U.S. patent application number 09/813820 was filed with the patent office on 2002-08-01 for collagen binding protein compositions and methods of use.
Invention is credited to Hook, Magnus, House-Pompeo, Karen, Patti, Joseph M..
Application Number | 20020102262 09/813820 |
Document ID | / |
Family ID | 21783969 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102262 |
Kind Code |
A1 |
Hook, Magnus ; et
al. |
August 1, 2002 |
Collagen binding protein compositions and methods of use
Abstract
Disclosed are the cna gene and cna-derived nucleic acid segments
from Staphylococcus aureus, and DNA segments encoding cna from
related bacteria. Also disclosed are Col binding protein (CBP)
compositions and methods of use. The CBP protein and antigenic
epitopes derived therefrom are contemplated for use in the
treatment of pathological infections, and in particular, for use in
the prevention of bacterial adhesion to Col. DNA segments encoding
these proteins and anti-(Col binding protein) antibodies will also
be of use in various screening, diagnostic and therapeutic
applications including active and passive immunization and methods
for the prevention of bacterial colonization in an animal such as a
human. These DNA segments and the peptides derived therefrom are
contemplated for use in the preparation of vaccines and, also, for
use as carrier proteins in vaccine formulations, and in the
formulation of compositions for use in the prevention of S. aureus
infection.
Inventors: |
Hook, Magnus; (Houston,
TX) ; Patti, Joseph M.; (Cumming, GA) ;
House-Pompeo, Karen; (Valdosta, GA) |
Correspondence
Address: |
LARSON & TAYLOR, PLC
1199 NORTH FAIRFAX STREET
SUITE 900
ALEXANDRIA
VA
22314
US
|
Family ID: |
21783969 |
Appl. No.: |
09/813820 |
Filed: |
March 22, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09813820 |
Mar 22, 2001 |
|
|
|
08856253 |
May 14, 1997 |
|
|
|
6288214 |
|
|
|
|
60017678 |
May 16, 1996 |
|
|
|
Current U.S.
Class: |
424/150.1 ;
530/388.4 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 39/00 20130101; C07K 16/1271 20130101; C07K 14/31 20130101;
A61P 31/04 20180101; C07K 2317/77 20130101 |
Class at
Publication: |
424/150.1 ;
530/388.4 |
International
Class: |
A61K 039/40; C07K
016/12 |
Goverment Interests
[0002] The United States Government has certain rights to the
present application pursuant to Grants HL473 13 and AI20624 from
the National Institutes of Health.
Claims
What is claimed is:
1. An isolated antibody which binds to a purified peptide
composition consisting essentially of the amino acid of SEQ ID
NO:4, wherein said antibody prevents S. aureus infection.
2. An antibody according to claim 1, wherein said antibody prevents
S. aureus infection in a human.
3. An antibody according to claim 1, wherein said antibody is
suitable for parenteral, oral, intranasal, subcutaneous, or
intravenous administration to an animal.
4. An antibody according to claim 1 wherein the antibody is a
monoclonal antibody.
5. An antibody according to claim 1 wherein the antibody is a
polyclonal antibody.
6. An isolated antisera containing an antibody according to claim
1.
7. An isolated antibody which binds to a purified peptide
composition encoded by a nucleic acid sequence consisting
essentially of the sequence of SEQ ID NO:3, wherein said antibody
prevents S. aureus infection.
8. An isolated antisera containing an antibody according to claim
7.
9. An isolated antibody which binds to a purified peptide
composition consisting essentially of the amino acid of SEQ ID
NO:4, wherein said antibody treats S. aureus infection when said
antibody is administered to an infected animal.
10. An antibody according to claim 9, wherein said antibody
prevents S. aureus infection in a human.
11. An antibody according to claim 9, wherein said antibody is
suitable for parenteral, oral, intranasal, subcutaneous, or
intravenous administration to an animal.
12. An antibody according to claim 9 wherein the antibody is a
monoclonal antibody.
13. An antibody according to claim 9 wherein the antibody is a
polyclonal antibody.
14. An isolated antisera containing an antibody according to claim
9.
15. An isolated antibody which binds to a purified peptide
composition encoded by a nucleic acid sequence consisting
essentially of the sequence of SEQ ID NO:3, wherein said antibody
treats S. aureus infection when said antibody is administered to an
infected animal.
16. An isolated antisera containing an antibody according to claim
15.
Description
[0001] The present application is a divisional application of U.S.
Ser. No. 08/856,253, filed May 14, 1997, which was based on U.S.
Provisional Application Ser. No. 60/017,678, filed May 16, 1996,
the entire content of which is incorporated herein by
reference.
1. BACKGROUND OF THE INVENTION
[0003] 1.1 Field Of The Invention
[0004] The present invention relates generally to the field of
molecular biology. More particularly, certain embodiments concern
methods and compositions comprising DNA segments, and proteins
derived from bacterial species. More particularly, the invention
provides can and can-derived nucleic acid compositions comprising a
collagen (Col) binding protein (CBP) from Staphylococcus aureus and
the corresponding peptide epitopes and protein sequences comprising
native and synthetically-modified Col binding site domains. Various
methods for making and using these DNA segments, DNA segments
encoding synthetically-modified ligand binding site domains, and
native and synthetic proteins are disclosed, such as, for example,
the use of DNA segments as diagnostic probes and templates for
protein production, and the use of proteins, fusion protein
carriers and peptides in various pharmacological and immunological
applications.
[0005] 1.2 Description Of The Related Art
[0006] 1.2.1 Colonization By Staphylococcus aureus
[0007] S. aureus cells can colonize many different host tissues and
cause various types of infections such as endocarditis, pneumonia,
wound infections, osteomyelitis, and septic arthritis. Adherence of
staphylococci to host tissues involves a family of adhesions that
recognize extracellular matrix components and which have been named
MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix
Molecules) (Patti et al., 1994a).
[0008] The expression of specific MSCRAMMs appears to be needed for
the colonization of different types of tissues. For example,
staphylococcal strains recovered from the joints of patients
diagnosed with septic arthritis or osteomyelitis almost invariably
express a CBP, whereas significantly fewer isolates obtained from
wound infections express this adhesin. (Switalski et al., 1993a)
Similarly, S. aureus strains isolated form the bones of patients
with osteomyelitis often have an MSCRAMM recognizing the
bone-specific protein, bone sialoprotein (BSP) (Ryden et al.,
1989).
[0009] The cloning, sequencing, and expression of a gene cna,
encoding a S. aureus CBP has been previously reported (Patti et
al., 1992). The cna gene encodes an 133-kDa adhesin that contains
structural features characteristic of surface proteins isolated
from Gram-positive bacteria. It has been demonstrated that the CBP
is required and sufficient for the adherence of S. aureus to
Col-coated artificial substrates as well as to cartilage, a tissue
rich in type II Col (Switalski et al., 1993a). All strains
expressing the CBP were able to adhere to cartilage, whereas those
strains lacking the MSCRAMMs did not adhere. Preincubation of S.
aureus with polyclonal antibodies raised against the purified
adhesin or saturation of the cartilage substrata with soluble
recombinant CBP resulted in a complete inhibition of bacterial
attachment (Switalski et al, 1993a).
[0010] S. aureus colonization of the articular cartilage within the
joint space appears to be an important factor contributing to the
development of septic arthritis. The importance of the CBP in the
pathogenesis of septic arthritis was examined by comparing the
virulence of two sets of S. aureus isogenic mutants in an animal
model (Patti et al, 1994b). Greater than 70% of mice injected with
CNA+strains (i.e. a clinical isolate expressing the CBP or a
negative strain into which the cna gene had been introduced)
developed clinical signs of arthritis, whereas less than 27% of the
animals showed symptoms of disease when injected with CNA strains
(i.e. a strain lacking the cna gene or a strain in which the cna
gene or a strain in which the cna gene had been inactivated through
homologous recombination). Taken together these results demonstrate
that the CBP plays an important role in the pathogenesis of septic
arthritis induced by S. aureus.
[0011] Recently, the ligand-binding site has been localized within
the N-terminal half of the CBP (Patti et al., 1993). By analyzing
the Col binding activity of recombinant proteins corresponding to
different segments of the MSCRAMM, a 168-amino-acid long protein
fragment (corresponding to amino acid residues 151-318) that had
appreciable Col binding activity was identified. Short truncations
of this protein in-the a N- or C terminus resulted in a loss of
ligand binding activity but also resulted in conformational changes
in the protein as indicated by circular dichroism spectroscopy.
These results raised the possibility that the ligand-binding
activity but also resulted in conformational changes in the protein
as indicated by circular dichroism spectroscopy. These results
raised the possibility the ligand-binding site of the MSCRAMM is
contained within a short segment of amino acids and that flanking
sequences are required for the proper folding of these residues in
the ligand-binding site.
[0012] 1.2.2 Role of S. aureus CBP in Human Disease
[0013] Hematogenously acquired bacterial arthritis remains a
serious medical problem. This rapidly progressive and highly
destructive joint disease is difficult to eradicate with less than
50% of the infected patients failing to recover without serious
joint damage. S. aureus is the predominant pathogen isolated from
adult patients with hematogenous (primary) and secondary
osteomyelitis (Waldvogel and Vasey 1980), while also causing up to
90% of the cases of acute hematogenous osteomyelitis in otherwise
healthy children (Cole 1982). Scanning electron microscopy studies
have shown S. aureus to be intimately associated with cartilage and
bone tissue retrieved from the site of infection. Additional
microscopic evidence suggests the predominate attachment and
subsequent colonization of cartilaginous rather than synovial
surfaces (Voytek et al. 1988). An analysis of S. aureus strains
isolated from patients diagnosed with osteomyelitis and septic
arthritis revealed that almost all of the isolates contained a Col
adhesin. In contrast, only one-third of the S. aureus strains
isolated from patients with soft tissue infections expressed the
Col adhesin (Switalski, Patti et al. 1993). These observations
suggest that cell surface expression of the Col adhesin is an
important virulence factor in staphylococcal mediated osteomyelitis
and septic arthritis Moreover, it has been observed that cartilage
degradation following staphylococcal joint infection can be
attributed to a direct interaction between bacteria and cartilage
(Smith et al. 1982) in addition to the inflanunatory response of
the host (Smith et al. 1987). Individuals who require more than 6
days for the synovial fluid to become free of microorganisms
typically result in poor clinical outcome (Ho and Su 1982). Poor
outcomes include permanent disability with limited motion or
persistent pain in the affected joint.
[0014] Therefore, by inhibiting the initial attachment of bacteria
to cartilage, the Likelihood of subsequent joint destruction may be
diminished.
[0015] 1.3 Deficiencies in the Prior Art
[0016] It is clear that while several approaches to the treatment
of bacterial diseases have experienced some success, many problems
remain, including antibiotic resistance, variability of antigens
between species and species variation through mutation of antigens,
as well as the need to protect susceptible groups such as young
children, the elderly and other immunocomprornised patients. Thus,
there exists an immediate need for an effective treatment for S.
aureus infection, and vaccines against this pathogen.
2. SUMMARY OF THE INVENTION
[0017] The present invention overcomes one or more of these and
other drawbacks inherent in the prior art by providing novel
compositions and methods for their use in the treatment of S.
aureus infection using non-antibiotic strategies. Disclosed are
methods for the use of novel peptide and antibody compositions in
the treatment of S. aureus infection mediated by the inhibition of
bacterial binding to the host cell ECM component, Col. Also
disclosed are methods for active and passive immunization against
S. aureus and related species using novel native and
site-specifically-altered CBP compositions and CBP-derived epitopic
peptides from bacterial species. Particular aspects of the
invention relate to novel nucleic acid segments encoding these
peptides and epitopes, and methods for the use of such nucleic acid
segments in a variety of diagnostic and therapeutic regimens. Also
obtained are peptide compositions derived from CBP which comprise
the Col binding domain. Using crystal structure analyses, the
inventors have developed site-specific mutations in CBP-encoding
DNA segments which give rise to altered Col binding domains.
[0018] In important embodiments methods and compositions are
obtained for inhibiting the binding of S. aureus to Col. These
compositions are useful in the prevention of bacterial adhesion to
extracellular matrix components such as Col, and in the inhibition
of bacterial colonization to collagenous substrata. In addition,
the invention encompasses the use of CBP or cna gene sequences to
produce antibodies which protect against staphylococcal
infections.
[0019] In another embodiment, the invention relates to a method of
preventing a S. aureus-mediated disease in an animal. The method
generally involves identifying an animal suspected of infection
with S. aureus and administering to the animal a collagen binding
protein or antibody composition effective to prevent the disease in
the animal.
[0020] In a further embodiment, the invention relates to a method
of increasing phagocytosis of an S. aureus cell by a macrophage
cell. The method generally involves providing to the macrophage
cell a pharmaceutically-acceptable collagen binding protein or
antibody composition in an amount effective to increase the
phagocytosis of the bacteria by the macrophage.
[0021] Preferably, the collagen binding protein composition
comprise and amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or
SEQ ID NO:6, or the antibody specifically binds to the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
[0022] In a related embodiment, the invention relates to a method
of enhancing intracellular killing of an S. aureus cell in a
macrophage cell. This method comprises providing to a macrophage
cell a pharnaceutically-acceptable collagen binding protein or
antibody composition in an amount effective to enhance the
intracellular killing of the S. aureus cell in the macrophage.
[0023] 2.1 CNA Nucleic Acid Compositions
[0024] The invention provides nucleic acid sequences encoding CBP.
As used herein, a gene encoding CBP means a nucleic acid sequence
encoding Col binding protein. A preferred nucleic acid sequence
encoding a CBP gene is the nucleotide sequence of SEQ ID NO:1, or a
strain variant or an active fragment thereof. It is expected that
the gene encoding CBP will vary in nucleic acid sequence from
strain to strain, but that the variation in nucleic acid sequence
will not preclude hybridization between sequences encoding CBP of
each strain under strict hybridization conditions.
[0025] As used herein, a strain variant of CBP means any
polypeptide encoded, in whole or in part, by a nucleic acid
sequence which hydridizes under strict hybridization conditions to
a nucleic acid sequence from any of SEQ ID NO:1, SEQ ID NO:3, and
SEQ ID NO:5 encoding the M17, M31, and M55 epitopes of a S. aureus
CBP, respectively. The amino acid sequence of M17, MD31, and M55
peptides is given in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6
respectively. One of skill in the art will understand that strain
variants of CBP include those proteins encoded by nucleic acid
sequences which may be amplified using a nucleic acid sequence of
any of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
[0026] In related embodiments, the invention also comprises strain
variants of CBP and the cna gene(s) encoding CBPs. Strain variants
are those nucleic acid compositions and polypeptide compositions
isolated from strains of S. aureus and related gram-positive
bacteria which express CBPs, and which adhere to Col
substrates.
[0027] Aspects of the invention concern the identification of such
strain variants using diagnostic methods and kits described herein.
In particular, methods utilizing cna gene sequences as nucleic acid
hybridization probes and/or anti-CBP antibodies in western blots or
related analyses are useful for the identification of such strain
variants. The identify of potential strain variants of CBP may be
confirmed by Col binding assays, e.g., by blot analysis with
labeled Col, or alternatively by the demonstrating the ability of
the strain-variant CBP to lessen or prevent adherence of S. aureus
and related bacteria Col.
[0028] As used herein, a CBP is a protein which confers protection
against staphylococcal or streptococcal infection. A CBP or
fragments thereof may prevent or lessen adhesion of S. aureus to
Col, or prevent or lessen adhesion the severity of any of the
disorders associated with S. aureus infection, including sepsis,
skin lesions, septic arthritis, endocarditis, mastitis, pneumonia,
neurological disorders, and other diseases which result from the
colonization of S. aureus or related organisms to Col-containing
substrata. An important aspect of the present invention concerns
isolated DNA segments and recombinant vectors encoding CBP, and the
creation and use of recombinant host cells through the application
of DNA technology, that express CBP gene and CBP-derived gene
products. As such, the invention concerns DNA segment comprising an
isolated gene that encodes a protein or peptide that includes an
amino acid sequence essentially as set forth by a contiguous
sequence from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. These DNA
segments are exemplified in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID
NO:5, respectively. Compositions that include a purified protein
that has an amino acid sequence essentially as set forth by the
amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 are
also encompassed by the invention.
[0029] Regarding the novel protein CBP epitopes, the present
invention concerns DNA segments, that can be isolated from
virtually any bacterial source, that are free from total genomic
DNA and that encode proteins or peptides having CBP-like activity.
DNA segments encoding CBP-like species may prove to encode
proteins, polypeptides, subunits, functional domains, and the
like.
[0030] As used herein, the term "DNA segment" refers to a DNA
molecule that has been isolated free of total genornic DNA of a
particular species. Therefore, a DNA segment encoding CBP refers to
a DNA segment that contains CBP coding sequences yet is isolated
away from, or purified free from, total genomic DNA of the species
from which the DNA segment is obtained. Included within the term
"DNA segment", are DNA segments and smaller fragments of such
segments, and also recombinant vectors, including, for example,
plasmids, cosmids, phagemids, phage, viruses, and the like.
[0031] Similarly, a DNA segment comprising an isolated or purified
CBP gene refers to a DNA segment including CBP coding sequences
and, in certain aspects, regulatory sequences, isolated
substantially away from other naturally occurring genes or protein
encoding sequences. In this respect, the term "gene" is used for
simplicity to refer to a functional protein, polypeptide or peptide
encoding unit. As will be understood by those in the art, this
functional term includes both genomic sequences, extra-genomnic and
plasmid-encoded sequences and smaller engineered gene segments that
express, or may be adapted to express, proteins, polypeptides or
peptides. Such segments may be naturally isolated, or modified
synthetically by the hand of man.
[0032] "Isolated substantially away from other coding sequences"
means that the gene of interest, in this case, a gene encoding CBP,
forms the significant part of the coding region of the DNA segment,
and that the DNA segment does not contain large portions of
naturally-occurring coding DNA, such as large chromosomal fragments
or other functional genes or polypeptide coding regions. Of course,
this refers to the DNA segment as originally isolated, and does not
exclude genes or coding regions later added to the segment by the
hand of man.
[0033] In particular embodiments, the invention concerns isolated
DNA segments and recombinant vectors incorporating DNA sequences
that encode a CBP species that includes within its amino acid
sequence an amino acid sequence essentially as set forth in SEQ ID
NO:2, SEQ ID NO:4, or SEQ ID NO:6. In other particular embodiments,
the invention concerns isolated DNA segments and recombinant
vectors incorporating DNA sequences that include within their
sequence a nucleotide sequence essentially as set forth in SEQ ID
NO:1, SEQ ID NO:3, or SEQ ID NO:5.
[0034] The term "a sequence essentially as set forth in SEQ ID
NO:2, SEQ ID NO:4, or SEQ ID NO:6" means that the sequence
substantially corresponds to a portion of SEQ ID NO:2, SEQ ID NO:4,
or SEQ ID NO:6 and has relatively few amino acids that are not
identical to, or a biologically functional equivalent of, the amino
acids of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. The term
"biologically functional equivalent" is well understood in the art
and is further defined in detail herein (for example, see
Illustrative Embodiments). Accordingly, sequences that have between
about 70% and about 80%; or more preferably, between about 81% and
about 90%; or even more preferably, between about 91% and about
99%; of amino acids that are identical or functionally equivalent
to the amino acids of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 will
be sequences that are "essentially as set forth in SEQ ID NO:2, SEQ
ID NO:4, or SEQ ID NO:6".
[0035] In certain other embodiments, the invention concerns
isolated DNA segments and recombinant vectors that include within
their sequence a nucleic acid sequence essentially as set forth in
SEQ ID NO:I, SEQ ID NO:3, or SEQ ID NO-5. The term "essentially as
set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5" is used in
the same sense as described above and means that the nucleic acid
sequence substantially corresponds to a portion of SEQ ID NO:1, SEQ
ID NO:3, or SEQ ID NO:5 and has relatively few codons that are not
identical, or functionally equivalent, to the codons of SEQ ID
NO:I, SEQ ID NO:3, or SEQ ID NO:5. Again, DNA segments that encode
proteins exhibiting CBP-like activity will be most preferred.
[0036] It will also be understood that amino acid and nucleic acid
sequences may include additional residues, such as additional N- or
C-terminal amino acids or 5' or 3' sequences, and yet still be
essentially as set forth in one of the sequences disclosed herein,
so long as the sequence meets the criteria set forth above,
including the maintenance of biological protein activity where
protein expression is concerned. The addition of terminal sequences
particularly applies to nucleic acid sequences that may, for
example, include various non-coding sequences flanking either of
the 5' or 3' portions of the coding region or may include various
upstream or downstream regulatory or structural genes.
[0037] Naturally, the present invention also encompasses DNA
segments that are complementary, or essentially complementary, to
the sequence set forth in SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID
NO:5. Nucleic acid sequences that are "complementary" are those
that are capable of base-pairing according to the standard
Watson-Crick complementarity rules. As used herein, the term
"complementary sequences" means nucleic acid sequences that are
substantially complementary, as may be assessed by the same
nucleotide comparison set forth above, or as defined as being
capable of hybridizing to the nucleic acid segment of SEQ ID NO:I,
SEQ ID NO:3, or SEQ ID NO:5, under relatively stringent conditions
such as those described herein.
[0038] The nucleic acid segments of the present invention,
regardless of the length of the coding sequence itself, may be
combined with other DNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol. For example, nucleic acid fragments may be prepared that
include a short contiguous stretch identical to or complementary to
SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO:5, such as about 14
nucleotides, and that are up to about 10,000 or about 5,000 base
pairs in length, with segments of about 3,000 being preferred in
certain cases. DNA segments with total lengths of about 2,000,
about 1,000, about 500, about 200, about 100 and about 50 base
pairs in length (including all intermediate lengths) are also
contemplated to be useful.
[0039] It will be readily understood that "intermediate lengths",
in these contexts, means any length between the quoted ranges, such
as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32,
etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,
152, 153, etc.; including all integers through the 200-500;
500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000
ranges, up to and including sequences of about 12,001, 12,002,
13,001, 13,002 and the like.
[0040] It will also be understood that this invention is not
limited to the particular nucleic acid sequences disclosed in SEQ
ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or to the amino acid
sequences disclosed in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
Recombinant vectors and isolated DNA segments may therefore
variously include the CBP epitope encoding regions themselves,
coding regions bearing selected alterations or modifications in the
basic coding region, or they may encode larger polypeptides that
nevertheless include CBP-derived coding regions or may encode
biologically functional equivalent proteins or peptides that have
variant amino acids sequences.
[0041] The DNA segments of the present invention encompass
biologically functional equivalent CBP-derived proteins and
peptides, in particular those CBP and related proteins isolated
from prokaryotic sources, and particularly bacteria. DNA segments
isolated from species of staphylococci and streptococci and related
bacteria which are shown to bind Col are particularly preferred for
use in the methods disclosed herein. Such sequences may arise as a
consequence of codon redundancy and functional equivalency that are
known to occur naturally within nucleic acid sequences and the
proteins thus encoded. Alternatively, functionally equivalent
proteins or peptides may be created via the application of
recombinant DNA technology, in which changes in the protein
structure may be engineered, based on considerations of the
properties of the amino acids being exchanged. Changes designed by
man may be introduced through the application of site-directed
mutagenesis techniques, e.g., to introduce improvements to the
antigenicity of the protein or to test mutants in order to examine
activity at the molecular level.
[0042] If desired, one may also prepare fusion proteins and
peptides, e.g., where the CBP or CBP-derived coding regions are
aligned within the same expression unit with other proteins or
peptides having desired functions, such as for purification or
immunodetection purposes (e.g., proteins that may be purified by
affinity chromatography and enzyme label coding regions,
respectively).
[0043] 2.2 Recombinant Expression of CBP and CBP-derived
Epitopes
[0044] The present invention also concerns recombinant host cells
for expression of an isolated cna gene, or for a DNA sequence
encoding one or more epitopic peptides derived from a cna-encoding
protein. It is contemplated that virtually any host cell may be
employed for this purpose, but certain advantages may be found in
using a bacterial host cell such as E. coli, S. typhimurium, B.
subtilis, or S. aureus, S. dysgalactiae, S. pyogenes or other
Gram-positive species. Expression in eukaryotic cells is also
contemplated such as those derived from yeast, insect, or mammalian
cell lines. These recombinant host cells may be employed in
connection with "overexpressing" CBP proteins, that is, increasing
the level of expression over that found naturally in S. aureus.
[0045] Proteins of amino acid sequence derived, from or similar to,
CBP are expected to have affinity for Col and can be purified from
other constituents of S. aureus or recombinant host cells by
chromatography on matrices containing Col, so-called "affinity
chromatography." CBPs may also be purified by methodologies not
relying on affinity for Col such as ion exchange chromatography,
size exclusion chromatography, metal chelation chromatography, or
the like. Buffer, detergent, and other conditions may be dissimilar
from those optimal for "affinity chromatography." In a preferred
embodiment, an affinity matrix comprising Type II Col or a related
Col type (e.g., Type I, Type III, Type V, Type IX, etc.) may be
used for the isolation of CBPs from solution, or alternatively,
isolation of intact bacteria expressing CBPs, or even membrane
fragments of bacteria expressing CBPs.
[0046] A particular aspect of this invention provides novel ways in
which to utilize recombinant CBPs or CBP-derived peptides, nucleic
acid segments encoding these peptides, recombinant vectors and
formed host cells comprising cna or cna-derived DNA segments,
recombinant vectors and transformed host cells comp rising cna or
cna-derived DNA segments, and recombinant vectors and transformed
host cells comprising S. aureus cna-derived DNA segments. In
particular embodiments, genetically-engineered nucleic acid
segments and CBP proteins are obtained which have altered Col
binding site domains. Using the methods disclosed herein, the
inventors have developed site-specifically altered CBPs which have
reduced affinity for Col. As is well known to those of skill in the
art, many such vectors and host cells are readily available, one
particular detailed example of a suitable vector for expression in
mammalian cells is that described in U.S. Pat. No. 5,168,050,
incorporated herein by reference. However, there is no requirement
that a highly purified vector be used, so long as the coding
segment employed encodes a protein or peptide of interest (e.g., a
CBP, and particularly a CBP from S. aureus, or related bacterium,
and does not include any coding or regulatory sequences that would
have an adverse effect on cells). Therefore, it will also be
understood that useful nucleic acid sequences may include
additional residues, such as additional non-coding sequences
flanking either of the 5' or 3' portions of the coding region or
may include various regulatory sequences.
[0047] After identifying an appropriate epitope-encoding nucleic
acid molecule, it may be inserted into any one of the many vectors
currently known in the art, so that it will direct the expression
and production of the protein or peptide epitope of interest (e.g,
a CBP from S. aureus) when incorporated into a host cell. In a
recombinant expression vector, the coding portion of the DNA
segment is positioned under the control of a promoter. The promoter
may be in the form of the promoter which is naturally associated
with a CBP-encoding nucleic acid segment, as may be obtained by
isolating the -5' non-coding sequences located upstream of the
coding segment, for example, using recombinant cloning and/or PCRTM
technology, in connection with the compositions disclosed herein.
Direct amplification of nucleic acids using the PCRTM technology of
U.S. Pat. Nos. 4,683,195 and 4,683,202 (herein incorporated by
reference) are particularly contemplated to be useful in such
methodologies.
[0048] In certain embodiments, it is contemplated that particular
advantages will be gained by positioning the CBP-encoding DNA
segment under the control of a recombinant, or heterologous,
promoter. As used herein, a recombinant or heterologous promoter is
intended to refer to a promoter that is not normally associated
with a cna or cna-like gene segment in its natural environment.
Such promoters may include those normally associated with other
MSCRAMM-encoding genes, and/or promoters isolated from any other
bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will
be important to employ a promoter that effectively directs the
expression of the DNA segment in the particular cell containing the
vector comprising the CBP-encoding nucleic acid segment.
[0049] The use of promoter and cell type combinations for protein
expression is generally known to those of skill in the art of
molecular biology, for example, see Sambrook et al., 1989. The
promoters employed may be constitutive, or inducible, and can be
used under the appropriate conditions to direct high level
expression of the introduced DNA segment, such as is advantageous
in the large-scale production of recombinant proteins or
peptides.
[0050] Prokaryotic expression of nucleic acid segments of the
present invention may be performed using methods known to those of
skill in the art, and will likely comprise expression vectors and
promotor sequences such as those obtained by tac, trp, lac, lacUV5
or T7. When expression of the recombinant CBP proteins is desired
in eukaryotic cells, a number of expression systems are available
and known to those of skill in the art. An exemplary eukaryotic
promoter system contemplated for use in high-level expression is
the Pichia expression vector system (Pharmacia LKB
Biotechnology).
[0051] In connection with expression embodiments to prepare
recombinant CBP and peptides, it is contemplated that longer DNA
segments will most often be used, with DNA segments encoding the
entire CBP or functional domains, epitopes, ligand binding domains,
subunits, etc. being most preferred. However, it will be
appreciated that the use of shorter DNA segments to direct the
expression of CBP peptides or epitopic core regions, such as may be
used to generate anti-CBP antibodies, also falls within the scope
of the invention. DNA segments that encode peptide antigens from
about 15 to about 100 amino acids in length, or more preferably,
from about 15 to about 50 amino acids in length are contemplated to
be particularly useful. Exemplary DNA segments encoding peptide
epitopes of the Col-binding protein which the inventors have shown
to be useful in preventing the binding to Col include the
polynucleotides disclosed in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID
NO:5.
[0052] The cna gene and cna-derived DNA segments may also be used
in connection with somatic expression in an animal or in the
creation of a transgenic animal. Again, in such embodiments, the
use of a recombinant vector that directs the expression of the full
length or one or more active CBP epitopes is particularly
contemplated. Expression of cna transgene in animals is
particularly contemplated to be useful in the production of
anti-CBP antibodies for use in passive immunization methods for
prevention of staphylococcal and streptococcal adhesion to Col.
[0053] The use of recombinant promoters to achieve protein
expression is generally known to those of skill in the art of
molecular biology, for example, see Sambrook et al, (1989). The
promoters employed may be constitutive, or inducible, and can be
used under the appropriate conditions to direct high level or
regulated expression of the introduced DNA segment. For eukaryotic
expression, the currently preferred promoters are those such as
CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter in
combination with the SV40 enhancer. In preferred embodiments, the
expression of recombinant CBPs is carried out using prokaryotic
expression systems, and in particular bacterial systems such as E.
coli. Such prokaryotic expression of nucleic acid segments of the
present invention may be performed using methods known to those of
skill in the art, and will likely comprise expression vectors and
promotor sequences such as those obtained by tac, trp, lac, lacUV5
or T7 promoters.
[0054] For the expression of CBP and CBP-derived epitopes, once a
suitable clone or clones have been obtained, whether they be native
sequences or genetically-modified, one may proceed to prepare an
expression system for the recombinant preparation of CBP or
CBP-derived peptides. The engineering of DNA segment(s) for
expression in a prokaryotic or eukaryotic system may be performed
by techniques generally known to those of skill in recombinant
expression. It is believed that virtually any expression system may
be employed in the expression of CBP or CBP-derived epitopes.
[0055] Alternatively, it may be desirable in certain embodiments to
express CBP or CBP-derived epitopes in eukaryotic expression
systems. The DNA sequences encoding the desired CBP or CBP-derived
epitope (either native or mutagenized) may be separately expressed
in bacterial systems, with the encoded proteins being expressed as
fusions with b-galactosidase, ubiquitin, Schistosoma japonicum
glutathione S-transferase, S. aureus Protein A, maltose binding
protein, and the like. It is believed that bacterial expression
will ultimately have advantages over eukaryotic expression in terms
of ease of use and quantity of materials obtained thereby.
[0056] It is proposed that transformation of host cells with DNA
segments encoding such epitopes will provide a convenient means for
obtaining CBP or CBP-derived peptides. Genomic sequences are
suitable for eukaryotic expression, as the host cell will, of
course, process the genomic transcripts to yield functional MRNA
for translation into protein.
[0057] It is similarly believed that almost any eukaryotic
expression system may be utilized for the expression of CBP and
CBP-derived epitopes, e.g., baculovirus-based, glutamine
synthase-based or dihydrofolate reductase-based systems may be
employed. In preferred embodiments it is contemplated that plasmid
vectors incorporating an origin of replication and an efficient
eukaryotic promoter, as exemplified by the eukaryotic vectors of
the pCMV series, such as pCMV5, will be of most use.
[0058] For expression in this manner, one would position the coding
sequences adjacent to and under the control of the promoter. It is
understood in the art that to bring a coding sequence under the
control of such a promoter, one positions the 5' end of the
transcription initiation site of the transcriptional reading frame
of the protein between about 1 and about 50 nucleotides
"downstream" of (i.e., 3' of) the chosen promoter.
[0059] Where eukaryotic expression is contemplated, one will also
typically desire to incorporate into the transcriptional unit which
includes nucleic acid sequences encoding CBP or CBP-derived
peptides, an appropriate polyadenylation site (e.g., 5'-AATAAA-3')
if one was not contained within the original cloned segment.
Typically, the poly-A addition site is placed about 30 to 2000
nucleotides "downstream" of the termination site of the protein at
a position prior to transcription termination.
[0060] It is contemplated that virtually any of the commonly
employed host cells can be used in connection with the expression
of the CBP and CBP-derived epitopes in accordance herewith.
Examples include cell lines typically employed for eukaryotic
expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK,
COS-7, RIN and MDCK cell lines.
[0061] It is further contemplated that the CBP or epitopic peptides
derived from native or recombinant CBPs may be "overexpressed",
i.e., expressed in increased levels relative to its natural
expression in human cells, or even relative to the expression of
other proteins in a recombinant host cell containing CBP-encoding
DNA segments. Such overexpression may be assessed by a variety of
methods, including radiolabeling and/or protein purification.
However, simple and direct methods are preferred, for example,
those involving SDS/PAGE and protein staining or Western blotting,
followed by quantitative analyses, such as densitometric scanning
of the resultant gel or blot. A specific increase in the level of
the recombinant protein or peptide in comparison to the level in
natural CBP-producing cells is indicative of overexpression, as is
a relative abundance of the specific protein in relation to the
other proteins produced by the host cell and, e.g., visible on a
gel.
[0062] As used herein, the term "engineered" or "recombinant" cell
is intended to refer to a cell into which a recombinant gene, such
as a gene encoding a CBP or CBP-derived epitope has been
introduced. Therefore, engineered cells are distinguishable from
naturally occurring cells which do not contain a recombinantly
introduced gene. Engineered cells are thus cells having a gene or
genes introduced through the hand of man. Recombinantly introduced
genes will either be in the form of a single structural gene, an
entire genomic clone comprising a structural gene and flanking DNA,
or an operon or other functional nucleic acid segment which may
also include genes positioned either upstream and/or downstream of
the promotor, regulatory elements, or structural gene itself, or
even genes not naturally associated with the particular structural
gene of interest.
[0063] Where the introduction of a recombinant version of one or
more of the foregoing genes is required, it will be important to
introduce the gene such that it is under the control of a promoter
that effectively directs the expression of the gene in the cell
type chosen for engineering. In general, one will desire to employ
a promoter that allows constitutive (constant) expression of the
gene of interest. Commonly used constitutive eukaryotic promoters
include viral promotors such as the cytomegalovirus (CMV) promoter,
the Rous sarcoma long-terminal repeat (LTR) sequence, or the SV40
early gene promoter. The use of these constitutive promoters will
ensure a high, constant level of expression of the introduced
genes. The inventors have noticed that the level of expression from
the introduced genes of interest can vary in different clones, or
genes isolated from different strains or bacteria. Thus, the level
of expression of a particular recombinant gene can be chosen by
evaluating different clones derived from each transfection study;
once that line is chosen, the constitutive promoter ensures that
the desired level of expression is permanently maintained. It may
also be possible to use promoters that are specific for cell type
used for engineering, such as the insulin promoter in insulinoma
cell lines, or the prolactin or growth hormone promoters in
anterior pituitary cell lines.
[0064] 2.3 Immunological Detection Of CBP And Bacteria Expressing
CBPs
[0065] A further aspect of the invention is the preparation of
immunological compositions, and in particular anti-CBP antibodies
for diagnostic and therapeutic methods relating to the detection
and treatment of infections caused by S. aureus and related
Gram-positive species. In preferred embodiments, antibody
compositions are disclosed which bind to the site-specifically
altered recombinant CBPs described in the present invention. Also
disclosed are antibodies which recognize specific native and
synthetically-mutated Col binding domain epitopes within the CBPs.
The inventors contemplate such antibodies to be useful in both
diagnostic screening assays, processes for purifying Col, detecting
anti-CBP antibodies, as well as their use in passive immunization
of an animal to prevent bacterial sepsis, colonization, or binding
to the ECM component Col.
[0066] 2.4 Methods For Producing An Immune Response
[0067] Also disclosed is a method of generating an immune response
in an animal. The method generally involves administering to an
animal a pharmaceutical composition comprising an immunologically
effective amount of a peptide composition disclosed herein.
Preferred peptide compositions include the peptide disclosed in any
of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
[0068] The invention also encompasses CBP and CBP-derived peptide
antigen compositions together with pharmaceutically-acceptable
excipients, carriers, diluents, adjuvants, and other components,
such as additional peptides, antigens, or outer membrane
preparations, as may be employed in the formulation of particular
vaccines.
[0069] The nucleic acid sequences of the present invention encode
CBP and are useful to generate pure recombinant CBP for
administration to a host. Such administration is useful as a
vaccine to induce therapeutic antibodies that prevent adherence of
S. aureus to the host's tissues.
[0070] It is shown herein that antisera raised against and reactive
with CBP inhibits binding, promotes, phagocytosis, and enhances
intracellular killing by macrophages. Thus, it is contemplated that
administration of antibodies reactive with CBP to at-risk subjects
will be effective for prophylaxis of, and in the case of infected
subjects for therapy of. bacterial infection.
[0071] Antibodies may be of several types including those raised in
heterologous donor animals or human volunteers immunized with CBPs,
monoclonal antibodies (mAbs) resulting from hybridomas derived from
fusions of B cells from CBP-immunized animals or humans with
compatible myeloma cell lines, so-called "humanized" mAbs resulting
from expression of gene fusions of combinatorial determining
regions of mAb-encoding genes from heterologous species with genes
encoding human antibodies, or CBP-reactive antibody-containing
fractions of plasma from human donors. It is contemplated that any
of the techniques described above might be used for the vaccination
of subjects for the purpose of antibody production. Optimal dosing
of such antibodies is highly dependent upon the pharmacokinetics of
the specific antibody population in the particular species to be
treated. It is contemplated that the duration of dosing maintaining
anti-CBP levels at these inhibitory antibody concentrations would
be for at least four to eight weeks following presumptive exposure
to S. aureus, or throughout the duration of symptoms of disease and
for at least four to eight weeks after cessation of these
symptoms.
[0072] Using the peptide antigens described herein, the present
invention also provides methods of generating an immune response,
which methods generally comprise administering to an animal, a
pharmaceutically-accepta- ble composition comprising an
immunologically effective amount of a CBP peptide composition.
Preferred animals include mammals, and particularly humans. Other
preferred animals include murines, bovines, equines, porcines,
canines, and felines. The composition may include partially or
significantly purified CBP peptide epitopes, obtained from natural
or recombinant sources, which proteins or peptides may be
obtainable naturally or either chemically synthesized, or
alternatively produced in vitro from recombinant host cells
expressing DNA segments encoding such epitopes. Smaller peptides
that include reactive epitopes, such as those between about 10 and
about 50, or even between about 50 and about 100 amino acids in
length will often be preferred. The antigenic proteins or peptides
may also be combined with other agents, such as other peptide or
nucleic acid compositions, if desired.
[0073] By "immunologically effective amount" is meant an amount of
a peptide composition that is capable of generating an immune
response in the recipient animal. This includes both the generation
of an antibody response (B cell response), and/or the stimulation
of a cytotoxic immune response (T cell response). The generation of
such an immune response will have utility in both the production of
useful bioreagents, e.g., CTLs and, more particularly, reactive
antibodies, for use in diagnostic embodiments, and will also have
utility in various prophylactic or therapeutic embodiments.
Therefore, although these methods for the stimulation of an immune
response include vaccination regimens designed to prevent or lessen
significant infections caused by bacteria expressing a CBP, and
treatment regimens that may lessen the severity or duration of any
infection, it will be understood that achieving either of these end
results is not necessary for practicing these aspects of the
invention. Such treatment methods may be used particularly for the
treatment of infections caused by pathogens such as S. aureus,
related species, and other bacteria which express CBPs and adhere
to Col.
[0074] Further means contemplated by the inventors for generating
an immune response in an animal includes administering to the
animal, or human subject, a pharmaceutically-acceptable composition
comprising an immunologically effective amount of a nucleic acid
composition encoding a CBP epitope, or an immunologically effective
amount of an attenuated live organism that includes and expresses
such a nucleic acid composition. The "immunologically effective
amounts" are those amounts capable of stimulating a B cell and/or T
cell response.
[0075] Immunoformulations of this invention, whether intended for
vaccination, treatment, or for the generation of antibodies useful
in the detection of S. aureus and related bacteria, the prevention
of bacterial adhesion, or in the case of bacterial colonization,
promotion of bacterial adhesion to ECM components such as Col, may
comprise native, or synthetically-derived antigenic peptide
fragments from these proteins. As such, antigenic functional
equivalents of the proteins and peptides described herein also fall
within the scope of the present invention. An "antigenically
functional equivalent" protein or peptide is one that incorporates
an epitope that is immunologically cross-reactive with one or more
epitopes derived from any of the particular MSCRAMM proteins
disclosed (e.g., CBPs), and particularly the CBP of S. aureus.
Antigenically functional equivalents, or epitopic sequences, may be
first designed or predicted and then tested, or may simply be
directly tested for cross-reactivity.
[0076] In still further embodiments, the present invention concerns
immunodetection methods and associated kits. It is contemplated
that the proteins or peptides of the invention may be employed to
detect antibodies having reactivity therewith, or, alternatively,
antibodies prepared in accordance with the present invention, may
be employed to detect CBP or peptides. Either type of kit may be
used in the to immunodetection of compounds, present within
clinical samples, that are indicative of infections caused by
gram-positive bacteria expressing a CBP, and in particular S.
aureus. The kits may also be used in antigen or antibody
purification, as appropriate.
[0077] In general, the preferred immunodetection methods will
include first obtaining a sample suspected of containing a
CBP-reactive antibody, such as a biological sample from a patient,
and contacting the sample with a first CBP or peptide under
conditions effective to allow the formation of an immunocomplex
(primary immune complex). One then detects the presence of any
primary immunocomplexes that are formed.
[0078] Contacting the chosen sample with the CBP or peptide under
conditions effective to allow the formation of (primary) immune
complexes is generally a matter of simply adding the protein or
peptide composition to the sample. One then incubates the mixture
for a period of time sufficient to allow the added antigens to form
immune complexes with, i.e., to bind to, any antibodies present
within the sample. After this time, the sample composition, such as
a tissue section, ELISA plate, dot blot or western blot, will
generally be washed to remove any non-specifically bound antigen
species, allowing only those specifically bound species within the
immune complexes to be detected.
[0079] The detection of immunocomplex formation is well known in
the art and may be achieved through the application of numerous
approaches known to the skilled artisan and described in various
publications, such as, e.g., Nakamura et al. (1987), incorporated
herein by reference. Detection of primary immune complexes is
generally based upon the detection of a label or marker, such as a
radioactive, fluorescent, biological or enzymatic label, with
enzyme tags such as alkaline phosphatase, urease, horseradish
peroxidase and glucose oxidase being suitable. The particular
antigen employed may itself be linked to a detectable label,
wherein one would then simply detect this label, thereby allowing
the amount of bound antigen present in the composition to be
determined.
[0080] Alternatively, the primary immune complexes may be detected
by means of a second binding ligand that is linked to a detectable
label and that has binding affinity for the first protein or
peptide. The second binding ligand is itself often an antibody,
which may thus be termed a "secondary" antibody. The primary immune
complexes are contacted with the labeled, secondary binding ligand,
or antibody, under conditions effective and for a period of time
sufficient to allow the formation of secondary immune complexes.
The secondary immune complexes are then generally washed to remove
any non-specifically bound labeled secondary antibodies and the
remaining bound label is then detected.
[0081] For diagnostic purposes, it is proposed that virtually any
sample suspected of containing the antibodies of interest may be
employed. Exemplary samples include clinical samples obtained from
a patient such as blood or serum samples, bronchoalveolar fluid,
ear swabs, sputum samples, middle ear fluid or even perhaps urine
samples may be employed. This allows for the diagnosis of
infections caused by Gram-positive bacterium, and in particular, S.
aureus. Furthermore, it is contemplated that such embodiments may
have application to nonclinical samples, such as in the titering of
antibody samples, in the selection of hybridomas, the detection of
anti-CBP antibodies in a sample, the purification of CBPs, and the
prevention of bacterial adhesion to Col, and the like.
Alternatively, the clinical samples may be from veterinary sources
and may include such domestic animals as cattle, sheep, and goats.
Samples from feline, canine, and equine sources may also be used in
accordance with the methods described herein.
[0082] In related embodiments, the present invention contemplates
the preparation of kits that may be employed to detect the presence
of CBP-specific antibodies in a sample. Generally speaking, kits in
accordance with the present invention will include a suitable
protein or peptide together with an immunodetection reagent, and a
means for containing the protein or peptide and reagent.
[0083] The immunodetection reagent will typically comprise a label
associated with a CBP or peptide, or associated with a secondary
binding ligand. Exemplary ligands might include a secondary
antibody directed against the first CBP or peptide or antibody, or
a biotin or avidin (or streptavidin) ligand having an associated
label. Detectable labels linked to antibodies that have binding
affinity for a human antibody are also contemplated, e.g., for
protocols where the first reagent is a CBP peptide that is used to
bind to a reactive antibody from a human sample. Of course, as
noted above, a number of exemplary labels are known in the art and
all such labels may be employed in connection with the present
invention. The kits may contain antigen or antibody-label
conjugates either in fully conjugated form, in the form of
intermediates, or as separate moieties to be conjugated by the user
of the kit.
[0084] The container means will generally include at least one
vial, test tube, flask, bottle, syringe or other container means,
into which the antigen may be placed, and preferably suitably
allocated. Where a second binding ligand is obtained, the kit will
also generally contain a second vial or other container into which
this ligand or antibody may be placed. The kits of the present
invention will also typically include a means for containing the
vials in close confinement for commercial sale, such as, e.g,
injection or blow-molded plastic containers into which the desired
vials are retained.
[0085] 2.5 Methods for Inhibiting Bacterial Adhesion to COL
[0086] In addition, the CBP is useful as an agent to block S.
aureus adherence to Col. In a preferred embodiment of the
invention, a therapeutically effective dose of a CBP-derived
epitope is administered to a subject to prevent or block adhesion
of S. aureus to the host's tissues by conventional methods. The CBP
composition is preferably systemically administered, (i.e. by oral,
intravenous, and/or parenteral routes) but may also be applied
topically, e.g., to a localized tissue site, wound, lesion, or any
other location where the prevention of S. aureus adhesion is
desired. The term therapeutically effective dose means that amount
of a CBP composition which is sufficient to lessen or prevent
adherence of S. aureus is to a subject or to neutralize the known
deleterious effects of S. aureus infection. Such a dose may readily
be determined by known clinical, diagnostic, and pharmacological
methods. Absent adhesion of the bacteria to the tissues, the
disease-inducing effects of the microorganism are halted, thus the
CBP of the present invention is useful as a therapeutic agent to
prevent adhesion of S. aureus and thereby lessen or prevent disease
induced by this microorganism.
[0087] 2.6 Methods for Identifying Inhibtors of COL Binding by
CBPs
[0088] In a preferred embodiment, the novel recombinant
polypeptides of the present invention are used in methods to
isolate, identify, and characterize compositions which inhibit the
binding of Col to CBPs. Such inhibitors are useful in the
prevention of bacterial adhesion to extracellular matrices
containing Col. These inhibitors provide a non-antibiotic strategy
for the prevention of bacterial infection in an animal,
particularly through the inhibition of Col binding to proteins
located on the bacterial cell surface. In particular, the inventors
contemplate the use of CBP and CBP-derived peptides to define and
screen small molecule inhibitors. A particular utility of these
inhibitors is the prevention of bacterial adhesion to exposed
collagenous tissues or to Col accumulating on artificial joints,
medical implants, and other surgical devices which may be
susceptible to Col coating and subsequent bacterial adherence to
the Col-coated surfaces. Likewise, the availability of the crystal
structure of the CBP also now permits for the first time design of
peptidemimetics which may also serve to inhibit the binding of CBP
to Col.
[0089] 2.7 Hybridzation Embodiments
[0090] In addition to their use in directing the expression of CBP
and CBP-derived epitopic peptides, the nucleic acid sequences
disclosed herein also have a variety of other uses. For example,
they also have utility as probes or primers in nucleic acid
hybridization embodiments. As such, it is contemplated that nucleic
acid segments that comprise a sequence region that consists of at
least a 14 nucleotide long contiguous sequence that has the same
sequence as, or is complementary to, a 14 nucleotide long
contiguous sequence of any of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID
NO:5 will find particular utility. Longer contiguous identical or
complementary sequences, e.g, those of about 20, 30, 40, 50, 100,
200, 500, 1000 (including all intermediate lengths) and even up to
fill length sequences will also be of use in certain
embodiments.
[0091] The ability of such nucleic acid probes to specifically
hybridize to CBP-encoding sequences will enable them to be of use
in detecting the presence of complementary sequences in a given
sample. However, other uses are envisioned, including the use of
the sequence information for the preparation of mutant species
primers, or primers for use in preparing other genetic
constructions.
[0092] Nucleic acid molecules having sequence regions consisting of
contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of
100-200 nucleotides or so, identical or complementary to SEQ ID
NO:1, SEQ ID NO:3, or SEQ ID NO:5, are particularly contemplated as
hybridization probes for use in, e.g., Southern and Northern
blotting. This would allow CBP structural or regulatory genes to be
analyzed, both in diverse cell types and also in various bacterial
cells. The total size of fragment, as well as the size of the
complementary stretch(es), will ultimately depend on the intended
use or application of the particular nucleic acid segment Smaller
fragments will generally find use in hybridization embodiments,
wherein the length of the contiguous complementary region may be
varied, such as between about 14 and about 100 nucleotides, but
larger contiguous complementarity stretches may be used, according
to the length complementary sequences one wishes to detect.
[0093] The use of a hybridization probe of about 14-25 nucleotides
in length allows the formation of a duplex molecule that is both
stable and selective. Molecules having contiguous complementary
sequences over stretches greater than 14 bases in length are
generally preferred, though, in order to increase stability and
selectivity of the hybrid, and thereby improve the quality and
degree of specific hybrid molecules obtained. One will generally
prefer to design nucleic acid molecules having gene-complementary
stretches of 15 to 25 contiguous nucleotides, or even longer where
desired.
[0094] Hybridization probes may be selected from any portion of any
of the sequences disclosed herein. All that is required is to
review the sequence set forth in SEQ ID NO:1, SEQ ID NO:3, and SEQ
ID NO:5, and to select any continuous portion of the sequence, from
about 14-25 nucleotides in length up to and including the full
length sequence, that one wishes to utilize as a probe or primer.
The choice of probe and primer sequences may be governed by various
factors, such as, by way of example only, one may wish to employ
primers from towards the termini of the total sequence.
[0095] The process of selecting and preparing a nucleic acid
segment that includes a contiguous sequence from within SEQ ID
NO:1, SEQ ID NO:3, or SEQ IDNO:5 may alternatively be described as
preparing a nucleic acid fragment. Of course, fragments may also be
obtained by other techniques such as, e.g., by mechanical shearing
or by restriction enzyme digestion. Small nucleic acid segments or
fragments may be readily prepared by, for example, directly
synthesizing the fragment by chemical means, as is commonly
practiced using an automated oligonucleotide synthesizer. Also,
fragments may be obtained by application of nucleic acid
reproduction technology, such as the PCR.TM. technology of U.S.
Pat. No. 4,683,202 (incorporated herein by reference), by
introducing selected sequences into recombinant vectors for
recombinant production, and by other recombinant DNA techniques
generally known to those of skill in the art of molecular
biology.
[0096] Accordingly, the nucleotide sequences of the invention may
be used for their ability to selectively form duplex molecules with
complementary stretches of the entire cna gene or gene fragments.
Depending on the application envisioned, one will desire to employ
varying conditions of hybridization to achieve varying degrees of
selectivity of probe towards target sequence. For applications
requiring high selectivity, one will typically desire to employ
relatively stringent conditions to form the hybrids, e.g., one will
select relatively low salt and/or high temperature conditions, such
as obtained by about 0.02 M to about 0.15 M NaCl at temperatures of
50.degree. C. to 70.degree. C. Such selective conditions tolerate
little, if any, mismatch between the probe and the template or
target strand, and would be particularly suitable for isolating CBP
genes.
[0097] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
CBP-encoding sequences from related species, functional
equivalents, or the like, less stringent hybridization conditions
will typically be needed in order to allow formation of the
heteroduplex. In these circumstances, one may desire to employ
conditions such as about 0.15 M to about 0.9 M salt, at
temperatures ranging from 20.degree. C. to 55.degree. C.
Cross-hybridizing species can thereby be readily identified as
positively hybridizing signals with respect to control
hybridizations. In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
[0098] In certain embodiments, it will be advantageous to employ
nucleic acid sequences of the present invention in combination with
an appropriate means, such as a label, for determining
hybridization. A wide variety of appropriate indicator means are
known in the art, including fluorescent, radioactive, enzymatic or
other ligands, such as avidin/biotin, which are capable of giving a
detectable signal. In preferred embodiments, one will likely desire
to employ a fluorescent label or an enzyme tag, such as urease,
alkaline phosphatase or peroxidase, instead of radioactive or other
environmental undesirable reagents. In the case of enzyme tags,
calorimetric indicator substrates are known that can be employed to
provide a means visible to the human eye or spectrophotometrically,
to identify specific hybridization with complementary nucleic
acid-containing samples.
[0099] In general, it is envisioned that the hybridization probes
described herein will be useful both as reagents in solution
hybridization as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the test DNA (or RNA) is
adsorbed or otherwise affixed to a selected matrix or surface. This
fixed, single-stranded nucleic acid is then subjected to specific
hybridization with selected probes under desired conditions. The
selected conditions will depend on the particular circumstances
based on the particular criteria required (depending, for exarnple,
on the G+C content, type of target nucleic acid source of nucleic
acid, size of hybridization probe, etc.). Following washing of the
hybridized surface so as to remove nonspecifically bound probe
molecules, specific hybridization is detected, or even quantitated,
by means of the label.
[0100] 2.8 Antibody Compositions
[0101] In a preferred embodiment, administration of a
therapeutically effective dose of CBP to a subject induces in the
subject antibodies which bind and neutralize S. aureus present in
the subject, thereby preventing the deleterious effects of this
microorganism. Alternatively, anti-CBP epitope antibodies generated
in a first host animal provide antibodies which can be administered
to a second subject for passive immunization or treatment against
S. aureus infection. Such antibodies are also useful as a
diagnostic screen for the presence of S. aureus in a test sample,
using conventional immunoassay techniques.
[0102] In the present invention, novel nucleic acid sequences are
disclosed which encode site-specifically modified CBPs of S.
aureus. These synthetic variants are prepared by the methods
disclosed herein, and encode CBPs having modified Col binding
domains.
[0103] In certain aspects, the present invention concerns novel
antibody compositions which inhibit Col binding to bacteria. In
particular, antibodies to native and synthetically-modified
epitopes from CBPs have been developed which inhibit Col binding to
CBPs both in vitro and in vivo. In particular, proteins, peptides
and peptide epitopes have been produced to provide vaccine
compositions useful in the prevention of bacterial infection and
antibody compositions useful in the prevention of Col binding to
such organisms.
[0104] In other embodirnents, the present invention encompasses
antibody compositions which enhance Col binding to bacterial cells.
These aspects provide methods and compositions for producing
bacterial colonization of an animal host with attenuated, or
avirulent bacterial strains expressing cell surface CBP
epitopes.
[0105] In one aspect, the invention discloses an antibody that
interacts with a CBP domain of a bacterial cna gene product, and
particularly, a CBP domain of an S. aureus cna gene product Such
antibody may be monoclonal, or preferably polyclonal. In another
aspect, the invention discloses an antibody which inhibits
bacterial adhesion, and the binding of the gene product to Col.
[0106] Also disclosed is a method for detecting a bacterium
expressing a CBP in a sample. The method generally involves
obtaining a sample suspected of containing a bacterium expressing
such a protein, then contacting the sample with an antibody
composition disclosed herein, and detecting the formation of immune
complexes. In preferred embodiments, the bacterium is S. aureus, S.
dysgalactiae, S. pyogenes, on a related species of Gram-positive
bacteria.
[0107] Other aspects of the invention include methods of inhibiting
bacterial colonization, and particularly colonization by S. aureus,
S. dysgalactiae, S. pyogenes, on a related species of Gram-positive
bacteria, in an animal by administering to the animal an antibody
of the present invention which prevents or significantly reduces
the binding of Col to the CBP expressed by the bacteria.
Administration of the antibody composition may be prophylactically
prior to and/or following diagnosis of infection or other
multisystemic disorders caused by bacterial infection which may
involve the skin, joints, heart, and central nervous system. The
administration may also be made in passive immunization protocols
designed to prevent and/or ameliorate systemic infections by
susceptible pathogens, and in particular, to ameliorate the effects
of infections by pathogenic streptococci staphylococci, and related
Gram-positive bacteria.
[0108] 2.9 Nucleic Acid Segments and Vectors
[0109] The present invention includes proteins expressed from genes
encoding a CBP such as that protein expressed from the DNA insert
of recombinant clones comprising site-specifically modified CBPs
from S. aureus. Also included are strain variants of the cna gene
derived from S. aureus which also encode proteins capable of
binding Col, which may hybridize to cna DNAs under conditions of
moderate or high stringency, or which may serve as templates for
gene amplification by PCRTM using oligonucleotide primers derived
from cna or cna-derived nucleic acid sequences. It is understood
that these variants may include genes containing codons not
identical in nucleotide sequence to those of the cna gene of S.
aureus, but encoding the same, or functionally equivalent amino
acid, as is expected by those practiced in the art who understand
the degeneracy of the genetic code.
[0110] These variants may also include those genes similar to the
cna gene from S. aureus, but having codons specifying relatively
few amino acids that are different from those of the protein(s)
encoded by S. aureus, or having somewhat fewer or greater numbers
of these codons. Accordingly such sequences include those that have
between about 60% and about 80%; or more preferably, between about
81% and about 90%; or even more preferably, between about 91% and
about 99%; of amino acids that are identical or functionally
equivalent to those of protein(s) encoded by S. aureus cna.
[0111] It is also understood that amino acid sequences and nucleic
acid sequences may include additional residues, such as additional
N- or C-terminal amino acids, or 5' or 3' nucleic acid sequences,
and yet still be as set forth herein, so long as the sequence meets
the criteria set forth above including the expression of a CBP
protein. These additional sequences may, for example, include
various transcriptional promoters, enhancers, or terminators,
various secretion-directing leader peptides, various amino acid
sequences directing posttranslational modifications, amino acids or
peptides which may facilitate isolation and purification of CBP(s),
and the like. Naturally, alterations and additions to these
sequences will be made given consideration of the cell type,
organism, or animal that will be chosen for expression of
CBP(s).
[0112] 2.10 Vaccine Formulation
[0113] It is expected that to achieve an "immunologically effective
formulation" it may be desirable to administer CBPs to the human or
animal subject in a pharmaceutically acceptable composition
comprising an immunologically effective amount of CBPs mixed with
other excipients, carriers, or diluents which may improve or
otherwise alter stimulation of B cell and/or T cell responses, or
immunologically inert salts, organic acids and bases,
carbohydrates, and the like, which promote stability of such
mixtures. Immunostimulatory excipients, often referred to as
adjuvants, may include salts of aluminum (often referred to as
Alurns), simple or complex fatty acids and sterol compounds,
physiologically acceptable oils, polymeric carbohydrates,
chemically or genetically modified protein toxins, and various
particulate or emulsified combinations thereof. CBPs or peptides
within these mixtures, or each variant if more than one are
present, would be expected to comprise about 0.0001 to 1.0
milligrams, or more preferably about 0.001 to 0.1 milligrams, or
even more preferably less than 0.1 milligrams per dose.
[0114] It is also contemplated that attenuated organisms may be
engineered to express recombinant CBP gene products and themselves
be delivery vehicles for the invention. Particularly preferred are
attenuated bacterial species such as Mycobacterium, and in
particular M. bovis, M. smegmatis, or BCG. Alternatively, pox-,
polio-, adeno-, or other viruses, and bacteria such as Salmonella,
Shigella, Listeria, Streptococcus species may also be used in
conjunction with the methods and compositions disclosed herein.
[0115] The naked DNA technology, often referred to as genetic
immunization, has been shown to be suitable for protection against
infectious organisms. Such DNA segments could be used in a variety
of forms including naked DNA and plasmid DNA, and may administered
to the subject in a variety of ways including parenteral, mucosal,
and so-called microprojectile-based "gene-gun" inoculations. The
use of cna nucleic acid compositions of the present invention in
such immunization techniques is thus proposed to be useful as a
vaccination strategy against streptococcal and staphylococcal
infection.
[0116] It is recognized by those skilled in the art that an optimal
dosing schedule of a vaccination regimen may include as many as
five to six, but preferably three to five, or even more preferably
one to three administrations of the immunizing entity given at
intervals of as few as two to four weeks, to as long as five to ten
years, or occasionally at even longer intervals.
[0117] 2.11 Recombinant Host Cells and Vectors
[0118] Particular aspects of the invention concern the use of
plasmid vectors for the cloning and expression of recombinant
peptides, and particular peptide epitopes comprising either native,
or site-specifically mutated CBP Col-binding site epitopes. The
generation of recombinant vectors, transformation of host cells,
and expression of recombinant proteins is well-known to those of
skill in the art. Prokaryotic hosts are preferred for expression of
the peptide compositions of the present invention. An example of a
preferred prokaryotic host is E. coli and in particular, E. coli
strains ATCC 69791, BL21(DE3), JM101, XL1-Blue.TM., RR1. LE392, B,
.chi..sup.1776 (ATCC 31537), and W3110 (F.sup.-, .lambda..sup.-,
prototrophic, ATCC 273325). Alternatively, other Enterobacteriaceae
species such as Salmonella typhimurium and Serratia marcescens, or
even other Gram-negative hosts including various Pseudomonas
species may be used in the recombinant expression of the genetic
constructs disclosed herein. Alternatively streptococci and
staphylococci species may be used to express these constructs, and
in particular, S. aureus, S. pyogenes, and S. dysgalactiae.
[0119] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli may be typically transformed using vectors such as
pBR322, or any of its derivatives (Bolivar et al., 1977). pBR322
contains genes for ampicillin and tetracycline resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
[0120] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lambda.GEM.TM.-11 may be utilized
in making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0121] Those promoters most commonly used in recombinant DNA
construction include the .beta.-lactamase (penicillinase) and
lactose promoter systems (Chang et al., 1978; Itakura et al., 1977;
Goeddel et al., 1979) or the tryptophan (trp) promoter system
(Goeddel et al., 1980). The use of recombinant and native microbial
promoters is well-known to those of skill in the art, and details
concerning their nucleotide sequences and specific methodologies
are in the public domain, enabling a skilled worker to construct
particular recombinant vectors and expression systems for the
purpose of producing compositions of the present invention.
[0122] In addition to the preferred embodiment expression in
prokaryotes, eukaryotic microbes, such as yeast cultures may also
be used in conjunction with the methods disclosed herein.
Saccharomyces cerevisiae, or common bakers' yeast is the most
commonly used among eukaryotic microorganisms, although a number of
other species may also be employed for such eukaryotic expression
systems. For expression in Saccharomyces, the plasmid YRp7, for
example, is commonly used (Stinchcomb et al., 1979; Kingsman et
al., 1979; Tschemper et al., 1980). This plasmid already contains
the trpL gene which provides a selection marker for a mutant strain
of yeast lacking the ability to grow in tryptophan, for example
ATCC 44076 or PEP4-1 (Jones, 1977). The presence of the trpL lesion
as a characteristic of the yeast host cell genome then provides an
effective environment for detecting transformation by growth in the
absence of tryptophan.
[0123] Suitable promoting sequences in yeast vectors include the
promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) or
other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978),
such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. In constructing suitable expression plasmids, the
termination sequences associated with these genes are also ligated
into the expression vector 3' of the sequence desired to be
expressed to provide polyadenylation of the MRNA and termination.
Other promoters, which have the additional advantage of
transcription controlled by growth conditions are the promoter
region for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Any plasmid vector containing a yeast-compatible
promoter, an origin of replication, and termination sequences is
suitable.
[0124] In addition to microorganisms, cultures of cells derived
from multicellular organisms may also be used as hosts in the
routine practice of the disclosed methods. In principle, any such
cell culture is workable, whether from vertebrate or invertebrate
culture. However, interest has been greatest in vertebrate cells,
and propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure in recent years. Examples of such useful
host cell lines are VERO and HeLa cells, Chinese hamster ovary
(CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines.
Expression vectors for such cells ordinarily include (if necessary)
an origin of replication, a promoter located in front of the gene
to be expressed, along with any necessary ribosome binding sites,
RNA splice sites, polyadenylation site, and transcriptional
terminator sequences.
[0125] For use in mammalian cells, the control functions on the
expression vectors are often obtained by viral material. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early
and late promoters of SV40 virus are particularly useful because
both are obtained easily from the virus as a fragment which also
contains the SV40 viral origin of replication (Fiers et al, 1978).
Smaller or larger SV40 fragments may also be used, provided there
is included the approximately 250 bp sequence extending from the
HindIII site toward the BgII site located in the viral origin of
replication. Further, it is also possible, and often desirable, to
utilize promoter or control sequences normally associated with the
desired gene sequence, provided such control sequences are
compatible with the host cell systems.
[0126] The origin of replication may be obtained from either by
construction of the vector to include an exogenous origin, such as
may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV,
BPV) source, or may be obtained by the host cell chromosomal
replication mechanism. If the vector is integrated into the host
cell chromosome, the latter is often sufficient It will be further
understood that certain of the polypeptides may be present in
quantities below the detection limits of the Coomassie brilliant
blue staining procedure usually employed in the analysis of
SDS/PAGE gels, or that their presence may be masked by an inactive
polypeptide of similar M.sub.r. Although not necessary to the
routine practice of the present invention, it is contemplated that
other detection techniques may be employed advantageously in the
visualization of particular polypeptides of interest.
Immunologically-based techniques such as Western blotting using
enzymatically-, radiolabel-, or fluorescently-tagged antibodies
described herein are considered to be of particular use in this
regard. Alternatively, the peptides of the present invention may be
detected by using antibodies of the present invention in
combination with secondary antibodies having affinity for such
primary antibodies. This secondary antibody may be enzymatically-
or radiolabeled, or alternatively, fluorescently-, or colloidal
gold-tagged. Means for the labeling and detection of such two-step
secondary antibody techniques are well-known to those of skill in
the art.
3. BRIEF DESCRIPTION OF THE DRAWINGS
[0127] The drawings form part of the present specification and are
included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to
one or more of these drawings in combination with the detailed
description of specific embodiments presented herein. The file of
this Patent contains at least one drawing executed in color. Copies
of this Patent with color drawing(s) will be provided by the Patent
and Trademark Office upon request and payment of the necessary
fee.
[0128] FIG. 1. Folding diagram of CBD(169-318). Arrows represent
strands of .beta.-sheets, cylinders represents .alpha.-helices.
Strands A, B, H, D, and E form .beta.-sheet I.
[0129] Strands F, G, C, I, and J form .beta.-sheet II.
[0130] FIGS. 2A and 2B. Two views of the Col-binding domain
CBD(169-318) represented as a ribbon diagram of secondary
structure. Strands A, B, H, D, and E form .beta.-sheet I, strands
F, G, C, I, and J form .beta.-sheet II. N denotes amino-terminus, C
denotes carboxy-terminus.
[0131] FIG. 3A. Connolly's molecular surface of the Col-binding
domain viewing the groove on .beta.-sheet I. The texture highlights
the surface area of residues within 6A from Col probes.
[0132] FIG. 3B. A model of bound Col based on the docking search.
Same view as in FIG. 3A with the Col triple-helix in gold; only
main chains are shown.
[0133] FIG. 4. A stereo view of the Col-binding site on the
.beta.-sheet I.
[0134] FIG. 5A is a graphic representation of the enhancement of
macrophage killing enabled by immunization with M55 in accordance
with the present invention.
[0135] FIG. 5B is a graphic representation of the enhancement of
phagocytosis enabled by immunization with M55 in accordance with
the present invention.
[0136] FIG. 6 is a graphic representation of studies determining
the serum level of antibodies against the collagen adhesin peptides
of the present invention.
[0137] FIG. 7A is a graphic representation of the results of
studies to detect human antibodies to M55 in accordance with the
present invention.
[0138] FIG. 7B is a graphic representation of the results of
studies to detect human antibodies to M55 in accordance with the
present invention.
[0139] FIG. 8 is a graphic representation of the protective
effective in mice against a lethal S. aureus challenge by passive
immunization with rat anti-M55 in accordance with the present
invention.
4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0140] The technology described herein is used to develop methods
and compositions that specifically interfere with bacterial
adhesion and the subsequent colonization host tissues, thus
resulting in the prevention of infection, and the prevention of
diseases caused by bacteria which express CBPs on the cell surface.
The technology is broadly applicable, has the potential to increase
the effectiveness of antibiotic therapy in many situations, and
replace antibiotic therapy in a number of other applications. The
technology is expected to be especially effective in treatment
regimens for staphylococcal and streptococcal infections, and as a
cost-effective prophylaxis for prevention of related diseases. The
elucidation of the crystal structure of the CBP by the inventors
represents a monumental advance in the medical arts, and
particularly in the field of infectious disease diagnosis and
treatment by providing critical information necessary for
identifying compositions which interfere with, or block completely,
the binding of Col to CBPS. Such inhibitors are therefore useful in
the prevention of bacterial adhesion to Col-containing matrices.
The inventors have identified the ligand-binding site in the S.
aureus CBP and a 25-amino-acid peptide has been characterized that
directly inhibits the binding of S. aureus to .sup.125I-labeled
type II Col. Furthermore, site-directed mutagenesis of the CBP has
revealed 2 specific residues critical for ligand binding activity.
The invention has obtained for the first time, novel compositions
for use in methods to identify inhibitors of the interaction
between Col and the CBP both in vitro and in vivo.
[0141] 4.1 The Role of CBP as a Virulence Factor
[0142] To determine the importance of the Col adhesin as a
virulence factor in staphylococcal induced septic arthritis, two
classes of mutants have been constructed. In the first class of
mutants, the isolated Col adhesin gene. cna, was inactivated in a
S. aureus clinical isolate obtained from a patient with
osteomvelitis. In the second type of mutant, the active cna gene
was introduced into a S. aureus strain that lacked the gene.
[0143] The virulence of the two classes of S. aureus mutants have
been compared with their respective parent strains using a newly
developed and characterized murine model of septic arthritis
(Bremell et al., 1991). In this model, mice display
histopathological signs of arthritis peaking approximately 2-3
weeks post-injection, both with regard to intensity and extension
of arthritis, and leveling off thereafter. Clinically estimated
signs of arthritis correlate closely to the histopathological
evaluation (Bremell et al., 1992). Tail lesions with inflammatory
cells invading and destroying the disk and the bone tissue occur
within 4 weeks after inoculation in approximately 50% of the mice.
Furthermore, the arthritic mice often display a tremendous IL-6
driven polyclonal B-cell activation (Bremell et al., 1992).
[0144] The results showed that mice injected intravenously with S.
aureus strains expressing the Col adhesin were much more likely to
develop arthritis compared to mice injected with the S. aureus
mutant strains. Moreover, serum levels of IgG1 and IL-6 were
dramatically elevated in mice injected with the CNA clinical
isolate compared to mice injected with the CNA mutant or saline
(Patti et al., 1994). Taken together these data demonstrate that
the Col adhesin is an important virulence factor in septic
arthritis induced by staphylococci.
[0145] 4.2 MSCRAMMs
[0146] Bacterial adherence to host tissue involves specific
microbial surface adhesins of which a subfamily termed MSCRAMMs
(Patti et al., 1994; Patti and Hook, 1994) specifically recognize
ECM components. Many pathogenic bacteria have been shown to
specifically recognize and bind to various components of the ECM in
an interaction which appears to represent a host tissue
colonization mechanism.
[0147] MSCRAMMs (on the bacterial cell surface) and ligands (within
the host tissue) are the molecules that interact in a lock and key
fashion resulting in the adherence of bacteria to the host.
Complete blockage of microbial adhesion is not required to prevent
infection. It is only necessary to keep the bacterial inoculum
below a critical mass. Several strategies have been developed which
are particularly useful in combating bacterial infections, such as
infection by streptococcal and staphylococcal species, by
preventing bacterial adhesion to Col substrata including the ECM of
susceptible host cells. Such strategies are contemplated to be
useful in the diagnosis, treatment, and prophylaxis of such
infections.
[0148] 4.3 Extracellular Matrix
[0149] The ECM contains numerous glycoproteins and proteoglycans
which, through inter- and intramolecular interactions, form
insoluble matrices. The ECM has a structural function in the
tissues but also affects the cellular physiology of the organism.
Perhaps the best characterized biological functions of the ECM are
related to its ability to serve as a substrata for the adhesion of
host tissue cells. This process involves the integrins, a family of
heterodimeric (.alpha./.beta.) cell surface receptors which
recognize specific structures in many of the ECM proteins. It is
clear that many bacteria also have taken advantage of the ECM as a
substrate for adhesion. Like most eukaryotic tissue cells, many
bacteria have developed several parallel adhesion mechanisms and
this apparent redundancy may reflect the importance of host tissue
adherence for the prosperity of the bacteria.
[0150] The adherence of microbes to various cell-surface and ECM
components has been widely reported (Abraham et al., 1983; Coburn
et al., 1993; Froman et al., 1984; Isaacs, 1994; Maxe et al., 1986;
Van Nhieu and Isber, 1993). The present invention has identified a
new bacterial MSCRAMM which promotes bacterial adhesion to Col and
other proteoglycans which are structurally similar to Col, which
are found in conjunction with ECM components such as Col.
[0151] 4.4 Collagen
[0152] Collagenous proteins are the major constituents of the ECM
(Bornstein and Sage, 1980). Most Cols are synthesized
intracellularly as precursor molecules and undergo extensive
posttranslational processing prior to secretion and incorporation
into the ECM or other Col-rich tissues such as cartilage
(Ramachandran and Reddi, 1976). To date over 18 different type of
Cols have been defined, and they are loosely categorized into five
groups (Vanderrest and Garrone, 1991). These groups are:
[0153] 1) Col types I, II, III, V, and XI which participate in
quarter-staggered fibrils;
[0154] 2) Col types XII, XIV, and IX which are fibril-associated
with interrupted triple helices;
[0155] 3) Col types IV, VIII, and X which form sheets;
[0156] 4) Col type VI which forms beaded filaments; and
[0157] 5) Col type VII, which forms anchoring fibrils.
[0158] The Col network in skin is composed predominantly of Cols
type I and type III. Col can inhibit transforming growth factor
beta activity (TGF-.beta.) (Yamaguchi et al., 1990) and inactivate
the complement component C1q (Krumdieck et al., 1992) and has been
proposed to act as an anti-inflammatory agent through these
interactions.
[0159] 4.5 CNA-encoding Nucleic Acid Segments
[0160] As used herein, the term "CBP gene" is used to refer to a
cna gene or DNA coding region that encodes a protein, polypeptide
or peptide that is capable of binding Col, or a related ECM
component.
[0161] The definition of a "CBP gene", as used herein, is a gene
that hybridizes, under relatively stringent hybridization
conditions (see, e.g., Maniatis et al., 1982), to DNA sequences
presently known to include CBP-encoding gene sequences. It will, of
course, be understood that one or more than one genes encoding CBPs
or peptides may be used in the methods and compositions of the
invention. The nucleic acid compositions and methods disclosed
herein may entail the administration of one, two, three, or more,
genes or gene segments. The maximum number of genes that may be
used is limited only by practical considerations, such as the
effort involved in simultaneously preparing a large number of gene
constructs or even the possibility of eliciting a significant
adverse cytotoxic effect.
[0162] In using multiple genes, they may be combined on a single
genetic construct under control of one or more promoters, or they
may be prepared as separate constructs of the same of different
types. Thus, an almost endless combination of different genes and
genetic constructs may be employed. Certain gene combinations may
be designed to, or their use may otherwise result in, achieving
synergistic effects on formation of an immune response, or the
development of antibodies to gene products encoded by such nucleic
acid segments, or in the production of diagnostic and treatment
protocols for streptococcal or staphylococcal infection, and in
particular, infection with S. aureus, S. dysgalactiae, and S.
pyogenes. Any and all such combinations are intended to fall within
the scope of the present invention. Indeed, many synergistic
effects have been described in the scientific literature, so that
one of ordinary skill in the art would readily be able to identify
likely synergistic gene combinations, or even gene-protein
combinations.
[0163] It will also be understood that, if desired, the nucleic
segment or gene could be administered in combination with further
agents, such as, e.g., proteins or polypeptides or various
pharmaceutically active agents. So long as genetic material forms
part of the composition, there is virtually no limit to other
components which may also be included, given that the additional
agents do not cause a significant adverse effect upon contact with
the target cells or tissues.
[0164] 4.6 Therapeutic and Diagnostic Kits Comprising CBP
Compositions
[0165] Therapeutic kits comprising, in suitable container means, a
CBP composition of the present invention in a pharmaceutically
acceptable formulation represent another aspect of the invention.
The CBP composition may be native CBP, truncated CBP,
site-specifically mutated CBP, or CBP-encoded peptide epitopes, or
alternatively antibodies which bind native CBP, truncated CBP,
site-specifically mutated CBP, or CBP-encoded peptide epitopes. In
other embodiments, the CBP composition may be nucleic acid segments
encoding native CBP, truncated CBP, site-specifically mutated CBP,
or CBP-encoded peptide epitopes. Such nucleic acid segments may be
DNA or RNA, and may be either native, recombinant, or mutagenized
nucleic acid segments.
[0166] The kits may comprise a single container means that contains
the CBP composition. The container means may, if desired, contain a
pharmaceutically acceptable sterile excipient, having associated
with it, the CBP composition and, optionally, a detectable label or
imaging agent. The container means may itself be a syringe,
pipette, or other such like apparatus, from which the CBP
composition may be applied to a tissue site, skin lesion, wound
area, or other site of bacterial infection. However, the single
container means may contain a dry, or lyophilized, mixture of a CBP
composition, which may or may not require pre-wetting before
use.
[0167] Alternatively, the kits of the invention may comprise
distinct container means for each component. In such cases, one
container would contain the CBP composition, either as a sterile
protein, RNA or DNA solution or in a lyophilized form, and the
other container would include the carrier, which may or may not
itself be solid or in a sterile solution, or be in a gelatinous,
liquid or other form.
[0168] The kits may also comprise a second or third container means
for containing a sterile, pharmaceutically acceptable buffer,
diluent or solvent. Such a solution may be required to formulate
the CBP component into a more suitable form for application to the
body, e.g., as a topical preparation, or alternatively, in oral,
parenteral, or intravenous forms. It should be noted, however, that
all components of a kit could be supplied in a dry form
(lyophilized), which would allow for "wetting" upon contact with
body fluids. Thus, the presence of any type of pharmaceutically
acceptable buffer or solvent is not a requirement for the kits of
the invention. The kits may also comprise a second or third
container means for containing a pharmaceutically acceptable
detectable imaging agent or composition.
[0169] The container means will generally be a container such as a
vial, test tube, flask, bottle, syringe or other container means,
into which the components of the kit may placed. The components may
also be aliquoted into smaller containers, should this be desired.
The kits of the present invention may also include a means for
containing the individual containers in close confinement for
commercial sale, such as, e.g, injection or blow-molded plastic
containers into which the desired vials or syringes are
retained.
[0170] Irrespective of the number of containers, the kits of the
invention may also comprise, or be packaged with, an instrument for
assisting with the placement of the CPB composition within the body
of an animal. Such an instrument may be a syringe, pipette,
forceps, or any such medically-approved delivery vehicle.
[0171] 4.7 Affinity Chromatography
[0172] Affinity chromatography is generally based on the
recognition of a protein by a substance such as a ligand or an
antibody. The column material may be synthesized by covalently
coupling a binding molecule, such as an activated dye, for example
to an insoluble matrix. The column material is then allowed to
adsorb the desired substance from solution. Next, the conditions
are changed to those under which binding does not occur and the
substrate is eluted. The requirements for successful affinity
chromatography are:
[0173] 1) that the matrix must specifically-adsorb the molecules of
interest;
[0174] 2) that other contaminants remain unadsorbed;
[0175] 3) that the ligand must be coupled without altering its
binding activity;
[0176] 4) that the ligand must bind sufficiently tight to the
matrix; and
[0177] 5) that it must be possible to elute the molecules of
interest without destroying them.
[0178] A preferred embodiment of the present invention is an
affinity chromatography method for purification of antibodies from
solution wherein the matrix contains CBPs or peptide epitopes
derived from CBPs, covalently-coupled to a Sepharose CL6B or
CL4B.
[0179] This matrix binds the antibodies of the present invention
directly and allows their separation by elution with an appropriate
gradient such as salt, GuHCl, pH, or urea. Another preferred
embodiment of the present invention is an affinity chromatography
method for the purification of CBPs and peptide epitopes from
solution. In this case, the matrix may be an antibody specific for
CBP or alternatively a composition having affinity for CBPs. The
amino acid compositions of the present invention are thus bound
directly, and this allows their subsequent purification by elution
from the column with a suitable buffer as described above.
[0180] 4.8 Methods of Nucleic Acid Delivery and DNA
Transfection
[0181] In certain embodiments, it is contemplated that the nucleic
acid segments disclosed herein will be used to transfect
appropriate host cells. Technology for introduction of DNA into
cells is well-known to those of skill in the art. Four general
methods for delivering a nucleic segment into cells have been
described:
[0182] (1) chemical methods (Graham and VanDerEb, 1973);
[0183] (2) physical methods such as microinjection (Capecchi,
1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985)
and the gene gun (Yang et al., 1990);
[0184] (3) viral vectors (Clapp, 1993; Eglitis and Anderson, 1988);
and
[0185] (4) receptor-mediated mechanisms (Curiel et al., 1991;
Wagner et al., 1992).
[0186] 4.9 Liposomes and Nanocapsules
[0187] In certain embodiments, the inventors contemplate the use of
liposomes and/or nanocapsules for the introduction of particular
peptides or nucleic acid segments into host cells. In particular,
the malonyltyrosyl and phosphotyrosyl peptides of the present
invention may be formulated for delivery in solution with DMSO or
encapsulated in liposomes.
[0188] Such formulations may be preferred for the introduction of
pharmaceutically-acceptable formulations of the nucleic acids,
peptides, and/or antibodies disclosed herein. The formation and use
of liposomes is generally known to those of skill in the art (see
for example, Couvreur et al., 1977; 1988 which describes the use of
liposomes and nanocapsules in the targeted antibiotic therapy of
intracellular bacterial infections and diseases). Recently,
liposomes were developed with improved serum stability and
circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen
and Choun, 1987).
[0189] Liposomes have been used successfully with a number of cell
types that are normally resistant to transfection by other
procedures including T cell suspensions, primary hepatocyte
cultures and PC 12 cells (Muller et al., 1990). In addition,
liposomes are free of the DNA length constraints that are typical
of viral-based delivery systems. Liposomes have been used
effectively to introduce genes, drugs (Heath and Martin, 1986;
Heath et al., 1986; Balazsovits et al., 1989), radiotherapeutic
agents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; 1
990b), viruses (Faller and Baltimore, 1984), transcription factors
and allosteric effectors (Nicolau and Gersonde, 1979) into a
variety of cultured cell lines and animals. In addition, several
successful clinical trails examining the effectiveness of
liposome-mediated drug delivery have been completed
(Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier et al.,
1988). Furthermore, several studies suggest that the use of
liposomes is not associated with autoimrnmune responses, toxicity
or gonadal localization after systemic delivery (Mori and Fukatsu,
1992).
[0190] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 A,
containing an aqueous solution in the core.
[0191] Liposomes bear many resemblances to cellular membranes and
are contemplated for use in connection with the present invention
as carriers for the peptide compositions. They are widely suitable
as both water- and lipid-soluble substances can be entrapped, i.e.,
in the aqueous spaces and within the bilayer itself, respectively.
It is possible that the drug-bearing liposomes may even be employed
for site-specific delivery of active agents by selectively
modifying the liposomal formulation.
[0192] In addition to the teachings of Couvreur et al. (1977;
1988), the following information may be utilized in generating
liposomal formulations. Phospholipids can form a variety of
structures other than liposomes when dispersed in water, depending
on the molar ratio of lipid to water. At low ratios the liposome is
the preferred structure. The physical characteristics of liposomes
depend on pH, ionic strength and the presence of divalent cations.
Liposomes can show low permeability to ionic and polar substances,
but at elevated temperatures undergo a phase transition which
markedly alters their permeability. The phase transition involves a
change from a closely packed, ordered structure, known as the gel
state, to a loosely packed, less-ordered structure, known as the
fluid state. This occurs at a characteristic phase-transition
temperature and results in an increase in permeability to ions,
sugars and drugs.
[0193] In addition to temperature, exposure to proteins can alter
the permeability of liposomes. Certain soluble proteins such as
cytochrome c bind, deform and penetrate the bilayer, thereby
causing changes in permeability. Cholesterol inhibits this
penetration of proteins, apparently by packing the phospholipids
more tightly. It is contemplated that the most useful liposome
formations for antibiotic and inhibitor delivery will contain
cholesterol.
[0194] The ability to trap solutes varies between different types
of liposomes. For example, MLVs are moderately efficient at
trapping solutes, but SUVs are extremely inefficient. SUVs offer
the advantage of homogeneity and reproducibility in size
distribution, however, and a compromise between size and trapping
efficiency is offered by large unilamellar vesicles (LUVs). These
are prepared by ether evaporation and are three to four times more
efficient at solute entrapment than MLVs.
[0195] In addition to liposome characteristics, an important
determinant in entrapping compounds is the physicochemical
properties of the compound itself Polar compounds are trapped in
the aqueous spaces and nonpolar compounds bind to the lipid bilayer
of the vesicle. Polar compounds are released through permeation or
when the bilayer is broken, but nonpolar compounds remain
affiliated with the bilayer unless it is disrupted by temperature
or exposure to lipoproteins. Both types show maximum efflux rates
at the phase transition temperature.
[0196] Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with- cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. It often
is difficult to determine which mechanism is operative and more
than one may operate at the same time.
[0197] The fate and disposition of intravenously injected liposomes
depend on their physical properties, such as size, fluidity and
surface charge. They may persist in tissues for hours or days,
depending on their composition, and half lives in the blood range
from minutes to several hours. Larger liposomes, such as MLVs and
LUVs, are taken up rapidly by phagocytic cells of the
reticuloendothelial system, but physiology of the circulatory
system restrains the exit of such large species at most sites. They
can exit only in places where large openings or pores exist in the
capillary endotheliurn, such as the sinusoids of the liver or
spleen. Thus, these organs are the predominate site of uptake. On
the other hand, SUVs show a broader tissue distribution but still
are sequestered highly in the liver and spleen. In general, this in
vivo behavior limits the potential targeting of liposomes to only
those organs and tissues accessible to their large size. These
include the blood, liver, spleen, bone marrow and lymphoid
organs.
[0198] Targeting is generally not a limitation in terms of the
present invention. However, should specific targeting be desired,
methods are available for this to be accomplished. Antibodies may
be used to bind to the liposome surface and to direct the antibody
and its drug contents to specific antigenic receptors located on a
particular cell-type surface. Carbohydrate determinants
(glycoprotein or glycolipid cell-surface components that play a
role in cell-cell recognition, interaction and adhesion) may also
be used as recognition sites as they have potential in directing
liposomes to particular cell types. Mostly, it is contemplated that
intravenous injection of liposomal preparations would be used, but
other routes of administration are also conceivable.
[0199] Alternatively, the invention provides for
pharmaceutically-acceptab- le nanocapsule formulations of the
peptides of the present invention. Nanocapsules can generally
entrap compounds in a stable and reproducible way (Henry-Michelland
et al., 1987). To avoid side effects due to intracellular polymeric
overloading, such ultrafine particles (sized around 0.1 .mu.m)
should be designed using polymers able to be degraded in vivo.
Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these
requirements are contemplated for use in the present invention, and
such particles may be are easily made, as described (Couvreur et
al., 1984; 1988).
[0200] 4.10 Methods for Preparing Antibody Compositions
[0201] In another aspect, the present invention contemplates an
antibody that is immunoreactive with a polypeptide of the
invention. As stated above, one of the uses for CBPs and
CBP-derived epitopic peptides according to the present invention is
to generate antibodies. Reference to antibodies throughout the
specification includes whole polyclonal and monoclonal antibodies
(mAbs), and parts thereof, either alone or conjugated with other
moieties. Antibody parts include Fab and F(ab).sub.2 fragments and
single chain antibodies. The antibodies may be made in vivo in
suitable laboratory animals, by imrunnizing donors (preferably
humans), or in vitro using recombinant DNA techniques. In a
preferred embodiment, an antibody is a polyclonal antibody. Means
for preparing and characterizing antibodies are well known in the
art (See, e.g., Harlow and Lane, 1988).
[0202] Briefly, a polyclonal antibody is prepared by immunizing an
animal with an immunogen comprising a polypeptide of the present
invention and collecting antisera from that immunized animal. A
wide range of animal species can be used for the production of
antisera. Typically an animal used for production of anti-antisera
is a rabbit, a mouse, a rat, a hamster, a goat, or a guinea pig.
Because of the relatively large blood volume of rabbits, a rabbit
is a preferred choice for production of polyclonal antibodies.
[0203] Antibodies, both polyclonal and monoclonal, specific for CBP
and CBP-derived epitopes may be prepared using conventional
immunization techniques, as will be generally known to those of
skill in the art. A composition containg antigenic epitopes of
particular CBPs can be used to immunize one or more experimental
animals, such as a rabbit or mouse, which will then proceed to
produce specific antibodies against CBP peptide. Polyclonal
antisera may be obtained, after allowing time for antibody
generation, simply by bleeding the animal and preparing serum
samples from the whole blood.
[0204] The amount of immunogen composition used in the production
of polyclonal antibodies varies upon the nature of the immunogen,
as well as the animal used for immunization. A variety of routes
can be used to administer the immunogen (subcutaneous,
intramuscular, intradermal, intravenous and intraperitoneal). The
production of polyclonal antibodies may be monitored by sampling
blood of the immunized animal at various points following
immunization. A second, booster injection, also may be given. The
process of boosting and titering is repeated until a suitable titer
is achieved. When a desired level of immunogenicity is obtained,
the immunized animal can be bled and the serum isolated and stored,
and/or the animal can be used to generate mAbs (below).
[0205] One of the important features obtained by the present
invention is a polyclonal sera that is relatively homogenous with
respect to the specificity of the antibodies therein. Typically,
polyclonal antisera is derived from a variety of different
"clones," i.e., B-cells of different lineage. mAbs, by contrast,
are defined as coming from antibody-producing cells with a common
B-cell ancestor, hence their "mono" clonality.
[0206] When peptides are used as antigens to raise polyclonal sera,
one would expect considerably less variation in the clonal nature
of the sera than if a whole antigen were employed. Unfortunately,
if incomplete fragments of an epitope are presented, the peptide
may very well assume multiple (and probably non-native)
conformations. As a result, even short peptides can produce
polyclonal antisera with relatively plural specificities and,
unfortunately, an antisera that does not react or reacts poorly
with the native molecule.
[0207] Polyclonal antisera according to present invention is
produced against peptides that are predicted to comprise whole,
intact epitopes. It is believed that these epitopes are, therefore,
more stable in an immunologic sense and thus express a more
consistent immunologic target for the immune system. Under this
model, the number of potential B-cell clones that will respond to
this peptide is considerably smaller and, hence, the homogeneity of
the resulting sera will be higher. In various embodiments, the
present invention provides for polyclonal antisera where the
clonality, i.e., the percentage of clone reacting with the same
molecular determinant, is at least 80%. Even higher clonality -90%,
95% or greater - is contemplated.
[0208] To obtain mAbs, one would also initially immunize an
experimental animal, often preferably a mouse, with a
CBP-containing composition. One would then, after a period of time
sufficient to allow antibody generation, obtain a population of
spleen or lymph cells from the animal. The spleen or lymph cells
can then be fused with cell lines, such as human or mouse myeloma
strains, to produce antibody-secreting hybridomas. These hybridomas
may be isolated to obtain individual clones which can then be
screened for production of antibody to the desired peptide.
[0209] Following immunization, spleen cells are removed and fused,
using a standard fusion protocol with plasmacytoma cells to produce
hybridomas secreting mAbs against CBP. Hybridomas which produce
rnAbs to the selected antigens are identified using standard
techniques, such as ELISA and Western blot methods. Hybridoma
clones can then be cultured in liquid media and the culture
supernatants purified to provide the CBP-specific mabs.
[0210] It is proposed that the mabs of the present invention will
also find useful application in immunochemical procedures, such as
ELISA and Western blot methods, as well as other procedures such as
immunoprecipitation, immunocytological methods, etc. which may
utilize antibodies specific to CBPs. In particular, CBP antibodies
may be used in immunoabsorbent protocols to purify native or
recombinant CBPs or CBP-derived peptide species or synthetic or
natural variants thereof.
[0211] The antibodies disclosed herein may be employed in antibody
cloning protocols to obtain cDNAs or genes encoding CBPs from other
species or organisms, or to identify proteins having significant
homology to CBP. They may also be used in inhibition studies to
analyze the effects of CBP in cells, tissues, or whole aninals.
Anti-CBP antibodies will also be useful in immunolocalization
studies to analyze the distribution of bacteria expressing CBPs
during cellular infection, for example, to determine the cellular
or tissue-specific distribution of streptococci and staphylococci
under different physiological conditions. A particularly useful
application of such antibodies is in purifying native or
recombinant CBPs, for example, using an antibody affinity column.
The operation of all such immunological techniques will be known to
those of skill in the art in light of the present disclosure.
[0212] 4.11 Recombinant Expression of CBP
[0213] Recombinant clones expressing the cna nucleic acid segments
may be used to prepare purified recombinant CBP (rCBP), purified
rCBP-derived peptide antigens as well as mutant or variant
recombinant protein species in significant quantities. The selected
antigens, and variants thereof, are proposed to have significant
utility in diagnosing and treating infections caused by S. aureus,
S. pyogenes and S. dysgalactiae. For example, it is proposed that
rCBPs, peptide variants thereof, and/or antibodies against such
rCBPs may also be used in immunoassays to detect S. aureus, S.
pyogenes and S. dysgalactiae cells or as vaccines or
immunotherapeutics to treat S. aureus, S. pyogenes and S.
dysgalactiae infections, and to prevent bacterial adhesion to ECM
components such as Col in the same manner as native CBP
compositions.
[0214] Additionally, by application of techniques such as DNA
mutagenesis, the present invention allows the ready preparation of
so-called "second generation" molecules having modified or
simplified protein structures. Second generation proteins will
typically share one or more properties in common with the
full-length antigen, such as a particular antigenic/immunogenic
epitopic core sequence. Epitopic sequences can be obtained on
relatively short molecules prepared from knowledge of the peptide,
or encoding DNA sequence information. Such variant molecules may
not only be derived from selected immunogenic/antigenic regions of
the protein structure, but may additionally, or alternatively,
include one or more functionally equivalent amino acids selected on
the basis of similarities or even differences with respect to the
natural sequence. This is particularly desirable in the preparation
of blocking antibodies which prevent bacterial adhesion to Col, as
outlined herein.
[0215] 4.12 Antibody Compositions and Formulations Thereof
[0216] Means for preparing and characterizing antibodies are well
known in the art (See, e.g., Harlow and Lane (1988); incorporated
herein by reference). The methods for generating mAbs generally
begin along the same lines as those for preparing polyclonal
antibodies. Briefly, a polyclonal antibody is prepared by
immunizing an animal with an immunogenic composition in accordance
with the present invention and collecting antisera from that
immunized animal. A wide range of animal species can be used for
the production of antisera. Typically the animal used for
production of anti-antisera is a rabbit, a mouse, a rat, a hamster,
a guinea pig or a goat. Because of the relatively large blood
volume of rabbits, a rabbit is a preferred choice for production of
polyclonal antibodies.
[0217] As is well known in the art, a given composition may vary in
its immunogenicity. It is often necessary therefore to boost the
host immune system, as may be achieved by coupling a peptide or
polypeptide immunogen to a carrier. Exemplary and preferred
carriers are keyhole limpet hemocyanin (KLH) and bovine serum
albumin (BSA). Other albumins such as ovalbumin, mouse serum
albumin or rabbit serum albumin can also be used as carriers. Means
for conjugating a polypeptide to a carrier protein are well known
in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hy-
droxysuccinimide ester, carbodiimide and bis-biazotized
benzidine.
[0218] mAbs may be readily prepared through use of well-known
techniques, such as those exemplified in U.S. Pat. No. 4,196,265,
incorporated herein by reference. Typically, this technique
involves immunizing a suitable animal with a selected immunogen
composition, e.g., a purified or partially purified protein,
polypeptide or peptide. The immunizing composition is administered
in a manner effective to stimulate antibody producing cells.
Rodents such as mice and rats are preferred animals, however, the
use of rabbit, sheep or frog cells is also possible. The use of
rats may provide certain advantages (Goding, 1986), but mice are
preferred, with the BALB/c mouse being most preferred as this is
most routinely used and generally gives a higher percentage of
stable fusions.
[0219] Following immunization, somatic cells with the potential for
producing antibodies, specifically B-lymphocytes (B-cells), are
selected for use in the mAb generating protocol. These cells may be
obtained from biopsied spleens, tonsils or lymph nodes, or from a
peripheral blood sample. Spleen cells and peripheral blood cells
are preferred, the former because they are a rich source of
antibody-producing cells that are in the dividing plasmablast
stage, and the latter because peripheral blood is easily
accessible. Often, a panel of animals will have been immunized and
the spleen of animal with the highest antibody titer will be
removed and the spleen lymphocytes obtained by homogenizing the
spleen with a syringe. Typically, a spleen from an immunized mouse
contains approximately about 5.times.10.sup.7 to about
2.times.10.sup.8 lymphocytes.
[0220] The antibody-producing B lymphocytes from the immunized
animal are then fused with cells of an immortal myeloma cell,
generally one of the same species as the animal that was immunized.
Myeloma cell lines suited for use in hybridoma-producing fusion
procedures preferably are non-antibody-producing, have high fusion
efficiency, and enzyme deficiencies that render them incapable of
growing in certain selective media which support the growth of only
the desired fused cells (hybridomas).
[0221] Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (Goding, 1986; Campbell, 1984).
For example, where the immunized animal is a mouse, one may use
P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U,
MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use
R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,
GM1500-GRG2,LICR-LON-HMy2 and UC729-6 are all useful in connection
with human cell fusions.
[0222] One preferred murine myeloma cell is the NS-1 myeloma cell
line (also termed P3-NS-1-Ag4-1), which is readily available from
the NIGMS Human Genetic Mutant Cell Repository by requesting cell
line repository number GM3573. Another mouse myeloma cell line that
may be used is the 8-azaguanine-resistant mouse murine myeloma
SP2/0non-producer cell line.
[0223] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually comprise mixing
somatic cells with myeloma cells in a 2:1 ratio, though the ratio
may vary from about 20:1 to about 1:1, respectively, in the
presence of an agent or agents (chemical or electrical) that
promote the fusion of cell membranes. Fusion methods using Sendai
virus have been described (Kohler and Milstein, 1975; 1976), and
those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by
Gefter et al. (1977). The use of electrically induced fusion
methods is also appropriate (Goding, 1986).
[0224] Fusion procedures usually produce viable hybrids at low
fiequencies, about 1.times.10.sup.-6 to about 1.times.10.sup.-8 .
However, this does not pose a problem, as the viable, fused hybrids
are differentiated from the parental, unfused cells (particularly
the unfused myeloma cells that would normally continue to divide
indefinitely) by culturing in a selective medium. The selective
medium is generally one that contains an agent that blocks the de
novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides (HAT medium). Where azaserine is used, the media is
supplemented with hypoxanthine.
[0225] The preferred selection medium is HAT. Only cells capable of
operating nucleotide salvage pathways are able to survive in HAT
medium. The myeloma cells are defective in key enzymes of the
salvage pathway e.g., hypoxanthine phosphoribosyl transferase
(HPRT), and they cannot survive. The B-cells can operate this
pathway, but they have a limited life span in culture and generally
die within about two weeks. Therefore, the only cells that can
survive in the selective media are those hybrids formed from
myeloma and B-cells.
[0226] This culturing provides a population of hybridomas from
which. specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants (after about two to three weeks) for the
desired reactivity. The assay should be sensitive, simple and
rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity
assays, plaque assays, dot immunobinding assays, and the like.
[0227] The selected hybridomas would then be serially diluted and
cloned into individual antibody-producing cell lines, which clones
can then be propagated indefinitely to provide mAbs. The cell lines
may be exploited for mAb production in two basic ways. A sample of
the hybridoma can be injected (often into the peritoneal cavity)
into a histocompatible animal of the type that was used to provide
the somatic and myeloma cells for the original fusion. The injected
animal develops tumors secreting the specific mAb produced by the
fused cell hybrid. The body fluids of the animal, such as serum or
ascites fluid, can then be tapped to provide mAbs in high
concentration. The individual cell lines could also be cultured in
vitro, where the mabs are naturally secreted into the culture
medium from which they can be readily obtained in high
concentrations. mAbs produced by either means may be further
purified, if desired, using filtration, centrifugation and various
chromatographic methods such as HPLC or affinity
chromatography.
[0228] 4.13 Immunoassays
[0229] As noted, it is proposed that native and
synthetically-derived peptides and peptide epitopes of the
invention will find utility as immunogens, e.g., in connection with
vaccine development, or as antigens in immunoassays for the
detection of reactive antibodies. Turning first to immunoassays, in
their most simple and direct sense, preferred immunoassays of the
invention include the various types of enzyme linked immunosorbent
assays (ELISAs), as are known to those of skill in the art.
However, it will be readily appreciated that the utility of
CBP-derived proteins and peptides is not limited to such assays,
and that other useful embodiments include RIAs and other non-enzyme
linked antibody binding assays and procedures.
[0230] In preferred ELISA assays, proteins or peptides
incorporating CBP, rCBP, or CBP-derived protein antigen sequences
are immobilized onto a selected surface, preferably a surface
exhibiting a protein affinity, such as the wells of a polystyrene
microtiter plate. After washing to remove incompletely adsorbed
material, one would then generally desire to bind or coat a
nonspecific protein that is known to be antigenically neutral with
regard to the test antisera, such as bovine serum albumin (BSA) or
casein, onto the well. This allows for blocking of nonspecific
adsorption sites on the immobilizing surface and thus reduces the
background caused by nonspecific binding of antisera onto the
surface.
[0231] After binding of antigenic material to the well, coating
with a non-reactive material to reduce background, and washing to
remove unbound material, the immobilizing surface is contacted with
the antisera or clinical or biological extract to be tested in a
manner conducive to immune complex (antigen/antibody) formation.
Such conditions preferably include diluting the antisera with
diluents such as BSA, bovine gamma globulin (BGG) and phosphate
buffered saline (PBS)/Tween.TM.. These added agents also tend to
assist in the reduction of nonspecific background. The layered
antisera is then allowed to incubate for, e.g., from 2 to 4 h, at
temperatures preferably on the order of about 250 to about
27.degree. C. Following incubation, the antisera-contacted surface
is washed so as to remove non-immunocomplexed material. A preferred
washing procedure includes washing with a solution such as
PBS/Tween.TM., or borate buffer.
[0232] Following formation of specific immunocomplexes between the
test sample and the bound antigen, and subsequent washing, the
occurrence and the amount of immunocomplex formation may be
determined by subjecting the complex to a second antibody having
specificity for the first. Of course, in that the test sample will
typically be of human origin, the second antibody will preferably
be an antibody having specificity for human antibodies. To provide
a detecting means, the second antibody will preferably have an
associated detectable label, such as an enzyme label, that will
generate a signal, such as color development upon incubating with
an appropriate chromogenic substrate. Thus, for example, one will
desire to contact and incubate the antisera-bound surface with a
urease or peroxidase-conjugated anti-human IgG for a period of time
and under conditions that favor the development of immunocomplex
formation (e.g., incubation for 2 h at room temperature in a
PBS-containing solution such as PBS-Tween.TM.).
[0233] After incubation with the second enzyme-tagged antibody, and
subsequent to washing to remove unbound material, the amount of
label is quantified by incubation with a chromogenic substrate such
as urea and bromocresol purple or
2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and
H.sub.2O.sub.2, in the case of peroxidase as the enzyme label.
Quantitation is then achieved by measuring the degree of color
generation, e.g., using a visible spectrum spectrophotometer.
[0234] ELISAs may be used in conjunction with the invention. In one
such ELISA assay, proteins or peptides incorporating antigenic
sequences of the present invention are immobilized onto a selected
surface, preferably a surface exhibiting a protein affinity such as
the wells of a polystyrene microtiter plate. After washing to
remove incompletely adsorbed material, it is desirable to bind or
coat the assay plate wells with a nonspecific protein that is known
to be antigenically neutral with regard to the test antisera such
as bovine serum albumin (BSA), casein or solutions of powdered
milk. This allows for blocking of nonspecific adsorption sites on
the immobilizing surface and thus reduces the background caused by
nonspecific binding of antisera onto the surface.
[0235] 4.14 Immunoprecipitation
[0236] The anti-CBP antibodies of the present invention are
particularly useful for the isolation of CBP antigens by
immunoprecipitation. Immunoprecipitation involves the separation of
the target antigen component from a complex mixture, and is used to
discriminate or isolate min amounts of protein. For the isolation
of cell-surface localized proteins such as CBP, peptides must be
solubilized from the bacterial cell wall by treatment with enzymes
such as lysozyme, lysostaphin or mutanolysin, or alternatively,
into detergent micelles. Nonionic salts are preferred, since other
agents such as bile salts, precipitate at acid pH or in the
presence of bivalent cations.
[0237] In an alternative embodiment the antibodies of the present
invention are useful for the close juxtaposition of two antigens.
This is particularly useful for increasing the -localized
concentration of antigens, e.g., enzyme-substrate pairs.
[0238] In a related embodiment, antibodies of the present invention
are useful for promoting the binding of Col to cna gene products.
Such binding is readily measured by monitoring ligand binding using
well-known procedures. Detection of the binding may be accomplished
by using radioactively labeled antibodies or alternatively,
radioactively-labeled Col. Alternatively, assays employing
biotin-labeled antibodies are also well-known in the art as
described (Bayer and Wilchek, 1980). 4.15 WESTERN BLOTS
[0239] The compositions of the present invention will find great
use in immunoblot or western blot analysis. The anti-CBP antibodies
may be used as high-affinity primary reagents for the
identification of proteins immobilized onto a solid support matrix,
such as nitrocellulose, nylon or combinations thereof. In
conjunction with immunoprecipitation, followed by gel
electrophoresis, these may be used as a single step reagent for use
in detecting antigens against which secondary reagents used in the
detection of the antigen cause an adverse background. This is
especially useful when the antigens studied are immunoglobulins
(precluding the use of immunoglobulins binding bacterial cell wall
components), the antigens studied cross-react with the detecting
agent, or they migrate at the same relative molecular weight as a
cross-reacting signal. Immunologically-based detection methods in
conjunction with Western blotting (including enzymatically-,
radiolabel-, or fluorescently-tagged secondary antibodies against
the toxin moiety) are considered to be of particular use in this
regard.
[0240] 4.16 Vaccines
[0241] The present invention contemplates vaccines for use in both
active and passive immunization embodiments. Immunogenic
compositions proposed to be suitable for use as a vaccine may be
prepared most readily directly from the novel immunogenic proteins
and/or peptide epitopes described herein. Preferably the antigenic
material is extensively dialyzed to remove undesired small
molecular weight molecules and/or lyophilized for more ready
formulation into a desired vehicle.
[0242] The preparation of vaccines that contain peptide sequences
as active ingredients is generally well understood in the art, as
exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;
4,599,230; 4,596,792; and 4,578,770, all incorporated herein by
reference. Typically, such vaccines are prepared as injectables,
either as liquid solutions or suspensions, solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. The preparation may also be emulsified. The active
immunogenic ingredient is often mixed with excipients that are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like and combinations thereof
In addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, or adjuvants that enhance the effectiveness of
the vaccines.
[0243] A composition comprising CBP or CBP-derived proteins and/or
native or modified epitopic peptides therefrom may also be the
basis for human vaccines. The preparation of such compositions that
are essentially free from endotoxin can be achieved by following
the published methodology, for example, U.S. Pat. No. 4,271,147
(incorporated herein by reference) discloses methods for the
preparation of Neisseria meningitidis membrane proteins for use in
vaccines.
[0244] CBP and CBP-derived epitope-based vaccines may be
conventionally administered parenterally, by injection, for
example, either subcutaneously or intramuscularly. Additional
formulations that are suitable for other modes of administration
include suppositories and, in some cases, oral formulations. For
suppositories, traditional binders and carriers may include, for
example, polyalkalene glycols or triglycerides: such suppositories
may be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1-2%. Oral formulations include
such normally employed excipients as, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate and the like. These
compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
contain 10-95% of active ingredient, preferably 25-70%.
[0245] The proteins may be formulated into the vaccine as neutral
or salt forms. Pharmaceutically acceptable salts, include the acid
addition salts (formed with the free amino groups of the peptide)
and those that are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups may also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2ethylarnino ethanol, histidine, procaine, and the
like.
[0246] The vaccines may be administered in a manner compatible with
the dosage formulation, and in such amount as will be
therapeutically effective and immununogenic. The quantity to be
administered depends on the subject to be treated, including, e.g.,
the capacity of the individual's immune system to synthesize
antibodies, and the degree of protection desired. Precise amounts
of active ingredient required to be administered will be readily
determinable by the skilled practitioner. However, suitable dosage
ranges are of the order of several hundred micrograms active
ingredient per vaccination. Suitable regimes for initial
administration and booster shots are also variable, but are
typified by an initial administration followed by subsequent
inoculations or other administrations.
[0247] The manner of application may be varied widely. Any of the
conventional methods for administration of a vaccine are
applicable. These are believed to include oral application on a
solid physiologically acceptable base or in a physiologically
acceptable dispersion, parenterally, by injection or the like. The
dosage of the vaccine will depend on the route of administration
and will vary according to the size of the host.
[0248] Various methods of achieving adjuvant effect for the vaccine
includes use of agents such as aluminum hydroxide, or phosphate
(alum), commonly used as 0.05 to 0.1 percent solution in phosphate
buffered saline, admixture with synthetic polymers of sugars
(Carbopol.RTM.) used as 0.25% solution, aggregation of the protein
in the vaccine by heat treatment with temperatures ranging between
about 70.degree. and about 101.degree. C. for 30 sec to 2 min
periods respectively. Aggregation by reactivating with pepsin
treated F(ab) antibodies to albumin, mixture with bacterial cells
such as C. parvum or endotoxins or lipopolysaccharide components of
gram-negative bacteria, emulsion in physiologically acceptable oil
vehicles such as mannide monooleate (Aracel-A.TM.) or emulsion with
20% solution of a perfluorocarbon (Fluosol-DA.TM.) used as a block
substitute may also be employed.
[0249] In many instances, it will be desirable to have multiple
administrations of the vaccine, usually not exceeding 6
vaccinations, more usually not exceeding 4 vaccinations and
preferably one or more, usually at least about 3 vaccinations. The
vaccinations will normally be at from 2 to 12 week intervals, more
usually from 3 to 5 week intervals. Periodic boosters at intervals
of 1-5 years, usually 3 years, will be desirable to maintain
protective levels of the antibodies. The course of the immunization
may be followed by assays for antibodies for the supernatant
antigens. The assays may be performed by labeling with conventional
labels, such as radionuclides, enzymes, fluorescers, and the like.
These techniques are well known and may be found in a wide variety
of Patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and
3,949,064, (each specifically incorporated herein by reference, as
illustrative of these types of assays).
[0250] Of course, in light of the new technology on DNA
vaccination, it will be understood that virtually all such
vaccination regimens will be appropriate for use with DNA vectors
and constructs, as described (Ulmer et al., 1993; Tang et al, 1992;
Cox et a., 1993; Fynan et al., 1993; Wang et al., 1993; Whitton et
al., 1993, each incorporated herein by reference). In addition to
parenteral routes of DNA inoculation, including intramuscular and
intravenous injections, mucosal vaccination is also contemplated,
as may be achieved by administering drops of DNA compositions to
the nares or trachea. It is particularly contemplated that a
gene-gun could be used to deliver an effectively immunizing amount
of DNA to the epidermis (Fynan et al., 1993).
[0251] The present invention contemplates vaccines for use in both
active and passive immunization embodiments. Immunogenic
compositions, proposed to be suitable for use as a vaccine, may be
prepared most readily directly from immunogenic peptides prepared
in a manner disclosed herein. Preferably the antigenic material is
extensively dialyzed to remove undesired small molecular weight
molecules and/or lyophilized for more ready formulation into a
desired vehicle. The preparation of vaccines which contain peptide
sequences as active ingredients is generally well understood in the
art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903;
4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated
herein by reference. Typically, such vaccines are prepared as
injectables. Either as liquid solutions or suspensions: solid forms
suitable for solution in, or suspension in, liquid prior to
injection may also be prepared. The preparation may also be
emulsified. The active immunogenic ingredient is often mixed with
excipients which are pharmaceutically acceptable and compatible
with the active ingredient. Suitable excipients are, for example,
water, saline, dextrose, glycerol, ethanol, or the like and
combinations thereof. In addition, if desired, the vaccine may
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, or adjuvants which enhance
the effectiveness of the vaccines.
[0252] 4.17 Pharmaceutical Formulation
[0253] The pharmaceutical compositions disclosed herein may be
orally administered, for example, with an inert diluent or with an
assimilable edible carrier, or they may be enclosed in hard or soft
shell gelatin capsule, or they may be compressed into tablets, or
they may be incorporated directly with the food of the diet. For
oral therapeutic administration, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 0.1% of active compound. The percentage of
the compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 60% of the weight of the
unit The amount of active compounds in such therapeutically useful
compositions is such that a suitable dosage will be obtained.
[0254] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit For instance,
tablets, pills, or capsules may be coated with shellac, sugar or
both. A syrup of elixir may contain the active compounds sucrose as
a sweetening agent methyl and propylparabens as preservatives, a
dye and flavoring, such as cherry or orange flavor. Of course, any
material used in preparing any dosage unit form should be
pharmaceutically pure and substantially non-toxic in the amounts
employed. In addition, the active compounds may be incorporated
into sustained-release preparation and formulations.
[0255] The active compounds may also be administered parenterally
or intraperitoneally. Solutions of the active compounds as free
base or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0256] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0257] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0258] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0259] For oral prophylaxis the polypeptide may be incorporated
with excipients and used in the form of non-ingestible mouthwashes
and dentifrices. A mouthwash may be prepared incorporating the
active ingredient in the required amount in an appropriate solvent,
such as a sodium borate solution (Dobell's Solution).
Alternatively, the active ingredient may be incorporated into an
antiseptic wash containing sodium borate, glycerin and potassium
bicarbonate. The active ingredient may also be dispersed in
dentifrices, including: gels, pastes, powders and slurries. The
active ingredient may be added in a therapeutically effective
amount to a paste dentifrice that may include water, binders,
abrasives, flavoring agents, foaming agents, and humectants.
[0260] The phrase "pharmaceutically-acceptable" refers to molecular
entities and compositions that do not produce an allergic or
similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as an
active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
[0261] The composition can be formulated in a neutral or salt
form.
[0262] Pharmaceutically-acceptable salts, include the acid addition
salts (formed with the free amino groups of the protein) and which
are formed with inorganic acids such as, for example, hydrochloric
or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug release capsules
and the like.
[0263] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biologics standards.
[0264] 4.18 Screening Assays
[0265] Host cells that have been transformed could be used in the
screening of natural and artificially derived compounds or mixtures
to select those that are capable of complexing with the CBP and
CBP-derived proteins of the present invention. This could be useful
in the search for compounds that inhibit or otherwise disrupt, or
even enhance the ability of the microorganism to bind Col. It is
contemplated that effective pharmaceutical agents could be
developed by identifying compounds that complex with the particular
CBP epitopes, including, for example, compounds isolated from
natural sources, such as plant, animal and marine sources, and
various synthetic compounds. Natural or man-made compounds that may
be tested in this manner could also include various minerals and
proteins, peptides or antibodies.
[0266] 4.19 Epitopic Core Sequences
[0267] The present invention is also directed to protein or peptide
compositions, free from total cells and other peptides, which
comprise a purified protein or peptide which incorporates an
epitope that is immunologically cross-reactive with one or more of
the antibodies of the present invention.
[0268] As used herein, the term "incorporating an epitope(s) that
is immunologically cross-reactive with one or more anti-CBP
antibodies" is intended to refer to a peptide or protein antigen
which includes a primary, secondary or tertiary structure similar
to an epitope located within a CBP polypeptide. The level of
similarity will generally be to such a degree that monoclonal or
polyclonal antibodies directed against the CBP polypeptide will
also bind to, react with, or otherwise recognize, the
cross-reactive peptide or protein antigen. Various immunoassay
methods may be employed in conjunction with such antibodies, such
as, for example, Western blotting, ELISA, RIA, and the like, all of
which are known to those of skill in the art.
[0269] The identification of CBP epitopes such as those derived
from cna or cna-like gene products and/or their functional
equivalents, suitable for use in vaccines is a relatively
straightforward matter. For example, one may employ the methods of
Hopp, as taught in U.S. Pat. No. 4,554,101, incorporated herein by
reference, which teaches the identification and preparation of
epitopes from amino acid sequences on the basis of hydrophilicity.
The methods described in several other papers, and software
programs based thereon, can also be used to identify epitopic core
sequences (see, for example, Jameson and Wolf, 1988; Wolf et al.,
1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these
"epitopic core sequences" may then be readily incorporated into
peptides, either through the application of peptide synthesis or
recombinant technology.
[0270] Preferred peptides for use in accordance with the present
invention will generally be on the order of about 5 to about 25
amino acids in length, and more preferably about 8 to about 20
amino acids in length. It is proposed that shorter antigenic
peptide sequences will provide advantages in certain circumstances,
for example, in the preparation of vaccines or in immunologic
detection assays. Exemplary advantages include the ease of
preparation and purification, the relatively low cost and improved
reproducibility of production, and advantageous
biodistribution.
[0271] It is proposed that particular advantages of the present
invention may be realized through the preparation of synthetic
peptides which include modified and/or extended
epitopic/immunogenic core sequences which result in a "universal"
epitopic peptide directed to CBP and CBP-related sequences, or
other domains which bind Color related proteoglycans. It is
proposed that these regions represent those which are most likely
to promote T-cell or B-cell stimulation in an animal, and, hence,
elicit specific antibody production in such an animal.
[0272] An epitopic core sequence, as used herein, is a relatively
short stretch of amino acids that is "complementary" to, and
therefore will bind, antigen binding sites on CBP epitope-specific
antibodies. Additionally or alternatively, an epitopic core
sequence is one that will elicit antibodies that are cross-reactive
with antibodies directed against the peptide compositions of the
present invention. It will be understood that in the context of the
present disclosure, the term "complementary" refers to amino acids
or peptides that exhibit an attractive force towards each other.
Thus, certain epitope core sequences of the present invention may
be operationally defined in terms of their ability to compete with
or perhaps displace the binding of the desired protein antigen with
the corresponding protein-directed antisera.
[0273] In general, the size of the polypeptide antigen is not
believed to be particularly crucial, so long as it is at least
large enough to carry the identified core sequence or sequences.
The smallest useful core sequence expected by the present
disclosure would generally be on the order of about 5 amino acids
in length, with sequences on the order of 8 or 25 being more
preferred. Thus, this size will generally correspond to the
smallest peptide antigens prepared in accordance with the
invention. However, the size of the antigen may be larger where
desired, so long as it contains a basic epitopic core sequence.
[0274] The identification of epitopic core sequences is known to
those of skill in the art, for example, as described in U.S. Pat.
No. 4,554,101, incorporated herein by reference, which teaches the
identification and preparation of epitopes from amino acid
sequences on the basis of hydrophilicity. Moreover, numerous
computer programs are available for use in predicting antigenic
portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et
al., 1988). Computerized peptide sequence analysis programs (e.g.,
DNAStar.RTM. software, DNAStar, Inc., Madison, WI) may also be
useful in designing synthetic CBP peptides and peptide analogs in
accordance with the present disclosure.
[0275] The peptides obtained from by this invention are ideal
targets for use as vaccines or immunoreagents for the treatment of
various staphylococcal- or streptococcal-related diseases, and in
particular, those caused by species which contain CBP and
CBP-encoding genes, and hence those which express either cna or
cna-like gene product(s) on the cell surface and in turn interact
with ECM components such as Col to promote bacterial adhesion to
host cells. In this regard, particular advantages may be realized
through the preparation of synthetic peptides that include
epitopic/immunogenic core sequences. These epitopic core sequences
may be identified as hydrophilic and/or mobile regions of the
polypeptides or those that include a T-cell motif. It is known in
the art that such regions represent those that are most likely to
promote B cell or T cell stimulation, and, hence, elicit specific
antibody production.
[0276] In the case of preventing bacterial adhesion, the
preparation of epitopes which produce antibodies which inhibit the
interaction of a Col-specific gene product and Col or proteoglycans
which are structurally similar to Col are particularly
desirable.
[0277] To confirm that a protein or peptide is immunologically
cross-reactive with, or a biological functional equivalent of, one
or more epitopes of the disclosed peptides is also a
straightforward matter. This can be readily determined using
specific assays, e.g., of a single proposed epitopic sequence, or
using more general screens, e.g., of a pool of randomly generated
synthetic peptides or protein fragments. The screening assays may
be employed to identify either equivalent antigens or
cross-reactive antibodies. In any event, the principle is the same,
ie., based upon competition for binding sites between antibodies
and antigens.
[0278] Suitable competition assays that may be employed include
protocols based upon immunohistochemical assays, ELISAs, RIAs,
Western or dot blotting and the like. In any of the competitive
assays, one of the binding components, generally the known element,
such as the CBP-derived peptide, or a known antibody, will be
labeled with a detectable label and the test components, that
generally remain unlabeled, will be tested for their ability to
reduce the amount of label that is bound to the corresponding
reactive antibody or antigen.
[0279] As an exemplary embodiment, to conduct a competition study
between a CBP and any test antigen, one would first label CBP with
a detectable label, such as, e.g., biotin or an enzymatic,
radioactive or fluorogenic label, to enable subsequent
identification. One would then incubate the labeled antigen with
the other, test, antigen to be examined at various ratios (e.g.,
1:1, 1:10 and 1:100) and, after mixing, one would then add the
mixture to an antibody of the present invention. Preferably, the
known antibody would be immobilized, e.g., by attaching to an ELISA
plate. The ability of the mixture to bind to the antibody would be
determined by detecting the presence of the specifically bound
label.
[0280] This value would then be compared to a control value in
which no potentially competing (test) antigen was included in the
incubation.
[0281] The assay may be any one of a range of immunological assays
based upon hybridization, and the reactive antigens would be
detected by means of detecting their label, e.g., using
streptavidin in the case of biotinylated antigens or by using a
chromogenic substrate in connection with an enzymatic label or by
simply detecting a radioactive or fluorescent label. An antigen
that binds to the same antibody as CBP, for example, will be able
to effectively compete for binding to and thus will significantly
reduce CBP binding, as evidenced by a reduction in the amount of
label detected.
[0282] The reactivity of the labeled antigen, e.g., a CBP
composition, in the absence of any test antigen would be the
control high value. The control low value would be obtained by
incubating the labeled antigen with an excess of unlabeled CBP
antigen, when competition would occur and reduce binding. A
significant reduction in labeled antigen reactivity in the presence
of a test antigen is indicative of a test antigen that is
"cross-reactive", ie., that has binding affinity for the same
antibody. "A significant reduction", in terms of the present
application, may be defined as a reproducible (i.e., consistently
observed) reduction in binding.
[0283] In addition to the peptidyl compounds described herein, the
inventors also contemplate that other sterically similar compounds
may be formulated to mimic the key portions of the peptide
structure. Such compounds, which may be termed peptidomimetics, may
be used in the same manner as the peptides of the invention and
hence are also functional equivalents. The generation of a
structural functional equivalent may be achieved by the techniques
of modeling and chemical design known to those of skill in the art.
It will be understood that all such sterically similar constructs
fall within the scope of the present invention.
[0284] Syntheses of epitopic sequences, or peptides which include
an antigenic epitope within their sequence, are readily achieved
using conventional synthetic techniques such as the solid phase
method (e.g., through the use of a commercially-available peptide
synthesizer such as an Applied Biosysterns Model 430A Peptide
Synthesizer). Peptide antigens synthesized in this manner may then
be aliquoted in predetermined amounts and stored in conventional
manners, such as in aqueous solutions or, even more preferably, in
a powder or lyophilized state pending use.
[0285] In general, due to the relative stability of peptides, they
may be readily stored in aqueous solutions for fairly long periods
of time if desired, e.g., up to six months or more, in virtually
any aqueous solution without appreciable degradation or loss of
antigenic activity. However, where extended aqueous storage is
contemplated it will generally be desirable to include agents
including buffers such as Tris or phosphate buffers to maintain a
pH of about 7.0 to about 7.5. Moreover, it may be desirable to
include agents which will inhibit microbial growth, such as sodium
azide or Merthiolate. For extended storage in an aqueous state it
will be desirable to store the solutions at 4.degree. C., or more
preferably, frozen. Of course, where the peptides are stored in a
lyophilized or powdered state, they may be stored virtually
indefinitely, e.g., in metered aliquots that may be rehydrated with
a predetermined amount of water (preferably distilled) or buffer
prior to use.
[0286] 4.20 Site-Specific Mutagenesis
[0287] Site-specific mutagenesis is a technique useful in the
preparation of individual peptides, or biologically functional
equivalent proteins or peptides, through specific mutagenesis of
the underlying DNA. The technique, well-known to those of skill in
the art, further provides a ready ability to prepare and test
sequence variants, for example, incorporating one or more of the
foregoing considerations, by introducing one or more nucleotide
sequence changes into the DNA. Site-specific mutagenesis allows the
production of mutants through the use of specific oligonucleotide
sequences which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent nucleotides, to provide a
primer sequence of sufficient size and sequence complexity to form
a stable duplex on both sides of the deletion junction being
traversed. Typically, a primer of about 14 to about 25 nucleotides
in length is preferred, with about 5 to about 10 residues on both
sides of the junction of the sequence being altered.
[0288] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by various publications. As
will be appreciated, the technique typically employs a phage vector
which exists in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage. These phage are readily
commercially-available and their use is generally well-known to
those skilled in the art. Double-stranded plasmids are also
routinely employed in site directed mutagenesis which eliminates
the step of transferring the gene of interest from a plasmid to a
phage.
[0289] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector or melting
apart of two strands of a double-stranded vector which includes
within its sequence a DNA sequence which encodes the desired
peptide. An oligonucleotide primer bearing the desired mutated
sequence is prepared, generally synthetically. This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli cells, and clones are
selected which include recombinant vectors bearing the mutated
sequence arrangement
[0290] The preparation of sequence variants of the selected
peptide-encoding DNA segments using site-directed mutagenesis is
obtained from as a means of producing potentially useful species
and is not meant to be limiting as there are other ways in which
sequence variants of peptides and the DNA sequences encoding them
may be obtained. For example, recombinant vectors encoding the
desired peptide sequence may be treated with mutagenic agents, such
as hydroxylamine, to obtain sequence variants. Specific details
regarding these methods and protocols are found in the teachings of
Maloy, 1990; Maloy etal., 1994; Segal, 1976; Prokop and Bajpai,
1991; Kuby, 1994; and Maniatis etal., 1982, each incorporated
herein by reference, for that purpose.
[0291] The PCR.TM.-based strand overlap extension (SOE) (Ho et al.,
1989) for site-directed mutagenesis is particularly preferred for
site-directed mutagenesis of the nucleic acid compositions of the
present invention. The techniques of PCR.TM. are well-known to
those of skill in the art, as described hereinabove. The SOE
procedure involves a two-step PCR.TM. protocol, in which a
complementary pair of internal primers (B and C) are used to
introduce the appropriate nucleotide changes into the wild-type
sequence. In two separate reactions, flanking PCRTM primer A
(restriction site incorporated into the oligo) and primer D
(restriction site incorporated into the oligo) are used in
conjunction with primers B and C, respectively to generate PCR.TM.
products AB and CD. The PCRTM products are purified by agarose gel
electrophoresis and the two overlapping PCR.TM. fragments AB and CD
are combined with flanking primers A and D and used in a second
PCR.TM. reaction The amplified PCR.TM. product is agarose gel
purified, digested with the appropriate enzymes, ligated into an
expression vector, and transformed into E. coli JM101, XL1-Blue.TM.
(Stratagene, La Jolla, Calif.), JM105, or TG1 (Carter et al., 1985)
cells. Clones are isolated and the mutations are confirmed by
sequencing of the isolated plasmids.
[0292] 4.21 Biological Functional Equivalents
[0293] Modification and changes may be made in the structure of the
peptides of the present invention and DNA segments which encode
them and still obtain a functional molecule that encodes a protein
or peptide with desirable characteristics. The following is a
discussion based upon changing the amino acids of a protein to
create an equivalent, or even an improved, second-generation
molecule. The amino acid changes may be achieved by changing the
codons of the DNA sequence, according to the following codon
table:
1TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine
Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K
AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0294] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Since it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like
properties. It is thus contemplated by the inventors that various
changes may be made in the peptide sequences of the disclosed
compositions, or corresponding DNA sequences which encode said
peptides without appreciable loss of their biological utility or
activity.
[0295] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982,
incorporate herein by reference). It is accepted that the relative
hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like. Each amino acid has been assigned a hydropathic index on the
basis of their hydrophobicity and charge characteristics (Kyte and
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0296] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e., still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
which are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred. It is also understood
in the art that the substitution of like amino acids can be made
effectively on the basis of hydrophilicity. U.S. Pat. No.
4,554,101, incorporated herein by reference, states that the
greatest local average hydrophilicity of a protein, as governed by
the hydrophilicity of its adjacent amino acids, correlates with a
biological property of the protein.
[0297] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0+1);
[0298] glutamate (+3.0+1); serine (+0.3); asparagine (+0.2);
glutamine (+0.2); glycine (0); threonine (-0.4); proline
(-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
It is understood that an amino acid can be substituted for another
having a similar hydrophilicity value and still obtain a
biologically equivalent, and in particular, an immunologically
equivalent protein. In such changes, the substitution of amino
acids whose hydrophilicity values are within .+-.2 is preferred,
those which are within .+-.1 are particularly preferred, and those
within .+-.0.5 are even more particularly preferred.
[0299] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0300] 4.22 Bacterial MSCRAMMS (Adhesins) as Vaccine Candidates
[0301] Historically, studies on bacterial adherence have focused
primarily on Gram-negative bacteria, which express a wide variety
of fimbrial adhesive proteins (designated adhesins) on their cell
surface (Falkow et al. 1992). These adhesins recognize specific
glycoconjugates exposed on the surface of host cells (particularly
epithelial layers).
[0302] Employing the lectin-like structures in attachment allows
the microorganism to efficiently colonize the epithelial surfaces,
this provides the bacteria an excellent location for replication
and also the opportunity to disseminate to neighboring host
tissues. In many cases it has been demonstrated that immunization
with pilus adhesins can elicit protection against microbial
challenge, such as in Hemophilus influenza induced otitis media in
a chinchilla model (Sirakova et al. 1994), Moraxella bovis in
experimentally induced infectious bovine keratoconjunctivitis
(Lepper et al. 1995), and E. coli induced diarrhea in rabbits
(McQueen et al. 1993). In most cases, immunization with adhesins
leads to the production of immune antibodies that prevent infection
by inhibiting bacterial attachment and colonization, as well as
enhancing bacterial opsonophagocytosis and antibody-dependent
complement -mediated killing.
[0303] The use of molecules that mediate the adhesion of pathogenic
microbes to host tissue components as vaccine components is
emerging as a critical step in the development of future vaccines.
Because bacterial adherence is the critical first step in the
development of most infections, it is an attractive target for the
development of novel vaccines. An increased understanding of the
interactions between MSCRAMMs and host tissue components at the
molecular level coupled with new techniques in recombinant DNA
technology have laid the foundation for a new generation of subunit
vaccines. Entire or specific domains of MSCRAMMs, either in their
native or site-specifically altered forms, can now be produced.
Moreover, the ability to mix and match MSCRAMMs from different
microorganisms creates the possibility of designing a single
vaccine that will protect against multiple bacteria.
[0304] The recent clinical trials with a new subunit vaccine
against whooping cough, consisting of the purified Bordatella
pertussis MSCRAMMs filamentous hemagglutinin and pertactin, in
addition to an inactivated pertussis toxin, are a prime example of
the success of this type of approach. Several versions of the new
acellular vaccine were shown to be safe and more efficacious than
the old vaccine that contained whole bacterial cells (Greco et al.
1996; Gustaffson et al. 1996).
[0305] 4.23 CURRENT S. AUREUS VACCINE COMPONENTS
[0306] The development of penicillin to combat S. aureus was a
major advance in infection control and treatment. Unfortunately,
penicillin-resistant organisms quickly emerged and the need for new
antibiotics was paramount. With the introduction of every new
antibiotic, S. aureus has been able to counter with
beta-lactamases, altered penicillin-binding proteins, and mutated
cell membrane proteins allowing the bacterium to persist.
Consequently, methicillin-resistant S. aureus (MRSA) and multidrug
resistant organisms have emerged and established major footholds in
hospitals and nursing homes around the world.
[0307] Immunity to S. aureus infections remains poorly understood.
Typically, healthy humans and animals exhibit a high degree of
innate resistance to S. aureus infections. Protection is attributed
to intact epithelial and mucosal barriers and normal cellular and
humoral responses. Titers of antibodies to S. aureus components are
elevated after severe infections (Ryding et al. 1995), however to
date there is no serological evidence of a correlation between
antibody titers and human immunity.
[0308] Over the past several decades live, heat-killed, and
formalin fixed preparations of S. aureus cells have been tested as
vaccines to prevent staphylococcal infections. A multicenter
clinical trial was designed to study the effects of a commercial
vaccine, consisting of a staphylococcus toxoid and whole killed
staphylococci, on the incidence of peritonitis, exit site
infection, and S. aureus nasal carriage among continuous peritoneal
dialysis patients (Poole-Warren et al. 1991). Although immunization
with the vaccine elicited an increase in the level of specific
antibodies to S. aureus, the incidence of peritonitis was
unaffected. Similarly, immunization of rabbits with whole cells of
S. aureus could not prevent or modify any stage in the development
of experimental endocarditis, reduce the incidence of renal
abscess, or lower the bacterial load in infected kidneys (Greenberg
et al. 1987).
[0309] Currently there is no FDA approved vaccine for the
prevention of S. aureus infections (Foster 1991). However, a S.
aureus vaccine (StaphVAX), based on the capsular polysaccharide, is
currently being developed by NABI (North American Biologicals
Inc.). This vaccine consists of type 5 or type 8 capsular
polysaccharides conjugated to Pseudomonas aeruginosa exotoxin A
(rEPA). The vaccine is designed to induce type-specific opsonic
antibodies and enhance opsonophagocytosis of (Karakawa et al.
1988). Using a refined lethal challenge mouse model (Fattom et al.
1996) it has been shown that intraperitoneal infusion of type 5
specific IgG reduces the mortality of mice inoculated
intraperitoneally with S. aureus. The type 5 capsular
polysaccharide-rEPA vaccine has also been used to vaccinate
seventeen patients with end-stage renal disease (Welch et al.
1996). Geometric means (GM) IgG antibody levels to the type 5
conjugate increased between 13 and 17-fold after the first
immunization, however no additional increases could be detected
after additional injections. Interestingly, the GM IgM levels of
the vaccinated patients were significantly lower than control
individuals. Supported by the animal studies, the vaccine has
recently completed a Phase II trial in continuous ambulatory
peritoneal dialysis patients. The clinical trial showed the vaccine
to be safe but ineffective in preventing staphylococcal infections
(NABI SEC FORM 10-K405, Dec. 31, 1995). Two possible explanations
for the inability of Staph VAX to prevent infections related to
peritoneal dialysis in vaccinated patients are that the
immunogenicity of the vaccine was too low due to suboptimal vaccine
dosing or that antibodies in the bloodstream are unable to affect
infection in certain anatomic areas, such as the peritoneum.
[0310] 4.24 Use Of The S. Aureus CBP As A Vaccine Component
[0311] 4.24.1 Sepsis
[0312] Gram-positive bacteria related sepsis is on the increase. In
fact between one-third and one-half of all cases of sepsis are
caused by gram-positive bacteria, particularly S. aureus and S.
epidermidis. In the United States, it can be estimated that over
200,000 patients will develop gram-positive related sepsis this
year. Using a mouse model (Bremell et al. 1991) the inventors have
clearly demonstrated that active immunization with M55 domain (SEQ
ID NO:6) of the Col-binding MSCRAMM can protect mice against sepsis
induced death. Mice were immunized subcutaneously with either M55
or a control antigen (bovine serum albumin) and then challenged
intravenously with S. aureus. Eighty-three percent (35/42) of the
mice immunized with M55 survived compared to only 27% of the BSA
immunized mice (12/45). This is a compilation of 3 separate
studies.
[0313] 4.25 Production Of A Prototype Multivalent MSCRAMM-Based
Vaccine
[0314] A series of recombinant proteins, representing domains from
the Col, Fn, and Fbg-binding MSCRAMMs, were overexpressed in E.
coli and affinity purified by metal chelating chromatography as
previously described (Joh et al. 1994; McDevitt et al., 1994, Patti
et al., 1995): (1) amino acids contained in the recombinant CBP
M17; SEQ ID NO:2; (2) amino acids contained in the recombinant
Fib-binding MSCRAMM (pCF33), and (3) amino acids contained in the
recombinant fibronectin-binding MSCRAMM (pQD).
5. EXAMPLES
[0315] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
5.1 Example 1
Critical Residues In The Ligand Binding Site Of The S. Aureus
CBP
[0316] A discrete Col-binding site has been identified within the
S. aureus Col adhesion that is located in a region between amino
acids Asp.sup.209 and Tyr.sup.233. Polyclonal antibodies raised
against a recombinant form of the Col adhesin inhibited the binding
of type II Col to S. aureus. When overlapping synthetic peptides
mimicking segments of the adhesion fragment were tested for their
ability to neutralize the inhibitory activity of the antibody only
one peptide, CBD4 was found to be active. CBD4 bound directly to
Col and at high concentrations inhibited the binding of Col to S.
aureus. A synthetic peptide derivative of CBD4 lacking 2
carboxyl-terrninal residues (Asn.sup.232, Tyr.sup.233) had minimal
inhibitory activity. The importance of these residues for Col
binding was confirmed by biospecific interaction analysis. Mutant
adhesin proteins N.sup.232.fwdarw.A and Y.sup.233.fwdarw.A
exhibited dramatic changes in Col binding activity. The dominant
dissociation rate for the binding of mutant adhesin protein
N.sup.232.fwdarw.A to immobilized Col II decreased almost 10-fold,
while the Y.sup.233.fwdarw.A and the double mutant exhibited even
more significant decreasesin affinity and apparent binding ratio
when compared to the wild type protein.
[0317] 5.1.1 Polyclonal IGG Against MSCRAMM Fragments Inhibit COL
Binding to S. aureus
[0318] Studies were conducted to identify the ligand-binding site
of the S. aureus CBP by examining the binding activity of
recombinant MSCRAMM fragments of progressively decreasing size.
Truncations beyond a 168-amino-acid long segment, CBD(151-318),
resulted in loss of Col binding activity but also affected the
folding of the resulting proteins as indicated by CD spectroscopy.
Thus it is possible that the ligand-binding site is contained
within a short segment of CBD(151-318), but due to the improper
folding of the protein the Col-binding site is not in an active
form. To explore this possibility, an antibody
inhibition-neutralization approach was developed. A similar
strategy was used successfully to monitor the purification of the
native CBP from S. aureus cells (Switalski et al., 1989). To
generate an inhibiting antibody, CBD(151-297), a recombinant
version of the largest segment that did not bind Col and exhibited
an altered conformation, was used as an antigen. In this way,
generating inhibiting antibodies which recognize conformational
dependent epitopes would be minimized. An inhibiting monoclonal
antibody generated against a biologically active CBP recognized a
conformational dependent epitope and was of limited use in
identifying the binding site.
[0319] Rabbits were immunized with CBD(157-297) as described. Sera
were also collected prior to immunization and tested for reactivity
to CBD(151-297). The reactivity of the antiserum with different
segments of CBD(151-297) was tested in an ELISA using a series of
eight 25-amino-acid long synthetic peptides with partially
overlapping sequences as targets. Purified IgG reacted strongly
with peptides 2, 3, 5, 6, and 7 and weakly with peptides 1, 4, and
8. When preimmune IgG was tested with the CBD peptides, little
reaction could be detected. The relative immunological reactivity
of the different peptides correlated closely with their antigenic
index using the algorithm of Jameson and Wolf (1988).
[0320] Purified .alpha.CBD(151-297) IgG inhibited the binding of S.
aureus to .sup.125I-labeled Col in a dose-dependent manner. The
amount of .sup.1251-Col bound by 108 bacterial cells was reduced
over 50% by 5 .mu.g and essentially completely inhibited by 10
.mu.g of the purified immune IgG. Conversely, antibodies purified
from preimmune sera did not possess significant inhibitory
activity. These results suggest that the .alpha.CBD(151-297)
antibodies recognize epitopes at or close to the active site of the
MSCRAMM, thereby inhibiting or sterically interfering with Col
binding.
[0321] 5.1.2 Synthetic peptides Neutralize Inhibitory activity of
.alpha.CBD(151-297) IGG
[0322] The different synthetic CBD peptides described above and
shown to react with the .alpha.CBD(151-297) IgG were assayed for
their potential to neutralize the inhibitory activity of the
antibody. The .alpha.CBD(151-297) IgG (12 .mu.g) was preincubated
with a single dose (100 .mu.g) of each peptide in step one. The S.
aureus cells were then added and the preincubation continued.
Finally, the .sup.125I-labeled type II Col was added. Peptide CBD4
neutralized 68% of .alpha.CBD(151-297) IgG inhibitory activity,
while the other peptides tested had little or no effect. These
results suggested that only antibodies recognizing epitopes present
in CBD4 were able to inhibit Col binding to bacteria. Although
other peptide sequences were more immunogenic than CBD4, the
antibodies recognizing the corresponding epitopes were not
inhibitory. These data suggest that the ligand-binding site of the
MSCRAMM is located close to or within the sequence covered by
peptide CBD4.
[0323] To further investigate the interaction between peptide CBD4
and .alpha.CBD(151-297) IgG and its affect on Col binding, a fixed
concentration of the antibody was incubated with an increasing
amount of peptide CBD4. To make the assay more sensitive, an
.alpha.CBD(151-297) IgG concentration (12 .mu.g/ml) was chosen
which resulted in a 50% reduction in Col binding to 10.sup.8 S.
aureus cells. Thus, a relatively small reduction in inhibition
could be easily detected. At low concentrations, the peptide
appears to neutralize the inhibitory activity, and in this study
100 .mu.g of peptide CBD4 restored the level of Col binding to S.
aureus observed in absence of .alpha.CBD(151-297) IgG. Somewhat
surprisingly, addition of more peptide CBD4 resulted in a
dose-dependent decrease in Col binding to S. aureus.
[0324] 5.1.3 Peptide Cbd4 Directly Inhibits COL Binding to S.
aureus
[0325] To assess the role of amino acids 209-233 in Col binding,
peptide CBD4 was tested for the ability to directly inhibit the
binding of .sup.125-Col to S. aureus. Peptide CBD7 which reacted
strongly with .alpha.CBD(151-297) IgG in the ELISA assay was also
tested. When increasing amounts of peptide CBD4 were incubated with
.sup.125I-Col prior to the addition of S. aureus, binding to Col
was inhibited in a dose-dependent manner. Five .mu.M CBD4 inhibited
binding by over 50%. Peptide CBD7 had no inhibitory effect when it
was preincubated with .sup.125I-Col. These data suggest that
peptide CBD4 can bind soluble .sup.125I-Col and that CBD4 contains
the residues that represent a Col-binding site within the MSCRAMM
protein.
[0326] 5.1.4 Critical CBD4 Residues Required for COL Binding
Activity
[0327] To identify residues within CBD4 necessary for Col binding
synthesized several smaller overlapping peptides were synthesized.
A series of peptides that contained a 2 amino-terminal residues and
one peptide that contained a 2 amino acid deletion at the carboxyl
terminus were made. The peptides (10 .mu.M) were assayed for their
ability to inhibit the binding of .sup.1251I-labeled Col to S.
aureus. Peptide CBD4 inhibited Col binding by 76%, whereas all
peptides containing amino-terminal truncations had little activity
at the concentration tested. These data indicate that when as few
as 5 residues are removed from the active site peptide, the ability
to bind Col is lost. Moreover, deletion of only the 2
carboxyl-terminal amino acids causes a complete loss of biological
activity. This suggests that amino acids at the C terminus of CBD4,
Asn.sup.232 and Tyr.sup.233 or both are integral member(s) of the
active site of CBP.
[0328] 5.1.5 COL Binding Activity of Defined Mscramm Mutants
[0329] The importance of amino acid residues Asn.sup.232 and Tyr
.sup.33 in the MSCRAMM for Col binding was examined by creating
specific mutants of CBD (30-529) and characterizing the
interactions of these mutants with immobilized type II Col using
the BlAcore. In the mutations made, the identified amino acid
residues were replaced individually, (N.sup.232 .fwdarw.A,
Y.sup.233 .fwdarw.A) or as a pair, (N.sup.232
.fwdarw.A:Y.sup.233.fwdarw.A) with alanine, a residue that is not
expected to interfere with the existing secondary structure. The
corresponding base changes were made using overlap extension
PCR.TM. as described and the sequence changes confirmed
experimentally. The recombinant proteins containing the mutations
were purified to homogeneity by metal ion chelating chromatography.
Structural analyses of the isolated proteins by near- and far-UV CD
spectroscopy suggested that no significant changes in secondary or
tertiary structure had occurred as a result of the mutations.
[0330] Under the conditions described CBD (30-529) exhibits complex
multiphasic interactions when analyzed using the BIAcore. True
equilibrium was not obtained during the injection time period due
to the slow dissociation rate (k.sub.off). The association phase
exhibits multiphasic binding with an interaction characterized by a
fast k.sub.off apparent at the start of the injection followed by a
second phase characterized by a much slower k.sub.off. The
dissociation phase gives information about the k.sub.off, and this
rate is independent of the number of binding sites, analyte
concentration, and flow rate. Analysis of these data indicates at
least a three-component dissociation with the two fastest rates
greater than 10.sup.- 2S.sup.-1 and the slowest k.sub.off at
5.times.10.sup.-4 S.sup.-1. The association phase contains the
information to determine the association rate k.sub.off the
interaction, but it is also influenced by the dissociation rate,
the number of binding sites for each interaction, and the
concentration of the analyte. Because of the complexity of this
interaction and the absence of measurable equilibrium data, it was
not possible to determine the binding constant (K.sub.D), apparent
binding ratio (BR.sub.app), and k.sub.on.
[0331] In a comparison of the three mutant proteins with wild type
CBD(30-529), it is readily apparent that each of the introduced
mutations affected the Col binding properties of the generated
proteins. While the shape of the binding sensorgram remains
essentially the same for mutant N.sup.232.fwdarw.A, analysis of the
dissociation phase indicates that the slowest dissociation rate has
increased almost 10-fold. Although a disassociation constant
(K.sub.D) cannot be determined, it appears that the affinity of
this mutant for type II Col has also been influenced by this
mutation. Both the Y.sup.233.fwdarw.A and the double mutant bind
immobilized type II. Analysis of the data obtained with the double
mutant produces a monophasic binding constant when evaluated by
either Scatchard analysis or Equation 1. Additionally, the
BR.sub.app has decreased to approximately two to three high
affinity sites.
[0332] From the Biosensor results it is apparent that residues
Asn.sup.232 and Tyr.sup.233 are important for both the affinity and
the specificity of CBD(30-529's binding to type II Col. There does
not, however, seem to be an additive effect of the double mutation
when compared to the single mutations.
5.2 EXAMPLE 2
Structure of the COL-binding Domain from the S. aureus CBP
[0333] The structural basis for host tissue targeting by S. aureus
presented here reveals that the Col-binding domain CBD(151-318) is
well-designed to interact with triple-helical Col structures. The
binding interface of the domain is built along a groove on a
concave .beta.-sheet and has considerable geometrical and chemical
complementarity to the Col helical segment containing four repeats
of Gly-Pro-Hyp or Gly-Pro-Pro per chain. Mutational analysis has
confined the putative Col binding site, and suggests that the
simple docking model may have more general significance. In this
model, the Col triple helix itself is a major recognition-element
for the bacterial adhesin containing complementary binding site.
This provides a structural explanation for the earlier observations
of the MSCRAMM's specificity for triple-helical structures
(Speziale et al., 1986). In addition, the suggested binding site on
the adhesin appears to be versatile, in that it allows appropriate
diversity, but restricted by structural complementarity. The
generality of this model must await structural analysis of other
proteins that bind to the Col triple-helix.
[0334] 5.2.1 Methods
[0335] 5.2.1.1 Crystallization and Data Collection
[0336] The recombinant polypeptide CBD(151-318) was crystallized by
the hanging drop vapor diffusion method using PEG 4000 as
precipitant, 50 mM HEPES buffer between pH 6.2 and 6.9, and the
detergent n-octyl-.beta.-D-glucopyranoside. The crystals belong to
trigonal space group P3.sub.221 or the enantiomorph P3.sub.121. The
unit cell parameters are a=74.0 .ANG., =74.0 .ANG., c=56.7 .ANG.,
.alpha.=90.degree., .beta.=90.degree., .gamma.=120.degree.. There
is one molecule per asymmetric unit with estimated solvent content
of 44%. Diffraction data were collected at room temperature on a
Siemens Histar area detector and processed with X-GEN from
Molecular Simulations Inc. A number of potential heavy atom
derivative data sets were collected, however, only HgCl.sub.2 and
K.sub.2PtCl.sub.4 derivatives were useable for MIR phasing.
[0337] 5.2.1.2 Structure Solution and Refinement
[0338] The phasing solution with the right handedness was
consistent with the space group P3.sub.221 and provided an
interpretable map. The solvent flattened MIR map at 3 .ANG.
resolution revealed major features of the structure and was
suitable for both chain tracing and sequence alignment. From the
deduced sequence of 168 amino acids, 150 residues between 169-318
were aligned to the map. The initial model was refined at 2.4 .ANG.
resolution with X-PLOR (Brunger, 1992) using conjugate gradient
minimization, MIR phases constraints, and F>2.sigma..sub.F. At
this stage, the R-factor and R-free were 34.3% and 41.4%,
respectively. The model was further improved by several cycles of
simulated annealing refinement and manual model building. Before
adding water molecules, isotropic temperature factors of individual
non-hydrogen atoms were refined and the R-factor and R-free dropped
to 25.3% and 31.3%, respectively, for the resolution shell 10.0-2.0
.ANG.. Water molecules were fitted to FO-FC maps at the 3.sigma.
level. Several final rounds of conjugate gradient minimization and
manual model building resulted in the R-factor of 20.0% and the
R-free of 24.9%. Atomic coordinates of the crystal structure will
be deposited in Protein Data Bank.
[0339] 5.2.1.3 Docking Search
[0340] Briefly, dot surfaces (Connolly, 1993) with normal vectors
are calculated for the target and the probe, respectively, and
divided in surface cubes and interior cubes. The nearest atom to
each dot determines the chemical propensity represented by a simple
six-color code. Rotation space of the `cubed` probe is sampled and
for each rotation step all integer translations of the probe cubes
to the stationary target cubes are calculated. Each translation of
the probe is scored according to the match of normal vectors,
compatibility of color codes, and overlapping interior cubes.
Clustered solutions with the best scores are averaged and the
corresponding rotations and translations applied to atomic
coordinates of the probe.
[0341] 5.2.1.4 CD Spectra
[0342] All CD spectra were collected using a Jasco J720
spectropolarimeter calibrated with a 0.1% (wt./vol.)
10-camphorsulfonic acid-d solution. Spectra were measured at
25.degree. C. and 5 scans were averaged. A 0.05 cm path length cell
was used for near-UV (250-320 nrm) CD and a 1 cm path length cell
was used for far-UV (190-250 nm) CD.
[0343] 5.2.2 Structure Determination
[0344] The crystal structure of CBD(151-318) was determined using
conventional heavy atom/multiple isomorphous replacement MR)
methods and was refined to a crystallographic R-factor of 20%
(R.sub.free=24.9%) using diffraction data between 10.0 and 2.0
.ANG. resolution (Table 2). The refined model includes 150 amino
acids between residues 169-318 and 74 water molecules. The root
mean square deviation (RMS) from ideal bond lengths is 0.012 .ANG.,
and RMS deviation from ideal angles is 1.625.degree.. The model
scores high in PROCHECK (Laskowski et al., 1993) analysis and a
Ramachandran plot of .phi...omega. conformation angles has no
outliers. The electron density was of good quality throughout the
structure and there were eight disordered exterior side chains in
the final model. No electron density was observed for N-terminal
residues 151-168.
[0345] 5.2.3 Structure Description
[0346] The molecular structure of the recombinant Col-binding
domain is very compact with approximate dimensions
55.times.35.times.25 .ANG.. The polypeptide chain of CBD(169-318)
is folded to a `jelly-roll` topological pattern (FIG. 1)
(Richardson, 1981). The secondary structure of the domain consists
of 53% .beta.-sheet, 39% coil, and 8% helix. There are no disulfide
bridges or free cysteines. The entire domain is essentially
composed of two .beta.-sheets, parallel to each other, and two
small .alpha.-helices (FIG. 2). .beta.-sheets I and II form a
sandwich with a largely hydrophobic interior. The exposed side of
the .beta.-sheet I has a concave trend, whereas .beta.-sheet II has
a convex face. There are five antiparallel strands in each
.beta.-sheet; strands D and J have breaks with less defined
secondary structure. A small two-turn a-helix is present in the
crossover between strands E and F on the wider side of the
sandwich. A single .alpha.-helical turn is apparent in the
connection between strands G and H. Other connections adopt various
forms of coiled structure. Three charged residues, K176, D209 and
E301, are buried in the interior of the molecule and all are well
defined in electron density maps. K176 and D209 are bridged through
N293 by hydrogen bonds and residue E301 is hydrogen bonded to S199.
Crystal packing results in large solvent channels, about 35 .ANG.
in diameter, along the three-fold screw axes. Domains are packed
around the screw axes exposing most of the .beta.-sheet II to the
solvent channels.
[0347] The Connolly's molecular surface (Connolly, 1983) of the
CBD(169-318) crystal structure shows an apparent groove on the
.beta.-sheet I (FIG. 3A) indicating a putative Col binding region.
This groove is about 10 .ANG. wide and spans diagonally across
strands D, H and B in the approximate direction T221-N196. With the
exception of the disordered N-terminal residues 169 and 170 of a
symmetry-related molecule, there are no significant short
intermolecular contacts for the residues in and around the groove.
The exterior residues on .beta.-sheet I, specifically K280, R189,
F191, Y175, E197, S235, Y233, N225, T227, and K198 (FIG. 3A)
delimit and form the walls of the groove. Residues N193, N223,
S274, and N278, with accessible surface area less than 20
.ANG..sup.2, are buried in the groove; whereas, N196 and S276 are
also inside the groove but relatively more exposed (FIG. 3A). The
only hydrophobic residues in the putative Col binding region are
V172, L181, and F191. Charged residues D179, E197, K198, D218, and
K280 are located around the edges of the groove along with Y175 and
Y233, whose phenolic rings are oriented towards the interior of the
groove. Conformations of some side chains are stabilized by
hydrogen bonds, for example R189 is hydrogen-bonded to D1 79, and
K198 to S274. No tightly bound water molecules are found on the
.beta.-sheet I.
[0348] The peptide sequence D209-Y233 was previously shown to be
essential for Col-binding activity (Patti et al., 1995). In the
crystal structure, this sequence spans strand D and portions of
strands C and E with exterior residues T221, N223, N225, T227, and
Y233 in the putative Col binding region. In addition, the mutation
of a residue 233Y.fwdarw.A in the 55-kDa domain A of the Col
MSCRAMM has been shown dramatically reduced Col-binding activity
(Patti et al., 1995).
[0349] 5.2.4 COL Docking
[0350] Previous studies have shown that the Col binding MSCRAMM on
S. aureus binds to several sites in different Col types (Patti et
al., 1993) but recognizes collagens in a triple helical form
(Speziale et al., 1986); therefore, the Col-binding site within the
MSCRAMM, should have provisions for direct interaction with a
triple-helical motif. Collagens are glycosylated to various degree
depending on the tissue. The possibility of Col binding was tested
through the carbohydrate by cocrystallization and soaking of
CBD(151-318) with glucose, galactose, and lactose, respectively.
Although high-uality isomorphous crystals were obtained, difference
Fourier maps did not indicate any appreciable binding of the
carbohydrates. The basic triple-helical conformation, approximately
15 .ANG. in diameter, consists of three supercoiled polyproline II
helices that require glycine residues to be present in every third
position in the sequence. This results in a (GLY-X-Y).sub.n
repeating pattern in which the X and Y positions are frequently
refined crystal structure of CBD(172-318) with the disordered
N-terminal residues 169-171 excluded. The docking was performed as
a full 6-dimensional search using the matching cubes algorithm
(Jiang and Kim, 1991) implemented in the program SoftDock. The top
sixteen solutions were evaluated in each search and solutions with
the best scores were consistently found along the groove on P-sheet
I in the direction T221-N196. Col helices were always oriented in
this direction from the N- to C-terminus. The docking score became
dramatically worse, when forced to the opposite direction.
[0351] Apparently, the Col `screw` matches the adhesin `nut` in
this site (FIG. 3B). Variations of the probe lengths, helical
parameters, and proline ring conformations and interchanging of Pro
and Hyp at the Y position of the probe had minimal effects on the
final `docked` positions. Both visual and computational analyses
indicated no serious conflicts in chemical propensities and atomic
distances between the binding site and Col probes. All successfully
docked complexes are energy-minimizable in X-PLOR (Brunger, 1992)
with a small loss of regular helicity and subtle conformational
changes in receptor side chains.
[0352] 5.2.5 Mutational Analysis
[0353] Mutational analysis was used to evaluate the putative
binding site defined by the docking search. Surface residues that
exhibited a decrease in their solvent accessible area were targeted
(Table 3 and FIG. 4). The surface area covered by the docked Col is
about 1630 .ANG..sup.2, which is 22% of the total
solvent-accessible surface of CBD(172-318). There are 19 residues
on the interface that exhibited a decrease in solvent accessible
area by more than 10 .ANG..sup.2. Nine of these residues were
mutated as well as two additional residues outside the putative
binding region. The single-site lysine and alanine mutations were
designed to disrupt the surface of the putative Col binding groove
of wild type CBD(15 1-318). The mutant proteins did not exhibit
significant changes in either the near-UV or far-UV circular
dichroism spectra when compared to the recombinant wild type;
affirming that the various mutations did not have a measurable
effect on the overall conformation. The MSCRAMM recognizes generic
triple-helical structures (Speziale et al., 1986; Sakakibara et
al., 1973; Heidemann and Roth, 1982), therefore, changes in the
low-affinity dissociation constants were used to evaluate
alterations in the binding of the mutant proteins (House-Pompeo et
al., 1994).
[0354] For mutants 212Q.fwdarw.A, 232N.fwdarw.A, 212T.fwdarw.K, and
225N.fwdarw.K (Table 3) the Col-binding activity, expressed as one
apparent dissociation constant K.sub.D, did not differ
significantly from that of wild type CBD(151-318). Residue Q212 is
located on the .beta.-sheet II, far from the suggested binding site
and was mutated as a control residue. This exhibited no significant
change in affinity for the Col. The side chain of N232 is not part
of the binding groove (FIG. 3A and FIG. 4) and does not exhibit a
decrease in solvent accessibility on docking of the Col. Consistent
with this, the mutation 232N.fwdarw.A in CBD(151-318) had a very
small effect on Col binding. Residues T221 and N225 are marginal
residues of the binding groove with relatively small side chains.
Modeling studies show that the lysine side chains in positions 221
and 225 may adopt conformations which do not interfere with Col
probes and point into the solvent region.
[0355] Site-specific alterations of residues N193, N223, and N278
to lysine residues resulted in recombinant proteins with a
significantly reduced affinity for Col. These sites are in the
interior of the binding groove and, according to the docking model,
are practically buried by the bound Col. Consequently, the lysine
side chains have less conformational freedom in these sites and may
sterically prevent the adhesin from assuming the proper position
along the Col helix. Significantly higher dissociation constant for
193N.fwdarw.K compared to the other two mutations could be
attributed to its proximity to the putative Col binding groove
`wall` and critical residues Y175 and F191.
[0356] Mutations in positions Y233, F191, R189 and Y175 resulted in
proteins with extremely low affinity for Col compared to wild type
CBD(151-318), suggesting that these are some of the major
determinants for Col binding. All four mutation sites involve
large, relatively exposed, residues which form the side walls of
the putative binding groove. The largest decrease in
solvent-accessible area on Col docking is for Y233 and modeling
studies have suggested a number of contacts with Col probes for
this residue. Although residues R189 and Y175 exhibited relatively
fewer contacts with docked Col probes, mutation of these residues
essentially destroys Col binding with KD values approaching the
millimolar range. The results for these two residues may be
reflecting the simplicity of the probe used in modeling, and
possible optimizations in `docking` it. It is important to note
that although generic peptides were used in the docking studies,
biosensor analysis of the site-specific mutants demonstrated a high
degree of correlation with native Col type II.
[0357] The mutational analysis shows two general trends. The
Lys/Ala mutation of large residues forming the walls of the binding
groove dramatically affects the affinity of the adhesin to Col.
Mutation of smaller residues found inside the groove or close to
its walls exhibit moderate to no effect on the binding. Overall,
the results of mutational analysis are in good agreement with
interactions seen in the Col-binding model derived from systematic
docking of the generic Col probes. Binding of native Col will
involve some non-proline residues in X/Y positions. Further
modeling studies indicate that, in addition to recognizing the
generic triplets, the binding site of CBD(151-318) can also
accommodate other small non-proline residues. The predominant
distribution of small polar residues (Thr, Asn, and Ser) in the
binding groove might possibly mimic the essential hydration in
stabilizing hydroxyprolines. In addition, they can sterically
facilitate the binding of suggested diverse residues in Col
sequence. Thus, it is possible that specific Col sequences may bind
better than the generic triplets used in docking studies, and may
account for the observed higher affinity binding sites on Col II
(Patti et al., 1993).
2TABLE 2 DATA COLLECTION AND PHASING STATISTICS OF CBD(151-318)
Native HgCl.sub.2 (I) HgCl.sub.2 (II) K.sub.2PtCl.sub.4 Internal
scaling No. of crystals 3 1 1 1 Observed reflections 104150 6505
10133 9040 Unique reflections 13094 3495 5588 3208 Completeness (%)
97.2 92.6 85.0 85.0 Resolution (.ANG.) 2.0 3.0 2.5 3.0
R.sub.sym.sup.1 (%) 7.8 7.0 5.8 3.2 Derivative scaling Resolution
(.ANG.) 3.0 3.0 3.0 R.sub.merge.sup.2 (%) 19.1 43.0 16.0 No. of
sites 4 5 2 Phasing power.sup.3 2.09 2.73 1.83 R.sub.CULLIS.sup.4
(%) 51.1 39.1 58.1 R.sub.KRAUT.sup.5 (%) 7.6 18.4 6.5 Figure of
merit.sup.6 0.442 0.390 0.423 .sup.1R.sub.SYM =
.SIGMA..sub.h.SIGMA..sub.l .vertline.I(h) - I.sub.i(h).vertline. /
.SIGMA.h .SIGMA..sub.hI(h), where I.sub.i(h) and 1(h) are the i-th
and the mean measurements of the intensity of reflection h.
.sup.2R.sub.MERGE = .SIGMA..sub.h .vertline.F.sub.p(h) -
F.sub.PH(h).vertline. / .SIGMA. F.sub.p(h), where F.sub.p(h) and
F.sub.PH(h) are observed native and scaled derivative structure
factors of the reflection h. .sup.3Phasing power is the ratio of
the root mean square (RMS) deviation of the calculated heavy-atom
structure amplitudes to the RMS lack of closure. .sup.4R.sub.CULLIS
= .SIGMA. .vertline..vertline..vertline.F.sub.PH.vertl-
ine..sub.OBS + .vertline.F.sub.P.vertline..sub.OBS -
.vertline.F.sub.H.vertline..sub.CALC.vertline. / .SIGMA.
.vertline..vertline.F.sub.PH.vertline..sub.OBS +
.vertline.F.sub.P.vertli- ne..sub.OBS.vertline., where F.sub.PH and
F.sub.P are the observed structure factor amplitudes for the
heavy-atom derivative and the native data sets, with the sum taken
over all centric reflections, and F.sub.H is the heavy-atom
structure factor. .sup.5R.sub.KRAUT = .SIGMA.
.vertline..vertline.F.sub.PH.vertline..sub.OBS - .SIGMA.
.vertline.F.sub.PH.vertline..sub.CALC.vertline. /
.SIGMA..vertline.F.sub.- PH.vertline..sub.OBS, with the sum taken
over all acentric reflections. .sup.6The overall figure of merit is
0.662.
[0358]
3 TABLE 3 CBD K.sub.D (mM) BR CBD(151-318) 31 10 212Q.fwdarw.A 34
10 221T.fwdarw.K 34 9 225N.fwdarw.K 30 10 232N.fwdarw.A 38 12
278N.fwdarw.K 50 9 223N.fwdarw.K 53 10 193N.fwdarw.K 140 12
233Y.fwdarw.A 195 11 191F.fwdarw.A 199 10 189R.fwdarw.A >350
>13 175Y.fwdarw.K >460 >20 Apparent dissociation constants
(K.sub.D) and approximate binding ratios (BR) for binding of
CBD(151-318) and corresponding mutants to Col type II as determined
by biosensor analysis. Serial dilutions of each protein, wild type
and mutants respectively, were passed over the covalently
immobilized Type II Col [Sigma]. Equilibrium binding response after
10 seconds of injection was used to calculate the constants (Patti
et al., 1995; House-Pompeo et al., 1994).
5.3 EXAMPLE 3
Passive Immunization Using Epitopes of MSCRAMMS
[0359] Underlined amino acids are encoded in the vector pQE.TM.-30
(Qiagen Inc. Chatsworth, Calif.)
4 5.3.1 S. AUREUS COL-BINDING MSCRAMM DERIVATIVE M17 (SEQ ID NO:2)
MRGSHHHHHHGSITSGNKSTNVTVHKSEAGTSSVFYYKTGDMLPEDTTHV
RWFLNINNEKSYVSKDITIKDQIQGGQQLDLSTLNINVTGTHSNYYSGQS
AITDFEKAFPGSKITVDNTKNTIDVTIPQGYGSYNSFSINYKTKITNEQQ KEFVNNSQA
(GenBank accession number of entire cna gene is M81736)
[0360]
5 5.3.2 S. AUREUS CBP EPITOPE M17 DNA (SEQ ID NO:1)
ATAACATCTGGGAATAAATCAACGAATGTTACGGTTCATAAAAGTGAAGCGGGAACAAGTAGTGTTTTC
TATTATAAAACGGGAGATATGCTACCAGAAGATACGACACATGTACGATGGTTTTTAAAT-
ATTAACAAT GAAAAAAGTTATGTATCGAAAGATATTACTATAAAGGATCAGATTCAA-
GGTGGACAGCAGTTAGATTTA AGCACATTAAACATTAATGTGACAGGTACACATAGC-
AATTATTATAGTGGACAAAGTGCAATTACTGAT TTTGAAAAAGCCTTTCCAGGTTCT-
AAAATAACTGTTGATAATACGAAGAACACAATTGATGTAACAATT
CCACAAGGCTATGGGTCATATAATAGTTTTTCAATTAACTACAAAACCAAAATTACGAATGAACAGCAA
AAAGAGTTTGTTAATAATTCACAAGCT
[0361]
6 5.3.3 S. AUREUS COL-BINDING MSCRAMM DERIVATIVE M31 (SEQ ID NO:4)
MRGSHHHHHHGSDDKVATITSGNKSTNVTVHKSEAGTSSVFYYKTGDMLP
EDTTHVRWFLNINNEKSYVSKDITIKDQIQGGQQLDLSTLNINVTGTHSN
YYSGQSAITDFEKAFPGSKITVDNTKNTIDVTIPQGYGSYNSFSINYKTK
ITNEQQKEFVNNSQAWYQEHGKEEVNGKSFNHTVHNINANAGIEGTVKGE LKVLKQDKDTK
(GenBank accession number of entire cna gene is M81736)
[0362]
7 3.3.4 S. AUREUS CBP EPITOPE M31 DNA (SEQ ID NO:3)
GACGATAAAAATGGAAAAATACAAAATGGTGACATGATTAAAGTGGCATGGCCGACAAGCGGTA
CAGTAAAGATAGAGGGTTATAGTAAAACAGTACCATTAACTGTTAAAGGTGAACAGGTGGGTCA
AGCAGTTATTACACCAGACGGTGCAACAATTACATTCAATGATAAAGTAGAAAAATT- AAGTGAT
GTTTCGGGATTTGCAGAATTTGAAGTACAAGGAAGAAATTTAACGCAAAC- AAATACTTCAGATG
ACAAAGTAGCTACGATAACATCTGGGAATAAATCAACGAATGT- TACGGTTCATAAAAGTGAAGC
GGGAACAAGTAGTGTTTTCTATTATAAAACGGGAGA- TATGCTACCAGAAGATACGACACATGTA
CGATGGTTTTTAAATATTAACAATGAAAA- AAGTTATGTATCGAAAGATATTACTATAAAGGATC
AGATTCAAGGTGGACAGCAGTTAGATTTAAGCACATTAAACATTAATGTGACAGGTACACATAG
CAATTATTATAGTGGACAAAGTGCAATTACTGATTTTGAAAAAGCCTTTCCAGGTTCTAAAATA
ACTGTTGATAATACGAAGAACACAATTGATGTAACAATTCCACAAGGCTATGGGTCATA- TAATA
GTTTTTCAATTAACTACAAAACCAAAATTACGAATGAACAGCAAAAAGAGTT- TGTTAATAATTC
ACAAGCTTGGTATCAAGAGCATGGTAAGGAAGAAGTGAACGGGAA- ATCATTTAATCATACTGTG
CACAATATTAATGCTAATGCCGGTATTGAAGGTACTGT- AAAAGGTGAATTAAAAGTTTTAAAAC
AGGATAAAGATACCAAG
[0363]
8 5.3.5 S. AUREUS COL-BINDING MSCRAMM DERIVATIVE M55 (SEQ ID NO:6)
MRGSHHHHHHGSARDISSTNVTDLTVSPSKIEDGGKTTVKMTFDDKNGKI
QNGDMIKVAWPTSGTVKIEGYSKTVPLTVKGEQVGQAVITPDGATITFND
KVEKLSDVSGFAEFEVQGRNLTQTNTSDDKVATITSGNKSTNVTVHKSEA
GTSSVFYYKTGDMLPEDTTHVRWFLNINNEKSYVSKDITIKDQIQGGQQL
DLSTLNINVTGTHSNYYSGQSAITDFEKAFPGSKITVDNTKNTIDVTIPQ
GYGSYNSFSINYKTKITNEQQKEFVNNSQAWYQEHGKEEVNGKSFNHTVH
NINANAGIEGTVKGELKVLKQDKDTKAPIANVKFKLSKKDGSVVKDNQKE
IEIITDANGIANIKALPSGDYILKEIEAPRPYTFDKDKEYPFTMKDTDNQ
GYFTTIENAKAIEKTKDVSAQKVWEGTQKVKPTIYFKLYKQDDNQNTTPV
DKAEIKKLEDGTTKVTWSNLPENDKNGKAIKYLVKEVNAQGEDTTPEGYT KKENGLVVTNTE
(GenBank accession number of entire cna gene is M81736)
[0364]
9 5.3.6 S. AUREUS CBP EPITOPE M55 DNA (SEQ ID NO:5)
GCACGAGATATTTCATCAACGAATGTTACAGATTTAACTGTATCACCGTCTAAGATAGAAGAT
GGTGGTAAAACGACAGTAAAAATGACGTTCGACGATAAAAATGGAAAAATACAAAATGGTGAC
ATGATTAAAGTGGCATGGCCGACAAGCGGTACAGTAAAGATAGAGGGTTATAGTAAAACA- GTA
CCATTAACTGTTAAAGGTGAACAGGTGGGTCAAGCAGTTATTACACCAGACGGT- GCAACAATT
ACATTCAATGATAAAGTAGAAAAATTAAGTGATGTTTCGGGATTTGCA- GAATTTGAAGTACAA
GGAAGAAATTTAACGCAAACAAATACTTCAGATGACAAAGTA- GCTACGATAACATCTGGGAAT
AAATCAACGAATGTTACGGTTCATAAAAGTGAAGCG- GGAACAAGTAGTGTTTTCTATTATAAA
ACGGGAGATATGCTACCAGAAGATACGACA- CATGTACGATGGTTTTTAAATATTAACAATGAA
AAAAGTTATGTATCGAAAGATATT- ACTATAAAGGATCAGATTCAAGGTGGACAGCAGTTAGAT
TTAAGCACATTAAACATTAATGTGACAGGTACACATAGCAATTATTATAGTGGACAAAGTGCA
ATTACTGATTTTGAAAAAGCCTTTCCAGGTTCTAAAATAACTGTTGATAATACGAAGAACACA
ATTGATGTAACAATTCCACAAGGCTATGGGTCATATAATAGTTTTTCAATTAACTACAAA- ACC
AAAATTACGAATGAACAGCAAAAAGAGTTTGTTAATAATTCACAAGCTTGGTAT- CAAGAGCAT
GGTAAGGAAGAAGTGAACGGGAAATCATTTAATCATACTGTGCACAAT- ATTAATGCTAATGCC
GGTATTGAAGGTACTGTAAAAGGTGAATTAAAAGTTTTAAAA- CAGGATAAAGATACCAAGGCT
CCTATAGCTAATGTAAAATTTAAACTTTCTAAAAAA- GATGGATCAGTTGTAAAGGACAATCAA
AAAGAAATTGAGATTATAACAGATGCAAAC- GGTATTGCTAATATTAAAGCGTTGCCTAGTGGA
GACTATATTTTAAAAGAAATAGAG- GCGCCACGACCGTATACATTTGATAAGGATAAAGAATAT
CCGTTTACTATGAAAGATACAGATAATCAGGGATATTTTACGACTATTGAAAATGCAAAAGCG
ATAGAAAAAACAAAAGATGTTTCTGCTCAAAAGGTTTGGGAAGGCACTCAAAAAGTGAAACCA
ACGATTTATTTCAAGTTGTACAAACAAGATGACAATCAAAATACAACACCAGTAGACAAA- GCA
GAGATTAAAAAATTAGAAGATGGAACGACAAAAGTGACATGGTCTAATCTTCCG- GAAAATGAC
AAAAATGGCAAGGCTATTAAATATTTAGTTAAAGAAGTAAATGCTCAA- GGTGAAGATACAACA
CCAGAAGGATATACTAAAAAAGAAAATGGTTTAGTGGTTACT- AATACTGAA
[0365]
10 5.3.7 S. AUREUS FIB-BINDING MSCRAMM DERIVATIVE PCF33 (SEQ ID
NO:7): MRGSHHHHHHGSMVAADAPAAGTDITNQLTNVTVGIDSGTTVYPHQAGY-
VKLNYGFSVPN SAVKGDTFKITVPKELNLNGVTSTAKVPPIMAGDQVLANGVIDSDG-
NVIYTFTDYVNTKD DVKATLTMPAYIDPENVKKTGNVTLATGIGSTTANKTVLVDYE-
KYGKFYNLSIKGTIDQI DKTNNTYRQTIYVNPSGDNVIAPVLTGNLKPNTDSNALID-
QQNTSIKVYKVDNAADLSES YFVNPENFEDVTNSVNITFPNPNQYKVEFNTPDDQIT-
TPYIVVVNGHIDPNSKGDLALRS TLYGYNSNIIWRSMSWDNEVAFNNGSGSGDGIDK-
PVVPEQPDEQA (GenBank accession number of entire clfA gene is
Z18852)
[0366]
11 5.3.8 S. AUREUS FIBRONECTIN-BINDING MSCRAMM DERIVATIVE (SEQ ID
NO:8) MRGSHHHHHHGSEGGQNSGNQSFEEDTEEDKPKYEQGGNIVDIDFDSVPQ-
IHGQNKGNQS FEEDTEKDKPKYEHGGNIIDIDFDSVPHIHGFNKHTEIIEEDTNKDK-
PSYQFGGHNSVDF EEDTLPKVSGQNEFDIKLN (GenBank accession number of
entire fnbA gene is I28324)
[0367] 5.3.9 Use of the Purified Hyperimmune Polyclonal Rabbit
Anti-MSCRAMM IGG in Passive immunization
[0368] 5.3.9.1 Bovine Mastitis
[0369] Several studies have suggested that epithelial cell damage
and the exposure of ECM molecules within the teat canal orifice and
mammary gland are critical factors leading to the development of
mastitis (Gudding et al. 1984; Olmsted and Norcross 1992; Cifrian
et al. 1995) S. aureus adhesion to mammary gland tissue is regarded
as the first step in the development of mastitis. Therefore,
adhesins that mediate S. aureus attachment to bovine mammary gland
tissues are critical targets for the development of blocking
antibodies. Hyperimmune polyclonal antibodies generated against
several MSCRAMMs have been analyzed for their ability to inhibit S.
aureus strain M60 attachment to cultured bovine mammary secretory
epithelial cells. A dose dependent decrease in the adherence of S.
aureus was demonstrated with the rabbit polyclonal anti-MSCRAMM IgG
to the CBPs of SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:8.
[0370] 5.3.9.2 Experimental Details
[0371] Briefly, 5.times.10.sup.4 secretory epithelial cells, in 100
.mu.l culture medium, were added to flat-bottom Col coated 96-well
plates and grown to confluence at 37.degree. C. The S. aureus
strains were grown overnight in trypticase soy broth (TSB) at
37.degree. C. The overnight culture was diluted into fresh TSB and
grown until the culture reached the exponential phase, the
organisms were harvested and resuspended into 3 ml fresh culture
media. The bacteria (3.1.times.10.sup.8) were incubated with
increasing amounts of anti-MSCRAMM IgG for 30 min at 37.degree. C.
The pre-treated bacteria were then added to was added to the cell
monolayers (4.1.times.10.sup.6 epithelial cells) and incubated for
3 hr at 37.degree. C. The monolayers were then washed five times
with phosphate buffered saline. The monolayers were fixed with
methanol and stained with Giemsa. Twenty fields (100X objective)
were examined in each monolayer for the presence of adherent
bacteria
[0372] 5.3.10 Peritonitis
[0373] Patients undergoing dialysis (continuous ambulatory
peritoneal dialysis, hemodialysis) present an increased risk for
the development of staphylococcal infections. Using an animal model
of peritonitis (Menzies and Kernodle 1996) we have demonstrated
that passive immunization with a single subcutaneous dose of
anti-MSCRAMM IgG can protect mice against S. aureus intraperitoneal
challenge. When mice were challenged with S. aureus, only
twenty-seven percent (10/37) of the mice passively immunized with
anti-MSCRAMM IgG became infected Conversely, 76% of the mice
passively immunized with normal rabbit serum became infected.
*Compilation of 3 separate studies.
[0374] Male NIH Swiss mice, 6-8 weeks of age weighing 18-22 g
(Harlan Sprague Dawley, Indianapolis, Ind.) were used. Anti-MSCRAMM
IgG was administered as a 0.25 ml subcutaneous (s.c.) injection
into the thigh of the mice. Control mice received an equivalent
amount of normal rabbit IgG (Sigma Chemical Co. St. Louis, Mo.).
Forty-eight hr post immunization, mice were challenged
intraperitoneally (ip.) with S. aureus. The bacterial strain was
prepared by growing overnight in brain heart infusion broth (Becton
Dickinson Microbiology Systems, Cockeysville, Md.), washing twice
in PBS, and resuspending in PBS, adjusting by light transmission to
a concentration of 6.times.10.sup.8 cfu/ml. Mice were inoculated
with 0.5 ml of the bacterial suspension. An aliquot of the
bacterial suspension was plated on sheep blood agar to determine
the exact cfu/ml.
[0375] Mice were sacrificed 48 hr post bacterial inoculation. Both
kidneys from each mouse were aseptically excised, weighed, placed
in a sterile bag containing 0.5 ml of PBS, and homogenized for 30
sec using a Tekmar Tissumizer (Tekmar, Cincinnati, Ohio). The
homogenate was serially diluted in sterile water and plated on
sheep blood agar plates and incubated at 37.degree.C. The plates
were counted 18-24 hr later. Homogenates of kidneys from mice
containing <100 cfu/ml were considered negative. The results are
shown in Table 4.
12TABLE 4 Normal Rabbit IgG # infected Total Anti-MSCRAMM Rabbit
IgG mice/total IgG Dose (mg/mouse) # infected mice/total mice (%)
mice (%) 0.3 5/6 (83%) 4/5 (80%) 0.6 1/6 (17%) 4/6 (67%) 1.63 8/23
(35%).sup.a 16/21 (76%) 3.50 1/8 (13%).sup.b 8/10 (80%) .sup.aP =
0.006 .sup.bP = 0.005
[0376] 5.3.11 Demonstration of Therapeutic Efficacy in the
Pneumonia Model
[0377] Staphylococcus aureus is a life-threatening agent of
nosocomial pneumonia in immunocompromised patients. Often S. aureus
isolated from these patients exhibit wide-spectrum resistance to
antibiotics, particularly methicillin. An experimental mouse model
of staphylococcal pneumonia (Ramisse et al., 1993) was used to
assess the ability of anti-MSCRAMM IgG to protect neutropenic mice
against S. aureus mediated pneumonia. Mice that had been treated
intranasally with anti-MSCRAMM IgG 3 hours after bacterial
inoculation had over a 1000-fold reduction in the amount of
bacteria recovered from their lungs (p<0.01) compared to the PBS
control treated animals.
[0378] 5.3.11.1 Experimental Details
[0379] Female 4 week-old BALB/c mice were treated intravenously
with cyclophosphamide at 150 mg/kg at day 4 and 75 mg/kg at day 1
before bacterial challenge. Cyclophosphamide induces a transient
neutropenia in the mice. S. aureus were grown in trypticase soy
broth (TSB) overnight at 37.degree. C. The cultures were washed and
adjusted to .ANG. 6.3.times.10.sup.6 cfu/ml in PBS. An aliquot of
the bacterial suspension was plated on sheep blood agar to
determine the exact cfu/ml. The mice were anesthetized and were
inoculated with 50 .mu.l of the bacterial suspension. Three hours
after the bacterial challenge each mouse received either 7.5 .mu.g
anti-MSCRAMM IgG, 75 .mu.g anti-MSCRAMM IgG, or PBS intranasally.
To check colonization of the lungs by the bacteria, 5 mice per time
point are sacrificed. Bacterial counts are determined from lung
homogenates (lungs are carefully dissected from the main bronchia)
serially diluted in PBS by plating 100 .mu.l-aliquots on trypticase
soy agar and counting the cfu after incubation for 24 hr at
37.degree. C. Bacterial counts were expressed as the mean (.+-.SE).
Statistical significance was determined by Student's t test. The
results are shown in Table 5.
13 TABLE 5 Kinetics of pulmonary infection (cfu/ml) Treatment 3 hr
24 hr 48 hr Study 1 PBS - control 4.80 .+-. 0.09 6.40 .+-. 0.30
7.93 .+-. 0.20 7.5 .mu.g anti-MSCRAMM IgG/ 4.94 .+-. 0.14 6.33 .+-.
0.38 7.04 .+-. 0.12 mouse 75 .mu.g anti-MSCRAMM IgG/ 5.20 .+-. 0.08
6.03 .+-. 0.70 5.53 .+-. 0.80.sup.a mouse Study 2 PBS - control
5.30 .+-. 0.03 7.15 .+-. 0.08 7.98 .+-. 0.11 7.5 .mu.g anti-MSCRAMM
IgG/ ND 5.50 .+-. 0.54 5.52 .+-. 0.40 mouse .sup.ap < 0.01 ND =
not done.
[0380] 5.3.12 Passive Immunization Using CBP Epitopes
[0381] In separate studies, rats were immunized with M55 or BSA as
a control as described herein. They were bled and the IgG fraction
obtained by ammonium sulfate precipitation. The IgG fraction (16
mg) was administered to mice I day before IV challenge with S.
aureus. These passive immunization data confirm the efficacy of the
active immunization--ie., antibodies directed against M55 of the
CBP protect against lethal doses of S. aureus (FIG. 8).
5.4 EXAMPLE 4
Animal Studies Involving the S. aureus CBP
[0382] Corneal infection is a leading cause of visual loss and is a
major public health problem in the U.S. Bacterial keratitis results
when microbial virulence factors overcome host defense mechanisms.
The successful corneal pathogen attaches to the comeal surface,
avoiding the clearance mechanisms of the tear film. Specific
bacterial surface proteins adhere to specific components of the
cornea, such as fibronectin, fibrinogen, and Col. Specific
microbial adhesins mediate this adherence by sophisticated
interaction with host molecules.
[0383] S. aureus MSCRAMMs have been studied using various animal
models. These MSCRAMMS are localized to the surface of S. aureus
and interact with ECM components with high affinity and high
specificity. MSCRAMMS recognizing fibronectin, fibrinogen, and Col
have been previously described with respect to structural
organization, ligand-binding domains, importance in host
colonization and invasion, and biological roles as virulence
factors.
[0384] The inventors have utilized both in vitro and in vivo
studies to determine the role of CBP in the pathophysiology of
infectious keratitis.
[0385] 5.4.1 COL Binding by Ocular Isolates
[0386] Twenty clinical isolates of S. aureus from the Cullen Eye
Institute at the Baylor College of Medicine (Houston, Tex.) were
selected randomly. Each was isolated from a previous case of
bacterial keratitis with standard clinical techniques. Type II Col
was labeled with .sup.125I by the chloramine-T method. A 5-ml
culture of each strain was grown overnight in Brain-heart infusion
broth. Cells were centrifuged and resuspended in 1 ml of PBS. For
each assay, 50 .mu.l of bacterial cells (approximately
5.times.10.sup.8 cfu/ml) was incubated with 400 .mu.l of PBS with
0.1% BSA and 0.1% Tween 80.RTM. and 5.times.10.sup.4 cpm of the
125I-labeled type II Col. Tubes were incubated at room temperature
for 1 hr and rotated end-over end. The reaction was stopped by the
addition of 3 ml of ice-cold PBS containing 0.1% Tween 80.RTM. and
the tubes were immediately centrifuged at 1500.times.g for 20 min.
After aspiration of the supernatant, the pellet containing
bacterial cells was analyzed for radioactivity in a gamma counter.
Triplicate samples were analyzed, and background values
representing radioactivity recovered in the tubes incubated in the
absence of bacteria was subtracted.
[0387] Analysis of the clinical isolates suggested that binding to
Col is an "all-or-none" phenomenon. Thirty percent (6/20) of the S.
aureus isolates bound to labeled Col. An additional 18 S. aureus
strains isolated from the vitreous of patients with bacterial
keratitis were also analyzed for Col binding activity.
Interestingly, 72% (13/18) of those strains bound Col. Taken
together these data suggest that Col binding is an important
virulence determinant in the pathogenesis of S. aureus eye
infections.
[0388] 5.4.2 Animal Studies Involving S. aureus Keratitis
[0389] In subsequent studies, the inventors have examined whether
ability to bind Col was important in production of microbial
keratitis in the rabbit. Previous animal models of S. aureus
keratitis have required direct intrastromal injection to induce
infection. Microbial adhesion to damaged corneal tissue is most
likely the initiating event in the development keratitis, therefore
to more closely mimic the natural course of disease an animal model
was developed that doesn't involve intrastromal injection. To
determine whether the S. aureus Col MSCRAMM can be considered a
virulence factor in keratitis a rabbit model developed for this
study. Soft contact lenses were placed in culture media of S.
aureus strain Phillips (CBP+) and its isogenic mutant derivative
PH100 (CBP). The contact lenses were incubated for 24 h in the
culture media that contained approximately 10.sup.8 bacteria. This
resulted in 1.times.10.sup.6 bacteria bound to the contact lens for
PH100 and 2.times.10.sup.5 organisms bound to the lens for strain
Phillips. This difference was not statistically significant. The
soft contacts were washed in PBS to remove any loosely bound
organisms and were then were placed onto the de-epithelialized
corneas of New Zealand White rabbits. The nictitating membrane of
the rabbits was removed to prevent dislocation of the contact lens
and the eyelids were sutured closed with 7-0 Vicryl.RTM.. In a
blinded trial a total of 16 rabbits, 8 in each group were used.
Eyes were opened after 48 h of contact lens placement. Comeal
scrapings were performed and placed onto blood agar to confirm the
presence of infection and corneas were removed for histological
examination. One of the rabbits assigned to CBP- group developed
spontaneous dehiscence of the tarsorrhaphy closure and loss of the
contact lens, so the animal was removed from study. Seventy-five
percent (6/8) of the rabbits with contact lenses incubated with
strain Phillips (CBP.sup.+) developed clinical microbial keratitis,
as evidenced by dense, central suppurative stromal keratitis.
Cultures confirmed the presence of S. aureus in all infected cases.
Conversely, 0/7 rabbits with contact lenses incubated with the
PH100 developed suppurative keratitis, though in all cases the
epithelial defect remained. This difference in rate of keratitis
was statistically significant (Fisher's exact test p =0.006).
[0390] The efficacy of a small molecule inhibitor based on the 3-D
structure of CBD(151-318) can be tested using the bacterial
keratitis model. After the contact lens has been incubated with S.
aureus and washed to remove any loosely bound organisms the
contaminated contact is placed in a solution that contains the
inhibitor. The inhibitor will bind to the active site thereby
saturating the Col MSCRAMM thereby preventing the bacteria from
adhering to the exposed Col fibers in the damaged cornea. In
addition to preventing S. aureus bacterial keratitis, the inhibitor
would be added to solutions commonly used by eye banks to prevent
infections in recipients of donor lenses.
5.5 EXAMPLE 5
Mouse Model of Septic Arthritis
[0391] Using the mouse model of septic arthritis (Bremell et al.,
1992), the inventors have studied the use of recombinant domains,
CBD(I 51-297) and CBD(61-343), of the S. aureus Col MSCRAMM as a
vaccine components. 18 mice were vaccinated with 100.mu.g of
protein (GST-61-343) in Freund's complete adjuvant (FCA) on day
days -34, -21, -9. 25 control mice were injected with PBS-FCA on
the same days. The mice were challenged with an intravenous
injection of S. aureus strain Phillips on day 0. The mice immunized
with GST-61-343 had a 50% reduction in arthritis compared to the
control mice. Additional studies have also been performed which use
different domains of the S. aureus Col MSCRAMM.
5.6 EXAMPLE 6
Evidence That Only Particular CBP Epitopes Confer Protection
Against S. aureus Infection
[0392] The following example shows the effective use of CBP
epitopes as vaccine components, and compositions useful in
conferring protection to an animal against infection by S.
aureus.
[0393] 5.6.1 Mouse Sepsis Model--CBP Epitopes Utilized
[0394] M17 contains amino acids 151-297 of the full length CBP (SEQ
ID NO:2).
[0395] M31 contains amino acids 61-343 of the fill length CBP (SEQ
ID NO:4).
[0396] M55 contains amino acids 30-531 of the full length CBP (SEQ
ID NO:6).
[0397] The DNA segments encodingM1 7, M3 1, and M55 epitopes are
disclosed in SEQ ID NO: 1, SEQ ID NO:3, and SEQ ID NO:5,
respectively.
[0398] 5.6.2 Immunization Schedule
[0399] Day -31: 100.mu.g/mouse of a CBP Epitope (i.e. M17, M31, or
M55) or BSA emulsified with Freund's Complete adjuvant (Difco
Laboratories) as a control.
[0400] Day -18: 100.mu.g/mouse of a CBP Epitope (ie. M17, M31, or
M55) or BSA dissolved in sterile phosphate buffered saline
(PBS)
[0401] Day -7: 100.mu.g/mouse of a CBP Epitope (i.e. M17, M31, or
M55) or BSA dissolved in PBS
[0402] Day 0: inoculate intravenously S. aureus strain Phillips
(which expresses CBP) (Dose: 2.8.times.107 cfu/mouse)
[0403] Day 14: end of study; all surviving mice were
euthanized.
5.6.2 RESULTS
[0404] 10/15 mice died in the M17 Col-binding MSCRAMM group
[0405] 12/14 mice died in the M31 Col-binding MSCRAMM group
[0406] 4/11 mice died in the M55 Col-binding MSCRAMM group
[0407] All 15 of 15 mice died in the BSA control group.
[0408] In a control study, M55 and BSA immunized mice were
challenged with a strain of S. aureus that does not express the
Col-binding MSCRAMM. The mortality in the M55 group was 50%
compared to 30% in the BSA group. Importantly, these data indicated
that protection is directly related to antibodies generated against
the M55 portion of the Col binding MSCRAMM.
[0409] In sharp contrast to the previously-identified full-length
sequence which does not confer protection against sepsis in an in
vivo animal model, these data clearly show that M55 (containing
only the A domain) was highly effective in protecting against
sepsis.
[0410] Surprisingly, while all of the CBP fragments (M17 and M31)
are contained within the M55 protein sequence, only M55 is highly
protective.
5.7 Example 7
Opsonization, Phagocytosis and Intracellular Killing Of
Bacteria
[0411] The phagocytosis test was done by a modification of the
previously described method (Lissner et al. 1983). Briefly,
peritoneal macrophages were collected from the peritoneal cavity by
injecting 3 ml of ice cold medium (Iscovs medium containing 10%
fetal calf serum, and 100 .mu.g/ml of gentamycin) ip., after one
minute massage of the abdomen the macrophage containing medium was
aspirated. The macrophages were washed and adjusted to
2.times.10.sup.6 cells/ml, seeded in 200 .mu.l volumes in 24-well
plates (Nunc, Roskilde, Denmark) and left at room temperature for
90 min. Five-hundred .mu.l of cell culture medium was added to each
well and the cells were incubated for 24 hours in 37.degree. C. The
medium was then removed and replaced by 500 .mu.l of a medium free
of antibiotics and the cells were then incubated overnight at
37.degree. C. The next day the staphylococci were opsonized for 30
min at 4.degree. C. with either a) 50% heat inactivated sera of
mice that have been hyperimmunized with M55 or b) with sera from
BSA hyperimmunized mice or alternatively c) with sera from mice
that have gone through infection with strain Phillips without
previous immunization. Five-hundred .mu.l of opsonized
staphylococci were added to the wells in a concentration of
1.4.times.10.sup.7 bacterial/ml. After 50 min of incubation, the
macrophages were washed 3 times in Iscovs in order to remove
non-ingested bacteria. Thereafter, the macrophages were analyzed
either directly after bacterial incubation or 4 hours later. To
those cultures that were incubated for 4 hours, Iscoves medium with
the minimal inhibitory concentration of gentamycin suitable for S.
aureus strain Phillips (5 .mu.g/ml) was added to avoid
extracellular replication of bacteria. The macrophages were lysed
with distilled water for 20 minutes, and the lysate diluted 1/1,
1/10, 1/100, and 1/1000 was cultured on 5% horse blood agar plates.
The plates were incubated overnight and the number of bacteria
counted.
[0412] 5.7.1 In Vitro Assays
[0413] In vitro assays were performed to assess the impact of
specific antibodies to collagen adhesion on phagocytosis and
intracellular killing capacity. Collagen adhesion expressing
Phillips strain was opsonized with either serum containing M55
specific antibodies or serum containing BSA antibodies, or
alternatively serum from naive mice that have gone through
infection with Phillips.
[0414] The results (FIG. 5A) show clearly that intracellular
killing of S. aureus Phillips strain is moderately enhanced by a
previous infection with the same strain (p-0.037). In contrast,
opsonization of staphylococci with serum from M55 mice immunized
(but not infected) mice displayed significantly enhanced
intracellular killing capacity as compared to control serum
(p=0.009). The phagocytic capacity was only modestly affected by
opsonization of bacteria with serum containing M55 antibodies and
significantly affected when the bacteria were opsonized with serum
of Phillips strain infected mice (FIG. 5B).
5.8 Example 8
Detection of CBP Antibodies In Mice Infected With S. Aureus
[0415] In this study, 15 mice were experimentally infected with a
sub-lethal does of S. aureus that expresses the Col-binding
MSCRAMM. Anti-M55 (Col MSCRAMM) IgG could not be detected in the
sera from the infected animals. Conversely, animals immunized with
M55 and infected with a sub-lethal dose S. aureus had anti-M55 IgG
(Col MSCRAMM) titers of 32.times.10.sup.9 units/ml.
[0416] These data indicate that during the normal course of
infection, the CBP is protected from immunological recognition. The
induction of polyclonal B-cell activation, as a consequence of
infection with S. aureus will down regulate specific immune
responses to many bacterial cell wall components, including the
native CBP.
[0417] 5.8.1 Experimental Details
[0418] Serum level of specific antibodies against the collagen
adhesion peptide M55 was measured by an enzyme-linked immunosorbent
assay (ELISA). 96-well microplates (Nunc) were coated overnight at
4.degree. C. with 2 .mu.g/ml of M55 peptide. Blocking was done with
0.5% ovalbumin (Sigma Chemical Co., St. Louis, MO) dissolved in
0.05 M Tris (pH 7.7). Sera, biotinylated antibodies, the
ExtrAvidin-peroxidase (Sigma) were all diluted in 0.05 M Tris
(pH7.4)-0.015 M NaCl. The plates were incubated overnight at
4.degree. C. with sera, washed and incubated stepwise with
biotinylated goat anti-mouse IgG antibody (Jackson Immuno Research
Laboratories, Inc., West Grove, PA), ExtrAvidin-peroxidase (0.5
.mu.g/ml; Sigma) and ABTS substrate. The A405 was measured in a
Titertec Multiscan photometer (Flow Laboratories, McLean, Va.).
Similar ELISA procedure as described above was employed to detect
antibodies to M17, M31, as well as to the Bi domain of collagen
adhesion. OVA was used as control solid antigen.
[0419] In addition, 96-well plates were precoated with
poly-L-lysine (Sigma) and then coated with either 1.5.times.107 S.
aureus strains Phillips or LS-1. After blocking procedure sera from
immunized mice and unimmunized but infected mice as well as naive
mouse sera, were incubated. The development steps were then made as
described above. The data are shown in FIG. 6.
5.9 Example 9
Detection of Antibodies To Col-Binding MSCRAMMs In Human Infected
By S. Aureus
[0420] This example describes the results of studies to detect
antibodies to the Col binding MSCRAMM.
[0421] Sera from 34 patients clinically diagnosed as having S.
aureus infections were examined by ELISA. IgG that recognized the
immobilized M55 (Col MSCRAMM) could be detected only when
exceedingly high levels of antigen were used in the assay. Thus,
despite the fact that these patients have had clinically diagnosed
S. aureus infections, only a minor increase in .alpha.-CBP titers
could be detected. These data (FIG. 7A and FIG. 7B) suggest that S.
aureus infection may induce a polyclonal B-Cell activation that
downregulates the human immune response to certain microbial cell
wall components, including the CBP.
6. REFERENCES
[0422] The following literature citations as well as those cited
above are incorporated in pertinent part by reference herein for
the reasons cited in the above text:
[0423] U.S. Pat. No. 3,791,932.
[0424] U.S. Pat. No. 3,949,064.
[0425] U.S. Pat. No. 4,174,384.
[0426] U.S. Pat. No. 4,196,265.
[0427] U.S. Pat. No. 4,271,147.
[0428] U.S. Pat. No. 4,554,101.
[0429] U.S. Pat. No. 4,578,770.
[0430] U.S. Pat. No. 4,596,792.
[0431] U.S. Pat. No. 4,599,230.
[0432] U.S. Pat. No. 4,599,231.
[0433] U.S. Pat. No. 4,601,903.
[0434] U.S. Pat. No. 4,608,251.
[0435] U.S. Pat. No. 4,683,195.
[0436] U.S. Pat. No. 4,683,202.
[0437] Abraham et al., "Adherence of Streptococcus pyogenes,
Escherichia coli, and Pseudomonas aeruginosa to Fibronectin-Coated
and Uncoated Epithelial Cells," Infect. Immun., 41:1261-1268,
1983.
[0438] Aleljung et al.,
[0439] Curr. Microbiol., 28:231-236, 1994.
[0440] Allen and Choun, "Large Unilamellar Liposomes with Low
Uptake into the Reticuloendothelial System," FEBS Lett., 223:4246,
1987.
[0441] Allen et al., J. Bact., 173:916-920, 1991.
[0442] Bayer and Wilchek, "The use of the avidin-biotin complex as
a tool for molecular biology," In: Methods of Biochemical Analysis,
Glick, D., John Wiley and Sons, New York, 1980.
[0443] Bella et al., "Crystal and Molecular Structure of a Col-Like
Peptide at 1.9 .ANG. Resolution," Science, 266:75-81, 1994.
[0444] Bidanset, Guidry, Rosenberg, Choi, Timpl, Hook, "Binding of
the Proteoglycan Decorin to Collagen Type VI," J. Biol. Chem.,
267:5250-5256, 1992.
[0445] Bolivar et al., Gene, 2:95, 1977.
[0446] Bremell et al, Infect. Immun., 59(8):2615-2623, 1991.
[0447] Bremell et al., Infect. Immun., 62(7):2976-2985, 1992.
[0448] Brunger et al., Science, 235:458460, 1987.
[0449] Brunger, "X-PLOR Manual," Version 3.1, Yale Univ., New
Haven, Conn., 1992.
[0450] Campbell, "Monoclonal Antibody Technology, Laboratory
Techniques in Biochemistry and Molecular Biology," Burden and Von
Knippenberg, Eds., Elsevier, Amsterdam, 13:75-83, 1984.
[0451] Capecchi, "High efficiency transformation by direct
microinjection of DNA into cultured mammalian cells," Cell,
22(2):479-488, 1980.
[0452] Carret et al., Ann Inst. Pasteur Microbiol., 136A:241-45,
1985.
[0453] Carter et al., Nucl. Acids Res., 12:4431-4443, 1985.
[0454] Chang et al., Nature, 375:615, 1978.
[0455] Chen et al., "An energetic evaluation of a "Smith" collagen
microfibril model," J. Protein Chem., 10:535-542, 1991.
[0456] China et al., Infect. Immun., 61(8):3129-3136, 1993.
[0457] Chou and Fasman, "Conformational Parameters for Amino Acids
in Helical, P-Sheet, and Random Coil Regions Calculated from
Proteins," Biochemistry, 13(2):211-222, 1974b.
[0458] Chou and Fasman, "Empincal Predictions of Protein
Conformation," Ann. Rev. Biochem., 47:251-276, 1978b.
[0459] Chou and Fasman, "Prediction of .beta.-Turns," Biophys. J.,
26:367-384, 1979.
[0460] Chou and Fasman, "Prediction of Protein Conformation,"
Biochemistry, 13(2):222-245, 1974a
[0461] Clapp, "Somatic gene therapy into hematopoietic cells.
Current status and future implications," Clin. Perinatol.,
20(1):155-168, 1993.
[0462] Cole et al., J. Bone and Joint Surg, 64B:218, 1982.
[0463] Connolly, "Analytical Molecular Surface Calculation," J.
Appl. Cryst., 16:548-558, 1983.
[0464] Connolly, "The molecular surface package," J. Mol. Graphics,
11:139-141,1993.
[0465] Couvreur et al., "Nanocapsules, a New Lysosomotropic
Carrier," FEBS Lett., 84:323-326, 1977.
[0466] Couvreur, "Polyalkyleyanoacrylates as Colloidal Drug
Carriers," Crit. Rev. Ther. Drug Carrier Syst., 5:1-20, 1988.
[0467] Cox et al., J. Virol., 67(9):5664-5667, 1993.
[0468] Curiel et al., "Adenovirus enhancement of
transferrin-polylysine-me- diated gene delivery," Proc. Natl. Acad
Sci. USA, 88(19):8850-8854, 1991.
[0469] Emody et al., J. Bact., 171(12):6674-6679, 1989.
[0470] Fields et al., Biopolymers, 33:1695-1707, 1993.
[0471] Fiers et al., Nature, 273:113, 1978.
[0472] FRAMBO, Siemens Analytical X-Ray Instruments, 6300
Enterprise Lane, Madison, Wis. 53719, USA, 1989.
[0473] Froman et al., "Binding of Escherichia coli to Fibronectin:
A Mechanism of Tissue Adherence," J. Biol. Chem., 259:14899-14905,
1984.
[0474] Fromm etal., "Expression of genes transferred into monocot
and dicot plant cells by electroporation," Proc. Natl. Acad. Sci.
USA, 82(17):5824-5828, 1985.
[0475] Furey and Swaminathan, Amer. Crystal. Assoc. Meet. Abstr.,
18:73, 1990.
[0476] Fynan et al., "DNA vaccines: protective immunizations by
parenteral, mucosal, and gene gun inoculations," Proc. Natl. Acad
Sci. USA, 90(24):11478-11482, 1993.
[0477] Gabizon and Papahadjopoulos, "Liposomes formulations with
prolonged circulation time in blood and enhanced uptake by tumors,"
Proc. Natl. Acad. Sci. USA, 85:6949-6953, 1988.
[0478] Goding, "Monoclonal Antibodies: Principles and Practice,"
2nd Edition, Academic Press, Orlando, Fla., pp. 60-74, 1986.
[0479] Goeddel et al., Nature, 281:544, 1979.
[0480] Goeddel et al., Nucl Acids Res., 8:4057, 1980.
[0481] Goldenberg, Arthritis Rheum., 32:496-502, 1989.
[0482] Graham and van der Eb, "Transformation of rat cells by DNA
of human adenovirus 5," Virology, 54(2):536-539, 1973.
[0483] Granfors et al., J. Infect. Dis., 141:424429, 1980.
[0484] Granfors et al., N. Engl. J. Med, 320:216-221, 1989.
[0485] Harlow and Lane, "Antibodies: a Laboratory Manual," Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988.
[0486] Hay, "Cell Biology of Extracellular Matrix," New York,
Plenum Press, 1991.
[0487] Hay, Cell Biology ofExtracellular Matrix, 2nd ed., Plenum
Press, New York, 1991.
[0488] Hedborn and Heinegard, J. Biol. Chem., 264:6898-6905,
1989.
[0489] Heidemann and Roth, "Synthesis and Investigation of Collagen
Model Peptides," Adv. Polym. Sci., 43:143-203, 1982.
[0490] Henry-Michelland et al., "Attachment of Antibiotics to
Nanoparticles; Preparation, Drug-Release and Antimicrobial Activity
in vitro,"
[0491] Int. J. Pharm., 35:121-127, 1987.
[0492] Hess et al., J. Adv. Enzyme Reg., 7:149, 1968.
[0493] Hitzeman et al., J. Biol. Chem., 255:2073, 1980.
[0494] Ho and Su, J. Amer. Med Assoc., 247:797-800, 1982,
[0495] Ho et al., "Site-Directed Mutagenesis by Overlap Extension
Using the Polymerase Chain Reaction,"
[0496] Gene, 77:51-59, 1989.
[0497] Hochuli et al., J. Chromatogr., 411:177-184, 1988.
[0498] Hodel et al., Acta Crystallogr., A48:851-858, 1992.
[0499] Holderbaum et al., Collagen Rel. Res., 5:261-271, 1985.
[0500] Holland et al., Biochemistry, 17:4900, 1978.
[0501] Hornick etal., Infect. Immun, 60(4):1577-1588, 1992.
[0502] House-Pompeo, Boles, and Hook, "Characterization of
Bacterial Adhesin Interactions with Extracellular Matrix Components
Utilizing Biosensor Technology," METHODS: A Companion to Meth in
Enzym., 6:134-142, 1994.
[0503] Ingham, Brew, Migliorini, J. Biol. Chem., 264:16977-16980,
1989.
[0504] Jameson and Wolf, Comp. Appl. Biosci., 4:181-186,-1988.
[0505] Jiang and Kim, "Soft Docking: Matching of Molecular Surface
Cubes," J. Mol. Biol., 219:79-102, 1991.
[0506] Joh et al., Biochemistry, 33(20):6086-6092, 1994.
[0507] Johnson, Anal Biochem., 206:215-225, 1992.
[0508] Jones et al., Acta Cryst, A47:110-119, 1991.
[0509] Jones, Genetics, 85:12 1977.
[0510] Kern et al., Eur. J. Biochem., 215:151-159, 1993.
[0511] Kingsman et al., Gene, 7:141, 1979.
[0512] Kohler and Milstein, Eur. J. Immunol., 6:511-519, 1976.
[0513] Kohler and Milstein, Nature, 256:495497, 1975.
[0514] Kostzynska et al., FEMS Microbiol. Lett., 59:229-234,
1989.
[0515] Krumdieck et al., "The Proteoglycan Decorin Binds C1q and
Inhibits the Activity of the C1 Complex," J. Immunol.,
149:3695-3701, 1992.
[0516] Kuby, "Immunology," 2nd Edition, W. H. Freeman &
Company, New York, 1994.
[0517] Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982.
[0518] Lantz et al., J. Bacteriol., 173(14):42634270, 1991.
[0519] Laskowski et al., "PROCHECK: a program to check the
stereochemical quality of protein structures," J. Appl. Cryst.,
26:283-291, 1993.
[0520] Laskowski et al., J. Appl. Cryst., 26:283-291, 1993.
[0521] Liu and Gibbons, Oral Micro. And Immun., 5(3):131-136,
1990.
[0522] Liu et al., Oral Micro. and Immun., 5(3):143-148, 1990.
[0523] Malmquist, Nature, 361:186-187, 1993.
[0524] Maloy, et al., "Microbial Genetics," 2nd Edition, Jones and
Bartlett Publishers, Boston, Mass., 1994.
[0525] Maniatis et al., "Molecular Cloning: a Laboratory Manual,"
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982.
[0526] Maxe et al., "Specific Attachment of Staphylococcus aureus
to Immobilized Fibronectin," Infect. Immun., 54:695-704, 1986.
[0527] McRee, Practical Protein Crystallography, Academic Press,
1993.
[0528] Merilahti-Palo, Ann Rheum. Dis., 50:87-90, 1991.
[0529] Miettinen et al., Kidney, 43:592-600, 1993.
[0530] Mumby, Raugi, Bornstein, J. Cell Biol., 98:646-652,
1984.
[0531] Nakamura et al., "Enzyme Immunoassays: Heterogenous and
Homogenous Systems," Chapter 27, 1987.
[0532] Nemethy et al., "Energy parameters in Polypeptides. 10.
Improved Geometrical Parameters and Nonbonded Interactions for Use
in the ECEPP/3 Algorithm, with Application to Proline-Containing
Peptides," J. Phys. Chem., 96:6472-6484, 1992.
[0533] Nicholls et al., Proteins: Struct. Funct. Genet.,
11:281-296, 1991.
[0534] Nowicki et al., J. Exp. Med., 178(6):2115-2121, 1993.
[0535] O'Shannessy, Anal. Biochem., 212:457468, 1993.
[0536] Oldberg et al., EMBO J., 8:2601-2606, 1989.
[0537] Patthy, J. Mol. Biol, 198:567-577, 1987.
[0538] Patti et al., "Critical Residues in the Ligand-binding Site
of the Staphylococcus aureus Collagen-binding Adhesin (MSCRAMM),"
J. Biol. Chem., 270:12005-12011, 1995.
[0539] Patti et al., "Molecular Characterization and Expression of
a Gene Encoding a Staphylococcus aureus Collagen Adhesin," J. Biol.
Chem., 267:47664772, 1992.
[0540] Patti et al., "The Staphylococcus aureus Collagen Adhesin is
a Virulence Determinant in Experimental Septic Arthritis," Infect.
Immun, 62:152-161, 1994.
[0541] Patti et al., Biochemistry, 32(42):11428-11435, 1993.
[0542] Patti etal., Biol. Chem., 270:12005-12011, 1995.
[0543] Patti et al, J. Biol. Chem., 267(7):47664772, 1992.
[0544] Patti, Allen, McGavin, Hook, Annu. Rev. Microbiol.L,
48:585-617. 1994.
[0545] Patti, Boles, Hook, "Identification and Biochemical
Characterization of the Ligand Binding Domain of the Collagen
Adhesin from Staphylococcus aureus," Biochemistry, 32:11428-11435,
1993.
[0546] Pilar Fernandez, Selmin, Martin, Yamada, Pfaffle, Deutzmann,
Mollenhauer, von der Mark, J. Biol Chem., 263:5921-5925, 1988.
[0547] Pilz et al., Infect. Immun., 60:189-195, 1992.
[0548] Porath et al., Nature, 258:598-599, 1975
[0549] Prokop and Bajpai, "Recombinant DNA Technology I" Ann N.Y.
Acad. Sci., Vol. 646, 1991.
[0550] Ramachandran and Reddi, "Biochemistry of Collagen," Plenum
Press, New York, 1976.
[0551] Richardson, "The anatomy and taxonomy of protein structure,"
Advances in Protein Chemistry, 34:167-339, 1981.
[0552] Richardson, Advances in Protein Chemisty, 34:297-306,
Academic Press, 1981.
[0553] Rubin, Hook, brink, Timpl, "Substrate Adhesion of Rat
Hepatocytes: Mechanism of Attachment to Collagen Substrates," Cell,
24:463-470, 1981.
[0554] Rydn et al., Eur. J. Biochem., 184:331-336, 1989.
[0555] Sack, J. Mol. Graphics, 6:224-225, 1988.
[0556] Sakakibara et al., "Synthesis of (Pro-Hyp-Gly).sub.n of
defined molecular weights. Evidence for the stabilization of
collagen triple helix by hydroxypyroline," Biochim. Biophys. Acta,
303:198-202, 1973.
[0557] Sambrook et al., "Molecular Cloning: A Laboratory Manual,"
2nd Edition, Chapter 12.6, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989.
[0558] Sanger, Proc. Natl. Acad Sci. USA, 74:5463-5467, 1977.
[0559] Santoro, Cell, 46:913-920, 1986.
[0560] Scatchard, Ann. N.Y. Acad. Sci., 51:660-672, 1949.
[0561] Schulze-Koops et al., Infect. Immun., 60:2153-2159,
1992.
[0562] Schulze-Koops et al., Infect. Immun, 61(6):2513-2519,
1993.
[0563] Segal and Traub, "Polymers of Tripeptides as Collagen
Models. VI. Synthesis and Structural Investigation of
Poly(L-alanyl-L-prolyl-glycine)- ," J. Mol. Biol., 43:487-496,
1969.
[0564] Segal, "Biochemical Calculations," 2nd Edition, John Wiley
and Sons, New York, 1976.
[0565] Smith et al., Arthritis Rheum, 25(4):441446, 1982.
[0566] Smith et al., J. Bone & Joint, 69(7):1063-1068,
1987.
[0567] Speziale et al., "Binding of Collagen to Staphylococcus
aureus," Cowan 1, J. Bacteriol., 167:77-81, 1986.
[0568] Speziale et al., FEMS Microbiol. Lett., 48:47-51, 1987.
[0569] Speziale et al., J. Bacteriol., 167:77-81, 1986.
[0570] Stinchcomb et al., Nature, 282:39, 1979.
[0571] Switalski et al., "A collagen receptor on Staphylococcus
aureus strains isolated from patients with septic arthritis
mediates adhesion to cartilage," Mol. Microbiol, 7:99-107,
1993.
[0572] Switalski et al., Infect. Immun., 61(10):41 194125,
1993.
[0573] Switalski et al., J. Biol. Chem., 264(35):21080-21086,
1989.
[0574] Switalski et al, Mol. Microbiol, 7(1):99-107, 1993.
[0575] Takada and Hemler, J. Cell Biol., 109:397-407, 1989.
[0576] Takagi etal., J Biol. Chem., 266:5575-5579, 1991.
[0577] Takagi, Biochem, 31:8530-8534, 1992.
[0578] Tamm et al., Mol. Microbiol., 10:995-1011, 1993.
[0579] Tang et al., Nature, 356:152-154, 1992.
[0580] Tarkkanen et al., Mol. Microbiol., 4(8):1353-1361, 1990.
[0581] Trust, et al., Mol. Microbiol., 7(4):593-600, 1993.
[0582] Tschemperetal., Gene, 10:157, 1980.
[0583] Ulmer et al., "Heterologous Protection Against Influenza by
Injection of DNA Encoding a Viral Protein," Science, 259:1745-1749,
1993.
[0584] Van Nhieu et al., "Bacterial Internalization Medicated by b1
Chain Integrins is Determined by Ligand Affinity and Receptor
Density," EMBO J., 12:1887-1895, 1993.
[0585] Vandenberg et al., "Characterization of a Type IV Collagen
Major Cell Binding Site with Affinity to the .alpha.1.beta.1 and
the .alpha.2.beta.1 Integrins," J. Cell Biol, 113:1475-1483 ,
1991.
[0586] Vanderrest and Garrone, FASEB J., 5:2814-2823, 1991.
[0587] Velge, Parasitology, 97:255-268, 1988.
[0588] Voytek et al, Biomaterials, 9:107-110, 1988.
[0589] Wagner et al., "Coupling of adenovirus to
transferrin-polylysine/DN- A complexes greatly enhances
receptor-mediated gene delivery and expression of transfected
genes," Proc. Natl. Acad. Sci. USA, 89(13):6099-6103, 1992.
[0590] Waldvogel et al., N. Eng J. Med, 303:360-369, 1980.
[0591] Wang et al., J Exp. Med., 177:699, 1993.
[0592] Wang et al., J. Immunol., 150:3022,1993.
[0593] Westerlund et al., Mol. Microbiol., 3:329-337, 1989.
[0594] Whitton et al, J. Virol., 67(1):348-352, 1993.
[0595] Wolfet al., Compu. Appl. Biosci., 4(1):187-91, 1988.
[0596] Wong and Neumann, "Electric field mediated gene transfer,"
Biochim. Biophys. Res. Commun, 107(2):584-587, 1982.
[0597] Woody, Peptides, Polypeptides, and Proteins, New York,
Wiley, 1974.
[0598] Yamaguchi et al., "Negative Regulation of Transforming
Growth Factor-b by the Proteoglycan Decorin," Nature, (London),
346:281-284, 1990.
[0599] Yang and Russel, Proc. Natl. Acad Sci. USA, 87:4144-4148,
1990.
[0600] Bremell, T., Lange, S., Yacoub, A., Rydn, C. and Tarkowski,
A. (1991) Experimental Staphylococcus aureus arthritis in mice.
Infect. Immun. 59(8): 2615-2623.
[0601] Cifrian, E., Guidry, A. J., O'Brien, C. N. and Marquardt, W.
W. (1995) Effect of alpha-toxin and capsular exoploysaccharide on
the adherence of Staphylococcus aureus to cultered teat, ductal,
and secretory mammary epithelial cells. Res. Vet. Sci.
58:20-25.
[0602] Falkow, S., Isberg, R. R. and Portnoy, D. A. (1992) The
Interaction of Bacteria with Mammalian Cells. Annual Review of Cell
Biology 8:333-363.
[0603] Fattom, A. I., Sarwar, J., Ortiz, A. and Naso, R. (1996) A
Staphylococcus aureus capsular polysaccharide (CP) vaccine and
CP-specific antibodies protect mice against bacterial challenge.
Infect Immun 64(5):1659-1665.
[0604] Foster, T. J. (1991) Potential for Vaccination Against
Infections Caused by Staphylococcus aureus. Vaccine
9(4):221-227.
[0605] Greco, D., Salmaso, S., Mastrantonio, P., Giuliano, M.,
Tozzi, A., et al. (1996) A controlled trial of two acellular
vaccines and one whole-cell vaccine against pertussis. N. Engl. J
Med. 334:341-348.
[0606] Greenberg, D. P., Ward, J. I. and Bayer, A. S. (1987)
Influence of Staphylococcus aureus antibody on experimental
endocarditis in rabbits. Infect. Immun. 55:3030-3034.
[0607] Gudding, R., McDonald, J. D. and Cheville, N. F. (1984)
Pathogenesis of Staphylococcus aureus mastitis: bacteriological,
histological, and ultrstructural pathological findings. Am. J. Vet.
Sci. 45:2525-2531.
[0608] Gustaffson, L., Hallander, H. O., Olin, P., Reizenstein, E.
and Storsaeter, J. (1996) A controlled trial of a two-component
acellular, a five-component acellular, and a whole-cell pertussis
vaccine. N. Engl. J. Med 334:349-355.
[0609] Joh, H. J., House-Pompeo, K., Patti, J. M., Gurusiddappa, S.
and Hook, M. (1994) Fibronectin receptors from gram-positive
bacteria: Comparison of active sites. Biochemistry
33(20):6086-6092.
[0610] Karakawa, W. W., Sutton, A., Schneerson, R., Karpas, A. and
Vann, W. F. (1988) Capsular antibodies induce type-specific
phagocytosis of capsulated Staphylococcus aureus by human
polymorphonuclear leukocytes. Infect. Immun. 56:1090-1094.
[0611] Lepper, A. W. D., Atwell, J. L., Lehrbach, P. R.,
Schwartzkoff, C. L., Egerton, J. R., et al. (1995) The protective
efficacy of cloned Moraxella bovis pill in monovalent and
multivalent vaccine formulations against experimentally induced
infectious bovine keratoconjunctivitis (IBK). Vet Microbiol
45(2-3):129-138.
[0612] McDevitt, D., Francois, P., Vaudaux, P. and Foster, T. J.
(1994) Molecular characterization of the clumping factor
(fibrinogen receptor) of Staphylococcus aureus. Mol. Micro. 11(2):
237-248.
[0613] McQueen, C. E., Boedeker, E. C., Reid, R., Jarboe, D., Wolf,
M., et al. (1993) Pili in microspheres protect rabbits from
diarrhoea induced by E. coli strain RDEC-1. Vaccine 11:201-206.
[0614] Menzies, B. E. and Kemodle, D. S. (1996) Passive
immunization with antiserum to a nontoxic alpha-toxin mutant from
Staphylococcus aureus is protective in a murine model. Infect Immun
64(5): 1839-1841.
[0615] Olmsted, S. B. and Norcross, N. L. (1992) Effect of specific
antibody on adherence of Staphylococcus aureus to bovine mammary
epithelial cells. Infect. Immun. 60(1): 249-256.
[0616] Patti, J. M., House-Pompeo, K., Boles, J. O., Garza, N.,
Gurusiddappa, G., et al. (1995) Critical residues in the ligand
binding site of the Staphylococcus aureus Col-binding adhesin
(MSCRAMM). J. Biol. Chem. 270:12005-12001.
[0617] Poole-Warren, L. A., Hallett, M. D., Hone, P. W., Burden, S.
H. and Farrell, P. C. (1991) Vaccination for prevention of CAPD
associated staphylococcal infection: results of a prospective
multicentre clinical trial. Clinical Nephrology 35:198-206.
[0618] Ramisse, F., Szatanik, M., Binder, P. and Alonso, J. M.
(1993) Passive Local Immunotherapy of Experimental Staphylococcal
Pneumonia with Human Intravenous Immunoglobulin. Journal of
Infectious Diseases 168(4):1030-1033.
[0619] Ryding, U., Christensson, B., Soderquist, B. and Wadstrom,
T. (1995) Antibody response to Staphylococcus aureus Col binding
protein in patients with S-aureus septicaemia and Col binding
properties of corresponding strains. J. Med Microbiol 43(5):
328-334.
[0620] Sirakova, T., Kolattukudy, P. E., Murwin, D., Billy, J.,
Leake, E., et al. (1994) Role of Fimbriae Expressed by Nontypeable
Haemophilus Influenzae in Pathogenesis of and Protection Against
Otitis Media and Relatedness of the Fimbrin Subunit to Outer
Membrane Protein a Infect. Immun. 62(5): 2002-2020.
[0621] Welch, P. G., Fattom, A., Moore, J., Schneerson, R.,
Shiloach, J., et al. (1996) Safety and immunogenicity of
Staphylococcus aureus type 5 capsular polysaccharide-pseudomonas
aeruginosa recombinant exoprotein A conjugate vaccine in patients
on hemodialysis. J Amer Soc Nephrol 7(2): 247-253.
Sequence CWU 1
1
* * * * *