Note: Descriptions are shown in the official language in which they were submitted.
CA 02581746 2007-03-23
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Staplzylococcus aureus Isd Protein-based Anti Infectives
Cross Reference to Related Applications
This application claims priority to U.S. Provisional Application No.
60/621,921,
which was filed on October 25, 2004, the contents of which are hereby
incorporated by
reference in their entirety.
Background
Iron i s an absolute r equirement for t he g rowth of m ost m icroorganisms,
with t he
possible exceptions of lactobacilli (Archibald (1983) FEMS Microbiol. Lett.
19:29-32) and
Borrelia burgdorferi (Posey and Gherardini (2000) Science 288:1651-1653).
Despite being
the fourth most abundant element on the Earth's crust, iron is frequently a
growth-limiting
nutrient. In aerobic environments and at physiological pH, iron is present in
the ferric
(Fe3) state and forms insoluble hydroxide and oxyhydroxide precipitates.
Mammals
overcome iron restriction by possessing high-affinity iron-binding
glycoproteins such as
transferrin and lactoferrin that serve to solubilize and deliver iron to host
cells (Weinberg
(1999) Emerg. Infect. Dis. 5:346-352). This results in a further restriction
of free
extracellular iron, and accordingly, the concentration of free iron in the
human body is
estimated to be 10"18 M, a concentration that is several orders lower than
that required to
support a productive bacterial infection (Braun et al., (1998) Bacterial iron
transport:
mechanisms, genetics, and regulation, p. 67-145. In A. Sigel and H. Sigel
(ed.), Metal Ions
in Biological Systems, vol. 35. Iron transport and storage in microorganisms,
plants, and
animals. Marcel Dekker, Inc., New York). Sequestration o f iron is an
important innate
defense against bacterial infection (Skaar and Schneewind (2004) Microbes and
Infect.
6:390-397).
To overcome iron restriction, bacteria have evolved several different
mechanisms to
acquire this essential nutrient. For example, members of the Pasteurellaceae
may express
receptors for the recognition of iron-loaded forms of transferrin and
lactoferrin (Gray-Owen
and Schryvers, (1996) Trends Microbiol. 4:185-91). One of the most common iron
acquisition mechanisms, though, is through the use of low molecular weight,
high-affinity
iron chelators, termed siderophores, and cognate cell envelope receptors that
serve to
actively internalize ferric-siderophore complexes. Many siderophores are able
to
successfully compete with transferrin and lactoferrin for host iron. Indeed,
the expression
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of ferric-siderophore uptake systems are critical virulence factors in
bacteria such as
septicemic E. coli (Williams (1979) Infect. Immun. 26:925-932), Vibrio
anguillarum (Crosa
et al., (1980) Infect. Immun. 27:897-902), Erwinia chrysantherni (Enard et
al., (1988) J.
Bacteriol. 170:2419-2426) and Pseudomonas aeruginosa (Meyer et al., (1996)
Infect.
Immun. 64:518-523).
Staphylococcus aureus (S. aureus) possesses several different iron-regulated
ABC
transporters, including those encoded by the sstABCD (Morrissey et al., (2000)
Infect.
Immun. 68:6281-6288), sirABC (Heinrichs et al., (1999) J. Bacteriol. 181:1436-
1443; Dale
et al., (2004) J. Bacteriol., In press), fhuCBG (Sebulsky et al., (2000) J.
Bacteriol.
182:4394-4400), and sbn (Dale et al., (2004) Infect. Immun. 72:29-37) operons.
While the
transported substrates are unknown for the sst and sir systems, the fhuCBG
genes, in
concert with fhuDl and fliuD2 (Sebulsky and Heinrichs (2001) J. Bacteriol.
183:4994-
5000), are involved in the acquisition - of iron(III)-hydroxamate complexes.
Several
members of the staphylococci, including numerous coagulase-negative
staphylococci
(CoNS) and strains of S. aureus, produce siderophores. Two of these
siderophores,
staphyloferrin A (Konetschny-Rapp et al., (1990) Eur. J. Biochem. 191:65-74;
Meiwes et
al., (1990) FEMS Microbiol. Lett. 67:201-206) and staphyloferrin B (Dreschel
et al., (1993)
BioMetals. 6:185-192; Haag et al., (1994) FEMS Microbiol. Lett. 115:125-130),
are of the
polycarboxylate class, while the third, aureochelin (Courcol et al., (1997)
Infect. Immun.
65:1944-1948), is chemically uncharacterized. Further, an additional
siderphore, encoded
by the sbn operon, appears to be specific for S. aureus and may be a key
determinant in the
virulence of S. aureus in comparison to other CoNS strains (Dale et al.,
(2004) Infect.
Immun. 72:29-37).
The most abundant source of iron in the human body, however, is sequestered in
heme-containing proteins (hemoproteins). Heme is a cyclic molecule that
contains a single
iron. atom bound to four-ring nitrogen atoms of porphyrins. Hemoproteins are
responsible
for numerous cellular functions, and hemoglobin and myoglobin are the most
abundant
heme-containing proteins in mammals. As iron is e ssential for the survival of
bacterial
pathogens, bacteria have acquired several mechanisms to scavenge iron from
free heme and
hemoproteins. S. aureus, in particular, can acquire iron from heme or
hemoproteins
through the Iron-regulated surface determinant (Isd) system. The Isd system
comprises
cell-surface proteins that can bind and transport heme across the bacterial
cell wall as well
as at least one cytoplasmic protein that can extract iron from heme (Mazmanian
et al.,
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(2003) Science 299:906-909; Clarke et al., (2004) Mol. Microbiol. 51:1509-
1519). Further,
Isd proteins, specifically IsdA appears to bind a broad spectrum of
extracellular matrix
proteins, including but not limited to, fibrinogen and fibronectin (Clarke et
al., (2004) Mol.
Microbiol. 51:1509-1519) as well as transferrin (Taylor and Heinrichs (2002)
Mol.
Microbiol. 43:1603-1614) and hemin (Mazmanian et al., (2003) Science 299:906-
909).
S. aureus is a prevalent human pathogen that causes a wide range of infections
ranging from minor skin and wound infections to more serious sequelae such as
endocarditis, osteomyelitis and septicemia (Archer (1998) Clin. Infect. Dis.
26:1179-1181).
The ability of S. aureus to invade and colonize many tissues may be ascribed
to its capacity
to express several virulence factors such as fibronectin-, elastin- and
collagen-binding
proteins that aid i n t issue a dherence, and multiple e xotoxins and
proteases that result i n
tissue destruction and bacterial dissemination. The ability of this bacterium
to acquire iron
during in vivo growth is also likely important to its pathogenesis, and
several research
groups have characterized several different genes whose products are involved,
in the
binding and/or transport of host iron compounds (Mazmanian et al., (2003)
Science
299:906-9; Modun et al., (1998) Infect. Immun. 66:3591-3596; Taylor and
Heinrichs
(2002) Mol. Microbiol. 43:1603-1614).
Initially, penicillin could be used to treat even the worst S. aureus
infections.
However, the emergence of penicillin-resistant strains of S. aureus has
reduced the
effectiveness of penicillin in treating S. aureus infections and most strains
of S. aureus
encountered in hospital infections today do not respond to penicillin.
Penicillin-resistant
strains of S. aureus produce a beta-lactamase, which converts penicillin to
pencillinoic acid,
and thereby destroys antibiotic activity. Furthermore, the beta-lactamase
encoding gene
often is p ropagated episomally, typically on a plasmid, a nd often i s only o
ne o f several
genes on an episomal element that, together, confer multidrug resistance.
Methicillins, introduced in the 1960s, largely overcame the problem of p
enicillin
resistance in S. aureus. These compounds conserve the portions of penicillin
responsible
for antibiotic activity and modify or alter other portions that make
penicillin a good
substrate f or inactivating 1 actamases. H owever, methicillin resistance has
emerged in S.
aureus, along with resistance to many other antibiotics effective against this
organism,
including aminoglycosides, tetracycline, chloramphenicol, macrolides and
lincosamides. In
fact, methicillin-resistant strains of S. aureus generally are multiply drug
resistant.
Methicillian-resistant S. aureus (MRSA) has become one of the most important
nosocomial
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pathogens worldwide and poses serious infection control problems. Today, many
strains
are multiresistant against virtually all antibiotics with the exception of v
ancomycin-type
glycopeptide antibiotics. Drug resistance of S. aureus infections poses
significant treatment
difficulties, which are likely to get much worse unless new therapeutic agents
are
developed. Thus, there is an urgent unmet medical need for new and effective
therapeutic
agents to treat S. aureus infections.
Summary of tlze Invention
The present invention is based, at least in part, on the identification and
characterization of Isd (iron-regulated surface determinant) proteins, IsdA,
IsdB, and IsdC,
which are part of the Isd system involved in the internalization of iron from
heme and
hemoproteins in S. aureus. IsdA, IsdB, and IsdC are expressed on the cell
surface of S.
aureus and are important for iron-restricted growth and survival in vivo. As a
result, IsdA,
IsdB, and IsdC proteins are attractive vaccine targets whose inhibition may,
lead to
compromised bacterial growth in vivo. Further, IsdA, IsdB, and IsdC proteins
are attractive
drug targets that can be used in screening assays to identify S. aur=eus
specific antibiotics.
In one aspect, the invention features Isd protein-based vaccines. In an
exemplary
embodiment, an Isd based vaccine comprises an IsdA polypeptide and a
pharmaceutically
acceptable carrier. In one embodiment, the IsdA polypeptide comprises the full-
length
amino acid sequence of SEQ ID NO: 3. In another embodiment, the IsdA
polypeptide is a
peptide of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of
SEQ ID NO: 3. In
another embodiment, the Isd vaccine comprises an IsdB polypeptide and a
pharmaceutically
acceptable carrier. In certain embodiments, the IsdB polypeptide comprises the
full-length
amino acid sequence of SEQ ID NO: 6. In another embodiment, the IsdB
polypeptide is a
peptide of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of
SEQ ID NO: 6. In
another embodiment, the Isd vaccine comprises an IsdC polypeptide and a
pharmaceutically
acceptable carrier. In certain embodiments, the IsdC polypeptide comprises the
full-length
amino acid sequence of SEQ ID NO: 9. In another embodiment, the IsdC
polypeptide is a
peptide of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of
SEQ ID NO: 9.
The vaccine composition may be formulated into an injectable formulation and
may further
comprise an adjuvant.
In another aspect, the invention features novel antibiotics including
antibodies,
antisense nucleic a cids, and s iRNAs that inhibit iron uptake in S
tapylococcus aureus (S.
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aureus). The invention features antibodies against IsdA, IsdB and/or IsdC. In
certain
embodiments, antibodies against an I sdA p olypeptide may b e g enerated
against the full-
length recombinant amino acid sequence of SEQ ID NO: 3 or against a peptide of
at least 5,
10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO: 3. Antibodies
against an
IsdB polypeptide be generated against the full-length recombinant amino acid
sequence of
SEQ ID NO: 6 or against a peptide of at least 5, 10, 15, 20, 25, 30, 35, 40,
45, or 50 amino
acids of SEQ ID NO: 6. Antibodies against an IsdC polypeptide be generated
against the
full-length recombinant amino acid sequence of SEQ ID NO: 9 or against a
peptide of at
least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of SEQ ID NO: 9.
Antibodies
against an Isd polypeptide may be monoclonal or polyclonal. Antibodies against
an Isd
polypeptide may be forniulated into an injectable formulation and may be
administered as
anti-bacterial treatments.
In a further aspect, the invention features screening a ssays for identifying
agents
that inhibit or otherwise interfere with the expression level and/or function
of any of the Isd
proteins. In an exemplary embodiment, the invention features screening assays
for agents
that inhibit the expression and/or function of IsdA. In one embodiment, the
assay is a
binding assay and an agent that binds to an isd gene product and thereby
interferes with its
biochemical function is a candidate S. aureus specific antibiotic. In another
embodiment,
the assay is an expression assay and an agent that reduces the expression
level of an Isd
polypeptide is a candidate S. aureus specific antibiotic.
In a further aspect, Isd proteins may be expressed on , Gram-positive
bacteria,
including but not limited to, S. aureus, Corynebacterium diphtheriae, Listeria
monocytogenes, and Bacillus antlaracis. Thus, vaccines and inhibitors that
target Isd
proteins, as described herein, may be used to treat numerous virulent Gram-
positive
bacterial strains that cause disease in mammals.
Further features and advantages of the instant disclosed inventions will now
be
discussed in conjunction with the following Detailed Description and Claims.
Brief Description of the Dratvitags
Figure 1 shows (A) the nucleic acid sequence encoding IsdA (SEQ ID NO: 1), (B)
the reverse complement of SEQ ID NO: 1 (SEQ ID NO: 2), and (C) the
corresponding
amino acid sequence for IsdA (SEQ ID NO: 3).
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Figure 2 shows (A) the nucleic acid sequence encoding IsdB (SEQ ID NO: 4), (B)
the reverse complement of SEQ ID NO: 4 (SEQ ID NO: 5), and (C) the
corresponding
amino acid sequence for IsdB (SEQ ID NO: 6).
Figure 3 shows (A) the nucleic acid sequence encoding IsdC (SEQ ID NO: 7), (B)
the reverse complement of SEQ ID NO: 7 (SEQ ID NO: 8), and (C) the
corresponding
amino acid sequence for IsdC (SEQ ID NO: 9).
Figure 4 is an SDS-PAGE gel that shows whole cell lysates from S. aureus grown
in
iron-rich media and iron-depleted media.
Figure 5 is a graph showing S. aureus counts recovered from kidneys of mice 6
days
following inj ection 107 bacteria into the tail vein.
Figure 6 is a table showing that wild-type S. aureus Isd proteins bound to
heme can
survive under conditions of increased hydrogen peroxide (H202) compared to S.
aureus
strains where isdA, isdB, isdC were knocked-out (/, indicates greater than 90%
survival of
bacteria).
Figures 7A and 7B are SDS-PAGE gels showing proteins from wild type and isdA
knockout S. aureus (A) stained with coomassie (for total protein) and (b)
stained with
TMBZ (tetramethylbenzidine).
Detailed Description of the Invention
1. General
As described herein, the internalization of iron through the uptake of heme is
a
virulence property that may be attenuated when isd genes, such as isdA, isdB,
and isdC are
knocked out. Further, as described herein, heme-bound Isd proteins may serve
an additional
role in promoting S. aureus survival in the host. Heme-bound Isd proteins
appear to serve
as an oxidative buffer that protects cells form the detrimental effects of
free radicals.
Therefore, mutants lacking expression of Isd proteins are more susceptible to
challenges
with hydrogen peroxide whereas wild type S. aur=eus can survive in higher
concentrations of
hydrogen peroxide.
The Isd proteins, as described herein, are essential for S. auf-eus infection
in vivo
and are highly expressed during S. aureus infection. As S. aureus enters a
host, it
encounters an environment that is iron-limited and Isd protein expression is
subsequently
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up regulated. In the iron-limited host, Isd expression likely remains up
regulated as the S.
aureus scavenge for iron. IsdA, in particular, as described herein is
immunodominant,
since a 1:4000 dilution of serum from convalescent patients (i.e., patients
suffering from S.
aureus infections) reacted positively in Western immunoblots with 4 micrograms
of
purified IsdA protein. Thus, Isd proteins are attractive targets for vaccine
development.
Antigenic peptides of Isd proteins may be used as vaccine targets to generate
an effective
immune response against S. aureus. Further, inhibiting the function of Isd
proteins using an
Isd specific antibody, antisense RNA, siRNA or small molecule inhibitor may be
an
effective way of attenuating the virulence of S. aureus.
The Isd proteins described herein are expressed on S. aureus. In further
embodiments, Isd proteins may be expressed on other Gram-positive bacteria.
Non-limiting
examples of Gram-positive pathogens expressing Isd proteins include S. aureus,
Corynebacterium diphtlaeriae, Listeria inonocytogenes, and Bacillus
antlaracis. Thus;
vaccines and inhibitors that target Isd proteins, as described herein, may be
usedfo treat
other virulent Gram-positive bacterial strains that cause disease in mamrnals.
2. D('.f ItitiOlts
For convenience, the meaning of certain terms and phrases employed' in the
specification, examples, and appended claims are provided below. Unless
defined
otherwise, all technical and scientific terms used herein have the same
meaning as
commonly understood by one of ordinary skill in the art to which this
invention belongs.
The articles "a" and "an" are used herein to refer to one or to more than one
(i.e., to
at least one) of the grammatical object of the article. By way of example, "an
element"
means one element or more than one element.
As used herein, the term "adjuvant" refers to a substance that elicits an
enhanced
immune response when used in combination with a specific antigen.
The term "agent" is used herein to denote a chemical compound, a mixture of
chemical compounds, a biological macromolecule (such as a nucleic acid, an
antibody, a
protein or portion thereof, e.g., a peptide), or an extract made from
biological materials
such as bacteria, plants, fungi, or animal (particularly mammalian) cells or
tissues.
Screening assays described herein below may identify agents. Such agents may
be
inhibitors or antagonists of Isd mediated iron uptake in Staplaylococcus
aureus. The
activity of such agents may render it suitable as a"therapeutic agent" which
is a
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biologically, physiologically, or pharmacologically active substance (or
substances) that
acts locally or systemically in a subject.
The t erms " antagonist" or "inhibitor" refer t o an agent that down r
egulates (e.g.,
suppresses or inhibits) at least one bioactivity of a protein. An antagonist
may be a
compound which inhibits or decreases the interaction between a protein and
another
molecule, e.g., a target peptide or enzyme substrate. An antagonist may also
be a compound
that down regulates expression of a gene or which reduces the amount of
expressed protein
present.
As used herein the term "antibody" refers to an immunoglobulin and any antigen-
binding portion of an immunoglobulin (e.g., IgG, IgD, IgA, IgM and IgE) i.e.,
a
polypeptide that contains an antigen-binding site, which specifically binds
("immunoreacts
with") an antigen. Antibodies can comprise at least one heavy (H) chain and at
least one
light (L) chain interconnected by at least one disulfide bond. The term "VH"
refers to a
heavy chain variable region of an antibody. The term "VL" refers to a light
chain variable
region of an antibody. In exemplary embodiments, the term "antibody"
specifically covers
monoclonal and polyclonal antibodies. A "polyclonal antibody" refers to an
antibody,
which has been derived from the sera of animals immunized with an antigen or
antigens. A
"monoclonal antibody" refers to an antibody produced by a single clone of
hybridoma cells.
Techniques for generating monoclonal antibodies include, but are not limited
to, the
hybridoma technique (see Kohler & Milstein (1975) Nature 256:495-497); the
trioma
technique; the human B-cell hybridoma technique (see Kozbor, et al. (1983)
Immunol.
Today 4:72), the EBV hybridoma technique (see Cole, et al., 1985 In:
Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) and phage
display.
Polyclonal or monoclonal antibodies can be further manipulated or modified to
generate chimeric or humanized antibodies. "Chimeric antibodies" are encoded
by
immunoglobulin genes that have been genetically engineered so that the light
and heavy
chain genes are composed of immunoglobulin gene segments belonging to
different
species. For example, substantial portions of the variable (V) segments of the
genes from a
mouse monoclonal antibody, e.g., obtained as described herein, may be joined
to substantial
portions of human constant (C) segments. Such a chimeric antibody is likely to
be less
antigenic to a human than a mouse monoclonal antibody.
As used herein, the term "humanized antibody" (HuAb) refers to a chimeric
antibody with a framework region substantially identical (i.e., at least 85%)
to a human
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framework, having CDRs from a non-human antibody, and in which any constant
region
has at least about 85-90%, and preferably about 95% polypeptide sequence
identity to a
human immunoglobulin constant region. See, for example, PCT Publication WO
90/07861
and European Patent No. 0451216. All parts of such a HuAb, except possibly the
CDRs,
are substantially identical to corresponding parts of one or more native human
immunoglobulin sequences. The term "framework region" as used herein, refers
to those
portions of immunoglobulin light and heavy chain variable regions that are
relatively
conserved (i.e., other than the CDRs) among different immunoglobulins in a
single species,
as defined by Kabat, et al. (1987) Sequences of Proteins of Immunologic
Interest, 4th Ed.,
US D ept. Health and H uman Services. Human constant r egion DNA sequences can
b e
isolated in accordance with well known procedures from a variety of human
cells, but
preferably from immortalized B cells. The variable regions or CDRs for
producing
humanized antibodies may be derived from monoclonal antibodies capable of
binding to the
antigen, and will be produced in any convenient mammalian source, including
mice, rats,
rabbits, or other vertebrates.
The term "antibody" also encompasses antibody fragments. Examples of antibody
fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies and
any
antibody fragment that has a primary structure consisting of one uninterrupted
sequence of
contiguous amino acid residues, including without limitation: single-chain Fv
(scFv)
molecules, single chain polypeptides containing only one light chain variable
domain, or a
fragment thereof that contains the three CDRs of the light chain variable
domain, without
an a ssociated h eavy c hain m oiety and (3) single c hain p olypeptides
containing o nly one
heavy chain variable region, or a fragment thereof containing the three CDRs
of the heavy
chain variable region, without an associated light chain moiety; and
multispecific or
multivalent structures formed from antibody fragments. In an antibody fragment
comprising one or more heavy chains, the heavy chain(s) can contain any
constant domain
sequence (e.g., CH1 in the IgG isotype) found in a non-Fc region of an intact
antibody,
and/or can contain any hinge region sequence found in an intact antibody,
and/or can
contain a leucine zipper sequence fused to or situated in the hinge region
sequence or the
constant domain sequence of the heavy chain(s). Suitable leucine zipper
sequences include
the jun and fos leucine zippers taught by Kostelney et al., (1992) J.
Immunol., 148: 1547-
1553 and the GCN4 leucine zipper described in U.S. Patent No. 6,468,532. Fab
and F(ab')2
fragments lack the Fc fragment of intact antibody and are typically produced
by proteolytic
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cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin
(to produce
F(ab')Z fragments).
An antibody "specifically binds" to an antigen or an epitope of an antigen i f
the
antibody binds preferably to the antigen over most other antigens. For
example, the
antibody may have less than about 50%, 20%, 10%, 5%, 1% or 0.1% cross-
reactivity
toward one or more other epitopes.
The term "conservative substitutions" refers to changes between amino acids of
broadly similar molecular properties. For example, interchanges within the
aliphatic group
alanine, valine, leucine and isoleucine can be considered as conservative.
Sometimes
substitution of glycine for one of these can also be considered conservative.
Other
conservative interchanges include t hose w ithin t he aliphatic g roup a
spartate a nd g lutamate;
within the amide group asparagine and glutamine; within the hydroxyl group
serine and
threonine; within the aromatic group phenylalanine, tyrosine and tryptophan;
within the basic
group lysine, arginine and histidine; and within the sulfur-containing group
methionine and
cysteine. Sometimes substitution within the group methionine and leucine can
also be
considered conservative. Preferred conservative substitution groups are
aspartate-glutamate;
asparagine-glutamine; valine-leucine-isoleucine; alanine-valine; valine-
leucine-isoleucine-
methionine; phenylalanine-tyrosine; phenylalanine-tyrosine-tryptophan; lysine-
arginine; and
histidine- lysine-arginine.
An " effective a mount" is a n amount sufficient t o p roduce a b eneficial o
r d esired
clinical result upon treatment. An effective amount can be administered to a
patient in one
or more doses. In terms of treatment, an effective amount is an amount that is
sufficient to
decrease an infection in a patient. Several factors are typically taken into
account when
determining an appropriate dosage to achieve an effective amount. These
factors include
age, sex and weight of the patient, the condition being treated, the severity
of the condition
and the form and effective concentration of the agent administered.
The term "epitope" refers to that region of an antigen to which an antibody
binds
preferentially and specifically. A monoclonal antibody binds preferentially to
a single
specific epitope of a molecule that can be molecularly defined. An epitope of
a particular
protein may be constituted by a limited number of amino acid residues, e.g. 5 -
15 residues
that are either in a linear or non-linear organization on the protein.
"Equivalent" when used to describe nucleic acids or nucleotide sequences
refers to
nucleotide sequences encoding functionally equivalent polypeptides. Equivalent
nucleotide
CA 02581746 2007-03-23
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sequences will include sequences that differ by one or more nucleotide
substitution,
addition or deletion, such as an allelic variant; and will, therefore, include
sequences that
differ due to the degeneracy of the genetic code. For example, nucleic acid
variants may
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.
"Homology" o r a lternatively "identity" r efers to s equence s imilarity
between two
peptides or between two nucleic acid molecules. Homology may be determined by
comparing a position in each sequence, which may be aligned for purposes of
comparison.
When a position in the compared sequence is occupied by the same base or amino
acid,
then the molecules are homologous at that position. A degree of homology
between
sequences is a function of the number of matching or homologous positions
shared, by the
sequences. The t erm "percent i dentical" r efers t o s equence identity
between two, amino
acid sequences or between two nucleotide sequences. Identity may be determined
by
comparing a position in each sequence, which may be aligned for purposes of
comparison.
When an equivalent position in the compared sequences is occupied by the same
base or
amino acid, then the molecules are identical at that position; when the
equivalent site is
occupied by the same or a similar amino acid residue (e.g., similar in steric
and/or
electronic nature), then the molecules may be referred to as homologous
(similar) at that
position. Expression as a percentage of homology, similarity, or identity
refers to a
function of the number of identical or similar amino acids at positions shared
by the
compared sequences. Various aligmnent algorithms and/or programs may be used,
including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as a part of
the
GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and
may be
used with, e.g., default settings. ENTREZ is available through the National
Center for
Biotechnology Information, National Library of Medicine, National Institutes
of Health,
Bethesda, Md. In one embodiment, the percent identity of two sequences may be
determined by the GCG program with a gap weight of 1, e.g., each amino acid
gap is
weighted as if it were a single amino acid or nucleotide mismatch between the
two
sequences. Other techniques for alignment are described in Methods in
Enzymology, vol.
266: Computer Methods for Macromolecular Sequence Analysis (1996), ed.
Doolittle,
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Academic Press, Inc., a division of Harcourt Brace & Co., San Diego,
California, USA.
Preferably, an alignment program that permits gaps in the sequence is utilized
to align the
sequences. The Smith-Waterman is one type of algorithm that permits gaps in
sequence
aligmnents. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program
using the
Needleman and Wunsch alignment method may be utilized to align sequences. An
alternative search strategy uses MPSRCH software, which runs on a MASPAR
computer.
MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively
parallel
computer. This approach improves the ability to pick up distantly related
matches, and is
especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-
encoded
amino acid sequences may be used to search both protein and DNA databases.
Databases
with individual sequences are d escribed i n Methods i n Enzynaology, ed.
Doolittle, s upra.
Databases include Genbank, EMBL, and DNA Database of Japan (DDBJ).
As used herein, the term. "infection" refers to an invasion and the
multiplication of
microorganisms such as S. aureus in body tissues, which may be clinically
unapparent or
result in local cellular injury due to competitive metabolism, toxins,
intracellular replication
or antigen antibody response. The infection may remain localized, subclinical
and
temporary if the body's defensive mechanisms are effective. A local infection
may persist
and spread by extension to become an acute, subacute or chronic clinical
infection or
disease state. A local infection may also become systemic when the
microorganisms gain
access to the lymphatic or vascular system.
The terms "iron-regulated surface determinant system" or "Isd system" as used
herein, refers to the S. aureus Isd locus, which comprises numerous genes
encoded by five
different transcriptional units that, together, encode a putative heme uptake
system. The
five transcriptional units are isdA, isdB, isdCDEFsrtBisdG, isdH, and isdl.
Transcription of
isd genes is regulated by environment iron on the control of the Fur promoter.
Four of the
proteins encoded by the Isd locus, IsdA, IsdB, IsdC, and IsdH, are covalently
anchored to
the cell wall by an amide linkage between the C-terminal end of the
polypeptide chain and
peptidoglycan. IsdA, IsdB, and IsdH are characterized as having a C-terminal
sorting
signal referred to as an LPXTG motif (i.e., a motif recognized by sortase A).
IsdC is
characterized as having a C-terminal sorting signal referred to as an NPQTN
motif (i.e., a
motif recognized by sortase B in S. aureus). IsdA, IsdB, and IsdH are anchored
to the cell
wall by a sortase A (srtA), a membrane anchored transpeptidase that cleaves
cell surface
proteins at the LPXTG motif and catalyzes the formation of the amide bond
between the
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polypeptide and peptidoglycan. IsdC is anchored to the c ell wall by sortase B
(srtB), a
transpeptidase similar to sortase A. Other proteins encoded by the Isd locus,
include IsdD,
IsdE, and IsdF, which are putative membrane translocation factors, and IsdG
and Isdl,
which are cytoplasmic heme-iron binding proteins, that may be involved in
extracting iron
from heme.
"IsdA polypeptide" as used herein refers to iron-regulated surface determinant
A.
The sequence of IsdA polypeptide is as set forth in SEQ ID NO: 3 and is
encoded by SEQ
ID NO: 1. The term also encompasses any fragments, variants, analogs,
agonists, chemical
derivatives, functional derivatives or functional fragments of an IsdA
polypeptide. "IsdA
immunogens" are IsdA polypeptides, which are capable of eliciting an immune
response in
a subject.
"IsdB polypeptide" as used herein refers to iron-regulated surface determinant
B.
The sequence of IsdB polypeptide is as .set.forth in SEQ ID NO: 6 and is
encoded by SEQ
ID NO: 4. The term also encompasses any fragments, variants, analogs,
agonists, chemical
derivatives, functional derivatives or functional fragments of an IsdB
polypeptide. "IsdB
immunogens" are IsdB polypeptides, which are capable of eliciting an immune
response in
a subject.
"IsdC polypeptide" as used herein refers to iron-regulated surface determinant
C.
The sequence of IsdC polypeptide is as set forth in SEQ ID NO: 9 and is
encoded by SEQ
ID NO: 7. The term also encompasses any fragments, variants, analogs,
agonists, chemical
derivatives, functional derivatives or functional fragments of an IsdC
polypeptide. "IsdC
immunogens" are IsdC polypeptides, which are capable of eliciting an inunune
response in
a subject.
"Label" and "detectable label" refer to a molecule capable of detection
including,
but not limited to radioactive isotopes, fluorophores, chemiluminescent
moieties, enzymes,
enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions,
ligands (e.g.,
biotin or haptens) and the like. "Fluorophore" refers to a substance or a
portion thereof
which is capable of exhibiting fluorescence in the detectable range.
Particular examples of
appropriate labels include fluorescein, rhodamine, dansyl, umbelliferone,
Texas red,
luminol, NADPH, alpha- or beta-galactosidase and horseradish peroxidase.
As used herein with respect t o genes, the t erm "mutant" r efers to a gene,
which
encodes a mutant protein. As used herein with respect to proteins, the term
"mutant" means
a protein, which does not perform its usual or normal physiological role. S.
aureus
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polypeptide mutants may be produced by amino acid substitutions, deletions or
additions.
The substitutions, deletions, or additions may involve one or more residues.
Especially
preferred among these are substitutions, additions and deletions, which alter
the properties
and activities of a S. aureus protein.
The terms "polynucleotide", and "nucleic acid" are used interchangeably to
refer to
a polymeric form of nucleotides of any length, either deoxyribonucleotides or
ribonucleotides, or analogs thereof. The following are non-limiting examples
of
polynucleotides: coding or non-coding regions of a gene or gene fragment, loci
(locus)
defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer
RNA,
ribosomal RNA, ribozymes, cDNA, antisense nucleic acids, recombinant
polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA
of any sequence, nucleic acid probes, and primers. A polynucleotide may
comprise
modified nucleotides, such as methylated nucleotides_ and nucleotide analogs.
If present,
modifications to the nucleotide structure may be imparted before or after
assembly of the
polymer. The sequence of nucleotides may be interrupted by non-nucleotide
components.
A polynucleotide may be further modified after polymerization, such as by
conjugation
with a labeling component. The term "recombinant" polynucleotide means a
polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, which
either does not
occur in nature or is linked to another polynucleotide in a non-natural
arrangement. An
"oligonucleotide" refers to a single stranded polynucleotide having less than
about 100
nucleotides, less than about, e.g., 75, 50, 25, or 10 nucleotides.
The terms "polypeptide", "peptide" and "protein" (if single chain) are used
interchangeably herein to refer to polymers of amino acids. The polymer may be
linear or
branched, it may comprise modified amino acids, and it may be interrupted by
non-amino
acids. The terms also encompass an amino acid polymer that has been modified;
for
example, disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation,
or any other manipulation, such as conjugation with a labeling component. As
used herein
the term "amino acid" refers to either natural and/or unnatural or synthetic
amino acids,
including glycine and both the D or L optical isomers, and amino acid analogs
and
peptidomimetics.
The term "small molecule" refers to a compound, which has a molecular weight
of
less than about 5 kD, less than about 2.5 kD, less than about 1.5 kD, or less
than about 0.9
kD. Small molecules may be, for example, nucleic acids, peptides,
polypeptides, peptide
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WO 2006/059247 PCT/IB2005/004126
nucleic acids, peptidomimetics, carbohydrates, lipids or other organic (carbon
containing)
or inorganic molecules. Many pharmaceutical companies have extensive libraries
of
chemical and/or biological mixtures, often fungal, bacterial, or algal
extracts, which can be
screened with any of the assays of the invention. The term "small organic
molecule" refers
to a small molecule that is often identified as being an organic or medicinal
compound, and
does not include molecules that are exclusively nucleic acids, peptides or
polypeptides.
The term "specifically hybridizes" refers to detectable and specific nucleic
acid
binding. Polynucleotides, oligonucleotides and nucleic acids of the invention
selectively
hybridize t o n ucleic a cid s trands u nder h ybridization and w ash
conditions t hat m inimize
appreciable amounts of detectable binding to nonspecific nucleic acids.
Stringent
conditions may be used to achieve selective hybridization conditions as known
in the art
and discussed herein. Generally, the nucleic acid sequence homology between
the
polynucleotides, oligonucleotides, and nucleic acids of the invention and a
nucleic acid
sequence of interest will be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,
95%,
98%, 99%, or more. In certain instances, hybridization and washing conditions
are
performed under stringent conditions according to conventional h ybridization
procedures
and as described further herein.
The terms "stringent conditions" or "stringent hybridization conditions" refer
to
conditions, which promote specific hybridization between two complementary
polynucleotide strands so as to form a duplex. Stringent conditions may be
selected to be
about 5 C lower than the thermal melting point (Tm) for a given polynucleotide
duplex at a
defined ionic strength and pH. The length of the complementary polynucleotide
strands
and their GC content will determine the Tm of the duplex, and thus the
hybridization
conditions necessary for obtaining a desired specificity of hybridization. The
Tm is the
temperature (under defined ionic strength and pH) at which 50% of a
polynucleotide
sequence hybridizes to a perfectly matched complementary strand. In certain
cases it may
be desirable to increase the stringency of the hybridization conditions to be
about equal to
the Tm for a particular duplex.
A variety of techniques for estimating the Tm are available. Typically, G-C
base
pairs in a duplex are estimated to contribute about 3 C to the Tm, while A-T
base pairs are
estimated to contribute about 2 C, up to a theoretical maximum of about 80-100
C.
However, more sophisticated models of Tm are available in which G-C stacking
interactions, solvent effects, the desired assay temperature and the like are
taken into
CA 02581746 2007-03-23
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account. For example, probes can be designed to have a dissociation
temperature (Td) of
approximately 60 C, using the forniula: Td =(((((3 x#GC) + (2 x #AT)) x 37) -
562)/#bp) -
5; where #GC, #AT, and #bp are the number of guanine-cytosine base pairs, the
number of
adenine-thymine base pairs, and the number of total base pairs, respectively,
involved in the
formation of the duplex.
Hybridization may be carried out in 5xSSC, 4xSSC, 3xSSC, 2xSSC, 1xSSC or
0.2xSSC for at least about 1 hour, 2 hours, 5 hours, 12 hours, or 24 hours.
The temperature
of the hybridization may be increased to adjust the stringency of the
reaction, for example,
from about 25 C (room temperature), to about 4 5 C, 50 C, 55 C, 60 C, or 65 C.
The
hybridization reaction may also include another agent affecting the
stringency, for example,
hybridization conducted in the presence of 50% formamide increases the
stringency of
hybridization at a defined temperature.
The hybridization reaction may be followed by. a single wash.step, or two or
more
wash steps, which may be at the same or a different salinity and temperature.
For example,
the t emperature of the wash may b e increased to a djust t he stringency from
a bout 2 5 C
(room temperature), to about 45 C, 50 C, 55 C, 60 C, 65 C, or higher. The wash
step may
be conducted in the presence of a detergent, e.g., 0.1 or 0.2% SDS. For
example,
hybridization may be followed by two wash steps at 65 C each for about 20
minutes in
2xSSC, 0.1 % S DS, a nd optionally two a dditional wash s teps a t 6 5 C each
for a bout 2 0
minutes in 0.2xSSC, 0.1%SDS.
Exemplary stringent hybridization conditions include overnight hybridization
at
65 C in a solution comprising, or consisting of, 50% formamide, lOxDenhardt
(0.2%
Ficoll, 0.2% Polyvinylpyrrolidone, 0.2% bovine serum albumin) and 200 g/ml of
denatured carrier DNA, e.g., sheared salmon sperm DNA, followed by two wash
steps at
65 C each for about 20 minutes in 2xSSC, 0.1% SDS, and two wash steps at 65 C
each for
about 20 minutes in 0.2xSSC, 0.1%SDS.
Hybridization may consist of hybridizing two nucleic acids in solution, or a
nucleic
acid in solution to a nucleic acid attached to a solid support, e.g., a
filter. When one nucleic
acid is on a solid support, a prehybridization step may be conducted prior to
hybridization.
Prehybridization may be carried out for at least about 1 hour, 3 hours or 10
hours in the
same solution and at the same temperature as the hybridization solution
(without the
complementary polynucleotide strand).
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Appropriate stringency conditions are known to those skilled in the art or may
be
determined experimentally by the skilled artisan. See, for example, Current
Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-12.3.6; Sambrook et
al., 1989,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y; S.
Agrawal
(ed.) Methods in Molecular Biology, volume 20; Tijssen (1993) Laboratory
Techniques in
biochemistry and molecular biology-hybridization with nucleic acid probes,
e.g., part I
chapter 2 "Overview of principles of hybridization and the strategy of nucleic
acid probe
assays", Elsevier, New York; and Tibanyenda, N. et al., Eur. J. Biochem.
139:19 (1984)
and Ebel, S. et al., Biochem. 31:12083 (1992).
. The tenn "substantially homologous" when used in connection with a nucleic
acid
or amino acid sequences, refers to sequences which are substantially identical
to or similar
in sequence with each other, giving rise to a homology of conformation and
thus to
retention, to a useful degree, of one or more biological (including
immunological)
activities. The term is not intended to imply a common evolution of the
sequences.
A"subject" refers to a male or female mammal, including humans.
A"variant" of an Isd polypeptide refers to a molecule, which is substantially
similar
to IsdA, IsdB, of IsdC. Variant peptides may be covalently prepared by direct
chemical
synthesis of the variant peptide, using methods well known in the art.
Variants of Isd
polypeptides may further include, for example, deletions, insertions or
substitutions of
residues within the amino acid sequence. Any combination of deletion,
insertion, and
substitution may also be made to arrive at the final construct, provided that
the final
construct possesses the desired activity. These variants may be prepared by
site-directed
mutagenesis, (as exemplified by Adelman et al., DNA 2: 183 (1983)) of the
nucleotides in
the DNA encoding the peptide molecule thereby producing DNA encoding the
variant and
thereafter expressing the DNA in recombinant cell culture. The variants
typically exhibit
the same qualitative biological activity as wild type Isd polypeptides. It is
known in the art
that one may also synthesize all possible single amino acid substitutions of a
known
polypeptide (Geysen et al., Proc. Nat. Acad. Sci. (USA) 18:3998-4002 (1984)).
While the
effects of different substitutions are not always additive, it is reasonable
to expect that two
favorable or neutral single substitutions at different residue positions in an
Isd polypeptide
can safely be combined without losing any Isd protein activity. Methods for
the preparation
of degenerate polypeptides are as described in Rutter, U.S. Pat. No.
5,010,175; Haughter et
al., Proc. Nat. Acad. Sci. (USA) 82:5131-5135 (1985); Geysen et al., Proc.
Nat. Acad. Sci.
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(USA) 18:3998-4002 (1984); W086/06487; and W086/00991. In devising a
substitution
strategy, a person of ordinary skill would determine which residues to vary
and which
amino acids or classes of amino acids are suitable replacements. One may also
take into
account studies of sequence variations in families or naturally occurring
homologous
proteins. Certain amino acid substitutions are more often tolerated than
others, and these
are often correlated with similarities in size, charge, etc., between the
original amino acid
and its replacement. Insertions or deletions of amino acids may also be made,
as described
above. The substitutions are preferably conservative, see, e.g., Schulz et
al., Principle of
Protein Structure (Springer-Verlag, New York (1978)); and Creighton, Proteins:
Structure
and Molecular Properties (W. H. Freeman & Co., San Francisco (1983)); both of
which are
hereby incorporated by reference in their entireties.
A "chemical derivative" of an Isd polypeptide can contain additional chemical
moieties not normally part of the IsdA, IsdB, or IsdC amino acid sequences.
Such chemical
modiftcations may be introduced into an Isd polypeptide by reacting targeted
amino acid
residues of the polypeptide with an organic derivatizing agent that is capable
of reacting
with selected side chains or terminal residues. Amino terminal residues can be
reacted with
succinic or other carboxylic acid anhydrides. Other suitable reagents for
derivatizing alpha-
amino-containing r esidues i nclude a mido-esters such as methyl p
icolinimidate; p yridoxal
phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-
methylisourea;
2,4-pentanedione; and transaminase-catalase reacted with glyoxylate. Specific
modifications of tyrosyl residues per se have been studied extensively, with
particular
interest in introducing spectral labels into tyrosyl residues by reaction with
aromatic
diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and
tetranitromethane are use to form 0-acetyl tyrosyl species and 3-nitro
derivatives,
respectively. Carboxyl side groups such as aspartyl or glutamyl can be
selectively modified
by reaction with carbodiimides (R'N--C--N--R') such as 1-cyclohexy-3-[2-
morpholinyl-(4-
ethyl)] carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl
and glutaminyl
residues by reaction with ammonium ions.
A "vector" is a self-replicating nucleic acid molecule that transfers an
inserted
nucleic acid molecule into and/or between host cells. The term includes
vectors that
function primarily for insertion of a nucleic acid molecule into a cell,
replication of vectors
that function primarily for the replication of nucleic acid, and expression
vectors that
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function for transcription and/or translation of the DNA or RNA. Also included
are vectors
that provide more than one of the above functions. As used herein, "expression
vectors"
are defined as polynucleotides which, when introduced into an appropriate host
cell, can be
transcribed and translated into a polypeptide(s). An "expression system"
usually connotes a
suitable host cell comprised o f an e xpression v ector t hat can function to
y ield a d esired
expression product.
3. Isd Genes
Three genes of the Isd 1 ocus, isdA, isdB, and isdC, encode cell surface
proteins,
which are covalently anchored to the S. aureus cell wall. Figures 1-3 provide
the nucleic
acid sequences of isdA (SEQ ID NO: 1), isdB (SEQ ID NO: 4), and isdC (SEQ ID
NO: 7).
Nucleic acids of the present invention may also comprise, consist of or
consist
essentially of any of the isd nucleotide sequences described herein. Yet other
nucleic acids
comprise, consist of or consist essentially of a nucleotide sequence that has
at least about
70%, 80%, 90%, 95%, 98% or 99% identity or homology with an isd gene.
Substantially
homologous sequences may be identified using stringent hybridization
conditions.
Isolated nucleic acids which differ from the nucleic acids of the invention
due to
degeneracy in the genetic code are also within the scope of the invention. For
example, a
number of amino acids are designated by more than one triplet. Codons that
specify the
same amino acid, or synonyms (for example, CAU and CAC are synonyms for
histidine)
may result in "silent" mutations which do not affect the amino acid sequence
of the protein.
However, it is expected that DNA sequence polymorphisms that do lead to
changes in the
amino acid sequences of the polypeptides of the invention will exist. One
skilled in the art
will appreciate that these variations in one or more nucleotides (from less
than 1% up to
about 3 or 5% or possibly more of the nucleotides) of the nucleic acids
encoding a
particular protein of the invention may exist among a given species due to
natural allelic
variation. Any and all such nucleotide variations and resulting amino acid
polymorphisms
are within the scope of this invention.
Nucleic a cids encoding proteins which have amino acid s equences
evolutionarily
related to a polypeptide disclosed herein are provided, wherein
"evolutionarily related to",
refers to proteins having different amino acid sequences which have arisen
naturally (e.g.,
by allelic variance or by differential splicing), as well as mutational
variants of the proteins
of the invention which are derived, for example, by combinatorial mutagenesis.
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Fragments of the polynucleotides of the invention encoding a biologically
active
portion of the subject polypeptides are also provided. As used herein, a
fragment of a
nucleic acid encoding an active portion of a polypeptide disclosed herein
refers to a
nucleotide sequence having fewer nucleotides than the nucleotide sequence
encoding the
full length a mino acid s equence o f a p olypeptide o f t he invention, a nd
which encodes a
given polypeptide that retains at least a portion of a biological activity of
the full-length Isd
protein as defined herein, or alternatively, which is functional as a
modulator of the
biological activity of the full-length protein. For example, such fragments
include a
polypeptide containing a domain of the full-length protein from which the
polypeptide is
derived that mediates the interaction of the protein with another molecule
(e.g., polypeptide,
DNA, RNA, etc.).
Nucleic acids provided herein may also contain linker sequences, modified
restriction endonuclease sites and other sequences useful for molecular
cloning, expression
or purification of such recombinant polypeptides.
A nucleic acid encoding an Isd polypeptide provided herein may be obtained
from
mRNA or genomic DNA from any organism in accordance with protocols described
herein,
as well as those generally known to those skilled in the art. A cDNA encoding
a
polypeptide of the invention, for example, may be obtained by isolating total
mRNA from
an organism, for example, a bacteria, virus, mammal, etc. Double stranded
cDNAs may
then be prepared from the total mRNA, and subsequently inserted into a
suitable plasmid or
bacteriophage vector using any one of a number of known techniques. A gene
encoding a
polypeptide of the invention may also be cloned using established polymerase
chain
reaction techniques in accordance with the nucleotide sequence information
provided by the
invention. In one aspect, methods for amplification of a nucleic acid of the
invention, or a
fragment thereof may comprise: (a) providing a pair of single stranded
oligonucleotides,
each of which is at least eight nucleotides in length, complementary to
sequences of a
nucleic acid of the invention, and wherein the sequences to which the
oligonucleotides are
complementary are at least ten nucleotides apart; and (b) contacting the
oligonucleotides
with a sample comprising a nucleic acid comprising the nucleic acid of the
invention under
conditions which permit amplification of the region located between the pair
of
oligonucleotides, thereby amplifying the nucleic acid.
Isd proteins may be expressed from recombinant vectors, host cells containing
the
recombinant vectors and methods of producing the encoded S. aureus
polypeptides.
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Appropriate vectors may be introduced into host cells using well-known
techniques such as
infection, transduction, transfection, transvection, electroporation and
transformation. The
vector may be, for example, a phage, plasmid, viral or retroviral vector.
Retroviral vectors
may be replication competent or replication defective. In the latter case,
viral propagation
generally will occur only in complementing host cells.
The vector may contain a selectable marker for propagation in a host.
Generally, a
plasmid vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in
a complex with a charged lipid. If the vector is a virus, it may be packaged
in vitro using an
appropriate packaging cell line and then transduced into host cells.
Preferred vectors comprise 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.
In certain embodiments,. the vectors provide for specific expression, which
may be
inducible and/or cell type-specific. Particularly preferred among such vectors
are those
inducible by e nvironmental factors t hat are easy to manipulate, such as
temperature and
nutrient additives.
Expression vectors useful in the present invention include chromosomal-,
episomal-
and virus-derived vectors, e.g., vectors derived from bacterial plasmids,
bacteriophage,
yeast episomes, yeast chromosomal elements, viruses such as baculoviruses,
papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and
retroviruses, and vectors derived from combinations thereof, such as cosmids
and
phagemids.
The DNA insert should be operatively linked to an appropriate promoter, such
as the
phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40
early and late
promoters and promoters of retroviral LTRs, to name a few. Other suitable
promoters will
be known to the skilled artisan. The expression constructs may 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
may preferably include a translation-initiating site at the beginning and a
termination codon
(UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be
translated.
As indicated, the expression vectors will preferably include at least one
selectable
marker. Such markers include dihydrofolate reductase or neomycin resistance
for
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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
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.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE9,
pQE10 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript
vectors,
pNH8A, pNHl6a, pNH18A, 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..
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, the trp promoter and the xyI/tet chimeric
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.
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 Metlzods
in Molecular
Biology (1986)).
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.
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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.
Coding sequences for a polypeptide of interest may be incorporated as a part
of a
fusion gene including a nucleotide sequence encoding a different polypeptide.
The present
invention contemplates an isolated nucleic acid comprising a nucleic acid of
the invention
and at least one heterologous sequence encoding a heterologous peptide linked
in frame to
the nucleotide sequence of the nucleic acid of the invention so as to encode a
fusion protein
comprising the heterologous polypeptide. The heterologous polypeptide may be
fused to
(a) the C-terminus of the polypeptide encoded by the nucleic acid of the
invention, (b) the
N-terminus of the polypeptide, or (c) the C-terminus and the N-terminus of the
polypeptide.
In certain instances, the heterologous sequence encodes a polypeptide
permitting. the
detection, isolation, solubilization and/or stabilization of the polypeptide
to which it is
fused. In still other embodiments, the heterologous sequence encodes a
polypeptide
selected from the group consisting of a poly-His tag, myc, HA, GST, protein A,
protein G,
calmodulin-binding peptide, thioredoxin, maltose-binding protein, poly
arginine, poly-His-
Asp, FLAG, a portion of an immunoglobulin protein, and a transcytosis peptide.
Fusion expression systems can be useful when it is desirable to produce an
immunogenic fragment of a polypeptide of the invention. For example, the VP6
capsid
protein of rotavirus may be used as an immunologic carrier protein for
portions of
polypeptide, either in the monomeric form or in the form of a viral particle.
The nucleic
acid sequences corresponding to the portion of a polypeptide of the invention
to which
antibodies are to be raised may be incorporated into a fusion gene construct
which includes
coding sequences for a late vaccinia virus structural protein to produce a set
of recombinant
viruses expressing fusion proteins comprising a portion of the protein as part
of the virion.
The Hepatitis B surface antigen may also be utilized in this role as well.
Similarly,
chimeric constructs coding for fusion proteins containing a portion of a
polypeptide of the
invention and the poliovirus capsid protein may be created to enhance
immunogenicity
(see, for example, EP Publication NO: 0259149; and Evans et al., (1989) Nature
339:385;
Huang et al., (1988) J. Virol. 62:3855; and Schlienger et al., (1992) J.
Virol. 66:2).
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WO 2006/059247 PCT/IB2005/004126
Fusion proteins may facilitate the expression and/or purification of proteins.
For
example, a p olypeptide of the invention may be generated as a glutathione-S-
transferase
(GST) fusion protein. Such GST fusion proteins may be used to simplify
purification of a
polypeptide of the invention, such as through the use of glutathione-
derivatized matrices
(see, for example, Current Protocols in Molecular Biology, eds. Ausubel et
al., (N.Y.: John
Wiley & Sons, 1991)). In another embodiment, a fusion gene coding f or a
purification
leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at
the N-terminus
of the desired portion of the recombinant protein, may allow purification of
the expressed
fusion protein by affinity chromatography using a Ni2+ metal resin. The
purification leader
sequence may then be subsequently removed by treatment with enterokinase to
provide the
purified protein (e.g., see Hochuli et al., (1987) J. Chromatography 411: 177;
and
Janknecht et al., PNAS USA 88:8972).
Techniques for making fusion g enes a re well known. E ssentially, the j
oining of
various DNA fragments coding for different polypeptide sequences is performed
in
accordance with conventional techniques, employing blunt-ended or stagger-
ended termini
for ligation, r estriction enzyme digestion to provide for appropriate t
ermini, filling-in of
cohesive ends as appropriate, alkaline phosphatase treatment to avoid
undesirable joining,
and enzymatic ligation. In another embodiment, the fusion gene may be
synthesized by
conventional techniques including automated DNA synthesizers. Alternatively,
PCR
amplification of gene fragments may be carried out using anchor primers which
give rise to
complementary overhangs between two consecutive gene fragments which may
subsequently be annealed to generate a chimeric gene sequence (see, for
example, Current
Protocols in Moleculaf-Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
In other embodiments, nucleic acids of the invention may be immobilized onto a
solid surface, including, plates, microtiter plates, slides, beads, particles,
spheres, films,
strands, precipitates, gels, sheets, tubing, containers, capillaries, pads,
slices, etc. The
nucleic acids of the invention may be immobilized onto a chip as part of an
array. The
array may comprise one or more polynucleotides of the invention as described
herein. In
one embodiment, the chip comprises one or more polynucleotides of the
invention as part
of an array of polynucleotide sequences.
Another aspect relates to the use of nucleic acids of the invention in
"antisense
therapy". As used herein, antisense therapy refers to administration or in
situ generation of
oligonucleotide probes or their derivatives which specifically hybridize or
otherwise bind
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WO 2006/059247 PCT/IB2005/004126
under cellular conditions with the cellular mRNA and/or genomic DNA encoding
one of
the polypeptides of the invention so as to inhibit expression of that
polypeptide, e.g., by
inhibiting transcription and/or translation. The binding may be by
conventional base pair
complementarity, or, for example, in the case of binding to DNA duplexes,
through specific
interactions in the major groove of the double helix. In general, antisense
therapy refers to
the range of techniques generally employed in the art, and includes any
therapy which relies
on specific binding to oligonucleotide sequences.
The oligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent transport agent, hybridization-
triggered cleavage
agent, etc. An antisense molecule can be a "peptide nucleic acid" (PNA). PNA
refers to an
antisense molecule or anti-gene agent which comprises an oligonucleotide of at
least about
5 nucleotides in length linked to a peptide backbone of amino acid residues
ending in
lysine. The terminal lysine confers solubility to the compositiori: " PNAs
preferentially bind
complementary single stranded DNA or RNA and stop transcript elongation, and
may be
pegylated to extend their lifespan in the cell.
An antisense construct of the present invention may be delivered, for example,
as an
expression plasmid which, when transcribed in the cell, produces RNA which is
complementary to at least a unique portion of the mRNA which encodes a
polypeptide of
the invention. Alternatively, the antisense construct may be an
oligonucleotide probe which
is generated ex vivo and which, when introduced into the cell causes
inhibition of
expression by hybridizing with the mRNA and/or genomic sequences encoding a
polypeptide of the invention. Such oligonucleotide probes may be modified
oligonucleotides which are resistant to endogenous nucleases, e.g.,
exonucleases and/or
endonucleases, and are therefore stable in vivo. Exemplary nucleic acid
molecules for use
as antisense oligonucleotides are phosphoramidate, phosphothioate and
methylphosphonate
analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775).
Additionally,
general approaches to constructing oligomers useful in antisense therapy have
been
reviewed, for example, by van der Krol et al., (1988) Biotechniques 6:958-976;
and Stein et
al., (1988) CazacerRes 48:2659-2668.
In a further aspect, double stranded small interfering RNAs (siRNAs), and
methods
for administering the same are provided. siRNAs decrease or block gene
expression.
While not wishing to be bound by theory, it is generally thought that siRNAs
inhibit gene
expression by mediating sequence specific mRNA degradation. RNA interference
(RNAi)
CA 02581746 2007-03-23
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is the process of sequence-specific, post-transcriptional gene silencing,
particularly in
animals and plants, initiated by double-stranded RNA (dsRNA) that is
homologous in
sequence to the silenced gene (Elbashir et al. Nature 2001; 411(6836): 494-8).
Accordingly, it is understood that siRNAs and long dsRNAs having substantial
sequence
identity to all or a portion of a polynucleotide of the present invention may
be used to
inhibit the expression of a nucleic acid of the invention.
Alternatively, siRNAs that decrease or block the expression the Isd
polypeptides
described herein may be determined by testing a plurality of siRNA constructs
against the
target gene. Such siRNAs against a target gene may be chemically synthesized.
The
nucleotide sequences of the individual RNA strands are selected such that the
strand has a
region of complementarity to the target gene to be inhibited (i.e., the
complementary RNA
strand c omprises a n ucleotide sequence that is c omplementary to a region of
an mRNA
transcript that is formed during expression of the target gene, or its
processiing products, or
a region of a (+) strand virus). The step of synthesizing the RNA strand may
involve solid-
phase synthesis, wherein individual nucleotides are joined end to end through
the formation
of internucleotide 3'-5' phosphodiester bonds in consecutive synthesis cycles.
Provided herein are siRNA molecules comprising a nucleotide sequence
consisting
essentially of a sequence of an isd nucleic acid as described herein. An siRNA
molecule
may c omprise t wo s trands, e ach s trand comprising a n ucleotide s equence
that i s a t least
essentially complementary to each other, one of which corresponds essentially
to a
sequence of a target gene. The sequence that corresponds essentially to a
sequence of a
target gene is referred to as the "sense target sequence" and the sequence
that is essentially
complementary thereto is referred to as the "antisense target sequence" of the
siRNA. The
sense and antisense target sequences may be from about 15 to about 30
consecutive
nucleotides long; from about 19 to about 25 consecutive nucleotides; from
about 19 to 23
consecutive nucleotides or about 19, 20, 21, 22 or 23 nucleotides long. The
length of the
sense and antisense sequences is determined so that an siRNA having sense and
antisense
target sequences of that length is capable of inhibiting expression of a
target gene,
preferably without significantly inducing a host interferon response.
SiRNA target sequences may be predicted using any of the aligorithms provided
on
the world wide web at the mmcmanus with the extension
web.mit. edu/mmcmanus/www/home 1.2files/siRNAs.
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WO 2006/059247 PCT/IB2005/004126
The sense target sequence may be essentially or substantially identical to the
coding
or a non-coding portion, or combination thereof, of a target nucleic acid. For
example, the
sense target sequence may be essentially complementary to the 5' or 3'
untranslated region,
promoter, intron or exon of a target nucleic acid or complement thereof. It
can also be
essentially complementary to a region encompassing the border between two such
gene
regions.
The nucleotide base composition of the sense target sequence can be about 5 0
/o
adenines (As) and thymidines (Ts) and 50% cytidines (Cs) and guanosines (Gs).
Alternatively, the base composition can be at least 50% Cs/Gs, e.g., about
60%, 70% or
80% of Cs/Gs. Accordingly, the choice of sense target sequence may be based on
nucleotide base composition. Regarding the accessibility of target nucleic
acids by
siRNAs, such can be determined, e.g., as described in Lee et al. (2002) Nature
Biotech.
'19:500. -" This approach involves the use of oligonucleotides that are
complementary to the -
target nucleic acids as probes to determine substrate accessibility, e.g., in
cell extracts.
After forming a duplex with the oligonucleotide 'probe, the substrate becomes
susceptible to
RNase H. Therefore, the degree of RNase H sensitivity to a given probe as
determined,
e.g., by PCR, reflects the accessibility of the chosen site, and may be of
predictive value for
how well a corresponding siRNA would perform in inhibiting transcription from
this target
gene. One may also use algorithms identifying primers for polyrnerase chain
reaction
(PCR) assays or for identifying antisense oligonucleotides for identifying
first target
sequences.
The sense and antisense target sequences are preferably sufficiently
complementary,
such that an siRNA comprising both sequences is able to inhibit expression of
the target
gene, i.e., to mediate RNA interference. For example, the sequences may be
sufficiently
complementary to permit hybridization under the desired conditions, e.g., in a
cell.
Accordingly, the s ense and a ntisense target sequences may be at 1 east about
95%, 9 7%,
98%, 99% or 100% identical and may, e.g., differ in at most 5, 4, 3, 2, 1 or 0
nucleotides.
Sense and antisense target sequences are also preferably sequences that are
not
likely to significantly interact with sequences other than the target nucleic
acid or
complement thereof. This can be confirmed by, e.g., comparing the chosen
sequence to the
other sequences in the genome of the target cell. Sequence comparisons can be
performed
according to methods known in the art, e.g., using the BLAST algorithm,
further described
herein. Of course, small scale experiments can also be performed to confirm
that a
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CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
particular first target sequence is capable of specifically inhibiting
expression of a target
nucleic acid and essentially not that of other genes.
siRNAs may also comprise sequences in addition to the sense and antisense
sequences. For example, an siRNA may be an RNA duplex consisting of two
strands of
RNA, in which at least one strand has a 3' overhang. The other strand can be
blunt-ended
or have an overhang. In the embodiment in which the RNA molecule is double
stranded and
both strands comprise an overhang, the length of the overhangs may be the same
or
different for each strand. In a particular embodiment, an siRNA comprises
sense and
antisense sequences, each of which are on one RNA strand, consisting of about
19-25
nucleotides which are paired and which have overhangs of from about 1 to about
3,
particularly about 2, nucleotides on both 3' ends of the RNA. In order to
further enhance
the stability of the RNA of the present invention, the 3' overhangs can be
stabilized against
degradation: In- one~ embodiment, the RNA is stabilized by including purine
nucleotides, "
such as adenosine or guanosine nucleotides. Alternatively, substitution of
pyrimidine
nucleotides by modified analogues, e.g., substitution of uridine 2 nucleotide
3' overhangs by
2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi. The
absence of a
2' hydroxyl significantly may also enhance the nuclease resistance of the
overhang at least
in tissue culture medium. RNA strands of siRNAs may have a 5' phosphate and a
3'
hydroxyl group.
In one embodiment, an siRNA molecule comprises two strands of RNA forming a
duplex. In another embodiment, an siRNA molecule consists of one RNA strand
forming a
hairpin loop, wherein the sense and antisense target sequences hybridize and
the sequence
between the two target sequences is a spacer sequence that essentially forms
the loop of the
hairpin s tructure. T he spacer s equence may b e any c ombination o f n
ucleotides and any
length provided that two c omplementary oligonucleotides linked by a spacer
having this
sequence can form a hairpin structure, wherein at least part of the spacer
forms the loop at
the closed end of the hairpin. For example, the spacer sequence can be from
about 3 to
about 30 nucleotides; from about 3 to about 20 nucleotides; from about 5 to
about 15
nucleotides; from about 5 to about 10 nucleotides; or from about 3 to about 9
nucleotides.
The sequence can be any sequence, provided that it does not interfere with the
formation of
a hairpin structure. In particular, the spacer sequence is preferably not a
sequence having
any significant homology to the first or the second target sequence, since
this might
interfere with the formation of a hairpin structure. The spacer sequence is
also preferably
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CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
not similar to other sequences, e.g., genomic sequences of the cell into which
the nucleic
acid will be introduced, since this may result in undesirable effects in the
cell.
A person of skill in the art will understand that when referring to a nucleic
acid, e.g.,
an RNA, the RNA may comprise or consist of naturally occurring nucleotides or
of
nucleotide derivatives that provide, e.g., more stability to the nucleic acid.
Any derivative
is permitted provided that the nucleic acid is capable of functioning in the
desired fashion.
For example, an siRNA may comprise nucleotide derivatives provided that the
siRNA is
still capable of inhibiting expression of the target gene.
For example, siRNAs may include one or more modified base and/or a backbone
modified for stability or for other reasons. For example, the phosphodiester
linkages of
natural RNA may be modified to include at least one of a nitrogen or sulphur
heteroatom.
Moreover, siRNA comprising unusual bases, such as inosine, or modified bases,
such as
tritylated bases, to name just two examples, can be used in the invention. It
will be
appreciated that a great variety of modifications have been made to RNA that
serve many
useful purposes known to those of skill in the art. The term siRNA as it is
employed herein
embraces such chemically, enzymatically or metabolically modified forms of
siRNA,
provided that it is derived from an endogenous template.
There is no limitation on the manner in which an siRNA may be synthesised.
Thus,
it may synthesized in vitro or in vivo, using manual and/or automated
procedures. In vitro
synthesis may be chemical or enzymatic, for example using cloned RNA
polymerase (e.g.,
T3, T7, SP6) for transcription of a DNA (or cDNA) template, or a mixture of
both.
SiRNAs may also be prepared by synthesizing each of the two strands, e.g.,
chemically, and
hybridizing the two strands to form a duplex. In vivo, the siRNA may be
synthesized using
recombinant techniques well known in the art (see e.g., Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I
and II
(D. N G lover ed. 1 985); Oligonucleotide Synthesis (M. J. Gait ed, 1984);
Nucleic Acid
Hybridisation (B. D. Hames & S. J. Higgins eds. 1984); Transcription and
Translation (B.
D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed.
1986);
Immobilised Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide
to
Molecular Cloning (1984); the series, Methods in Enzymology (Academic Press,
Inc.);
Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds.
1987, Cold
Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu and
Grossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987),
Immunochemical
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WO 2006/059247 PCT/IB2005/004126
Methods in Cell and Molecular Biology (Academic Press, London), Scopes,
(1987), Protein
Purification: Principles and Practice, Second Edition (Springer-Verlag,
N.Y.),and
Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell
eds 1986). For example, bacterial cells can be transformed with an expression
vector which
comprises the DNA template from which the siRNA is to be derived.
If synthesized outside the cell, the siRNA may be purified prior to
introduction into
the cell. Purification may be by extraction with a solvent (such as
phenol/chloroform) or
resin, precipitation (for example in ethanol), electrophoresis,
chromatography, or a
combination thereof. However, purification may result in loss of siRNA and may
therefore
be minimal or not carried out at all. The siRNA may be dried for storage or
dissolved in an
aqueous solution, which may contain buffers or salts to promote annealing,
and/or
stabilization of the RNA strands.
The double-stranded structure may be formed by a single self-complementary RNA
strand or two separate complementary RNA strands.
It is known that mammalian cells can respond to extracellular siRNA and
therefore
may h ave a transport mechanism for d sRNA (Asher e t a l. (1969) Nature 2 23
7 15-717).
Thus, siRNA may be administered extracellularly into a cavity, interstitial
space, into the
circulation of a mammal, or introduced orally. Methods for oral introduction
include direct
mixing of the RNA with food of the mammal, as well as engineered approaches in
which a
species that is used as food is engineered to express the RNA, then fed to the
mammal to be
affected. For e xample, food bacteria, s uch as L actococcus lactis, m ay b e
t ransformed to
produce the dsRNA (see W093/17117, W097/14806). Vascular or extravascular
circulation, the blood or lymph systems and the cerebrospinal fluid are sites
where the RNA
may be injected.
RNA may be introduced into the cell intracellularly. Physical methods of
introducing nucleic acids m ay also be used in this r espect. siRNA m ay be
administered
using the microinjection techniques described in Zernicka-Goetz et al. (1997)
Development
124, 1133-1137 and Wianny et al. (1998) C'laromosonza 107, 430-439.
Other physical methods of introducing nucleic acids intracellularly include
bombardment by particles covered by the siRNA, for example gene gun technology
in
which the siRNA is immobilized on gold particles and fired directly at the
site of wounding.
Thus, the invention provides the use of an siRNA in a gene gun for inhibiting
the
expression of a target gene. Further, there is provided a composition suitable
for gene gun
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
therapy comprising an siRNA and gold particles. An alternative physical method
includes
electroporation of cell membranes in the presence of the siRNA. This method
permits
RNAi on a large scale. Other methods known in the art for introducing nucleic
acids to
cells m ay b e u sed, such as 1 ipid-mediated c arrier transport, c hemical-
mediated transport,
such as calcium phosphate, and the like. siRNA may be introduced along with
components
that perform one or more of the following activities: enhance RNA uptake by
the cell,
promote annealing of the duplex strands, stabilize the annealed strands, or
otherwise
increase inhibition of the target gene.
Any known gene therapy technique can be used to administer the RNA. A viral
construct packaged into a viral particle would accomplish both efficient
introduction of an
expression construct i nto t he c ell and t ranscription o f s iRNA e ncoded
by the e xpression
construct. Thus, siRNA can also be produced inside a cell. Vectors, e.g.,
expression vectors
that comprise a nucleic acid encoding one or the two strands of an siRNA
molecule may be
used for that purpose. The nucleic acid may further comprise an antisense
sequence that is
essentially complementary to the sense target sequence. The nucleic acid may
further
comprise a spacer sequence between the sense and the antisense target
sequence. The
nucleic acid may further comprise a promoter for directing expression of the
sense and
antisense sequences in a cell, e.g., an RNA Polymerase II or III promoter and
a
transcriptional termination signal. The sequences may be operably linked.
In one embodiment a nucleic acid comprises an RNA coding region (e.g., sense
or
antisense target sequence) operably linked to an RNA polymerase III promoter.
The RNA
coding region can be immediately followed by a pol III terminator sequence,
which directs
termination of RNA synthesis by pol III. The pol III terminator sequences
generally have 4
or more consecutive thymidine ("T") residues. In a preferred embodiment, a
cluster of 5
consecutive T residues is used as the terminator by which pol III
transcription is stopped at
the second or third T of the DNA template, and thus only 2 to 3 uridine ("U")
residues are
added to the 3' end of the coding sequence. A variety of pol III promoters can
be used with
the invention, including for example, the promoter fragments derived from H1
RNA genes
or U6 snRNA genes of hunian or mouse origin or from any other species. In
addition, pol
III promoters can be modified/engineered to incorporate other desirable
properties such as
the ability to be induced by small chemical molecules, either ubiquitously or
in a tissue-
specific manner. For example, in one embodiment the promoter may be activated
by
tetracycline. In another embodiment the promoter may be activated by IPTG
(lacI system).
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siRNAs can be produced in cells by transforming cells with two nucleic acids,
e.g.,
vectors, each nucleic acid comprising an expressing cassette, each expression
cassette
comprising a promoter, an RNA coding sequence (one being a sense target
sequence and
the other being an antisense target sequence) and a termination signal.
Alternatively, a
single nucleic acid may comprise these two expression cassettes. In yet
another
embodiment, a nucleic acid encodes a single stranded RNA comprising a sense
target
sequence linked to a spacer linked to an antisense target sequence. The
nucleic acids may
be present in a vector, such as an expression vector, e.g., a eukaryotic
expression vector that
allows expression of the sense and antisense target sequences in cells into
which it is
introduced.
Vectors for producing siRNAs are described, e.g., in Paul et al. (2002) Nature
Biotechnology 29:505; Xia et al. (2002) Nature Biotechnology 20:1006; Zeng et
al. (2002)
Mol. Cell 9:1327; Thijn et al. (2002) Science 296:550; BMC Biotechnol. 2002
Aug
28;2(1):15; Lee et al. (2002) Nature Biotechnology 19: 500; McManus et al.
(20,02) RNA
8:842; Miyagishi et al. (2002) Nature Biotechnology 19:497; Sui et al. (2002)
PNAS
99:5515; Yu et al. (2002) PNAS 99:6047; Shi et al. (2003) Trends Genet.
19(1):9;
Gaudilliere et al. (2002) J. Biol. Claem. 277(48):46442; US2002/0182223; US
2003/0027783; WO 01/36646 and WO 03/006477. Vectors are also available
commercially. For example, the pSilencer is available from Gene Therapy
Systems, Inc.
and pSUPER RNAi system is available from Oligoengine.
Also provided herein are compositions comprising one or more siRNA or nucleic
acid encoding an RNA coding region of an siRNA. Compositions may be
pharmaceutical
compositions and comprise a pharmaceutically acceptable carrier. Compositions
may also
be provided in a device for administering the composition in a cell or in a
subject. For
example a composition may be present in a syringe or on a stent. A composition
may also
comprise agents facilitating the entry of the siRNA or nucleic acid into a
cell.
In general, the oligonucleotides may be synthesized using protocols known in
the
art, for example, as described in Caruthers et al., Metlaods in Enzymology
(1992) 211:3-19;
Thompson et al., International PCT Publication No. WO 99/54459; Wincott et
al., Nucl.
Acids Res. (1995) 23:2677-2684; Wincott et al., Methods Mol. Bio., (1997)
74:59; Brennan
et al., Biotechnol. Bioeng. (1998) 61:33-45; and Brennan, U.S. Pat. No.
6,001,311; each of
which is hereby incorporated by reference in its entirety herein. In general,
the synthesis of
oligonucleotides involves conventional nucleic acid protecting and coupling
groups, such as
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dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-
limiting
example, small scale syntheses are conducted on a Expedite 8909 RNA
synthesizer sold by
Applied Biosystems, Inc. (Weiterstadt, Germany), using ribonucleoside
phosphoramidites
sold by ChemGenes Corporation (Ashland Technology Center, 200 Homer Avenue,
Ashland, MA 01721, USA). Alternatively, syntheses can be performed on a 96-
well plate
synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.,
USA), or by
methods such as those described in Usman et al., J. Ain. Claem. Soc. (1987)
109:7845;
Scaringe et al., Nucl. Acids Res. (1990) 18:5433; Wincott et al., Nucl. Acids
Res. (1990)
23:2677-2684; and Wincott et al., Methods Mol. Bio. (1997) 74:59, each of
which is hereby
incorporated by reference in its entirety.
The nucleic acid molecules of the present invention may be synthesized
separately
and dsRNAs may be formed post-synthetically, for example, by ligation (Moore
et al.,
Science (1992) 256:9923; Draper et al., International.PCT publication No. WO
93/23569;
Shabarova et al., Nucl. Acids Res. (1991) 19:4247; Bellon et al., Nucleosides
&
Nucleotides (1997) 16:951; and Bellon et al., Bioconjugate Chem. (1997) 8:204;
or by
hybridization following synthesis and/or deprotection. The nucleic acid
molecules can be
purified by gel electrophoresis using conventional methods or can be purified
by high
pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality
of which is
hereby incorporated herein by reference) and re-suspended in water.
In another embodiment, the level of a particular mRNA or polypeptide in a cell
is
reduced by introduction of a ribozyme into the cell or nucleic acid encoding
such.
Ribozyme molecules designed to catalytically cleave mRNA transcripts can also
be
introduced into, or expressed, in cells to inhibit target gene expression
(see, e.g., Sarver et
al., 1990, Science 247:1222-1225 and U.S. Patent No. 5,093,246). One commonly
used
ribozyme motif is the hammerhead, for which the substrate sequence
requirements are
minimal. Design of the hammerhead ribozyme is disclosed in Usman et al.,
Current Opin.
Struct. Biol. (1996) 6:527-533. Usman also discusses the therapeutic uses of
ribozymes.
Ribozymes can also be prepared and used as described in Long et al., FASEB J.
(1993)
7:25; Symons, Ann. Rev. Biochem. (1992) 61:641; Perrotta et al., Biochem.
(1992) 31:16-
17; Ojwang et al., Proc. Natl. Acad. Sci. (USA) (1992) 89:10802-10806; and
U.S. Patent
No. 5,254,678. Ribozyme cleavage of HIV-I RNA is described in U.S. Patent No.
5,144,019; methods of cleaving RNA using ribozymes is described in U.S. Patent
No.
5,116,742; and methods for increasing the specificity of ribozymes are
described in U.S.
33
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
Patent No. 5,225,337 and Koizumi et al., Nucleic Acid Res. (1989) 17:7059-
7071.
Preparation and use of ribozyme fragments in a hammerhead structure are also
described by
Koizumi et al., Nucleic Acids Res. (1989) 17:7059-7071. Preparation and use of
ribozyme
fragments in a hairpin structure are described by Chowrira and Burke, Nucleic
Acids Res.
(1992) 20:2835. Ribozymes can also be made by rolling transcription as
described in
Daubendiek and Kool, Nat. Biotechnol. (1997) 15(3):273-277.
Gene expression can be reduced by targeting deoxyribonucleotide sequences
complementary to the regulatory region of the target gene (i.e., the gene
promoter and/or
enhancers) to form triple helical structures that prevent transcription of the
gene in target
cells in the body. (See generally, Helene (1991) Anticancer Drug Des.,
6(6):569-84;
Helene et al. (1992) Ann. NY. Acad. Sci., 660:27-36; and Maher (1992)
Bioassays
14(12):807-15).
In a further embodiment, RNA aptamers can be introduced into or expressed in a
cell. RNA aptamers are specific RNA ligands for proteins, such as for Tat and
Rev RNA
(Good et al. (1997) Gene Therapy 4: 45-54) that can specifically inhibit their
translation.
4. Isd Polypeptides
The S. aureus polypeptides, including IsdA (SEQ ID NO: 3), IsdB (SEQ ID NO:
6),
and IsdC (SEQ ID NO: 9) (Figures 1-3), described herein, include naturally
purified
products, products of chemical synthetic procedures, and products produced by
recombinant techniques from a prokaryotic or eukaryotic host cell, including
for example,
bacterial, yeast, higher plant, insect, and mammalian cells. In certain,
embodiments, the
polypeptides disclosed herein inhibit the function of Isd polypeptides.
Polypeptides may also comprise, consist of or consist essentially of any of
the amino
acid sequences described herein. Yet other polypeptides comprise, consist of
or consist
essentially of an amino acid sequence that has at least about 70%, 80%, 90%,
95%, 98% or
99% identity or homology with an Isd polypeptide. For example, polypeptides
that differ
from a sequence in a naturally occurring Isd protein in about 1, 2, 3, 4, 5 or
more amino acids
are also contemplated. The differences may be substitutions, e.g.,
conservative substitutions,
deletions or additions. The differences are preferably in regions that are not
significantly
conserved among different species. Such regions may be identified by aligning
the amino acid
sequences of Isd proteins from various species. These amino acids can be
substituted, e.g.,
with those found.in another species. Other amino acids that may be
substituted, inserted or
34
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
deleted at these or other locations can be identified by mutagenesis studies
coupled with
biological assays.
Proteins may also comprise one or more non-naturally occurring amino acids.
For
example, n onclassical amino acids or chemical amino a cid analogs c an be
introduced as a
substitution or addition into proteins. Non-classical amino acids include, but
are not limited
to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-
amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu,
epsilon-Ahx, 6-
amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,
ornithine,
norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,
cysteic acid, t-
butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine,
fluoro-amino
acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl
amino acids,
Nalpha-methyl amino acids, and amino acid analogs in general. Furthermore, the
amino acid
can be D (dextrorotary) or L (levorotary). Yet other proteins that are
encompassed herein are those that comprise- modified
amino acids. Exemplary proteins are derivative proteins that may be one
modified by
glycosylation, pegylation, phosphorylation or any similar process that retains
at least one
biological function of the protein from which it was derived.
Proteins may be used as a substantially pure preparation, e.g., wherein at
least about
90% of the protein in the preparation are the desired protein. Compositions
comprising at
least about 50%, 60%, 70%, or 80% of the desired protein may also be used.
The S. aureus 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. Proteins may be used as a
substantially pure
preparation, e.g., wherein at least about 90% of the protein in the
preparation are the desired
protein. Compositions comprising at least about 50%, 60%, 70%, or 80% of the
desired
protein may also be used.
Proteins may be denatured or non-denatured and may be aggregated or non-
aggregated as a result thereof. Proteins can be denatured according to methods
known in the
art.
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
In certain embodiments, an Isd polypeptide described herein may be a fusion
protein
containing a domain which increases its solubility and/or facilitates its
purification,
identification, detection, and/or structural characterization. Exemplary
domains, include, for
example, glutathione S-transferase (GST), protein A, protein G, calmodulin-
binding peptide,
thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly
His-Asp or
FLAG fusion proteins and tags. Additional exemplary domains include domains
that alter
protein localization in vivo, such as signal peptides, type III secretion
system-targeting
peptides, transcytosis domains, nuclear localization signals, etc. In various
embodiments, a
polypeptide of the invention may comprise one or more heterologous fusions.
Polypeptides
may contain multiple copies of the same fusion domain or may contain fusions
to two or more
different d omains. T he fusions m ay o ccur at t he N-terminus of t he p
olypeptide, at t he C-
terminus of the polypeptide, or at both the N- and C-terminus of the
polypeptide. It is also
within the scope of the invention to include linker sequences between a
polypeptide of the
invention and the fusion domain in order to facilitate construction of the
fusion protein or to
optimize protein expression or structural constraints of the fusion protein.
In another
embodiment, the polypeptide may be constructed so as to contain protease
cleavage sites
between the fusion polypeptide and polypeptide of the invention in order to
remove the tag
after protein expression or thereafter. Examples of suitable endoproteases,
include, for
example, Factor Xa and TEV proteases. A protein may also be fused to a signal
sequence.
For example, when prepared recombinantly, a nucleic acid encoding the peptide
may be linked
at its 5' end to a signal sequence, such that the protein is secreted from the
cell.
In certain embodiments, polypeptides of the invention may be synthesized
chemically, ribosomally in a cell free system, or ribosomally within a cell.
Chemical
synthesis of polypeptides of the invention may be carried out using a variety
of art
recognized methods, including stepwise solid phase synthesis, semi-synthesis
through the
conformationally-assisted re-ligation of peptide fragments, enzymatic ligation
of cloned or
synthetic peptide segments, and chemical ligation. Native chemical ligation
employs a
chemoselective reaction of two unprotected peptide segments to produce a
transient
thioester-linked intermediate. The transient thioester-linked intermediate
then
spontaneously undergoes a rearrangement to provide the full length ligation
product having
a native peptide bond at the ligation site. Full length ligation products are
chemically
identical to proteins produced by cell free synthesis. Full length ligation
products may be
refolded and/or oxidized, as allowed, to form native disulfide-containing
protein molecules.
36
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
(see e.g., U.S. Patent Nos. 6,184,344 and 6,174,530; and Muir et al., Curr.
Opin. Biotech.
(1993): vol. 4, p 420; Miller et al., Science (1989): vol. 246, p 1149;
Wlodawer et al.,
Science (1989): vol. 245, p 616; Huang et al., Biochenaistzy (1991): vol. 30,
p 7402;
Schnolzer, et al., Int. J Pept. Prot. Res. (1992): vol. 40, p 180-193;
Rajarathnam et al.,
Science (1994): vol. 264, p 90; R. E. Offord, "Chemical Approaches to Proteiri
Engineering", in Protein Design and the Development of New therapeutics and
Vaccines, J.
B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; Wallace
et al., J.
Biol. Chenz. (1992): vol. 267, p 3852; Abrahmsen et al., Biochemistry (1991):
vol. 30, p
4151; Chang, et al., Proc. Natl. Acad. Sci. USA (1994) 91: 12544-12548;
Schnlzer et al.,
Science (1992): vol., 3256, p 221; and Akaji et al., Chem. Plaarnn. Bull.
(Tokyo) (1985) 33:
184).
In certain embodiments, it may be advantageous to provide naturally-occurring
or
experimentally-derived homologs of a polypeptide of the invention. Such
homologs may.
function in a limited capacity as a modulator to promote or inhibit a subset
of the biological
activities of the naturally-occurring form of the polypeptide. Thus, specific
biological
effects may be elicited by treatment with a homolog of limited function, and
with fewer
side effects relative to treatment with agonists or antagonists which are
directed to all of the
biological activities of a polypeptide of the invention. For instance,
antagonistic homologs
may be generated which interfere with the ability of the wild-type polypeptide
of the
invention to associate with certain proteins, but which do not substantially
interfere with the
formation of complexes between the native polypeptide and other cellular
proteins.
Polypeptides may be derived from the full-length polypeptides of the
invention.
Isolated peptidyl portions of those polypeptides may be obtained by screening
polypeptides
recombinantly produced from the corresponding fragment of the nucleic acid
encoding such
polypeptides. In addition, fragments may be chemically synthesized using
techniques
known in the art such as conventional Merrifield solid phase f-Moc or t-Boc
chemistry. For
example, proteins may be arbitrarily divided into fragments of desired length
with no
overlap of the fragments, or may be divided into overlapping fragments of a
desired length.
The fragments may be produced (recombinantly or by chemical synthesis) and
tested to
identify those peptidyl fragments having a desired property, for example, the
capability of
functioning as a modulator of the polypeptides of the invention. In an
illustrative
embodiment, peptidyl portions of a protein of the invention may be tested for
binding
activity, as well as inhibitory ability, by expression as, for example,
thioredoxin fusion
37
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
proteins, each of which contains a discrete fragment of a protein of the
invention (see, for
example, U.S. Patents 5,270,181 and 5,292,646; and PCT publication
W094/02502).
In another embodiment, truncated polypeptides may be prepared. Truncated
polypeptides have from 1 to 20 or more amino acid residues removed from either
or both
the N- and C-termini. Such truncated polypeptides may prove more amenable to
expression, purification or characterization than the full-length polypeptide.
For example,
truncated polypeptides may prove more amenable than the full-length
polypeptide to
crystallization, to yielding high quality diffracting crystals or to yielding
an HSQC
spectrum with high intensity peaks and minimally overlapping peaks. In
addition, the use
of truncated polypeptides may also identify stable and active d omains of the
full-length
polypeptide that may be more amenable to characterization.
It is also possible to modify the structure of the polypeptides of the
invention for
such purposes as enhancing therapeutic or prophylactic efficacy, or stability
(e.g., ex vivo - -.-
shelf life, resistance to proteolytic degradation in vivo, etc.). Such
modified polypeptides,
when designed to retain at least one activity of the naturally-occurring form
of the protein,
are considered "functional equivalents" of the polypeptides described in more
detail herein.
Such modified polypeptides may be produced, for instance, by amino acid
substitution,
deletion, or addition, which substitutions may consist in whole or part by
conservative
amino acid substitutions.
For instance, it is reasonable to expect that an isolated conservative amino
acid
substitution, such as replacement of a leucine with an isoleucine or, valine,
an aspartate with
a glutamate, a threonine with a serine, will not have a major affect on the
biological activity
of the resulting molecule. Whether a change in the amino acid sequence of a
polypeptide
results in a functional homolog may be readily determined by assessing the
ability of the
variant polypeptide to produce a response similar to that of the wild-type
protein.
Polypeptides in which more than one replacement has taken place may readily be
tested in
the same manner.
Methods of generating sets of combinatorial mutants of polypeptides of the
invention are provided, as well as truncation mutants, and is especially
useful for
identifying p otential variant s equences ( e.g., h omologs). T he purpose of
s creening such
combinatorial libraries is to generate, for example, homologs which may
modulate the
activity of a polypeptide of the invention, or alternatively, which possess
novel activities
altogether. Combinatorially-derived h omologs m ay b e g enerated w hich have
a s elective
38
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
potency relative to a naturally-occurring protein. Such homologs may be used
in the
development of therapeutics.
Likewise, mutagenesis may give rise to homologs which have intracellular half-
lives dramatically different than the corresponding wild-type protein. For
example, the
altered protein may be rendered either more stable or less stable to
proteolytic degradation
or other cellular process which result in destruction of, or otherwise
inactivation of the
protein. Such homologs, and the genes which encode them, may be utilized to
alter protein
expression by modulating the half-life of the protein. As above, such proteins
may be used
for the development of therapeutics or treatment.
In similar fashion, protein homologs may be generated by the present
combinatorial
approach t o a ct as antagonists, in that they a re able t o i nterfere w ith
the activity o f the
corresponding wild-type protein.
In a representative embodiment of this method, the amino acid sequences for a
population of protein homologs are aligned, preferably to promote the highest
homology
possible. Such a population of variants may include, for example, homologs
from one or
more species, or homologs from the same species but which differ due to
mutation. Amino
acids which appear at each position of the aligned sequences are selected to
create a
degenerate set of combinatorial sequences. In certain embodiments, the
combinatorial
library is produced by way of a degenerate library of genes encoding a library
of
polypeptides which each include at least a portion of potential protein
sequences. For
instance, a mixture of synthetic oligonucleotides may be enzymatically ligated
into gene
sequences such that the degenerate set of potential nucleotide sequences are
expressible as
individual polypeptides, or alternatively, as a set of larger fusion proteins
(e.g. for phage
display).
There are many ways by which the library of potential homologs may be
generated
from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate
gene
sequence may be carried out in an automatic DNA synthesizer, and the synthetic
genes may
then be ligated into an appropriate vector for expression. One purpose of a
degenerate set
of genes is to provide, in o ne m ixture, a 11 of the s equences e ncoding t
he desired s et of
potential protein sequences. The synthesis of degenerate oligonucleotides is
well known in
the art (see for example, Narang (1983) Tetrahedron 39:3; Itakura et al.
(1981)
Recombiraarat DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,
Amsterdam: Elsevier pp. 273-289; Itakura et al. (1984) Annu. Rev. Biochem.
53:323;
39
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WO 2006/059247 PCT/IB2005/004126
Itakura et al. (1984) Scierzce 198:1056; Ike et al., (1983) Nucleic Acid Res.
11:477). Such
techniques have been employed in the directed evolution of other proteins
(see, for
example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS
USA
89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990)
PNAS USA
87: 6378-6382;. as well as U.S. Patent Nos: 5,223,409, 5,198,346, and
5,096,815).
Alternatively, other forms of mutagenesis may be utilized to generate a
combinatorial library. For example, protein homologs (both agonist and
antagonist forms)
may be generated and isolated from a library by screening using, for example,
alanine
scanning mutagenesis and the like (Ruf et al. (1994) Biochemistry 33:1565-
1572; Wang et
al. (1994) J. Biol. Chena. 269:3095-3099; Balint et al. (1993) Gene 137:109-
118; Grodberg
et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima et al. (1993) J. Biol.
Clzem.
268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838; and
Cunningham et
al., (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et
al. (1993)
Virology 193:653-660; Brown et al. (1992) Mol. Cell Biol. 12:2644-2652;
McKnight et al.
(1982) Science 232:316); by saturation mutagenesis (Meyers et al. (1986)
Science
232:613); by PCR mutagenesis (Leung et al. (1989) Method Cell Mol Biol 1:11-
19); or by
random mutagenesis (Miller et al. (1992) A Short Course in Bacterial Genetics,
CSHL
Press, Cold Spring Harbor, NY; and Greener et al. (1994) Strategies in Mol
Biol 7:32-34).
Linker scanning mutagenesis, particularly in a combinatorial setting, is an
attractive method
for identifying truncated forms of proteins that are bioactive.
A wide r ange of techniques are known i n the art f or s creening g ene
products of
combinatorial libraries made by point mutations and truncations, and for
screening cDNA
libraries for gene products having a certain property. Such techniques will be
generally
adaptable for rapid screening of the gene libraries generated by the
combinatorial
mutagenesis of protein homologs. T he most widely used techniques for
screening large
gene libraries typically comprises cloning the gene library into replicable
expression
vectors, transforming appropriate cells with the resulting library of vectors,
and expressing
the combinatorial genes under conditions in which detection of a desired
activity facilitates
relatively easy isolation of the vector encoding the gene whose product was
detected.
In a n i llustrative e mbodiment of a s creening assay, c andidate c
ombinatorial gene
products are displayed on the surface of a cell and the ability of particular
cells or viral
particles to bind to the combinatorial gene product is detected in a "panning
assay". For
instance, the gene library may be cloned into the gene for a surface membrane
protein of a
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
bacterial cell (Ladner et al., WO 88/06630; Fuchs et al., (1991)
Bio/Technology 9:1370-
1371; and Goward et al., (1992) TIBS 18:136-140), and the resulting fusion
protein detected
by panning, e.g. using a fluorescently labeled molecule which binds the cell
surface protein,
e.g., FITC-substrate, to score for potentially functional homologs. Cells may
be visually
inspected and separated under a fluorescence microscope, or, when the
morphology of the
cell permits, separated by a fluorescence-activated cell sorter. This method
may be used to
identify substrates or other polypeptides that can interact with a polypeptide
of the
invention.
In similar fashion, the gene library may be expressed a s a fusion protein on
the
surface of a viral particle. For instance, in the filamentous phage system,
foreign peptide
sequences may be expressed on the surface of infectious phage, thereby
conferring two
benefits. First, because these phage may be applied to affinity matrices at
very high
concentrations, a large number of phage may be screened at one time. Second,
because
each infectious phage displays the combinatorial gene product on its surface,
if a particular
phage is recovered from an affinity matrix in low yield, the phage may be
amplified by
another round of infection. The group of almost identical E. coli filamentous
phages M13,
fd, and fl are most often used in phage display libraries, as either of the
phage gIII or gVIII
coat proteins may be used to generate fusion proteins without disrupting the
ultimate
packaging of the viral particle (Ladner et al., PCT publication WO 90/02909;
Garrard et al.,
PCT publication WO 92/09690; Marks et al., (1992) J. Biol. Chenz. 267:16007-
16010;
Griffiths et al., (1993) EMBO J. 12:725-734; Clackson et al., (1991) Nature
352:624-628;
and Barbas et al., (1992) PNAS USA 89:4457-4461). Other phage coat proteins
may be
used as appropriate.
The polypeptides disclosed herein may be reduced to generate mimetics, e.g.
peptide or non-peptide agents, which are able to mimic binding of the
authentic protein to
another cellular partner. Such mutagenic techniques as described above, as
well as the
thioredoxin system, are also particularly useful for mapping the determinants
of a protein
which participates in a protein-protein interaction with another protein. To
illustrate, the
critical residues of a protein which are involved in molecular recognition of
a substrate
protein may be determined and used to generate peptidomimetics that may bind
to the
substrate protein. The peptidomimetic may then be used as an inhibitor of the
wild-type
protein by binding to the substrate and covering up the critical residues
needed for
interaction with the wild-type protein, thereby preventing interaction of the
protein and the
41
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WO 2006/059247 PCT/IB2005/004126
substrate. By employing, for example, scanning mutagenesis to map the amino
acid
residues of a protein which are involved in binding a substrate polypeptide,
peptidomimetic
compounds may be generated which mimic those residues in binding to the
substrate.
For instance, derivatives of the Isd proteins described herein may be
chemically
modified peptides and peptidomimetics. Peptidomimetics are compounds based on,
or
derived from, peptides and proteins. Peptidomimetics can be obtained by
structural
modification of known peptide sequences using unnatural amino acids,
conformational
restraints, isosteric replacement, and the like. The subject peptidomimetics
constitute the
continum of structural space between peptides and non-peptide synthetic
structures;
peptidomimetics may be useful, therefore, in delineating pharmacophores and in
helping to
translate peptides into nonpeptide compounds with the activity of the parent
peptides.
Moreover, mimetopes of the subject peptides can be provided. Such
peptidomimetics can have such attributes.as_being non-hydrolyzable (e.g.,
increased
stability against proteases or other physiological conditions which degrade
the
corresponding peptide), increased specificity and/or potency for stimulating
cell
differentiation. For illustrative purposes, non-hydrolyzable peptide analogs
of such
residues may be generated using benzodiazepine (e.g., see Freidinger et al.,
in Peptides:
Chenaistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988),
azepine (e.g., see Huffman et al., in Peptides: Chemistry and Biology, G.R.
Marshall ed.,
ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings
(Garvey et
al., in Peptides: Chernistry and Biology, G.R. Marshall ed., ESCOM Publisher:
Leiden,
Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.,(1986) J.
Med. Chem.
29:295; and Ewenson et al., in Peptides: Structure and Function (Proceedings
of the 9th
American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), (3-turn
dipeptide
cores (Nagai et a l., (1985) Tetrahedron L ett 26:647; and Sato e t a l.
(1986) J Chem Soc
Perkin Trans 1:1231), and (3-aminoalcohols (Gordon e t al. (1985) B iochenz
Bioplays R es
Commun 126:419; and Dann et al. (1986) Biochem Bioplays Res Commun 134:71).
In addition to a variety of sidechain replacements which can be carried out to
generate peptidomimetics, the description specifically contemplates the use of
conformationally restrained mimics of peptide secondary structure. Numerous
surrogates
have been developed for the amide bond of peptides. Frequently exploited
surrogates for
the amide bond include the following groups (i) trans-olefins, (ii)
fluoroalkene, (iii)
methyleneamino, (iv) phosphonamides, and (v) sulfonamides.
42
CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
O
N
H
amide bond
Examples of Surrogates:
F
N
H
trans olefin fluoroalkene methyleneamino
0 Q\ O
N~ N
OH H
H
phosphonamide sulfonamide
Additionally, peptidomimietics based on more substantial modifications of the
backbone of a peptide can be used. Peptidomimetics which fall in this category
include (i)
retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called peptoids).
O R2
H~N\
R1 H O
dipeptide
Examples of analogs:
O H R2 O RR2
~N N~ ~N H I ~r\
~
1 O R1 O
retro-inverso N-alkyl glycine
43
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WO 2006/059247 PCT/IB2005/004126
Furthermore, the methods of combinatorial chemistry are being brought to bear,
on
the development of new peptidomimetics. For example, one embodiment of a so-
called
"peptide morphing" strategy focuses on the random generation of a library of
peptide
analogs that comprise a wide range of peptide bond substitutes.
O R2
H~
N
R1 H O
dipeptide
peptide
morphing
R2
H new backbone \
element
Rl O
In an exemplary embodiment, the peptidomimetic can be derived as a retro-
inverso
analog of the peptide. Such retro-inverso analogs can be made according to the
methods
known in the art, such as that described by the Sisto et al. U.S. Patent
4,522,752. A retro-
inverso analog can be generated as described, e.g., in WO 00/01720. It will be
understood
that a mixed peptide, e.g., including some normal peptide linkages, may be
generated. As a
general guide, sites which are most susceptible to proteolysis are typically
altered, with less
susceptible amide linkages b eing optional f or mimetic switching. The final
product, o r
intermediates thereof, can be purified by HPLC.
Peptides may comprise at least one amino a cid or every amino acid that is a D
stereoisomer. Other peptides may comprise at least one amino acid that is
reversed. The
amino acid that .is reversed may be a D stereoisomer. Every amino acid of a
peptide may
be reversed and/or every amino acid may be a D stereoisomer.
In another illustrative embodiment, a peptidomimetic can be derived as a retro-
enantio analog of a peptide. Retro-enantio analogs such as this can be
synthesized with
commercially available D-amino acids (or analogs thereof) and standard solid-
or solution-
phase peptide-synthesis techniques, as described, e.g., in WO 00/01720. The
final product
may be purified by HPLC to yield the pure retro-enantio analog.
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In still another illustrative embodiment, trans-olefin derivatives can be made
for the
subject peptide. Trans-olefin analogs can be synthesized according to the
method of Y.K.
Shue et al. (1987) Tetrahedron Letters 28:3225 and as described in WO
00/01720. It is
further possible to couple pseudodipeptides synthesized by the above method to
other
pseudodipeptides, to make peptide analogs with several olefinic
functionalities in place of
amide functionalities.
Still another class of peptidomimetic derivatives include the phosphonate
derivatives. The synthesis of such phosphonate derivatives can be adapted from
known
synthesis schemes. See, for example, Loots et al. in Peptides: Chemistr.y and
Biology,
(Escom Science Publishers, Leiden, 1988, p. 118); Petrillo et al. in Peptides:
Structure and
Function (Proceedings of the 9th American Peptide Symposium, Pierce Chemical
Co.
Rockland, IL, 1985).
Many other peptidomimetic structures are known in the art and can be readily
adapted for use in the subject peptidomimetics. To illustrate, a
peptidomimetic may
incorporate the 1-azabicyclo[4.3.0]nonane surrogate ( see Kim et al. (1997) J.
Org. Chem.
62:2847), or an N-acyl piperazic acid (see Xi et al. (1998) J. Am. Chem. Soc.
120:80), or a
2-substituted piperazine moiety as a constrained amino acid analogue (see
Williams et al.
(1996) J. Med. Chem. 39:1345-1348). In still other embodiments, certain amino
acid
residues can be replaced with aryl and bi-aryl moieties, e.g., monocyclic or
bicyclic
aromatic or heteroaromatic nucleus, or a biaromatic, aromatic-heteroaromatic,
or
biheteroaromatic nucleus.
The subject peptidomimetics can be optimized by, e.g., combinatorial synthesis
techniques combined with high throughput screening.
Moreover, other examples of mimetopes include, but are not limited to, protein-
based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic
acid-
based compounds, natural organic compounds, synthetically derived organic
compounds,
anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof. A
mimetope can
be obtained by, for example, screening libraries of natural and synthetic
compounds for
compounds capable of inhibiting cell survival and/or tumor growth. A mimetope
can also
be obtained, for example, from libraries of natural and synthetic compounds,
in particular,
chemical or combinatorial libraries (i.e., libraries of compounds that differ
in sequence or
size but that have the same building blocks). A mimetope can also be obtained
by, for
example, rational drug design. In a rational drug design procedure, the three-
dimensional
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structure of a compound of the present invention can be analyzed by, for
example, nuclear
magnetic resonance (NMR) or x-ray crystallography. The three-dimensional
structure can
then be used to predict structures of potential mimetopes by, for example,
computer
modelling. The predicted mimetope structures can then be produced by, for
example,
chemical synthesis, recombinant DNA technology, or by isolating a mimetope
from a
natural source (e.g., plants, animals, bacteria and fungi).
"Peptides, variants and derivatives thereof' or "peptides and analogs thereof'
are
included in "peptide therapeutics" and is intended to include any of the
peptides or
modified forms thereof, e.g., peptidomimetics, described herein. Preferred
peptide
therapeutics decrease cell survival or increase apoptosis. For example, they
may decrease
cell survival or increase apoptosis by a factor of at least about 2 fold, 5
fold, 10 fold, 30 fold
or 100 fold, as determined, e.g., in an assay described herein.
The activity of an Isd protein, fragment, or variant thereof may be assayed
using an
appropriate substrate or binding partner or other reagent suitable to test for
the suspected
activity as described below.
In another embodiment, the activity of a polypeptide may be determined by
assaying for the level of expression of RNA and/or protein molecules.
Transcription levels
may be determined, for example, using Northern blots, hybridization to an
oligonucleotide
array or by assaying for the level of a resulting protein product. Translation
levels may be
determined, for example, using Western blotting or by identifying a detectable
signal
produced by a protein product (e.g., fluorescence, luminescence, enzymatic
activity, etc.).
Depending on the particular situation, it may be desirable to detect the level
of transcription
and/or translation of a single gene or of multiple genes.
Alternatively, it may be desirable to measure the overall rate of DNA
replication,
transcription and/or translation in a cell. In general this may be
accomplished by growing
the cell in the presence of a detectable metabolite which is incorporated into
the resultant
DNA, RNA, or protein product. For example, the rate of DNA synthesis may be
determined by growing cells in the presence of BrdU which is incorporated into
the newly
synthesized DNA. The amount of BrdU may then be determined histochemically
using an
anti-BrdU antibody.
In other embodiments, p olypeptides o f t he invention may be immobilized onto
a
solid surface, including, microtiter plates, slides, beads, fihns, etc. The
polypeptides of the
invention may be immobilized onto a "chip" as part of an array. An array,
having a
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plurality of addresses, may comprise one or more polypeptides of the invention
in one or
more of those addresses. In one embodiment, the chip comprises one or more
polypeptides
of the invention as part of an array of polypeptide sequences.
In other embodiments, polypeptides of the invention may be immobilized onto a
solid
surface, including, plates, microtiter plates, slides, beads, particles,
spheres, rilms, strands,
precipitates, gels, sheets, tubing, containers, capillaries, pads, slices,
etc. The polypeptides of
the invention may be immobilized onto a "chip" as part of an array. An array,
having a
plurality of addresses, may comprise one or more polypeptides of the invention
in one or more
of those addresses. In one embodiment, the chip comprises one or more
polypeptides of the
invention as part of an array.
5. Isd Vaccines
IsdA, IsdB, and IsdC polypeptides are cell surface proteins expressed by S.
aureus
and are essential for full virulence in vivo (shown using a mouse model of '
kidney
infection). Further, IsdA is immunodominant as anti-IsdA antibodies are
detected in
convalescent human sera. Thus, the IsdA, IsdB, and/or IsdC polypeptides may be
used as a
vaccine tlierapy to treat S. aureus infections.
IsdA, IsdB, and/or IsdC polypeptides or polynucleotides may be formulated into
a
vaccine and administered to a subject to induce an immune response (e.g.
cellular or
humoral) against IsdA, IsdB, and/or IsdC in that subject.
An exemplary IsdA protein for inclusion in a vaccine is the full length IsdA
polypeptide or an IsdA peptide. In certain embodiments, recombinant IsdA
protein will be
used in a vaccine. In alternate embodiments, IsdB or IsdC protein used as a
vaccine may be
full-length IsdB or IsdC, a peptide fragment of IsdB or IsdC, or recombinant
IsdB or IsdC
protein.
Isd peptides that are antigenic and used as a vaccine may be identified using
a
variety of methods. In one approach, peptides containing antigenic sequences
may be
selected on the basis of generally accepted criteria of potential antigenicity
and/or exposure.
Such criteria include the hydrophilicity and relative antigenic index, as
determined by
surface exposure analysis of p roteins. T he d etermination o f a ppropriate c
riteria i s w ell-
known to one of skill in the art, and has been described, for example, by Hopp
et al., Proc
Natl Acad Sci USA 1981; 78: 3824-8; Kyte et al., JMoI Biol 1982; 157: 105-32;
Emini, J
Virol 1985; 55: 836-9; Jameson et al., CA BIOS 1988; 4: 181-6; and Karplus et
al.,
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Naturwissenschaften 1985; 72: 212-3. Amino acid domains predicted by these
criteria to be
surface exposed may be selected preferentially over domains predicted to be
more
hydrophobic.
Portions of IsdA, IsdB and/or IsdC determined to be antigenic may be
chemically
synthesized by methods known in the art from individual amino acids. Suitable
methods
for synthesizing protein fragments are described by Stuart and Young in "Solid
Phase
Peptide Synthesis," Second Edition, Pierce Chemical Company (1984).
Alternatively, antigenic linear epitope(s) of IsdA, IsdB or IsdC may be
identified by
minotope analysis with a corresponding Isd antibody. Briefly, for mimotope
analysis a
polypeptide will be subdivided into overlapping fragments. For example,
overlapping 15
amino acid peptides will be synthesized to cover the entire length of the full-
length
polypeptide. Each 15 amino acid peptide may overlap by three amino acids.
Alternatively,
amino acid peptide fragments may be designed in tandem order to cover the
entire linear
amino acid sequence. Each peptide may then biotinylated and allowed to bind to
15 strepavidin-coated wells in 96-well plates. The reactivity of various
antisera may be
detected by enzyme-linked immunosorbent assay (ELISA). After blocking non-
specific
binding, an anti-Isd antibody may be added to each well, followed by* the
sequential
addition of peroxidase-conjugated secondary antibody, and peroxidase
substrate. Anti-Isd
antibodies may b e affinity purified a nti-full-length r ecombinant IsdA or a
ffinity p urified
anti-IsdA peptide. A lternatively, anti-Isd a ntibodies may be against IsdB or
IsdC. The
optical density of each well may be read at 450 nm and duplicate or triplicate
wells may be
averaged. The average value obtained from a similar ELISA using control serum
(i.e.,
preimmune serum) may be subtracted from the test immunoglobulin values and the
resultant values may be plotted to determine which linear epitopes are
recognized by the
immunoglobulin(s).
Further, competitive binding assays using synthetic peptides representing
linear
eptitopes may be used to determine antigenic fragments. In certain
embodiments,
antigenic fragments may inhibit uptake of labeled iron.
Also p rovided herein are D NA vaccines comprising n ucleotide s equences,
which
encode IsdA, IsdB, and/or IsdC peptides. Exemplary DNA vaccines encode two or
more
IsdA peptides. Alternate DNA vaccines may encode two or more IsdB or IsdC
peptides or
any combination of two or more IsdA, IsdB, or IsdC peptides. The efficacy of
candidate
vaccines (peptide or DNA) may be tested in appropriate animal models such as
rats, mice,
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WO 2006/059247 PCT/IB2005/004126
guinea pigs, monkeys and baboons. A protective or positive effect of the
vaccine should be
reflected by reduced fertility in the experimental animals.
Nucleic acids encoding IsdA, IsdB, or IsdC immunogens may be obtained by
polymerase chain reaction (PCR), amplification of gene segments from genomic
DNA,
cDNA, RNA (e.g. by RT-PCR), or cloned sequences. PCR primers are chosen, based
on
the known sequences of the genes or cDNA, so that they result in the
amplification of
relatively unique fragments. Computer programs may be used in the design of
primers with
required specificity and optimal amplification purposes. See e.g., Oligo
version 5.0
(National Biosciences). Factors which apply to the design and selection of
primers for
amplification are described for example, by Rylchik, W. (1993) "Selection of
Primers for
Polymerase Chain Reaction." In Methods in Molecular Biology, vol. 15, White B.
ed.,
Humana Press, Totowa, N.J. Sequences may be obtained from GenBank or other
public
sources. Alternatively, the nucleic acids of this invention may also be
synthesized by
standard m ethods known i n t he art, e.g. b y u se of an automated D NA
synthesizer (such
synthesizers are commercially available from Biosearch, Applied Biosystems,
etc).
Suitable cloning vectors for expressing Isd polypeptides in a host or in a
cell may be
constructed according to standard techniques as described above.
Isd immunogens may alternatively be prepared from enzymatic cleavage of intact
Isd polypeptides. Examples of proteolytic enzymes include, but are not limited
to, trypsin,
chymotrypsin, pepsin, papain, V8 protease, subtilisin, plasmin, and thrombin.
Intact
polypeptides can be incubated with one or more proteinases simultaneously or
sequentially.
Alternatively, or in addition, intact Isd polypeptides can be treated with
disulfide reducing
agents. Peptides may then be separated from each other by techniques known in
the art,
including but not limited to, gel filtration chromatography, gel
electrophoresis, and reverse-
phase HPLC.
6. Isd Antibodies atzd Uses tlzereof
To produce antibodies against IsdA, IsdB, and/or IsdC, host animals may be
injected with full-length Isd polypeptides or with Isd peptides. Hosts may be
injected with
peptides of different lengths encompassing a desired target sequence. For
example, peptide
antigens that are at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90,
95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 o r 150 amino acids m ay
b e u sed.
Alternatively, if a portion of an Isd protein defines an epitope, but is too
short to be
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antigenic, it may be conjugated to a carrier molecule in order to produce
antibodies. Some
suitable carrier molecules include keyhole limpet hemocyanin, Ig sequences,
TrpE, and
human or bovine serum albumen. Conjugation may be carried out by methods known
in
the art. One such method is to combine a cysteine residue of the fragments
with a cysteine
residue on the carrier molecule.
In addition, antibodies to three-dimensional epitopes, i.e., non-linear
epitopes, may
also be prepared, based on, e.g., crystallographic data of proteins.
Antibodies obtained from
that injection may be screened against the short antigens of IsdA, IsdB or
IsdC. Antibodies
prepared against an Isd peptide may be tested for activity against that
peptide as well as the
full length Isd protein. Antibodies may have affinities of at-:.~-ast about 10-
6M, 10"7M, 10"
8M, 10-9M, 10-10M, 10-"M or 10"12M toward the Isd peptide and/or the full
length Isd
protein.
Suitable cells f or t he DNA sequences and host cells for antibody expression
and
secretion can be obtained from a number of sources, including the American
TypeCulture
Collection ("Catalogue of Cell Lines and Hybridomas" 5th edition (1985)
Rockville, Md.,
U.S.A.).
Polyclonal and monoclonal antibodies may be produced by methods known in the
art. Monoclonal antibodies may be produced by hybridomas prepared using known
procedures including the immunological method described by Kohler and
Milstein, Nature
1975; 256: 495-7; and Campbell in "Monoclonal Antibody Technology, The
Production
and Characterization of Rodent and Human Hybridomas" in Burdon et al., Eds.
Laboratory
Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science
Publishers, Amsterdam (1985); as well as by the recombinant DNA method
described by
Huse et al, Science (1989) 246: 1275-8 1.
Methods of antibody purification are well known in the art. See, for example,
Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, N.Y. Purification methods may include salt precipitation (for
example, with
ammonium sulfate), ion exchange chromatography (for example, on a cationic or
anionic
exchange column run at neutral pH and eluted with step gradients of increasing
ionic
strength), gel filtration chromatography (including gel filtration HPLC), and
chromatography on affinity resins such as protein A, protein G,
hydroxyapatite, and anti-
antibody. Antibodies may also be purified on affinity columns according to
methods
known in the art.
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Other embodiments include functional equivalents o f antibodies, and include,
for
example, chimerized, humanized, and single chain antibodies as well as
fragments thereof.
Methods of producing functional equivalents are disclosed in PCT Application
WO
93/21319; European Patent Application No. 239,400; PCT Application WO
89/09622;
European Patent Application 388,745; and European Patent Application EP
332,424.
Functional equivalents include polypeptides with amino acid sequences
substantially the same as the amino acid sequence of the variable or
hypervariable regions
of the antibodies of the invention. "Substantially the same" amino acid
sequence is defined
herein as a sequence with at least 70%, preferably at least about 80%, and
more preferably
at least 90% homology to another amino acid sequence as determined by the
FASTA search
method in accordance with Pearson and Lipman, (1988) Proc Natl Acd Sci USA 85:
2444-
8.
Chimerized antibodies may have , constant regions derived substantially or
exclusively from human antibody constant regions and variable regions derived
substantially or exclusively from the sequence of the variable region from a
mammal other
than a human. Humanized antibodies may have constant regions and variable
regions
other than the complement determining regions (CDRs) derived substantially or
exclusively
from the corresponding human antibody regions and CDRs derived substantially
or
exclusively from a mammal other than a human.
Suitable mammals other than a human may include any mammal from which
monoclonal antibodies may be made. Suitable examples of mammals other than a
human
may include, for example, a rabbit, rat, mouse, horse, goat, or primate.
Antibodies to IsdA, IsdB or IsdC may be prepared as described above use as an
anti-
infective. In other embodiments, antibodies that recognize functional Isd
fragments may
also be used in random peptide phage display technology (Eidne et al., Biol
Reprod.
63(5):1396-402 (2000)). Briefly, fifteen or twelve-mer random peptide phage
display
libraries can be used to determine peptides that might interact with
functional Isd peptides
by competitive displacement of Fab fragments of Isd antibodies. For this,
fixed S. aureus
cells are allowed to adhere to wells in multiwell plates, and immunostaining
for IsdA, IsdB
or IsdC may then be evaluated in the absence and presence of unique and random
peptides
expressed by the phage library. Once the competitive peptides are identified
by amino acid
sequence analysis, increased amounts of peptide can be synthesized and used as
alternative
molecular antagonists to antibodies directed against functional fragments.
Another
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alternative is to screen small molecule libraries for their ability to
competitively displace
Fab fragments to functional IsdA, IsdB, or IsdC fragments. Molecular
antagonists
identified in this manner may be used to neutralize the effect of antibodies
generated by an
immune response to the Isd polypeptide or polynucleotide vaccine.
In a further embodiment, the antibodies to IsdA, IsdB, or IsdC (whole
antibodies or
antibody fragments) may be conjugated to a biocompatible material, such as
polyethylene
glycol molecules (PEG) according to methods well known to persons of skill in
the art to
increase the antibody's half-life. See for example, U.S. Patent No. 6,468,532.
Functionalized PEG polymers are available, for example, from Nektar
Therapeutics.
Commercially available PEG derivatives include, but are not limited to, amino-
PEG, PEG
amino acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated
PEG,
PEG-propionic acid, PEG amino acids, PEG succinimidyl succinate, PEG
succinimidyl
propionate, succinimidyl ester of carboxymethylated PEG, succinimidyl
carbonate of PEG,
succinimidyl esters of amino acid PEGs, PEG-oxycarbonylimidazole, PEG-
nitrophenyl
carbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEG vinylsulfone,
PEG-
maleimide, PEG-orthopyridyl-disulfide, heterofunctional PEGs, PEG vinyl
derivatives,
PEG silanes, and PEG phospholides. The reaction conditions for coupling these
PEG
derivatives will vary depending on the polypeptide, the desired degree of
PEGylation, and
the PEG derivative utilized. Some factors involved in the choice of PEG
derivatives
include: the desired point of attachment (such as lysine or cysteine R-
groups), hydrolytic
stability and reactivity of the derivatives, stability, toxicity and
antigenicity of the linkage,
suitability for analysis, etc.
7. Plzarmaceutical Conapositions
Purified IsdA, IsdB, or IsdC polypeptides and nucleic acids may be formulated
and
introduced as a vaccine through oral, intradennal, intramuscular,
intraperitoneal,
intravenous, subcutaneous, intranasal; intravaginal, and via scarification
(i.e., scratching
through the top layers of skin, e.g., using a bifurcated needle) or any other
standard route of
immunization. Isd polypeptides may further be orally delivered as a vaccine by
enteric-
coated capsules, which will dissolve in the gut and taken up by antigen
presenting cells in
Peyer's patches. Oral delivery of Isd polypeptides may supplement injections
of Isd
polypeptides.
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Further, S. a ureus a nti-Isd antibodies, i sd a ntisense n ucleic a cids and
s iRNAs, as
described herein may be administered by various means, depending on their
intended use,
as is well known in the art. For example, if such S. aureus antagonist
compositions are to
be administered orally, they may be fonnulated as tablets, capsules, granules,
powders or
syrups. Alternatively, formulations of the present invention may be
administered
parenterally as injections (intravenous, intramuscular or subcutaneous), drop
infusion
preparations or suppositories. For application by the ophthalmic mucous
membrane route,
compositions o f the present invention may be formulated as eyedrops or eye o
intments.
These formulations may be prepared by conventional means, and, if desired, the
compositions may be mixed with any conventional additive, such as an
excipient, a binder,
a d isintegrating a gent, a lubricant, a corrigent, a solubilizing agent, a
suspension aid, an
emulsifying agent or a coating agent.
In formulations of the subject invention, wetting agents, emulsifiers and
lubricants,
such as sodium lauryl sulfate and magnesium stearate, as well as coloring
agents, release
agents, coating agents, sweetening, flavoring and perfuming agents,
preservatives and
antioxidants may be present in the formulated agents.
Subject compositions may be suitable for oral, nasal, topical (including
buccal and
sublingual), rectal, vaginal, aerosol and/or parenteral administration. The
formulations may
conveniently be presented in unit dosage form and may be prepared by any
methods well
known in the art of pharmacy. The amount of composition that may be combined
with a
carrier material to produce a single dose vary depending upon the subject
being treated, and
the particular mode of administration.
Methods of preparing these formulations include the step of bringing into
association compositions of the present invention with the carrier and,
optionally, one or
more accessory i ngredients. I n g eneral, the formulations are p repared by u
niformly a nd
intimately bringing into association agents with liquid carriers, or finely
divided solid
carriers, or both, and then, if necessary, shaping the product.
Formulations suitable for oral administration may be in the form of capsules,
cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and
acacia or
tragacanth), powders, granules, or as a solution or a suspension in an aqueous
or non-
aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as
an elixir or syrup,
or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose
and acacia), each
containing a predetermined amount of a subject composition thereof as an
active ingredient.
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Compositions of the present invention may also be administered as a bolus,
electuary, or
paste.
In solid dosage forms for oral administration (capsules, tablets, pills,
dragees,
powders, granules and the like), the subject composition is mixed with one or
more
pharmaceutically acceptable carriers, such as sodium citrate or dicalcium
phosphate, and/or
any of the following: (1) fillers or extenders, such as starches, lactose,
sucrose, glucose,
mannitol, and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose,
alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)
humectants, such as
glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate,
potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate; (5) solution
retarding agents,
such as paraffin; (6) absorption accelerators, such as quatemary ammonium
compounds; (7)
wetting agents, such as, for example, acetyl alcohol and glycerol
monostearate; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc,
calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and
mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets and pills,
the
compositions may also comprise buffering agents. S olid c ompositions of a
similar type
may also be employed as fillers in soft and hard-filled gelatin capsules using
such
excipients as lactose or milk sugars, as well as high molecular weight
polyethylene glycols
and the like.
A tablet may be made by compression or molding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared using binder (for
example,
gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent,
preservative,
disintegrant (for example, sodium starch glycolate or cross-linked sodium
carboxymethyl
cellulose), surface-active or dispersing agent. Molded tablets may be made by
molding in a
suitable machine a mixture of the subject composition moistened with an inert
liquid
diluent. Tablets, and other solid dosage forms, such as dragees, capsules,
pills and
granules, may optionally be scored or prepared with coatings and shells, such
as enteric
coatings and other coatings well known in the pharmaceutical-formulating art.
Liquid dosage forms for oral administration include pharmaceutically
acceptable
emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the
subject composition, the liquid dosage forms may contain inert diluents
commonly used in
the art, such as, for example, water or other solvents, solubilizing agents
and emulsifiers,
such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,
benzyl alcohol,
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benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed,
groundnut, corn, germ, olive, castor and sesame oils), glycerol,
tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Suspensions, in addition to the subject composition, may contain suspending
agents
as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan
esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-
agar and
tragacanth, and mixtures thereof.
Formulations for rectal or vaginal administration may be presented as a
suppository,
which may be prepared by mixing a subject composition with one or more
suitable non-
irritating excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol,
a suppository wax or a salicylate, and which is solid at room temperature, but
liquid at body
temperature and, therefore, will melt in the body cavity and release the
active agent.
Formulations, which are suitable for vaginal administration also include
pessaries, tampons,
creams, gels, pastes, foams or spray formulations containing such carriers as
are known in
the art to be appropriate.
Dosage forms for transdermal administration of a subject composition includes
powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches
and inhalants.
The active component may be mixed under sterile conditions with a
pharmaceutically
acceptable carrier, and with any preservatives, buffers, or propellants, which
may be
required.
The ointments, pastes, creams and gels may contain, in addition to a subject
composition, excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid,
talc and zinc oxide, or mixtures thereof.
Powders and sprays may contain, in addition to a subject composition,
excipients
such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and
polyamide
powder, or mixtures of these substances. Sprays may additionally contain
customary
propellants, such as chlorofluorohydrocarbons and volatile unsubstituted
hydrocarbons,
such as butane and propane.
Compositions of the present invention may alternatively be administered by
aerosol.
This is accomplished by preparing an aqueous aerosol, liposomal preparation or
solid
particles containing the compound. A non-aqueous (e.g., fluorocarbon
propellant)
suspension could be used. Sonic nebulizers may be used because they minimize
exposing
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the agent to shear, which may result in degradation of the compounds contained
in the
subject compositions.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or
suspension of a subject composition with conventional pharmaceutically
acceptable carriers
and stabilizers. The carriers and stabilizers vary with the requirements of
the p articular
subject composition, but typically include non-ionic surfactants (Tweens,
Pluronics, or
polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters,
oleic acid,
lecithin, amino acids such as glycine, buffers, salts, sugars or sugar
alcohols. Aerosols
generally are prepared from isotonic solutions.
In addition, Isd based vaccines may be administered parenterally as injections
(intravenous, intramuscular or subcutaneous). The vaccine compositions of the
present
invention may optionally contain one or more adjuvants. Any suitable a djuvant
can be
.. used, s uch a s a luminum hydroxide, aluminum phosphate, plant and animal
oils, and the
like, with the amount of adjuvant depending on the nature of the particular
adjuvant
employed. In addition, the anti-infective vaccine compositions may also
contain at least
one stabilizer, such as carbohydrates such as sorbitol, mannitol, starch,
sucrose, dextrin, and
glucose, as well a s p roteins s uch a s albumin or c asein, a nd b uffers
such a s alkali metal
phosphates and the like. Preferred adjuvants include the SynerVaxTM adjuvant.
Pharmaceutical compositions of this invention suitable for parenteral
administration
comprise a subject composition in combination with one or more
pharmaceutically-
acceptable s terile isotonic a queous or n on-aqueous s olutions, d
ispersions, s uspensions o r
emulsions, or sterile powders which may be reconstituted into sterile
injectable solutions or
dispersions just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes
which render the formulation isotonic with the blood of the intended recipient
or
suspending or thickening agents.
Examples of suitable aqueous and non-aqueous carriers, which may be employed
in
the pharmaceutical compositions of the invention, include water, ethanol,
polyols (such as
glycerol, propylene glycol, polyethylene glycol, and the like), and suitable
mixtures thereof,
vegetable oils, such as olive oil, and injectable organic esters, such as
ethyl oleate. Proper
fluidity may be maintained, for example, by the use of coating materials, such
as lecithin,
by the maintenance of the required particle size in the case of dispersions,
and by the use of
surfactants.
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Further, Isd inununogens or Isd antibodies of the present invention may be
encapsulated in liposomes and administered via injection. Commercially
available
liposome delivery systems are available from Novavax, Inc. of Rockville, Md.,
commercially available under the name NovasomesTM. These 1 iposomes are
specifically
formulated for immunogen or antibody delivery. In an embodiment of the
invention,
NovasomesTM containing Isd peptides or antibody molecules bound to the surface
of these
non-phospholipid positively charged liposomes may be used.
The pharmaceutical compositions described herein may be used to prevent or
treat
conditions or dieseases resulting from S. aureus infections including, but not
limited to a
furuncle, chronic furunculosis, impetigo, acute osteomyelitis, pneumonia,
endocarditis,
scalded skin syndrome, toxic shock syndrome, and food poisoning.
8. Exemplar,y screeiaing assays for ifahibitors of Isd
In general, agents or compounds capable of reducing pathogenic virulence by
interfering with iron-regulated surface determinants (Isd) can be identified
using the instant
disclosed assays to screen large libraries of both natural product or
synthetic (or semi-
synthetic) extracts or chemical libraries. Those skilled in the field of drug
discovery and
development will understand that the precise source of agents (e.g., test
extracts or
compounds) is not critical to the screening procedures of the invention.
Accordingly,
virtually any number of chemical extracts or compounds can be screened using
the methods
described herein. Examples of such agents, extracts, or compounds include, but
are not
limited to, plant-, fungal-, prokaryotic- or animal-based extracts,
fermentation broths, and
synthetic compounds, as well as modification of existing compounds. Numerous
methods
are also available for generating random or directed synthesis (e.g., semi-
synthesis or total
synthesis) of any number of chemical compounds, including, but not limited to,
saccharide-,
lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound
libraries are
commercially available from Brandon Associates (Merrimack, NH) and Aldrich
Chemical
(Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form
of bacterial,
fungal, plant, and animal extracts are commercially available from a number of
sources,
including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch
Oceangraphics
Institute (Ft. Pierce, Fla.), and PharmnaMar, U.S.A. (Cambridge, MA). In
addition, natural
and synthetically produced libraries are produced, if desired, according to
methods known
in the art, for example, by standard extraction and fractionation methods.
Furthermore, if
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desired, any library or compound is readily modified using standard chemical,
physical, or
biochemical methods.
In addition, those skilled in the art of drug discovery and development
readily
understand that methods for dereplication (e.g., taxonomic dereplication,
biological
dereplication, and chemical dereplication, or any combination thereof) or the
elimination of
replicates or repeats of materials already known for their anti-pathogenic
activity should be
employed whenever possible.
When a crude extract is found to have an anti-pathogenic or anti-virulence
activity,
or a binding activity, further fractionation of the positive lead extract is
necessary to isolate
chemical constituents responsible for the observed effect. Thus, the goal of
the extraction,
fractionation, and purification process is the careful characterization and
identification of a
chemical entity within the crude extract having anti-pathogenic activity.
Methods of
fractionation and purification of such heterogeneous extracts are known in the
art. If
desired, compounds shown to be useful agents for the treatment of
pathogenicity are
chemically modified according to methods known in the art.
Potential inhibitors or antagonists of Isd encoded polypeptides may include
organic
molecules, peptides, peptide mimetics, polypeptides, and antibodies that bind
to a nucleic
acid sequence or polypeptide of the invention and thereby inhibit or
extinguish its activity.
Potential antagonists also include small molecules that bind to and occupy the
binding site
of the polypeptide thereby preventing binding to cellular binding molecules,
such that
normal biological activity is prevented. Other potential antagonists include
antisense
molecules.
Further, S. aureus anti-Isd antagonists as identified by the screening assays
described herein may be administered by various means, depending on their
intended use,
as described above.
8.1 Interaction Assays
Purified and recombinant IsdA, IsdB, and IsdC polypeptides may be used to
develop
assays to screen for agents that bind to an Isd gene product, and disrupt a
protein-protein
interaction. Potential inhibitors or antagonists of IsdA, IsdB, or IsdC may
include small
organic molecules, peptides, polypeptides, peptide mimetics, and antibodies
that bind to
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either IsdA, IsdB, or IsdC and therelby reduce or extinguish its activity.
In certain embodiments, an agent may be identified that binds to an Isd
polypeptide
and inhibits the uptake of iron comprising the steps of (i) contacting the Isd
polypeptide
with an appropriate interacting molecule in the presence of an agent under
conditions
permitting the interaction between the Isd polypeptide and the interacting
molecule in the
absence of an agent, and (ii) determining the level of interaction between the
Isd
polypeptide and the interacting molecule, wherein a different level of
interaction between
the Isd polypeptide and the interacting molecule in the presence of the agent
relative to the
absence of the agent indicate that the agent inhibits the interaction between
the Isd
polypeptide and the interacting molecule.
In another embodiment, an agent may be identified that disrupts the
interaction
between an Isd polypeptide and an interacting molecule. In an exemplary
binding assay, a
reaction mixture may be generated to include at least a biologically active
portion of either
IsdA, IsdB, or IsdC, an agent(s) of interest, and an appropriate interacting
molecule. An
exemplary interacting molecule may be a hemoprotein, hemin, transferrin,
fibrinogen or
fibronectin. In an exemplary embodiment, the agent of interest is an antibody
against a
particular Isd polypeptide. Binding of an antibody to an Isd polypeptide may
inhibit the
function of the Isd polypeptide in binding heme or a hemoprotein. Detection
and
quantification of an interaction of a particular Isd polypeptide with an
appropriate
interacting molecule provides a means for determining an agent's efficacy at
inhibiting the
interaction. The efficacy of the agent can be assessed by generating dose
response curves
from data obtained using various concentrations of the test agent. Moreover, a
control
assay can also be performed to provide a baseline for comparison. In the
control assay, the
interaction of a particular Isd polypeptide with an appropriate interacting
molecule may be
quantitated in the absence of the test agent.
Interaction between a particular Isd polypeptide and an appropriate
interacting
molecule may be detected by a variety of techniques. Modulation of the
formation of
complexes can be quantitated using, for example, detectably labeled proteins
such as
radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides, by
immunoassay, or by chromatographic detection.
The measurement of the interaction of a particular Isd protein with the
appropriate
interacting molecule may be observed directly using surface plasmon resonance
technology
in optical biosensor devices. This method is particularly useful for measuring
interactions
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with larger (>5 kDa) polypeptides and can be adapted to screen for inhibitors
of the protein-
protein interaction.
Alternatively, it will be desirable to immobilize a particular Isd polypeptide
or the
appropriate interacting molecule to facilitate separation of complexes from
uncomplexed
forms of one or both of the proteins, as well as to accommodate automation of
the assay.
Binding of a particular Isd protein to the interacting molecule for example,
in the presence
and absence of a candidate agent, can be accomplished in any vessel suitable
for containing
the reactants. Examples include microtitre plates, test tubes, and micro-
centrifuge tubes. In
one embodiment, a fusion protein can be provided which adds a domain that
allows the
protein to be bound to a matrix. For example, glutathione-S-transferase/IsdA
(GST/IsdA)
fusion proteins can be adsorbed, onto glutathione sepharose beads (Sigma
Chemical, St.
Louis, Mo.) or glutathione derivatized microtitre plates, which are then
combined with, for
example, an 35S-labeled interacting molecule,.and the testagent, and the
mixture incubated
under conditions conducive to complex formation, for example, at physiological
conditions
for salt and pH, though slightly more stringent conditions may be desired.
Following
incubation, the beads are washed to remove any unbound label, and the matrix
immobilized
and radiolabel determined directly (e.g., beads placed in scintillant), or in
the supematant
after the complexes are subsequently dissociated. Alternatively, the complexes
can be
dissociated from the matrix, separated by SDS-PAGE, and the level of
interacting molecule
found in the bead fraction quantitated from the gel using standard
electrophoretic
techniques.
Other techniques for immobilizing proteins and other molecules on matrices are
also
available for use in the subject assay. For instance, either a particular Isd
protein or the
appropriate interacting molecule can be immobilized utilizing conjugation of
biotin and
streptavidin. For instance, biotinylated IsdA, IsdB, or IsdC can be prepared
from biotin-
NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g.,
biotinylation
kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated
96 well plates (Pierce Chemical). Alternatively, antibodies reactive with
either IsdA, IsdB,
or I sdC but w hich d o n ot i nterfere w ith the i nteraction b etween t he
polypeptide and t he
interacting molecule, can be derivatized to the wells of the plate, and IsdA,
IsdB, or IsdC
may be trapped in the wells by antibody conjugation. As above, preparations of
an
interacting molecule and a test compound may be incubated in the polypeptide-
presenting
wells of the plate, and the amount of complex trapped in the well can be
quantitated in the
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presence or absence of a test agent. Exemplary methods for detecting such
complexes, in
addition to those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the interacting
molecule or
enzyme-linked assays, which rely on detecting an enzymatic activity associated
with the
interacting molecule.
For example, an enzyme can be chemically conjugated or provided as a fusion
protein with the interacting molecule. To illustrate, the interacting molecule
can be
chemically cross-linked or genetically fused with horseradish peroxidase, and
the amount of
polypeptide trapped in the complex can be assessed with a chromogenic
substrate of the
enzyme, for example, 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l-
napthol.
Likewise, a fusion protein comprising the polypeptide and glutathione-S-
transferase can be
provided, and complex formation quantitated by detecting the GST activity
using 1-chioro-
2,4-dinitrobenzene (Habig et al. (1974) J. Biol. .Chem. 249:7130).
8.2 Expression assays
In a further embodiment, antagonists of iron uptake may affect the expression
of
isdA, isdB, and isdC nucleic acid or protein. In this screen, S. aureus cells
may be treated
with a compound(s) of interest, and then assayed for the effect of the
compound(s).on isdA,
isdB, and isdC nucleic acid or protein expression.
In certain embodiments, an agent may be identified that inhibits the
expression of an
Isd polypeptide in Staphylococcus aureus comprising the step of (i) culturing
a wild type
Staplaylococcus aureus strain in the presence or absence of said agent;
and(ii) comparing
the expression of Isd polypeptides wherein a greater reduction. in the
expression of Isd
polypeptides in cells treated with said agent indicates that said agent
inhibits the expression
of Isd polypeptides in Staphylococcus aureus.
In an alternate embodiment, an agent may be identified that inhibits the
expression
of an isd nucleic acid in Staphylococcus aureus comprising the step of (i)
culturing a wild
type Staplaylococcus aureus strain in the presence or absence of said agent;
and (ii)
comparing the expression of isd nucleic acids wherein a greater reduction in
the expression
of i sd n ucleic acids i n cells treated w ith said a gent i ndicates that
said a gent i nhibits t he
expression of isd nucleic acids in Staphylococcus aureus.
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For example, total RNA can be isolated from S. aureus cells cultured in the
presence
or absence of test agents, using any suitable technique such as the single-
step guanidinium-
thiocyanate-phenol-chloroform method described in Chomczynski et al. (1987)
Araal.
Biochem. 162:156-159. The expression of isdA, isdB, or isdC may then be
assayed by any
appropriate method such as Northern blot analysis, 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).
Northern blot analysis can be performed as described in Harada et al. (1990)
Cell
63:303-312. Briefly, total RNA is prepared from S. aureus cells cultured in
the presence of
a test agent. 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 S. aureus isdA, isdB, or isdC DNA sequence may be labeled
according
to any appropriate method (such as the 32P-multiprimed DNA labeling system
(Amersham))
and used as probe. After hybridization overnight, the filter is washed and
exposed to x-ray
film. Moreover, a control can also be performed to provide a baseline for
comparison. In
the control, the expression of isdA, isdB, or isdC in S. aureus may be
quantitated in the
absence of the test agent.
Alternatively, the levels of mRNA encoding IsdA, IsdB, and IsdC polypeptides
may
also be assayed, for e.g., using the RT-PCR method described in Makino et al.
(1990)
Technique 2:295-301. Briefly, this method involves adding total RNA isolated
from S.
aureus cells cultured in the presence of a test agent, 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 P CR 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 may
be quantified using an imaging analyzer. RT and PCR reaction ingredients and
conditions,
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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 (Dieffenbach et al. eds., Cold Spring Harbor Lab Press, 1995). A
control can also
be performed to provide a baseline for comparison. In the control, the
expression of isdA,
isdB, or isdC in S. aureus may be quantitated in the absence of the test
agent.
Alternatively, the expression of IsdA, IsdB, and IsdC polypeptides may be
quantitated following the treatment of S. aureus cells with a test agent using
antibody-based
methods such as immunoassays. Any suitable immunoassay can be used, including,
without limitation, competitive and non-competitive assay systems using
techniques such as
western blots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel
diffusion
precipitin reactions, immunodiffusion assays, agglutination assays, complement-
fixation
assays, immunoradiometric assays, fluorescent immunoassays and protein A
immunoassays.
For example, IsdA, IsdB, or IsdC polypeptides can be detected in a sample
obtained
from S. aureus cells treated with a test agent, by means of a two-step
sandwich assay. In
the first step, a capture reagent (e.g., either a IsdA, IsdB, or IsdC
antibody) is used to
capture the specific polypeptide. The capture reagent can optionally be
immobilized on a
solid phase. In the second step, a directly or indirectly labeled detection
reagent is used to
detect the captured marker. In one embodiment, the detection reagent is an
antibody. The
amount of IsdA, IsdB, or IsdC, polypeptide present in S. aureus cells treated
with a test
agent can be calculated by reference to the amount present in untreated S.
aureus cells.
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 (12sI,
121I), carbon (14C),
sulphur (35S), tritium (3H).
Examples of suitable fluorescent labels include 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.
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Examples of suitable enzyme labels include malate dehydrogenase,
staphylococcal
nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-
glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase,
asparaginase,
glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-
phosphate
dehydrogenase, glucoamylase, and acetylcholine esterase. Examples of
chemiluminescent
labels include a luminol label, an isoluminol 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.
Exemplificatiofa
The invention, having been generally described, may be more readily understood
by
reference to the following examples, which are included merely for purposes of
illustration
of certain aspects and embodiments of the present invention, and are not
intended to limit
the invention in any way.
Example 1: Expression of IsdA, IsdB and IsdC proteins
IsdA, IsdB, and IsdC proteins are expressed under iron-limiting conditions as
shown
in Figure 4 (S. aureus - Fe). The SDS-PAGE gel shown in Figure 4 illustrates
that the
IsdA, I sdB, a nd IsdC p roteins are three of t he most p redominant iron
regulated proteins
expressed by S. aureus. These proteins are not expressed when the S. a ureus
cells are
cultured in iron-rich media (S. aureus + Fe) and are, therefore, by inference
likely all highly
expressed ira vivo.
Overexpression of IsdA, IsdB, and IsdC, as well as IsdE, as fusions in E. coli
results
in highly colored lysates. Absorptions and magnetic circular dichroism
spectroscopy was
used to conrirm that this coloration was due to the ability of the proteins to
scavenge
different forms of protoporphyrin and heme from within the E. coli cytoplasm,
confirming
their role in heme binding.
Example 2: Generation of isd gene knockout mutants
Further, the coding regions of isdA, isdB and isdC were interrupted
individually to
generate strains that contain a single mutation in each of the isd genes. The
isdA coding
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region was interrupted by inserting a cassette encoding resistance to
tetracycline. The isdB
coding region was interrupted by inserting a cassette encoding resistance to
erythromycin.
The isdC coding region was interrupted by inserting a cassette encoding
resistance to
kanamycin. Each mutation was then moved into the same genetic background using
phage
transduction procedures and selected for using the appropriate r esistance as
described in
Sebulsky et al., (2001) J. Bacteriol. 183:4994-5000. Further, strains
containing mutations
knocking out two or more of the isd genes (e.g., a strain mutated in isdA,
isdB, and isdC
may also be generated.
Example 3: Survival studies in the mouse model of kidney infection
Female Swiss-Webster mice, weighing 2 5 g, were purchased from Charles River
Laboratories Canada, Inc., and housed in microisolator cages. Bacteria were
grown
overnight in Tryptic Soy Broth (TSB), harvested and washed three times in
sterile saline.
Pilot experiments demonstrated that S. aureus Newman colonized mice better in
this model
than did RN6390, and that the optimal amount of S. aureus Newman to inject
into the tail
vein to obtain an acute, but non-lethal kidney infection was 1 x 107 CFU.
Bacteria,
suspended in sterile saline, were administered intravenously via the tail
vein. The number
of viable bacteria injected were conftrmed by plating serial dilutions of the
inoculum on
TSB. On day six post-injection, mice were sacrificed and kidneys were
aseptically
removed. Using a PowerGen 700 Homogenizer, kidneys were homogenized for 45
seconds
in sterile PBS containing 0.1% Triton X-100 and homogenate dilutions were
plated on
TSB-agar to enumerate viable bacteria. Data presented are the log CFU
recovered per
mouse.
Results indicate that mutations in either IsdA alone, or in a strain carrying
mutations
in all of IsdA, IsdB, and IsdC attenuate S. aureus virulence using a murine
kidney abscess
model of S. aureus infection. Interestingly, after 6 days post-infection,
recovered mutant
bacteria are 90% decreased from the numbers recovered from the wildtype, thus
indicating
that these proteins, when expressed on the bacterial cell surface, play a
essential role in the
fitness o f t he bacteria during infection. This also indicates then that
inhibition of these
proteins in vivo could either prevent infection by Isd-expressing bacteria
(i.e., in the case of
an Isd-based vaccine) or could result in clearance of the Isd-expressing
bacteria once
infection was initiated.
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Example 4: Survival of S. aureus under increasing hydrogen peroxide
concentration
Isd proteins bound to heme appear to act as an oxidative buffer that protects
S.
aureus cells from the detrimental effects of free radicals. A direct
comparison of Newman
strains incubated in the presence of heme to Newman strains deleted for IsdA,
IsdB, and
IsdC incubated in the presence of heme shows that mutant cells were not able
to survive
increased concentrations of hydrogen peroxide (Figure 6). Thus, mutants
lacking the
expression of several Isd proteins are more susceptible to challenge with
hydrogen
peroxide.
Figure 7 shows the expression of IsdA plus and minus heme in both wild type S.
aureus and S.aureus isdA::kmc run on an SDS-PAGE gel stained with (A)
Coomassie and
(B) TMBZ (tetramethylbenzidine). Catalase activity associated with the heme-
bound form
of IsdA cleaves the TMBZ compound to yield a colored reaction product. Thus,
heme-
bound IsdA has catalase activity that may help resist the oxidative killing by
phagocytes.
Example 5: Isd Vaccines
A vaccine comprising recombinant IsdA polypeptide can establish protective
immunity in mice against systemic and localized S. aureus infection.
Recombinant IsdA
protein may be prepared using standard techniques. Groups of 12-15 Swiss-
Webster mice
(25 g) can be u sed f or all inununization experiments and injected i
ntraperitoneally (IP).
Mice can be boosted with subsequent injections at various different time
points. Sera can
be monitored over the course of the experiment for anti-IsdA antibody titres.
On
approximately day 30, mice can be challenged intravenously with 1 x 107 S.
aureus and
monitored for a further 7 days. We have previously shown that injection with
this number
of live organisms results in non-fatal kidney infections. Mice can be
sacrificed at various
time points post infection to monitor the number of organisms infecting the
kidney tissue.
Passive immunization experiments can also be performed using sera collected
from
previously immunized mice to examine their effectiveness at preventing
infection in other
groups of mice. Similar immunization experiments can be conducted with IsdB
and IsdC
polypeptides.
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Example 6: IsdA, IsdB, and IsdC Antibodies
A. Preparation of Monoclonal antibodies against full-length Isd proteins
BALB/c mice can be immunized initially via intraperitoneal injections with
full-
length recombinant IsdA, IsdB, or IsdC and later boosted similarly with native
IsdA, IsdB,
or IsdC approximately six weeks later. The mice can be immunized with an
appropriate
adjuvant. Mouse serum can be obtained approximately ten days after the second
injection
and t hen tested for a nti-HRP a ctivity v ia ELISA. T he mice whose serum
exhibits h igh
levels of anti-HRP activity can be chosen for cell fusion. Spleens can be
collected from
these mice and cell suspensions prepared by perfusion with Dulbecco's Modified
Eagle
Medium (DMEM).
Spleen cell suspension containing B-lymphocytes and macrophages can be
prepared
by perfusion of the spleen. The cell suspension can be washed and collected by
centrifugation; myeloma cells can also be washed in this manner. Live cells
can be counted
and the cells can be placed into a 37 C water bath. One mL of 50% polyethylene
glycol
(PEG) can be added to DMEM. The Balb/c spleen cells can be fused with SP 2/0-
Ag 14
mouse myeloma cells by PEG and the resultant hybridomas can be grown in
hypoxanthine
(H), aminopterin (A) and thymidine (T) (HAT) selected tissue culture media
plus 20% fetal
calf serum. The surviving cells can be allowed to grow to confluence. The
spent culture
medium can be checked for antibody titer, specificity, and affinity. The cells
can be
incubated in the PEG for one to 1.5 minutes at 37 C, after which the PEG was
diluted by
the slow addition of DMEM media. The cells can be pelleted and 35 to 40 mL of
DMEM
containing 10% fetal bovine serum may be added. The cells can then be
dispensed into
tissue culture plates and incubated overnight in a 37 C, 5% C02, humidified
incubator.
The next day, DMEM-FCS containing hypoxanthine (H), aminopterin (A) and
thymidine (T) medium (HAT medium) can be added to each well. The concentration
of
HAT in the medium to be added can be twice the final concentration required,
i.e., Hfiõal =1
times 104M; Afna1=4 times 10'7M; and Tfiõa1=1.6 times 10-5M.
Subsequently, the plates can be incubated with HAT medium every three to four
days for two weeks. Fused c ells c an be then cultured in DMEM-FCS containing
HAT
medium. As fused cells become 1/2 to 3/4 confluent on the bottom of the wells,
supernatant tissue c ulture f luid can be t aken and tested for IsdA, I sdB,
or I sdC specific
antibodies by ELISA. Positive wells can be cloned by limiting dilution over
macrophage or
thymocyte feeder plates, and cultured in DMEM-FCS. Cloned wells can be tested
and
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CA 02581746 2007-03-23
WO 2006/059247 PCT/IB2005/004126
recloned three times before a statistically significant monoclonal antibody
can be obtained.
Spent culture media can be tested from the antibody-producing clones.
B. Preparation of Polyclonal antibodies against full-length Isd proteins
Unconjugated purified recombinant IsdA, IsdB and/of IsdC can be used as an
antigen to immunize two rabbits. Briefly, 1 mg of recombinant IsdA, IsdB, or
IsdC can be
resuspended in 1 ml of phosphate buffered saline and emulsified with an equal
volume of
Complete Freund's Adjuvant and approximately 1 ml (half of the total volume)
can be
injected into each rabbit intraperitoneally. A second and third immunization
can follow two
and three weeks later, using Incomplete Freund's Adjuvant. Sera may be tested
using
enzyme-linked immunosorbent assays (ELISA) to determine recombinant IsdA, IsdB
or
IsdC s pecific a ntibody titers. A nti-recombinant I sdA, I sdB, a nd/or I sdC
c ontaining s era
that exhibits high titer based on ELISA results can be purified by affinity
chromatography
on a Sepharose column conjugated with corresponding recombinant Isd
polypeptide. Anti-
recombinant IsdA, IsdB, and IsdC immunoglobulin can be tested for the ability
to attenuate
the virulence of S. aureus infection.
Example 7: Expression Assays
Assays to screen for agents that disrupt the expression of IsdA in S. aureus
can be
conducted as follows. Wild type S. aureus cells can be cultured overnight in
tryptic soy
broth ( TSB) (Difco) in the p resence o r a bsence o f a test agent. Following
2 4 hours of
culture, the cells can be washed in 1X PBS (phosphate buffered saline) and
then lysed at
37 C using 10 g of lysostaphin in STE (0.1 M NaC1, 10 mM Tris-HCl [pH 8.0], 1
mM
EDTA [pH 8.0]). The cell lysates can then be transferred to anti-IsdA antibody
precoated
plates and incubated for 45 to 60 minutes at room temperature. As a control,
cell lysates
from untreated S. aureus cells can be used. After three washes with water, a
secondary
antibody conjugated to either alkaline phosphatase (AP) or horseradish
peroxidase (HRP)
can be added and incubated for one hour. The plate can then be washed to
separate the
bound from the free antibody complex. A chemiluminescent substrate (alkaline
phosphatase or Super Signal luminol solution from Pierce for horseradish
peroxidase) can
be u sed t o detect b ound a ntibody. A m icroplate 1 uminometer can b e u sed
t o d etect the
chemiluminescent s ignal. The absence of the signal i n s amples o f c ell
lysates obtained
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WO 2006/059247 PCT/IB2005/004126
from cells treated with test agent may indicate that the test agent inhibits
the expression of
IsdA. Similar expression assays may also be conducted for IsdB and IsdC.
The practice of the present invention may employ, unless otherwise indicated,
conventional techniques of cell biology, cell culture, molecular biology,
transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the skill of
the art.
Such techniques are described in the literature. See, for example, Molecular
Cloning.= A
Laboratory Manual, 2d Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring
Harbor
Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985);
Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent
No: 4,683,195;
Nucleic Acid Hybridizatiozz (B. D. Hames & S. J. Higgins eds. 1984);
Trazzscription And
Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Anizzzal Cells
(R. I.
- Freshney, Alan R. Liss, Inc., 1987); hnmobilized Cells And Enzymes (IRL
Press, 1986); B.
Perbal, A Practical Guide To Molecular Clonizzg (1984); the treatise, Methods
In
Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Maznmalian
Cells (J.
H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods
In
Enzymology, Vols. 154 and 155 (Wu et al. eds.), Inzzzzunochemical Metlzods In
Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987);
Handbook
Of Experimental Imnzunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,
eds., 1986);
Antibodies: A Laboratory Manual, and Aninzal Cell Culture (R. I. Freshney, ed.
(1987)),
Manipulating tlze Mouse Enzbryo, (Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, N.Y., 1986).
Incorporation by Reference
All publications and patents mentioned herein are hereby incorporated by
reference
in their entirety as if each individual publication or patent was specifically
and individually
indicated to be incorporated by reference. In case of conflict, the present
application,
including any derinitions herein, will control.
Equivaleuts
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following claims.
69
