease associated borreliae contrary to the plasmid-
encoded
major outer surface proteins A, B, and C which exhibit a significant species
and strain
dependent genetic and antigenic polymorphism (Barbour 1986, Jonsson et al.,
1992,
Wilske et al., 19931. Furthermore, the level of similarity and identity
between the
deduced amino acid sequence of the P13 protein from different borrelia strains
further shows that this protein can be useful as a vaccine against Lyme
disease as
well as a target for diagnostic use.
In order to identify DNA segments encoding the P13 proteins, purified protein
was
isolated from B. burgdorferi 8313 by preparative SDS-PAGE for subsequent use
in
amino acid sequencing. Attempts to N-terminally sequence the purified protein
by
standard techniques were unsuccessful. The protein was therefore subjected to
V8
protease cleavage. After protease cleavage the peptide was transferred to poly-
vinylene diffusable membranes, and sequence analysis was performed using
standard
sequencing techniques (Matsudaira, 19871. A 25 amino acid sequence was
identified
(SEO. ID NO: 11.
DNA libraries were prepared by restriction enzyme digestion of DNA prepared
from
the strains B, burgdorferi B31, B. afzeiii ACAI and B. garinii IP90.
Codons for the amino acid sequence obtained, SEQ ID NO: 1, were selected by
reverse translation based on ( 11 conclusion that codons containing A or T
were
favoured and (2) knowledge of published DNA sequences for several Bb proteins.
A
choice favouring A or T containing codons was based on the observation that
the G
+ C content of Bb is only 28-35% (Burman et al., 1990). Two oligonucleotides
were
synthesized having the sequences shown in in SEQ ID N0: 2 and SEQ, ID N0:3.
These were used as primers in a PCR reaction with DNA prepared from B.
burgdorferi
B31 as template. The amplified fragment was sequenced, SEQ ID NO: 4, and
verified
to code for the amino acid sequence, SEQ l0 NO: 1.
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A DNA probe, designated Y7 (SEa ID N0:7), was designed and used to screen the
DNA library prepared from B. burgdorferi B31 in an attempt to identify DNA
encoding
the P13 protein from this Bb species. This attempt proved unsuccessful.
An Rsal restriction site identified in the DNA sequence of the PCR fragment
was used
in a further attempt to clone the P13 gene. Bb DNA was digested with Rsal and
the
fragments cloned into a pUC plasmid. Further PCR amplification using the
sequence
identified surrounding the Rsal site yielded DNA fragments which were found to
code
for the P13 protein.
The identified sequence of the P13 gene from B. burgdon'eri B31 was used to
design
PCR primers which were subsequently used to clone the P13 gene from B. afzeiii
ACAI and B. garinii IP90.
The P13 protein which has been cloned by the inventors of the present
invention has
been shown to have a molecular weight of about 19,000 as calculated from the
deduced amino acid sequence of the full-length protein but a molecular weight
of
about 14,000 as determined by MS but nevertheless to be identical to a protein
from
Bb which has an apparent molecular weight in SDS-PAGE of 13 kDa. This
difference
can be explained by post-translational modifications of the P13 protein. This
is in
accordance with the observation that it was not possible by standard methods
to
obtain an N-terminal amino acid sequence of P13 protein prepared from Bb.
The deduced amino acid sequences of P13 from B. burgdorferi B31, B, afzelii
ACAI
and B. garinii IP90 were analysed and it was found that the N-terminal regions
of the
deduced amino acid sequences are typical of the signal peptides of bacterial
proteins.
These leader peptide sequences are typical of exported proteins with a basic
residue
followed by a hydrophobic and a potential leader peptidase I cleavage site
according
to the criteria established by von Heijne (19861.
Amino acid sequences resembling the signal sequences of bacterial lipoproteins
can
also be found in the N-terminal region of the deduced amino acid sequences.
The N-
terminal methionine is followed by a hydrophobic region and a signal peptidase
II
recognition sequence. The signal sequences, Leu Ala Thr Phe Cys for B.
burgdon'eri
B31, Leu Leu Ala Phe Cys for B. afzeiii ACAI and Leu Val Ile Phe Cys for B.
garinii
IP90, differed somewhat from the consensus signal peptidase II recognition
sequence
(Leu Xaa Xaa Cys) found in most bacteria, but resembled the cleavage sequence
Leu
Ser lle Ser Cys of the outer surface protein D fOspD) of Bb and Leu Met Ile
Gly Cys
of the variable major proteins Vmp7 and Vmp21 of B. hermsii. These surface
anti-
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gens have been shown to be lipoproteins (Norris et al., 1992; Burman et al.,
1990).
The presence of this leader sequence may imply that mature P13 proteins are
trans-
located across the cytoplasmic membrane and are anchored to the cytoplasmic
mem-
brane and/or outer membranes via fatty acids associated with an N-terminal
cysteinyl
residue. Lipidated forms of the outer surface protein A (OspA) from Bb have
been
shown to be more immunogenic that non-lipidated forms of OspA (Erdile et al.,
1993?.
However, it should be understood that when the terms "13 kDa protein" or "13
kDa
antigen" or "13 kDa polypeptide" are used in the present specification and
claims,
this is an alternative designation of the P13 polypeptide.
Antigenicity of the P13 protein was verified by immunisation of a rabbit.
Antiserum
collected from rabbits injected with the P13 protein prepared from B,
burgdorferi
8313 was found to recognise the P13 protein of B. burgdorferi B31, B. afzeiii
ACA/,
and B. garinii IP90. There was no apparent reactivity of the antiserum with
B. hermsii, B. crocidurae, B. anserina.
The nucleic acid segments of the present invention encode antigenic amino acid
sequences associated with Bb. These sequences are important for their ability
to
selectively hybridise with complementary stretches of Bb gene segments.
Varying
conditions of hybridization may be desired, depending on the application
envisioned
and the selectivity of the probe toward the target sequence. Where a high
degree of
selectivity is desired, one may employ relatively stringent conditions to form
the
hybrids, such as relatively low salt and/or high temperature conditions. Under
these
conditions, little mismatch between the probe and template or target strand is
tolerated. Less stringent conditions might be employed when, for example, one
desires to prepare mutants or to detect mutants when significant divergence
exists.
In clinical diagnostic embodiments, nucleic acid segments of the present
invention
may be used in combination with an appropriate means, such as a label, to
determine
hybridization with DNA of a pathogenic organism. Typical methods of detection
might
utilise, for example, radioactive species, enzyme-active or other marker
ligands such
as avidinibiotin, which are detectable directly or indirectly. In preferred
diagnostic
embodiments, one will likely desire to employ an enzyme tag such as alkaline
phos-
phatase or peroxidase rather than radioactive or other reagents that may have
un-
desirable environmental effects. Enzyme tags, for example, often utilise
colorimetric
indicator substrates that are readily detectable spectrophotometrically, many
in the
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visible wavelength range. Luminescent substrates could also be used for
increased
sensitivity.
Hybridisable DNA segments may include any of a number of segments of the dis
closed DNA. For example, relatively short segments of at least 12 or so base
pairs
may be employed, or, more preferably when probes are desired, longer segments
of
at least 20, at least 30, and at least 40 base pairs, depending on the
particular
applications desired. Shorter segments are preferred as primers in molecular
amplifi-
cation techniques such as PCR, while some of the longer segments are generally
preferable for blot hybridizations. It should be pointed out, however, that
while
sequences disclosed for the DNA segments of the present invention are defined
by
SEO ID N0: 18, SECT ID N0: 20 and SEQ ID NO: 22, a certain amount of variation
or
base substitution would be expected, e.g., as may be found in mutants or
strain
variants, but which do not significantly affect hybridization characteristics.
Such
variations, including base modifications occurring naturally or otherwise, are
intended
to be included within the scope of the present invention.
While the Bb P13 antigens of the present invention have been disclosed in
terms of
the specific amino acid sequences SECT ID N0: 19, SEC2 ID N0: 21 and SECT ID
NO:
23, it is nonetheless contemplated that the amino acid sequences will be found
to
vary from species to species and from isolate to isolate. Moreover, it is
quite clear
that changes may be made in the underlying amino acid sequence through, e.g.,
site-
directed mutagenesis of the DNA coding sequence, in a way that will not negate
its
antigenic capability.
The invention also relates to at least partially purified antigenic Bb
proteins or poly-
peptides which are capable of producing an in vivo immunogenic response when
challenged with Bb. These proteins may comprise all or part of the amino acid
se-
quence encoded by the DNA disclosed herein. Particularly preferred antigenic
proteins
have the amino acid sequence shown in SECT ID NO: 19, SECT ID NO: 21 and SEO,
ID
NO: 23. Post-translationally modified forms of these antigenic proteins are
also the
subject of this invention. These proteins as well as their epitopes will be
useful in
connection with vaccine development, and as antigens) in immunoassays for
detec-
tion of Bb antibodies in biological fluids such as serum, seminal or vaginal
fluids,
urine, saliva, body exudates and the like.
In other aspects, the invention concerns recombinant vectors such as plasmids,
phages or viruses, which comprise DNA segments in accordance with the
invention,
for use in replicating such sequences or even for the expression of encoded
antigenic
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peptides or proteins. Vectors or plasmids may be used to transform a selected
host
cell. In preparing a suitable vector for transforming a cell, desired DNA
segments
from any of several Bb sources may be used, including genomic fragments, cDNA
or
synthetic DNA. In the practice of the present invention, an expression vector
may
incorporate at least part of the DNA sequence of SEQ ID NO: 18, SEa ID NO: 20
and
SEa ID NO: 22, encoding one or more epitopic segments of the disclosed
antigens of
the present invention.
Expression vectors may be constructed to include any of the DNA segments
herein-
above disclosed. Such DNA might encode an antigenic protein specific for
virulent
strains of Bb or even hybridization probes for detecting Bb nucleic acids in
samples.
longer or shorter DNA segments could be used, depending on the antigenic
protein
desired. Epitopic regions of the disclosed proteins of the present invention
expressed
or encoded by the disclosed DNA could be included as relatively short segments
of
DNA. A wide variety of expression vectors are possible including, for example,
DNA
segments encoding reporter gene products useful for identification of
heterologous
gene products and/or resistance genes such as antibiotic resistance genes
which may
be useful in identifying transformed cells.
Recombinant vectors such as those described are particularly preferred for
t~ansform-
ing bacterial host cells. Accordingly, a method is disclosed for preparing
transformed
bacterial host cells that generally includes the steps of selecting a suitable
bacterial
host cell, preparing a vector containing a desired DNA segment and
transforming the
selected bacterial host cell. Several types of bacterial host cells may be
employed,
including Bb, E. coli, B. subtilis, and the like as well as prokaryotic host
cells.
Transformed cells may be selected using various techniques, including
screening by
differential hybridization, differential display techniques, identification of
fused re-
porter gene products, resistance markers, anti-antigen antibodies and the
like. After
identification of an appropriate clone, it may be selected and cultivated
under con-
ditions appropriate to the circumstances, as for example, conditions favouring
expression or, when DNA is desired, replication conditions.
Another aspect of the invention involves the preparation of antibodies and
vaccines
from the antigenic P13 proteins or epitopic regions of these proteins encoded
by the
disclosed DNA. It is expected that the sensitivity and specificity of an
antibody
response to these P13 proteins and their epitopes will be superior to the
response
that has been obtained from other Bb antigens that are not associated with
virulence.
Previous work with several Bb antigens isolated from both virulent and
avirulent
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strains indicated low sensitivity when immunofluorescence and ELISA assays
were
employed, especially during early stages of infection.
In both immunodiagnostics and vaccine preparation, it is often possible and
indeed
5 more practical to prepare antigens from segments of a known immunogenic
protein or
polypeptide. Certain epitopic regions may be used to produce responses similar
to
those produced by the entire antigenic polypeptide. Potential antigenic or
immuno-
genic regions may be identified by any of a number of approaches, e.g.,
Jameson-
Wolf or Kyte-Doolittle antigenicity analyses or Hopp and Woods ( 1981 )
hydropho-
10 bicity analysis (see, e.g., Kyte and Doolittle, 1982, or U.S. Patent No.
4,554,101).
Hydrophobicity analysis assigns average hydrophilicity values to each amino
acid
residue, and from these values average hydrophilicities can be calculated and
regions
of greatest hydrophilicity determined. Using one or more of these methods,
regions of
predicted antigenicity may be derived from the amino acid sequence of the
disclosed
P13 polypeptides. Proposed epitopic regions from the disclosed P13 antigens
include
the sequences corresponding to amino acid residues 7-15, 21-24, 29-35, 83-92,
126-135 and 162-167 in SEQ ID N0: 19; amino acid residues 7-14, 20-23, 28-35,
82-89, 125-134 and 162-166 in SEQ ID NO: 21; and amino acid residues 6-14, 18-
21, 27-34, 79-92, 125-133 and 161-165 in SEQ 1D NO: 23.
Antigenic epitopes can also be determined using different experimental
procedures
known to the skilled person. For example, the DNA encoding the P13
polypeptides
can be digested with restriction enzymes in such a manner that DNA fragments
encoding specific parts of the P13 poiypeptide are obtained. These DNA
fragments
can be expressed in a suitable expression system. The P13 polypeptide
fragments
obtained can be analysed with monoclonal or polyclona! antibodies obtained by
immunisation with the full-length form of the P13 polypeptide or fragments
thereof,
or with Lyme disease patient sera to obtain information about immunogenicity
and
the presence of epitopes. Fragments found to be positive in such a simple
screening
assay where they react specifically with e.g. polyclonals raised against P13
are thus
suitable candidates for a multitude of the applications where full-length P13
would
also be suitable.
A similar approach can be used where random mutations are introduced in the
nucleotide sequence encoding P13, and only the expression products which
retain a
suitable reactivity with e.g. P13 positive polyclonal antibodies will be used
as candi-
dates for further applications.
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11
Peptide fragments for the identification of epitopes can also be obtained by
synthetic
methods. Suitable peptides can be synthetised based on the amino acid sequence
of
the P13 polypeptides, e.g. peptides with amino acid sequences identical to
conse-
cutive fragments from the N-terminus to the C-terminus of the P13 polypeptide
can
be synthetised on solid-phase media. Such custom-made peptide libraries can be
obtained from commercial sources.
Finally, another way of simply identifying epitopes is to digest a polypeptide
antigen
with a known amino acid sequence with endo- and exopeptidases. The obtained
frag-
ments are tested against antibodies directed against the whole polypeptide,
and by
way of deduction, the precise location of the linear epitopes can be
determined. A
variation of this method involves the recombinant production of subfragments
(cf. the
abovel of the full-length polypeptide followed by the same test procedure.
It is contemplated that the antigens and immunogens of the invention will be
useful in
providing the basis for one or more assays to detect antibodies against Bb.
Previous
assays have used whole Bb as the antigen. Sera from normal individuals not
exposed
to Bb often contain antibodies that react with Bb antigens, in particular
antigens that
have epitopes in common with other bacteria. It is necessary to adjust assay
condi-
tions or the diagnostic threshold of reactivity to avoid false positive
reactions due to
these cross-reactive antibodies in normal sera. These adjustments may in turn
de-
crease the sensitivity of the assay and lead to false negative reactions,
particularly in
the early stages of Bb infection. Assays using the disclosed P13 proteins or
antigenic
polypeptides thereof are expected to give superior results both in terms of
sensitivity
and selectivity when compared to assays that use whole Bb or even purified
flagella
in either an indirect ELISA or an antibody capture ELISA format. Western
immuno-
blots based on reactions with such antigens (whole Bb, flagella and the like)
have
been difficult to interpret due to the presence of antibodies in sera from
unexposed
individuals. These antibodies cross-react with Bb antigens, most particularly
the 41
kDa flagellin and the 60 kDa common antigen protein. Generally, assays which
use
whole organisms or purified flagella tend to contain antigens with epitopes
that will
cross-react with other bacterial antigens. For example, the N and C terminal
regions
of the Bb flagellin possess 52-55°6 sequence identity with the
Salmonella typhimu-
rium and Bacillus subtiiis sequences (Wallich et al., 1990), exemplifying the
highly
conserved nature of flagellin structure. The 60 kDa Bb protein is likewise
5896 homo-
logous with the E. coli protein (Shanafelt et al., 1991 ). Such cross-
reactivity is not
likely with the disclosed P13 antigens, which are apparently unique to Bb.
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It is further anticipated that recombinantly derived P13 Bb proteins will be
particularly
preferred for detecting Bb infections. Unexposed individuals should have a low
reacti-
vity to one or more epitopes of the P13 proteins thereby making it possible to
use
lower dilutions of serum and increase sensitivity. Using a combination of more
than
one of these unique antigens may also enhance sensitivity without sacrificing
speci-
ficity.
Preferred immunoassays are contemplated as including various types of enzyme
linked immunoassays (ELISAs), immunoblot techniques, and the like, known in
the
art. However, it readily appreciated that utility is not limited to such
assays, and
useful embodiments include RIAs and other non-enzyme linked antibody binding
assays or procedures.
Yet another aspect of the invention is a method of detecting Bb nucleic acid
in a
sample. The presence of Bb nucleic acid in the sample may be indicated by the
presence of the polypeptide products which it encodes. The method therefore in-
cludes detecting the presence of at least a portion of any of the polypeptides
herein
disclosed. Suitable detection methods include, for example, immunodetection
rea-
gents, PCR amplification, and hybridization.
Yet another aspect of the invention includes one or more primers capable of
priming
amplification of the disclosed DNA of SEQ ID N0: 18, SEQ ID NO: 20 and SEQ ID
NO: 22. Such primers are readily generated taking into account the base
sequence of
the DNA segment of SEa ID N0: 18, SEQ ID NO: 20 and SEQ ID NO: 22, the dis-
closed DNA, or deriving a base sequence from the amino acid sequence of a
purified
polypeptide encoded by the DNA. Primers are analogous to hybridization probes,
but
are generally relatively short DNA segments, usually about 7-20 nucleotides.
Methods of diagnosing Lyme disease are also included in the invention. In one
embo-
diment, an antibody-based method includes obtaining a sample from a patient
sus-
pected of having Lyme disease, exposing that sample to one or more epitopes of
the
Bb protein which is encoded by the DNA disclosed, and finally determining a
reac-
tivity of the antibody with one or more epitopes of a Bb protein that may be
in the
sample. The reactivity measured is indicative of the presence of Lyme disease.
Typical samples obtainable from a patient include human serum, plasma, whole
blood, cerebrospinal fluid, seminal or vaginal fluids, exudates and the like.
Several variations of antibody-based methods are contemplated for development;
for
example, an indirect ELISA using the P13 proteins or other Bb proteins as an
antigen.
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13
The P13 proteins may be produced in large quantities by recombinant DNA
vectors
already disclosed and purified. Optimum concentration of the antigen could be
deter-
mined by checker board titration and diagnostic potential of the P13 proteins
assay
examined further by testing serum from mice at different stages of infection
and
infected with different strains of Bb. These results could indicate the
relative time
course for serum conversion for each of the assays and would also show whether
infection with different strains causes variation in anti-P13 protein titers.
Likewise, reactive epitopes of the P13 polypeptides are contemplated as useful
either
as antigens in an ELISA assay or to inhibit the reaction of antibodies toward
intact
P13 proteins bound to a well. Epitopic peptides could be generated by
recombinant
DNA techniques previously disclosed or by synthesis of peptides from
individual
amino acids. In either case, reaction with a given peptide would indicate
presence of
antibodies directed against one or more epitopes. In addition to its
diagnostic poten-
tial, this method is seen as being particularly effective in characterising
monoclonal
antibodies against the P13 proteins and other virulence associated proteins.
In a further aspect, the present invention concerns a kit for the detection of
Bb anti-
gens, the kit including a protein or peptide which includes an epitope
thereof, to-
gether with means for detecting a specific immunoreaction between an antibody
and
its corresponding antigen. Examples of suitable means include labels attached
directly
to the antigen or antibody, a secondary antibody having specificity for human
Ig, or
protein A or protein G. Alternatively, avidin-biotin mediated Staphylococcus
aureus
binding could be used. For example, the monoclonal antibody may be
biotinylated so
as to react with avidin complexed with an enzyme or a fluorescent compound.
A particular kit embodiment of the invention concerns detection of antibodies
against
the described Bb P13 antigens, epitopes thereof as represented by portions of
the
amino acid sequences, or closely related proteins or peptides, such as
epitopes asso-
ciated with other virulence-associated proteins detected by comparison of low-
pas-
sage, virulent and high-passage, avirutent strains of Bb. The antigen for the
kids) con-
sists of the Bb P13 proteins or portions thereof produced by a recombinant DNA
vector in E. coli or another bacterial or non-bacterial host. Alternatively,
the antigen
may be purified directly from Bb or manufactured as a synthetic peptide.
Samples for
the assays may be body fluids or other tissue samples from humans or animals.
The
presence of reactive antibodies in the samples may be demonstrated by antibody
binding to antigen followed by detection of the antibody-antigen complex by
any of a
number of methods, inctuding ELISA, TRIFMA (time-resolved immunofluorometric
assay, RIA, fluorescence, agglutination or precipitation reactions,
nephelometry, or
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14
any of these assays using avidin-biotin reactions. The degree of reactivity
may be
assessed by comparison to control samples, and the degree of reactivity used
as a
measure of present or past infection with Bb. The assays) could also be used
to
monitor reactivity during the course of Lyme disease, e.g., to determine the
efficiency
of therapy.
In still further embodiments, the invention contemplates a kit for the
detection of Bb
nucleic acids in the sample, wherein the kit includes one or more nucleic acid
probes
specific for the P13 genes, together with means for detecting a specific
hybridization
between such a probe and Bb nucleic acid, such as an associated label.
LEGENDS TO THE FIGURES
Figure 1 A, 1 B and 1 C. Analysis of Borreiia proteins.
A: Effect of Protease K treatment on Bb cells. Coomassie-blue stained 15 % SDS-
PAGE gel showing protein profiles of whole cell (WC), B-fraction (BF) and
Proteinase
K (PK) treated cells from B. burgdorferi B31, B. afzeiii ACA1 and B. garinii
IP90.
B: The Western Blot corresponding to Figure 1 A probed with the rabbit
polyclonal
antiserum raised against the 13 kDa protein prepared from B. burgdorferi 8313.
C: Comparison of phenotypic expression of the 13 kDa protein in Borrelia
species.
Western Blot of SDS-PAGE separated proteins from B. hermsii and B. crocidurae
probed with the rabbit polyclonal antiserum raised against the 13 kDa protein
prepared from B. burgdorferi 8313.
Arrows indicate the position of 13 kDa protein. Mw - molecutar weight
standard, kD -
kilodalton.
Figure 2A and 2B. Demonstration of outer membrane association of the 13 kDa
protein.
A: Electron micrographs of immunogold-stained cells from B. burgdorferi B31.
B: Electron micrographs of immunogold-stained cells from B. burgdorferi 8313.
Monoclonal antibody 1566 was used as the primary antibody.
Figure 3A and 3B. Analysis of membrane Fraction B.
A: Coomassie-blue stained 15°Yo SDS-PAGE gel showing protein profiles
of Fraction B
(BFI prepared from cells from B. burgdorferi B31, B. afzeiii ACA1 and B,
garinii IP90.
B: The corresponding Western Blot probed with the monoclonal antibody 1566.
Arrows indicate the position of 13 kDa protein. Mw - molecular weight
standard, kD -
kilodalton.
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Figure 4. Antigenicity plot.
Antigenicity plot according to Jameson-Wolf of the deduced amino acid sequence
of
P13 from A) B. burgdorferi B31, B) B. afzelii ACA1 and C) B. garinii IP90.
5
Figure 5A and 5B. Gene localisation analysis of the P13 gene.
A: Separation of total DNA prepared from B. burgdorferi B31, B. burgdorferi
8313,
B. afzeiii ACA1 and B. garinii IP90 by pulse-field agarose gel electrophoresis
(AGE).
B: The corresponding Southern blot using an a-32P labelled probe prepared by
PCR
10 amplification of a part of the P13 gene.
Figure 6. Southern blot.
Total DNA from B, burgdorferi, B. hermsii, B. crocodurae, and B. anserina was
digested with EcoRl and separated by AGE. DNA was transferred to a Hybond-N
15 membrane. The filter was probed with a PCR fragment obtained by
amplification
using primers Y9 fSEQ ID:7) and Y7R fSEQ ID:61. Hybridization temperature was
55°C. In general as described in section 9.2.
Figure 7A and 7B. Expression of recombinant P13 in E. coil.
A: Coomassie-blue stained 15~o SDS-PAGE gel showing protein profiles of whole
cell
lysates of E, coil transfected with plasmid pLY313F and plasmid pLY313T.
B: The corresponding Western Blot probed with the monoclonal antibody 1566.
pGEX-2T, E. coli transfected with the control plasmid.
B-fract.B313, B-fraction prepared from B. burgdorferi 8313.
Mw - molecular weight standard, kD - kilodalton.
Figure 8A and 8B. Constructs for DNA vaccination.
A: Schematic representation of the insert of plasmid pLY-H used in DNA
vaccination
for the expression of recombinant P13.
B: Schematic representation of the insert of ptasmid pLY-HA used in DNA
vaccination
for the expression of fusion of recombinant P13 and recombinant OspA.
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16
Figure 9. Electron mass spectrum of purified P13 protein.
The molecular masses of the three major components are indicated above the
peaks.
DETAILED DESCRIPTION OF THE INVENTION
Nucleic acid fragments of the invention
The present invention relates to the utility of Bb associated nucleic acid
fragments as
diagnostic or preventive tools in Lyme disease as well as for the preparation
of P13
and useful P13 analogues.
In a first aspect the present invention therefore relates to an isolated
nucleic acid
fragment encoding a polypeptide fragment which exhibits a substantial
immunological
reactivity with a rabbit polyclonal antibody raised against a polypeptide
having an
apparent molecular weight of 13 kDa as determined by SDS-PAGE and subsequent
visualization, said polypeptide being derived from Borrelia burgdorferi 8313
and con-
sisting of the amino acid sequence of SEQ ID N0: 19 or a post-translationally
modi-
fied form thereof, said rabbit polyclonal antibody exhibiting substantially no
immuno-
logical reactivity with proteins from at least 95% of spirochaetes randomly
selected
from the group consisting of Borrelia hermsii, Borreiia crocidurae, Borrelia
anserina,
and Borreiia hispanica.
By the term "nucleic acid fragment" as used herein is meant a fragment of DNA
or
RNA, but also of PNA (cf. Nielsen et al., 1991 ), having a length of at least
two joined
nucleotides. It will be understood that although the disclosed nucleic acid
fragments
of the present invention are DNA fragments, it may be desirable to employ an
RNA
fragment in e.g. a viral vector, the genome of which is natively composed of
RNA.
For the purposes of preparing e.g. probes for hybridization assays as
described
below, PNA fragments may prove useful, as these artificial nucleic acids have
been
demonstrated to exhibit very dynamic hybridization properties.
The term "a substantial immunological reactivity" is meant to designate a
marked
immunological binding between an antibody/antiserum on the one hand, and on
the
other hand an antigen, under well-defined conditions with respect to
physicochemical
parameters as well as concentrations of antigens and antibodies. Thus, a
substantial
immunologicai reactivity should be clearly distinguishable from a non-specific
inter-
action between an antibody/antiserum and an antigen. This distinction can for
in-
stance be made by reacting the antibody/antiserum with a known concentration
of an
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17
antigen which has previously been shown not to react with the
antibody/antiserum,
and then using this reaction as a negative control. A positive control could
suitably be
the reaction between the antibodyiantiserum and the same concentration of the
anti-
gen used for the immunisation resulting in the production of the
antibody/antiserum.
In such an assay, an antigen resulting in a relative signal of at least 1096
(calculated
as Sm (Sp-S")~100, where Sm is the measured signal, Sp the positive control
signal, and
S" the negative control signal) is regarded as having a substantial
immunoiogical reac-
tivity. An antigen exhibiting "substantially no immunoiogical reactivity" is
therefore
defined as an antigen giving a signal of less than 10 r6.
By the terms "present" and "substantially absent", when referring to amino
acid
sequences and polypeptides in bacteria, is meant that the concentration of the
amino
acid sequence/polypeptide in a bacterium where it is "present" is at least 100
times
higher than in a bacterium where it is substantially absent. However, it is
preferred
that the ratio of the concentrations are at least 1000, and more preferred at
least
10,000, 100,000 or even higher. It is especially preferred that no
concentration of
the amino acid sequenceipolypeptide can be observed in the bacterium from
where it
is substantially absent.
Although the data presented herein demonstrate that there is no cross-
reactivity
between antigens from Borreiia hermsii, Borrelia crocidurae, Borreiia
anserina, or
Borreiia hispanica and the disclosed polypeptides, it is conceivable that a
few isolates
of these bacteria will exhibit some cross-reactivity. As can be deduced from
the
above it is expected that the cross-reactivity will be less than 5°~
(since there is no
reactivity with at least 95°rb of randomly chosen Borrelia hermsii,
Borrelia crocidurae,
Borreiia anserina, or Borrelia hispanical, and according to the invention this
cross-
reactivity may be even lower, such as at the most 496 and 3 r6, preferably at
the
most 2%, such as 1 °~. According to the invention the cross-reactivity
is most pre-
ferred at most 1/2%, such as 0°~6. In such a case there will be no
substantial
immunologicai reactivity between the rabbit antiserum mentioned above and
whole
cell preparations of Borrelia hermsii, Borreiia crocidurae, Borrelia anserina,
or Borrelia
hispanica.
When using the term "cross-reactivity" is herein meant the phenomenon that two
species exhibit a common feature which is detected in a reaction. In the
present
context the term cross-reactivity is used for similar reactions in antigen-
antibody
interactions as well as in hybridization interactions. Hence, the above-cited
consi-
derations concerning cross-reactivity of polypeptides apply for all cross-
reactions
between on the one hand the polypeptides/DNA fragments of the invention and on
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18
the other hand material from Borrelia hermsii, Borrelia crocidurae, Borrelia
anserina,
and Borrelia hispanica; this is also true for the quantitative assessment of
whether
cross-reactivity is present or not.
Nucleic acid fragments of the invention useful as hybridization probes and/or
primers
are not necessarily those fragments encoding immunologically useful
polypeptides.
Therefore the invention also relates to nucleic acid fragments which hybridise
readily
under highly stringent hybridization conditions with a DNA fragment having a
nucleotide sequence selected from the group consisting of SEQ ID NO: 18, SEQ
ID
NO: 20, and SEQ ID N0: 22, or with a DNA fragment complementary thereto, but
exhibit no substantial hybridization when the hybridization conditions are
highly
stringent with genomic DNA from at Least 95% of spirochaetes randomly selected
from the group consisting of Borreiia hermsii, Borrelia crocidurae, Borrelia
anserina,
and Borrelia hispanica. The term "highly stringent" when used in conjunction
with
hybridization conditions is as defined in the art, i.e. 5-10°C under
the melting point
Tm, cf. Sambrook et al, 1989, pages 11.45-11.49.
Interesting nucleic acid fragments of the invention encode a polypeptide
fragment
comprising an amino acid sequence comprised in a polypeptide, said polypeptide
being present in whole cell preparations of Borrelia burgdorferi 831, Borrelia
burg-
dorferi 8313, Borrelia garinii IP90, and/or Borrelia afzelii ACA/ but being
substantially
absent from whole cell preparations of at least 95 % of randomly selected
Borreiia
hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica. It is
preferred that
said polypeptide is a protein having an apparent molecular weight of 13 kDa,
and it is
still more preferred that the encoded polypeptide fragment comprises at least
one
epitope (such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18,
19, 20, or at least 25 epitopesl being present in whole cell preparations of
Borrelia
burgdorferi B31, Borreiia burgdorferi 8313, Borrelia garinii IP90, or Borrelia
afzelii
ACA/ but being substantially absent from whole cell preparations of at least
95% of
randomly selected Borrelia hermsii, Borrelia crocidurae, Borreiia anserina,
and Borrelia
hispanica. This at least one epitope is preferably one from a protein having
an appa-
rent molecular weight of 13 kDa.
By the terms "epitope" and "epitopic region" is meant the spatial part of an
antigen
responsible for the specific binding to the antigen-binding part of an
antibody. It goes
without saying that the identification of epitopes of the disclosed antigens
will facili-
tate the production of polypeptides which exhibit marked antigenicity thus
making
them interesting with respect to diagnosis of Borreliosis and vaccination
against
infections with Bb; identification of epitopes has been discussed above.
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19
Preferred nucleic acid fragments of the invention are DNA fragments,
especially those
which have nucleotide sequences with a sequence identity of at least 7096 with
SEO.
ID NO: 18, SEa ID NO: 20, or SEO. ID N0: 22 or with subsequences thereof of at
least 12 nucleotides. However, the degree of sequence identity may be even
higher
such as at least 75°~, 8096, 859b, 8796, and 8996. It is preferred that
the degree of
sequence identity is at least 9096, such as 92%, 9496 or 9596, and especially
pre-
ferred are DNA fragments with a sequence identity of at least 9696 with SEQ ID
NO:
18, SEa ID NO: 20, or SEQ ID NO: 22. Especially for high accuracy
hybridization
assays, a total sequence identity is necessary, and therefore preferred. Other
pre-
ferred nucleotide acid fragments of the invention are those which encode a
polypep-
tide of the invention /cf. the below discussions concerning these polypeptides
and
their degree of sequence identity with the amino acid sequences disclosed
herein
which has an amino acid sequence exhibiting a sequence homology of at least
50°~
with SEQ ID NO: 19, SEQ ID N0: 21, or SEQ ID NO: 23 or with subsequences
thereof having a length of at least 10 amino acid residues. Also the sequence
identity
of the encoded polypeptide fragment is preferably higher, such as at least
60%, at
least 70%, at least 75%, at least 8096, at least 8596, at least 90%, at least
92%, at
least 9496, at least 950, and at least 9696.
The term "identity" is, with respect to nucleotide fragments such as DNA
fragments,
intended to indicate the identity between the nucleotides in question between
which
the identity is to be established, in the match with respect to nucleotide
composition
and position in the DNA fragments. With respect to polypeptides and fragments
thereof described herein, the term means an identity between the amino acids
in
question between which the homology is to be established, in the match with
respect
to amino acid composition and their position in the poiypeptides. The term
"sequence
identity" thus indicates a quantitative measure of the degree of homology
between
two amino acid sequences or between two nucleotide sequences of equal length:
The
~N~f-Ndrj~100
sequence identity can be calculated as N,~f 1, wherein Nd" is the total
number of non-identical residues in the two sequences when aligned and wherein
N",
is the number of residues in one of the sequences. Hence, the DNA sequence
AGTCAGTC will have a sequence identity of 7596 with the sequence AATCAATC
(N~,=2 and N,~,=81.
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Considerations similar to those given above for the immunological reactivity
and
cross-reactivity of antigens can be applied for the distinction between a
nucleic acid
fragment which "hybridizes readily" and a fragment which "exhibits
substantially no
hybridization" under high stringency conditions; i.e. a nucleic acid which
hybridizes
5 readily should exhibit at least 1096 of a true positive signal.
As discussed in the examples, putative epitopes have been identified in the
P13 pro-
tein sequences. Therefore, especially preferred nucleic acid fragments of the
inven-
tion are those comprising a sequence encoding a polypeptide which comprises at
10 least one amino acid sequence selected from the group consisting of amino
acid
residues 19-27, 33-36, 41-47, 95-104, 138-147 and 174-179 in SEQ !D N0: 19;
amino acid residues 19-26, 32-35, 40-47, 94-101, 137-146, and 174-178 in SEQ
ID
NO: 21; and amino acid residues 18-26, 30-33, 39-46, 91-104, 137-145 and 173-
177 in SEO. ID N0: 23, all of which have been identified as putative epitopic
regions.
Highly preferred nucleic acid fragments are those which encode mature P13,
i.e.
nucleic acid fragments which encode a protein having an apparent molecular
weight
of 13 kDa which is present in whole cell preparations of Borrelia burgdorferi
B31,
Borrelia burgdorferi 8313, Borrelia garinii IP90, or Borrelia afzelii ACAI but
which is
substantially absent from whole cell preparations of at least 9596 of randomly
se-
lected Borreiia hermsii, Borrelia crocidurae, Borreiia anserina, and Borrelia
hispanica.
Since this 13 kDa protein has been demonstrated in fraction B of Bb, it is
preferred
that this encoded protein is present in fraction B from Borrelia burgdorferi
B31, Borre-
lia burgdorferi 8313, Borrelia garinii IP90, or Borrelia afzeiii ACAI.
Analysis of the effects of exposure of whole Bb cells to proteinase K has now
de-
monstrated that such Bb cells lose their reactivity with antibodies against
the 13 kDa
polypeptide and that fraction B of Bb cells are devoid of the 13 kDa protein.
Hence, it
must be concluded that P13 is a surface exposed polypeptide in Bb, and
therefore
preferred nucleic acid fragments are those which encode a surface exposed
protein of
Borrelia burgdorferi B31, Borrelia burgdon'eri 8313, Borrelia garinii IP90, or
Borrelia
afzelii ACAI.
Highly preferred embodiments of the nucleic acid fragments of the invention
comprise
a nucleotide sequence encoding a polypeptide fragment which includes an amino
acid
sequence selected from the group consisting of SEQ ID NOs: 19, 21, and 23, and
of
these, those which comprise a nucleotide sequence selected from the group
consist-
ing of SEQ iD NOs: 18, 20, and 22 are especially preferred. Most preferred
nucleic
acid fragments of the invention are those which consist of a nucleotide
sequence
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21
encoding a poiypeptide fragment consisting of an amino acid sequence selected
from
the group consisting of SEQ ID NOs: 19, 21, and 23, and the three most
preferred
are those which consist of the nucleotide sequence selected from the group
consist-
ing of SEa ID NOs: 18, 20, and 22.
Analogues of the disclosed nucleic acid fragments which also form part of the
inven-
tion are nucleic acid fragments which are fused to at least one other nucleic
acid
fragment which encodes a fusion partner. This fusion partner can be a protein
en-
hancing the immunogenicity of the fused protein relative to a protein w'tthout
the
encoded fusion partner. Such encoded proteins may e.g. be lipoproteins, e.g.
the
outer membrane lipoprotein from E. coli and OspA from Borreiia burgdorferi
sensu
lato; viral proteins, e.g. from Hepatitis B surface antigen, Hepatitis B core
antigen,
and the influenza virus non-structural protein NS1 (cf. EP-A-0 366 238 and WO
88/01875); immunoglobulin binding proteins, e.g. protein A, protein G, and the
syn-
thetic ZZ-peptide (cf. EP-A-0 243 333 and US 5,411,7321; T-cell epitopes; or B-
cell
epitopes.
Other nucleic acid fragments to form part of a nucleic acid fragment of the
invention
encoding a fusion polypeptide are those encoding polypeptides which facilitate
expression and/or purification of the fused peptide. Such encoded polypeptides
could,
according to the invention, be bacterial fimbrial proteins, e:g, the pilus
components
pilin and papA; protein A; the ZZ-peptide; the maltose binding protein;
glutathione S-
transferase; ~i-galactosidase; calmodulin binding protein; or poly-histidine.
Other nucleic acid fragments to form part of a nucleic acid fragment of the
invention
encoding a fusion polypeptide are those encoding polypeptides which facilitate
modu-
lation in drug resistance of the host thereby facilitating selection of the
host harbour-
ing the plasmid. Such hosts could, according to the invention, be a bacterium,
a
yeast, a protozoan, or a cell derived from a multicellular organism selected
from a
fungus, an insect cell, a plant cell, and a mammalian cell.
Of course, also fusion partners derived from P13 are interesting and these
could be
those which have the same amino acid sequence as at least one amino acid
sequence
selected from the group consisting of amino acid residues 19-27, 33-36, 41-47,
95-
104, 138-147 and 174-179 in SEQ ID NO: 19; amino acid residues 19-26, 32-35,
40-47, 94-101, 137-146, and 174-178 in SEQ ID NO: 21; and amino acid residues
18-26, 30-33, 39-46, 91-104, 137-145 and 173-177 in SEa ID NO: 23
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It will be understood from the above that various analogues and subsequences
of the
nucleic acids disclosed in the sequence listing herein are interesting aspects
of the
invention, as are nucleic acid fragments encoding fused polypeptides including
poly-
peptides encoded by nucleic acid fragments of the invention.
The term "analogue" with regard to the nucleic acid fragments of the invention
is
intended to indicate a nucleotide sequence which encodes a polypeptide
identical or
substantially identical to a polypeptide encoded by a nucleic acid fragment of
the
invention (SEa ID NOs: 18, 20, and 221.
It is well known that the same amino acid may be encoded by various colons,
the
colon usage being related, infer alia, to the preference of the organisms in
question
expressing the nucleotide sequence. Thus, one or more nucleotides or colons of
a
nucleic acid fragment of the invention may be exchanged by others which, when
expressed, result in a polypeptide identical or substantially identical to the
poly-
peptide encoded by the nucleic acid fragment in question.
Also, the term "analogue" is used in the present context to indicate a nucleic
acid
fragment or a nucleic acid sequence of a similar nucleotide composition or
sequence
as the nucleic acid sequence encoding the amino acid sequence having the
immuno-
logical properties discussed above, allowing for minor variations which do not
have
an adverse effect on the biological function and/or immunogenicity as compared
to
the disclosed polypeptides, or which give interesting and useful novel binding
proper-
ties or biological functions and immunogenicities etc. of the analogue. The
analogous
nucleic acid fragment or nucleic acid sequence may be derived from an animal
or a
human or may be partially or completely of synthetic origin as described
herein. The
analogue may also be derived through the use of recombinant nucleic acid tech-
niques.
Furthermore, the terms "analogue" and "subsequence" are intended to allow for
variations in the sequence such as substitution, insertion including introns),
addition,
deletion and rearrangement of one or more nucleotides, which variations do not
have
any substantial effect on the polypeptide encoded by a nucleic acid fragment
or a
subsequence thereof. The term "substitution" is intended to mean the
replacement of
one or more nucleotides in the full nucleotide sequence with one or more
different
nucleotides, "addition" is understood to mean the addition of one or more
nucleotides
at either end of the full nucleotide sequence, "insertion" is intended to mean
the
introduction of one or more nucleotides within the full nucleotide sequence,
"dele-
tion" is intended to indicate that one or more nucleotides have been deleted
from the
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23
full nucleotide sequence whether at either end of the sequence or at any
suitable
point within it, and "rearrangement" is intended to mean that two or more
nucleotide
residues have been exchanged with each other.
Polvpeptide fragments of the invention
The present invention relates to the utility of Bb associated antigenic
proteins as
diagnostic or preventive tools in Lyme disease. Proteins have been indentified
as
associated only (or predominantly) with virulent isolates of Bb, providing a
basis for
several types of diagnostic tests for Lyme disease, including immunodiagnostic
and
nucleic acid identification, such as those based on amplification procedures.
All these
embodiments rely on the availability of the P13 proteins and their analogues.
Another part of the invention therefore pertains to a polypeptide fragment
which
exhibits a substantial immunological reactivity with a polyclonal rabbit
antibody raised
against a polypeptide having an apparent molecular weight of 13 kDa as
determined
by SDS PAGE, followed by visualization, and being derived from Borrelia
burgdorferi
8313, said polypeptide comprising the amino acrd sequence 1-167 of SEQ ID NO:
19, said polyclonal rabbit antibody exhibiting substantially no immunological
reactivity
with whole cell preparations from at least 95°~ of randomly selected B.
hermsii, B.
crocidurae, B, anserina, or B. hispanica, with the proviso that said
polypeptide is
essentially free from other Borreiia-derived antigens when it is identical in
amino acid
sequence to a 13 kDa surface exposed polypeptide which can be extracted from
Borrelia burgdorferi sensu lato.
As mentioned above, the 13 kDa polypeptide has previously been identified in
SDS
gels. However, the 13 kDa polypeptide has never been purified to homogeneity,
let
alone been cloned and sequenced. The present invention is therefore the first
to
provide the 13 kDa polypeptide in a form which is totally free from
contaminating and
potentially harmful (for e.g. vaccine purposes) Borrelia antigens, i.a. the 13
kDa
polypeptide in a substantially pure form. The present invention is also the
first to pro-
vide useful variants of the 13 kDa polypeptide (such variants including
subsequences
of the polypeptide as well as analogues wherein changes have been made to the
native amino acid sequence). It should be noted that it is highly problematic
to purify
the native 13 kDa antigen to homogeneity since it is a membrane protein; it is
well-
known to the skilled person in protein purification that membrane proteins
present
special problems. However, upon the provision of recombinant or synthetic P13
as
disclosed herein, it has become possible to readily prepare P13 in a form free
of other
Borrelia antigens and it has also become possible to prepare variants of P13
which
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24
were not available without access to knowledge of the genetic material
encoding the
protein.
The polypeptide fragment of the invention is otherwise precisely as described
above
when discussing the nucleic acid fragments of the invention and all
discussions
pertaining to polypeptide fragments encoded by the nucleic acid fragments of
the
invention apply mutatis mutandis to the polypeptide fragments of the
invention.
Hence, all considerations regarding the presence or absence of the polypeptide
frag-
ments and their epitopes in various borrelial species as well as other
considerations,
apply to the polypeptide fragments of the invention.
Therefore, also analogues of the P13 polypeptides of the invention are
embraced by
the present invention. When using the terms "analogue" and "subsequence" in
connection with polypeptides is meant any poiypeptide having the same immuno-
logical characteristics as the polypeptides of the invention described above
with
respect to the ability to confer an equivalent and increased resistance to
infections
with Borreiia burgdorferi sensu lato through immune responses against P13.
Thus,
included is also a polypeptide from different sources, such as other bacteria
or even
from eukaryotic cells.
The terms "analogue" and "subsequence" with regard to a polypeptide of the
inven-
tion are also used in the present context to indicate a protein or polypeptide
of a
similar amino acid composition or sequence as the characteristic amino acid
sequen-
ces shown in SEQ ID NOs: 19, 21 and 23, allowing for minor variations which do
not
have an adverse effect on the ligand binding properties and/or biological
function
and/or immunogenicity, or which may give interesting and useful novel binding
pro-
perties or biological functions and immunogenicities etc. The analogous
polypeptide
or protein may be derived from other microorganisms, cells, or animals and the
analogue may also be derived through the use of recombinant DNA techniques as
described herein.
The invention also comprises polypeptides which can be the product of post-
trans-
lational modifications of the polypeptides of the invention described above.
By the
term "post-translational modification" with regard to a polypeptide of the
invention is
meant any modification or processing of the full-length polypeptide that can
occur
during the production of the peptides in Bb, or in the case of recombinant
polypep-
tides in the production of the polypeptides in a host cell. These
modifications include,
but are not limited to, the processing by various peptidases, such as signal
pepti-
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Bases, and modifications such as glycosylations, phosphorylations,
acetylations,
formylations, acylations, palmitylations, sulphations and lipidations.
Furthermore, in the present context the term "immunologically equivalent"
means
5 that the analogue or subsequence of the polypeptide is functionally
equivalent to the
polypeptide with respect to the ability of evoking a protective immune
response
against Borrelia burgdorferi sensu lato infections.
The term "protective immune response" has its usual meaning, i.e. that the
immune
10 response evoked by the polypeptide in question protects the person
immunized from
contracting Lyme disease, or that the immune response evoked by the
polypeptide at
least confers a substantially increased resistance to infections with Borrelia
burgdor-
feri sensu lato.
15 Finally, also fusion polypeptides as described above are part of the
invention and this
is also true for all considerations relating to fusion partners etc. which are
discussed
above when dealing with the nucleic acid fragments of the invention.
Vectors, host cells and cell lines of the invention
Having provided the genetic information relating to the P13 proteins, the
invention
also allows for the preparation of P13 and variants thereof by means of
genetic
engineering. Useful tools in this connection are cloning and expression
vectors and
therefore another important part of the invention is a non-borrelial vector
carrying the
nucleic acid fragment according to the invention and described in detail
above. Such
a vector of the invention is preferably capable of autonomous replication.
Preferred
vectors are selected from the group consisting of a plasmid, a phage, a
cosmid, a
mini-chromosome, and a virus.
Even though plasmid vectors are often preferred because of their relative ease
of use,
vectors which, when introduced in a host cell, are integrated in the host cell
genome
are especially preferred due to the increased stability of the obtained
transformed
cells.
In view of the discussion below, a preferred vector of the invention
comprises, in the
5'~3' direction and in operable linkage, a promoter for driving expression of
the
nucleic acid fragment of the invention, a nucleic acid sequence encoding a
leader
peptide enabling secretion of or integration into the membrane of the
polypeptide, the
nucleic acid fragment according to the invention, and a nucleic acid sequence
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26
encoding a terminator. It is preferred that the promoter drives expression in
a euka-
ryotic cell and also that the leader peptide enables secretion from or
integration into
the membrane of a mammalian cell.
The invention also relates to a transformed cell carrying the vector of the
invention
and capable of replicating the nucleic acid fragment according to the
invention. Such
a transformed cell is preferably a microoganism selected from a bacterium, a
yeast, a
protozoan, or a cell derived from a multicellular organism selected from a
fungus, an
insect cell, a plant cell, and a mammalian cell. Especially preferred host
cells are
bacteria of the genera Escherichia, Bacillus or Salmonella. E. coli is
preferred. Host
cells which are capable of mediating a post-transiational modification
important for
the biological function of the polypeptide of the invention are also a part of
the
invention.
For the purposes of production of recombinant P13 and variants thereof, a
stable cell
line is preferred and therefore the invention also relates to a stable cell
line producing
the polypeptide of the invention, which carries a vector of the invention, and
which
expresses the nucleic acid fragment of the invention.
In general, of course, prokaryotes are preferred for the initial cloning of
DNA sequen-
ces and constructing the vectors useful in the invention. For example, in
addition to
the particular strains mentioned in the specific disclosure below, one may, by
way of
example, mention strains such as E. coli K12 strain 294 (ATCC No. 31446), E.
coli B,
and E. coli X 1776 IATCC No. 31537). These examples are, of course, intended
to
be illustrative rather than limiting.
Prokaryotes are preferred for expression. The aforementioned strains, as well
as
E. coli W3110 tF-, lambda-, prototrophic, ATCC No. 2733251, bacilli such as
Bacillus
subtilis, or other enterobacteriaceae such as Salmonella typhimurium or
Serratia mar-
cescens, and various Pseudomonas species may be used.
In general, plasmid vectors containing replicon and control sequences which
are
derived from species compatible with the host cell are used in connection with
these
hosts. The vector ordinarily carries a replication site, as well as marking
sequences
which are capable of providing phenotypic selection in transformed cells. For
exam-
ple. E. coli is typically transformed using pBR322, a plasmid derived from an
E. coli
species (see, e.g., Bolivar et al., 1977). The pBR322 plasmid contains genes
for
ampicillin and tetracycline resistance and thus provides easy means for
identifying
transformed cells. The pBR plasmid or another microbial piasmid or phage must
also
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27
contain, or be modified to contain, promoters which can be used by the micro-
organism for expression.
The promoters most commonly used in recombinant DNA construction include the
(i-
lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978;
Itakura
et al., 1977; Goeddel et al., 1979) and a tryptophan (trp) promoter system (EP-
B-
0 036 7761. While these are the most commonly used, other microbial promoters
have been discovered and utilised, and details concerning their nucleotide
sequences
have been published, enabling a skilled worker to ligate them functionally
with plas-
mid vectors (Siebenlist et al., 19801. Certain genes from prokaryotes may be
ex-
pressed efficiently in E. coli from their own promoter sequences, thus
avoiding the
need for addition of another promoter by artificial means.
In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may
also be
used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly
used among eukaryotic microorganisms, although a number of other strains are
commonly available. For expression in Saccharomyces, the plasmid YRp7, for
exam-
ple, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979;
Tschumper
et al., 1980). This pfasmid already contains the trpl gene which provides a
selection
marker for a mutant strain of yeast lacking the ability to grow in tryptophan,
for
example ATCC No. 44076 or PEP4-1 (Jones, 19771. The use of the trpl lesion as
a
characteristic of the yeast host cell genome then provides an effective
environment
for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-
phospho-
glycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et
al.,
1968; Holland et al., 19781, such as enolase, glyceraldehyde-3-phosphate
dehydro-
genase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-
phos-
phate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable
expression plasmids, the termination sequences associated with these genes are
also
ligated into the expression vector 3' of the sequence desired to be expressed
to
provide polyadenylation of the mRNA and termination.
Other promoters, which have the additional advantage of transcription
controlled by
growth conditions, are the promoter region for alcohol dehydrogenase 2,
isocyto-
chrome C, acid phosphatase, degradative enzymes associated with nitrogen meta-
bolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and
enzymes responsible for maltose and galactose utilisation. Any plasmid vector
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28
containing a yeast-compatible promoter, origin of replication and termination
sequences is suitable.
In addition to microorganisms, cultures of cells derived from multicelluiar
organisms
may also be used as hosts. In principle, any such cell culture is workable,
whether
from vertebrate or invertebrate culture. However, interest has been greatest
in verte-
brate cells, and propagation of vertebrate cells in culture (tissue culture)
has become
a routine procedure in recent years. Examples of such useful host cell lines
are VERO
and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7,
293
and MDCK cell lines.
Expression vectors for such cells ordinarily include (if necessary) an origin
of repli-
cation, a promoter located in front of the gene to be expressed, along with
any
necessary ribosome binding sites, RNA splice sites, polyadenylation site, and
tran-
scriptional terminator sequences.
For use in mammalian cells, the control functions on the expression vectors
are often
provided by viral material. For example, commonly used promoters are derived
from
polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV401. The early
and
late promoters of SV40 virus are particularly useful because both are obtained
easily
from the virus as a fragment which also contains the SV40 viral origin of
replication
(Fiers et al., 19781. Smaller or larger SV40 fragments may also be used,
provided
there is included the approximately 250 by sequence extending from the Hindlll
site
toward the Bgll site located in the viral origin of replication. Further, it
is also pos-
sible, and often desirable, to utilise promoter or control sequences normally
asso-
ciated with the desired gene sequence, provided such control sequences are com-
patible with the host cell systems.
An origin of replication may be provided either by construction of the vector
to
include an exogenous origin, such as may be derived from SV40 or other viruses
(e.g., Polyoma, Adeno, VSV, BPVI or may be provided by the host cell
chromosomal
replication mechanism. If the vector is integrated into the host cell
chromosome, the
latter is often sufficient.
Methods of producing the polypeptides of the invention which are themselves
part of
the invention thus comprise the following steps:
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29
- culturing a transformed cell or a stable cell line according to the
invention under
conditions facilitating the expression of the poiypeptide by the cell or cell
line,
and
- harvesting the poiypeptide, and optionally subjecting the polypeptide to
post-
translational modification(s1;
or, alternatively,
- synthesising the polypeptide by solid-phase peptide synthesis or by liquid-
phase
peptide synthesis.
The latter approach is preferred with the present technology when the
polypeptide
fragment is relatively short, but of course it cannot be excluded that these
techniques
will ultimately be refined so as to allow feasible production of longer
fragments.
The need for post-translational modifications exists because certain
polypeptides are
prepared in the above-described manner lacking for instance a fatty-acylation
of an
amino acid residue, or the polypeptide has for some reason been prepared in an
elongated version which should be cleaved before the polypeptide will prove
func-
tional. The optional post-translational modifications thus preferably involve
lipidation
or glycosylation when these modifications have not been accomplished by means
of
the preparative procedure itself. Applicable methods for accomplishing
lipidation
andlor glycosylation are well-known to the skilled person. Other post-
translational
modifications include cleavage or elongation of the obtained product. In some
instances, the host cell or cell line also processes the translation product
so as to
obtain a processed polypeptide.
Preparation of useful variants of P13
The present invention has addressed the cloning of nucleic acids encoding
certain
antigenic polypeptides related to the P13 proteins.
A method of preparing variants of the P13 antigens is site-directed
mutagenesis. This
technique is useful in the preparation of individual peptides, or biologically
functional
equivalent proteins or peptides, derived from the P13 antigen sequences,
through
specific mutagenesis of the underlying DNA. The technique further provides a
ready
ability to prepare and test sequence variants, for example, incorporating one
or more
of the foregoing considerations, by introducing one or more nucleotide
sequence
CA 02300365 2000-02-10
WO 99/12960 PCT/IB98/01424
changes into the DNA. Site-specific mutagenesis allows the production of
mutants
through the use of specific oligonucleotide sequences which encode the DNA se-
quence of the desired mutation, as well as a sufficient number of adjacent
nucleo-
tides, to provide a primer sequence of sufficient size and sequence complexity
to
5 form a stable duplex on both sides of the deletion junction being traversed.
Typically,
a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to
10
residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the
art as
10 exemplified by publications (Adelman et al., 1983). As will be appreciated,
the tech-
nique typically employs a phage vector which exists in both a single stranded
and
double stranded form. Typical vectors useful in site-directed mutagenesis
include
vectors such as the M13 phage (Messing et al., 1981). These phages are readily
commercially available and their use is generally well known to those skilled
in the
15 art.
In general, site-directed mutagenesis in accordance herewith is performed by
first
obtaining a single-stranded vector which includes within its sequence a DNA se-
quence which encodes the P13 antigens. An oligonucleotide primer bearing the
20 desired mutated sequence is prepared, generally synthetically, for example
by the
method of Crea et al. ( 19781. This primer is then annealed with the single
stranded
vector, and subjected to DNA polymerising enzymes such as E. toll polymerase I
Klenow fragment, in order to complete the synthesis of the mutation-bearing
strand.
Thus, a heteroduplex is formed wherein one strand encodes the original non-
mutated
25 sequence and the second strand bears the desired mutation. This
heteroduplex vector
is then used to transform appropriate cells, such as E. toll cells, and clones
are se-
lected which include recombinant vectors bearing the mutated sequence arrange-
ment.
30 The preparation of sequence variants of the selected P13 genes using site-
directed
mutagenesis is provided as a means of producing potentially useful species of
the
P13 genes and is not meant to be limiting as there are other ways in which
sequence
variants of the P13 genes may be obtained. For example, recombinant vectors
com-
prising the desired P13 genes may be treated with mutagenic agents to obtain
se-
quence variants (see, e.g., a method described by Eichenlaub, 1979) for the
muta-
genesis of plasmid DNA using hydroxylamine.
Further, another embodiment of the invention is a nucleic acid fragment
substantially
identical to a nucleic acid fragment of the invention which can be provided
e.g. by
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31
dual hybridisation. This method employs a nucleic acid fragment which
specifically
hybridizes under stringency hybridization conditions to a complementary
nucleic acid
fragment which in turn specifically hybridizes under stringency hybridization
condi-
tions to a third nucleic acid fragment encoding a polypeptide comprising the
amino
acid sequences of the invention. This third nucleic acid fragment will thus be
sub-
stantially identical to initial nucleic acid fragment.
Vaccine Preparation and Use
Part of the present invention contemplates vaccine preparation and use.
General
concepts related to methods of preparation and use are discussed as applicable
to
preparations and formulations with the disclosed P13 antigens, its epitopes
and
subfragments thereof. in general, a vaccine of the invention comprises an
amount of
a polypeptide of the invention or produced according to the invention, said
amount
being effective to confer substantially increased resistance to infections
with Borreiia
burgdorferi sensu lato in an animal, including a human being, the polypeptide
being
formulated in combination with a pharmaceutically acceptable carrier, diluent
or
vehicle and the vaccine optionally further comprising an adjuvant. Further,
the vac-
cine is generally used in a method of immunizing an animal, including a human
being,
against infections with Borrelia burgdorferi sensu iato, the method comprising
admini-
staring an immunogenically effective amount of the vaccine to the animal.
Preparation of vaccines which contain peptide sequences as active ingredients
is
generally well understood in the art, as exemplified by U.S. Patents
4,608,251;
4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated
herein by reference. In general terms, the preparation of the vaccines of the
invention
is accomplished by admixing
- a polypeptide of the invention or prepared by the method thereof, and
- a pharmaceutically acceptable carrier, vehicle, or diluent, and optionally
- an adjuvant.
Typically, such vaccines are prepared as injectables either as liquid
solutions or
suspensions; solid forms suitable for solution in, or suspension in, liquid
prior to
injection may also be prepared. The preparation may also be emulsified. The
active
immunogenic ingredient is often mixed with excipients which are
pharmaceutically
acceptable and compatible with the active ingredient. Suitable excipients are,
for
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32
example, water, saline, dextrose, glycerol, ethanol, or the like, and
combinations
thereof. In addition, if desired, the vaccine may contain minor amounts of
auxiliary
substances such as wetting or emulsifying agents, pH buffering agents, or
adjuvants
which enhance the effectiveness of the vaccines.
Suitable carriers are selected from the group consisting of a polymer to which
the
polypeptide(s) is/are bound by hydrophobic non-covalent interaction, such as a
plastic, e.g. polystyrene, or a polymer to which the polypeptide(s) is/are
covalently
bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin,
ovalbumin or keyhole limpet haemocyanin. Suitable vehicles are selected from
the
group consisting of a diluent and a suspending agent.
The vaccines are conventionally administered parenterally, by injection, for
example,
either subcutaneously or intramuscularly. Additional formulations which are
suitable
for other modes of administration include suppositories and, in some cases,
oral
formulations. For suppositories, traditional binders and carriers may include,
for exam-
ple, polyalkalene glycols or triglycerides; such suppositories may be formed
from mix-
tures containing the active ingredient in the range of 0.5°r6 to 10%,
preferably 1-296.
Oral formulations include such normally employed excipients as, for example,
phar-
maceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccha-
rine, cellulose, magnesium carbonate, and the like. These compositions take
the form
of solutions, suspensions, tablets, pills, capsules, sustained release
formulations or
powders and contain 10-950 of active ingredient, preferably 25-70~.
The proteins may be formulated into the vaccine as neutral or salt forms.
Pharmaceu-
tically acceptable salts include acid addition salts /formed with the free
amino groups
of the peptide) which are formed with inorganic acids such as, for example,
hydro-
chloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic,
and the like. Salts formed with the free carboxyl groups may also be derived
from
inorganic bases such as, for example, sodium. potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-
ethylamino ethanol, histidine, procaine, and the like.
The vaccines are administered in a manner compatible with the dosage
formulation,
and in such an amount as will be therapeutically effective and immunogenic.
The
quantity to be administered depends on the subject to be treated, including,
e.g., the
capacity of the individual's immune system to synthesise antibodies, and the
degree
of protection desired. Precise amounts of active ingredient required to be
admini-
stered depend on the judgement of the practitioner. However, suitable dosage
ranges
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33
are of the order of several hundred micrograms of active ingredient per
vaccination.
For example, suitable dosages can be in the range of 1-1000 N.g, such as
between 2
and 750 wg, between 5 and 500 fig, between 7.5 and 250 ~cg, between 10 and 150
N.g, between 10 and 100 fig, between 10 and 75 fig, and between 10 and 50 pg.
Suitable regimes for initial administration and booster shots are also
variable but are
typified by an initial administration followed by subsequent inoculations or
other
administrations.
The manner of application may be varied widely. Any of the conventional
methods
for administration of a vaccine are applicable. These are believed to include
oral appli
cation on a solid physiologically acceptable base or in a physiologically
acceptable
dispersion, parenterally, by injection or the like. The dosage of the vaccine
will
depend on the route of administration and will vary according to the size of
the host.
Various methods of achieving adjuvant effect for the vaccine include use of
agents
such as aluminium hydroxide or phosphate (aluml, commonly used as 0.05 to 0.1
percent solution in phosphate buffered saline, admixture with synthetic
polymers of
sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in
the
vaccine by heat treatment with temperatures ranging between 70°C and
101 °C for
30 second to 2 minute periods, respectively. Aggregation by.reactivating with
pepsin
treated (Fab) antibodies to albumin, mixture with bacterial cells such as C.
parvum or
endotoxins or lipopolysaccharide components of gram-negative bacteria,
emulsion in
physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A)
or
emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a
block
substitute may also be employed. The adjuvant is preferably selected from the
group
consisting of dimethyldioctadecylammonium bromide (DDA), Quil A, poly I:C,
Freund's incomplete adjuvant, IFN-y, IL-2, IL-12, monophosphoryl lipid A
(MPL), and
muramyl dipeptide (MDPI.
In many instances, it will be desirable to have multiple administrations of
the vaccine,
usually not exceeding six vaccinations. more usually not exceeding tour
vaccinations
and preferably one or more, usually at least about three vaccinations. The
vaccina-
tions will normally be at from two to twelve week intervals, more usually from
three
to five week intervals. Periodic boosters at intervals of 1-5 years, usually
three years,
will be desirable to maintain levels of the antibodies. The course of the
immunisation
may be followed by assays for antibodies for the supernatant antigens. The
assays
may be performed by labelling with conventional labels, such as radionuclides,
enzymes, fluorescers, and the like. These techniques are well known and may be
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34
found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932;
4,174,384
and 3,949,064, which are illustrative of these types of assays.
The polypeptide of the invention can also be part of a multi-component or
combina-
lion vaccine, which is also an important part of the invention. Such a vaccine
con-
tains
an amount of the polypeptide fragment of the invention or of a polypeptide
fragment prepared according to the invention, the amount of the polypeptide
fragment being effective to confer substantially increased resistance to infec-
lions with Borrelia burgdorferi sensu lato in an animal, including a human
being;
and
at least one further Borrelia antigen,
the polypeptide fragment and the antigen being formulated in combination with
a
pharmaceutically acceptable carrier, vehicle, or diluent and the vaccine
optionally
further comprising an adjuvant. All components of such a vaccine apart from
the at
least one further Borrelia antigen are as described in detail herein. With
respect to the
at least one further Borrelia antigen, it is preferred that it is selected
from the group
consisting of OspA, OspB, OspC, OspD, OspE, OspF, OspG, PC, Oms28, Oms45,
Oms 66, decorin binding protein (dbpl, LpLA7, EppA, T5, S1, 26 kDa, 39 kDa, 66
kDa, 79 kDa, 85 KDa, and 110 kDa antigen.
Another variant of a combination vaccine of the invention comprises at least
two
non-identical polypeptide fragments of the present invention or at least two
non-
identical polypeptide fragments prepared by the method of the invention, the
vaccine
comprising an amount of the polypeptides effective to confer substantially
increased
resistance to infections with Borrelia burgdorferi sensu lato in an animal,
including a
human being, in combination with a pharmaceutically acceptable carrier,
vehicle, or
diluent, the vaccine optionally further comprising an adjuvant. Also in this
case, all
components of the vaccine have been described elsewhere herein.
Another known way of achieving a suitable immune response in a vaccinated
animal
is by employing a so-called live vaccine which i.a. triggers both a B- and a T-
cell
mediated immune response, and therefore the invention also pertains to such a
vac-
cine comprising a non-pathogenic microorganism carrying and being capable of
ex-
pressing the nucleic acid fragment of the invention so as to produce the
polypeptide
of the invention, the live vaccine being effective in conferring increased
resistance to
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WO 99/12960 PCT/IB98/01424
infection with Borrelia burgdorferi sensu lato in an animal, including a human
being.
Preferred non-pathogenic microorganisms are selected from the group consisting
of
Mycobacterium bovis BCG, Salmonella typhi, Salmonella typhimurium, Salmonella
paratyphi, Staphylococcus aureus, and Listeria monocytogenes.
5
DNA vaccination
The invention also contemplates the use of disclosed nucleic acid segments in
the
construction of expression vectors or plasmids and use in host cells with a
view to
10 vaccination of the individual housing the host cells. Hence, the invention
also pertains
to a vaccine comprising a nucleic acid fragment or a vector of the invention;
the vac-
cine effecting in vivo expression of antigens by an animal, including a human
being,
to whom the vaccine has been administered, the amount of expressed antigens
being
effective to confer substantially increased resistance to infections with
Borrelia burg-
15 dorferi sensu lato in an animal, including a human being. The related
vaccination
method consists of administering an amount of this vaccine which is effective
to
confer an increased resistance to such infections upon the mammal to which it
has
been administered.
20 The following is a general discussion relating to such use of nucleic acid
fragments
and the particular considerations in practising this aspect of the invention.
Direct injection of plasmid DNA has become a simple and effective method of
vacci-
nation against a variety of infectious diseases (see, e.g., Ulmer et al.,
19931. It is
25 potentially more potent and longer tasting than recombinant protein
vaccination
because it elicits both a humoral as well as a cellular immune response.
The present invention also provides for a DNA-based vaccine or immunological
com-
position against Lyme disease (e.g., Borrelia burgdorferi, afzelii, or
gariniil which can
30 elicit an immunological response, which can confer protection, even up to
100°~, in
mice against challenge with an infectious strain of Borrelia burgdorferi. An
exemplary
plasmid of the invention contains the human cytomegalovirus immediate early
pro-
moter driving expression of the P13 protein. To facilitate expression in
eukaryotic
cells, the natural leader sequence of the gene encoding P13 has been replaced
with
35 the human tissue plasminogen activator leader sequence.
Protection can be demonstrated in mice by injecting, intramuscularly, naked
plasmid
DNA and subsequently challenging with Bb spirochaetes. Following vaccination
sera
CA 02300365 2000-02-10
WO 99/12960 PCT/IB98/01424
36
will contain high titers of antibody to P13 which will inhibit spirochaete
growth in
vitro.
Thus, a DNA vaccine or immunological composition, expressing a P13 antigen,
from
Borrelia burgdorferi, Borrelia afzelii or Borrelia garinii or any combination
thereof, can
protect mice against infection by a Borreiia genospecies, the etiologic agent
of Lyme
disease. The composition is thus useful for eliciting a protective response in
a host
susceptible to Lyme Disease, as well as for eliciting antigens and antibodies.
which
are also useful in and of themselves.
Therefore, as discussed above, the invention in a general sense preferably
provides
methods for immunising, or vaccinating, or eliciting an immunological response
in a
host, such as a host susceptible to Lyme disease, e.g., a mammalian host,
against
Borrelia and accordingly Lyme Disease, by administering DNA encoding a P13
anti-
gen, for instance DNA encoding P13 from Borreiia burgdorferi, Borrelia
afzeiii, Borrelia
garinii antigen or combinations thereof, in a suitable carrier or diluent,
such as saline;
and the invention provides plasmids and compositions for performing the
method, as
well as methods for making the plasmids, and uses of the expression products
of the
plasmids, as well as antibodies elicited thereby.
From present dog and human trials based on efficiency studies with mice
(Erdile et
al., 1993; USSN 08/373,455), it is clear that mice are now a suitable animal
model
with respect to Borrelia and Lyme disease for extrapolation to domestic
animals,
humans, and other animals susceptible to Lyme disease or Borrelia infection
(e.g.,
wild animals such as deer).
In the present invention, the DNA encoding P13 or an immunologically active
frag-
ment thereof can be administered in dosages and by techniques well known to
those
skilled in the medical or veterinary arts taking into consideration such
factors as the
age, sex, weight, species and condition of the particular patient, and the
route of
administration. DNA encoding P13 or an immunologically active fragment thereof
can
be administered alone, or can be co-administered or sequentially administered
with
other Bb antigens, or with DNA encoding other Bb antigens; and the DNA
encoding
P13 or an immunologically active fragment thereof can be sequentially
administered,
e.g., each spring as the "Lyme Disease season" is about to begin.
As broadly discussed above, the invention also pertains to plasmids comprising
DNA
including P13 encoding DNA for expression by eukaryotic cells. The DNA, from
upstream to downstream (5' to 3'), can comprise: DNA encoding a promoter for
CA 02300365 2000-02-10
WO 99/12960 PGT/IB98/01424
37
driving expression in eukaryotic cells, DNA encoding a leader peptide which
enables
secretion of a prokaryotic protein sequence from a mammalian cell, DNA
encoding a
P13 antigen (or antigens) or an immunologically active fragment thereof, DNA
en-
coding other Bb antigens such as OspA, OspB, OspC or OspD .or an
immunologically
active fragment thereof, and DNA encoding a terminator.
For instance, the promoter can be a eukaryotic viral promoter such as a herpes
virus
promoter, e.g., human cytomegalovirus promoter DNA.
The DNA encoding a leader peptide which enables secretion of a prokaryotic
protein
sequence from a mammalian cell is any DNA encoding any suitable leader for
this
purpose such as DNA encoding a eukaryotic, preferably mammalian, leader
sequence;
for instance, DNA encoding a leader peptide of a peptide hormone, or, for
example,
of insulin, renin, Factor VIII, TPA, and the like, with DNA encoding human
tissue
plasminogen activator ITPAI leader peptide being presently preferred.
The human cytomegalovirus promoter can be an immediate early human cytomegalo-
virus promoter such as HCMV-IE. As to HCMV promoters, reference is made to
U.S.
Patents Nos. 5,168,062 and 5,385,839. The plasmid of the invention can contain
the HCMV-IE gene 5' untranslated region (UTR~ which includes Intron A. This se-
quence can be 3' to the HCMV-IE promoter and 5' to the activator portion of
the 5'
UTR sequence and leader peptide. The TPA sequence can be derived from the TPA
gene and can encode a portion of the 5' UTR and leader peptide from that gene.
The
5' UTR of TPA may increase eukaryotic cell expression.
The transcriptional terminator sequence can be any suitable terminator, such
as a
eukaryotic terminator, for instance, DNA encoding a terminator for a mammalian
peptide, with the BGH terminator being presently preferred.
The plasmid can be in admixture with any suitable carrier, diluent or
excipient such as
sterile water, physiological saline, and the like. Of course, the carrier,
diluent or exci-
pient should not disrupt or damage the plasmid DNA.
The plasmid can be administered in any suitable manner. The plasmid can be in
a
composition suitable for the manner of administration. The compositions can
include:
liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral,
intragastric
administration and the like, such as solutions, suspensions, syrups, elixirs;
and liquid
preparations for parenteral, subcutaneous, intradermal, intramuscular,
intravenous
administration, and the like, such as sterile solutions, suspensions or
emulsions, e.g.,
CA 02300365 2000-02-10
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38
for administration by injection. Intramuscular administration and compositions
therefor are presently preferred.
The plasmids of the invention can be used for in vitro expression of antigens
by
eukaryotic cells. Recovery of such antigens can be by any suitable techniques;
for
instance, techniques analogous to the recovery techniques employed in the docu-
ments cited herein (such as the applications cited under Related Applications
and the
documents cited therein).
The thus expressed antigens can be used in immunological, antigenic or vaccine
compositions, with or without an immunogenicity-enhancing adjuvant ("expressed
antigen compositions"1. Such compositions can be administered in dosages and
by
techniques well known to those skilled in the medical or veterinary arts
taking into
consideration such factors as age, sex, weight, species, condition of the
particular
patient, and the route of administration. These compositions can be
administered
alone or with other compositions, and can be sequentially administered, e.g.,
each
spring as the "Lyme Disease season" is about to begin.
The route of administration for the expressed antigen compositions can be
oral,
nasal, anal, vaginal, perorat, intragastric, parenteral, subcutaneous,
intradermat,
intramuscular, intravenous, and the like.
The expressed antigen compositions can be solutions, suspensions, emulsions,
syrups, elixirs, capsules (including "gelcaps" - a gelatin capsule containing
a liquid
antigen or fragment thereof - preparations), tablets, hard-candy-like
preparations, and
the like. The expressed antigen compositions may contain a suitable carrier,
diluent,
or excipient such as sterile water, physiological saline, glucose or the like.
The com-
positions can also be lyophilised. The compositions can contain auxiliary
substances
such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling
or
viscosity enhancing additives, preservatives, flavouring agents, colours, and
the like,
depending upon the route of administration and the preparation desired.
Standard
texts, such as Remington: The Science and Practice of Pharmacy, 19t" ed.,
Gennaro,
AR, 1995, incorporated herein by reference, may be consulted to prepare
suitable
preparations, without undue experimentation.
Suitable dosages for plasmid compositions and for expressed antigen
compositions
can also be based upon the examples below, and upon the documents cited
herein.
For example, suitable dosages can be in the range of 1-1000 wg, such as
between 2
and 750 fig, between 5 and 500 pg, between 7.5 and 250 fig, between 10 and 150
*rB
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WO 99/12960 PCT/IB98/014Z4
39
fig, between 10 and 100 fig, between 10 and 75 pg, and between 10 and 50 fig,
in
expressed antigen compositions. In plasmid compositions, the dosage should be
a
sufficient amount of plasmid to elicit a response analogous to the expressed
antigen
compositions; or expression analogous to dosages in expressed antigen
compositions.
For instance, suitable quantities of plasmid DNA in plasmid compositions can
be 0.1
to 2 mg, preferably 1-10 N.g.
Thus, in a broad sense, the invention further provides a method comprising
admini-
stering a composition containing plasmid DNA including DNA encoding a P13
antigen
or antigens, for expression of the antigen or antigens in vivo for eliciting
an immuno-
logical, antigenic or vaccine (protective) response by a eukaryotic cell; or
for ex vivo
or in vitro expression (that is, the cell can be a cell of a host susceptible
to Lyme Dis-
ease, i.e., the administration can be to a host susceptible to Lyme Disease
such as a
mammal, e.g., a human; or the cell can be an ex vivo or in vitro celll. The
invention
further provides a composition containing a P13 antigen or antigens from
expression
of the plasmid DNA by a eukaryotic cell, in vitro or ex vivo, and methods for
admini-
stering such compositions to a host mammal susceptible to Lyme disease to
elicit a
response.
Since the methods can stimulate an immune or immunological response, the
inventive
methods can be used for merely stimulating an immune response (as opposed to
also
being a protective response) because the resulting antibodies (without
protection) are
nonetheless useful. From eliciting antibodies, by techniques well-known in the
art,
monoclonal antibodies can be prepared and the monoclonal antibodies can be em-
ployed in well known antibody binding assays, diagnostic kits or tests to
determine
the presence or absence of a P13 antigen or to determine whether an immune re-
sponse to the bacteria has simply been stimulated. The monoclonal antibodies
can
also be employed in recovery or testing procedures, for instance, in
immunoadsorp-
tion chromatography to recover or isolate a P13 antigen.
To prepare the inventive plasmids, the DNA therein is preferably iigated
together to
form a plasmid. For instance, the promoter, leader sequence, antigen and
terminator
DNA is preferably isolated, purified and ligated together in a 5' to 3'
upstream to
downstream orientation. A three-way ligation, as exemplified below, is
presently
preferred.
CA 02300365 2000-02-10
WO 99/12960 PCT/IB98/01424
Nucleic Acid Hybridisation Embodiments
Also contemplated within the scope of the present invention is the use of the
dis-
closed DNA as a hybridization probe. While particular examples are provided to
5 illustrate such use, the following provides a general background for
hybridization
applications taking advantage of the disclosed nucleic acid sequences of the
invention.
As mentioned, in certain aspects, the DNA sequence information provided by the
10 invention allows for the preparation of relatively short DNA (or RNA)
sequences
having the ability to specifically hybridise to Bb gene sequences. In these
aspects,
nucleic acid probes of an appropriate length are prepared based on a
consideration of
the sequence, e.g., SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22 or derived
from flanking regions of these genes. The ability of such nucleic acid probes
to speci-
15 fically hybridise to the Bb gene sequences lend them particular utility in
a variety of
embodiments. Most importantly, the probes can be used in a variety of
diagnostic
assays for detecting the presence of pathogenic organisms in a given sample.
How-
ever, either uses are envisioned, including the use of the sequence
information for the
preparation of mutant species primers, or primers for use in preparing other
genetic
20 constructs.
To provide certain of the advantages in accordance with the invention, the
preferred
nucleic acid sequence employed for hybridization studies or assays includes
sequen-
ces that are complementary to at least a 10 to 40, or so, nucleotide stretch
of the
25 selected sequence, such as that shown in SEa ID N0: 18, SEQ ID NO: 20 and
SEQ
ID NO: 22. A size of at least 10 nucleotides in length helps to ensure that
the frag-
ment will be of sufficient length to form a duplex molecule that is both
stable and
selective. Molecules having complementary sequences over stretches more than
10
bases in length are generally preferred, though, in order to increase
stability and
30 selectivity of the hybrid, and thereby improve the quality and degree of
specific
hybrid molecules obtained. Thus, one will generally prefer to design nucleic
acid
molecules having gene-complementary stretches of 15 to 20 nucleotides, or even
longer where desired. Such fragments may be readily prepared by, for example,
directly synthesising the fragment by chemical means, by application of
nucleic acid
35 reproduction technology, such as the PCR technology of U.S. Patent
4,803,102, or
by introducing selected sequences into recombinant vectors for recombinant pro
duction.
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41
The present invention will find particular utility as the basis for diagnostic
hybridiza-
tion assays for detecting Bb-specific RNA or DNA in clinical samples.
Exemplary
clinical samples that can be used in the diagnosis of infections are thus any
samples
which could possibly include nucleic acid, including samples from tissue,
blood,
serum, urine or the like. A variety of tissue hybridization techniques and
systems are
known which can be used in connection with the hybridization aspects of the
inven-
tion, including diagnostic assays such as those described in Falkow et al.,
U.S. Patent
4,358,535.
Accordingly, the nucleotide sequences of the invention are important for their
ability
to selectively form duplex molecules with complementary stretches of Bb gene
seg-
ments. Depending on the application envisioned, one will desire to employ
varying
conditions of hybridization to achieve varying degrees of selectivity of the
probe
toward the target sequence. For applications requiring a high degree of
selectivity,
one will typically desire to employ relatively stringent conditions to form
the hybrids,
for example, one will select relatively low salt and/or high temperature
conditions,
such as provided by 0.02M-0.15M NaCI at temperatures of 50°C to
70°C. These
conditions are particularly selective and tolerate little, if any, mismatch
between the
probe and the template or target strand.
Of course, for some applications, for example where one desires to prepare
mutants
employing a mutant primer strand hybridised to an underlying template, less
stringent
hybridization conditions are called for in order to allow formation of the
heteroduplex.
In these circumstances, one would desire to employ conditions such as 0.15M-
0.9M
salt, at temperatures ranging from 20°C to 55°C. In any case, it
is generally appre-
ciated that conditions can be rendered more stringent by the addition of
increasing
amounts of formamide which serves to destabilise the hybrid duplex in the same
manner as increased temperature. Thus, hybridization conditions can be readily
mani-
pulated, and will thus generally be a method of choice depending on the
desired
results.
In clinical diagnostic embodiments, nucleic acid sequences of the present
invention
are used in combination with an appropriate means, such as a label, for
determining
hybridization. A wide variety of appropriate indicator means are known in the
art,
including radioactive, enzymatic or other ligands, such as avidin/biotin,
which are
capable of giving a detectable signal. In preferred diagnostic embodiments,
one will
likely desire to employ an enzyme tag such as alkaline phosphatase or
peroxidase,
instead of radioactive or other environmentally undesirable reagents. In the
case of
enzyme tags, colorimetric indicator substrates are known which are employed to
CA 02300365 2000-02-10
WO 99/12960 PCT/IB98/01424
42
provide a means visible to the human eye or spectrophotometrically to identify
spe-
cific hybridization with pathogenic nucleic acid-containing samples.
Luminescent
substrates, which give off light upon enzymatic degradation, could also be
employed
and may provide increased sensitivity.
In general, it is envisioned that the hybridization probes described herein
will be use-
ful both as reagents in solution hybridization as well as in embodiments
employing a
solid phase. In embodiments involving a solid phase, the test DNA (or RNA)
from
suspected clinical samples, such as exudates, body fluids (e.g., amniotic
fluid, cere-
brospinal fluid) or even tissues, is adsorbed or otherwise affixed to a
selacted matrix
or surface. This fixed, single-stranded nucleic acid is then subjected to
specific hybri-
dization with selected probes under desired conditions. The selected
conditions will
depend on the particular circumstances based on the particular criteria
required
(depending, for example, on the G + C content, type of target nucleic acid,
source of
nucleic acid, size of hybridization probe, etc.). Following washing of the
hybridised
surface so as to remove non-specifically bound probe molecules, specific
hybridi-
zation is detected, or even quantified, by means of the label.
Furthermore, it is envisioned that synthetic single stranded nucleotides can
be pro-
duced (by a series of photolitograpic and chemical steps) on a solid phase
based on
nucleic acid sequences or the complementary sequence of the invention and
sequen-
ces comprised thereof. Single-stranded nucleic acid fragments from suspected
clinical
samples, such as exudates, body fluids (e.g., amniotic fluid, cerebrospinal
fluid) or
even tissues are then subjected to specific hybridisation under desired
conditions.
These single-stranded nucleic acid fragments are labeled for detection prior
to hybri-
disation. The selected conditions for hybridisation will depend on the
particular cir-
cumstances based on the particular criteria required (depending, for example,
on the
G + C content, type of target nucleic acid, source of nucleic acid, size of
hybridization
probe, etc.). Following washing of the hybridised surface so as to remove non-
speci-
fically bound probe molecules, specific hybridization is detected, or even
quantified,
by means of the label.
The invention discloses a DNA segment encoding an antigenic Bb protein.
Detection
of that DNA or various parts thereof is expected to provide the basis for a
useful
amplification assay. One method of detecting the P13 antigen genes is based on
selective amplification of known portions of the gene. A particular method
utilises
PCR amplification, using any one of a number of primers that could be prepared
from
knowledge of the nucleic acid sequence of SEO, ID NO: 18, SEt1 ID NO: 20 and
SEQ
ID NO: 22. Generally, such primers are relatively short, e.g., 7-28 base pairs
in
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WO 99/12960 PCT/IB98/01424
43
length, and may be derived from the respective sense or anti-sense strands of
the
disclosed DNA segment. Synthesis of these primers may utilise standard
phosphor-
amidite chemistry (Beaucage et al., 1981 ).
In summary, this part of the invention relates to a diagnostic composition
adapted for
the determination of Borrelia burgdorferi sensu lato in a sample, the
composition com-
prising an amount of the nucleic acid fragment of the invention which is
effective to
detectably bind to a nucleic acid fragment from Borrelia burgdorferi sensu
lato pre-
sent in the sample, the composition optionally comprising a detectable label.
Further, another embodiment of the invention is a method of determining the
pre-
sence of Borrelia burgdorferi sensu lato nucleic acids in a sample, comprising
incu-
bating the sample with the nucleic acid fragment of the invention, and
detecting the
presence of hybridized nucleic acids resulting from the incubation.
Alternatively, such
a method comprises subjecting the nucleic acid fragment of the invention to a
mole-
cular amplification reaction, such as PCR, and detecting the presence of
amplified
nucleic acid which is specific for Borrelia burgdorferi sensu lato.
Finally, the invention also provides a diagnostic kit comprising
a nucleic acid fragment of the invention and a means for detecting the binding
between the nucleic acid fragment and nucleic acid bound thereto, or
a set of nucleic acid primers which, when used in a molecular amplification
procedure together with the nucleic acid fragment of the invention, will
result in
specific amplification of said nucleic acid fragment, and a means for
detecting
the amplified nucleic acid fragment.
Diagnostic immunoloaical embodiments
Antibodies could be produced and used for screening strains for protein
expression,
for determining structural location and for examining bactericidal activity of
anti-
bodies against these proteins. Means and measures for producing both
monoclonal
and polyclonal antibodies against P13 are easily applied by the skilled person
on the
basis of the teachings herein.
It is contemplated that several assays for Lyme disease may be developed using
any
of the P13 proteins or its epitopes, the corresponding DNA encoding the
protein,
functionally similar proteins and their epitopes, or by detection of the
appropriate
*rB
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WO 99/12960 PCT/IB98/01424
44
mRNA. An indirect ELISA assay could be used with the P13 protein or other
antigenic
proteins. These methods are similar in principle to those previously described
(Mag-
narelli et al., 1989; Craft et al., 1984; Bergstrt3m et al., 1991 ). Reactive
epitopes
representing portions of the P13 protein sequences could be utilised in an
analogous
manner.
Another promising assay is the microcapsule agglutination technique (MCAT)
(Arimit-
su et al., 1991 ). In this procedure, microscopic polystyrene beads are coated
with Bb
antigen and incubated with dilutions of patient serum. After overnight
incubation at
4°C, the agglutination patterns are determined. Using whole Bb as
antigen, the MCAT
has been shown to be highly discriminatory between Lyme disease patients and
healthy individuals, with little overlap in agglutination titre, although
false positive
reactions have been obtained with rheumatoid arthritis patients (Anderson et
al.,
1988) and leptospirosis samples (Barbour, 1988) An assay using P13 protein
alone or
in combination with other antigens such as the 94 kDa, 30 kDa and 21 k0a
antigens
should be feasible. Such a combination may increase sensitivity of the assay.
In summary, an embodiment of this part of the invention is a diagnostic
composition
adapted for the determination of Borrelia burgdorferi sensu lato in a sample,
the com-
position comprising the polypeptide of the invention or prepared thereby, the
amount
of the polypeptide being effective to detectably react with antibodies present
in the
sample, the antibodies being directed against Borrelia burgdorferi sensu lato,
the com-
position optionally comprising a detectable label, e.g. as described above.
Related to
this is another embodiment of the invention, i.e. a method of determining the
pre-
sence of antibodies directed against Borrelia burgdorferi sensu lato in a
sample,
comprising incubating the sample with the polypeptide of the invention or
prepared
by the method of the invention, and detecting the presence of bound antibody
resulting from the administration or incubation.
Finally, this part of the invention also pertains to a diagnostic kit
comprising a poly
peptide of the invention and a means for detecting the polypeptide with
antibody
bound thereto.
EXAMPLES
Bacterial strains and culture conditions. Borreiia strains used in this study
were the
following: strain B31 of B. burgdorferi, a tick isolate from North America
(ATCC
35210); strain ACAI of B. afzelii, a human skin isolate from Sweden (I~sbrink
et al.,
CA 02300365 2000-02-10
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19841; strain IP90 of B. garinii, a tick isolate from the Asian Russia
(Kryuchechnikov
et al., 1988); strain B. burgdorferi 8313, a mutant of B. burgdorferi B31
lacking
OspA, OspB, OspC and OspD (Sadziene et al., 19931.
5 Also used were three relapsing fever borreliae species, B. hermsii, B.
crocidurae, and
B. hispanica, as well as B. anserina, the causative agent of avian
borreliosis.
Borreliae were grown in BSK ll medium (Barbour, 1984) and the cells were
harvested
in late-log phase by centrifugation at 5,000 rpm for 20 min.
The Escherichia colt strains DHSa and BL21 were used for transformation with
the
recombinant plasmids in DNA cloning and gene expression experiments,
respectively.
E. colt strains were grown in Luria broth medium (Gibco BRL, Gaithersburg, MD)
supplemented, when required, with carbenicillin (Sigma, St. Louis, MO) at 50
p,g/ml.
Monoclonal antibodies 1566 and 7D4 were obtained from Dr. Alan G. Barbour (Sad-
ziene et al., 19941
DNA fragments were sequenced by the dideoxy chain termination method, with ABI
PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit, with AmpIiTaq~ DNA
Polymerase, FS. The sequence fragments were assembled using the GCG software
for UNIX computer.
EXAMPLE 1
Preparation of Bb proteins, sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGED, and Wesiem blot
1.1 Preparation of Bb proteins.
For the whole-cell protein preparations, bacteria harvested from 250 mi of BSK
II
medium were washed twice with phosphate-buffered saline-5mM MgCl2 (PBS-Mg).
The pellet was suspended in 2 ml of PBS, sonicated and the supernatant was
collected after centrifugation at 10,000 rpm for 30 min.
The subcellular fraction of borreliae outer membrane components (designated
Fraction
B) was prepared as described elsewhere (WO 90/04411 ). Briefly, cells
harvested
from 1.5 I of the culture were washed three times with 10 mM Tris-HCI (pH
7.4),
150 mM NaCI and 5 mM MgCl2 (TSM buffed. Octyl-[3-D-glucopyranoside (OGP)
(Sigma St. Louis, MO) was added to a final concentration of 2°6 in 10
ml TSM buffer
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WO 99/12960 PCT/IB98/01424
46
and the suspension was incubated at 37°C for 60 min. The cell lysate
was centri-
fuged and the supernatant was incubated at 56°C for 30 min. The
precipitate was
removed by centrifugation at 20,000 rpm for 30 min at 37°C, and the
supernatant
was dialysed against water at 4°C for 2 days. The precipitate (Fraction
B) formed in
the dialysis bag was recovered by centrifugation at 20,000 rpm for 30 min at
25°C.
1.2 Separation of proteins by SDS-PAGE.
Bacterial proteins were separated by 15% SDS-PAGE essentially according to
Laemmli (1970). Subsequently, gels were either stained with Coomassie Blue R-
250
(CB) (Sigma, St Louis, MO), silver-staining (BioRad Hercules, CAI, or were
subjected
to Western blotting.
1.3 Western blotting.
The proteins were transferred to a PVDF-membrane (BioRad, Hercules, CA) by
elec-
troblotting at 0.8 mAicm2 for 1 h. The non-specific binding was blocked by
immer-
sing the filter for 2 h into 5~o non-fat milk powder (Semper, Stockholm,
Sweden) in
PBS containing 0.05°6 Tween-20 (PBS-Tl. Primary or secondary
antibodies were
diluted with 2.5 ~o milk-powder in PBS-T, and both incubations of the filter
for 1 h
were followed by washing in PBS-T. In a developing reaction the substrate for
the
alkaline phosphatase conjugate was 5-bromo-4-chloro-3-indolyl phosphate IBCIP)
(Sigma, St. Louis, MO).
EXAMPLE 2
Preparation of antiserum against the 13 kDa antigen
2.1 Purification of the 13 kDa antigen.
A 13 kDa protein was purified by 15% SDS-PAGE of Fraction B obtained from the
B. burgdorferi 8313 spirochaetes. The appropriate band was visualised by
staining
the gel with 250 mM KCI in ice-cold water without fixation in MeOH and acetic
acid.
Elution of protein from the gel was performed in a Schleicher and Schuell
Biotrap in
15 mM NH4HC03 (200V for 8 hr).
2.2 Immunisation of rabbits.
A mixture of eluted protein and protein from a crushed SDS-PAGE gel of approxi-
mately 100 L~g of the 13 kDa protein prepared as described above was
homogenised
and used in each of four immunisations of one rabbit performed at one and two
(for
the last immunisation) months intervals. Serum samples were obtained during a
5-
month period, and serum was diluted 1:1,000 when used for Western blot
analysis.
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47
EXAMPLE 3
Cell surface proteolysis of Borrelia cells
3.1 Protease treatment of Borrelia cells.
Cell surface proteolysis of Bb cells was conducted as previously described f
Barbour et
al., 19841. Briefly, washed spirochetes were resuspended in PBS-Mg at a
concentra-
tion of 2 x 109 cells/ml. To 950 ~,I of the cell suspension was added 50 ~I of
one of
the following: distilled water, proteinase K (Sigma, St Louis, MOI (4 mg/ml in
water)
or trypsin (Gibco BRL, Gaithersburg, MD) ( 1 mgiml in 10'3 M HCI?. After
incubation
for 40 min at 20°C, the proteolytic treatment was stopped by the
addition of 10 ~,I
from a solution of the peptidase inhibitor phenyfmethylsulfonyl fluoride
(PMSF)
(Sigma, St. Louis, M0) (50 mg of PMSF per 1 ml of isopropanoll, and the cells
were
centrifuged and washed twice with PBS-Mg. The pellets were resuspended in TSM
buffer. One-third of the cell suspension of each preparation was subjected to
the
whole cell protein extraction by boiling in SDS-PAGE sample buffer. The
remaining
part of the suspensions was used to prepare the subcellular fraction of the
barrelial
outer membrane components, Fraction B, as described above.
3.2 Analysis of the protease treated Borrelia cells.
The result of the protease treatment of Bb cells as analysed by SDS-PAGE is
pre-
sented in Figure 1 A, and followed by Western Blot, Figure 1 B. As seen in the
CB
stained protein profiles of the whole-cell lysates (Figure 1 A~, proteinase K
conside-
rably affected the minor protein with an apparent molecular weight of 13 kDa.
The
protein composition of the subcellular fractions of outer membrane components
(Fraction B) recovered from protease treated and untreated spirochaetes, was
also
investigated. The 13 kDa protein was shown to constitute a substantial part of
the
Fraction B obtained from the Bb cells. In the Fraction B derived from the
proteinase K
treated cells, the 13 kDa protein was entirely absent.
The finding that protease treatment eliminates the 13 kDa protein clearly
shows that
the 13 kDa protein is surface exposed, and most probably associated with the
outer
membrane of the Bb cells.
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EXAMPLE 4
Immunogold labelling of Borrelia cells
The monoclonal antibody 1566 raised against the 13 kDa protein was used as the
primary antibody for immunogold staining of intact B. burgdorferi B31 and B.
burg-
dorferi 8313. Cells from strain B31 (Figure 2A) were labelled to a minor
extent than
cells from the strain 8313 (Figure 2B). This was probably due to the presence
of
outer surface proteins, i.e. OspA, OspB, OspC, OspD, on the surface of the B31
cells. The labelling was confined to the outer surface membrane for both
strains
indicating that the 13 kDa protein is an outer surface protein.
EXAMPLE 5
Expression of the 13 kDa protein in different Borrelia species
5.1 SDS-PAGE analysis.
The CB stained SDS-PAGE of the whole-cell protein preparations of Lyme disease
borreliae is shown in Figure 1 A. The 13 kDa protein was present in the whole-
cell
preparations (WC) and enriched in the membrane fraction (BF) of B. burgdorferi
B31,
B. afzelii ACAI, and B. garinii IP90. The PAGE revealed no major differences
among
the borrelial strains in respect of either apparent molecular weight or
expression level
of the 13 kDa protein. In the analogous preparations from B. hermsii, B.
crocidurae
and B. anserina, no visible band corresponding to the 13 kDa protein was
detectable.
5.2 Western blotting.
In Western blot analysis of Fraction B prepared from B. burgdorferi B31, B.
afzelii
ACAI and B. garinii IP90 (Figure 3A and 38), the 13 kDa protein of B.
burgdorferi B31
reacted with the monoclonal antibody 1566. The monoclonal antibody failed to
recognise the 13 kDa protein from B. afzelii ACAI, and B. garinii IP90
indicating that
the antibody is directed against a variable epitope.
However, polyclonal rabbit antiserum (described in Example 2.2) was able to re-
cognise the 13 kDa protein from all three Lyme Disease species, i.e. B.
burgdorferi
B31, B. afzelii ACAI and B. garinii IP90 (Figure 1 B).
In another Western blot analysis the rabbit antiserum raised against the 13
kDa
protein prepared from B. burgdorferi 8313 did not recognise a 13 kDa protein
or any
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proteins of similar molecular weight from B. henrsii or B. crocidurae (Figure
1 C), or
B. anserina or B. hispanica (data not shown).
These data indicate that 13 kDa protein is unique for Lyme disease borreliae.
Con-
s versely, it was shown recently that the ospC gene homologues and OspC-
related
proteins are present in Borrelia species not associated with Lyme borreliosis
(Marconi
et al., 1993).
EXAMPLE 6
Isolation and N terminal amino acid sequencing of the 13 kDa protein
6.1 Amino acid sequencing.
The 13 kDa protein band was isolated and cut from a SDS-PAGE gel and eluted in
a
Biotrap as described above, Example 2.1. N-terminal amino acid sequencing of
the
purified 13 kDa protein was attempted but no sequence was obtained. It was con-
cluded that the N-terminus of the 13 kDa protein was blocked. Therefore, the
purified
protein was digested with Staphylococcus aureus V8 protease. The protein
cleavage
resulted in two fragments of about equal size. As one of the fragments is
blocked,
only one can be sequenced. After cleavage the fragments were transferred to a
PVDF
membrane (Biorad, Hercules, CA) by soaking the membrane in the protein
solution
over night. N-terminal amino acid sequence analysis was performed on a 477A
sequenator /Applied Biosystems, Foster City, CAI at Ume~ University.
N-terminal amino acid sequence of the peptide fragment obtained by protease
cleavage of the 13 kDa protein, recovered from the Fraction B of B.
burgdorferi
B313, resulted in the following sequence
TSKQDPIVPFLLNLFLGFGIGSFAQ, (SEQ ID NO: 1 )
6.2 Design of oligonucleotide probe.
The sequence of the 25 amino acid fragment was used to design two oligonucleo-
tides, one designated Y5.2 (SEQ ID N0: 2), and one designated Y6.2 (SEC1 ID
N0: 31.
Codons for the amino acid sequence obtained, SEQ ID NO: 1, were selected by
reverse translation based on (1 ) conclusion that codons containing A or T
were
favoured and (2) knowledge of published DNA sequences for several Bb proteins.
A
choice favouring A or T containing codons was based on the observation that
the G
+ C content of Bb is only 28-35% (Burman et al., 1990). These oligonucleotides
were used in a PCR reaction with DNA prepared from B. burgdarferi B31 as
template
*rB
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and a 74 by fragment was obtained. The PCR fragment was cloned into the T-
vector
(Novagen, Madison, WII and sequenced, SEa ID NO: 4. It was verified that the
obtained PCR fragment coded for the N-terminal amino acid sequence of the
peptide
fragment obtained after protease cleavage of the 13 kDa protein, SEa ID NO: 1.
5 Based on the sequence of the PCR fragment an oligonucieotide designated Y7
(SEQ
ID NO: 5) was designed. This oligonucleotide was to be used as a probe.
EXAMPLE 7
10 Preparation of Bb DNA libraries
7.1 Extraction of DNA.
Spirochetes harvested from 400 ml of culture of B, burgdorferi B31, B.
burgdorferi
8313, B. afzelii ACAI, and B. garinii IP90 were washed twice with 50 mM Tris-
HCl
15 (pH = 7.4) and resuspended in 10 ml of buffer containing 50 mM Tris-HCI (pH
= 7.41,
25% sucrose, and 50 mM EDTA. The cells were iysed by adding SDS to a final
concentration of 296, lysozyme (Sigma, St. Louis, MO) (1.5 mglml), proteinase
K
(Sigma, St. Louis, MO) (0.1 mg/ml), and RNAase A (Sigma, St Louis, MO) (10
p,glml).
The DNA was extracted with buffered phenol and ethanol precipitated.
7.2 Construction of DNA libraries.
Restriction enzymes were obtained from Boehringer, Mannheim, Germany. 100 ng
of
borrelial DNA prepared as described above was completely or partially digested
using
EcoRl and Xbal restriction endonucleases separately. For the partial
digestions, 1 U of
restriction endonuclease was incubated with 100 ng of DNA for 10 min. at
37°C.
Twenty nanograms of appropriately digested pUC19 (Pharmacia, Uppsaia, Sweden)
vector was used for legations.
EXAMPLE 8
Cloning and sequencing of the gene encoding the 13 kDa protein
8.1 Screening of a DNA library prepared from B. burgdorferi.
The recombinant plasmids were transformed into competent E. colt DHSa cells.
Initially, a B. burgdorferi B31 and 8313 EcoRl digested DNA library was
screened
with the DNA probe Y7 (SEQ ID N0: 51. This screening did not result in any
positive
clones.
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An Rsal restriction site was identified in the sequence of the PCR fragment,
SEQ ID
NO: 4. This site was used in a further attempt to clone the gene encoding the
13 kDa
protein as described below. DNA prepared from B. burgdorferi B31 was cut with
Rsal
and the fragment ligated into Hincil digested pUC18 (Pharmacia, Uppsala,
Sweden)
plasmid. The oligonucleotide designated Y7 corresponding to the sequence down-
stream of the Rsal site, SEQ ID NO: 5, was used together with pUC primers
forward
and reversed (Pharmacia, Uppsala, Sweden) in a PCR reaction to obtain a DNA
frag-
ment corresponding to the downstream part of the gene coding for the 13 kDa
pro-
tein. Another oligonucleotide designated Y7R corresponding to the sequence up-
stream of the Rsal site, SEa ID NO: 6, was constructed and used together with
the
pUC primers forward and reversed in a PCR reaction with EcoRl digested DNA as
template to obtain a DNA fragment corresponding to the upstream part of the
gene
coding for the 13 kDa protein. However, the obtained total sequence coded for
a
protein with a calculated molecular weight of 7 kDa. This did not correspond
to the
expected molecular weight of the full-length DNA fragment coding for the 13
kDa
protein.
Therefore, a new oligonucleotide designated Y9, SEQ ID N0: 7, was designed and
used together with the oligonucleotide Y7R, SECZ ID NO: 6, to generate a new
PCR
fragment. This PCR fragment was used as a probe to screen a library of Xbal
digested DNA prepared from B. burgdorferi B31 in an attempt to isolate a full-
length
DNA fragment encoding the 13 kDa protein.
A recombinant plasmid designated pLY-100 recovered from one positive E. coli
DHSa
clone was isolated. The DNA insert of this piasmid was sequenced and found to
com-
prise a gene fragment containing 537 by including an ATG start codon followed
by
an open reading frame (ORF), SEQ ID N0: 18.
8.2 Sequencing of a gene encoding the 13 kDa protein from B. afzeln" ACAI and
B. ga~nn" IP90.
The full-length 13 kDa protein gene was retrieved by PCR amplification
followed by
ligation into a pT7Blue vector (Novagen, Madison, WI). Multiple amplifications
were
used to ensure that the DNA Taq polymerase did not introduce any errors in the
sequence.
The following primer pairs and number of clones was used to obtain the full-
length
and double stranded sequence of the gene encoding the 13 kDa protein in B.
afzelii
ACAI and B. garinii IP90.
# of B. afzelii ACAI Primer pairs° Primer pairs'
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clones
1 Y9-Y18 SEa ID N0:7 - SEQ ID NO:
13
1 Y9-CMV3 SEQ ID N0:7 - SEO, ID NO:
17
1 Y9-Y10 SEa ID N0:7 - SEQ ID N0:8
3 Y12-Y10 SEQ ID NO: 10 - SEa ID N0:8
2 Y13-CMV3 SEQ ID NO: 11 - SEa ID NO:
17
# of B. garinii Primer pairs Primer pairs
IP90
CIOneSa
2 Y9-CMV2 SEQ ID N0:7 - SEQ ID NO:
16
1 Y12-Y10 SEQ ID NO: 10 - SEa ID N0:8
1 Y11-Y10 SEQ ID N0:9 - SEn ID N0:8
1 Y13-Y10 SEa ID NO: 11 - SEQ ID N0:8
3 Y31-CMV2 SEa ID NO: 14 - SEa ID NO:
16
a Clones obtained by different PCR amplifications
Primer pairs used in different PCR amplifications
Primer pairs used identified by SEQ ID numbers
8.3 Sequence analysis.
Sequence analyses were performed using the University of Wisconsin GCG
Sequence
Analysis Software Version 7.2 for UNIX computer. Search in protein sequence
data-
bases was performed at the NCBI using the BLAST network service.
The nucleotide sequences of the gene encoding the 13 kDa protein of B.
burgdorferi
B31, B. afzeiii ACAI, B. garinii IP90, are shown in SEQ ID NO: 18, SEn iD NO:
20,
and SEQ ID NO: 22, respectively. The ATG start codon was followed by an ORF of
534 and 531 nucleotides for strains ACAI and IP90, respectively.
The nucleotide sequence of the gene encoding the 13 kDa protein of B.
burgdorferi
8313 was identical to the nucleotide sequence of the gene encoding the 13 kDa
protein of B. burgdorferi B31.
The deduced amino acid sequences of the 13 kDa protein of B. burgdorferi B31,
B. afzelii ACAI and B. garinii IP90 are presented in SEQ ID NO: 19, SEa ID NO:
21
and SEQ ID NO: 23. The deduced amino acid sequences of the full-length protein
consist of 179, 178 and 177 amino acids for the respective strain. The
computer
analysis predicted a potential leader peptidase II cleavage site between amino
acid
residues at position 12 and 13 (LXXF-C), and the N-terminal peak was found on
the
hydrophobicity plot (data not shown). Based on this and the fact that the
protein was
found to be N-terminally blocked, it could be concluded that the 13 kDa
protein is a
lipoprotein. However, no labetling of the 13 kDa protein with tritiated
palmitate could
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be detected in Bb. This might be explained by the relatively early location of
the
cysteine, i.e., position 13. Usually the lipoprotein acylation sites are
located within
the first 16-30 amino acids. Further computer analysis predicted a potential
signal
peptidase I cleavage site between amino acids 21 and 22 in both B. burgdorferi
B31
and B, afzelii ACAI, while two potential cleavage sites, between amino acids
19 and
20, and between amino acids 21 and 22, were identified in B. garinii IP90. Sur-
prisingly, the full-length P13 protein from the strains B31, ACAI and IP90
consisted
of, respectively, 179, 178 and 177 amino acids with a calculated molecular
weight
of 19107 Da, 19197 Da and 19311 Da. The cleavage of the respective leader se-
quences recognized by signal peptidase I would yield a mature P13 protein
composed
of: 158 amino acids with a calculated molecular mass of 16,772 Da for B.
burgdor-
feri B31; 157 amino acids with a molecular mass of 16,864 for B. afielii ACAI;
and
156 or 158 amino acids with molecular masses of 16,872 Da or 17,115 Da, respec-
tively for B. garinii IP90, in contrast to the apparent molecular weight of 13
kDa ob-
served on PAGE. It therefore seems likely that P13 is both post-
translationally pro-
cessed and modified. Further analysis, such as determination of C-terminal
amino
acid and total amino acid composition of the purified P13 protein, will be
necessary
to finally establish the nature of this modification and processing.
The amino acid sequence of the P13 protein from B. burgdorferi B31 was 87.996
and
87.5 % identical to the sequences from B. afzelii ACAI and B. garinii IP90,
respective-
ly. When compared with each other, the two latter strains showed
90.5°r6 identity.
The level of similarity and identity between the deduced amino acid sequence
of the
P13 protein from different Borrelia strains further shows that this protein
can be
useful as a vaccine against Lyme disease as well as a target for diagnostic
use.
The P13 proteins were examined for the sequence similarity to other known
proteins
in database libraries. There were no other sequences related significantly to
the P13
proteins. The best hit in an EMBL database search was a 95 residues long amino
acid
sequence encoded by the p11 gene on the Bb 49 kb linear plasmid. The encoded
amino acid sequence was 41.5% identical in sequence in a 82 amino acid overlap
(corresponding to amino acid residues -12 to 70 in SEQ ID N0: 191. For the
purposes
of reference, this best hits are included herein as SEQ ID NOs: 30 and 31. The
P13
gene sequence was compared to the recently completed B. burgdorferi B31 genome
(Fraser et al., 1997) and was found to be identical with an ORF called BB034.
Four
additional Bb proteins with a high degree of sequence identity to P13 were
also
found. Together with these putative peptides denoted BBA01, BBI31, BBH41 and
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BBGZ07, P13 belongs to a protein family designated paralogous family No. 5,
formerly
No. 48 (Fraser et al., 1997).
8.4 Antigenicity plot.
Potential antigenic regions of the deduced amino acid sequences of the P13
proteins
from B. burgdorferi B31, B. afzelii ACAI and B. garinii IP90 were identified
by calcula-
tion of the antigenic index using the algorithm of Jameson and Wolf (19881.
The
results are shown in Figure 4. Proposed epitopic regions having a high
antigenic index
are e.g. the amino acid sequences corresponding to amino acid residues 19-27,
33-
36, 41-47, 95-104, 138-147 and 174-179 in SEa ID NO: 19; amino acid residues
19-26, 32-35, 40-47, 94-101, 137-146, and 174-178 in SEO, ID N0: 21; and amino
acid residues 18-26, 30-33, 39-46, 91-104, 137-145 and 173-177 in SEQ ID NO:
23.
EXAMPLE 9
Localisation of the P13 protein gene
9.1 Separation of DNA by pulse-field agarose gel electrophoresis.
For the pulse-field AGE, the DNA prepared from B. burgdorferi B31, B,
burgdorferi
8313, B. afielii ACAI and B. garinii IP90 was recovered in 1 ~ agarose blocks
as
previously described (Ferdows and Barbour, 19891. One-dimensional AGE and
pulse-
field AGE were performed in 1 °rb agarose in TBE buffer. For the pulse-
field AGE pulse
times were 0.5 s for 30 min, 8 s for 30 min, 1 s for 3 h, 2 s for 3 h, 4 s for
6 h, 8 s
for 8 h at a constant current of 200 V, see Figure 5A.
9.2 Southern blotting.
Following depurination, denaturation and neutralisation of the gels, the DNA
was
transferred to a Hybond-N membrane (Amersham, Buckinghamshire, UK) by the
method of Southern (Sambrook et al., 1989), and cross-linked with UV light.
Filters
were pre-hybridised and hybridised in 0.25 M Na2HP04 (pH =7.2), 1 °rb
BSA, 1 mM
EDTA, 7% SDS for 1 h and 4 h, respectively, and washed for 15 min in 5xSSC,
0.1 °~6 SDS; followed by 15 min in 2xSSC, 0.1 % SDS; and 15 in 1 xSSC,
0.1 % SDS,
at the hybridisation temperature. The temperature was 60°C for probing
with a PCR
fragment obtained by amplification using the primers Y7R (SEQ ID N0: 6) and Y9
(SEQ ID NO: 71 (see abovei ta-3zP]dATP (Amersham, Buckinghamshire, UK), radio-
labeled by random primer technique.
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The hybridising band corresponded to the position of the 1 Mbp linear
chromosome
of Lyme disease borreliae, see Figure 5B.
EXAMPLE 10
5
Existence of a P13 homologue in related Borrelia species
10.1 DNA.
Total DNA from B. burgdorferi, B. hermsii, B. crocidurae, and B. anserina was
10 digested with EcoRl and separated by AGE.
10.2 Southern blotting.
Following depurination, denaturation and neutralisation of the gets, the DNA
was
transferred to a Hybond-N membrane (Amersham, Buckinghamshire, UK) by the
15 method of Southern (Sambrook et al., 1989), and cross-linked with UV light.
Filters
were pre-hybridised and hybridised in 0.25 M Na2HP04 (pH = 7.2), 1 °r6
BSA, 1 mM
EDTA, 7°~ SDS for 1 h and 4 h, respectively, and washed for 15 min in
5xSSC,
0.1 °r6 SDS; followed by 15 min in 2xSSC, 0.1 % SDS; and 15 in 1 xSSC,
0.1 °~6 SDS,
at the hybridisation temperature. The temperature was 55°C for probing
with a PCR
20 fragment obtained by amplification using the primers Y9 (SEQ ID NO: 7) and
Y7R
(SEQ ID NO: 6) (see above) [a-'2P]dATP (Amersham, Buckinghamshire, UKI, radio-
labeled by random primer technique.
There was no hybridization with either the DNA from the relapsing fever
Borrelia
25 species, B. hermsii, B. crocidurae, nor the avian borreliosis agent B.
anserina (Figure
6).
Furthermore, the P13 protein gene being localised to the chromosome of
borreliae
shows a higher degree of conservation among Lyme disease associated borreliae
30 contrary to the plasmid-encoded major outer surface proteins A, B, and C
which
exhibit a significant species and strain dependent genetic and antigenic
polymorphism
(Barbour 1986, Jonsson et al., 1992, Wilske et al., 19931.
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EXAMPLE 11
Expression of the P13 protein from B. burgdorferi B31 in E. coli
11.1 Expression of full-length P13.
Two oligonucleotide primers, Y14 (SEQ ID NO: 12) and Y33 (SEQ ID NO: 15), were
designed to anneal to the 5 ' end containing the iipidation signal and the 3 '
end of
the P13 gene from B. burgdorferi B31. The primers contained BamHl and EcoRl
restriction sites, respectively, and were used to amplify the P13 gene in the
PCR.
PCR amplification was performed using Ampli-Taq DNA polymerise (Perkin Elmer
Cetus, Norwalk, CT). The PCR product was then treated with the mentioned
restric-
tion enzymes, purified by AGE and ligated in fusion with GST (gtutathione S-
trans-
ferase) into the tic promoter based expression vector pGEX-2T (Pharmacia,
Uppsala,
Swedenl. The recombinant plasmid, designated pLY313F, was then used to trans-
form E. toll DHSa cells. E, toll DHSa cells containing the insert were grown
and
induced by adding isopropyl-~i-D-thiogalactopyranoside (IPTG) (Sigma, St.
Louis, MO)
to a final concentration of 1 mM to express the introduced P13 gene. The P13
gene
product was subsequently identified by SDS-PAGE (Figure 7A) and Western blot
with
monoclonal antibody 1566 against the P13 protein (Figure 7B).
11.2 Expression of truncated P13.
A similar procedure was used to obtain a truncated variant, i.e. lacking the
signal
peptide. An oligonucieotide primer, Y13 (SEa ID N0: 11 ), was designed to
anneal to
the 5 ' end without the lipidation signal sequence) of the P13 gene from B.
burgdor-
feri B31. The primer also contains a BamHl restriction site. The primer was
used
together with the primer Y33 (SEa ID N0: 15) in a PCR reaction to amplify the
part
of the P13 gene without the DNA sequence encoding the signal sequence. A recom-
binant plasmid designated pLY313T was prepared, E. toll transformed and grown
as
described above. The truncated P13 gene product was subsequently identified by
SDS-PAGE (Figure 7A) and Western blot with monoclonal antibody 1566 against
the
P13 protein (Figure 7B).
EXAMPLE 12
DNA vaccination
12.1 Preparation of DNA constructs.
To enable expression of B. burgdorferi B31 P13 in mammalian cells, the natural
leader
sequence of the P13 gene was replaced with the human tissue plasminogen
activator
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(hTPA) leader sequence and cloned into the expression vector VR1020 (Luke et
al.,
1997) to yield plasmid pLY-H (Figure SA). A similar plasmid enabling the
expression
of B. burgdorferi B31 P13 in translational fusion with OspA was designed pLY-
HA
(Figure 8B).
More specifically, the DNA encoding TPA 5' UTR and leader peptide, P13 and
OspA
were isolated from previous piasmid constructs or amplified. In pLY-H the TPA
signal
was isolated from VR2210 (Luke et al., 1997) by digestion with PsflIKpnl. The
P13
gene was PCR amplified from pLY100 using the primers L1 (SEQ ID NO: 24) and L2
(SEQ ID NO: 25). The P13 containing fragment was digested with KpnIIXbaI and
introduced together with the PstIIKpnI isolated TPA signal into VR1020
digested with
PstIIXbaI.
In pLY-HA the TPA signal was PCR amplified with the primers L5 (SEQ ID NO: 28)
and L6 (SEQ ID NO: 29). The P13 gene was PCR amplified from pLY100 using the
primers L3 (SEQ ID NO: 26) and L4 (SEQ ID NO: 27) The PCR fragments were
digested with the appropriate restriction enzymes. The OspA gene was isolated
from
VR2210 by digestion with KpnIIXbaI. All three fragments were combined in a
three
fragment ligation into the PsfIIXbaI digested VR1020 to yield pLY-HA.
12.2 Vaccination of mice.
Mice will be injected with the plasmids pLY-h and pLY-HA as well as a negative
control plasmid not containing a coding sequence for a Borrelia antigen. The
plasmid
and control DNA are diluted in standard saline. Three bilateral injections of
DNA will
be given at two week intervals at a dosage of 50 pg/ieg into the rectus
femoris
muscle.
12.3 Analysis of immune response.
Sera will be collected after each injection and analyzed by 1 ) antibody ELISA
and 2)
growth inhibition of spirochaetes.
RECTIFIED SHEET (RULE 91)
ISA/EP
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12.4 Challenge with Bb spirochaetes.
After the last injection, mice will be challenged with B. burgdorferi sensu
stricto N40
spirochaetes (same OspA serogroup as B31 ). Spirochetes will be either
injected
intradermally in the tail or by the tick challenge model (Telford et al.,
19931. Mice will
be sacrificed following the challenge. Bladder, heart, plasma, and cross-
cuttings of
the tibiotarsal joints will be cultured in growth medium. Cultures will be
examined for
the presence of spirochaetes by phase-contrast microscopy and scored as
negative if
no spirochaetes are seen in 50 high-power fields.
EXAMPLE 13
Attempted cloning of the P13 gene encoding the 13 kDa protein using monoclonal
antibodies.
The clone pMG2 was obtained from Dr. Michael Norgard's laboratory. This clone
had
been isolated from a library prepared from partial Sau3Al digested DNA of
Borreiia
burgdorferi 297 cloned into the BamHl site of the piasmid pGEX-1 (Pharmacial.
The
library had been screened with the monoclonal antibody 1566. After IPTG
induction
of E. colt DHSa cells transfected with the plasmid pMG2, a glutathione-S-
transferase
(GST) fusion protein reacting with the monoclonal antibody 1566 could be seen
in an
immunoblot experiment. The fusion protein had a molecular weight of about 36
kDa,
26 kDa for the fusion partner GST plus 10 kDa for the Bb protein fragment.
According to restriction mapping, this clone contained an approximately 300
base
pair insert. DNA sequencing showed an open reading frame of 251 bases. The
insert
was PCR amplified, oligolabelled, and used as a probe to screen libraries
prepared
from partial Hino'I il digested DNA from B. burgdorferi B31, B. afzelii ACAI,
and
B. garinii IP90, respectively.
Two positive clones were obtained from each Borrelia species and subsequently
sequenced. A complete sequence determination was obtained from B31 and IP90
and
a partial sequence was obtained from ACA1 (first half of the genet. The
nucleotide
sequences were found to encode a 27 kDa protein in size which did not coincide
with
the expected size of the 13 kDa protein from Bb.
Furthermore, in another expression experiment the expression in E. colt of
IPTG
induced pMG2 and the above mentioned Bb derived clones was studied using the
monoclonal antibody 7D4. After induction E. colt transfected with all clones
as well
as the negative control produced two proteins reacting with the antibody,
about 36
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kDa and 26 kDa in size. These results indicate that the monoclonal antibody
7D4 did
not specifically recognise the 13 kDa Bb protein but cross-reacted with other
proteins
produced by the E,coli cells under these conditions.
Later the identified 27 kDa protein has been verified to be encoded by
supercoiled
plasmids of Bb (Porcella et al., 19961.
EXAMPLE 14
Mass spectrometry analysis.
Molecular weight determinations of P13 were performed on a VG Platform II mass
spectrometer with a range of 2000 m/z equipped with an electrospray source
(Micromass, Altrincham, UKI. Prior to injection, the purified P13 preparation
was
precipitated with methanol:chloroform to remove any trace of SDS
contamination. A
P13 solution of 20 pmol/ml in water-acetonitrile (50:50 [vollvol]) was mixed
with 5%
formic acid and introduced directly into the electrospray source at a flow
rate of 5
ml/min. Calibration was performed by a separate introduction using horse heart
myoglobin (16,951.5 Dal. The MassLynx software was used to calculate the mole-
cular weight.
The mass spectrometry analysis (Figure 9) indicated a molecular weight of 13.9
kDa
compared to the deduced molecular weight of the mature (cleavedl P13 protein
of
16.8 kDa but in good agreement with the observed molecular weight of the
mature
P13 protein of 13 kDa as determined by gel-electrophoresis.
In addition, the whole molecule mass spectrum detected two minor peptide
popula-
tions increased with 267 Da increments from the major peak. These peptides pro-
bably represent further modified subsets of P13. Two possible post-
translational
modifications can be considered to generate the mass change of 267 Da. Stea-
roylation gives an average mass change of 266.5 Da while the addition of two
pentoses generates a mass change of 264.2 Da.
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1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Symbicom AB
(B) STREET: Tvistev~gen 48
(C) CITY: Umea
(E) COUNTRY: Sweden
(F) POSTAL CODE (ZIP): S-907 36
(ii) TITLE OF INVENTION: P13 antigens from Borrelia
(iii) NUMBER OF SEQUENCES: 31
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
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(C) STRANDEDNESS: single
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(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Borrelia burgdorferi
(B) STRAIN: B313
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Thr Ser Lys Gln Asp Pro Ile Val Pro Phe Leu Leu Asn Leu Phe Leu
1 5 10 15
Gly Phe Gly Ile Gly Ser Phe Ala Gln
20 25
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
ACNTCNAARC ARGAYCCNAT 20
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
TGNGCRAARC TNCCDATNCC 20
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
ACATCTAAGC AGGACCCTAT TGTACCATCT TTATTGAACC TTTTTTTAGG GTTTGGCATC 60
GGGAGCTTCG CCCA 74
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i
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(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
TGTACCATCT TTATTGAACC TTTTTTTAGG GTTT gq
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GATTTTTCAT TGGATCCCAG AATTTG 26
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i
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CTATACCAAC CGAATTCAAA TCCAAG . 26
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 9:
GGTTTTTATG GATCCACTTT T 21
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 15:
TAGAATTCAG CAATTGCAAT ACAG 24
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CACCCATTTT CTAGATAAAT AAAATTAATA GC 32
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(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 17:
ATAAAAGGTA CCATAGCTTT TTTTGAAAGA CAG 33
(2} INFORMATION FOR SEQ ID N0: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 759 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
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(A) ORGANISM: Borrelia burgdorferi
(B) STRAIN: B31
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:170..709
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
ATTGTTAAAA GAATTGAAAT TGATAATTTT ATGGTCAAAT CAAGAAGCTC TATTGGGAAG 60
CGAATTTCAA GCAATAATTT GAAAAAAGTT AAATTTAAAT AACTTTAAAA ACCTTTTTTA 120
AATTTCATTA ATATGCTACC ATAGTACCAG TTTTAATAAA GGGGTTTTT ATG AAT 175
Met Asn
1
AAA CTT TTA ATT TTT GTT TTG GCA ACC TTT TGT GTT TTT TCT AGC TTT 223
Lys Leu Leu Ile Phe Val Leu Ala Thr Phe Cys Val Phe Ser Ser Phe
10 15
GCT CAA GCT AAT GAT TCT AAA AAT GGT GCG TTT GGG ATG AGT GCT GGA 271
Ala Gln Ala Asn Asp Ser Lys Asn Gly Ala Phe Gly Met Ser Ala Gly
20 25 30
GAA AAA CTT TTG GTT TAT GAA ACT AGC AAG CAA GAT CCT ATT GTA CCA 319
Glu Lys Leu Leu Val Tyr Glu Thr Ser Lys Gln Asp Pro Ile Val Pro
35 40 45 50
TTT TTA TTG AAC CTT TTT TTA GGG TTT GGA ATA GGC TCC TTT GCT CAA 367
Phe Leu Leu Asn Leu Phe Leu Gly Phe Gly Ile Gly Ser Phe Ala Gln
55 60 65
GGA GAT ATT CTT GGA GGT TCT CTT ATT CTT GGA TTT GAT GCG GTT GGT 415
Gly Asp Ile Leu Gly Gly Ser Leu Ile Leu Gly Phe Asp Ala Val Gly
70 75 80
ATA GGG CTT ATA CTT GCG GGG GCT TAT TTG GAT ATC AAA GCG CTT GAT 463
Ile Gly Leu Ile Leu Ala Gly Ala Tyr Leu Asp Ile Lys Ala Leu Asp
85 90 95
GGT ATT ACT AAA AAA GCT GCT TTT CAA TGG ACT TGG GGT AAG GGA GTT 511
Gly Ile Thr Lys Lys Ala Ala Phe Gln Trp Thr Trp Gly Lys Gly Val
i
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100 105 110
ATG TTA GCA GGT GTG GTT ACT ATG GCT GTG ACA AGA TTA ACA GAA ATT 559
Met Leu Ala Gly Val Val Thr Met Ala Val Thr Arg Leu Thr Glu Ile
115 120 125 130
ATT CTT CCA TTT ACA TTT GCT AAT AGT TAT AAT AGG AAG CTA AAA AAT 607
Ile Leu Pro Phe Thr Phe Ala Asn Ser Tyr Asn Arg Lys Leu Lys Asn
135 140 145
AGC CTT AAT GTA GCT TTA GGA GGA TTT GAA CCT AGT TTT GAT GTT GCA 655
Ser Leu Asn Val Ala Leu Gly Gly Phe Glu Pro Ser Phe Asp Val Ala
150 155 160
ATG GGC CAA TCC AGT GCT CTT GGG TTT GAA CTG TCT TTC AAA AAA AGC 703
Met Gly Gln Ser Ser Ala Leu Gly Phe Glu Leu Ser Phe Lys Lys Ser
165 170 175
TAT TAA TTTTATTTAT TACAAAAATG GGTGATTGCA ATTCTGTATT GAAATGGGTG 759
Tyr
180
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 179 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 19:
Met Asn Lys Leu Leu Ile Phe Val Leu Ala Thr Phe Cys Val Phe Ser
1 5 10 15
Ser Phe Ala Gln Ala Asn Asp Ser Lys Asn Gly Ala Phe Gly Met Ser
20 25 30
Ala Gly Glu Lys Leu Leu Val Tyr Glu Thr Ser Lys Gln Asp Pro Ile
35 40 45
Val Pro Phe Leu Leu Asn Leu Phe Leu Gly Phe Gly Ile Gly Ser Phe
i
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50 55 60
Ala Gln Gly Asp Ile Leu Gly Gly Ser Leu Ile Leu Gly Phe Asp Ala
65 70 75 80
Val Gly Ile Gly Leu Ile Leu Ala Gly Ala Tyr Leu Asp Ile Lys Ala
85 90 g5
Leu Asp Gly Ile Thr Lys Lys Ala Ala Phe Gln Trp Thr Trp Gly Lys
100 105 110
Gly Val Met Leu Ala Gly Val Val Thr Met Ala Val Thr Arg Leu Thr
115 120 125
Glu Ile Ile Leu Pro Phe Thr Phe Ala Asn Ser Tyr Asn Arg Lys Leu
130 135 140
Lys Asn Ser Leu Asn Val Ala Leu Gly Gly Phe Glu Pro Ser Phe Asp
145 150 155 160
Val Ala Met Gly Gln Ser Ser Ala Leu Gly Phe Glu Leu Ser Phe Lys
165 170 175
Lys Ser Tyr
179
(2) INFORMATION FOR SEQ ID N0: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 862 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Borrelia afzelii
(B) STRAIN: ACAI
(ix) FEATURE:
*rB
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(A) NAME/KEY: CDS
(B) LOCATION:219..755
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 20:
GATTTTTCAT TGGATCCCAG AATTTGTAGA ATTTTCGACA AATAAAGACA TTATTAAAAG 60
AATTGAAATT GCTAATTTTA TGGTCAAATC AAGAAGCTCT ATTGGGAAGC GAATTTCAAG 120
TAATACTTTG AAAAAAGTTA AATTTAAATA GTTTTAAAAA CCTTTTTTAA ATTTCATTAA 180
TATGTTACTA TAATACCAGT TTTAATAAAG AGGTTTTT ATG AAT AAA TTT TTA 233
Met Asn Lys Phe Leu
1 5
ATT GTT GTT TTG CTA GCC TTT TGT GTT TTT TCT AGC TTT GCT CAA GCT 281
Ile Val Val Leu Leu Ala Phe Cys Val Phe Ser Ser Phe Ala Gln Ala
10 15 20
GAT GAT TCT AAA AGC GCT TTT AAT TTG GGA GCG GGA GAA AAA CTT TTA 329
Asp Asp Ser Lys Ser Ala Phe Asn Leu Gly Ala Gly Glu Lys Leu Leu
25 30 35
GCT TAT GAA ACT AGT AAG AAA GAT CCT ATT GTG CCA TTT TTA TTG AAC 377
Ala Tyr Glu Thr Ser Lys Lys Asp Pro Ile Val Pro Phe Leu Leu Asn
40 45 50
CTT TTT TTA GGG TTT GGA ATA GGT TCT TTT GCT CAA GGA GAT ATT CTT 425
Leu Phe Leu Gly Phe Gly Ile Gly Ser Phe Ala Gln Gly Asp Ile Leu
55 60 65
GGG GGT TTT CTT ATT CTT GGA TTT GAT GCA GTT GGT ATA GGG TTA ATA 473
Gly Gly Phe Leu Ile Leu Gly Phe Asp Ala Val Gly Ile Gly Leu Ile
70 75 80 85
CTT ACA GGA GCT TAT TTA GAT ATC AAA GCT CTT GAT AAG AAT GCT CCA 521
Leu Thr Gly Ala Tyr Leu Asp Ile Lys Ala Leu Asp Lys Asn Ala Pro
90 95 100
AAA GCC GCT TTT AAG TGG ACT TGG GGT AAG GGA ATG ATG TTG GCA GGT 569
Lys Ala Ala Phe Lys Trp Thr Trp Gly Lys Gly Met Met Leu Ala Gly
105 110 115
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GCA GTT ACT ATG GCT GTG ACA AGA TTG ACA GAA ATT ATT ATT CCG TTT 617
Ala Val Thr Met Ala Val Thr Arg Leu Thr Glu Ile Ile Ile Pro Phe
120 125 130
ACA TTT GCT AAT AGT TAT AAT AGG AAA CTG AAA AAT AGC CTT AAT ATA 665
Thr Phe Ala Asn Ser Tyr Asn Arg Lys Leu Lys Asn Ser Leu Asn Ile
135 140 145
GCT TTT GGA GGG TTT GAG CCT AGT TTT GAT ATT AAT ATG GGC CAA GCT 713
Ala Phe Gly Gly Phe Glu Pro Ser Phe Asp Ile Asn Met Gly Gln Ala
150 155 160 165
AGC GCT CTT GGG TTT GAA CTA TCT TTC AAA AAA AGT TAT TAA 755
Ser Ala Leu Gly Phe Glu Leu Ser Phe Lys Lys Ser Tyr
170 175
TTTTATTTTA TTATTAAAAT GAGTGATAGC AATTTTGTAT TGTGATTGCT CATTGTAATT 815
GAAAATTAGA GCTTTTGTTT ATTATTTATA TTTTATTTCT CTGCTAA 862
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Met Asn Lys Phe Leu Ile Val Val Leu Leu Ala Phe Cys Val Phe Ser
1 5 10 15
Ser Phe Ala Gln Ala Asp Asp Ser Lys Ser Ala Phe Asn Leu Gly Ala
20 25 30
Gly Glu Lys Leu Leu Ala Tyr Glu Thr Ser Lys Lys Asp Pro Ile Val
35 40 45
Pro Phe Leu Leu Asn Leu Phe Leu Gly Phe Gly Ile Gly Ser Phe Ala
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50 55 60
Gln Gly Asp Ile Leu Gly Gly Phe Leu Ile Leu Gly Phe Asp Ala Val
65 70 75 80
Gly Ile Gly Leu Ile Leu Thr Gly Ala Tyr Leu Asp Ile Lys Ala Leu
85 90 95
Asp Lys Asn Ala Pro Lys Ala Ala Phe Lys Trp Thr Trp Gly Lys Gly
100 105 110
Met Met Leu Ala Gly Ala Val Thr Met Ala Val Thr Arg Leu Thr Glu
115 120 125
Ile Ile Ile Pro Phe Thr Phe Ala Asn Ser Tyr Asn Arg Lys Leu Lys
130 135 140
Asn Ser Leu Asn Ile Ala Phe Gly Gly Phe Glu Pro Ser Phe Asp Ile
145 150 155 160
Asn Met Gly Gln Ala Ser Ala Leu Gly Phe Glu Leu Ser Phe Lys Lys
165 170 175
Ser Tyr
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 749 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Borrelia garinii
(B) STRAIN: IP90
(ix) FEATURE:
(A) NAME/KEY: CDS
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(B) LOCATION:192..725
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 22:
TAGAATTTTC AACAAATAAA GATATTGTTA AAAGAATTGA AATTGCTAAT TTTATGGTTA 60
AATCAAGAAG CTCTATTGGT AAGCGAATTT CGAGTAACAA TTTGAAAAAA GTTAAATTTA 120
AATAGTTCCA AAAGCCTTTT TTAAATTTCA TTAATATGCT ACCATAATAC CAGTTTAATA 180
AAGGGGTTTT T ATG AAT AAG TTT TTA ATT TTT ATT TTG GTA ATC TTT TGT 230
Met Asn Lys Phe Leu Ile Phe Ile Leu Val Ile Phe Cys
1 5 10
GCT TTT TCT AGT TTT GCT CAA GAT GAT TCT AAA AGC ACT TTT AAT CTG 278
Ala Phe Ser Ser Phe Ala Gln Asp Rsp Ser Lys Ser Thr Phe Rsn Leu
is zo 25
GGA GCG GGA GAA AAA TTT TTG GTT TAT GAA ACT AAT AAG AAA GAT TCT 326
Gly Ala Gly Glu Lys Phe Leu Val Tyr Glu Thr Asn Lys Lys Asp Ser
30 35 90 45
CTT GTA CCA TTT TTA TTG AAC CTT TTT TTA GGG TTC GGG ATA GGT TCT 374
Leu Val Pro Phe Leu Leu Asn Leu Phe Leu Gly Phe Gly Ile Giy Ser
50 55 60
TTT GCT CAA GGA GAT ATC CTT GGA GGT TCT CTT ATT CTT GGA TTT GAT 422
Phe Ala Gln Gly Asp Ile Leu Gly Gly Ser Leu Ile Leu Gly Phe Rsp
65 70 75
GCG GTT GGT ATA GGG TTA ATA CTT ACA GGA GCT TAT TTG GAC ATC AAG 470
Ala Val Gly Ile Gly Leu Ile Leu Thr Gly Ala Tyr Leu Asp Ile Lys
80 85 90
GAT TTT GAT AAT AAT GCT AAA AAA GCT GAT TTT AAG TGG ACT TGG GGT 5I8
Asp Phe Asp Asn Asn Ala Lys Lys Ala Asp Phe Lys Trp Thr Trp Gly
95 100 105
AAG GGA ATG ATG TTG GCA GGT GTG GTT ACT ATG GCT GTG ACA AGA TTG 566
Lys Gly Met Met Leu Ala Gly Val Val Thr Met Ala Val Thr Arg Leu
110 115 120 125
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ACA GAA ATT CTTCCA TTT TTT GCT AAT AAC AAG 614
GTT ACA AAT TAT AGG
Thr Glu Ile LeuPro Phe Phe Ala Asn TyrAsn ArgLys
Val Thr Asn
130 135 140
CTG AAA AAT CTTAAT ATA TTG GGA GGA GAGCCT AGTTTT 662
AGT GCC TTT
Leu Lys Asn LeuAsn Ile Leu Gly Gly GluPro SerPhe
Ser Ala Phe
145 150 155
GAT ATT AAC GGCCAA GCT GCT CTT GGT GGACTG TCTTTC 710
ATG AGT TTT
Rsp Ile Asn GlyGln Ala Ala Leu Gly GlyLeu SerPhe
Met Ser Phe
160 165 170
AAA AAA AGC TAATTTTATTTAT 74g
TAT CTAGAAAATG
GGTG
Lys Lys Ser
Tyr
175
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 177 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 23:
Met Asn Lys Phe Leu Ile Phe Ile Leu Val Ile Phe Cys Ala Phe Ser
1 5 10 15
Ser Phe Ala Gln Asp Asp Ser Lys Ser Thr Phe Asn Leu Gly Ala Gly
20 25 30
Glu Lys Phe Leu Val Tyr Glu Thr Asn Lys Lys Asp Ser Leu Val Pro
35 40 45
Phe Leu Leu Asn Leu Phe Leu Gly Phe Gly Ile Gly Ser Phe Ala Gln
50 55 60
Gly Asp Ile Leu Gly Gly Ser Leu Ile Leu Gly Phe Asp Ala Val Gly
65 70 75 g0
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Ile Gly Leu Ile Leu Thr Gly Ala Tyr Leu Asp Ile Lys Asp Phe Asp
85 90 95
Asn Asn Ala Lys Lys Ata Asp Phe Lys Trp Thr Trp Gly Lys Gly Met
100 105 110
Met Leu Ala Gly Val Val Thr Met Ala Val Thr Arg Leu Thr Glu Ile
115 120 125
Val Leu Pro Phe Thr Phe Ala Asn Asn Tyr Asn Arg Lys Leu Lys Asn
130 135 140
Ser Leu Asn Ile Ala Leu Gly Gly Phe Glu Pro Ser Phe Asp Ile Asn
145 150 155 160
Met Gly Gln Ala Ser Ala Leu Gly Phe Gly Leu Ser Phe Lys Lys Ser
165 170 175
Tyr
(2) INFORMATION FOR SEQ ID N0: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
TTGGCAGGTA CCTGTGTTTT TTCTAGCTTT GC 32
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:. DNA (synthetic)
i
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
CACCCATTTT CTAGATAAAT AAAATTAATA GC 32
(2) INFORMATION FOR SEQ ID N0: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
ATAAAAGGTA CCATAGCTTT TTTTGAAAGA CAG 33
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(Cy STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
TTGGCAGAAT TCTGTGTTTT TTCTAGCTTT GG 32
(2) INFORMATION FOR SEQ ID N0: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 28:
TCTTTTCTGC AGTCACCGTC G 21
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(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 29:
TTGCTTACAG AATTCGCTGG GCGAAACGAA 30
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 396 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 109...396
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
ACGAGCTCAA TCCAAACTTT ATTTGCTTGC AATAAATTAA TATTAATTTA TTATAAATTG 60
CGCTAATATT TTACTTGTCA AAACTTACCA TTAGGAGATA ATAAAAAC ATG AAA AAA 117
Met Lys Lys
1
ATT TTC ACA TTA ATA TTA ATT TTT GGG TTG ACA ATT GAA ATC TTT GCC 165
Ile Phe Thr Leu Ile Leu Ile Phe Gly Leu Thr Ile Glu Ile Phe Ala
10 15
ACA AAA GAC ACA CAA RAT AGA ATT GAA AAA GGC ATT GAA AGT TTT AAC 213
Thr Lys Asp Thr Gln Asn Arg Ile Glu Lys Gly Ile Glu Ser Phe Asn
20 25 30 35
AAA TAT GAT AAA GAG AAA AAA AAT CCA ATA GGG CCA TTC CTT TTA AAT 261
Lys Tyr Asp Lys Glu Lys Lys Asn Pro Ile Gly Pro Phe Leu Leu Asn
40 45 50
TTA TTT TTG CCC TTT GGA ATA GGA TCC TTT GTC CAA GGG GAT TAT ATT 309
Leu Phe Leu Pro Phe Gly Ile Gly Ser PheaVal Gln Gly Asp Tyr Ile
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55 60 65
GGT GGA GGC TCA GTG CTT GGA TTT AAT TTA TTA GGA GCA ATC CTT TGG 357
Gly Gly Gly Ser Val Leu Gly Phe Asn Leu Leu Gly Ala Ile Leu Trp
70 75 80
GAA CTG GAA TTA TTC TTA ATC ACC GAG AAA CAC AAT TAA 396
Glu Leu Glu Leu Phe Leu Ile Thr Glu Lys His Asn
85 90 95
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v} FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Met Lys Lys Ile Phe Thr Leu Ile Leu Ile Phe Gly Leu Thr Ile Glu
1 5 10 15
Ile Phe Ala Thr Lys Asp Thr Gln Asn Arg Ile Glu Lys Gly Ile Glu
20 25 30
Ser Phe Asn Lys Tyr Asp Lys Glu Lys Lys Asn Pro Ile Gly Pro Phe
35 q0 q5
Leu Leu Asn Leu Phe Leu Pro Phe Gly Ile Gly Ser Phe Val Gln Gly
50 55 60
Asp Tyr Ile Gly Gly Gly Ser Val Leu Gly Phe Asn Leu Leu Gly Ala
65 70 75 80
Ile Leu Trp Glu Leu Glu Leu Phe Leu Ile Thr Glu Lys His Asn
85 90 95
