U.S. patent application number 10/912362 was filed with the patent office on 2005-02-24 for enterococcus faecalis polynucleotides and polypeptides.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Bailey, Camella, Choi, Gil H., Hromockyj, Alex, Kunsch, Charles A..
Application Number | 20050043528 10/912362 |
Document ID | / |
Family ID | 27366427 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050043528 |
Kind Code |
A1 |
Choi, Gil H. ; et
al. |
February 24, 2005 |
Enterococcus faecalis polynucleotides and polypeptides
Abstract
The present invention relates to novel genes from E. faecalis
and the polypeptides they encode. Also provided as are vectors,
host cells, antibodies and methods for producing the same. The
invention further relates to screening methods for identifying
agonists and antagonists of E. faecalis polypeptide activity. The
invention additionally relates to diagnostic methods for detecting
Enterococcus nucleic acids, polypeptides and antibodies in a
biological sample. The present invention further relates to novel
vaccines for the prevention or attenuation of infection by
Enterococcus.
Inventors: |
Choi, Gil H.; (Rockville,
MD) ; Bailey, Camella; (Washington, DC) ;
Hromockyj, Alex; (Kalamazoo, MI) ; Kunsch, Charles
A.; (Norcross, GA) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
27366427 |
Appl. No.: |
10/912362 |
Filed: |
August 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10912362 |
Aug 6, 2004 |
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10206576 |
Jul 29, 2002 |
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10206576 |
Jul 29, 2002 |
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09071035 |
May 4, 1998 |
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6448043 |
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60066009 |
Nov 14, 1997 |
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60044031 |
May 6, 1997 |
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60046655 |
May 16, 1997 |
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Current U.S.
Class: |
536/23.7 |
Current CPC
Class: |
A61P 31/04 20180101;
C07K 14/24 20130101; C07K 14/315 20130101 |
Class at
Publication: |
536/023.7 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding any one of the amino acid
sequences of the polypeptides shown in Table 1; or (b) a nucleotide
sequence complementary to any one of the nucleotide sequences in
(a). (c) a nucleotide sequence at least 95% identical to any one of
the nucleotide sequences shown in Table 1; or, (d) a nucleotide
sequence at least 95% identical to a nucleotide sequence
complementary to any one of the nucleotide sequences shown in Table
1.
2. An isolated nucleic acid molecule of claim 1 comprising a
polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence
identical to a nucleotide sequence in (a) or (b) of claim 1.
3. An isolated nucleic acid molecule of claim 1 comprising a
polynucleotide which encodes an epitope-bearing portion of a
polypeptide in (a) of claim 1.
4. The isolated nucleic acid molecule of claim 3, wherein said
epitope-bearing portion of a polypeptide comprises an amino acid
sequence listed in Table 4.
5. A method for making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 1 into a vector.
6. A recombinant vector produced by the method of claim 5.
7. A host cell comprising the vector of claim 6.
8. A method of producing a polypeptide comprising: (a) growing the
host cell of claim 7 such that the protein is expressed by the
cell; and (b) recovering the expressed polypeptide.
9. An isolated polypeptide comprising a polypeptide selected from
the group consisting of: (a) a polypeptide consisting of one of the
complete amino acid sequences of Table 1; (b) a polypeptide
consisting of one the complete amino acid sequences of Table 1
except the N-terminal residue; (c) a fragment of the polypeptide of
(a) having biological activity; and (d) a fragment of the
polypeptide of (a) which binds to an antibody specific for the
polypeptide of (a).
10. An isolated antibody specific for the polypeptide of claim
9.
11. A polypeptide produced according to the method of claim 8.
12. An isolated polypeptide comprising an amino acid sequence at
least 95% identical to a sequence selected from the group
consisting of an amino acid sequence of any one of the polypeptides
in Table 1.
13. An isolated polypeptide antigen comprising an amino acid
sequence of an E. faecalis epitope shown in Table 4.
14. An isolated nucleic acid molecule comprising a polynucleotide
with a nucleotide sequence encoding a polypeptide of claim 9.
15. A hybridoma which produces an antibody of claim 10.
16. A vaccine, comprising: (1) one or more E. faecalis polypeptides
selected from the group consisting of a polypeptide of claim 9; and
(2) a pharmaceutically acceptable diluent, carrier, or excipient;
wherein said polypeptide is present, in an amount effective to
elicit protective antibodies in an animal to a member of the
Enterococcus genus.
17. A method of preventing or attenuating an infection caused by a
member of the Enterococcus genus in an animal, comprising
administering to said animal a polypeptide of claim 9, wherein said
polypeptide is administered in an amount effective to prevent or
attenuate said infection.
18. A method of detecting Enterococcus nucleic acids in a
biological sample comprising: (a) contacting the sample with one or
more nucleic acids of claim 1, under conditions such that
hybridization occurs, and (b) detecting hybridization of said
nucleic acids to the one or more Enterococcus nucleic acid
sequences present in the biological sample.
19. A method of detecting Enterococcus nucleic acids in a
biological sample obtained from an animal, comprising: (a)
amplifying one or more Enterococcus nucleic acid sequences in said
sample using polymerase chain reaction, and (b) detecting said
amplified Enterococcus nucleic acid.
20. A kit for detecting Enterococcus antibodies in a biological
sample obtained from an animal, comprising (a) a polypeptide of
claim 9 attached to a solid support; and (b) detecting means.
21. A method of detecting Enterococcus antibodies in a biological
sample obtained from an animal, comprising (a) contacting the
sample with a polypeptide of claim 9; and (b) detecting
antibody-antigen complexes.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/206,576, filed Jul. 29, 2002, which is a divisional of U.S.
application Ser. No. 09/071,035, filed May 4, 1998 (now U.S. Pat.
No. 6,448,043, issued Sep. 10, 2002), which is a nonprovisional of
and claims benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Application Nos. 60/046,655, filed May 16, 1997, 60/044,031, filed
May 6, 1997, and 60/066,009, filed Nov. 14, 1997. U.S. Provisional
Application No. 60/066,009, filed Nov. 14, 1997 is herein
incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING AND TABLE 1
[0002] This application refers to a "Sequence Listing" and Table 1
listed below, which are provided as electronic documents on two
identical compact discs (CD-R), labeled "Copy 1" and "Copy 2."
These compact discs each contain the following files, which are
hereby incorporated in their entirety herein:
1 Document File Name Size in bytes Date of Creation Sequence
PB369P1D2.seqlist.txt 1,476,601 Aug. 04, 2004 Listing Table 1
PB369P1D2.table1.txt 476,355 Aug. 04, 2004
FIELD OF THE INVENTION
[0003] The present invention relates to novel Enterococcus faecalis
genes (E. faecalis) nucleic acids and polypeptides. Also provided
are vectors, host cells and recombinant methods for producing the
same. Further provided are diagnostic methods for detecting
Enterococcus faecalis using probes, primers, and antibodies to the
E. faecalis nucleic acids and polypeptides of the present
invention. The invention further relates to screening methods for
identifying agonists and antagonists of E. faecalis polypeptide
activity and to vaccines using E. faecalis nucleic acids and
polypeptides.
BACKGROUND OF THE INVENTION
[0004] Enterococci have been recognized as being pathogenic for
humans since the turn of the century when they were first described
by Thiercelin in 1988 as microscopic organisms. The genus
Enterococcus includes the species Enterococcus faecalis or E.
faecalis which is the most common pathogen in the group, accounting
for 80-90 percent of all enterococcal infections. See Lewis et al.
(1990) Eur J. Clin Microbiol Infect Dis.9:111-117.
[0005] The incidence of enterococcal infections has increased in
recent years and enterococci are now the second most frequently
reported nosocomial pathogens. Enterococcal infection is of
particular concern because of its resistance to antibiotics. Recent
attention has focused on enterococci not only because of their
increasing role in nosocomial infections, but also because of their
remarkable and increasing resistance to antimicrobial agents. These
factors are mutually reinforcing since resistance allows
enterococci to survive in an environment in which antimicrobial
agents are heavily used; the hospital setting provides the
antibiotics which eliminate or suppress susceptible bacteria,
thereby providing a selective advantage for resistant organisms,
and the hospital also provides the potential for dissemination of
resistant enterococci via the usual routes of hand and
environmental contamination.
[0006] Antimicrobial resistance can be divided into two general
types, inherent or intrinsic property and that which is acquired.
The genes for intrinsic resistance, like other species
characteristics, appear to reside on the chromosome. Acquired
resistance results from either a mutation in the existing DNA or
acquisition of new DNA. The various inherent traits expressed by
enterococci include resistance to semisynthetic
penicillinase-resistant penicillins, cephalosporins, low levels of
aminoglycosides, and low levels of clindamycin. Examples of
acquired resistance include resistance to chloramphenicol,
erythromycin, high levels of clindamycin, tetracycline, high levels
of aminoglycosides, penicillin by means of penicillinase,
fluoroquinolones, and vancomycin. Resistance to high levels of
penicillin without penicillinase and resistance to fluoroquinolones
are not known to be plasmid or transposon mediated and presumably
are due to mutation(s).
[0007] Although the main reservoir for enterococci in humans is the
gastrointestinal tract, the bacteria can also reside in the
gallbladder, urethra and vagina.
[0008] E. faecalis has emerged as an important pathogen in
endocarditis, bacteremia, urinary tract infections (UTIs),
intraabdominal infections, soft tissue infections, and neonatal
sepsis. See Lewis et al. (1990) supra.. In the 1970s and 1980s
entercocci became firmly established as major nosocomial pathogens.
They are now the fourth leading cause of hospital-acquired
infection and the third leading cause of bacteremia in the United
States. Fatality ratios for enterococcal bactermia range from 12%
to 68%, with death due to enterococcal sepsis in 4 to 50% of these
cases. See T. G. Emori (1993) Clin. Microbiol. Rev. 6:428-442.
[0009] The ability of entercocci to colonize the gastrointestinal
tract, plus the many intrinsic and acquired resistance traits,
means that these organisms, which usually seem to have relatively
low intrinsic virulence, are given an excellent opportunity to
become secondary invaders. Since nosocomial isolates of entercocci
have displayed resistance to essentially every useful antimicrobial
agent, it will likely become increasingly difficult to successfully
treat and control enterococcal infections. Particularly when the
various resistance genes come together in a single strain, an event
almost certain to occur at some time in the future.
[0010] The etiology of diseases mediated or exacerbated by
Enterococcus faecalis, involves the programmed expression of E.
faecalis genes, and that characterizing these genes and their
patterns of expression would dramatically add to our understanding
of the organism and its host interactions. Knowledge of the E.
faecalis gene and genomic organization would improve our
understanding of disease etiology and lead to improved and new ways
of preventing, treating and diagnosing diseases. Thus, there is a
need to characterize the genome of E. faecalis and for
polynucleotides of this organism.
SUMMARY OF THE INVENTION
[0011] The present invention provides for isolated E. faecalis
polynucleotides and polypeptides shown in Table 1 and SEQ ID NO:1
through SEQ ID NO:496 (polynucleotide sequences having odd SEQ ID
NOs and polypeptide sequences having even SEQ ID NOs). One aspect
of the invention provides isolated nucleic acid molecules
comprising polynucleotides having a nucleotide sequence selected
from the group consisting of: (a) a nucleotide sequence shown in
Table 1; (b) a nucleotide sequence encoding any of the amino acid
sequences of the polypeptides shown in Table 1; and (c) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a) or (b). The invention further provides for
fragments of the nucleic acid molecules of (a), (b) & (c)
above.
[0012] 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) or (c) above, or a polynucleotide which
hybridizes under stringent hybridization conditions to a
polynucleotide in (a), (b) or (c) above. 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 E. faecalis polypeptide
having an amino acid sequence in (a) above.
[0013] 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. The
present invention further relates to the use of these vectors in
the production of E. faecalis polypeptides or peptides by
recombinant techniques.
[0014] The invention further provides isolated E. faecalis
polypeptides having an amino acid sequence selected from the group
consisting of an amino acid sequence of any of the polypeptides
described in Table 1 or fragments thereof.
[0015] 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 Table 1, as
well as polypeptides having an amino acid sequence at least 70%
identical, more preferably at least 75% 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.
[0016] The present invention further provides a single or
multi-component vaccine comprising one or more of the E. faecalis
polynucleotides or polypeptides described in Table 1, or fragments
thereof, together with a pharmaceutically acceptable diluent,
carrier, or excipient, wherein the E. faecalis polypeptide(s) are
present in an amount effective to elicit an immune response to
members of the Enterococcus genus, or at least E. faecalis, in an
animal. The E. faecalis polypeptides of the present invention may
further be combined with one or more immunogens of one or more
other Enterococcal or non-Enterococcal organisms to produce a
multi-component vaccine intended to elicit an immunological
response against members of the Enterococcus genus and, optionally,
one or more non-Enterococcal organisms.
[0017] The vaccines of the present invention can be administered in
a DNA form, e.g., "naked" DNA, wherein the DNA encodes one or more
Enterococcal polypeptides and, optionally, one or more polypeptides
of a non-Enterococcal organism. The DNA encoding one or more
polypeptides may be constructed such that these polypeptides are
expressed as fusion proteins.
[0018] The vaccines of the present invention may also be
administered as a component of a genetically engineered organism or
host cell. Thus, a genetically engineered organism or host cell
which expresses one or more E. faecalis polypeptides may be
administered to an animal. For example, such a genetically
engineered organism or host cell may contain one or more E.
faecalis polypeptides of the present invention intracellularly, on
its cell surface, or in its periplasmic space. Further, such a
genetically engineered organism or host cell may secrete one or
more E. faecalis polypeptides. The vaccines of the present
invention may also be co-administered to an animal with an immune
system modulator (e.g., CD86 and GM-CSF).
[0019] The invention also provides a method of inducing an
immunological response in an animal to one or more members of the
Enterococcus genus, preferably one or more isolates of the E.
faecalis species, comprising administering to the animal a vaccine
as described above.
[0020] The invention further provides a method of inducing a
protective immune response in an animal, sufficient to prevent,
attenuate, or control an infection by members of the Enterococcus
genus, preferably at least E. faecalis species, comprising
administering to the animal a composition comprising one or more of
the polynucleotides or polypeptides described in Table 1, or
fragments thereof. Further, these polypeptides, or fragments
thereof, may be conjugated to another immunogen and/or administered
in admixture with an adjuvant.
[0021] The invention further relates to antibodies elicited in an
animal by the administration of one or more E. faecalis
polypeptides of the present invention and to methods for producing
such antibodies and fragments thereof. The invention further
relates to recombinant antibodies and fragments thereof and to
methods for producing such antibodies and fragments thereof.
[0022] The invention also provides diagnostic methods for detecting
the expression of the polynucleotides of Table 1 by members of the
Enterococcus genus in an animal. One such method involves assaying
for the expression of a polynucleotide encoding E. faecalis
polypeptides 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 described
in Table 1) or indirectly (e.g., by assaying for antibodies having
specificity for amino acid sequences described in Table 1). The
expression of polynucleotides can also be assayed by detecting the
nucleic acids of Table 1. An example of such a method involves the
use of the polymerase chain reaction (PCR) to amplify and detect
Enterococcus nucleic acid sequences.
[0023] The present invention also relates to nucleic acid probes
having all or part of a nucleotide sequence described in Table 1
(odd SEQ ID NOs) which are capable of hybridizing under stringent
conditions to Enterococcus nucleic acids. The invention further
relates to a method of detecting one or more Enterococcus nucleic
acids in a biological sample obtained from an animal, said one or
more nucleic acids encoding Enterococcus 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 Enterococcus nucleic acid present in the biological
sample.
[0024] Other uses of the polypeptides of the present invention
include: inter alia, to detect E. aurues in immunoassays, as
epitope tags, as molecular weight markers on SDS-PAGE gels, as
molecular weight markers for molecular sieve gel filtration
columns, to generate antibodies that specificaly bind E. faecalis
polypeotides of the present invention for the detection E. faecalis
in immunoassays, to generate an immune response against E. faecalis
and other Enterococcus species, and as vaccines against E.
faecalis, other Enterococcus species and other bacteria
genuses.
[0025] Isolated nucleic acid molecules of the present invention,
particularly DNA molecules, are useful as probes for gene mapping
and for identifying E. faecalis in a biological samples, for
instance, by Southern and Northern blot analysis. Polynucleotides
of the present invention are also useful in detecting E. faecalis
by PCR using primers for a particular E. faecalis polynucleotide.
Isolated polynucleotides of the present invention are also useful
in making the polypeptides of the present invention.
DETAILED DESCRIPTION
[0026] The present invention relates to recombinant E. faecalis
nucleic acids and fragments thereof. The present invention further
relates to recombinant E. faecalis 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
Enterococcus, at least isolates of the E. faecalis genus. The
invention further relates to nucleic acid sequences which encode
antigenic E. faecalis polypeptides and to methods for detecting E.
faecalis nucleic acids and polypeptides in biological samples. The
invention also relates to antibodies specific for the polypeptides
and peptides of the present invention and methods for detecting
such antibodies produced in a host animal.
[0027] Definitions
[0028] The following definitions are provided to clarify the
subject matter which the inventors consider to be the present
invention.
[0029] 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 Enterococcus
which produce disease states in animals.
[0030] As used herein, the term "organism" means any living
biological system, including viruses, regardless of whether it is a
pathogenic agent.
[0031] As used herein, the term "Enterococcus" means any species or
strain of bacteria which is members of the genus Enterococcus. Such
species and strains are known to those of skill in the art, and
include those that are pathogenic and those that are not.
[0032] As used herein, the phrase "one or more E. faecalis
polypeptides of the present invention" means polypeptides
comprising the amino acid sequence of one or more of the E.
faecalis polypeptides described in Table 1 (even SEQ ID NOs). These
polypeptides may be expressed as fusion proteins wherein the E.
faecalis polypeptides of the present invention are linked to
additional amino acid sequences which may be of Enterococcal or
non-Enterococcal origin. This phrase further includes polypeptide
comprising fragments of the E. faecalis polypeptides of the present
invention. Additional definitions are provided throughout the
specification.
[0033] Explanation of Table 1
[0034] Table 1, provided on CD-R and incorporated by reference
herein, provides information describing genes which encode
polypeptides of E. faecalis. The table lists the gene identifier
which consists of the letters EF, which denote E. faecalis,
followed immediately by a three digit numeric code, which
arbitrarily number the E. faecalis genes of the present invention.
A number from 1 through 4 follows the three digit number. A number
1 represents the full length open reading frame of the gene
specified by the preceeding three digit number. A number 2
represents the full lenght polypeptide encoded by the gene
specified the preceeding three digit number. A number 3 represents
a polynucleotide fragment, of the gene represented by the
preceeding three digit number, used to produce an antigenic
polypeptide. A number 4 represents an antigenic polypeptide
fragement, of the gene represented by the preceeding three digit
number, used in the to stimulate an immune response or as a
vaccine. The nucleotide and amino acid sequences of each gene and
fragment are also shown in the Sequence Listing under the SEQ ID NO
listed in Table 1.
[0035] Explanation of Table 2
[0036] Table 2 lists accession numbers for the closest matching
sequences between the polypeptides of the present invention and
those available through GenBank and Derwent 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 1 lists the gene or ORF of the present invention. Column 2
lists the accession number of a "match" gene sequence in GenBank or
Derwent databases. Column 3 lists the description of 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 identity/similarity
with any polypeptide sequences of GenBank and Derwent 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 Derwent are represented more than
once.
[0037] Explanation of Table 3
[0038] The E. faecalis 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,
[0039] Explanation of Table 4
[0040] Table 4 lists residues comprising antigenic epitopes of
antigenic epitope-bearing fragments present in each of the full
length E. faecalis 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 3.11 for
the Power MacIntosh, DNASTAR, Inc., 1228 South Park Street Madison,
Wis.). E. faecalis 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 1 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.
[0041] Selection of Nucleic Acid Sequences Encoding Antigenic E.
faecalis Polypeptides
[0042] Sequenced E. faecalis genomic DNA was obtained from the E.
faecalis strain V586. The E. faecalis strain V586 was deposited 2
May 1997 at the ATCC, 10801 University Blvd. Manassas, Va.
20110-2209, and given accession number 55969.
[0043] Some ORFs contained in the subset of fragments of the E.
faecalis genome disclosed herein were derived through the use of a
number of screening criteria detailed below. The ORFs are bounded
at the amino terminus by a methionine or valine residue and usually
at the carboxy terminus by a stop codon.
[0044] Most of the selected sequences consist of complete ORFs. The
polypeptides that do not comprise a complete ORF can be determined
by determining whether the corresponding polynucleotide sequence
comprises a stop codon after the codon for the last amino acid
residue in the polypeptide sequence. 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 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.
[0045] 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 Enterococcus
faecalis ORFs for several aspects of potential immunogenicity. One
set of selection criteria are as follows:
[0046] 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, J. 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.
[0047] 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 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.
[0048] 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)).
[0049] 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, E. faecalis, 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. 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 (SEQ ID NO:497), where X is any amino
acid.
[0050] An algorithm for selecting antigenic and immunogenic
Enterococcus faecalis polypeptides including the foregoing criteria
was developed. The algorithm is similar to that described in U.S.
patent application Ser. No. 08/781,986, filed Jan. 3, 1997, which
is fully incorporated by reference herein. Use of the algorithm by
the inventors to select immunologically useful Enterococcus
faecalis 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.
[0051] Nucleic Acid Molecules
[0052] Sequenced E. faecalis genomic DNA was obtained from the E.
faecalis strainV586. As discussed elsewhere herein, polynucleotides
of the present invention readily may be obtained by routine
application of well known and standard procedures for cloning and
sequencing DNA. Detailed methods for obtaining libraries and for
sequencing are provided below, for instance. A wide variety of
Enterococcus faecalis strains that can be used to prepare E.
faecalis genomic DNA for cloning and for obtaining polynucleotides
and polypeptides of the present invention. A wide variety of
Enterococcus faecalis strains are available to the public from
recognized depository institutions, such as the American Type
Culture Collection (ATCC). It is recognized that minor variation is
the nucleic acid and amino acid sequence may be expected from E
faecalis strain to strain. The present invention provides for
genes, including both polynucleotides and polypeptides, of the of
the present invention from all the Enterococcus faecalis
strains.
[0053] 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., Foster City, Calif.), and all amino acid
sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined
as 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 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 from the amino acid sequence actually encoded by the
sequenced DNA molecule, beginning at the point of such an insertion
or deletion. In case of conflict between Table 1 and either the
nucleic acid sequence of the clones listed in Table 1 or the amino
acid sequence of the protein expressed by the clones listed in
Table 1, the clones listed in Table 1 are controlling. By
"nucleotide sequence" of a nucleic acid molecule or polynucleotide
is intended to mean either a DNA or RNA sequence. Using the
information provided herein, such as the nucleotide sequence in
Table 1, a nucleic acid molecule of the present invention encoding
a E. faecalis polypeptide may be obtained using standard cloning
and screening procedures, such as those for cloning DNAs using
genomic DNA as starting material. 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). Illustrative of the
invention, the nucleic acid molecule described in Table 1 was
discovered in a DNA library derived from a E. faecalis genomic
DNA.
[0054] 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, DNA 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.
[0055] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. This includes segments of DNA comprising the E.
faecalis polynucleotides of the present invention isolated from the
native chromosome. These fragments include both isolated fragments
consisting only of E. faecalis DNA and fragments comprising
heterologous sequences such as vector sequences or other foreign
DNA. 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.
[0056] 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 E. faecalis
polypeptides and peptides of the present invention (e.g.
polypeptides of Table 1). That is, all possible DNA sequences that
encode the E. faecalis 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).
[0057] 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 E. faecalis in a biological sample, for instance, by
PCR, Southern blot, Northern blot, or other form of hybridization
analysis.
[0058] 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, or the E. faecalis nucleotide
sequences contained in the plasmid clones listed in 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 3' nucleotide position, where the
first nucleotide for each nucleotide sequence in Table 1 is
position 1. That is, every combination of a 5' and 3' nucleotide
position that a fragment at least 10 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 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.
[0059] Further, the invention includes polynucleotides comprising
fragments specified by size, in nucleotides, rather than by
nucleotide positions. The invention includes any fragment size, in
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 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 1 or of the E.
faecalis nucleotide sequences of the plasmid 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 the invention. Additional preferred
nucleic acid fragments of the present invention include nucleic
acid molecules encoding epitope-bearing portions of E. faecalis
polypeptides identified in Table 4.
[0060] 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
Table 1 or the plasmid clones listed in Table 1. Any number of
fragments of nucleotide sequences in Table 1 or the plasmid 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.
[0061] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of a
polynucleotide in a nucleic acid molecules of the invention
described above, for instance, nucleotide sequences of Table 1 or
the E. faecalis sequences of the plasmid clones listed in Table 1.
By "stringent hybridization conditions" is intended overnight
incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0062] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides bases, and more
preferably at least about 20 nucleotides bases, still more
preferably at least about 30 nucleotides bases, and even more
preferably about 30-70 (e.g., 50) nucleotides bases of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above. By a portion of a polynucleotide of "at
least 20 nucleotides bases in length," for example, is intended 20
or more contiguous nucleotides bases nucleotides from the
nucleotide sequence of the reference polynucleotide (e.g., the
nucleotide sequence as shown in Table 1). Portions of a
polynucleotide which hybridizes to a nucleotide sequence in Table
1, which can be used as probes and primers, may also be precisely
specified by 5' and 3' base positions or by size in nucleotide
bases as described above or precisely excluded in the same
manner.
[0063] The nucleic acid molecules of the present invention include
those encoding the full length E. faecalis polypeptides of Table 1
and portions of the E. faecalis polypeptides of Table 1. Also
included in the present invention are nucleic acids encoding the
above full length sequences and further comprise additional
sequences, such as those encoding an added secretory leader
sequence, such as a pre-, or pro- or prepro- protein sequence.
Further included in the present invention are nucleic acids
encoding the above full length sequences and portions thereof and
further comprise additional heterologous amino acid sequences
encoded by nucleic acid sequences from a different source.
[0064] Also included in the present invention are nucleic acids
encoding the above protein sequences together with additional,
non-coding sequences, including for example, but not limited to
non-coding 5' and 3' sequences. These sequences include
transcribed, non-translated sequences that may play a role in
transcription, and mRNA processing, for example, ribosome binding
and stability of mRNA. Also included in the present invention are
additional coding sequences which provide additional
functionalities.
[0065] Thus, a nucleotide sequence encoding a polypeptide 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., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. 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 E. faecalis polypeptides of the present invention fused
to Fc at the N- or C-terminus.
[0066] Variant and Mutant Polynucleotides
[0067] The present invention further relates to variants of the
nucleic acid molecules which encode portions, analogs or
derivatives of a E. faecalis polypeptides of Table 1 and variant
polypeptides thereof including portions, analogs, and derivatives
of the E. faecalis polypeptides. 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. See, e.g., B. Lewin, Genes IV (1990).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0068] Such nucleic acid variants include those produced by
nucleotide substitutions, deletions, or additions. The
substitutions, deletions, or additions may involve one or more
nucleotides. The variants may be altered in coding regions,
non-coding regions, or both. Alterations in the coding regions 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 a E. faecalis protein of the present
invention or portions thereof. Also especially preferred in this
regard are conservative substitutions.
[0069] Such polypeptide variants include those produced by amino
acid substitutions, deletions or additions. The substitutions,
deletions, or additions may involve one or more residues.
Alterations 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 a E. faecalis protein of
the present invention or portions thereof. Also especially
preferred in this regard are conservative substitutions.
[0070] 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 them for production of E. faecalis polypeptides or peptides
by recombinant techniques.
[0071] The present application is 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 E. faecalis activity. This is because even where
a particular nucleic acid molecule does not encode a polypeptide
having E. faecalis 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 molecules of the
present invention that do not encode a polypeptide having E.
faecalis activity include, inter alia, isolating an E. faecalis
gene or allelic variants thereof from a DNA library, and detecting
E. faecalis mRNA expression samples, environmental samples,
suspected of containing E. faecalis by Northern Blot analysis.
[0072] 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 E. faecalis protein activity By "a polypeptide having E.
faecalis activity" is intended polypeptides exhibiting activity
similar, but not necessarily identical, to an activity of the E.
faecalis protein of the invention, as measured in a particular
biological assay suitable for measuring activity of the specified
protein.
[0073] Due to the degeneracy of the genetic code, one of ordinary
skill in the art will immediately 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 E. faecalis 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 that,
for such nucleic acid molecules that are not degenerate variants, a
reasonable number will also encode a polypeptide having E. faecalis
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.
[0074] 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.
[0075] 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 may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence encoding the E. faecalis polypeptide. In other words, to
obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% 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 1, the ORF (open reading frame), or
any fragment specified as described herein.
[0076] 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) 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 al. (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 length of the subject
nucleotide sequence, whichever is shorter.
[0077] 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 arrive 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.
[0078] 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
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.
[0079] Vectors and Host Cell
[0080] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
comprising the recombinant vectors, and the production of E.
faecalis polypeptides and peptides of the present invention
expressed by the host cells.
[0081] 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.
[0082] 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.
[0083] Preferred are vectors comprising cis-acting control regions
to the polynucleotide of interest. Appropriate trans-acting factors
may be supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0084] 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.
[0085] 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.
[0086] 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 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.
[0087] 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, kanamycin, 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 above-described host cells are known in the
art.
[0088] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE9, pQE10 available from Qiagen; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH 18A,
pNH46A available from Stratagene; pET series of vectors available
from Novagen; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
available from Pharmacia. Among preferred eukaryotic vectors are
pWLNEO, pSV2CAT, pOG44, pXT1 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.
[0089] Among known bacterial promoters suitable for use in the
present invention include the E. coli laci and lacZ promoters, the
T3, T5 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.
[0090] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals (for
example, Davis, et al., Basic Methods In Molecular Biology
(1986)).
[0091] Transcription of DNA encoding the polypeptides of the
present invention by higher 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
nucleotides 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 nucleotides 100 to 270, the cytomegalovirus
early promoter enhancer, the polyoma enhancer on the late side of
the replication origin, and adenovirus enhancers.
[0092] 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, for example, the amino
acid sequence KDEL. The signals may be endogenous to the
polypeptide or they may be heterologous signals.
[0093] 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 amino 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 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
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobulin 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, hIL5-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. (1995) J. Molec. Recogn. 8:52-58 and
Johanson, K. et al. (1995) J. Biol. Chem. 270 (16):9459-9471.
[0094] The E. faecalis polypeptides can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation 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 techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells.
[0095] Polypeptides and Fragments
[0096] The invention further provides an isolated E. faecalis
polypeptide having an amino acid sequence in Table 1, or a peptide
or polypeptide comprising a portion of the above polypeptides.
[0097] Variant and Mutant Polypeptides
[0098] To improve or alter the characteristics of E. faecalis
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.
[0099] N-Terminal and C-Terminal Deletion Mutants
[0100] 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 finction. For instance, Ron et al. J. Biol. Chem.,
268:2984-2988 (1993), reported modified KGF proteins that had
heparin binding 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 E. faecalis polypeptides
shown in Table 1, and polynucleotides encoding such
polypeptides.
[0101] Similarly, many examples of biologically functional
C-terminal deletion muteins are 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 E. faecalis 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.
[0102] 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 1, 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.
[0103] Further, the invention includes polypeptides comprising
fragments specified by size, in 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.
[0104] 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.
[0105] Other Mutants
[0106] In addition to N- and C-terminal deletion forms of the
protein discussed above, it also will be recognized by one of
ordinary skill in the art that some amino acid sequences of the E.
faecalis 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.
[0107] Thus, the invention further includes variations of the E.
faecalis polypeptides which show substantial E. faecalis
polypeptide activity or which include regions of E. faecalis
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 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.
[0108] 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 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.
[0109] Thus, the fragment, derivative, analog, or homolog of the
polypeptide of Table 1, or that encoded by the plasmids 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 E.
faecalis 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.
[0110] Thus, the E. faecalis polypeptides of the present invention
may include one or more amino acid substitutions, deletions, or
additions, either from natural mutations or human 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).
[0111] Amino acids in the E. faecalis 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.
[0112] 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.
[0113] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of the E. faecalis
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.
[0114] The invention further provides for isolated E. faecalis
polypeptides comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence of a full-length
E. faecalis polypeptide having the complete amino acid sequence
shown in Table 1; (b) the amino acid sequence of a full-length E.
faecalis 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 plasmids listed in Table 1; and
(d) the complete amino acid sequence excepting the N-terminal
methionine encoded by the plasmids 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.
[0115] 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.
[0116] A further embodiment of the invention relates to a
polypeptide which comprises the amino acid sequence of a E.
faecalis 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 E.
faecalis polypeptide, having at least one, but not more than 10, 9,
8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
[0117] 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.
[0118] 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 plasmids listed in Table 1 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) 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 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.
[0119] 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 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
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 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.
[0120] 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 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 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 purposes of the present invention.
[0121] 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 polypeptides of the present invention
that do not have E. faecalis 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.
[0122] 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 E. faecalis
protein expression or as agonists and antagonists capable of
enhancing or inhibiting E. faecalis protein function. Further, such
polypeptides can be used in the yeast two-hybrid system to
"capture" E. faecalis protein binding proteins which are also
candidate agonists and antagonists according to the present
invention. See, e.g., Fields et al. (1989) Nature 340:245-246.
[0123] Epitope-Bearing Portions
[0124] In another aspect, the invention provides peptides and
polypeptides comprising epitope-bearing portions of the E. faecalis
polypeptides of the present invention. These 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 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 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 antigenic
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.
[0125] 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 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 HA1 polypeptide chain, induced
antibodies that reacted with the HA 1 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.
[0126] 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 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 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 for purification of the mimicked protein, for instance, by
adsorption chromatography using methods known in the art.
[0127] 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, 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 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.
[0128] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate an enterococcal-specific immune
response or antibodies include portions of the amino acid sequences
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 isolated and purified antigenic
epitope-bearing fragments 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 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) 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 fragments 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 fragments of the present invention, wherein the
initiation codon is residue 1. Any number of the described
antigenic epitope-bearing fragments of the present invention may
also be excluded from the present invention in the same manner.
[0129] 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. Pat. 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.
[0130] 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 carrier, 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 .mu.g peptide or 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.
[0131] 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 all 20 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. Pat. No. 4,708,781 to Geysen
(1987) further describes this method of identifying a peptide
bearing an immunogenic epitope of a desired protein.
[0132] Further still, U.S. Pat. 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. Pat. 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. Pat. No. 5,480,971
to Houghten, R. A. et al. (1996) discloses linear
C.sub.1-C.sub.7-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 "Polypeptides and Fragments" is hereby
incorporated herein by reference.
[0133] 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 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 E. faecalis polypeptide or fragment thereof alone. See
Fountoulakis et al. (1995) J. Biochem. 270:3958-3964. Nucleic acids
encoding the above epitopes of E. faecalis polypeptides can also be
recombined with a gene of interest as an epitope tag to aid in
detection and purification of the expressed polypeptide.
[0134] Antibodies
[0135] E. faecalis protein-specific antibodies for use in the
present invention can be raised against the intact E. faecalis
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 carrier.
[0136] 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
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 E. faecalis
polypeptide or fragment thereof is prepared and purified to render
it substantially free of natural contaminants. Such a preparation
is then introduced into an animal in order to produce polyclonal
antisera of greater specific activity.
[0137] 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 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 maybe
produced by proteolytic cleavage, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
Alternatively, E. faecalis polypeptide-binding fragments, chimeric,
and humanized antibodies can be produced through the application of
recombinant DNA technology or through synthetic chemistry using
methods known in the art.
[0138] 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, therefore, it is possible to obtain an antibody
which binds to a second antibody. In accordance with this method,
E. faecalis 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
to bind to the E. faecalis polypeptide-specific antibody can be
blocked by the E. faecalis polypeptide antigen. Such antibodies
comprise anti-idiotypic antibodies to the E. faecalis
polypeptide-specific antibody and can be used to immunize an animal
to induce formation of further E. faecalis polypeptide-specific
antibodies.
[0139] Antibodies and fragments thereof of the present invention
may be described by the portion of a polypeptide of the present
invention recognized or specifically bound by the antibody.
Antibody binding fragments of a polypeptide of the present
invention may be described or specified in the same manner as for
polypeptide fragments 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-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 particularly
described fragment of a polypeptide of the present invention and
allows for the exclusion of the same.
[0140] Antibodies and fragments thereof of the present invention
may also be described or specified in terms of their
cross-reactivity. Antibodies and fragments that do not bind
polypeptides of any other species of Enterococcus other than E.
faecalis are included in the present invention. Likewise,
antibodies and fragments that bind only species of Enterococcus,
i.e. antibodies and fragments that do not bind bacteria from any
genus other than Enterococcus, are included in the present
invention.
[0141] Diagnostic Assays
[0142] The present invention further relates to methods for
assaying enterococcal infection in an animal by detecting the
expression of genes encoding enterococcal polypeptides of the
present invention. The methods comprise analyzing tissue or body
fluid from the animal for Enterococcus-specific antibodies, nucleic
acids, or proteins. Analysis of nucleic acid specific to
Enterococcus 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).
[0143] Where diagnosis of a disease state related to infection with
Enterococcus has already been made, the present invention is useful
for monitoring progression or regression of the disease state
whereby patients exhibiting enhanced Enterococcus gene expression
will experience a worse clinical outcome relative to patients
expressing these gene(s) at a lower level.
[0144] By "biological sample" is intended any biological sample
obtained from an animal, cell line, tissue culture, or other source
which contains Enterococcus 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
Enterococcus polypeptides or nucleic acids. Methods for obtaining
biological samples such as tissue are well known in the art.
[0145] The present invention is useful for detecting diseases
related to Enterococcus infections in animals. Preferred animals
include monkeys, apes, cats, dogs, birds, cows, pigs, mice, horses,
rabbits and humans. Particularly preferred are humans.
[0146] 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
Enterococcus polypeptides having sufficient 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, S1
nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0147] 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 is prehybridized in a solution
containing formamide, SSC, Denhardt's solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. A E. faecalis
polynucleotide sequence shown in Table 1 labeled according to any
appropriate method (such as the .sup.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 the
sections above and will preferably at least 15 nucleotides in
length.
[0148] S1 mapping can be performed as described in Fujita et al.
(1987) Cell 49:357-367. To prepare probe DNA for use in S1 mapping,
the sense strand of an above-described E. faecalis 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 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 Enterococcus polypeptides).
[0149] Levels of mRNA encoding Enterococcus 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 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 Enterococcus 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).
[0150] The polynucleotides of the present invention, including both
DNA and RNA, may be used to detect polynucleotides of the present
invention or Enterococcal species including E. faecalis using bio
chip technology. The present invention includes both high density
chip arrays (>1000 oligonucleotides per cm.sup.2) and low
density chip arrays (<1000 oligonucleotides per cm.sup.2). Bio
chips comprising arrays of polynucleotides of the present invention
may be used to detect Enterococcal species, including E. faecalis,
in biological and environmental samples and to diagnose an animal,
including humans, with an E. faecalis or other Enterococcal
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 differential pathogenic detection and diagnosis. The bio
chips can also be used to monitor an E. faecalis or other
Enterococcal 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 fragments,
i.e, by their 5' and 3' positions or length in contiguous base
pairs and include from. Methods and particular uses of the
polynucleotides of the present invention to detect Enterococcal
species, including E. faecalis, using bio chip technology include
those known in the art and those of: U.S. Pat. Nos. 5,510,270,
5,545,531, 5,445,934, 5,677,195, 5,532,128, 5,556,752, 5,527,681,
5,451,683, 5,424,186, 5,607,646, 5,658,732 and World Patent Nos.
WO/9710365, WO/9511995, WO/9743447, WO/9535505, each incorporated
herein in their entireties.
[0151] Biosensors using the polynucleotides of the present
invention may also be used to detect, diagnose, and monitor E.
faecalis or other Enterococcal species and infections thereof.
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 Enterococcal species, including E. faecalis,
using biosensors include those known in the art and those of: U.S.
Pat. Nos. 5,721,102, 5,658,732, 5,631,170, and World Patent Nos.
W097/35011, WO/9720203, each incorporated herein in their
entireties.
[0152] Thus, the present invention includes both bio chips and
biosensors comprising polynucleotides of the present invention and
methods of their use.
[0153] Assaying Enterococcus polypeptide levels in a biological
sample can occur using any art-known method, such as antibody-based
techniques. For example, Enterococcus 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 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 Enterococcus 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, which is based on the use of
cationic solid phases, quantitation of a Enterococcus polypeptide
can be accomplished using an isolated Enterococcus polypeptide as a
standard. This technique can also be applied to body fluids.
[0154] Other antibody-based methods useful for detecting
Enterococcus polypeptide gene expression include immunoassays, such
as the ELISA and the radioimmunoassay (RIA). For example, a
Enterococcus polypeptide-specific monoclonal antibodies can be used
both as an immunoabsorbent and as an enzyme-labeled probe to detect
and quantify a Enterococcus polypeptide. The amount of a
Enterococcus 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
lacobelli et al. (1988) Breast Cancer Research and Treatment
11:19-30. In another ELISA assay, two distinct specific monoclonal
antibodies can be used to detect Enterococcus 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.
[0155] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. The "one-step" assay involves
contacting the Enterococcus polypeptide with immobilized antibody
and, 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 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).
[0156] 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 (.sup.125I,
.sup.121I), carbon (.sup.14C), sulphur (.sup.35S), tritium
(.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0157] Further suitable labels for the Enterococcus
polypeptide-specific antibodies of the present invention are
provided below. Examples of suitable enzyme labels include malate
dehydrogenase, Enterococcal 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.
[0158] Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc,
.sup.109Pd, etc. .sup.111In is a preferred isotope where in vivo
imaging is used since its avoids the problem of dehalogenation of
the .sup.125I or .sup.131I-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, .sup.111In coupled to monoclonal
antibodies with 1-(P-isothiocyanatobenzy- l)-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.
[0159] Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, and .sup.56Fe.
[0160] Examples of suitable fluorescent labels include an
.sup.152Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine
label.
[0161] Examples of suitable toxin labels include, Pseudomonas
toxin, diphtheria toxin, ricin, and cholera toxin.
[0162] 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.
[0163] Examples of nuclear magnetic resonance contrasting agents
include heavy metal nuclei such as Gd, Mn, and iron.
[0164] Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al. (1976) Clin. Chim. Acta
70:1-31, and Schurs et al. (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.
[0165] In a related aspect, the invention includes a diagnostic kit
for use in screening serum containing antibodies specific against
E. faecalis infection. Such a kit may include an isolated E.
faecalis antigen comprising an epitope which is specifically
immunoreactive with at least one anti-E. faecalis 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
recombinantly produced or chemically synthesized peptide or
polypeptide antigen. The peptide or polypeptide antigen may be
attached to a solid support.
[0166] 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 antibody to the E. faecalis antigen can
be detected by binding of the reporter labeled antibody to the
anti-E. faecalis polypeptide antibody.
[0167] In a related aspect, the invention includes a method of
detecting E. faecalis infection in a subject. This detection method
includes reacting a body fluid, preferably serum, from the subject
with an isolated E. faecalis antigen, and examining the antigen for
the presence of 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.
[0168] The solid surface reagent employed in the above assays and
kits is prepared by known 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
conjunction with biotinylated antigen(s).
[0169] The polypeptides and antibodies of the present invention,
including fragments thereof, may be used to detect Enterococcal
species including E. faecalis 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 Enterococcal species,
including E. faecalis. Bio chip and biosensors of the present
invention may also comprise antibodies which specifically recognize
the polypeptides of the present invention to detect Enterococcal
species, including E. faecalis or specific polypeptides of the
present invention. Bio chips or biosensors comprising polypeptides
or antibodies of the present invention may be used to detect
Enterococcal species, including E. faecalis, in biological and
environmental samples and to diagnose an animal, including humans,
with an E. faecalis or other Enterococcal infection. Thus, the
present invention includes both bio chips and biosensors comprising
polypeptides or antibodies of the present invention and methods of
their use.
[0170] 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
polypeptide sequences of the present invention, for use in rapid
differential pathogenic detection and diagnosis. The bio chips of
the present invention may further comprise antibodies or fragments
thereof specific for other pathogens including bacteria, viral,
parasitic, and fungal polypeptide sequences, in addition to the
antibodies or fragments thereof of the present invention, for use
in rapid differential pathogenic detection and diagnosis. The bio
chips and biosensors of the present invention may also be used to
monitor an E. faecalis or other Enterococcal infection and to
monitor the genetic changes (amino 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 fragments, i.e, by their N-terminal and
C-terminal positions or length in contiguous amino acid residue.
Methods and particular uses of the polypeptides and antibodies of
the present invention to detect Enterococcal species, including E.
faecalis, or specific polypeptides using bio chip and biosensor
technology include those known in the art, those of the U.S. Pat.
Nos. and World Patent Nos. listed above for bio chips and
biosensors using polynucleotides of the present invention, and
those of: U.S. Pat. Nos. 5,658,732, 5,135,852, 5,567,301,
5,677,196, 5,690,894 and World Patent Nos. WO9729366, WO9612957,
each incorporated herein in their entireties.
[0171] Treatment:
[0172] Agonists and Antagonists--Assays and Molecules
[0173] The invention also provides a method of screening compounds
to identify those which enhance or block the biological activity of
the E. faecalis polypeptides of the present invention. The present
invention further provides where the compounds kill or slow the
growth of E. faecalis. The ability of E. faecalis antagonists,
including E. faecalis 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.
[0174] 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.
[0175] The antagonists may be employed for instance to inhibit
peptidoglycan cross bridge formation. Antibodies against E.
faecalis may be employed to bind to and inhibit E. faecalis
activity to treat antibiotic resistance. Any of the above
antagonists may be employed in a composition with a
pharmaceutically acceptable carrier.
[0176] Vaccines
[0177] 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 E. faecalis
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 Enterococcus genus than single
polypeptide vaccines.
[0178] Multi-component vaccines are known in the art to elicit
antibody production to numerous immunogenic components. See, e.g.,
Decker et al. (1996) J. Infect. Dis. 174:S270-275. 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.
See, e.g., Aristegui, J. et al. (1997) Vaccine 15:7-9.
[0179] The present invention in addition to single-component
vaccines includes multi-component vaccines. These vaccines comprise
more than one polypeptide, immunogen or antigen. Thus, a
multi-component vaccine would be a vaccine comprising more than one
of the E. faecalis polypeptides of the present invention.
[0180] 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 E. faecalis
polypeptides described in Table 1. For example, the E. faecalis
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 E. faecalis
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. (1997) Nature Biotech. 15:653-657;
Sirard, J. et al. (1997) Infect. Immun. 65:2029-2033; Chabalgoity,
J. et al. (1997) Infect. Immun. 65:2402-2412. 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.
[0181] A multi-component vaccine can also be prepared using
techniques known in the art by combining one or more E. faecalis
polypeptides of the present invention, or fragments thereof, with
additional non-Enterococcal 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 Enterococcus genus and
non-Enterococcal pathogenic agents.
[0182] The vaccines of the present invention also include DNA
vaccines. DNA vaccines are currently being developed for a number
of infectious diseases. See, et al., Boyer, et al. (1997) Nat. Med.
3:526-532; reviewed in Spier, R. (1996) Vaccine 14:1285-1288. Such
DNA vaccines contain a nucleotide sequence encoding one or more E.
faecalis polypeptides of the present invention oriented in a manner
that allows for expression of the subject polypeptide. For example,
the direct administration of plasmid DNA encoding B. burgdorgeri
OspA has been shown to elicit protective immunity in mice against
borrelial challenge. See, Luke et al. (1997) J. Infect. Dis.
175:91-97.
[0183] The present invention also relates to the administration of
a vaccine which is co-administered with a molecule capable of
modulating immune responses. Kim et al. (1997) Nature Biotech.
15:641-646, 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.
[0184] The vaccines of the present invention may be used to confer
resistance to Enterococcal infection by either passive or active
immunization. When the vaccines of the present invention are used
to confer resistance to Enterococcal 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 Enterococcal infection. When the vaccines
of the present invention are used to confer resistance to
Enterococcal 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 Enterococcus genus.
[0185] The ability to label antibodies, or fragments of antibodies,
with toxin molecules provides an additional method for treating
Enterococcal infections when passive immunization is conducted. In
this embodiment, antibodies, or fragments of antibodies, capable of
recognizing the E. faecalis polypeptides disclosed herein, or
fragments thereof, as well as other Enterococcus proteins, are
labeled with toxin molecules prior to their administration to the
patient. When such toxin derivatized antibodies bind to
Enterococcus cells, toxin moieties will be localized to these cells
and will cause their death.
[0186] The present invention thus concerns and provides a means for
preventing or attenuating a Enterococcal 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.
[0187] The administration of the vaccine (or the antisera which it
elicits) may be for either a "prophylactic" or "therapeutic"
purpose. When provided prophylactically, the compound(s) are
provided in advance of any symptoms of Enterococcal infection. The
prophylactic administration of the compound(s) serves to prevent or
attenuate any subsequent infection. When provided therapeutically,
the compound(s) is provided upon or after the detection of symptoms
which indicate that an animal may be infected with a member of the
Enterococcus genus. The therapeutic administration of the
compound(s) serves to attenuate any actual infection. Thus, the E.
faecalis 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.
[0188] 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 macromolecular
carrier. Example of such carriers 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, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988).
[0189] A composition is said to be "pharmacologically or
physiologically 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 is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient patient.
[0190] 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 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 E. faecalis polypeptides of the
present invention will normally be higher when administered to a
human than when administered to a non-human animal.
[0191] 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
antigen(s) from being rapidly catabolized after administration and
(2) they nonspecifically stimulate immune responses.
[0192] 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 oil adjuvants (for example, Freund's complete
and incomplete), mineral salts (for example, AlK(SO.sub.4).sub.2,
AlNa(SO.sub.4).sub.2, AlNH.sub.4(SO.sub.4), 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 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 AlK(SO.sub.4).sub.2, AlNa(SO.sub.4).sub.2,
and AlNH.sub.4(SO.sub.4). Examples of materials suitable for use in
vaccine compositions are provided in REMINGTON'S PHARMACEUTICAL
SCIENCES 1324-1341 (A. Osol, ed, Mack Publishing Co, Easton, Pa.,
(1980) (incorporated herein by reference).
[0193] 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, 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 liposome solution
containing the liquid dosage form. Suitable forms for suspending
liposomes include emulsions, suspensions, solutions, syrups, and
elixirs containing inert diluents 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.
[0194] Therapeutic compositions of the present invention can also
be administered in encapsulated form. For example, intranasal
immunization using vaccines encapsulated in biodegradable
microsphere composed of poly(DL-lactide-co-glycolide). See, Shahin,
R. et al. (1995) Infect. Immun. 63:1195-1200. Similarly, orally
administered encapsulated Salmonella typhimurium antigens can also
be used. Allaoui-Attarki, K. et al. (1997) Infect. Immun.
65:853-857. 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).
[0195] 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.
[0196] 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.
[0197] 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 .mu.g/ml per dose, more preferably 0.1-500
.mu.g/ml per dose, and most preferably 10-300 .mu.g/ml per
dose.
EXAMPLES
Example 1
Isolation of a Selected DNA Clone From the Deposited Sample of E.
faecalis
[0198] Three approaches can be used to isolate a E. faecalis clone
comprising a polynucleotide of the present invention from any E.
faecalis genomic DNA library. The E. faecalis strain V586 has been
deposited as a convienent source for obtaining a E. faecalis strain
although a wide varity of strains E. faecalis strains can be used
which are known in the art.
[0199] E. faecalis genomic DNA is prepared using the following
method. A 20 ml overnight bacterial culture grown in a rich medium
(e.g., Trypticase Soy Broth, Brain Heart Infusion broth or Super
broth), pelleted, washed two times with TES (3 OmM Tris-pH 8.0, 25
mM EDTA, 50 mM NaCl), and resuspended in 5 ml high salt TES (2.5M
NaCl). Lysostaphin is added to final concentration of approx 50
ug/ml and the mixture is rotated slowly 1 hour at 37 C to make
protoplast cells. The solution is then placed in incubator (or
place in a shaking water bath) and warmed to 55 C. Five hundred
micro liter of 20% sarcosyl in TES (final concentration 2%) is then
added to lyse the cells. Next, guanidine HCl is added to a final
concentration of 7M (3.69 g in 5.5 ml). The mixture is swirled
slowly at 55 C for 60-90 min (solution should clear). A CsCl
gradient is then set up in SW41 ultra clear tubes using 2.0 ml 5.7M
CsCl and overlaying with 2.85M CsCl. The gradient is carefully
overlayed with the DNA-containing GuHCl solution. The gradient is
spun at 30,000 rpm, 20 C 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.
[0200] In the first method, a plasmid is directly isolated by
screening a plasmid E. faecalis 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 .sup.32P-.gamma.-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, N.Y. (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 MOLECULAR
BIOLOGY (John Wiley and Sons, N.Y. 1989) or other techniques known
to those of skill in the art.
[0201] 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 E. faecalis
genomic DNA prep as a template. PCR is carried out under routine
conditions, for instance, in 25 .mu.l of reaction mixture with 0.5
ug of the above DNA template. A convenient reaction mixture is
1.5-5 mM MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each of dATP,
dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq
polymerase. Thirty five cycles of PCR (denaturation at 94.degree.
C. for 1 min; annealing at 55.degree. C. for 1 min; elongation at
72.degree. C. for 1 min) are 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.
[0202] Finally, overlapping oligos of the DNA sequences of Table 1
can be chemically synthesized and used to generate a nucleotide
sequence of desired length using PCR methods known in the art.
Example 2(a)
Expression and Purification Enterococcal polypeptides in E.
coli
[0203] The bacterial expression vector pQE60 was used for bacterial
expression of some of the polypeptide fragments used in the soft
tissue and systemic infection models discussed below. (QIAGEN,
Inc., 9259 Eton Avenue, Chatsworth, Calif., 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 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.times.His tag") covalently linked to the
carboxyl terminus of that polypeptide.
[0204] The DNA sequence encoding the desired portion of a E.
faecalis protein of the present invention was amplified from E.
faecalis genomic DNA using PCR oligonucleotide primers which anneal
to the 5' and 3' sequences coding for the portions of the E.
faecalis 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.
[0205] 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 E. faecalis
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 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.
[0206] The amplified E. faecalis DNA fragment and the vector pQE60
were digested with restriction enzymes which recognize the sites in
the primers and the digested DNAs were then ligated together. The
E. faecalis DNA was inserted into the restricted pQE60 vector in a
manner which places the E. faecalis protein coding region
downstream from the IPTG-inducible promoter and in-frame with an
initiating AUG and the six histidine codons.
[0207] The ligation mixture was transformed into competent E. coli
cells using standard procedures such as those described by Sambrook
et al., supra. E. coli strain M15/rep4, containing multiple copies
of the plasmid pREP4, which expresses the lac repressor and confers
kanamycin resistance ("Kanr"), was used in carrying out the
illustrative example described herein. This strain, which was only
one of many that are suitable for expressing a E. faecalis
polypeptide, is available commercially (QIAGEN, Inc., supra).
Transformants were identified by their ability to grow on LB agar
plates in the presence of ampicillin and kanamycin. Plasmid DNA was
isolated from resistant colonies and the identity of the cloned DNA
confirmed by restriction analysis, PCR and DNA sequencing.
[0208] Clones containing the desired constructs were grown
overnight ("O/N") in liquid culture in LB media supplemented with
both ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture was used to inoculate a large culture, at a dilution of
approximately 1:25 to 1:250. The cells were grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-.beta.-D-thiogalactopyranoside ("IPTG") was 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 were incubated further for 3 to 4 hours. Cells
then were harvested by centrifugation.
[0209] The cells were then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris was removed by
centrifugation, and the supernatant containing the E. faecalis
polypeptide was loaded onto a nickel-nitrilo-tri-acetic acid
("Ni-NTA") affinity resin column (QIAGEN, Inc., supra). Proteins
with a 6.times. His tag bind to the Ni-NTA resin with high affinity
were purified in a simple one-step procedure (for details see: The
QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the
supernatant was loaded onto the column in 6 M guanidine-HCl, pH 8,
the column was first washed with 10 volumes of 6 M guanidine-HCl,
pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and
finally the E. faecalis polypeptide was eluted with 6 M
guanidine-HCl, pH 5.
[0210] The purified protein was then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. 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 NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM imidazole.
Imidazole was removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein was stored at 40.degree. C. or frozen at -80.degree. C.
[0211] Some of the polypeptide of the present invention were
prepared using a non-denaturing protein purification method. For
these polypeptides, the cell pellet from each liter of culture was
resuspended in 25 mls of Lysis Buffer A at 4.degree. C. (Lysis
Buffer A=50 mM Na-phosphate, 300 mM NaCl, 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 was approximately 10-20 O.D./ml. The
suspension was then put through three freeze/thaw cycles from
-70.degree. C. (using a ethanol-dry ice bath) up to room
temperature. The cells were lysed via sonication in short 10 sec
bursts over 3 minutes at approximately 80W while kept on ice. The
sonicated sample was then centrifuged at 15,000 RPM for 30 minutes
at 4.degree. C. The supernatant was 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 was collected.
[0212] The pre-cleared flow-through was applied to a
nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column
(Qiagen, Inc., supra). Proteins with a 6.times. His tag bind to the
Ni-NTA resin with high affinity and can be purified in a simple
one-step procedure. Briefly, the supernatant was loaded onto the
column in Lysis Buffer A at 4.degree. C., the column was first
washed with 10 volumes of Lysis Buffer A until the A280 of the
eluate returns to the baseline. Then, the column was washed with 5
volumes of 40 mM Imidazole (92% Lysis Buffer A/8% Buffer B) (Buffer
B=50 mM Na-Phosphate, 300 mM NaCl, 10% Glycerol, 10 mM
2-mercaptoethanol, 500 mM Imidazole, pH of the final buffer should
be 7.5). The protein was eluted off of the column with a series of
increasing Imidazole solutions made by adjusting the ratios of
Lysis Buffer A to Buffer B. Three different concentrations were
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 were analyzed using 8%, 10% or 14% SDS-PAGE
depending on the protein size. The purified protein was then
dialyzed 2.times. against phosphate-buffered saline (PBS) in order
to place it into an easily workable buffer. The purified protein
was stored at 4.degree. C. or frozen at -80.degree..
[0213] The following alternative method may be used to purify E.
faecalis 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.degree. C.
[0214] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus 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.
[0215] The cells are then lysed by passing the solution through a
microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0216] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the E. faecalis polypeptide-containing supernatant is incubated
at 4.degree. C. overnight to allow further GuHCl extraction.
[0217] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0218] To clarify the refolded E. faecalis polypeptide solution, a
previously prepared tangential filtration unit equipped with 0.16
.mu.m 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, 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.
[0219] Fractions containing the E. faecalis 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, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the E. faecalis polypeptide (determined, for instance,
by 16% SDS-PAGE) are then pooled.
[0220] The resultant E. faecalis 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 .mu.g 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 according to
LAL assays.
Example 2(b)
Alternative Expression and Purification Enterococcal polypeptides
in E. coli
[0221] The vector pQE10 was 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.times. His tag") covalently linked to the amino
terminus of that polypeptide. The bacterial expression vector pQE10
(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311) was
used in this example. The components of the pQE10 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.times. His tag")) covalently linked to the
amino terminus.
[0222] The DNA sequences encoding the desired portions of a
polypeptide of Table 1 were amplified using PCR oligonucleotide
primers from genomic E. faecalis 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 facilitate cloning in the pQE10
vector were added to the 5' and 3' primer sequences,
respectively.
[0223] 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 invention. The 5'
primer was designed so the coding sequence of the 6.times. His tag
is aligned with the restriction site so as to maintain its reading
frame with that of E. faecalis 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.
[0224] The DNA sequences encoding the amino acid sequences of Table
1 may also be cloned and expressed as fusion proteins by a protocol
similar to that described directly above, wherein the pET-32b(+)
vector (Novagen, 601 Science Drive, Madison, Wis. 53711) is
preferentially used in place of pQE10.
[0225] The above methods are not limited to the polypeptide
fragments actually produced. The above method, like the methods
below, can be used to produce either full length polypeptides or
desired fragments thereof.
Example 2(c)
Alternative Expression and Purification of Enterococcal
polypeptides in E. coli
[0226] The bacterial expression vector pQE60 is used for bacterial
expression in this example (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311). 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.times.His tag.
[0227] The DNA sequence encoding the desired portion of the E.
faecalis amino acid sequence is amplified from an E. faecalis
genomic DNA prep the deposited DNA clones using PCR oligonucleotide
primers which anneal to the 5' and 3' nucleotide sequences
corresponding to the desired portion of the E. faecalis
polypeptides. Additional nucleotides containing restriction sites
to facilitate cloning in the pQE60 vector are added to the 5' and
3' primer sequences.
[0228] For cloning a E. faecalis 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 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.
[0229] The amplified E. faecalis 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 E. faecalis DNA into the restricted pQE60 vector places the
E. faecalis 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.
[0230] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described by Sambrook
et al. E. coli strain M15/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 E. faecalis
polypeptide, is available commercially (QIAGEN, Inc., supra).
Transformants are identified by their ability to grow on LB 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.
[0231] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.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 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.
[0232] To purify the E. faecalis polypeptide, the cells are then
stirred for 3-4 hours at 4.degree. C. in 6M guanidine-HCl, pH 8.
The cell debris is removed by centrifugation, and the supernatant
containing the E. faecalis polypeptide is dialyzed against 50 mM
Na-acetate buffer pH 6, supplemented with 200 mM NaCl.
Alternatively, the protein can be successfully refolded by
dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH
7.4, containing protease 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 E.
faecalis polypeptide. The purified protein is stored at 4.degree.
C. or frozen at -80.degree. C.
[0233] The following alternative method may be used to purify E.
faecalis 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.degree. C.
[0234] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus 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.
[0235] The cells were then lysed by passing the solution through a
microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0236] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the E. faecalis polypeptide-containing supernatant is incubated
at 4.degree. C. overnight to allow further GuHCl extraction.
[0237] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0238] To clarify the refolded E. faecalis polypeptide solution, a
previously prepared tangential filtration unit equipped with 0.16
.mu.m 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, 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.
[0239] Fractions containing the E. faecalis 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, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the E. faecalis polypeptide (determined, for instance,
by 16% SDS-PAGE) are then pooled.
[0240] The resultant E. faecalis 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 .mu.g 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 according to
LAL assays.
Example 2(d)
Cloning and Expression of E. faecalis in Other Bacteria
[0241] E. faecalis polypeptides can also be produced in: E.
faecalis using the methods of 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 al., U.S. Pat. No.
4,952,508.
Example 3
Cloning and Expression in COS Cells
[0242] A E. faecalis expression plasmid is made by cloning a
portion of the DNA encoding a E. faecalis polypeptide into the
expression vector pDNAI/Amp or pDNAIII (which can be obtained from
Invitrogen, Inc.). The expression vector pDNAI/amp contains: (1) an
E. coli origin of replication effective for propagation in E. coli
and other prokaryotic cells; (2) an 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 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.
[0243] A DNA fragment encoding a E. faecalis 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 E. faecalis
genomic DNA prep is amplified using primers that contain convenient
restriction sites, much as described above for construction of
vectors for expression of E. faecalis in E. coli. The 5' primer
contains a Kozak sequence, an AUG start codon, and nucleotides of
the 5' coding region of the E. faecalis polypeptide. The 3' primer,
contains nucleotides complementary to the 3' coding sequence of the
E. faecalis DNA, a stop codon, and a convenient restriction
site.
[0244] 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 SURE.TM. (Stratagene Cloning Systems, La Jolla,
Calif. 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 E. faecalis polypeptide
[0245] For expression of a recombinant E. faecalis polypeptide, COS
cells are transfected with an expression vector, as described
above, using DEAE-dextran, as described, for instance, by Sambrook
et al. (supra). Cells are incubated under conditions for expression
of E. faecalis by the vector.
[0246] Expression of the E. faecalis-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 .sup.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: 150 mM NaCl, 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.
Example 4
Cloning and Expression in CHO Cells
[0247] The vector pC4 is used for the expression of E. faecalis
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. 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 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 chromosome(s) of the
host cell.
[0248] 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
polyadenylation site of the rat preproinsulin gene. Other high
efficiency promoters can also be used for the expression, e.g., the
human .beta.-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 E. faecalis 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, G418 or
hygromycin. It is advantageous to use more than one selectable
marker in the beginning, e.g., G418 plus methotrexate.
[0249] 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 E. faecalis 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 E. faecalis
polypeptide is synthesized and used. A 3' primer, containing a
restriction site, stop codon, and nucleotides complementary to the
3' coding sequence of the E. faecalis 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.
[0250] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using a
lipid-mediated transfection agent such as Lipofectin.TM. or
LipofectAMINE..TM. (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 G418. The cells are seeded in alpha minus
MEM supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml
of methotrexate plus 1 mg/ml G418. 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
.mu.M, 2 .mu.M, 5 .mu.M, 10 mM, 20 mM). The same procedure is
repeated until clones are obtained which grow at a concentration of
100-200 .mu.M. Expression of the desired gene product is analyzed,
for instance, by SDS-PAGE and Western blot or by reversed phase
HPLC analysis.
Example 5
Quantitative Murine Soft Tissue Infection Model for E. faecalis
[0251] Compositions of the present invention, including
polypeptides and peptides, are assayed for their ability to
function as vaccines or to enhance/stimulate an immune response to
a bacterial species (e.g., E. faecalis) using the following
quantitative murine soft tissue infection model. Mice (e.g., NIH
Swiss female mice, approximately 7 weeks old) are first treated
with a biologically protective effective amount, or immune
enhancing/stimulating effective amount of a composition of the
present invention using methods known in the art, such as those
discussed above. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY
MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). An
example of an appropriate starting dose is 20 ug per animal.
[0252] The desired bacterial species used to challenge the mice,
such as E. faecalis, is grown as an overnight culture. The culture
is diluted to a concentration of 5.times.10.sup.8 cfu/ml, in an
appropriate media, mixed well, serially diluted, and titered. The
desired doses are further diluted 1:2 with sterilized Cytodex 3
microcarrier beads preswollen in sterile PBS (3 g/100 ml). Mice are
anesthetize briefly until docile, but still mobile and injected
with 0.2 ml of the Cytodex 3 bead/bacterial mixture into each
animal subcutaneously in the inguinal region. After four days,
counting the day of injection as day one, mice are sacrificed and
the contents of the abscess is excised and placed in a 15 ml
conical tube containing 1.0 ml of sterile PBS. The contents of the
abscess is then enzymatically treated and plated as follows.
[0253] The abscess is first disrupted by vortexing with sterilized
glass beads placed in the tubes. 3.0 mls of prepared enzyme mixture
(1.0 ml Collagenase D (4.0 mg/ml), 1.0 ml Trypsin (6.0 mg/ml) and
8.0 mls PBS) is then added to each tube followed by a 20 min.
incubation at 37 C. The solution is then centrifuged and the
supernatant drawn off. 0.5 ml dH20 is then added and the tubes are
vortexed and then incubated for 10 min. at room temperature. 0.5 ml
media is then added and samples are serially diluted and plated
onto agar plates, and grown overnight at 37 C. Plates with distinct
and separate colonies are then counted, compared to positive and
negative control samples, and quantified. The method can be used to
identify composition and determine appropriate and effective doses
for humans and other animals by comparing the effective doses of
compositions of the present invention with compositions known in
the art to be effective in both mice and humans. Doses for the
effective treatment of humans and other animals, using compositions
of the present invention, are extrapolated using the data from the
above experiments of mice. It is appreciated that further studies
in humans and other animals may be needed to determine the most
effective doses using methods of clinical practice known in the
art.
Example 6
Murine Systemic Neutropenic Model for E. faecalis Infection
[0254] Compositions of the present invention, including
polypeptides and peptides, are assayed for their ability to
function as vaccines or to enhance/stimulate an immune response to
a bacterial species (e.g., E. faecalis) using the following
qualitative murine systemic neutropenic model. Mice (e.g., NIH
Swiss female mice, approximately 7 weeks old) are first treated
with a biologically protective effective amount, or immune
enhancing/stimulating effective amount of a composition of the
present invention using methods known in the art, such as those
discussed above. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY
MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). An
example of an appropriate starting dose is 20 ug per animal.
[0255] Mice are then injected with 250-300 mg/kg cyclophosphamide
intraperitonially. Counting the day of C.P. injection as day one,
the mice are left untreated for 5 days to begin recovery of
PMNL'S.
[0256] The desired bacterial species used to challenge the mice,
such as E. faecalis, is grown as an overnight culture. The culture
is diluted to a concentration of 5.times.10.sup.8 cfu/ml, in an
appropriate media, mixed well, serially diluted, and titered. The
desired doses are further diluted 1:2 in 4% Brewer's yeast in
media.
[0257] Mice are injected with the bacteria/brewer's yeast challenge
intraperitonially. The Brewer's yeast solution alone is used as a
control. The mice are then monitored twice daily for the first week
following challenge, and once a day for the next week to ascertain
morbidity and mortality. Mice remaining at the end of the
experiment are sacrificed. The method can be used to identify
compositions and determine appropriate and effective doses for
humans and other animals by comparing the effective doses of
compositions of the present invention with compositions known in
the art to be effective in both mice and humans. Doses for the
effective treatment of humans and other animals, using compositions
of the present invention, are extrapolated using the data from the
above experiments of mice. It is appreciated that further studies
in humans and other animals may be needed to determine the most
effective doses using methods of clinical practice known in the
art.
[0258] 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.
[0259] 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 apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
Sequence CWU 0
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