Note: Descriptions are shown in the official language in which they were submitted.
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CA 02294568 1999-12-17
WO 98/59071 PCT/US98/12718
i
Lyme Disease Vaccines
s
Field of the Invention
The present invention relates to novel vaccines for the prevention or
attenuation of Lyme
disease. The invention further relates to isolated nucleic acid molecules
encoding antigenic
to polypeptides of Borrelia burgdorferi. Antigenic polypeptides are also
provided, as are vectors,
host cells and recombinant methods for producing the same. The invention
additionally relates to
diagnostic methods for detecting Borrelia gene expression.
15 Background of the Invention
Lyme disease (Steere, A.C., Proc. Natl. Acad. Sci. USA 91:2378-2383 ( 1991 )),
or Lyme
borreliosis, is presently the most common human disease in the United States
transmitted by an
arthropod vector (Center for Disease Control, Morbid. Mortal. Weekly Rep.
46(23):531-535
2o (1997)). Further, infection of house-hold pets, such as dogs, is a
considerable problem.
While initial symptoms often include a rash at the infection point, Lyme
disease is a
multisystemic disorder that may include arthritic, carditic, and neurological
manifestations. While
antibiotics are currently used to treat active cases of Lyme disease, B.
burgdorferi persists even
after prolonged antibiotic treatment. Further, B. burgdorferi can persist for
years in a mammalian
25 host in the presence of an active immune response (Straubinger, R. et al.,
J. Clin. Microbiol.
35:111-116 (1997); Steere, A., N. Engl. J. Med. 321:586-596 (1989)).
Lyme disease is caused by the related tick-home spirochetes classified as
Borrelia
burgdorferi sensu lato (including B. burgdorferi sensu stricto, B. afzelii, B.
garinii). Although
substantial progress has been made in the biochemical, ultrastructural, and
genetic characterization
30 of the organism, the spirochetal factors responsible for infectivity,
immune evasion and disease
pathogenesis remain largely obscure.
A number of antigenic B. burgdorferi cell surface proteins have been
identified. These
include the outer membrane surface proteins (Osp) OspA, OspB, OspC and OspD.
OspA and
OspB are encoded by tightly linked tandem genes which are transcribed as a
single transcriptional
- 35 unit (Brusca, J. et al., J. Bacteriol. 173:8004-8008 ( 1991 )). The most-
studied B. burgdorferi
membrane protein is OspA, a lipoprotein antigen expressed by borreliae in
resting ticks and the
most abundant protein expressed in vitro by most borrelial isolates (Barbour,
A.G., et al.,
Infection & Immunity 41:795-804 ( 1983); Howe, T.R., et al., Science 227:645 (
1985)).
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2
A number of different types of Lyme disease vaccines have been shown to induce
immunological responses. Whole-cell B. burgdorferi vaccines, for example, have
been shown to
induce both immunological responses and protective immunity in several animal
models
(Reviewed in Wormser, G., Clin. Infect. Dis. 21:1267-1274 (1995)). Further,
passive immunity
has been demonstrated in both humans and other animals using B. burgdorferi
specific antisera.
While whole-cell Lyme disease vaccines confer protective immunity in animal
models, use
of such vaccines presents the risk that responsive antibodies will produce an
autoimmune
response (Reviewed in Wormser, G., supra). This problem is at least partly the
result of the
production of B. burgdorferi specific antibodies which cross-react with
hepatocytes and both
to muscle and nerve cells. B. burgdorferi heat shock proteins and the 41-kd
flagellin subunit are
believed to contain antigens which elicit production of these cross-reactive
antibodies.
Single protein subunit vaccines for Lyme disease have also been tested. The
cell surface
proteins of B. burgdorferi are potential candidates for use in such vaccines
and several have been
shown to elicit protective immune responses in mammals (Probert, W. et al.,
Vaccine 15:15-19
(1997); Fikrig, E. et al., Infect. Immun. 63:1658-1662 (1995); Langerman S. et
al., Nature
372:552-556 ( 1994); Fikrig, E. et al., J. Immunol. 148:2256-2260 ( 1992)).
Experimental OspA
vaccines, for example, have demonstrated efficacy in several animal models
(Fikrig, E., et al.,
Proc. Natl. Acad. Sci. USA 89:5418-5421 (1992); Johnson, B.J., et al., Vaccine
13:1086-1094
( 1996); Fikrig, E., et al., Infect. Immun. 60:657-661 ( 1992); Chang, Y.F.,
et al., Infection &
Immunity 63:3543-3549 { 1995)), and OspA vaccines for human use are under
clinical evaluation
(Keller, D., et al., J. Am. Med. Assoc. 271:1764-1768 (1994); Van Hoecke, C.,
et al., Vaccine
14:1620-1626 (1996)). Passive immunity is also conferred by antisera
containing antibodies
specific for the full-length OspA protein. Further, vaccination with plasmid
DNA encoding OspA
has been demonstrated to elicit protective immune responses in mice (Luke, C.
et al., J. Infect.
Dis. 175:91-97 (1997); Zhong, W. et al., Eur. J. Immunol. 26:2749-2757
{1996)).
Recent immunofluorescence assay observations indicate that during tick
engorgement the
expression of OspA by borreliae diminishes (deSilva, A.M., et al., J. Exp.
Med. 183:271-275
(1996)) while expression of other proteins, exemplified by OspC, increases
(Schwan, T.G., et
al., Proc. Natl. Acad. Sci. USA 92:2909-2913 { 1985)). By the time of
transmission to hosts,
spirochetes in the tick salivary glands express little or no OspA. This down-
modulation of OspA
appears to explain the difficulties in demonstrating immune responses to this
antigen early in
infection following tick bites (Kalish, R.A., et al., Infect. Immun. 63:2228-
2235 (1995); Gern,
L., et al., J. Infect. Dis. 167:971-975 (1993); Schiable, U.E., et al.,
Immunol. Lett. 36:219-226
(1993)) or following challenge with limiting doses of cultured borreliae
(Schiable, U.E., et al.,
Immunol. Lett. 36:219-226 (1993); Barthold, S.W. and Bockenstedt, L.K.,
Infect. Immun.
61:4696-4702 (1993)).
Furthermore, OspA-specific antibodies are ineffective if administered after a
borrelial
challenge delivered by syringe (Schiable, U.E., et al., Proc. Natl. Acad. Sci.
USA 87:3768-3772
(1990)) or tick bite (deSilva, A.M.> et al., J. Exp. Med. 183:271-275 (
1996)). To be efficacious,
CA 02294568 1999-12-17
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3
OspA vaccines must elicit protective levels of antibody which are maintained
throughout periods
of tick exposure in order to block borrelia transmission from the arthropod
vector.
Vaccines in current use against other pathogens include in vivo-expressed
antigens which
could boost anamnestic responses upon infection, potentiate the action of
immune effector'cells
and complement, and inhibit key virulence mechanisms. OspC is both expressed
during infection
(Montgomery, R.R., et al., J. Exp. Med. 183:261-269 ( 1996)) and a target for
protective
immunity (Gilmore, R.D., et al., Infect. Immun. 64:2234-2239 (1996); Probert,
W.S. and
LeFebvre, R.B., Infect. Immun. 62:1920-1926 (1994); Preac-Mursic, V., et al.,
Infection
20:342-349 ( 1992)), but mice immunized with this protein were only protected
against challenge
to with the homologous borrelial isolate (Probert, W.S., et al., J. Infect.
Dis. 175:400-405 (1997)).
Identification of in vivo-expressed, and broadly protective, antigens of B.
burgdorferi has
remained elusive.
Summary of the Invention
The present invention provides isolated nucleic acid molecules comprising
polynucleotides
encoding the B. burgdorferi peptides having the amino acid sequences shown in
Table 1. Thus,
one aspect of the invention provides isolated nucleic acid molecules
comprising polynucleotides
having a nucleotide sequence selected from the group consisting o~ (a) a
nucleotide sequence
encoding any of the amino acid sequences of the full-length polypeptides shown
in Table 1; (b) a
nucleotide sequence encoding any of the amino acid sequences of the full-
length polypeptides
shown in Table 1 but minus the N-terminal methionine residue, if present; (c)
a nucleotide
sequence encoding any of the amino acid sequences of the truncated
polypeptides shown in Table
l; and (d) a nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), or
(c) above.
Further embodiments of the invention include isolated nucleic acid molecules
that
comprise a polynucleotide having a nucleotide sequence at least 90% identical,
and more
preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the
nucleotide sequences in
(a), (b), (c), or (d) above, or a polynucleotide which hybridizes under
stringent hybridization
conditions to a polynucleotide in {a), (b), (c), or (d) above. This
polynucleotide which hybridizes
3o does not hybridize under stringent hybridization conditions to a
polynucleotide having a
nucleotide sequence consisting of only A residues or of only T residues.
Additional nucleic acid
embodiments of the invention relate to isolated nucleic acid molecules
comprising polynucleotides
which encode the amino acid sequences of epitope-bearing portions of a B.
burgdorfera
polypeptide having an amino acid sequence in (a), (b), or (c) above.
The present invention also relates to recombinant vectors, which include the
isolated
nucleic acid molecules of the present invention, and to host cells containing
the recombinant
vectors, as well as to methods of making such vectors and host cells and for
using these vectors
for the production of B. burgdorferi polypeptides or peptides by recombinant
techniques.
The invention further provides isolated B. burgdorferi polypeptides having an
amino acid
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sequence selected from the group consisting of: (a) an amino acid sequence of
any of the full-
length polypeptides shown in Table 1; (b) an amino acid sequence of any of the
full-length
polypeptides shown in Table 1 but minus the N-terminal methionine residue, if
present; (c) an
amino acid sequence of any of the truncated polypeptides shown in Table 1; and
(d) an amino acid
sequence of an epitope-bearing portion of any one of the polypeptides of (a),
(b), or (c).
The polypeptides of the present invention also include polypeptides having an
amino acid
sequence with at least 70% similarity, and more preferably at least 75%, 80%,
85%, 90%, 95%,
96%, 97%, 98%, or 99% similarity to those described in (a), (b), (c), or (d)
above, as well as
polypeptides having an amino acid sequence at least 70% identical, more
preferably at least 75%
to identical, and still more preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% identical to
those above; as well as isolated nucleic acid molecules encoding such
polypeptides.
The present invention further provides a vaccine, preferably a mufti-component
vaccine
comprising one or more of the B. burgdorferi polypeptides shown in Table l, or
fragments
thereof, together with a pharmaceutically acceptable diluent, carrier, or
excipient, wherein the
B. burgdorferi polypeptide(s) are present in an amount effective to elicit an
immune response to
members of the Borrelia genus in an animal. The B. burgdorferi polypeptides of
the present
invention may further be combined with one or more immunogens of one or more
other borrelial
or non-borrelial organisms to produce a mufti-component vaccine intended to
elicit an
immunological response against members of the Borrelia genus and, optionally,
one or more non-
borrelial organisms.
The vaccines of the present invention can be administered in a DNA form, e.g.,
"naked"
DNA, wherein the DNA encodes one or more borrelial polypeptides and,
optionally, one or more
polypeptides of a non-borrelial organism. The DNA encoding one or more
polypeptides may be
constructed such that these polypeptides are expressed fusion proteins.
The vaccines of the present invention may also be administered as a component
of a
genetically engineered organism. Thus, a genetically engineered organism which
expresses one
or more B. burgdorferi polypeptides may be administered to an animal. For
example, such a
genetically engineered organism may contain one or more B. burgdorferi
polypeptides of the
present invention intracellularly, on its cell surface, or in its periplasmic
space. Further, such a
3o genetically engineered organism may secrete one or more B. burgdorferi
polypeptides.
The vaccines of the present invention may be co-administered to an animal with
an
immune system modulator (e.g., CD86 and GM-CSF).
The invention also provides a method of inducing an immunological response in
an animal
to one or more members of the Borrelia genus, e.g., B. burgdorferi sensu
stricto, B. afzelii, and
B. garinii, comprising administering to the animal a vaccine as described
above.
The invention further provides a method of inducing a protective immune
response in an
animal, sufficient to prevent or attenuate an infection by members of the
Borrelia genus,
comprising administering to the animal a composition comprising one or more of
the polypeptides
shown in Table l, or fragments thereof. Further, these polypeptides, or
fragments thereof, may
CA 02294568 1999-12-17
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be conjugated to another immunogen and/or administered in admixture with an
adjuvant.
The invention further relates to antibodies elicited in an animal by the
administration of one
or more B. burgdorferi polypeptides of the present invention.
The invention also provides diagnostic methods for detecting the expression of
genes of
5 members of the Borrelia genus in an animal. One such method involves
assaying for the
expression of a gene encoding Borrelia peptides in a sample from an animal.
This expression
may be assayed either directly (e.g., by assaying polypeptide levels using
antibodies elicited in
response to amino acid sequences shown in Table 1) or indirectly (e.g., by
assaying for
antibodies having specificity for amino acid sequences shown in Table 1 ). An
example of such a
method involves the use of the polymerase chain reaction (PCR) to amplify and
detect Borrelia
nucleic acid sequences.
The present invention also relates to nucleic acid probes having all or part
of a nucleotide
sequence shown in Table 1 which are capable of hybridizing under stringent
conditions to
Borrelia nucleic acids. The invention further relates to a method of detecting
one or more Borrelia
nucleic acids in a biological sample obtained from an animal, said one or more
nucleic acids
encoding Borrelia polypeptides, comprising:
a) contacting the sample with one or more of the above-described nucleic acid
probes,
under conditions such that hybridization occurs, and
b) detecting hybridization of said one or more probes to the Borrelia nucleic
acid present
2o in the biological sample.
Detailed Description
The present invention relates to recombinant antigenic B. burgdorferi
polypeptides and
fragments thereof. The invention also relates to methods for using these
polypeptides to produce
immunological responses and to confer immunological protection to disease
caused by members
of the genus Borrelia. The invention further relates to nucleic acid sequences
which encode
antigenic B. burgdorferi polypeptides and to methods for detecting Borrelia
nucleic acids and
polypeptides in biological samples. The invention also relates to Borrelia
specific antibodies and
methods for detecting such antibodies produced in a host animal.
Definitions
The following definitions are provided to clarify the subject matter which the
inventors
consider to be the present invention.
As used herein, the phrase "pathogenic agent" means an agent which causes a
disease state
or affliction in an animal. Included within this definition, for examples, are
bacteria, protozoans,
fungi, viruses and metazoan parasites which either produce a disease state or
render an animal
infected with such an organism susceptible to a disease state (e.g., a
secondary infection).
Further included are species and strains of the genus Borrelia which produce
disease states in
animals.
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As used herein, the term "organism" means any living biological system,
including
viruses, regardless of whether it is a pathogenic agent.
As used herein, the term "Borrelia" means any species or strain of bacteria
which is
members of the genus Borrelia. Included within this definition are Borrelia
burgdorferi sensu lato
(including B. burgdorferi sensu stricto, B. afzelii, B. garinii), B.
andersonii, B. anserina, B.
japonica, B. coriaceae, and other members of the genus Borrelia regardless of
whether they are
known pathogenic agents.
As used herein, the phrase "one or more B. burgdorferi polypeptides of the
present
invention" means the amino acid sequence of one or more of the B. burgdorferi
polypeptides
disclosed in Table 1. These polypeptides may be expressed as fusion proteins
wherein the
B. burgdorferi polypeptides of the present invention are linked to additional
amino acid
sequences which may be of borrelial or non-borrelial origin. This phrase
further includes
fragments of the B. burgdorferi polypeptides of the present invention.
As used herein, the phrase "full-length amino acid sequence" and "full-length
polypeptide"
refer to an amino acid sequence or polypeptide encoded by a full-length open
reading frame
(ORF). An ORF may be defined as a nucleotide sequence bounded by stop codons
which
encodes a putative polypeptide. An ORF may also be defined as a nucleotide
sequence within a
stop codon bounded sequence which contains an initiation codon (e.g., a
methionine or valine
codon) on the 5' end and a stop codon on the 3' end.
As used herein, the phrase "truncated amino acid sequence" and "truncated
polypeptide"
refer to a sub-sequence of a full-length amino acid sequence or polypeptide.
Several criteria may
also be used to define the truncated amino acid sequence or polypeptide. For
example, a truncated
polypeptide may be defined as a mature polypeptide (e.g., a polypeptide which
lacks a leader
sequence). A truncated polypeptide may also be defined as an amino acid
sequence which is a
portion of a longer sequence that has been selected for ease of expression in
a heterologous
system but retains regions which render the polypeptide useful for use in
vaccines (e.g., antigenic
regions which are expected to elicit a protective immune response).
Additional definitions are provided throughout the specification.
Explanation of Table 1
Table 1 lists B. burgdorferi nucleotide and amino acid sequences of the
present invention.
The nomenclature used therein is as follows:
"nt" refers to nucleotide sequences;
"aa" refers to amino acid sequences;
"f' refers to full-length nucleotide or amino acid sequences; and
"t" refers to truncated nucleotide or amino acid sequences.
Thus, for example, the designation "f101.aa" refers to the full-length amino
acid sequence
of B. burgdorferi polypeptide number 101. Further, "f101.nt" refers to the
full-length nucleotide
sequence encoding the full-length amino acid sequence of B. burgdorferi
polypeptide number
101.
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Explanation of Table 2
Table 2 lists accession numbers for the closest matching sequences between the
polypeptides of the present invention and those available through GenBank and
GeneSeq
databases. These reference numbers are the database entry numbers commonly
used by those of
skill in the art, who will be familar with their denominations. The
descriptions of the
numenclature for GenBank are available from the National Center for
Biotechnology Information.
Column I lists the gene or ORF of the present invention. Column 2 lists the
accession number of
a "match" gene sequence in GenBank or GeneSeq databases. Column 3 lists the
description of
l0 the "match" gene sequence. Columns 4 and 5 are the high score and smallest
sum probability,
respectively, calculated by BLAST. Polypeptides of the present invention that
do not share
significant identitylsimilarity with any polypeptide sequences of GenBank and
GeneSeq are not
represented in Table 2. Polypeptides of the present invention that share
significant
identity/similarity with more than one of the polypeptides of GenBank and
GeneSeq are
represented more than once.
Explanation of Table 3.
The B. burgdorferi polypeptides of the present invention may include one or
more conservative
amino acid substitutions from natural mutations or human manipulation as
indicated in Table 3.
Changes are preferably of a minor nature, such as conservative amino acid
substitutions that do
not significantly affect the folding or activity of the protein. Residues from
the following groups,
as indicated in Table 3, may be substituted for one another: Aromatic,
Hydrophobic, Polar, Basic,
Acidic, and Small,
Explanation of Table 4
Table 4 lists residues comprising antigenic epitopes of antigenic epitope-
bearing fragments
present in each of the full length B. burgdorferi polypeptides described in
Table 1 as predicted by
the inventors using the algorithm of Jameson and Wolf, (1988) Comp. Appl.
Biosci. 4:181-186.
The Jameson-Wolf antigenic analysis was performed using the computer program
PROTEAN
(Version 3.1 I for the Power Macintosh, DNASTAR, Inc., 1228 South Park Street
Madison,
WI). B. burgdorferi polypeptide shown in Table 1 may one or more antigenic
epitopes
comprising residues described in Table 4. It will be appreciated that
depending on the analytical
criteria used to predict antigenic determinants, the exact address of the
determinant may vary
slightly. The residues and locations shown described in Table 4 correspond to
the amino acid
sequences for each full length gene sequence shown in Table l and in the
Sequence Listing.
Polypeptides of the present invention that do not have antigenic epitopes
recognized by the
Jameson-Wolf algorithm are not represented in Table 2.
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Selection of Nucleic Acid Sequences Encoding Antigenic B. burgdorferi
Polypeptides
The present invention provides a select number of ORFs from those presented in
the
fragments of the Borrelia burgdorferi genome which may prove useful for the
generation of a
protective immune response. The sequenced B. burgdorferi genomic DNA was
obtained from a
sub-cultured isolate of ATCC Deposit No. 35210. The sub-cultured isolate was
deposited on
August 8, 1997 at the American Type Culture Collection, 12301 Park Lawn Drive,
Rockville,
Maryland 20852, and given accession number 202012.
Some ORFs contained in the subset of fragments of the B. burgdorferi genome
disclosed
herein were derived through the use of a number of screening criteria detailed
below. The ORFs
are generally bounded at the amino terminus by a methionine residue and at the
carboxy terminus
by a stop codon.
Many of the selected sequences do not consist of complete ORFs. Although a
polypeptide
representing a complete ORF may be the closest approximation of a protein
native to an organism,
it is not always preferred to express a complete ORF in a heterologous system.
It may be
challenging to express and purify a highly hydrophobic protein by common
laboratory methods.
Some of the polypeptide vaccine candidates described herein have been modified
slightly to
simplify the production of recombinant protein. For example, nucleotide
sequences which encode
highly hydrophobic domains, such as those found at the amino terminal signal
sequence, have
been excluded from some constructs used for in vitro expression of the
polypeptides.
Furthermore, any highly hydrophobic amino acid sequences occurring at the
carboxy terminus
have also been excluded from the recombinant expression constructs. Thus, in
one embodiment,
a polypeptide which represents a truncated or modified ORF may be used as an
antigen.
While numerous methods are known in the art for selecting potentially
immunogenic
polypeptides, many of the ORFs disclosed herein were selected on the basis of
screening all
theoretical Borrelia burgdorferi ORFs for several aspects of potential
immunogenicity. One set of
selection criteria are as follows:
1. Type I signal sequence: An amino terminal type I signal sequence generally
directs a
nascent protein across the plasma and outer membranes to the exterior of the
bacterial cell.
Experimental evidence obtained from studies with Escherichia coli suggests
that the typical type I
signal sequence consists of the following biochemical and physical attributes
(Izard, 3. W. and
Kendall, D. A. Mol. Microbiol. 13:765-773 ( 1994)). The length of the type I
signal sequence is
approximately 15 to 25 primarily hydrophobic amino acid residues with a net
positive charge in
the extreme amino terminus. In addition, the central region of the signal
sequence adopts an
alpha-helical conformation in a hydrophobic environment. Finally, the region
surrounding the
actual site of cleavage is ideally six residues long, with small side-chain
amino acids in the -1 and
-3 positions.
2. Type IV signal sequence: The type IV signal sequence is an example of the
several
types of functional signal sequences which exist in addition to the type I
signal sequence detailed
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above. Although functionally related, the type IV signal sequence possesses a
unique set of
biochemical and physical attributes (Strom, M. S. and Lory, S., J. Bacteriol.
174:7345-7351
( 1992)). These are typically six to eight amino acids with a net basic charge
followed by an
additional sixteen to thirty primarily hydrophobic residues. The cleavage site
of a type IV signal
sequence is typically after the initial six to eight amino acids at the
extreme amino terminus. In
addition, type IV signal sequences generally contain a phenylalanine residue
at the +1 site relative
to the cleavage site.
3. Lipoprotein: Studies of the cleavage sites of twenty-six bacterial
lipoprotein precursors
has allowed the definition of a consensus amino acid sequence for lipoprotein
cleavage. Nearly
three-fourths of the bacterial lipoprotein precursors examined contained the
sequence L-(A,S)-
(G,A)-C at positions -3 to +1, relative to the point of cleavage (Hayashi, S.
and Wu, H. C., J.
Bioenerg. Biomembr. 22:451-471 (1990)).
4. LPXTG motif. It has been experimentally determined that most anchored
proteins
found on the surface of gram-positive bacteria possess a highly conserved
carboxy terminal
sequence. More than fifty such proteins from organisms such as S. pyogenes, S.
mutans, B.
burgdorferi, S. pneumoniae, and others, have been identified based on their
extracellular location
and carboxy terminal amino acid sequence (Fischetti, V. A., ASM News 62:405-
410 ( 1996)).
The conserved region consists of six charged amino acids at the extreme
carboxy terminus
coupled to 15-20 hydrophobic amino acids presumed to function as a
transmembrane domain.
2o Immediately adjacent to the transmembrane domain is a six amino acid
sequence conserved in
nearly all proteins examined. The amino acid sequence of this region is L-P-X-
T-G-X, where X
is any amino acid.
An algorithm for selecting antigenic and immunogenic Borrelia burgdorferi
polypeptides
including the foregoing criteria was developed. The algorithm is similar to
that described in U.S.
patent application 08/781,986, filed January 3, 1997, which is fully
incorporated by reference
herein. Use of the algorithm by the inventors to select immunologically useful
Borrelia
burgdorferi polypeptides resulted in the selection of a number of the
disclosed ORFs.
Polypeptides comprising the polypeptides identified in this group may be
produced by techniques
standard in the art and as further described herein.
Nucleic Acid Molecules
The present invention provides isolated nucleic acid molecules comprising
polynucleotides
encoding the B. burgdorferi polypeptides having the amino acid sequences shown
in Table 1,
which were determined by sequencing the genome of B. burgdorferi deposited as
ATCC deposit
no. 202012 and selected as putative immunogens.
Unless otherwise indicated, all nucleotide sequences determined by sequencing
a DNA
molecule herein were determined using an automated DNA sequencer (such as the
Model 373
from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides
encoded by DNA
molecules determined herein were predicted by translation of DNA sequences
determined as
CA 02294568 1999-12-17
WO 98/59071 PCT/US98/12718
above. Therefore, as is known in the art for any DNA sequence determined by
this automated
approach, any nucleotide sequence determined herein may contain some errors.
Nucleotide
sequences determined by automation are typically at least about 90% identical,
more typically at
least about 95% to at least about 99.9% identical to the actual nucleotide
sequence of the
5 sequenced DNA molecule. The actual sequence can be more precisely determined
by other
approaches including manual DNA sequencing methods well known in the art. As
is also known
in the art, a single insertion or deletion in a determined nucleotide sequence
compared to the actual
sequence will cause a frame shift in translation of the nucleotide sequence
such that the predicted
amino acid sequence encoded by a determined nucleotide sequence will be
completely different
10 from the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the
point of such an insertion or deletion.
Unless otherwise indicated, each "nucleotide sequence" set forth herein is
presented as a
sequence of deoxyribonucleotides (abbreviated A, G , C and T). However, by
"nucleotide
sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA
molecule or
polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide,
the corresponding sequence of ribonucleotides (A, G, C and U), where each
thymidine
deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is
replaced by the
ribonucleotide uridine (U). For instance, reference to an RNA molecule having
a sequence of
Table 1 set forth using deoxyribonucleotide abbreviations is intended to
indicate an RNA molecule
2o having a sequence in which each deoxyribonucleotide A, G or C of Table 1
has been replaced by
the cor! esponding ribonucleotide A, G or C, and each deoxyribonucleotide T
has been replaced
by a ribonucleotide U.
Nucleic acid molecules of the present invention may be in the form of RNA,
such as
mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA
obtained by
cloning or produced synthetically. The DNA may be double-stranded or single-
stranded.
Single-stranded DNA or RNA may be the coding strand, also known as the sense
strand, or it
may be the non-coding strand, also referred to as the anti-sense strand.
By "isolated" nucleic acid molecules) is intended a nucleic acid molecule, DNA
or RNA,
which has been removed from its native environment. For example, recombinant
DNA molecules
contained in a vector are considered isolated for the purposes of the present
invention. Further
examples of isolated DNA molecules include recombinant DNA molecules
maintained in
heterologous host cells or purified {partially or substantially) DNA molecules
in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules
of the present
invention. Isolated nucleic acid molecules according to the present invention
further include such
molecules produced synthetically.
In addition, isolated nucleic acid molecules of the invention include DNA
molecules which
comprise a sequence substantially different from those described above but
which, due to the
degeneracy of the genetic code, still encode a B. burgdorferi polypeptides and
peptides of the
present invention (e.g. polypeptides of Table 1 ). That is, all possible DNA
sequences that encode
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11
the B. burgdorferi polypeptides of the present invention. This includes the
genetic code and
species-specific codon preferences known in the art. Thus, it would be routine
for one skilled in
the art to generate the degenerate variants described above, for instance, to
optimize codon
expression for a particular host (e.g., change codons in the bacteria mRNA to
those preferred by a
mammalian or other bacterial host such as E. coli).
The invention further provides isolated nucleic acid molecules having the
nucleotide
sequence shown in Table 1 or a nucleic acid molecule having a sequence
complementary to one of
the above sequences. Such isolated molecules, particularly DNA molecules, are
useful as probes
for gene mapping and for identifying B. burgdorferi in a biological sample,
for instance, by PCR,
to Southern blot, Northern blot, or other form of hybridization analysis.
The present invention is further directed to nucleic acid molecules encoding
portions or
fragments of the nucleotide sequences described herein. Fragments include
portions of the
nucleotide sequences of Table 1 at least 10 contiguous nucleotides in length
selected from any two
integers, one of which representing a 5' nucleotide position and a second of
which representing a
15 3' nucleotide position, where the first nucleotide for each nucleotide
sequence in Table I is
position 1. That is, every combination of a 5' and 3' nucleotide position that
a fragment at least
contiguous nucleotides in length could occupy is included in the invention.
"At least" means a
fragment may be 10 contiguous nucleotide bases in length or any integer
between 10 and the
length of an entire nucleotide sequence of Table 1 minus 1. Therefore,
included in the invention
2o are contiguous fragments specified by any 5' and 3' nucleotide base
positions of a nucleotide
sequences of Table 1 wherein the contiguous fragment is any integer between 10
and the length of
an entire nucleotide sequence minus 1.
Further, the invention includes polynucleotides comprising fragments specified
by size, in
nucleotides, rather than by nucleotide positions. The invention includes any
fragment size, in
25 contiguous nucleotides, selected from integers between 10 and the length of
an entire nucleotide
sequence minus 1. Preferred sizes of contiguous nucleotide fragments include
20 nucleotides, 30
nucleotides, 40 nucleotides, 50 nucleotides. Other preferred sizes of
contiguous nucleotide
fragments, which may be useful as diagnostic probes and primers, include
fragments 50-300
nucleotides in length which include, as discussed above, fragment sizes
representing each integer
30 between 50-300. Larger fragments are also useful according to the present
invention
corresponding to most, if not all, of the nucleotide sequences shown in Table
for of the B.
burgdorferi nucleotide sequences of the plasimd clones listed in Table 1. The
preferred sizes are,
of course, meant to exemplify not limit the present invention as all size
fragments, representing
any integer between 10 and the length of an entire nucleotide sequence minus
1, are included in
35 the invention. Additional preferred nucleic acid fragments of the present
invention include
nucleic acid molecules encoding epitope-bearing portions of B. burgdorferi
polypeptides
identified in Table 4.
The present invention also provides for the exclusion of any fragment,
specified by 5' and
3' base positions or by size in nucleotide bases as described above for any
nucleotide sequence of
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Table 1 or the plasimd clones listed in Table 1. Any number of fragments of
nucleotide sequences
in Table I or the plasimd clones listed in Table 1, specified by 5' and 3'
base positions or by size
in nucleotides, as described above, may be excluded from the present
invention.
Preferred nucleic acid fragments of the present invention also include nucleic
acid
molecules encoding epitope-bearing portions of the B. burgdorferi polypeptides
shown in Table
1. Such nucleic acid fragments of the present invention include, for example,
nucleic acid
molecules encoding polypeptide fragments comprising from about the amino
terminal residue to
about the carboxy terminal residue of each fragment shown in Table 4. The
above referred to
polypeptide fragments are antigenic regions of particular B. burgdorferi
polypeptides shown in
Table 1. Methods for determining other such epitope-bearing portions for the
remaining
polypeptides described in Table 1 are well known in the art and are described
in detail below.
In another aspect, the invention provides isolated nucleic acid molecules
comprising
polynucleotides which hybridize under stringent hybridization conditions to a
portion of a
polynucleotide in a nucleic acid molecule of the invention described above,
for instance, a nucleic
acid sequence shown in Table 1. By "stringent hybridization conditions" is
intended overnight
incubation at 42 C in a solution comprising: 50% formamide, 5x SSC ( 150 mM
NaCI, 15 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution,
10% dextran
sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing
the filters in
O.lx SSC at about 65 C.
By polynucleotides which hybridize to a "portion" of a polynucleotide is
intended
polynucleotides (either DNA or RNA) which hybridize to at least about 15
nucleotides (nt), and
more preferably at least about 20 nt, still more preferably at least about 30
nt, and even more
preferably about 30-70 nt of the reference polynucleotide. These are useful as
diagnostic probes
and primers as discussed above and in more detail below.
Of course, polynucieotides hybridizing to a larger portion of the reference
polynucleotide,
for instance, a portion 50-100 nt in length, or even to the entire length of
the reference
polynucleotide, are also useful as probes according to the present invention,
as are
polynucleotides corresponding to most, if not all, of a nucleotide sequence as
shown in Table 1.
By a portion of a polynucleotide of "at least 20 nt in length," for example,
is intended 20 or more
contiguous nucleotides from the nucleotide sequence of the reference
polynucleotide (e.g., a
nucleotide sequences as shown in Table I ). As noted above, such portions are
useful
diagnostically either as probes according to conventional DNA hybridization
techniques or as
primers for amplification of a target sequence by PCR, as described, for
instance, in Molecular
Cloning, A Laboratory Manual, 2nd. edition, Sambrook, J., Fritsch, E. F. and
Maniatis, T.,
eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989),
the entire
disclosure of which is hereby incorporated herein by reference.
Since nucleic acid sequences encoding the B. burgdorferi polypeptides of the
present
invention are provided in Table 1, generating polynucleotides which hybridize
to portions of these
sequences would be routine to the skilled artisan. For example, the
hybridizing polynucleotides
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13
of the present invention could be generated synthetically according to known
techniques.
As indicated, nucleic acid molecules of the present invention which encode B.
burgdorferi
polypeptides of the present invention may include, but are not limited to
those encoding the amino
acid sequences of the polypeptides by themselves; and additional coding
sequences which code
for additional amino acids, such as those which provide additional
functionalities. Thus, the
sequences encoding these polypeptides may be fused to a marker sequence, such
as a sequence
encoding a peptide which facilitates purification of the fused polypeptide. In
certain preferred
embodiments of this aspect of the invention, the marker amino acid sequence is
a hexa-histidine
peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among
others, many of which
l0 are commercially available. As described in Gentz et al., Proc. Natl. Acad
Sci. USA 86:821-824
( 1989), for instance, hexa-histidine provides for convenient purification of
the resulting fusion
protein.
Thus, the present invention also includes genetic fusions wherein the
B. burgdorferi nucleic acid sequences coding sequences provided in Table 1 are
linked to
additional nucleic acid sequences to produce fusion proteins. These fusion
proteins may include
epitopes of borrelial or non-borrelial origin designed to produce proteins
having enhanced
immunogenicity. Further, the fusion proteins of the present invention may
contain antigenic
determinants known to provide helper T-cell stimulation, peptides encoding
sites for
post-translational modifications which enhance immunogenicity (e.g.,
acylation), peptides which
facilitate purification (e.g., histidine "tag"), or amino acid sequences which
target the fusion
protein to a desired location (e.g., a heterologous leader sequence). For
instance, hexa-histidine
provides for convenient purification of the fusion protein. See Gentz et al. (
1989) Proc. Natl.
Acad. Sci. 86:821-24. The "HA" tag is another peptide useful for purification
which corresponds
to an epitope derived from the influenza hemagglutinin protein. See Wilson et
al. ( 1984) Cell
37:767. As discussed below, other such fusion proteins include the B.
burgdorferi polypeptides
of the present invention fused to Fc at the N- or C-terminus.
Post-translational modification of the full-length B. burgdorferi OspA protein
expressed in E. coli is believed to increase the immunogenicity of this
protein. Erdile, L. et al.,
Infect. Immun. 61:81-90 (1993). B. burgdorferi OspA when expressed in E. coli,
for example,
is post-translationally modified in at least two ways. First, a signal peptide
is cleaved; second,
lipid moieties are attached. The presence of these lipid moieties is believed
to confer enhanced
immunogenicity and results in the elicitation of a strong protective
immunological response.
Variant and Mutant Polynucleotides
The present invention thus includes nucleic acid molecules and sequences which
encode
fusion proteins comprising one or more B. burgdorferi polypeptides of the
present invention
fused to an amino acid sequence which allows for post-translational
modification to enhance
immunogenicity. This post-transiational modification may occur either in vitro
or when the fusion
protein is expressed in vivo in a host cell. An example of such a modification
is the introduction
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14
of an amino acid sequence which results in the attachment of a lipid moiety.
Such a lipid moiety
attachment site of OspA, which is lipidated upon expression in E. coli, has
been identified.
Bouchon, B. et al., Anal. Biochem. 246:52-61 ( 1997).
Thus, as indicated above, the present invention includes genetic fusions
wherein a
B. burgdorferi nucleic acid sequence provided in Table 1 is linked to a
nucleotide sequence
encoding another amino acid sequence. These other amino acid sequences may be
of borrelial
origin (e.g., another sequence selected from Table 1) or non-borrelial origin.
An example of such
a fusion protein is reported in Fikrig, E. et al., Science 250:553-556 ( 1990)
where an OspA-
glutathione-S-transferase fusion protein was produced and shown to elicit
protective immunity
1o against Lyme disease in immune competent mice.
The present invention further relates to variants of the nucleic acid
molecules of the present
invention, which encode portions, analogs or derivatives of the B. burgdorferi
polypeptides
shown in Table 1. Variants may occur naturally, such as a natural allelic
variant. By an "allelic
variant" is intended one of several alternate forms of a gene occupying a
given locus on a
chromosome of an organism. Genes 11, Lewin, B., ed., 3ohn Wiley & Sons, New
York ( 1985).
Non-naturally occurring variants may be produced using art-known mutagenesis
techniques.
Such variants include those produced by nucleotide substitutions, deletions or
additions.
The substitutions, deletions or additions may involve one or more nucleotides.
These variants
may be altered in coding regions, non-coding regions, or both. Alterations in
the coding regions
2o may produce conservative or non-conservative amino acid substitutions,
deletions or additions.
Especially preferred among these are silent substitutions, additions and
deletions, which do not
alter the properties and activities of the B. burgdorferi polypeptides
disclosed herein or portions
thereof. Also especially preferred in this regard are conservative
substitutions.
The present application is further directed to nucleic acid molecules at least
90%, 95%,
96%, 97%, 98% or 99% identical to a nucleic acid sequence shown in Table 1.
The above
nucleic acid sequences are included irrespective of whether they encode a
polypeptide having B.
burgdorferi activity. This is because even where a particular nucleic acid
molecule does not
encode a polypeptide having B. burgdorferi activity, one of skill in the art
would still know how
to use the nucleic acid molecule, for instance, as a hybridization probe. Uses
of the nucleic acid
3o molecules of the present invention that do not encode a polypeptide having
B. burgdorferi activity
include, inter alia, isolating an B. burgdorferi gene or allelic variants
thereof from a DNA library,
and detecting B. burgdorferi mRNA expression samples, environmental samples,
suspected of
containing B. burgdorferi by Northern Blot analysis.
Embodiments of the invention include isolated nucleic acid molecules
comprising a
polynucleotide having a nucleotide sequence at least 90% identical, and more
preferably at least
95%, 96%, 97%, 98% or 99% identical to {a) a nucleotide sequence encoding any
of the amino
acid sequences of the full-length polypeptides shown in Table l; (b) a
nucleotide sequence
encoding any of the amino acid sequences of the full-length polypeptides shown
in Table 1 but
minus the N-terminal methionine residue, if present; (c) a nucleotide sequence
encoding any of the
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amino acid sequences of the truncated polypeptides shown in Table 1; and (d) a
nucleotide
sequence complementary to any of the nucleotide sequences in (a), (b), or (c)
above.
Preferred, are nucleic acid molecules having sequences at least 90%, 95%, 96%,
97%,
98% or 99% identical to the nucleic acid sequence shown in Table 1, which do,
in fact, encode a
polypeptide having B. burgdorferi protein activity By "a polypeptide having B.
burgdorferi
activity" is intended polypeptides exhibiting activity similar, but not
necessarily identical, to an
activity of the B. burgdorferi protein of the invention, as measured in a
particular biological assay
suitable for measuring activity of the specified protein.
Due to the degeneracy of the genetic code, one of ordinary skill in the art
will immediately
10 recognize that a large number of the nucleic acid molecules having a
sequence at least 90%, 95%,
96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in Table 1
will encode a
polypeptide having B. burgdorferi protein activity. In fact, since degenerate
variants of these
nucleotide sequences all encode the same polypeptide, this will be clear to
the skilled artisan even
without performing the above described comparison assay. It will be further
recognized in the art
15 that, for such nucleic acid molecules that are not degenerate variants, a
reasonable number will
also encode a polypeptide having B. burgdorferi protein activity. This is
because the skilled
artisan is fully aware of amino acid substitutions that are either less likely
or not likely to
significantly effect protein function (e.g., replacing one aliphatic amino
acid with a second
aliphatic amino acid), as further described below.
2o The biological activity or function of the polypeptides of the present
invention are expected
to be similar or identical to polypeptides from other bacteria that share a
high degree of structural
identity/similarity. Tables 2 lists accession numbers and descriptions for the
closest matching
sequences of polypeptides available through Genbank and Derwent databases. It
is therefore
expected that the biological activity or function of the polypeptides of the
present invention will be
similar or identical to those polypeptides from other bacterial genuses,
species, or strains listed in
Table 2.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical" to
a reference nucleotide sequence of the present invention, it is intended that
the nucleotide sequence
of the polynucleotide is identical to the reference sequence except that the
polynucleotide sequence
3o may include up to five point mutations per each 100 nucleotides of the
reference nucleotide
sequence encoding the B. burgdorferi polypeptide. In other words, to obtain a
polynucleotide
having a nucleotide sequence at least 95% identical to a reference nucleotide
sequence, up to 5%
(5 of 100) of the nucleotides in the reference sequence may be deleted,
inserted, or substituted
with another nucleotide. The query sequence may be an entire sequence shown in
Table l, the
ORF (open reading frame), or any fragment specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or
polypeptide is at least
90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the
presence invention
can be determined conventionally using known computer programs. A preferred
method for
determining the best overall match between a query sequence (a sequence of the
present invention)
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16
and a subject sequence, also referred to as a global sequence alignment, can
be determined using
the FASTDB computer program based on the algorithm of Brutlag et al. See
Brutlag et ai.
( 1990) Comp. App. Biosci. 6:237-245. In a sequence alignment the query and
subject sequences
are both DNA sequences. An RNA sequence can be compared by first converting
U's to T's.
The result of said global sequence alignment is in percent identity. Preferred
parameters used in a
FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-
tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0,
Cutoff
Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the lenght
of the subject
nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3'
deletions,
not because of internal deletions, a manual correction must be made to the
results. This is because
the FASTDB program does not account for 5' and 3' truncations of the subject
sequence when
calculating percent identity. For subject sequences truncated at the 5' or 3'
ends, relative to the
query sequence, the percent identity is corrected by calculating the number of
bases of the query
sequence that are 5' and 3' of the subject sequence, which are not
matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is matched/aligned
is determined by
results of the FASTDB sequence alignment. This percentage is then subtracted
from the percent
identity, calculated by the above FASTDB program using the specified
parameters, to arnve at a
final percent identity score. This corrected score is what is used for the
purposes of the present
invention. Only nucleotides outside the 5' and 3' nucleotides of the subject
sequence, as
displayed by the FASTDB alignment, which are not matched/aligned with the
query sequence, are
calculated for the purposes of manually adjusting the percent identity score.
For example, a 90 nucleotide subject sequence is aligned to a 100 nucleotide
query
sequence to determine percent identity. The deletions occur at the 5' end of
the subject sequence
and therefore, the FASTDB alignment does not show a matched/alignment of the
first 10
nucleotides at 5' end. The 10 unpaired nucleotides represent 10% of the
sequence (number of
nucleotides at the 5' and 3' ends not matched/total number of nucleotides in
the query sequence)
so 10% is subtracted from the percent identity score calculated by the FASTDB
program. If the
remaining 90 nucleotides were perfectly matched the final percent identity
would be 90%. In
3o another example, a 90 nucleotide subject sequence is compared with a 100
nucleotide query
sequence. This time the deletions are internal deletions so that there are no
nucleotides on the 5'
or 3' of the subject sequence which are not matched/aligned with the query. In
this case the
percent identity calculated by FASTDB is not manually corrected. Once again,
only nucleotides
5' and 3' of the subject sequence which are not matched/aligned with the query
sequence are
manually corrected for. No other manual corrections are to made for the
purposes of the present
invention.
Vectors and Host Cells
The present invention also relates to vectors which include the isolated DNA
molecules of
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17
the present invention, host cells which are genetically engineered with the
recombinant vectors,
and the production of B. burgdorferi polypeptides or fragments thereof by
recombinant
techniques.
Recombinant constructs may be introduced into host cells using well known
techniques
such as infection, transduction, transfection, transvection, electroporation
and transformation.
The vector may be, for example, a phage, plasmid, viral or retroviral vector.
Retroviral vectors
may be replication competent or replication defective. In the latter case,
viral propagation
generally will occur only in complementing host cells.
The polynucleotides may be joined to a vector containing a selectable marker
for
propagation in a host. Generally, a plasmid vector is introduced in a
precipitate, such as a calcium
phosphate precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be
packaged in vitro using an appropriate packaging cell line and then transduced
into host cells.
Preferred are vectors comprising cis-acting control regions to the
polynucleotide of
interest. Appropriate traps-acting factors may be supplied by the host,
supplied by a
complementing vector or supplied by the vector itself upon introduction into
the host.
In certain preferred embodiments in this regard, the vectors provide for
specific
expression, which may be inducible and/or cell type-specific. Particularly
preferred among such
vectors are those inducible by environmental factors that are easy to
manipulate, such as
temperature and nutrient additives.
Expression vectors useful in the present invention include chromosomal-,
episomal- and
virus-derived vectors, e.g., vectors derived from bacterial plasmids,
bacteriophage, yeast
episomes, yeast chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia
viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and
retroviruses, and vectors
derived from combinations thereof, such as cosmids and phagemids.
The DNA insert should be operatively linked to an appropriate promoter, such
as the
phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40
early and late
promoters and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be
known to the skilled artisan. The expression constructs will further contain
sites for transcription
initiation, termination and, in the transcribed region, a ribosome binding
site for translation. The
3o coding portion of the mature transcripts expressed by the constructs will
preferably include a
translation initiating site at the beginning and a termination codon (UAA, UGA
or UAG)
appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker.
Such markers include dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture
and tetracycline or ampicillin resistance genes for culturing in E. coli and
other bacteria.
Representative examples of appropriate hosts include, but are not limited to,
bacterial cells, such
as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such
as yeast cells;
insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such
as CHO, COS and
Bowes melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the
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above-described host cells are known in the art.
18
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9,
available
from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNHBA, pNH
16a, pNH 18A,
pNH46A available from Stratagene; pET series of vectors available from
Novagen; and ptrc99a,
pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia. Among preferred
eukaryotic
vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene;
and
pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors
will be
readily apparent to the skilled artisan.
Among known bacterial promoters suitable for use in the present invention
include the E.
io coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter,
the lambda PR and PL
promoters and the trp promoter. Suitable eukaryotic promoters include the CMV
immediate early
promoter, the HSV thymidine kinase promoter, the early and late SV40
promoters, the promoters
of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and
metallothionein
promoters, such as the mouse metallothionein-I promoter.
15 Introduction of the construct into the host cell can be effected by calcium
phosphate
transfection, DEAF-dextran mediated transfection, cationic lipid-mediated
transfection,
electroporation, transduction, infection or other methods. Such methods are
described in many
standard laboratory manuals, such as Davis et al., Basic Methods In Molecular
Biology ( 1986).
Transcription of DNA encoding the polypeptides of the present invention by
higher
2o eukaryotes may be increased by inserting an enhancer sequence into the
vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 by that act to
increase transcriptional
activity of a promoter in a given host cell-type. Examples of enhancers
include the SV40
enhancer, which is located on the late side of the replication origin at by
100 to 270, the
cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side
of the replication
25 origin, and adenovirus enhancers.
For secretion of the translated polypeptide into the lumen of the endoplasmic
reticulum,
into the periplasmic space or into the extracellular environment, appropriate
secretion signals may
be incorporated into the expressed polypeptide. The signals may be endogenous
to the
polypeptide or they may be heterologous signals.
30 The polypeptide may be expressed in a modified form, such as a fusion
protein, and may
include not only secretion signals, but also additional heterologous
functional regions. For
instance, a region of additional amino acids, particularly charged anuno
acids, may be added to
the N-terminus of the polypeptide to improve stability and persistence in the
host cell, during
purification, or during subsequent handling and storage. Also, peptide
moieties may be added to
35 the polypeptide to facilitate purification. Such regions may be removed
prior to final preparation
of the polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or
excretion, to improve stability and to facilitate purification, among others,
are familiar and routine
techniques in the art. A preferred fusion protein comprises a heterologous
region from
immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464
533 (Canadian
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19
counterpart 2045869) discloses fusion proteins comprising various portions of
constant region of
immunoglobin molecules together with another human protein or part thereof. In
many cases, the
Fc part in a fusion protein is thoroughly advantageous for use in therapy and
diagnosis and thus
results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
On the other
hand, for some uses it would be desirable to be able to delete the Fc part
after the fusion protein
has been expressed, detected and purified in the advantageous manner
described. This is the case
when Fc portion proves to be a hindrance to use in therapy and diagnosis, for
example when the
fusion protein is to be used as antigen for immunizations. In drug discovery,
for example, human
proteins, such as, hILS-receptor has been fused with Fc portions for the
purpose of
high-throughput screening assays to identify antagonists of hIL-5. See
Bennett, D. et al., J.
Molec. Recogn. 8:52-58 (1995) and Johanson, K. et al., J. Biol. Chem. 270
(16):9459-9471
( 1995).
The B. burgdorferi polypeptides can be recovered and purified from recombinant
cell
cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid
extraction, anion or canon exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography, lectin chromatography and high performance liquid
chromatography ("HPLC"}
is employed for purification. Polypeptides of the present invention include
naturally purified
products, products of chemical synthetic procedures, and products produced by
recombinant
2o techniques from a prokaryotic or eukaryotic host, including, for example,
bacterial, yeast, higher
plant, insect and mammalian cells.
Polypeptides and Fragments
The invention further provides isolated polypeptides having the amino acid
sequences in
Table 1, and peptides or polypeptides comprising portions of the above
polypeptides. The terms
"peptide" and "oligopeptide" are considered synonymous (as is commonly
recognized) and each
term can be used interchangeably as the context requires to indicate a chain
of at least to amino
acids coupled by peptidyl linkages. The word "polypeptide" is used herein for
chains containing
more than ten amino acid residues. Ali oligopeptide and polypeptide formulas
or sequences
herein are written from left to right and in the direction from amino terminus
to carboxy terminus.
As discussed in detail below, immunization using B. burgdorferi sensu stricto
isolate B3 i
decorin-binding protein elicits the production of antiserum which confers
passive immunity
against Borrelia species and strains which express divergent forms of this
protein. Cassatt, D. et
al., Protection of Borrelia burgdorferi Infection by Antibodies to Decorin-
binding Protein, in
VACC1NES97, Cold Spring Harbor Press (1997), pages 191-195. Thus, some amino
acid
sequences of the B. burgdorferi polypeptides shown in Table 1 can be varied
without
significantly effecting the antigenicity of the polypeptides. If such
differences in sequence are
contemplated, it should be remembered that there will be critical areas on the
polypeptide which
determine antigenicity. In general, it is possible to replace residues which
do not form part of an
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antigenic epitope without significantly effecting the antigenicity of a
polypeptide.
Variant and Mutant Polypeptides
To improve or alter the characteristics of B. burgdorferi polypeptides of the
present
invention, protein engineering may be employed. Recombinant DNA technology
known to those
skilled in the art can be used to create novel mutant proteins or muteins
including single or
multiple amino acid substitutions, deletions, additions, or fusion proteins.
Such modified
polypeptides can show, e.g., enhanced activity or increased stability. In
addition, they may be
purified in higher yields and show better solubility than the corresponding
natural polypeptide, at
least under certain purification and storage conditions.
l0
N-Terminal and C-Terminal Deletion Mutants
It is known in the art that one or more amino acids may be deleted from the N-
terminus or
C-terminus without substantial loss of biological function. For instance, Ron
et al. J. Biol.
Chem., 268:2984-2988 ( 1993), reported modified KGF proteins that had heparin
binding
15 activity even if 3, 8, or 27 N-terminal amino acid residues were missing.
Accordingly, the
present invention provides polypeptides having one or more residues deleted
from the amino
terminus of the amino acid sequence of the B. burgdorferi polypeptides shown
in Table l, and
polynucleotides encoding such polypeptides.
Similarly, many examples of biologically functional C-terminal deletion
muteins are
20 known. For instance, Interferon gamma shows up to ten times higher
activities by deleting 8-10
amino acid residues from the carboxy terminus of the protein See, e.g.,
Dobeli, et al. ( 1988) J.
Biotechnology 7:199-216. Accordingly, the present invention provides
polypeptides having one
or more residues from the carboxy terminus of the amino acid sequence of the
B. burgdorferi
polypeptides shown in Table 1. The invention also provides polypeptides having
one or more
amino acids deleted from both the amino and the carboxyl termini as described
below.
The present invention is further directed to polynucleotide encoding portions
or fragments
of the amino acid sequences described herein as well as to portions or
fragments of the isolated
amino acid sequences described herein. Fragments include portions of the amino
acid sequences
of Table l, are at least 5 contiguous amino acid in length, are selected from
any two integers, one
of which representing a N-terminal position. The initiation codon of the
polypeptides of the
present inventions position 1. Every combination of a N-terminal and C-
terminal position that a
fragment at least 5 contiguous amino acid residues in length could occupy, on
any given amino
acid sequence of Table 1 is included in the invention. At least means a
fragment may be 5
contiguous amino acid residues in length or any integer between 5 and the
number of residues in a
full length amino acid sequence minus 1. Therefore, included in the invention
are contiguous
fragments specified by any N-terminal and C-terminal positions of amino acid
sequence set forth
in Table 1 wherein the contiguous fragment is any integer between 5 and the
number of residues
in a full length sequence minus 1.
Further, the invention includes polypeptides comprising fragments specified by
size, in
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21
amino acid residues, rather than by N-terminal and C-terminal positions. The
invention includes
any fragment size, in contiguous amino acid residues, selected from integers
between 5 and the
number of residues in a full length sequence minus 1. Preferred sizes of
contiguous polypeptide
fragments include about 5 amino acid residues, about 10 amino acid residues,
about 20 amino
acid residues, about 30 amino acid residues, about 40 amino acid residues,
about 50 amino acid
residues, about 100 amino acid residues, about 200 amino acid residues, about
300 amino acid
residues, and about 400 amino acid residues. The preferred sizes are, of
course, meant to
exemplify, not limit, the present invention as all size fragments representing
any integer between 5
and the number of residues in a full length sequence minus 1 are included in
the invention. The
present invention also provides for the exclusion of any fragments specified
by N-terminal and C-
terminal positions or by size in amino acid residues as described above. Any
number of
fragments specified by N-terminal and C-terminal positions or by size in amino
acid residues as
described above may be excluded.
The above fragments need not be active since they would be useful, for
example, in
immunoassays, in epitope mapping, epitope tagging, to generate antibodies to a
particular portion
of the protein, as vaccines, and as molecular weight markers.
Other Mutants
In addition to N- and C-terminal deletion forms of the protein discussed
above, it also will
2o be recognized by one of ordinary skill in the art that some amino acid
sequences of the B.
burgdorferi polypeptide can be varied without significant effect of the
structure or function of the
protein. If such differences in sequence are contemplated, it should be
remembered that there will
be critical areas on the protein which determine activity.
Thus, the invention further includes variations of the B. burgdorferi
polypeptides which
show substantial B. burgdorferi polypeptide activity or which include regions
of B. burgdorferi
protein such as the protein portions discussed below. Such mutants include
deletions, insertions,
inversions, repeats, and type substitutions selected according to general
rules known in the art so
as to have little effect on activity. For example, guidance concerning how to
make phenotypically
silent amino acid substitutions is provided. There are two main approaches for
studying the
3o tolerance of an amino acid sequence to change. See, Bowie, J. U. et al. (
1990), Science
247:1306-1310. The first method relies on the process of evolution, in which
mutations are either
accepted or rejected by natural selection. The second approach uses genetic
engineering to
introduce amino acid changes at specific positions of a cloned gene and
selections or screens to
identify sequences that maintain functionality.
These studies have revealed that proteins are surprisingly tolerant of amino
acid
substitutions. The studies indicate which amino acid changes are likely to be
permissive at a
certain position of the protein. For example, most buried amino acid residues
require nonpolar
side chains, whereas few features of surface side chains are generally
conserved. Other such
phenotypically silent substitutions are described by Bowie et al. (supra) and
the references cited
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22
therein. Typically seen as conservative substitutions are the replacements,
one for another,
among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the
hydroxyl residues Ser
and Thr, exchange of the acidic residues Asp and Glu, substitution between the
amide residues
Asn and Gln, exchange of the basic residues Lys and Arg and replacements among
the aromatic
residues Phe, Tyr.
Thus, the fragment, derivative, analog, or homolog of the polypeptide of Table
1, or that
encoded by the plaimds listed in Table 1, may be: (i) one in which one or more
of the amino acid
residues are substituted with a conserved or non-conserved amino acid residue
(preferably a
conserved amino acid residue) and such substituted amino acid residue may or
may not be one
encoded by the genetic code: or (ii) one in which one or more of the amino
acid residues includes
a substituent group: or (iii) one in which the B. bur~dorferi polypeptide is
fused with another
compound, such as a compound to increase the half life of the polypeptide (for
example,
polyethylene glycol): or (iv) one in which the additional amino acids are
fused to the above form
of the polypeptide, such as an IgG Fc fusion region peptide or leader or
secretory sequence or a
sequence which is employed for purification of the above form of the
polypeptide or a proprotein
sequence. Such fragments, derivatives and analogs are deemed to be within the
scope of those
skilled in the art from the teachings herein.
Thus, the B. burgdorferi polypeptides of the present invention may include one
or more
amino acid substitutions, deletions, or additions, either from natural
mutations or human
2o manipulation. As indicated, changes are preferably of a minor nature, such
as conservative amino
acid substitutions that do not significantly affect the folding or activity of
the protein (see Table 3).
Amino acids in the B. burgdorferi proteins of the present invention that are
essential for
function can be identified by methods known in the art, such as site-directed
mutagenesis or
alanine-scanning mutagenesis. See, e.g., Cunningham et al. (1989) Science
244:1081-1085.
The latter procedure introduces single alanine mutations at every residue in
the molecule. The
resulting mutant molecules are then tested for biological activity using
assays appropriate for
measuring the function of the particular protein.
Of special interest are substitutions of charged amino acids with other
charged or neutral
amino acids which may produce proteins with highly desirable improved
characteristics, such as
less aggregation. Aggregation may not only reduce activity but also be
problematic when
preparing pharmaceutical formulations, because aggregates can be immunogenic.
See, e.g.,
Pinckard et al., (1967) Clin. Exp. Immunol. 2:331-340; Robbins, et al., (1987)
Diabetes 36:838-
845; Cleland, et al., ( 1993) Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377.
The polypeptides of the present invention are preferably provided in an
isolated form, and
preferably are substantially purified. A recombinantly produced version of the
B. burgdorferi
polypeptide can be substantially purified by the one-step method described by
Smith et al. ( 1988)
Gene 67:31-40. Polypeptides of the invention also can be purified from natural
or recombinant
sources using antibodies directed against the polypeptides of the invention in
methods which are
well known in the art of protein purification.
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23
The invention further provides for isolated B. burgdorferi polypeptides
comprising an
amino acid sequence selected from the group consisting of: (a) the amino acid
sequence of a full-
length B. burgdorferi polypeptide having the complete amino acid sequence
shown in Table 1;
(b) the amino acid sequence of a full-length B. burgdorferi polypeptide having
the complete amino
acid sequence shown in Table 1 excepting the N-terminal methionine; (c) the
complete amino acid
sequence encoded by the plaimds listed in Table 1; and (d) the complete amino
acid sequence
excepting the N-terminal methionine encoded by the plaimds listed in Table 1.
The polypeptides
of the present invention also include polypeptides having an amino acid
sequence at least 80%
identical, more preferably at least 90% identical, and still more preferably
95%, 96%, 97%, 98%
or 99% identical to those described in (a), (b), (c), and (d) above.
Further polypeptides of the present invention include polypeptides which have
at least
90% similarity, more preferably at least 95% similarity, and still more
preferably at least 96%,
97%, 98% or 99% similarity to those described above.
A further embodiment of the invention relates to a polypeptide which comprises
the amino
acid sequence of a B. burgdorferi polypeptide having an amino acid sequence
which contains at
least one conservative amino acid substitution, but not more than 50
conservative amino acid
substitutions, not more than 40 conservative amino acid substitutions, not
more than 30
conservative amino acid substitutions, and not more than 20 conservative amino
acid
substitutions. Also provided are polypeptides which comprise the amino acid
sequence of a B.
burgdorferi polypeptide, having at least one, but not more than 10, 9, 8, 7,
6, 5, 4, 3, 2 or 1
conservative amino acid substitutions.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to
a query amino acid sequence of the present invention, it is intended that the
amino acid sequence
of the subject polypeptide is identical to the query sequence except that the
subject polypeptide
sequence may include up to five amino acid alterations per each 100 amino
acids of the query
amino acid sequence. In other words, to obtain a polypeptide having an amino
acid sequence at
least 95% identical to a query amino acid sequence, up to 5% of the amino acid
residues in the
subject sequence may be inserted, deleted, (indels) or substituted with
another amino acid. These
alterations of the reference sequence may occur at the amino or carboxy
terminal positions of the
reference amino acid sequence or anywhere between those terminal positions,
interspersed either
individually among residues in the reference sequence or in one or more
contiguous groups within
the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%,
95%, 96%, 97%,
98% or 99% identical to, for instance, the amino acid sequences shown in Table
1 or to the amino
acid sequence encoded by the plaimds listed in Table 1 can be determined
conventionally using
known computer programs. A prefer ed method for determining the best overall
match between a
query sequence (a sequence of the present invention) and a subject sequence,
also referred to as a
global sequence alignment, can be determined using the FASTDB computer program
based on the
algorithm of Brutlag et al., ( 1990) Comp. App. Biosci. 6:237-245. In a
sequence alignment the
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24
query and subject sequences are both amino acid sequences. The result of said
global sequence
alignment is in percent identity. Preferred parameters used in a FASTDB amino
acid alignment
are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,
Randomization Group
Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size
Penalty=0.05, Window Size=500 or the length of the subject amino acid
sequence, whichever is
shorter.
If the subject sequence is shorter than the query sequence due to N- or C-
terminal
deletions, not because of internal deletions, the results, in percent
identity, must be manually
corrected. This is because the FASTDB program does not account for N- and C-
terminal
to truncations of the subject sequence when calculating global percent
identity. For subject
sequences truncated at the N- and C-termini, relative to the query sequence,
the percent identity is
corrected by calculating the number of residues of the query sequence that are
N- and C-terminal
of the subject sequence, which are not matched/aligned with a corresponding
subject residue, as a
percent of the total bases of the query sequence. Whether a residue is
matched/aligned is
15 determined by results of the FASTDB sequence alignment. This percentage is
then subtracted
from the percent identity, calculated by the above FASTDB program using the
specified
parameters, to arrive at a final percent identity score. This final percent
identity score is what is
used for the purposes of the present invention. Only residues to the N- and C-
termini of the
subject sequence, which are not matched/aligned with the query sequence, are
considered for the
20 purposes of manually adjusting the percent identity score. That is, only
query amino acid
residues outside the farthest N- and C-terminal residues of the subject
sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100
residue query
sequence to determine percent identity. The deletion occurs at the N-terminus
of the subject
sequence and therefore, the FASTDB alignment does not match/align with the
first 10 residues at
25 the N-terminus. The 10 unpaired residues represent 10% of the sequence
(number of residues at
the N- and C- termini not matched/total number of residues in the query
sequence) so 10% is
subtracted from the percent identity score calculated by the FASTDB program.
If the remaining
90 residues were perfectly matched the final percent identity would be 90%. In
another example,
a 90 residue subject sequence is compared with a 100 residue query sequence.
This time the
30 deletions are internal so there are no residues at the N- or C-termini of
the subject sequence which
are not matched/aligned with the query. In this case the percent identity
calculated by FASTDB is
not manually corrected. Once again, only residue positions outside the N- and
C-terminal ends of
the subject sequence, as displayed in the FASTDB alignment, which are not
matched/aligned
with the query sequence are manually corrected. No other manual corrections
are to made for the
35 purposes of the present invention.
The above polypeptide sequences are included irrespective of whether they have
their
normal biological activity. This is because even where a particular
polypeptide molecule does not
have biological activity, one of skill in the art would still know how to use
the polypeptide, for
instance, as a vaccine or to generate antibodies. Other uses of the
poiypeptides of the present
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invention that do not have B. burgdorferi activity include, inter alia, as
epitope tags, in epitope
mapping, and as molecular weight markers on SDS-PAGE gels or on molecular
sieve gel
filtration columns using methods known to those of skill in the art.
As described below, the polypeptides of the present invention can also be used
to raise
polyclonal and monoclonal antibodies, which are useful in assays for detecting
B. burgdorferi
protein expression or as agonists and antagonists capable of enhancing or
inhibiting B.
burgdorferi protein function. Further, such polypeptides can be used in the
yeast two-hybrid
system to "capture" B. burgdorferi protein binding proteins which are also
candidate agonists and
antagonists according to the present invention. See, e.g., Fields et al.
(1989) Nature
10 340:245-246.
Epitope-Bearing Portions
In another aspect, the invention provides peptides and polypeptides comprising
epitope-bearing portions of the B. burgdorferi polypeptides of the present
invention. These
15 epitopes are immunogenic or antigenic epitopes of the polypeptides of the
present invention. An
"immunogenic epitope" is defined as a part of a protein that elicits an
antibody response when the
whole protein or polypeptide is the immunogen. These immunogenic epitopes are
believed to be
confined to a few loci on the molecule. On the other hand, a region of a
protein molecule to
which an antibody can bind is defined as an "antigenic determinant" or
"antigenic epitope." The
20 number of immunogenic epitopes of a protein generally is less than the
number of antigenic
epitopes. See, e.g., Geysen, et al. ( 1983) Proc. Natl. Acad. Sci. USA 81:3998-
4002.
Predicted antigenic epitopes are shown in Table 4, below. It is pointed out
that Table 4 only lists
amino acid residues comprising epitopes predicted to have the highest degree
of antigenicity. The
polypeptides not listed in Table 4 and portions of polypeptides not listed in
Table 4 are not
25 considered non-antigenic. This is because they may still be antigenic in
vivo but merely not
recognized as such by the particular algorithm used. Thus, Table 4 lists the
amino acid residues
comprising preferred antigenic epitopes but not a complete list. Amino acid
residues comprising
other anigenic epitopes may be determined by algorithms similar to the Jameson-
Wolf analysis or
by in vivo testing for an antigenic response using the methods described
herein or those known in
the art.
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e., that
contain a region of a protein molecule to which an antibody can bind), it is
well known in that art
that relatively short synthetic peptides that mimic part of a protein sequence
are routinely capable
of eliciting an antiserum that reacts with the partially mimicked protein.
See, e.g., Sutcliffe, et
al., (1983) Science 219:660-666. Peptides capable of eliciting protein-
reactive sera are frequently
represented in the primary sequence of a protein, can be characterized by a
set of simple chemical
rules, and are confined neither to immunodominant regions of intact proteins
(i.e., immunogenic
epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely
hydrophobic and
those of six or fewer residues generally are ineffective at inducing
antibodies that bind to the
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26
mimicked protein; longer, peptides, especially those containing proline
residues, usually are
effective. See, Sutcliffe, et al., supra, p. 661. For instance, 18 of 20
peptides designed
according to these guidelines, containing 8-39 residues covering 75% of the
sequence of the
influenza virus hemagglutinin HA 1 polypeptide chain, induced antibodies that
reacted with the
HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and
18/18 from the
rabies glycoprotein induced antibodies that precipitated the respective
proteins.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore useful
to raise antibodies, including monoclonal antibodies, that bind specifically
to a polypeptide of the
invention. Thus, a high proportion of hybridomas obtained by fusion of spleen
cells from donors
to immunized with an antigen epitope-bearing peptide generally secrete
antibody reactive with the
native protein. See Sutcliffe, et al., supra, p. 663. The antibodies raised by
antigenic
epitope-bearing peptides or polypeptides are useful to detect the mimicked
protein, and antibodies
to different peptides may be used for tracking the fate of various regions of
a protein precursor
which undergoes post-translational processing. The peptides and anti-peptide
antibodies may be
15 used in a variety of qualitative or quantitative assays for the mimicked
protein, for instance in
competition assays since it has been shown that even short peptides (e.g.,
about 9 amino acids)
can bind and displace the larger peptides in immunoprecipitation assays. See,
e.g., Wilson, et
al., ( 1984) Cell 37:767-778. The anti-peptide antibodies of the invention
also are useful fvr
purification of the mimicked protein, for instance, by adsorption
chromatography using methods
2o known in the art.
Antigenic epitope-bearing peptides and polypeptides of the invention designed
according
to the above guidelines preferably contain a sequence of at least seven, more
preferably at least
nine and most preferably between about 10 to about 50 amino acids (i.e. any
integer between 7
and 50) contained within the amino acid sequence of a polypeptide of the
invention. However,
25 peptides or polypeptides comprising a larger portion of an amino acid
sequence of a polypeptide
of the invention, containing about 50 to about 100 amino acids, or any length
up to and including
the entire amino acid sequence of a polypeptide of the invention, also are
considered
epitope-bearing peptides or polypeptides of the invention and also are useful
for inducing
antibodies that react with the mimicked protein. Preferably, the amino acid
sequence of the
30 epitope-bearing peptide is selected to provide substantial solubility in
aqueous solvents (i.e., the
sequence includes relatively hydrophilic residues and highly hydrophobic
sequences are
preferably avoided); and sequences containing proline residues are
particularly preferred.
Non-limiting examples of antigenic polypeptides or peptides that can be used
to generate
an Borrelia-specific immune response or antibodies include portions of the
amino acid sequences
35 identified in Table 1. More specifically, Table 4 discloses a list of non-
limiting residues that are
involved in the antigenicity of the epitope-bearing fragments of the present
invention. Therefore,
the present inventions provides for isolatd and purified antigenic epitope-
bearing fragements of
the polypeptides of the present invention comprising a peptide sequences of
Table 4. The
antigenic epitope-bearing fragments comprising a peptide sequence of Table 4
preferably contain a
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27
sequence of at least seven, more preferably at least nine and most preferably
between about 10 to
about 50 amino acids (i.e. any integer between 7 and SO) of a polypeptide of
the present
invention. That is, included in the present invention are antigenic
polypeptides between the
integers of 7 and 50 amino acid in length comprising one or more of the
sequences of Table 4.
Therefore, in most cases, the polypeptides of Table 4 make up only a portion
of the antigenic
polypeptide. All combinations of sequences between the integers of 7 and 50
amino acid in length
comprising one or more of the sequences of Table 4 are included. The antigenic
epitope-bearing
fragements may be specified by either the number of contiguous amino acid
residues or by
specific N-terminal and C-terminal positions as described above for the
polypeptide fragements of
the present invention, wherein the initiation codon is residue 1. Any number
of the described
antigenic epitope-bearing fragements of the present invention may also be
excluded from the
present invention in the same manner.
The epitope-bearing peptides and polypeptides of the invention may be produced
by any
conventional means for making peptides or polypeptides including recombinant
means using
nucleic acid molecules of the invention. For instance, an epitope-bearing
amino acid sequence of
the present invention may be fused to a larger polypeptide which acts as a
carrier during
recombinant production and purification, as well as during immunization to
produce anti-peptide
antibodies. Epitope-bearing peptides also may be synthesized using known
methods of chemical
synthesis. For instance, Houghten has described a simple method for synthesis
of large numbers
of peptides, such as 10-20 mg of 248 different 13 residue peptides
representing single amino acid
variants of a segment of the HA1 polypeptide which were prepared and
characterized (by
ELISA-type binding studies) in less than four weeks (Houghten, R. A. Proc.
Natl. Acad. Sci.
USA 82:5131-5135 (1985)). This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process
is further described in U.S. Patent No. 4,631,211 to Houghten and coworkers
(1986). In this
procedure the individual resins for the solid-phase synthesis of various
peptides are contained in
separate solvent-permeable packets, enabling the optimal use of the many
identical repetitive steps
involved in solid-phase methods. A completely manual procedure allows 500-1000
or more
syntheses to be conducted simultaneously (Houghten et al. ( 1985) Proc. Natl.
Acad. Sci.
82:5131-5135 at 5134.
Epitope-bearing peptides and polypeptides of the invention are used to induce
antibodies
according to methods well known in the art. See, e.g., Sutcliffe, et al.,
supra;; Wilson, et al.,
supra;; and Bittle, et al. ( 1985) J. Gen. Virol. 66:2347-2354. Generally,
animals may be
immunized with free peptide; however, anti-peptide antibody titer may be
boosted by coupling of
the peptide to a macromolecular Garner, such as keyhole limpet hemacyanin
(KLH) or tetanus
toxoid. For instance, peptides containing cysteine may be coupled to carrier
using a linker such
as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides
may be
coupled to carrier using a more general linking agent such as glutaraldehyde.
Animals such as
rabbits, rats and mice are immunized with either free or carrier-coupled
peptides, for instance, by
intraperitoneal and/or intradermal injection of emulsions containing about 100
p,g peptide or
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28
carrier protein and Freund's adjuvant. Several booster injections may be
needed, for instance, at
intervals of about two weeks, to provide a useful titer of anti-peptide
antibody which can be
detected, for example, by ELISA assay using free peptide adsorbed to a solid
surface. The titer of
anti-peptide antibodies in serum from an immunized animal may be increased by
selection of
anti-peptide antibodies, for instance, by adsorption to the peptide on a solid
support and elution of
the selected antibodies according to methods well known in the art.
Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a
protein that
elicit an antibody response when the whole protein is the immunogen, are
identified according to
methods known in the art. For instance, Geysen, et al. , supra, discloses a
procedure for rapid
concurrent synthesis on solid supports of hundreds of peptides of sufficient
purity to react in an
ELISA. Interaction of synthesized peptides with antibodies is then easily
detected without
removing them from the support. In this manner a peptide bearing an
immunogenic epitope of a
desired protein may be identified routinely by one of ordinary skill in the
art. For instance, the
immunologically important epitope in the coat protein of foot-and-mouth
disease virus was located
by Geysen et al. supra with a resolution of seven amino acids by synthesis of
an overlapping set
of all 208 possible hexapeptides covering the entire 213 amino acid sequence
of the protein.
Then, a complete replacement set of peptides in which x1120 amino acids were
substituted in turn
at every position within the epitope were synthesized, and the particular
amino acids conferring
specificity for the reaction with antibody were determined. Thus, peptide
analogs of the
epitope-bearing peptides of the invention can be made routinely by this
method. U.S. Patent No.
4,708,781 to Geysen ( 1987) further describes this method of identifying a
peptide bearing an
immunogenic epitope of a desired protein.
Further still, U.S. Patent No. 5,194,392, to Geysen ( 1990), describes a
general method
of detecting or determining the sequence of monomers (amino acids or other
compounds) which
is a topological equivalent of the epitope (i.e., a "mimotope") which is
complementary to a
particular paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Patent
No. 4,433,092, also to Geysen ( 1989), describes a method of detecting or
determining a
sequence of monomers which is a topographical equivalent of a ligand which is
complementary to
the ligand binding site of a particular receptor of interest. Similarly, U.S.
Patent No. 5,480,971
to Houghten, R. A. et al. ( 1996) discloses linear C,-C~-alkyl peralkylated
oligopeptides and sets
and libraries of such peptides, as well as methods for using such oligopeptide
sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor
molecule of interest. Thus, non-peptide analogs of the epitope-bearing
peptides of the invention
also can be made routinely by these methods. The entire disclosure of each
document cited in this
section on "PoIypeptides and Fragments" is hereby incorporated herein by
reference.
As one of skill in the art will appreciate, the polypeptides of the present
invention and the
epitope-bearing fragments thereof described above can be combined with parts
of the constant
domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins
facilitate purification and show an increased half-life in vivo. This has been
shown, e.g., for
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29
chimeric proteins consisting of the first two domains of the human CD4-
polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins.
(EPA 0,394,827; Traunecker et al. ( 1988) Nature 331:84-86. Fusion proteins
that have a
disulfide-linked dimeric structure due to the IgG part can also be more
efficient in binding and
neutralizing other molecules than a monomeric B. burgdorferi polypeptide or
fragment thereof
alone. See Fountoulakis et al. ( 1995) J. Biochem. 270:3958-3964. Nucleic
acids encoding the
above epitopes of B. burgdorferi polypeptides can also be recombined with a
gene of interest as
an epitope tag to aid in detection and purification of the expressed
polypeptide.
1o Antibodies
B. burgdorferi protein-specific antibodies for use in the present invention
can be raised
against the intact B. burgdorferi protein or an antigenic polypeptide fragment
thereof, which may
be presented together with a carrier protein, such as an albumin, to an animal
system (such as
rabbit or mouse) or, if it is long enough (at least about 25 amino acids),
without a Garner.
15 As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is
meant to
include intact molecules, single chain whole antibodies, and antibody
fragments. Antibody
fragments of the present invention include Fab and F(ab')2 and other fragments
including single-
chain Fvs (scFv) and disulfide-linked Fvs (sdFv). Also included in the present
invention are
chimeric and humanized monoclonal antibodies and polyclonal antibodies
specific for the
20 polypeptides of the present invention. The antibodies of the present
invention may be prepared by
any of a variety of methods. For example, cells expressing a polypeptide of
the present invention
or an antigenic fragment thereof can be administered to an animal in order to
induce the production
of sera containing polyclonal antibodies. For example, a preparation of B.
burgdorferi
polypeptide or fragment thereof is prepared and purified to render it
substantially free of natural
25 contaminants. Such a preparation is then introduced into an animal in order
to produce polyclonal
antisera of greater specific activity.
In a preferred method, the antibodies of the present invention are monoclonal
antibodies or
binding fragments thereof. Such monoclonal antibodies can be prepared using
hybridoma
technology. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold
3o Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:
MONOCLONAL
ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981 ). Fab and
F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such
as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
Alternatively, B. burgdorferi
polypeptide-binding fragments, chimeric, and humanized antibodies can be
produced through the
35 application of recombinant DNA technology or through synthetic chemistry
using methods known
in the art.
Alternatively, additional antibodies capable of binding to the polypeptide
antigen of the
present invention may be produced in a two-step procedure through the use of
anti-idiotypic
antibodies. Such a method makes use of the fact that antibodies are themselves
antigens, and that,
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therefore, it is possible to obtain an antibody which binds to a second
antibody. In accordance
with this method, B. burgdorferi polypeptide-specific antibodies are used to
immunize an animal,
preferably a mouse. The splenocytes of such an animal are then used to produce
hybridoma cells,
and the hybridoma cells are screened to identify clones which produce an
antibody whose ability
5 to bind to the B. burgdorferi polypeptide-specific antibody can be blocked
by the B. burgdorferi
polypeptide antigen. Such antibodies comprise anti-idiotypic antibodies to the
B. burgdorferi
polypeptide-specific antibody and can be used to immunize an animal to induce
formation of
further B. burgdorferi polypeptide-specific antibodies.
Antibodies and fragements thereof of the present invention may be described by
the
10 portion of a polypeptide of the present invention recognized or
specifically bound by the antibody.
Antibody binding fragements of a polypeptide of the present invention may be
described or
specified in the same manner as for polypeptide fragements discussed above.,
i.e, by N-terminal
and C-terminal positions or by size in contiguous amino acid residues. Any
number of antibody
binding fragments, of a polypeptide of the present invention, specified by N-
terminal and C-
15 terminal positions or by size in amino acid residues, as described above,
may also be excluded
from the present invention. Therefore, the present invention includes
antibodies the specifically
bind a particuarlly discribed fragement of a polypeptide of the present
invention and allows for the
exclusion of the same.
Antibodies and fragements thereof of the present invention may also be
described or
20 specified in terms of their cross-reactivity. Antibodies and fragements
that do not bind
polypeptides of any other species of Borrelia other than B. burgdorferi are
included in the present
invention. Likewise, antibodies and fragements that bind only species of
Borredia, i.e. antibodies
and fragements that do not bind bacteria from any genus other than Borrelia,
are included in the
present invention.
Diagnostic Assays
The present invention further relates to methods for assaying staphylococcal
infection in
an animal by detecting the expression of genes encoding staphylococcal
polypeptides of the
present invention. The methods comprise analyzing tissue or body fluid from
the animal for
Borrelia-specific antibodies, nucleic acids, or proteins. Analysis of nucleic
acid specific to
Borrelia is assayed by PCR or hybridization techniques using nucleic acid
sequences of the
present invention as either hybridization probes or primers. See, e.g.,
Sambrook et al. Molecular
cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.,
1989, page 54
reference); Eremeeva et al. ( 1994) J. Clin. Microbiol. 32:803-810 (describing
differentiation
among spotted fever group Rickettsiae species by analysis of restriction
fragment length
polymorphism of PCR-amplified DNA) and Chen et al. 1994 J. Clin. Microbiol.
32:589-595
(detecting B. burgdorferi nucleic acids via PCR).
Where diagnosis of a disease state related to infection with Borrelia has
already been
made, the present invention is useful for monitoring progression or regression
of the disease state
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31
whereby patients exhibiting enhanced Borrelia gene expression will experience
a worse clinical
outcome relative to patients expressing these genes) at a lower level.
By "biological sample" is intended any biological sample obtained from an
animal, cell
line, tissue culture, or other source which contains Borrelia polypeptide,
mRNA, or DNA.
Biological samples include body fluids (such as saliva, blood, plasma, urine,
mucus, synovial
fluid, etc.) tissues (such as muscle, skin, and cartilage) and any other
biological source suspected
of containing Borrelia polypeptides or nucleic acids. Methods for obtaining
biological samples
such as tissue are well known in the art.
The present invention is useful for detecting diseases related to Borrelia
infections in
to animals. Preferred animals include monkeys, apes, cats, dogs, birds, cows,
pigs, mice, horses,
rabbits and humans. Particularly preferred are humans.
Total RNA can be isolated from a biological sample using any suitable
technique such as
the single-step guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski et
al. (1987) Anal. Biochem. 162:156-159. mRNA encoding Borrelia polypeptides
having sufficient
15 homology to the nucleic acid sequences identified in Table 1 to allow for
hybridization between
complementary sequences are then assayed using any appropriate method. These
include
Northern blot analysis, S 1 nuclease mapping, the polymerise chain reaction
(PCR), reverse
transcription in combination with the polymerise chain reaction (RT-PCR), and
reverse
transcription in combination with the ligase chain reaction (RT-LCR).
20 Northern blot analysis can be performed as described in Harada et al. (
1990) Cell
63:303-312. Briefly, total RNA is prepared from a biological sample as
described above. For the
Northern blot, the RNA is denatured in an appropriate buffer (such as
glyoxal/dimethyl
sulfoxide/sodium phosphate buffer), subjected to agarose gel electrophoresis,
and transferred
onto a nitrocellulose filter. After the RNAs have been linked to the filter by
a UV linker, the filter
25 is prehybridized in a solution containing formamide, SSC, Denhardt's
solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. A B. burgdorferi polynucleotide
sequence shown in
Table 1 labeled according to any appropriate method (such as the 32P-
multiprimed DNA labeling
system (Amersham)) is used as probe. After hybridization overnight, the filter
is washed and
exposed to x-ray film. DNA for use as probe according to the present invention
is described in
30 the sections above and will preferably at least 15 nucleotides in length.
S 1 mapping can be performed as described in Fujita et al. ( 1987) Cell 49:357-
367. To
prepare probe DNA for use in S 1 mapping, the sense strand of an above-
described B. burgdorferi
DNA sequence of the present invention is used as a template to synthesize
labeled antisense DNA.
The antisense DNA can then be digested using an appropriate restriction
endonuclease to generate
35 further DNA probes of a desired length. Such antisense probes are useful
for visualizing
protected bands corresponding to the target mRNA {i.e., mRNA encoding Borrelia
polypeptides).
Levels of mRNA encoding Borrelia polypeptides are assayed, for e.g., using the
RT-PCR
method described in Makino et al. { 1990} Technique 2:295-301. By this method,
the
radioactivities of the "amplicons" in the polyacrylamide gel bands are
linearly related to the initial
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32
concentration of the target mRNA. Briefly, this method involves adding total
RNA isolated from
a biological sample in a reaction mixture containing a RT primer and
appropriate buffer. After
incubating for primer annealing, the mixture can be supplemented with a RT
buffer, dNTPs,
DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve
reverse transcription
of the RNA, the RT products are then subject to PCR using labeled primers.
Alternatively, rather
than labeling the primers, a labeled dNTP can be included in the PCR reaction
mixture. PCR
amplification can be performed in a DNA thermal cycler according to
conventional techniques.
After a suitable number of rounds to achieve amplification, the PCR reaction
mixture is
electrophoresed on a polyacrylamide gel. After drying the gel, the
radioactivity of the appropriate
bands (corresponding to the mRNA encoding the Borrelia polypeptides of the
present invention)
are quantified using an imaging analyzer. RT and PCR reaction ingredients and
conditions,
reagent and gel concentrations, and labeling methods are well known in the
art. Variations on the
RT-PCR method will be apparent to the skilled artisan. Other PCR methods that
can detect the
nucleic acid of the present invention can be found in PCR PRIMER: A LABORATORY
MANUAL (C.W. Dieffenbach et al. eds., Cold Spring Harbor Lab Press, 1995).
The polynucleotides of the present invention, including both DNA and RNA, may
be used
to detect polynucleotides of the present invention or Borrelia species
including B. burgdorferi
using bio chip technology. The present invention includes both high density
chip arrays (> 1000
oligonucleotides per cmz) and low density chip arrays (<1000 oligonucleotides
per cm2). Bio
chips comprising arrays of polynucleotides of the present invention may be
used to detect Borrelia
species, including B. burgdorferi, in biological and environmental samples and
to diagnose an
animal, including humans, with an B. burgdorferi or other Borrelia infection.
The bio chips of
the present invention may comprise polynucleotide sequences of other pathogens
including
bacteria, viral, parasitic, and fungal polynucleotide sequences, in addition
to the polynucleotide
sequences of the present invention, for use in rapid diffenertial pathogenic
detection and
diagnosis. The bio chips can also be used to monitor an B. burgdorferi or
other Borrelia
infections and to monitor the genetic changes (deletions, insertions,
mismatches, etc.) in response
to drug therapy in the clinic and drug development in the laboratory. The bio
chip technology
comprising arrays of polynucleotides of the present invention may also be used
to simultaneously
monitor the expression of a multiplicity of genes, including those of the
present invention. The
polynucleotides used to comprise a selected array may be specified in the same
manner as for the
fragements, i.e, by their S' and 3' positions or length in contigious base
pairs and include from.
Methods and particular uses of the polynucleotides of the present invention to
detect Borrelia
species, including B. burgdorferi, using bio chip technology include those
known in the art and
those of: U.S. Patent Nos. 5510270, 5545531, 5445934, 5677195, 5532128,
5556752,
5527681, 5451683, 5424186, 5607646, 5658732 and World Patent Nos. WO/9710365,
WO/9511995, WO/9743447, WO/9535505, each incorporated herein in their
entireties.
Biosensors using the polynucleotides of the present invention may also be used
to detect,
diagnose, and monitor B. burgdorferi or other Borrelia species and infections
thereof.
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33
Biosensors using the polynucleotides of the present invention may also be used
to detect particular
polynucleotides of the present invention. Biosensors using the polynucleotides
of the present
invention may also be used to monitor the genetic changes (deletions,
insertions, mismatches,
etc.) in response to drug therapy in the clinic and drug development in the
laboratory. Methods
and particular uses of the polynucleotides of the present invention to detect
Borrelia species,
including B. burgdorferi, using biosenors include those known in the art and
those of: U.S.
Patent Nos 5721102, 5658732, 5631170, and World Patent Nos. W097/35011,
WO/9720203,
each incorporated herein in their entireties.
Thus, the present invention includes both bio chips and biosensors comprising
1o polynucleotides of the present invention and methods of their use.
Assaying Borrelia polypeptide levels in a biological sample can occur using
any art-known
method, such as antibody-based techniques. For example, Borrelia polypeptide
expression in
tissues can be studied with classical immunohistological methods. In these,
the specific .
recognition is provided by the primary antibody (polyclonal or monoclonal) but
the secondary
15 detection system can utilize fluorescent, enzyme, or other conjugated
secondary antibodies. As a
result, an immunohistological staining of tissue section for pathological
examination is obtained.
Tissues can also be extracted, e.g., with urea and neutral detergent, for the
liberation of Borrelia
polypeptides for Western-blot or dot/slot assay. See, e.g., Jalkanen, M. et
al. ( 1985) J. Cell.
Biol. 101:976-985; Jalkanen, M. et al. (1987) J. Cell . Biol. 105:3087-3096.
In this technique,
2o which is based on the use of cationic solid phases, quantitation of a
Borrelia polypeptide can be
accomplished using an isolated Borrelia polypeptide as a standard. This
technique can also be
applied to body fluids.
Other antibody-based methods useful for detecting Borrelia polypeptide gene
expression
include immunoassays, such as the ELISA and the radioimmunoassay (RIA). For
example, a
25 Borrelia polypeptide-specific monoclonal antibodies can be used both as an
immunoabsorbent and
as an enzyme-labeled probe to detect and quantify a Borrelia polypeptide. The
amount of a
Borrelia polypeptide present in the sample can be calculated by reference to
the amount present in
a standard preparation using a linear regression computer algorithm. Such an
ELISA is described
in Iacobelli et al. ( 1988) Breast Cancer Research and Treatment 11:19-30. In
another ELISA
3o assay, two distinct specific monoclonal antibodies can be used to detect
Borrelia polypeptides in a
body fluid. In this assay, one of the antibodies is used as the
immunoabsorbent and the other as
the enzyme-labeled probe.
The above techniques may be conducted essentially as a "one-step" or "two-
step" assay.
The "one-step" assay involves contacting the Borrelia polypeptide with
immobilized antibody and,
35 without washing, contacting the mixture with the labeled antibody. The "two-
step" assay
involves washing before contacting the mixture with the labeled antibody.
Other conventional
methods may also be employed as suitable. It is usually desirable to
immobilize one component
of the assay system on a support, thereby allowing other components of the
system to be brought
into contact with the component and readily removed from the sample.
Variations of the above
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34
and other immunological methods included in the present invention can also be
found in Harlow
et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press,
2nd ed. 1988).
Suitable enzyme labels include, for example, those from the oxidase group,
which
catalyze the production of hydrogen peroxide by reacting with substrate.
Glucose oxidase is
particularly preferred as it has good stability and its substrate (glucose) is
readily available.
Activity of an oxidase label may be assayed by measuring the concentration of
hydrogen peroxide
formed by the enzyme-labeled antibody/substrate reaction. Besides enzymes,
other suitable labels
include radioisotopes, such as iodine ('ZSI, '2'I), carbon ('4C), sulphur
(3sS), tritium (~H), indium
("ZIn), and technetium (~'"Tc), and fluorescent labels, such as fluorescein
and rhodamine, and
biotin.
Further suitable labels for the Borrelia polypeptide-specific antibodies of
the present
invention are provided below. Examples of suitable enzyme labels include
malate dehydrogenase,
Borrelia nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase,
alpha-glycerol
phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,
catalase,
glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.
Examples of suitable radioisotopic labels include 3H, "'In,'ZSI,'3'h 32P,
3sS,'aC, s'Cr,
s'To, sBCo, s9Fe,'sSe, 's2Eu, 9°Y, 6'Cu, 2"Ci, z"At, 2'ZPb, 4'Sc,
'°9Pd, etc. "'In is a preferred
2o isotope where in vivo imaging is used since its avoids the problem of
dehalogenation of the "~I or
'3'I-labeled monoclonal antibody by the liver. In addition, this
radionucleotide has a more
favorable gamma emission energy for imaging. See, e.g., Perkins et al. ( 1985)
Eur. J. Nucl.
Med. 10:296-301; Carasquillo et al. (1987) J. Nucl. Med. 28:281-287. For
example, "'In
coupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTA has
shown little uptake
in non-tumors tissues, particularly the liver, and therefore enhances
specificity of tumor
localization. See, Esteban et al. (1987) J. Nucl. Med. 28:861-870.
Examples of suitable non-radioactive isotopic labels include 's'Gd, ssMn,
'6'Dy, s2Tr, and
sbFe.
Examples of suitable fluorescent labels include an's2Eu label, a fluorescein
label, an
3U isothiocyanate label, a rhodamine label, a phycoerythrin label, a
phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.
Examples of suitable toxin labels include, Pseudomonas toxin, diphtheria
toxin, ricin, and
cholera toxin.
Examples of chemiluminescent labels include a luminal label, an isoluminal
label, an
aromatic acridinium ester label, an imidazole label, an acridinium salt label,
an oxalate ester label,
a luciferin label, a luciferase label, and an aequorin label.
Examples of nuclear magnetic resonance contrasting agents include heavy metal
nuclei
such as Gd, Mn, and iron.
Typical techniques for binding the above-described labels to antibodies are
provided by
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Kennedy et al. ( 1976) Clin. Chim. Acta 70:1-31, and Schurs et aI. ( 1977)
Clin. Chim. Acta
81:1-40. Coupling techniques mentioned in the latter are the glutaraldehyde
method, the periodate
method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide
ester method,
all of which methods are incorporated by reference herein.
5 In a related aspect, the invention includes a diagnostic kit for use in
screening serum
containing antibodies specific against B. burgdorferi infection. Such a kit
may include an
isolated B. burgdorferi antigen comprising an epitope which is specifically
immunoreactive with
at least one anti-B. burgdorferi antibody. Such a kit also includes means for
detecting the
binding of said antibody to the antigen. In specific embodiments, the kit may
include a
to recombinantly produced or chemically synthesized peptide or polypeptide
antigen. The peptide or
polypeptide antigen may be attached to a solid support.
In a more specific embodiment, the detecting means of the above-described kit
includes a
solid support to which said peptide or polypeptide antigen is attached. Such a
kit may also
include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the
15 antibody to the B. burgdorferi antigen can be detected by binding of the
reporter labeled antibody
to the anti-B. burgdorferi polypeptide antibody.
In a related aspect, the invention includes a method of detecting B.
burgdorferi infection in
a subject. This detection method includes reacting a body fluid, preferably
serum, from the
subject with an isolated B. burgdorferi antigen, and examining the antigen for
the presence of
2o bound antibody. In a specific embodiment, the method includes a polypeptide
antigen attached to
a solid support, and serum is reacted with the support. Subsequently, the
support is reacted with
a reporter-labeled anti-human antibody. The support is then examined for the
presence of
reporter-labeled antibody.
The solid surface reagent employed in the above assays and kits is prepared by
known
25 techniques for attaching protein material to solid support material, such
as polymeric beads, dip
sticks, 96-well plates or filter material. These attachment methods generally
include non-specific
adsorption of the protein to the support or covalent attachment of the protein
, typically through a
free amine group, to a chemically reactive group on the solid support, such as
an activated
carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated
plates can be used in
3o conjunction with biotinylated antigen(s).
The polypeptides and antibodies of the present invention, including fragments
thereof,
may be used to detect Borrelia species including B. burgdorferi using bio chip
and biosensor
technology. Bio chip and biosensors of the present invention may comprise the
polypeptides of
the present invention to detect antibodies, which specifically recognize
Borreiia species, including
35 B. burgdorferi. Bio chip and biosensors of the present invention may also
comprise antibodies
which specifically recognize the polypeptides of the present invention to
detect Borrelia species,
including B. burgdorferi or specific polypeptides of the present invention.
Bio chips or
biosensors comprising polypeptides or antibodies of the present invention may
be used to detect
Borrelia species, including B. burgdorferi, in biological and environmental
samples and to
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36
diagnose an animal, including humans, with an B. burgdorferi or other Borrelia
infection. Thus,
the present invention includes both bio chips and biosensors comprising
polypeptides or
antibodies of the present invention and methods of their use.
The bio chips of the present invention may further comprise polypeptide
sequences of
other pathogens including bacteria, viral, parasitic, and fungal polypeptide
sequences, in addition
to the poiypeptide sequences of the present invention, for use in rapid
diffenertial pathogenic
detection and diagnosis. The bio chips of the present invention may further
comprise antibodies
or fragements thereof specific for other pathogens including bacteria, viral,
parasitic, and fungal
polypeptide sequences, in addition to the antibodies or fragements thereof of
the present
invention, for use in rapid diffenertial pathogenic detection and diagnosis.
The bio chips and
biosensors of the present invention may also be used to monitor an B.
burgdorferi or other
Borrelia infection and to monitor the genetic changes (amio acid deletions,
insertions,
substitutions, etc.) in response to drug therapy in the clinic and drug
development in the
laboratory. The bio chip and biosensors comprising polypeptides or antibodies
of the present
invention may also be used to simultaneously monitor the expression of a
multiplicity of
polypeptides, including those of the present invention. The polypeptides used
to comprise a bio
chip or biosensor of the present invention may be specified in the same manner
as for the
fragements, i.e, by their N-terminal and C-terminal positions or length in
contigious amino acid
residue. Methods and particular uses of the polypeptides and antibodies of the
present invention
to detect Borrelia species, including B. burgdorferi, or specific polypeptides
using bio chip and
biosensor technology include those known in the art, those of the U.S. Patent
Nos. and World
Patent Nos. listed above for bio chips and biosensors using polynucleotides of
the present
invention, and those of: U.S. Patent Nos. 5658732, 5135852, 5567301, 5677196,
5690894 and
World Patent Nos. W09729366, W09612957, each incorporated herein in their
entireties.
Treatment:
Agonists and Antagonists - Assays and Molecules
The invention also provides a method of screening compounds to identify those
which
enhance or block the biological activity of the B. burgdorferi polypeptides of
the present
invention. The present invention further provides where the compounds kill or
slow the growth
of B. burgdorferi. The ability of B. burgdorferi antagonists, including B.
burgdorferi ligands, to
prophylactically or therapeutically block antibiotic resistance may be easily
tested by the skilled
artisan. See, e.g., Straden et al. {1997) J Bacteriol. 179(1):9-16.
An agonist is a compound which increases the natural biological function or
which
functions in a manner similar to the polypeptides of the present invention,
while antagonists
decrease or eliminate such functions. Potential antagonists include small
organic molecules,
peptides, polypeptides, and antibodies that bind to a polypeptide of the
invention and thereby
inhibit or extinguish its activity.
The antagonists may be employed for instance to inhibit peptidoglycan cross
bridge
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37
formation. Antibodies against B. burgdorferi may be employed to bind to and
inhibit B.
burgdorferi activity to treat antibiotic resistance. Any of the above
antagonists may be employed
in a composition with a pharmaceutically acceptable carrier.
Vaccines
The present invention also provides vaccines comprising one or more
polypeptides of the
present invention. Heterogeneity in the composition of a vaccine may be
provided by combining
B. burgdorferi polypeptides of the present invention. Multi-component vaccines
of this type are
desirable because they are likely to be more effective in eliciting protective
immune responses
against multiple species and strains of the Borrelia genus than single
polypeptide vaccines. Thus,
as discussed in detail below, a mufti-component vaccine of the present
invention may contain one
or more, preferably 2 to about 20, more preferably 2 to about 15, and most
preferably 3 to about
8, of the B. burgdorferi polypeptides shown in Table 1, or fragments thereof.
Mufti-component vaccines are known in the art to elicit antibody production to
numerous
immunogenic components. Decker, M. and Edwards, K., J. Infect. Dis. 174:S270-
275 (1996).
In addition, a hepatitis B, diphtheria, tetanus, pertussis tetravalent vaccine
has recently been
demonstrated to elicit protective levels of antibodies in human infants
against all four pathogenic
agents. Aristegui, J. et al., Vaccine 15:7-9 (1997).
The present invention thus also includes mufti-component vaccines. These
vaccines
comprise more than one polypeptide, immunogen or antigen. An example of such a
multi-
component vaccine would be a vaccine comprising more than one of the B.
burgdorferi
polypeptides shown in Table 1. A second example is a vaccine comprising one or
more, for
example 2 to 10, of the B. burgdorferi polypeptides shown in Table 1 and one
or more, for
example 2 to I0, additional polypeptides of either borrelial or non-borrelial
origin. Thus, a multi-
2s component vaccine which confers protective immunity to both a borrelial
infection and infection
by another pathogenic agent is also within the scope of the invention.
As indicated above, the vaccines of the present invention are expected to
elicit a protective
immune response against infections caused by species and strains of Borrelia
other than B.
burgdorferi sensu stricto isolate B31 (ATCC Accession No. 35210).
Immunizations using
decorin-binding protein and OspA derived from one strain of B. burgdorferi has
been shown to
elicit the production of antiserum which confers passive immunity against
other strains of B.
burgdorferi. Cassatt, D. et al., Protection of Borrelia burgdorferi Infection
by Antibodies to
Decorin-binding Protein, in VACCINES97, Cold Spring Harbor Press (1997), pages
191-195.
Further, the inventors have found using an in vitro assay that antiserum
produced in response to
B. burgdorferi decorin-binding protein will kill several species of Borrelia.
The amino acid
sequences of decorin-binding protein expressed by different strains of B.
burgdorferi are
believed to diverge by as much as 25%. Thus, antisera elicited against decorin-
binding proteins
confers passive immunity against Borrelia expressing proteins having only 75%
or less amino
acid sequence similarity.
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38
Further within the scope of the invention are whole cell and whole viral
vaccines. Such
vaccines may be produced recombinantly and involve the expression of one or
more of the
B. burgdorferi polypeptides shown in Table 1. For example, the B. burgdorferi
polypeptides of
the present invention may be either secreted or localized intracellular, on
the cell surface, or in the
periplasmic space. Further, when a recombinant virus is used, the B.
burgdorferi polypeptides
of the present invention may, for example, be localized in the viral envelope,
on the surface of the
capsid, or internally within the capsid. Whole cells vaccines which employ
cells expressing
heterologous proteins are known in the art. See, e.g., Robinson, K. et al.,
Nature Biotech.
15:653-657 (1997); Sirard, J. et al., Infect. Immun. 65:2029-2033 (1997);
Chabalgoity, J. et al.,
Infect. Immun. 65:2402-2412 ( 1997). These cells may be administered live or
may be killed
prior to administration. Chabalgoity, J. et al., supra, for example, report
the successful use in
mice of a live attenuated Salmonella vaccine strain which expresses a portion
of a platyhelminth
fatty acid-binding protein as a fusion protein on its cells surface.
A mufti-component vaccine can also be prepared using techniques known in the
art by
1 S combining one or more B. burgdorferi polypeptides of the present
invention, or fragments
thereof, with additional non-borrelial components (e.g., diphtheria toxin or
tetanus toxin, and/or
other compounds known to elicit an immune response). Such vaccines are useful
for eliciting
protective immune responses to both members of the Borrelia genus and non-
borrelial pathogenic
agents.
The vaccines of the present invention also include DNA vaccines. DNA vaccines
are
currently being developed for a number of infectious diseases. Boyer, J et
al., Nat. Med. 3:526-
532 ( 1997); reviewed in Spier, R., Vaccine 14:1285-1288 ( 1996). Such DNA
vaccines contain a
nucleotide sequence encoding one or more B. burgdorferi polypeptides of the
present invention
oriented in a manner that allows for expression of the subject polypeptide.
The direct
administration of plasmid DNA encoding OspA has been shown to elicit
protective immunity in
mice against borrelial challenge. Luke, C. et al., J. Infect. Dis. 175:91-97 (
1997).
The present invention also relates to the administration of a vaccine which is
co-administered with a molecule capable of modulating immune responses. Kim,
J. et al., Nature
Biotech. 15:641-646 (1997), for example, report the enhancement of immune
responses produced
by DNA immunizations when DNA sequences encoding molecules which stimulate the
immune
response are co-administered. In a similar fashion, the vaccines of the
present invention may be
co-administered with either nucleic acids encoding immune modulators or the
immune modulators
themselves. These immune modulators include granulocyte macrophage colony
stimulating factor
(GM-CSF) and CD86.
The vaccines of the present invention may be used to confer resistance to
borrelial
infection by either passive or active immunization. When the vaccines of the
present invention are
used to confer resistance to borrelial infection through active immunization,
a vaccine of the
present invention is administered to an animal to elicit a protective immune
response which either
prevents or attenuates a borrelial infection. When the vaccines of the present
invention are used to
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39
confer resistance to borrelial infection through passive immunization, the
vaccine is provided to a
host animal (e.g., human, dog, or mouse), and the antisera elicited by this
antisera is recovered
and directly provided to a recipient suspected of having an infection caused
by a member of the
Borrelia genus.
The ability to label antibodies, or fragments of antibodies, with toxin
molecules provides
an additional method for treating borrelial infections when passive
immunization is conducted. In
this embodiment, antibodies, or fragments of antibodies, capable of
recognizing the
B. burgdorferi polypeptides disclosed herein, or fragments thereof, as well as
other Borrelia
proteins, are labeled with toxin molecules prior to their administration to
the patient. When such
toxin derivatized antibodies bind to Borrelia cells, toxin moieties will be
localized to these cells
and will cause their death.
The present invention thus concerns and provides a means for preventing or
attenuating a
borrelial infection resulting from organisms which have antigens that are
recognized and bound by
antisera produced in response to the polypeptides of the present invention. As
used herein, a
vaccine is said to prevent or attenuate a disease if its administration to an
animal results either in
the total or partial attenuation (i.e., suppression) of a symptom or condition
of the disease, or in
the total or partial immunity of the animal to the disease.
The administration of the vaccine (or the antisera which it elicits) may be
for either a
"prophylactic" or "therapeutic" purpose. When provided prophylactically, the
compounds) are
provided in advance of any symptoms of borrelial infection. The prophylactic
administration of
the compounds) serves to prevent or attenuate any subsequent infection. When
provided
therapeutically, the compounds) is provided upon or after the detection of
symptoms which
indicate that an animal may be infected with a member of the Borrelia genus.
The therapeutic
administration of the compounds) serves to attenuate any actual infection.
Thus, the
B. burgdorferi polypeptides, and fragments thereof, of the present invention
may be provided
either prior to the onset of infection (so as to prevent or attenuate an
anticipated infection) or after
the initiation of an actual infection.
The polypeptides of the invention, whether encoding a portion of a native
protein or a
functional derivative thereof, may be administered in pure form or may be
coupled to a
3o macromolecular carrier. Example of such Garners are proteins and
carbohydrates. Suitable
proteins which may act as macromolecular carrier for enhancing the
immunogenicity of the
polypeptides of the present invention include keyhole limpet hemacyanin (KLH)
tetanus toxoid,
pertussis toxin, bovine serum albumin, and ovalbumin. Methods for coupling the
polypeptides of
the present invention to such macromolecular carriers are disclosed in Harlow
et al., Antibodies:
A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New
York ( 1988), the entire disclosure of which is incorporated by reference
herein.
A composition is said to be "pharmacologically acceptable" if its
administration can be
tolerated by a recipient animal and is otherwise suitable for administration
to that animal. Such an
agent is said to be administered in a "therapeutically effective amount" if
the amount administered
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is physiologically significant. An agent is physiologically significant if its
presence results in a
detectable change in the physiology of a recipient patient.
While in all instances the vaccine of the present invention is administered as
a
pharmacologically acceptable compound, one skilled in the art would recognize
that the
5 composition of a pharmacologically acceptable compound varies with the
animal to which it is
administered. For example, a vaccine intended for human use will generally not
be co-
administered with Freund's adjuvant. Further, the level of purity of the B.
burgdorferi
polypeptides of the present invention will normally be higher when
administered to a human than
when administered to a non-human animal.
1o As would be understood by one of ordinary skill in the art, when the
vaccine of the
present invention is provided to an animal, it may be in a composition which
may contain salts,
buffers, adjuvants, or other substances which are desirable for improving the
efficacy of the
composition. Adjuvants are substances that can be used to specifically augment
a specific
immune response. These substances generally perform two functions: ( 1 ) they
protect the
15 antigens) from being rapidly catabolized after administration and (2) they
nonspecifically
stimulate immune responses.
Normally, the adjuvant and the composition are mixed prior to presentation to
the immune
system, or presented separately, but into the same site of the animal being
immunized. Adjuvants
can be loosely divided into several groups based upon their composition. These
groups include
20 oil adjuvants (for example, Freund's complete and incomplete), mineral
salts (for example,
AlK(S04)2, AINa(S04)2, A1NH4(S04), silica, kaolin, and carbon),
polynucleotides (for example,
poly IC and poly AU acids), and certain natural substances (for example, wax D
from
Mycobacterium tuberculosis, as well as substances found in Corynebacterium
parvum, or
Bordetella pertussis, and members of the genus Brucella. Other substances
useful as adjuvants
25 are the saponins such as, for example, Quil A. (Superfos A/S, Denmark).
Preferred adjuvants for
use in the present invention include aluminum salts, such as A1K(S04)2,
AINa(S04)2, and
A1NH4(S04). Examples of materials suitable for use in vaccine compositions are
provided in
Remington's Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton,
PA, pp. 1324-
1341 ( 1980), which reference is incorporated herein by reference).
3o The therapeutic compositions of the present invention can be administered
parenterally by
injection, rapid infusion, nasopharyngeal absorption (intranasopharangeally),
dermoabsorption,
or orally. The compositions may alternatively be administered intramuscularly,
or intravenously.
Compositions for parenteral administration include sterile aqueous or non-
aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are propylene
glycol,
35 polyethylene glycol, vegetable oils such as olive oil, and injectable
organic esters such as ethyl
oleate. Carriers or occlusive dressings can be used to increase skin
permeability and enhance
antigen absorption. Liquid dosage forms for oral administration may generally
comprise a
Iiposome solution containing the liquid dosage form. Suitable forms for
suspending liposomes
include emulsions, suspensions, solutions, syrups, and elixirs containing
inert diluents
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41
commonly used in the art, such as purified water. Besides the inert diluents,
such compositions
can also include adjuvants, wetting agents, emulsifying and suspending agents,
or sweetening,
flavoring, or perfuming agents.
Therapeutic compositions of the present invention can also be adn>inistered in
encapsulated form. For example, intranasal immunization of mice against
Bordetella pertussis
infection using vaccines encapsulated in biodegradable microsphere composed of
poly(DL-lactide-
co-glycolide) has been shown to stimulate protective immune responses. Shahin,
R. et al.,
Infect. Immun. 63:1195-1200 (1995). Similarly, orally administered
encapsulated Salmonella
typhimurium antigens have also been shown to elicit protective immunity in
mice. Allaoui-
Attarki, K. et al., Infect. Immun. 65:853-857 { 1997). Encapsulated vaccines
of the present
invention can be administered by a variety of routes including those involving
contacting the
vaccine with mucous membranes (e.g., intranasally, intracolonicly,
intraduodenally).
Many different techniques exist for the timing of the immunizations when a
multiple
administration regimen is utilized. It is possible to use the compositions of
the invention more
than once to increase the levels and diversities of expression of the
immunoglobulin repertoire
expressed by the immunized animal. Typically, if multiple immunizations are
given, they will be
given one to two months apart.
According to the present invention, an "effective amount" of a therapeutic
composition is
one which is sufficient to achieve a desired biological effect. Generally, the
dosage needed to
provide an effective amount of the composition will vary depending upon such
factors as the
animal's or human's age, condition, sex, and extent of disease, if any, and
other variables which
can be adjusted by one of ordinary skill in the art.
The antigenic preparations of the invention can be administered by either
single or multiple
dosages of an effective amount. Effective amounts of the compositions of the
invention can vary
from 0.01-1,000 pg/ml per dose, more preferably 0.1-500 p,g/ml per dose, and
most preferably
10-300 p.g/ml per dose.
Having now generally described the invention, the same will be more readily
understood
through reference to the following example which is provided by way of
illustration, and is not
intended to be limiting of the present invention, unless specified.
Examples
1. Preparation of PCR Primers and Amplification of DNA
Various fragments of the Borrelia burgdorferi genome, such as those of Table
l, can be
used, in accordance with the present invention, to prepare PCR primers for a
variety of uses. The
PCR primers are preferably at least 15 bases, and more preferably at least 18
bases in length.
When selecting a primer sequence, it is preferred that the primer pairs have
approximately the
same G/C ratio, so that melting temperatures are approximately the same. The
PCR primers and
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42
amplified DNA of this Example find use in the Examples that follow.
2. Isolation of a Selected DNA Clone From B. burgdorferi
Three approaches are used to isolate a B. burgdorferi clone comprising a
polynucleotide of
the present invention from any B. burgdorferi genomic DNA library. The B.
burgdorferi strain
B31PU has been deposited as a convienent source for obtaining a B. burgdorferi
strain although a
wide varity of strains B. burgdorferi strains can be used which are known in
the art.
B. burgdorferi genomic DNA is prepared using the following method. A 20m1
overnight
bacterial culture grown in a rich medium (e.g., Trypticase Soy Broth, Brain
Heart Infusion broth
or Super broth), pelleted, fished two times with TES (30mM Tris-pH 8.0, 25mM
EDTA, SOmM
NaCI), and resuspended in Sml high salt TES (2.SM NaCI). Lysostaphin is added
to final
concentration of approx 50ug/ml and the mixture is rotated slowly 1 hour at
37C to make
protoplast cells. The solution is then placed in incubator (or place in a
shaking water bath) and
warmed to SSC. Five hundred micro liter of 20% sarcosyl in TES (final
concentration 2%) is
then added to lyse the cells. Next, guanidine HCI is added to a final
concentration of 7M (3.69g
in 5.5 ml). The mixture is swirled slowly at 55C for 60-90 min (solution
should clear). A CsCI
gradient is then set up in SW41 ultra clear tubes using 2.Oml 5.7M CsCI and
overlaying with
2.85M CsCI. The gradient is carefully overlayed with the DNA-containing GuHCI
solution. The
gradient is spun at 30,000 rpm, 20C for 24 hr and the lower DNA band is
collected. The volume
is increased to 5 ml with TE buffer. The DNA is then treated with protease K (
10 ug/ml)
overnight at 37 C, and precipitated with ethanol. The precipitated DNA is
resuspended in a
desired buffer.
In the first method, a plasmid is directly isolated by screening a plasmid B.
burgdorferi
genomic DNA library using a polynucleotide probe corresponding to a
polynucleotide of the
present invention. Particularly, a specific polynucleotide with 30-40
nucleotides is synthesized
using an Applied Biosystems DNA synthesizer according to the sequence
reported. The
oligonucleotide is labeled, for instance, with 32P-y ATP using T4
polynucleotide kinase and
purified according to routine methods. (See, e.g., Maniatis et al., Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY (1982).) The
library is
transformed into a suitable host, as indicated above (such as XL-1 Blue
(Stratagene)) using
techniques known to those of skill in the art. See, e.g., Sambrook et al.
MOLECULAR
CLONING: A LABORATORY MANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel
et
al., CURRENT PROTOCALS IN MOLECULAR BIOLOGY (John Wiley and Sons, N.Y.
1989). The transformants are plated on 1.5% agar plates (containing the
appropriate selection
agent, e.g., ampicillin) to a density of about 150 transformants (colonies)
per plate. These plates
are screened using Nylon membranes according to routine methods for bacterial
colony screening.
See, e.g., Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL (Cold
Spring Harbor, N.Y. 2nd ed. 1989); Ausubel et al., CURRENT PROTOCALS IN
CA 02294568 1999-12-17
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43
MOLECULAR BIOLOGY (John Wiley and Sons, N.Y. 1989) or other techniques known
to
those of skill in the art.
Alternatively, two primers of 15-25 nucleotides derived from the 5' and 3'
ends of a
polynucleotide of Table 1 are synthesized and used to amplify the desired DNA
by PCR using a
B. burgdorferi genomic DNA prep as a template. PCR is carried out under
routine conditions, for
instance, in 25 pl of reaction mixture with 0.5 ug of the above DNA template.
A convenient
reaction mixture is 1.5-5 mM MgCl2, 0.01 % (w/v) gelatin, 20 p.M each of dATP,
dCTP, dGTP,
dTTP, 25 pmol of each primer and 0.25 Unit of Tag polymerase. Thirty five
cycles of PCR
(denaturation at 94°C for 1 min; annealing at 55°C for 1 min;
elongation at 72°C for 1 min) are
i o performed with a Perkin-Elmer Cetus automated thermal cycler. The
amplified product is
analyzed by agarose gel electrophoresis and the DNA band with expected
molecular weight is
excised and purified. The PCR product is verified to be the selected sequence
by subcloning and
sequencing the DNA product.
Finally, overlapping oligos of the DNA sequences of Table 1 can be chemically
15 synthesized and used to generate a nucleotide sequence of desired length
using PCR methods
known in the art.
3(a). Expression and Purification Borrelia polypeptides in E. coli
The bacterial expression vector pQE60 is used for bacterial expression of some
of the
20 polypeptide fragements of the present invention. (QIAGEN, Inc., 9259 Eton
Avenue,
Chatsworth, CA, 91311). pQE60 encodes ampicillin antibiotic resistance
("Ampr") and contains
a bacterial origin of replication ("ori"), an IPTG inducible promoter, a
ribosome binding site
("RBS"), six codons encoding histidine residues that allow affinity
purification using nickel-
nitrilo-tri-acetic acid ("Ni-NTA") affinity resin (QIAGEN, Inc., supra) and
suitable single
25 restriction enzyme cleavage sites. These elements are arranged such that an
inserted DNA
fragment encoding a polypeptide expresses that polypeptide with the six His
residues (i.e., a "6 X
His tag") covalently linked to the carboxyl terminus of that polypeptide.
The DNA sequence encoding the desired portion of a B. burgdorferi protein of
the present
invention is amplified from B. burgdorferi genomic DNA using PCR
oligonucleotide primers
30 which anneal to the 5' and 3' sequences coding for the portions of the B.
burgdorferi
polynucleotide shown in Table 1. Additional nucleotides containing restriction
sites to facilitate
cloning in the pQE60 vector are added to the 5' and 3' sequences,
respectively.
For cloning the mature protein, the 5' primer has a sequence containing an
appropriate
restriction site followed by nucleotides of the amino terminal coding sequence
of the desired B.
35 burgdorferi polynucleotide sequence in Table 1. One of ordinary skill in
the art would appreciate
that the point in the protein coding sequence where the 5' and 3' primers
begin may be varied to
amplify a DNA segment encoding any desired portion of the complete protein
shorter or longer
than the mature form. The 3' primer has a sequence containing an appropriate
restriction site
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44
followed by nucleotides complementary to the 3' end of the polypeptide coding
sequence of Table
1, excluding a stop codon, with the coding sequence aligned with the
restriction site so as to
maintain its reading frame with that of the six His codons in the pQE60
vector.
The amplified B. burgdorferi DNA fragment and the vector pQE60 are digested
with
restriction enzymes which recognize the sites in the primers and the digested
DNAs are then
ligated together. The B. burgdorferi DNA is inserted into the restricted pQE60
vector in a manner
which places the B. burgdorferi protein coding region downstream from the IPTG-
inducible
promoter and in-frame with an initiating AUG and the six histidine codons.
The ligation mixture is transformed into competent E. coli cells using
standard procedures
such as those described by Sambrook et al., supra.. E. coli strain M 15/rep4,
containing multiple
copies of the plasmid pREP4, which expresses the lac repressor and confers
kanamycin resistance
("Kanr"), is used in carrying out the illustrative example described herein.
This strain, which is
only one of many that are suitable for expressing a B. burgdorferi
polypeptide, is available
commercially (QIAGEN, Inc., supra). Transformants are identified by their
ability to grow on
LB agar plates in the presence of ampicillin and kanamycin. Plasmid DNA is
isolated from
resistant colonies and the identity of the cloned DNA confirmed by restriction
analysis, PCR and
DNA sequencing.
Clones containing the desired constructs are grown overnight ("O/N") in liquid
culture in
LB media supplemented with both ampicillin (100 p.g/ml) and kanamycin (25
~,g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of approximately
1:25 to 1:250. The cells
are grown to an optical density at 600 nm ("OD600" ) of between fl.4 and 0.6.
Isopropyl-~3-D-
thiogalactopyranoside ("IP1'G") is then added to a final concentration of 1 mM
to induce
transcription from the lac repressor sensitive promoter, by inactivating the
lacI repressor. Cells
subsequently are incubated further for 3 to 4 hours. Cells then are harvested
by centrifugation.
The cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCI, pH
8. The cell
debris is removed by centrifugation, and the supernatant containing the B.
burgdorferi
polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin column
(QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin
with high affinity
are purified in a simple one-step procedure (for details see: The
QIAexpressionist, 1995,
QIAGEN, Inc., supra). Briefly the supernatant is loaded onto the column in 6 M
guanidine-HCI,
pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCI, pH 8,
then washed
with 10 volumes of 6 M guanidine-HCl pH 6, and finally the B. burgdorferi
polypeptide is eluted
with 6 M guanidine-HCI, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-
buffered saline
(PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI. Alternatively, the
protein could be
successfully refolded while immobilized on the Ni-NTA column. The recommended
conditions
are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCI,
20% glycerol, 20
mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be
performed over
CA 02294568 1999-12-17
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a period of 1.5 hours or more. After renaturation the proteins can be eluted
by the addition of 250
mM immidazole. Immidazole is removed by a final dialyzing step against PBS or
50 mM sodium
acetate pH 6 buffer plus 200 mM NaCI. The purified protein is stored at
4° C or frozen at -80° C.
The polypeptide of the present invention are also prepared using a non-
denaturing protein
5 purification method. For these polypeptides, the cell pellet from each liter
of culture is
resuspended in 25 mls of Lysis Buffer A at 4°C (Lysis Buffer A = 50 mM
Na-phosphate, 300
mM NaCI, 10 mM 2-mercaptoethanol, 10% Glycerol, pH 7.5 with 1 tablet of
Complete EDTA-
free protease inhibitor cocktail (Boehringer Mannheim #1873580) per 50 ml of
buffer).
Absorbance at 550 nm is approximately 10-20 O.D./ml. The suspension is then
put through three
to freeze/thaw cycles from -70°C (using a ethanol-dry ice bath) up to
room temperature. The cells
are lysed via sonication in short 10 sec bursts over 3 minutes at
approximately 80W while kept on
ice. The sonicated sample is then centrifuged at 15,000 RPM for 30 minutes at
4°C. The
supernatant is passed through a column containing 1.0 ml of CL-4B resin to pre-
clear the sample
of any proteins that may bind to agarose non-specifically, and the flow-
through fraction is
15 collected.
The pre-cleared flow-through is applied to a nickel-nitrilo-tri-acetic acid
("Ni-NTA")
affinity resin column (Quiagen, Inc., supra). Proteins with a 6 X His tag bind
to the Ni-NTA
resin with high affinity and can be purified in a simple one-step procedure.
Briefly, the
supernatant is loaded onto the column in Lysis Buffer A at 4°C, the
column is first washed with
2o 10 volumes of Lysis Buffer A until the A280 of the eluate returns to the
baseline. Then, the
column is washed with 5 volumes of 40 mM Imidazole (92% Lysis Buffer A / 8%
Buffer B)
(Buffer B = 50 mM Na-Phosphate, 300 mM NaCI, 10% Glycerol, 10 mM 2-
mercaptoethanol,
500 mM Imidazole, pH of the final buffer should be 7.5). The protein is eluted
off of the column
with a series of increasing Imidazole solutions made by adjusting the ratios
of Lysis Buffer A to
25 Buffer B. Three different concentrations are used: 3 volumes of 75 mM
Imidazole, 3 volumes of
150 mM Imidazole, 5 volumes of 500 mM Imidazole. The fractions containing the
purified
protein are analyzed using 8 %, 10 % or 14% SDS-PAGE depending on the protein
size. The
purified protein is then dialyzed 2X against phosphate-buffered saline (PBS}
in order to place it
into an easily workable buffer. The purified protein is stored at 4° C
or frozen at -80°.
30 The following alternative method may be used to purify B. burgdorferi
expressed in E toll
when it is present in the form of inclusion bodies. Unless otherwise
specified, all of the
following steps are conducted at 4-10°C.
Upon completion of the production phase of the E. toll fermentation, the cell
culture is
cooled to 4-10°C and the cells are harvested by continuous
centrifugation at 15,000 rpm (Heraeus
35 Sepatech). On the basis of the expected yield of protein per unit weight of
cell paste and the
amount of purified protein required, an appropriate amount of cell paste, by
weight, is suspended
in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are
dispersed to a
homogeneous suspension using a high shear mixer.
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46
The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics,
Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then
mixed with NaCI
solution to a final concentration of 0.5 M NaCI, followed by centrifugation at
7000 x g for 15
min. The resultant pellet is washed again using 0.5M NaCI, 100 mM Tris, 50 mM
EDTA, pH
7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
hydrochloride
(GuHCI) for 2-4 hours. After 7000 x g centrifugation for 15 min., the pellet
is discarded and the
B. burgdorferi polypeptide-containing supernatant is incubated at 4°C
overnight to allow further
GuHCI extraction.
to Following high speed centrifugation (30,000 x g) to remove insoluble
particles, the
GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract with
20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA by vigorous
stirring.
The refolded diluted protein solution is kept at 4°C without mixing for
12 hours prior to further
purification steps.
15 To clarify the refolded B. burgdorferi polypeptide solution, a previously
prepared
tangential filtration unit equipped with 0.16 pm membrane filter with
appropriate surface area
(e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed.
The filtered sample
is loaded onto a cation exchange resin (e.g., Poros HS-50, Perceptive
Biosystems). The column
is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,
1000 mM, and
20 1500 mM NaCI in the same buffer, in a stepwise manner. The absorbance at
280 mm of the
effluent is continuously monitored. Fractions are collected and further
analyzed by SDS-PAGE.
Fractions containing the B. burgdorferi polypeptide are then pooled and mixed
with 4
volumes of water. The diluted sample is then loaded onto a previously prepared
set of tandem
columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak anion
(Poros CM-20,
25 Perseptive Biosystems) exchange resins. The columns are equilibrated with
40 mM sodium
acetate, pH 6Ø Both columns are washed with 40 mM sodium acetate, pH 6.0,
200 mM NaCI.
The CM-20 column is then eluted using a 10 column volume linear gradient
ranging from 0.2 M
NaCI, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH
6.5. Fractions
are collected under constant AZBO monitoring of the effluent. Fractions
containing the B.
30 burgdorferi polypeptide (determined, for instance, by 16% SDS-PAGE) are
then pooled.
The resultant B. burgdorferi polypeptide exhibits greater than 95% purity
after the above
refolding and purification steps. No major contaminant bands are observed from
Commassie blue
stained 16% SDS-PAGE gel when 5 pg of purified protein is loaded. The purified
protein is also
tested for endotoxin/LPS contamination, and typically the LPS content is less
than 0.1 ng/ml
35 according to LAL assays.
3(b). Alternative Expression and Purification Borrelia polypeptides in E.
CA 02294568 1999-12-17
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colt
47
Tthe vector pQElO is alternatively used to clone and express some of the
polypeptides of
the present invention for use in the soft tissue and systemic infection models
discussed below.
The difference being such that an inserted DNA fragment encoding a polypeptide
expresses that
polypeptide with the six His residues (i.e., a "6 X His tag") covalently
linked to the amino
terminus of that polypeptide. The bacterial expression vector pQElO (QIAGEN,
Inc., 9259 Eton
Avenue, Chatsworth, CA, 91311 ) was used in this example . The components of
the pQE 10
plasmid are arranged such that the inserted DNA sequence encoding a
polypeptide of the present
invention expresses the polypeptide with the six His residues (i.e., a "6 X
His tag")) covalently
l0 linked to the amino terminus.
The DNA sequences encoding the desired portions of a polypeptide of Table 1
were
amplified using PCR oligonucleotide primers from genomic B. burgdorferi DNA.
The PCR
primers anneal to the nucleotide sequences encoding the desired amino acid
sequence of a
polypeptide of the present invention. Additional nucleotides containing
restriction sites to
15 facilitate cloning in the pQElO vector were added to the 5' and 3' primer
sequences, respectively.
For cloning a polypeptide of the present invention, the 5' and 3' primers were
selected to
amplify their respective nucleotide coding sequences. One of ordinary skill in
the art would
appreciate that the point in the protein coding sequence where the 5' and 3'
primers begins may be
varied to amplify a DNA segment encoding any desired portion of a polypeptide
of the present
2o invention. The 5' primer was designed so the coding sequence of the 6 X His
tag is aligned with
the restriction site so as to maintain its reading frame with that of B.
burgdorferi polypeptide. The
3' was designed to include an stop codon. The amplified DNA fragment was then
cloned, and the
protein expressed, as described above for the pQE60 plasmid.
The DNA sequences of Table 1 encoding amino acid sequences may also be cloned
and
25 expressed as fusion proteins by a protocol similar to that described
directly above, wherein the
pET-32b(+) vector (Novagen, 601 Science Drive, Madison, WI 53711 ) is
preferentially used in
place of pQE 10.
The above methods are not limited to the polypeptide fragements actually
produced. The
above method, like the methods below, can be used to produce either full
length polypeptides or
3o desired fragements therof.
3(c). Alternative Expression and Purification of Borrelia polypeptides in
E. coli
The bacterial expression vector pQE60 is used for bacterial expression in this
example
35 (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 913 i 1 ). However, in
this example, the
polypeptide coding sequence is inserted such that translation of the six His
codons is prevented
and, therefore, the polypeptide is produced with no 6 X His tag.
The DNA sequence encoding the desired portion of the B. burgdorferi amino acid
sequence is amplified from an B. burgdorferi genomic DNA prep the deposited
DNA clones
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48
using PCR oligonucleotide primers which anneal to the 5' and 3' nucleotide
sequences
corresponding to the desired portion of the B. burgdorferi polypeptides.
Additional nucleotides
containing restriction sites to facilitate cloning in the pQE60 vector are
added to the 5' and 3'
primer sequences.
For cloning a B. burgdorferi polypeptides of the present invention, 5' and 3'
primers are
selected to amplify their respective nucleotide coding sequences. One of
ordinary skill in the art
would appreciate that the point in the protein coding sequence where the 5'
and 3' primers begin
may be varied to amplify a DNA segment encoding any desired portion of a
polypeptide of the
present invention. The 3' and 5' primers contain appropriate restriction sites
followed by
to nucleotides complementary to the 5' and 3' ends of the coding sequence
respectively. The 3'
primer is additionally designed to include an in-frame stop codon.
The amplified B. burgdorferi DNA fragments and the vector pQE60 are digested
with
restriction enzymes recognizing the sites in the primers and the digested DNAs
are then ligated
together. Insertion of the B. burgdorferi DNA into the restricted pQE60 vector
places the B.
burgdorferi protein coding region including its associated stop codon
downstream from the IPTG
inducible promoter and in-frame with an initiating AUG. The associated stop
codon prevents
translation of the six histidine codons downstream of the insertion point.
The ligation mixture is transformed into competent E. coli cells using
standard procedures
such as those described by Sambrook et al. E. coli strain MI5/rep4, containing
multiple copies of
2o the plasmid pREP4, which expresses the lac repressor and confers kanamycin
resistance
("Kanr"), is used in carrying out the illustrative example described herein.
This strain, which is
only one of many that are suitable for expressing B. burgdorferi polypeptide,
is available
commercially (QIAGEN, Inc., supra). Transformants are identified by their
ability to grow on
LB plates in the presence of ampiciliin and kanamycin. Plasmid DNA is isolated
from resistant
colonies and the identity of the cloned DNA confirmed by restriction analysis,
PCR and DNA
sequencing.
Clones containing the desired constructs are grown overnight ("O/N") in liquid
culture in
LB media supplemented with both ampicillin ( 100 ~,g/ml) and kanamycin (25
pg/ml). The O/N
culture is used to inoculate a large culture, at a dilution of approximately
1:25 to 1:250. The cells
are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
isopropyl-b-D-
thiogalactopyranoside {"IPTG") is then added to a final concentration of 1 mM
to induce
transcription from the lac repressor sensitive promoter, by inactivating the
lacI repressor. Cells
subsequently are incubated further for 3 to 4 hours. Cells then are harvested
by centrifugation.
To purify the B. burgdorferi polypeptide, the cells are then stirred for 3-4
hours at 4°C in
6M guanidine-HCI, pH 8. The cell debris is removed by centrifugation, and the
supernatant
containing the B. burgdorferi polypeptide is dialyzed against 50 mM Na-acetate
buffer pH 6,
supplemented with 200 mM NaCI. Alternatively, the protein can be successfully
refolded by
dialyzing it against 500 mM NaCI, 20°lo glycerol, 25 mM Tris/HCI pH
7.4, containing protease
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49
inhibitors. After renaturation the protein can be purified by ion exchange,
hydrophobic interaction
and size exclusion chromatography. Alternatively, an affinity chromatography
step such as an
antibody column can be used to obtain pure B. burgdorferi polypeptide. The
purified protein is
stored at 4° C or frozen at -80° C.
The following alternative method may be used to purify B. burgdorferi
polypeptides
expressed in E coli when it is present in the form of inclusion bodies. Unless
otherwise
specified, all of the following steps are conducted at 4-10°C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture is
cooled to 4-10°C and the cells are harvested by continuous
centrifugation at 15,000 rpm (Heraeus
to Sepatech). On the basis of the expected yield of protein per unit weight of
cell paste and the
amount of purified protein required, an appropriate amount of cell paste, by
weight, is suspended
in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are
dispersed to a
homogeneous suspension using a high shear mixer.
The cells ware then lysed by passing the solution through a microfluidizer
(Microfuidics,
15 Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then
mixed with NaCI
solution to a final concentration of 0.5 M NaCI, followed by centrifugation at
7000 x g for 15
min. The resultant pellet is washed again using O.SM NaCI, 100 mM Tris, 50 mM
EDTA, pH
7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
hydrochloride
20 (GuHCI) for 2-4 hours. After 7000 x g centrifugation for 15 min., the
pellet is discarded and the
B. burgdorferi polypeptide-containing supernatant is incubated at 4°C
overnight to allow further
GuHCl extraction.
Following high speed centrifugation (30,000 x g) to remove insoluble
particles, the
GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract with
20 volumes of
25 buffer containing SO mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA by vigorous
stirring.
The refolded diluted protein solution is kept at 4°C without mixing for
12 hours prior to further
purification steps.
To clarify the refolded B. burgdorferi polypeptide solution, a previously
prepared
tangential filtration unit equipped with 0.16 ~,m membrane filter with
appropriate surface area
30 (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is
employed. The filtered sample
is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive
Biosystems). The column
is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,
1000 mM, and
1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280
mm of the
effluent is continuously monitored. Fractions are collected and further
analyzed by SDS-PAGE.
35 Fractions containing the B. burgdorferi polypeptide are then pooled and
mixed with 4
volumes of water. The diluted sample is then loaded onto a previously prepared
set of tandem
columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak anion
(Poros CM-20,
CA 02294568 1999-12-17
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Perseptive Biosystems) exchange resins. The columns are equilibrated with 40
mM sodium
acetate, pH 6Ø Both columns are washed with 40 mM sodium acetate, pH 6.0,
200 mM NaCI.
The CM-20 column is then eluted using a 10 column volume linear gradient
ranging from 0.2 M
NaCI, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH
6.5. Fractions
are collected under constant AzBO monitoring of the effluent. Fractions
containing the B.
burgdorferi polypeptide (determined, for instance, by 16% SDS-PAGE) are then
pooled.
The resultant B. burgdorferi polypeptide exhibits greater than 95% purity
after the above
refolding and purification steps. No major contaminant bands are observed from
Commassie blue
stained 16% SDS-PAGE gel when 5 p,g of purified protein is loaded. The
purified protein is also
to tested for endotoxin/LPS contamination, and typically the LPS content is
less than 0.1 ng/ml
according to LAL assays.
3(d). Cloning and Expression of B. burgdorferi in Other Bacteria
B. burgdorferi polypeptides can also be produced in: B. burgdorferi using the
methods of
15 S. Skinner et al., ( 1988) Mol. Microbiol. 2:289-297 or J. I. Moreno (
1996) Protein Expr. Purif.
8(3):332-340; Lactobacillus using the methods of C. Rush et al., 1997 Appl.
Microbiol.
Biotechnol. 47(5):537-542; or in Bacillus subtilis using the methods Chang et
ai., U.S. Patent
No. 4,952,508.
20 4. Cloning and Expression in COS Cells
A B. burgdorferi expression plasmid is made by cloning a portion of the DNA
encoding a
B. burgdorferi polypeptide into the expression vector pDNAI/Amp or pDNAIII
(which can be
obtained from Invitrogen, Inc.). The expression vector pDNA1/amp contains: ( 1
) an E. coli
origin of replication effective for propagation in E. coli and other
prokaryotic cells; (2) an
25 ampicillin resistance gene for selection of plasmid-containing prokaryotic
cells; (3) an SV40 origin
of replication for propagation in eukaryotic cells; (4) a CMV promoter, a
polylinker, an SV40
intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA"
tag to facilitate
purification) followed by a termination codon and polyadenylation signal
arranged so that a DNA
can be conveniently placed under expression control of the CMV promoter and
operably linked to
3o the SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker.
The HA tag corresponds to an epitope derived from the influenza hemagglutinin
protein described
by Wilson et al. 1984 Cell 37:767. The fusion of the HA tag to the target
protein allows easy
detection and recovery of the recombinant protein with an antibody that
recognizes the HA
epitope. pDNAIII contains, in addition, the selectable neomycin marker.
35 A DNA fragment encoding a B. burgdorferi polypeptide is cloned into the
polylinker
region of the vector so that recombinant protein expression is directed by the
CMV promoter. The
plasmid construction strategy is as follows. The DNA from a B. burgdorferi
genomic DNA prep
is amplified using primers that contain convenient restriction sites, much as
described above for
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construction of vectors for expression of B. burgdorferi in E. coli. The 5'
primer contains a
Kozak sequence, an AUG start codon, and nucleotides of the 5' coding region of
the B.
burgdorferi polypeptide. The 3' primer, contains nucleotides complementary to
the 3' coding
sequence of the B. burgdorferi DNA, a stop codon, and a convenient restriction
site.
The PCR amplified DNA fragment and the vector, pDNAI/Amp, are digested with
appropriate restriction enzymes and then ligated. The ligation mixture is
transformed into an
appropriate E. coli strain such as SUREr"" (Stratagene Cloning Systems, La
Jolla, CA 92037),
and the transformed culture is plated on ampicillin media plates which then
are incubated to allow
growth of ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and
examined by restriction analysis or other means for the presence of the
fragment encoding the B.
burgdorferi polypeptide
For expression of a recombinant B. burgdorferi polypeptide, COS cells are
transfected
with an expression vector, as described above, using DEAF-dextran, as
described, for instance,
by Sambrook et al. (supra). Cells are incubated under conditions for
expression of B.
burgdorferi by the vector.
Expression of the B. burgdorferi-HA fusion protein is detected by
radiolabeling and
immunoprecipitation, using methods described in, for example Harlow et al.,
supra.. To this
end, two days after transfection, the cells are labeled by incubation in media
containing 35S-
cysteine for 8 hours. The cells and the media are collected, and the cells are
washed and the lysed
with detergent-containing RIPA buffer: 1 SO mM NaCI, 1 % NP-40, 0.1 % SDS, 1 %
NP-40, 0.5%
DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. (supra ). Proteins are
precipitated
from the cell lysate and from the culture media using an HA-specific
monoclonal antibody. The
precipitated proteins then are analyzed by SDS-PAGE and autoradiography. An
expression
product of the expected size is seen in the cell lysate, which is not seen in
negative controls.
5. Cloning and Expression in CHO Cells
The vector pC4 is used for the expression of B. burgdorferi polypeptide in
this example.
Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.
37146). The
plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
Chinese
hamster ovary cells or other cells lacking dihydrofolate activity that are
transfected with these
plasmids can be selected by growing the cells in a selective medium (alpha
minus MEM, Life
Technologies) supplemented with the chemotherapeutic agent methotrexate. The
amplification of
the DHFR genes in cells resistant to methotrexate (MTX) has been well
documented. See, e.g.,
Alt et al., 1978, J. Biol. Chem. 253:1357-1370; Hamlin et al., 1990, Biochem.
et Biophys.
3s Acta, 1097:107-143; Page et al., 1991, Biotechnology 9:64-68. Cells grown
in increasing
concentrations of MTX develop resistance to the drug by overproducing the
target enzyme,
DHFR, as a result of amplification of the DHFR gene. If a second gene is
linked to the DHFR
gene, it is usually co-amplified and over-expressed. It is known in the art
that this approach may
CA 02294568 1999-12-17
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52
be used to develop cell lines carrying more than 1,000 copies of the amplified
gene(s).
Subsequently, when the methotrexate is withdrawn, cell lines are obtained
which contain the
amplified gene integrated into one or more chromosomes) of the host cell.
Plasmid pC4 contains the strong promoter of the long terminal repeat (LTR) of
the Rouse
Sarcoma Virus, for expressing a polypeptide of interest, Cullen, et al. (
1985) Mol. Cell. Biol.
5:438-447; plus a fragment isolated from the enhancer of the immediate early
gene of human
cytomegalovirus (CMV), Boshart, et al., 1985, Cell 41:521-530. Downstream of
the promoter
are the following single restriction enzyme cleavage sites that allow the
integration of the genes:
Bam HI, Xba I, and Asp 718. Behind these cloning sites the plasmid contains
the 3' intron and
1o polyadenylation site of the rat preproinsulin gene. Other high efficiency
promoters can also be
used for the expression, e.g., the human B-actin promoter, the SV40 early or
late promoters or the
long terminal repeats from other retroviruses, e.g., HIV and HTLVI. Clontech's
Tet-Off and Tet-
On gene expression systems and similar systems can be used to express the B.
burgdorferi
polypeptide in a regulated way in mammalian cells (Gossen et al., 1992, Proc.
Natl. Acad. Sci.
USA 89:5547-5551. For the polyadenylation of the mRNA other signals, e.g.,
from the human
growth hormone or globin genes can be used as well. Stable cell lines carrying
a gene of interest
integrated into the chromosomes can also be selected upon co-transfection with
a selectable
marker such as gpt, 6418 or hygromycin. It is advantageous to use more than
one selectable
marker in the beginning, e.g., G418 plus methotrexate.
2o The plasmid pC4 is digested with the restriction enzymes and then
dephosphorylated
using calf intestinal phosphates by procedures known in the art. The vector is
then isolated from
a 1 % agarose gel. The DNA sequence encoding the B. burgdor feri polypeptide
is amplified using
PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the
desired portion of
the gene. A 5' primer containing a restriction site, a Kozak sequence, an AUG
start codon, and
nucleotides of the 5' coding region of the B. burgdorferi polypeptide is
synthesized and used. A
3' primer, containing a restriction site, stop codon, and nucleotides
complementary to the 3'
coding sequence of the B. burgdorferi polypeptides is synthesized and used.
The amplified
fragment is digested with the restriction endonucleases and then purified
again on a 1 % agarose
gel. The isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase.
E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are
identified that contain the
fragment inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used for
transfection. Five
p,g of the expression plasmid pC4 is cotransfected with 0.5 p,g of the plasmid
pSVneo using a
lipid-mediated transfection agent such as LipofectinT"" or LipofectAMnVE.T""
(LifeTechnologies
Gaithersburg, MD). The plasmid pSV2-neo contains a dominant selectable marker,
the neo gene
from Tn5 encoding an enzyme that confers resistance to a group of antibiotics
including 6418.
The cells are seeded in alpha minus MEM supplemented with 1 mg/ml 6418. After
2 days, the
cells are trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus
CA 02294568 1999-12-17
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53
MEM supplemented with 10, 25, or SO ng/ml of methotrexate plus 1 mg/ml 6418.
After about
10-14 days single clones are trypsinized and then seeded in 6-well petri
dishes or 10 ml flasks
using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM,
800 nM).
Clones growing at the highest concentrations of methotrexate are then
transferred to new 6=well
plates containing even higher concentrations of methotrexate ( 1 EtM, 2 ~t,M,
5 ~t,M, 10 mM, 20
mM). The same procedure is repeated until clones are obtained which grow at a
concentration of
100-200 pM. Expression of the desired gene product is analyzed, for instance,
by SDS-PAGE
and Western blot or by reversed phase HPLC analysis.
6. Immunization and Detection of Immune Responses
io
6(a). B. burgdorferi propagation
B. burgdorferi sensu stricto isolate B31 is propagated in tightly-closed
containers at 34°C
in modified Barbour-Stoenner-Kelly (BSKII) medium (Barbour, A.G., Yale J.
Biol. Med.
57:521-525 (1984)) overlaid with a 5%OZ/5%COZ/90%NZ gas mixture. Ceil
densities of these
15 cultures are determined by darkfield microscopy at 400X.
Immunization of Mice and Challenge with B. burgdorferi. For active
immunizations
BALB/cByJ mice (BALB, Jackson Laboratories) are injected intraperitoneally
(i.p.) at week 0
with 20 g of recombinant borrelial protein, or phosphate-buffered saline
(PBS), emulsified with
complete Freund's adjuvant (CFA), given a similar booster immunization in
incomplete Freund's
2o adjuvant (IFA) at week 4, and challenged at week 6. For challenge B.
burgdorferi are diluted in
BSKII from exponentially-growing cultures and mice are injected.subcutaneously
(s.c.) at the
base of the tail with 0.1 ml of these dilutions (typically 103-10°
borreliae; approximately 10-100
times the median infectious dose). Borreliae used for challenge are passaged
fewer than six times
in vitro. To assess infection, mice are sacrificed at 14-17 days post-
challenge, and specimens
25 derived from ear, bladder, and tibiotarsal joints are placed in BSKII plus
1.4% gelatin, 13 g/ml
amphotericin B, 1.5 glml phosphomycin, and 15 g/ml rifampicin, and borrelia
outgrowth at two
or three weeks is quantified by darkfield microscopy. Batches of BSKII are
qualified for
infection testing by confirming that they supported the growth of 1-5 cells of
isolate B31. In
some instances seroconversion for protein P39 reactivity is also used to
confirm infections (see
3o below). Others have previously shown that mice elicited antibodies to P39
when inoculated with
live borreliae by syringe or tick bite, but not with killed borreliae
{Simpson, W.J., et al., J. Clin.
Microbiol. 29:236-243 { 1991 )).
6(b). Immunoassays
35 Several immunoassay formats are used to quantify levels of borrelia-
specific antibodies
(ELISA and immunoblot), and to evaluate the functional properties of these
antibodies {growth
inhibition assay). The ELISA and immunoblot assays are also used to detect and
quantify
antibodies elicited in response to borrelial infection that react with
specific borrelial antigens.
Where antibodies to certain borrelial antigens are elicited by infection this
is taken as evidence that
CA 02294568 1999-12-17
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54
the borrelial proteins in question are expressed in vivo. Absence of infection-
derived antibodies
(seroconversion) following borrelial challenge is evidence that infection is
prevented or
suppressed. The immunoblot assay is also used to ascertain whether antibodies
raised against
recombinant borrelial antigens recognize a protein of similar size in extracts
of whole borreliae.
Where the natural protein is of similar, or identical, size in the immunoblot
assay to the
recombinant version of the same protein, this is taken as evidence that the
recombinant protein is
the product of a full-length clone of the respective gene.
Enzyme-Linked Immunosorbant Assay (ELISA). The ELISA is used to quantify
levels of
antibodies reactive with borrelial antigens elicited in response to
immunization with these borrelial
1o antigens. Wells of 96 well microtiter plates {Immunlon 4, Dynatech,
Chantilly, Virginia, or
equivalent) are coated with antigen by incubating 50 1 of 1 g/ml protein
antigen solution in a
suitable buffer, typically 0.1 M sodium carbonate buffer at pH 9.6. After
decanting unbound
antigen, additional binding sites are blocked by incubating 100 1 of 3% nonfat
milk in wash
buffer (PBS, 0.2% Tween 20, pH 7.4). After washing, duplicate serial two-fold
dilutions of sera
in PBS, Tween 20, 1 % fetal bovine serum, are incubated for 1 hr, removed,
wells are washed
three times, and incubated with horseradish peroxidase-conjugated goat anti-
mouse IgG. After
three washes, bound antibodies are detected with H202 and 2,2'-azino-di-(3-
ethylbenzthiazoline
sulfonate) (Schwan, T.G., et al., Proc. Natl. Acad. Sci. USA 92:2909-2913
(1985)) (ABTS~,
Kirkegaard & Perry Labs., Gaithersburg, MD) and A~5 is quantified with a
Molecular Devices,
2o Corp. (Memo Park, California) VmaxTM plate reader. IgG levels twice the
background level in
serum from naive mice are assigned the minimum titer of 1:100.
G(c). In Vitro Growth Inhibition Assay
Unlike other bacteria, borreliae can be killed by the binding of specific
antibodies to their
surface antigens. The mechanism for this in vitro killing or growth-inhibitory
effect is not
known, but can occur in the absence of serum complement, or other immune
effector functions.
Antibodies elicited in animals receiving immunizations with specific borrelial
antigens that result
in protection from borrelial challenge usually will directly kill borreliae in
vitro. Thus, the in vitro
growth inhibition assay also has a high predictive value for the protective
potency of the borrelial
3o antibodies, although exceptions, such as antibodies against OspC which are
weak at in vitro
growth inhibition, have been observed. Also, this assay can be used to
evaluate the serologic
conservation of epitope binding protective antibodies. A microwell antibody
titration assay
(Sadziene, A., et al., J. Infect. Dis. 167:165-172 (1993)) is used to evaluate
the growth inhibition
(GI) properties of antisera against recombinant borrelial antigens against the
homologous B31
isolate, and against various strains of borrelia. Briefly, 105 borrelia in 100
1 BSKII are added to
serial two-fold dilutions of sera in 100 1 BSKII in 96-well plates, and the
plates are covered and
incubated at 34°C in a 5%OZ/5%CO~/90%NZ gas mixture for 72 h prior to
quantification of
borrelia growth by darkfield microscopy.
CA 02294568 1999-12-17
WO 98/59071 PCTNS98/12718
6(d). Sodiumdodecylsulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) and Immunoblotting
Using a single well format, total borrelial protein extracts, recombinant
borrelial antigen,
or recombinant P39 samples (2 g of purified protein, or more for total
borrelial extracts) are
5 boiled in SDS/2-ME sample buffer before electrophoresis through 3%
acrylamide stacking gels,
and resolving gels of higher acrylamide concentration, typically 10-15%
acrylamide monomer.
Gels are electro-blotted to nitrocellulose membranes and lanes are probed with
dilutions of
antibody to be tested for reactivity with specific borrelial antigens,
followed by the appropriate
secondary antibody-enzyme (horseradish peroxidase) conjugate. When it is
desirable to confirm
10 that the protein had transferred following electro-blotting, membranes are
stained with Ponceau S.
Immunoblot signals from bound antibodies are detected on x-ray film as
chemiluminescence using
ECLTM reagents (Amersham Corp., Arlington Heights, Illinois).
6(e). Detection of Borrelia mRNA expression
15 Northern blot analysis is carried out using methods described by, among
others,
Sambrook et al., supra. to detect the expression of the B. burgdorferi
nucleotide sequences of the
present invention in animal tissues. A cDNA probe containing an entire
nucleotide sequence
shown in Table 1 is labeled with 32P using the rediprimeTM DNA labeling system
(Amersham Life
Science), according to manufacturer's instructions. After labeling, the probe
is purified using a
20 CHROMA SPIN-100TM column (Clontech Laboratories, Inc.), according to
manufacturer's
protocol number PT1200-1. The purified labeled probe is then used to detect
the expression of
Borrelia mRNA in an animal tissue sample.
Animal tissues, such as blood or spinal fluid, are examined with the labeled
probe using
ExpressHybTM hybridization solution (Clontech) according to manufacturer's
protocol number
25 PT1190-1. Following hybridization and washing, the blots are mounted and
exposed to film at -
70 C overnight, and films developed according to standard procedures.
The disclosure of all publications (including patents, patent applications,
journal articles,
laboratory manuals, books, or other documents) cited herein are hereby
incorporated by reference
in their entireties.
30 The present invention is not to be limited in scope by the specific
embodiments described
herein, which are intended as single illustrations of individual aspects of
the invention.
Functionally equivalent methods and components are within the scope of the
invention, in
addition to those shown and described herein and will become apparant to those
skilled in the art
from the foregoing description and accompanying drawings. Such modifications
are intended to
35 fall within the scope of the appended claims.
Provisional Application Serial No. 60/057,483 filed 3 September 1997 is
incorporated by
reference herein in its entirety.
s
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