U.S. patent application number 13/229575 was filed with the patent office on 2012-05-24 for compositions and methods for the removal of biofilms.
This patent application is currently assigned to Nationwide Children's Hospital, Inc.. Invention is credited to Lauren O. Bakaletz, Steven D. Goodman.
Application Number | 20120128701 13/229575 |
Document ID | / |
Family ID | 44654515 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120128701 |
Kind Code |
A1 |
Goodman; Steven D. ; et
al. |
May 24, 2012 |
COMPOSITIONS AND METHODS FOR THE REMOVAL OF BIOFILMS
Abstract
Methods of breaking down a biofilm or inhibiting, preventing or
treating a microbial infection that produces a biofilm are
disclosed, which involves administration of a polypeptide that has
one or more HMG-box domains to a subject suffering from the
infection or having the biofilm. By competing with microbial
proteins that bind to DNA scaffold in the biofilm, these
polypeptides destabilize the biofilm leading to destruction and
removal of the biofilm by the immune system.
Inventors: |
Goodman; Steven D.; (Los
Angeles, CA) ; Bakaletz; Lauren O.; (Columbus,
OH) |
Assignee: |
Nationwide Children's Hospital,
Inc.
University of Southern California
|
Family ID: |
44654515 |
Appl. No.: |
13/229575 |
Filed: |
September 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61381377 |
Sep 9, 2010 |
|
|
|
Current U.S.
Class: |
424/184.1 ;
514/2.4; 514/2.6; 514/2.7; 514/2.8 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 31/04 20180101; Y02A 50/481 20180101; A61K 38/1709 20130101;
A61P 37/04 20180101; Y02A 50/471 20180101; A61P 1/02 20180101; A61P
43/00 20180101; Y02A 50/473 20180101; A61P 27/16 20180101; A01N
37/46 20130101; Y02A 50/401 20180101; Y02A 50/30 20180101 |
Class at
Publication: |
424/184.1 ;
514/2.7; 514/2.6; 514/2.8; 514/2.4 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A01P 1/00 20060101 A01P001/00; A61P 31/04 20060101
A61P031/04; A61K 39/00 20060101 A61K039/00; A01N 37/46 20060101
A01N037/46 |
Claims
1. A method for inhibiting, competing or titrating the binding of a
DNABII polypeptide or protein to a microbial DNA, comprising
contacting the DNABII polypeptide or protein or the microbial DNA
with a polypeptide comprising an HMG-box domain, thereby
inhibiting, competing or titrating the binding of the DNABII
protein or polypeptide to the microbial DNA.
2. A method for inhibiting, preventing or breaking down a microbial
biofilm, comprising contacting the biofilm with a polypeptide
comprising an HMG-box domain, thereby inhibiting, preventing or
breaking down the microbial biofilm.
3. The method of claim 1, wherein the contacting is in vitro or in
vivo.
4. A method of inhibiting, preventing or breaking down a biofilm in
a subject, comprising administering to the subject an effective
amount of a polypeptide comprising an HMG-box domain, thereby
inhibiting, preventing or breaking down the microbial biofilm.
5. A method for inhibiting, preventing or treating a microbial
infection that produces a biofilm in a subject, comprising
administering to the subject an effective amount of a polypeptide
comprising an HMG-box domain, thereby inhibiting, preventing or
treating a microbial infection that produces the biofilm in the
subject.
6. The method of claim 1, wherein the polypeptide comprising an
HMG-box domain comprises one or more of: (a) an isolated or
recombinant protein HMGB1 or a fragment thereof that comprises one
or more HMG-box domains; (b) an isolated or recombinant protein
HMGB2 or a fragment thereof that comprises one or more HMG-box
domains; (c) an isolated or recombinant protein HMGB3 or a fragment
thereof that comprises one or more HMG-box domains; (d) an isolated
or recombinant protein HMGB4 or a fragment thereof that comprises
one or more HMG-box domains; or (e) a polypeptide that is at least
about 70% identical to any of (a), (b), (c) or (d).
7. The method of claim 1, wherein the polypeptide comprising an
HMG-box domain comprises an isolated or recombinant protein HMGB1,
a polypeptide that is at least about 70% identical to HMGB1, or a
fragment thereof that comprises one or more HMG-box domains.
8. The method of claim 6, wherein the isolated or recombinant
protein is selected from the group of: a mammalian protein that is
or is not post-translationally modified, a mammalian protein that
is or is not post-translationally alkylated or a protein expressed
in a non-mammalian system.
9. The method of claim 8, wherein the mammalian protein is a human
protein.
10. The method of claim 4, further comprising administering to the
subject an effective amount of one or more of an antimicrobial, an
antigenic peptide or an adjuvant.
11. The method of claim 4, wherein the subject is a non-human
animal or a human patient.
12. The method of claim 4, wherein the polypeptide is administered
by a method comprising topically, transdermally, sublingually,
rectally, vaginally, ocularly, subcutaneous, intramuscularly,
intraperitoneally, urethrally, intranasally, by inhalation or
orally.
13. The method of claim 4, wherein the subject is a pediatric
patient and the polypeptide is administered in a formulation for
the pediatric patient.
14. The method of claim 4, wherein the biofilm comprises microbial
DNA from a microorganism identified in Table 1.
15. The method of claim 4, wherein the polypeptide is administered
locally to the microbial infection.
16. A method for inducing or providing an immune response in a
subject in need thereof, comprising administering to the subject an
effective amount of a polypeptide comprising an HMG-box domain.
17. The method of claim 16, wherein the administration is local to
where the immune response is desired.
18. The method of claim 16, wherein the polypeptide comprising an
HMG-box domain comprises one or more of: (a) an isolated or
recombinant protein HMGB1 or a fragment thereof that comprises one
or more HMG-box domains; (b) an isolated or recombinant protein
HMGB2 or a fragment thereof that comprises one or more HMG-box
domains; (c) an isolated or recombinant protein HMGB3 or a fragment
thereof that comprises one or more HMG-box domains; (d) an isolated
or recombinant protein HMGB4 or a fragment thereof that comprises
one or more HMG-box domains; or (e) a polypeptide that is at least
about 70% identical to any of (a), (b), (c) or (d).
19. The method of claim 16, wherein the polypeptide comprising an
HMG-box domain comprises an isolated or recombinant protein HMGB1,
a polypeptide that is at least about 70% identical to HMGB1, or a
fragment thereof that comprises one or more HMG-box domains.
20. The method of claim 18, wherein the isolated or recombinant
protein is a mammalian protein.
21. The method of claim 20, wherein the mammalian protein is a
human protein.
22. The method of claim 16, wherein the subject is a non-human
animal or a human patient.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Ser. No. 61/381,377, filed Sep. 9,
2010, the contents of which are incorporated by reference in their
entirety into the current disclosure.
FIELD OF THE INVENTION
[0002] This invention generally relates to the methods and
compositions to lessen and/or cure clinical or industrial bacterial
biofilms.
BACKGROUND
[0003] Bacteria persisting in a biofilm in the human body cause
about two-thirds of all chronic/recurrent diseases. These biofilms
are comprised of bacteria protected by an outer "slime" that is
often comprised primarily of DNA which prevents the innate and
adaptive immune systems, antibiotics and other antibacterial agents
from gaining access to the bacteria inside the biofilm. Biofilms
make it extremely difficult to clear the infection from the body.
Furthermore, biofilms can act as a reservoir for future acute
infections often with lethal consequences.
[0004] At least one protein from the DNABII family of proteins is
found in all known eubacteria and are naturally found outside of
the bacterial cell. While they elicit a strong innate immune
response, host subjects fail to naturally produce specific antibody
to family members as a result of infection. The major problem with
bacterial biofilms is the inability of the host immune system
and/or antibiotics and other antimicrobials to gain access to the
bacteria protected within the biofilm.
[0005] Biofilms are present in an industrial setting as well. For
example, biofilms are implicated in a wide range of petroleum
process problems, from the production field to the gas station
storage tank. In the field, sulfate reducing biofilm bacteria
produce hydrogen sulfide (soured oil). In the process pipelines,
biofilm activity develops slimes which impede filters and orifices.
Biofilm and biofilm organisms also cause corrosion of pipeline and
petroleum process equipment. These problems can be manifested
throughout an oil or gas production facility to the point where
fouling and corrosive biofilm organisms have even been found on the
surfaces of final product storage tanks.
[0006] In the home, biofilms are found in or on any surface that
supports microbial growth, e.g., in drains, on food preparation
surfaces, in toilets and in swimming pools and spas.
[0007] Biofilms are implicated in a wide range of water processes,
both domestic and industrial. They can grow on the surface of
process equipment and impede the performance of the equipment, such
as degradation of heat transfer or plugging of filters and
membranes. Biofilms growing on cooling tower fill can add enough
weight to cause collapse of the fill. Biofilms cause corrosion of
even highly specialized stainless steels. Biofilms in a water
process can degrade the value of a final product. Biofilms growing
in drinking water distribution systems can harbor potential
pathogenic organisms, corrosive organisms or bacteria that degrade
the aesthetic quality of the water.
[0008] Thus, a need exists to break through the protective barrier
of biofilms to treat or kill the associated bacterial infections
and clear them from surfaces and in water systems. This invention
satisfies this need and provides related advantages as well.
SUMMARY
[0009] It is discovered herein that polypeptides that have one or
more HMG-box domains(s), such as HMGB1, can interfere with the
structure of extracellular DNA scaffold inside biofilms. By
competing with microbial proteins that bind to the DNA scaffold in
the biofilm, these polypeptides destabilize the biofilm, leading to
destruction and removal of the biofilm by the host immune
system.
[0010] HMG-box domain(s) enable a protein to bind non-B-type DNA
conformations such as kinked or unwound DNA structures. HMG-box
domain containing proteins, such as HMGB1, HMGB2, HMGB3 and HMGB4,
serve important intracellular functions. HMGB1, for instance, binds
to DNA structures that are "pre-bent" and is believed to function
in many types of DNA metabolism, e.g., RAG1/2 mediated
immunoglobulin recombination. Moreover, HMGB1 proteins are known to
be found extracellularly and are released by necrotic but not
apoptotic cells as part of the innate immune system. It is observed
herein that HMGB1, when added to bacterial biofilm communities,
altered DNA based lattice in the biofilms. The altered DNA based
lattice can then allow access of the host immune system to the
biofilm, permitting the host immune system to clear the
biofilm.
[0011] The method for using this technology is provided herein. A
HMG-box domain containing polypeptide can be used as a therapeutic
to destabilize the extracellular DNA shroud of bacterial biofilms.
Bacteria that cannot form functional biofilms are more readily
cleared by the remainder of the host's immune system.
[0012] Accordingly, one embodiment of the present disclosure
provides a method for inhibiting, competing or titrating the
binding of a DNABII polypeptide or protein to a microbial DNA,
comprising contacting the DNABII polypeptide or protein or the
microbial DNA with a polypeptide comprising an HMG-box domain,
thereby inhibiting, competing or titrating the binding of the
DNABII protein or polypeptide to the microbial DNA.
[0013] Another embodiment of the present disclosure provides a
method for inhibiting, preventing or breaking down a microbial
biofilm, comprising contacting the biofilm with a polypeptide
comprising an HMG-box domain, thereby inhibiting, preventing or
breaking down the microbial biofilm. In some aspects, the
contacting is in vitro or in vivo.
[0014] Yet another embodiment of the present disclosure provides a
method of inhibiting, preventing or breaking down a biofilm in a
subject, comprising administering to the subject an effective
amount of a polypeptide comprising an HMG-box domain, thereby
inhibiting, preventing or breaking down the microbial biofilm.
[0015] Also provided, in another embodiment, is a method for
inhibiting, preventing or treating a microbial infection that
produces a biofilm in a subject, comprising administering to the
subject an effective amount of a polypeptide comprising an HMG-box
domain, thereby inhibiting, preventing or treating a microbial
infection that produces the biofilm in the subject.
[0016] In an aspect of any of the above embodiments, the
polypeptide comprising an HMG-box domain comprises one or more
of:
[0017] (a) an isolated or recombinant protein HMGB1 or a fragment
thereof that comprises one or more HMG-box domains;
[0018] (b) an isolated or recombinant protein HMGB2 or a fragment
thereof that comprises one or more HMG-box domains;
[0019] (c) an isolated or recombinant protein HMGB3 or a fragment
thereof that comprises one or more HMG-box domains;
[0020] (d) an isolated or recombinant protein HMGB4 or a fragment
thereof that comprises one or more HMG-box domains; or
[0021] (e) a polypeptide that is at least about 70% identical to
any of (a), (b), (c) or (d).
[0022] In another aspect, the polypeptide comprising an HMG-box
domain comprises an isolated or recombinant protein HMGB1, a
polypeptide that is at least about 70% identical to HMGB1 or a
fragment thereof that comprises one or more HMG-box domains.
[0023] In some aspects, the isolated or recombinant protein is a
mammalian protein. In a particular aspect, the mammalian protein is
a human protein.
[0024] Any of the above method can further comprise administering
to the subject an effective amount of one or more of an
antimicrobial, an antigenic peptide or an adjuvant. The subject, in
one aspect, is a non-human animal or a human patient.
[0025] The polypeptide is administered by a method comprising
topically, transdermally, sublingually, rectally, vaginally,
ocularly, subcutaneous, intramuscularly, intraperitoneally,
urethrally, intranasally, by inhalation or orally.
[0026] In some aspects, the subject is a pediatric patient and the
polypeptide is administered in a formulation for the pediatric
patient.
[0027] In any of the above embodiments, the biofilm can comprise
microbial DNA from a microorganism identified in Table 1.
TABLE-US-00001 TABLE 1 Examples of bacterial strains that can
generate biofilms S. sobrinus S. pyogenes S. gordonii Challis S.
agalactiae S. mutans S. pneumoniae S. gallolyticus S. aureus S.
epidermidis E. coli H. influenza Salmonella enteric serovar typhi
Aggregatibacter actinomycetemcomitans YP_003255304 P. gingivalis N.
gonorrhoeae N. meningitides NMB_1302 P. aeruginosa H. pylori B.
burgdorferi Moraxella catarrhalis V. cholera El Tor Burkholderia
cenocepacia Burkholderia pseudomallei Mycobacterium tuberculosis
Mycobacterium smegmatis Treponema denticola Treponema palladium
Nichols Prevotella melaninogenica Prevotella intermedia Bordetella
pertusis Tohama Enterococcus faecalis
[0028] In one embodiment, the polypeptide is administered locally
to the microbial infection.
[0029] In one embodiment, the present disclosure provides a method
for inducing or providing an immune response in a subject in need
thereof, comprising administering to the subject an effective
amount of a polypeptide comprising an HMG-box domain. In another
embodiment, the administration is local to where the immune
response is desired.
[0030] In one aspect, the polypeptide comprising an HMG-box domain
comprises one or more of:
[0031] (a) an isolated or recombinant protein HMGB1 or a fragment
thereof that comprises one or more HMG-box domains;
[0032] (b) an isolated or recombinant protein HMGB2 or a fragment
thereof that comprises one or more HMG-box domains;
[0033] (c) an isolated or recombinant protein HMGB3 or a fragment
thereof that comprises one or more HMG-box domains;
[0034] (d) an isolated or recombinant protein HMGB4 or a fragment
thereof that comprises one or more HMG-box domains; or
[0035] (e) a polypeptide that is at least about 70% identical to
any of (a), (b), (c) or (d).
[0036] In a particular aspect, the polypeptide comprising an
HMG-box domain comprises an isolated or recombinant protein HMGB1,
a polypeptide that is at least about 70% identical to HMGB1 or a
fragment thereof that comprises one or more HMG-box domains.
[0037] The isolated or recombinant protein can be a mammalian
protein or in a particular aspect, a human protein. The subject, in
some aspects, is a non-human animal or a human patient.
[0038] Also provided is a kit comprising any one or more agent of
the group
[0039] (a) an isolated or recombinant protein HMGB1 or a fragment
thereof that comprises one or more HMG-box domains;
[0040] (b) an isolated or recombinant protein HMGB2 or a fragment
thereof that comprises one or more HMG-box domains;
[0041] (c) an isolated or recombinant protein HMGB3 or a fragment
thereof that comprises one or more HMG-box domains;
[0042] (d) an isolated or recombinant protein HMGB4 or a fragment
thereof that comprises one or more HMG-box domains; or
[0043] (e) a polypeptide that is at least about 70% identical to
any of (a), (b), (c) or (d) and instructions for use in breaking
down a biofilm or inhibiting, preventing or treating a microbial
infection that produces a biofilm. In one embodiment, the kit
further comprises one or more of an adjuvant, an antigenic peptide
or an antimicrobial. In yet another embodiment, the kit further
comprises a carrier selected from the group of a liquid carrier, a
pharmaceutically acceptable carrier, a solid phase carrier, a
pharmaceutically acceptable carrier, an implant, a stent, a paste,
a gel, a dental implant or a medical implant.
[0044] Yet another embodiment of the present disclosure provides
use of a polypeptide of the group of:
[0045] (a) an isolated or recombinant protein HMGB1 or a fragment
thereof that comprises one or more HMG-box domains;
[0046] (b) an isolated or recombinant protein HMGB2 or a fragment
thereof that comprises one or more HMG-box domains;
[0047] (c) an isolated or recombinant protein HMGB3 or a fragment
thereof that comprises one or more HMG-box domains;
[0048] (d) an isolated or recombinant protein HMGB4 or a fragment
thereof that comprises one or more HMG-box domains; or
[0049] (e) a polypeptide that is at least about 70% identical to
any of (a), (b), (c) or (d) in the manufacture of a medicament for
breaking down a biofilm or inhibiting, preventing or treating a
microbial infection that produces a biofilm.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIG. 1 are Western blot gel pictures each with a different
antibody indicating the recognized proteins. Polyclonal antisera to
HMGB1 fail to crossreact with DNABII proteins members and
polyclonal antisera to DNABII family members fail to crossreact
with HMGB1.
[0051] FIG. 2 are Western blot gel pictures showing the binding
specificity of goat anti-human HMGB1 antibodies and that HMGB1 is
found in naive serum.
[0052] FIG. 3 presents confocal microscopy images of 40h in vitro
NTHI biofilms not treated (left) or treated (right) with anti-HMGB1
antibodies at 24 hours. The images show that reduction of HMGB1
found in naive serum by the antibody caused enhanced biofilm
growth, shown as thicker biofilm at lower right as compared to a
thinner one at lower left.
[0053] FIG. 4 includes gel images showing detection of HMGB1 in
mammalian naive serum by Western blot. The arrows indicate the
detected HMGB1 in each sample. Note that HMGB1 had a His tag.
Doublet observed in HMGB1 lanes was also present in example blot on
specification sheet.
[0054] FIG. 5 presents confocal microscopy images of NTHI biofilms
treated with different concentrations of HMGB1 and shows that HMGB1
dose-dependently inhibited biofilm formation. Control: in sterile
medium sBHI (BHI with 2 mg heme/mL and 2 mg b-NAD/mL).
[0055] FIG. 6 presents dual labeling images of HMBG1 and IHF in
bronchoalveolar lavage (BAL): A, labeling of HMGB1 with Alexafluor
488 conjugated antibodies; B, labeling of IHF with Alexafluor 594
conjugated antibodies; C, merged image of (A) and (B) showing
localization of both antibodies. DAPI was psuedocolored white in
all images.
[0056] FIG. 7 presents microscopy images showing HMGB1 and IHF
labeling of biomass formed by NTHI in the middle ear of a
chinchilla. The images are from serial sections of an OCT embedded
biomass co-labeled for HMGB1 and IHF using goat anti-HMGB1 (diluted
1:25) and rabbit anti-IHF (diluted 1:200). Labeling was detected
using Donkey anti-Goat AlexaFluor 488 and Donkey anti-rabbit
AlexaFluor 594. dsDNA was stained with DAPI and pseudocolored
white.
[0057] FIG. 8 shows that HMGB1 was detected periodically along the
length of dsDNA strands. It was also found to be in close proximity
of IHF at junctions.
[0058] FIG. 9 presents different z-plane images of the same section
of the slide. HMGB1 and IHF are both detected at the junction of
strands of dsDNA and are in close proximity.
[0059] FIG. 10 presents an electromobility shift assay of HMGB1
bound to synthetic DNA Holliday junctions. Images show that HMGB1
failed to stabilize Holliday junction structural integrity with
increasing temperature.
DETAILED DESCRIPTION
[0060] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices and materials are now
described. All technical and patent publications cited herein are
incorporated herein by reference in their entirety. Nothing herein
is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0061] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of tissue culture,
immunology, molecular biology, microbiology, cell biology and
recombinant DNA, which are within the skill of the art. See, e.g.,
Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory
Manual, 3.sup.rd edition; the series Ausubel et al. eds. (2007)
Current Protocols in Molecular Biology; the series Methods in
Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991)
PCR 1: A Practical Approach (IRL Press at Oxford University Press);
MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and
Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005)
Culture of Animal Cells: A Manual of Basic Technique, 5.sup.th
edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.
4,683,195; Hames and Higgins eds. (1984) Nucleic Acid
Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames
and Higgins eds. (1984) Transcription and Translation; Immobilized
Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical
Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene
Transfer Vectors for Mammalian Cells (Cold Spring Harbor
Laboratory); Makrides ed. (2003) Gene Transfer and Expression in
Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical
Methods in Cell and Molecular Biology (Academic Press, London); and
Herzenberg et al. eds (1996) Weir's Handbook of Experimental
Immunology.
[0062] All numerical designations, e.g., pH, temperature, time,
concentration and molecular weight, including ranges, are
approximations which are varied (+) or (-) by increments of 1.0 or
0.1, as appropriate, or alternatively by a variation of +/-15%, or
alternatively 10%, or alternatively 5% or alternatively 2%. It is
to be understood, although not always explicitly stated, that all
numerical designations are preceded by the term "about". It also is
to be understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
[0063] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a polypeptide"
includes a plurality of polypeptides, including mixtures
thereof.
[0064] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
do not exclude others. "Consisting essentially of" when used to
define compositions and methods, shall mean excluding other
elements of any essential significance to the combination for the
intended use. Thus, a composition consisting essentially of the
elements as defined herein would not exclude trace contaminants
from the isolation and purification method and pharmaceutically
acceptable carriers, such as phosphate buffered saline,
preservatives and the like. "Consisting of" shall mean excluding
more than trace elements of other ingredients and substantial
method steps for administering the compositions of this invention.
Embodiments defined by each of these transition terms are within
the scope of this invention.
[0065] A "biofilm" intends a thin layer or an organized community
of microorganisms that at times can adhere to the surface of a
structure, that may be organic or inorganic, together with the
polymers; such as DNA; that they secrete and/or release. The
biofilms are very resistant to microbiotics and antimicrobial
agents. They live on gingival tissues, teeth and restorations,
causing caries and periodontal disease, also known as periodontal
plaque disease. They also cause chronic middle ear infections.
Biofilms can also form on the surface of dental implants, stents,
catheter lines and contact lenses. They grow on pacemakers, heart
valve replacements, artificial joints and other surgical implants.
The Centers for Disease Control estimate that over 65% of
nosocomial (hospital-acquired) infections are caused by biofilms.
Fungal biofilms also frequently contaminate medical devices. They
cause chronic vaginal infections and lead to life-threatening
systemic infections in people with hobbled immune systems. Biofilms
also are involved in numerous diseases. For instance, cystic
fibrosis patients have Pseudomonas infections that often result in
antibiotic resistant biofilms.
[0066] A "DNABII polypeptide or protein" intends a DNA binding
protein or polypeptide that is composed of DNA-binding domains and
thus have a specific or general affinity for DNA. In one aspect,
they bind DNA in the minor grove. Non-limiting examples of DNABII
proteins are an integration host factor (IHF) protein and a
histone-like protein from E. coli strain U93 (HU). Other DNA
binding proteins that can be associated with the biofilm include
DPS (Genbank Accession No.: CAA49169), H-NS (Genbank Accession No.:
CAA47740), Hfq (Genbank Accession No.: ACE63256), CbpA (Genbank
Accession No.: BAA03950) and CbpB (Genbank Accession No.:
NP.sub.--418813).
[0067] An "integration host factor" or "IHF" protein is a bacterial
protein that is used by bacteriophages to incorporate their DNA
into the host bacteria. These are DNA binding proteins that
function in genetic recombination as well as in transcription and
translational regulation. They also bind extracellular microbial
DNA. The genes that encode the IHF protein subunits in E. coli are
himA (Genbank accession No.: POA6X7.1) and himD (POA6Y1.1) genes.
Homologs for these genes are found in other organisms, and peptides
corresponding to these genes from other organisms can be found in
Table 1.
[0068] "HMGB1" is a high mobility group box (HMGB) 1 protein that
is reported to bind to and distort the minor groove of DNA and is
an example of an interfering agent. Recombinant or isolated protein
and polypeptide are commercially available from Atgenglobal,
ProSpecBio, Protein1 and Abnova.
[0069] "HU" or "histone-like protein from E. coli strain U93"
refers to a class of heterodimeric proteins typically associated
with E. coli. HU proteins are known to bind DNA junctions. Related
proteins have been isolated from other microorganisms. The complete
amino acid sequence of E. coli HU was reported by Laine et al.
(1980) Eur. J. Biochem. 103(3):447-481. Antibodies to the HU
protein are commercially available from Abcam.
[0070] "Microbial DNA" intends single or double stranded DNA from a
microorganism that produces a biofilm.
[0071] "Inhibiting, preventing or breaking down" a biofilm intends
the prophylactic or therapeutic reduction in the structure of a
biofilm. In one aspect, the terms "inhibiting, competing or
titrating" intend a reduction in the formation of the DNA/protein
matrix (for example as shown in FIG. 1) that is a component of a
microbial biofilm.
[0072] A "bent polynucleotide" intends a double strand
polynucleotide that contains a small loop on one strand which does
not pair with the other strand and any polynucleotide where the end
to end distance is reduced beyond natural thermal fluctations i.e.
that is bending beyond the persistence length of 150 bp for native
B-form double stranded DNA. In some embodiments, the loop is from 1
base to about 20 bases long, or alternatively from 2 bases to about
15 bases long, or alternatively from about 3 bases to about 12
bases long, or alternatively from about 4 bases to about 10 bases
long, or alternatively has about 4, 5, or 6, or 7, or 8, or 9 or 10
bases.
[0073] A "subject" of diagnosis or treatment is a cell or an animal
such as a mammal or a human. Non-human animals subject to diagnosis
or treatment and are those subject to infections or animal models,
for example, simians, murines, such as, rats, mice, chinchilla,
canine, such as dogs, leporids, such as rabbits, livestock, sport
animals and pets.
[0074] The term "protein", "peptide" and "polypeptide" are used
interchangeably and in their broadest sense refer to a compound of
two or more subunit amino acids, amino acid analogs or
peptidomimetics. The subunits may be linked by peptide bonds. In
another embodiment, the subunit may be linked by other bonds, e.g.,
ester, ether, etc. A protein or peptide must contain at least two
amino acids and no limitation is placed on the maximum number of
amino acids which may comprise a protein's or peptide's sequence.
As used herein the term "amino acid" refers to either natural
and/or unnatural or synthetic amino acids, including glycine and
both the D and L optical isomers, amino acid analogs and
peptidomimetics.
[0075] The term "isolated" or "recombinant" as used herein with
respect to nucleic acids, such as DNA or RNA, refers to molecules
separated from other DNAs or RNAs, respectively that are present in
the natural source of the macromolecule as well as polypeptides.
The term "isolated or recombinant nucleic acid" is meant to include
nucleic acid fragments which are not naturally occurring as
fragments and would not be found in the natural state. The term
"isolated" is also used herein to refer to polynucleotides,
polypeptides and proteins that are isolated from other cellular
proteins and is meant to encompass both purified and recombinant
polypeptides. In other embodiments, the term "isolated or
recombinant" means separated from constituents, cellular and
otherwise, in which the cell, tissue, polynucleotide, peptide,
polypeptide, protein, antibody or fragment(s) thereof, which are
normally associated in nature. For example, an isolated cell is a
cell that is separated from tissue or cells of dissimilar phenotype
or genotype. An isolated polynucleotide is separated from the 3'
and 5' contiguous nucleotides with which it is normally associated
in its native or natural environment, e.g., on the chromosome. As
is apparent to those of skill in the art, a non-naturally occurring
polynucleotide, peptide, polypeptide, protein, antibody or
fragment(s) thereof, does not require "isolation" to distinguish it
from its naturally occurring counterpart.
[0076] It is to be inferred without explicit recitation and unless
otherwise intended, that when the present invention relates to a
polypeptide, protein, polynucleotide or antibody, an equivalent or
a biologically equivalent of such is intended within the scope of
this invention. As used herein, the term "biological equivalent
thereof" is intended to be synonymous with "equivalent thereof"
when referring to a reference protein, antibody, polypeptide or
nucleic acid, intends those having minimal homology while still
maintaining desired structure or functionality. Unless specifically
recited herein, it is contemplated that any polynucleotide,
polypeptide or protein mentioned herein also includes equivalents
thereof. For example, an equivalent intends at least about 70%
homology or identity, or alternatively about 80% homology or
identity and alternatively, at least about 85%, or alternatively at
least about 90%, or alternatively at least about 95% or
alternatively 98% percent homology or identity and exhibits
substantially equivalent biological activity to the reference
protein, polypeptide or nucleic acid. In another aspect, the term
intends a polynucleotide that hybridizes under conditions of high
stringency to the reference polynucleotide or its complement.
[0077] A polynucleotide or polynucleotide region (or a polypeptide
or polypeptide region) having a certain percentage (for example,
80%, 85%, 90% or 95%) of "sequence identity" to another sequence
means that, when aligned, that percentage of bases (or amino acids)
are the same in comparing the two sequences. The alignment and the
percent homology or sequence identity can be determined using
software programs known in the art, for example those described in
Current Protocols in Molecular Biology (Ausubel et al., eds. 1987)
Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default
parameters are used for alignment. A preferred alignment program is
BLAST, using default parameters. In particular, preferred programs
are BLASTN and BLASTP, using the following default parameters:
Genetic code=standard; filter=none; strand=both; cutoff=60;
expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH
SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR. Details of these programs
can be found at the following Internet address:
ncbi.nlm.nih.gov/cgi-bin/BLAST.
[0078] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are homologous at that position. A
degree of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences. An
"unrelated" or "non-homologous" sequence shares less than 30%
identity or alternatively less than 25% identity, less than 20%
identity, or alternatively less than 10% identity with one of the
sequences of the present invention.
[0079] "Homology" or "identity" or "similarity" can also refer to
two nucleic acid molecules that hybridize under stringent
conditions to the reference polynucleotide or its complement.
[0080] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein
binding, or in any other sequence-specific manner. The complex may
comprise two strands forming a duplex structure, three or more
strands forming a multi-stranded complex, a single self-hybridizing
strand, or any combination of these. A hybridization reaction may
constitute a step in a more extensive process, such as the
initiation of a PCR reaction, or the enzymatic cleavage of a
polynucleotide by a ribozyme.
[0081] Examples of stringent hybridization conditions include:
incubation temperatures of about 25.degree. C. to about 37.degree.
C.; hybridization buffer concentrations of about 6.times.SSC to
about 10.times.SSC; formamide concentrations of about 0% to about
25%; and wash solutions from about 4.times.SSC to about
8.times.SSC. Examples of moderate hybridization conditions include:
incubation temperatures of about 40.degree. C. to about 50.degree.
C.; buffer concentrations of about 9.times.SSC to about
2.times.SSC; formamide concentrations of about 30% to about 50%;
and wash solutions of about 5.times.SSC to about 2.times.SSC.
Examples of high stringency conditions include: incubation
temperatures of about 55.degree. C. to about 68.degree. C.; buffer
concentrations of about 1.times.SSC to about 0.1.times.SSC;
formamide concentrations of about 55% to about 75%; and wash
solutions of about 1.times.SSC, 0.1.times.SSC, or deionized water.
In general, hybridization incubation times are from 5 minutes to 24
hours, with 1, 2, or more washing steps, and wash incubation times
are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate
buffer. It is understood that equivalents of SSC using other buffer
systems can be employed.
[0082] As used herein, the terms "treating," "treatment" and the
like are used herein to mean obtaining a desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms
of completely or partially preventing a disorder or sign or symptom
thereof and/or may be therapeutic in terms of a partial or complete
cure for a disorder and/or adverse effect attributable to the
disorder.
[0083] To "prevent" intends to prevent a disorder or effect in
vitro or in vivo in a system or subject that is predisposed to the
disorder or effect. An example of such is preventing the formation
of a biofilm in a system that is infected with a microorganism
known to produce one.
[0084] "Pharmaceutically acceptable carriers" refers to any
diluents, excipients or carriers that may be used in the
compositions of the invention. Pharmaceutically acceptable carriers
include ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances, such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat. Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, Mack Publishing Company, a
standard reference text in this field. They are preferably selected
with respect to the intended form of administration, that is, oral
tablets, capsules, elixirs, syrups and the like and consistent with
conventional pharmaceutical practices.
[0085] "Administration" can be effected in one dose, continuously
or intermittently throughout the course of treatment. Methods of
determining the most effective means and dosage of administration
are known to those of skill in the art and will vary with the
composition used for therapy, the purpose of the therapy, the
target cell being treated and the subject being treated. Single or
multiple administrations can be carried out with the dose level and
pattern being selected by the treating physician. Suitable dosage
formulations and methods of administering the agents are known in
the art. Route of administration can also be determined and method
of determining the most effective route of administration are known
to those of skill in the art and will vary with the composition
used for treatment, the purpose of the treatment, the health
condition or disease stage of the subject being treated and target
cell or tissue. Non-limiting examples of route of administration
include oral administration, nasal administration, injection and
topical application.
[0086] The term "effective amount" refers to a quantity sufficient
to achieve a beneficial or desired result or effect. In the context
of therapeutic or prophylactic applications, the effective amount
will depend on the type and severity of the condition at issue and
the characteristics of the individual subject, such as general
health, age, sex, body weight, and tolerance to pharmaceutical
compositions. In the context of an immunogenic composition, in some
embodiments the effective amount is the amount sufficient to result
in a protective response against a pathogen. In other embodiments,
the effective amount of an immunogenic composition is the amount
sufficient to result in antibody generation against the antigen. In
some embodiments, the effective amount is the amount required to
confer passive immunity on a subject in need thereof. With respect
to immunogenic compositions, in some embodiments the effective
amount will depend on the intended use, the degree of
immunogenicity of a particular antigenic compound, and the
health/responsiveness of the subject's immune system, in addition
to the factors described above. The skilled artisan will be able to
determine appropriate amounts depending on these and other
factors.
[0087] In the case of an in vitro application, in some embodiments
the effective amount will depend on the size and nature of the
application in question. It will also depend on the nature and
sensitivity of the in vitro target and the methods in use. The
skilled artisan will be able to determine the effective amount
based on these and other considerations. The effective amount may
comprise one or more administrations of a composition depending on
the embodiment.
[0088] The agents and compositions can be used in the manufacture
of medicaments and for the treatment of humans and other animals by
administration in accordance with conventional procedures, such as
an active ingredient in pharmaceutical compositions.
[0089] An agent of the present invention can be administered for
therapy by any suitable route of administration. It will also be
appreciated that the preferred route will vary with the condition
and age of the recipient and the disease being treated.
[0090] An example of a solid phase support include glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,
natural and modified celluloses, polyacrylamides, gabbros and
magnetite. The nature of the carrier can be either soluble to some
extent or insoluble. The support material may have virtually any
possible structural configuration so long as the coupled molecule
is capable of binding to a polynucleotide, polypeptide or antibody.
Thus, the support configuration may be spherical, as in a bead or
cylindrical, as in the inside surface of a test tube or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. or alternatively polystyrene
beads. Those skilled in the art will know many other suitable
carriers for binding antibody or antigen or will be able to
ascertain the same by use of routine experimentation.
[0091] As used herein, an "antibody" includes whole antibodies and
any antigen binding fragment or a single chain thereof. Thus the
term "antibody" includes any protein or peptide containing molecule
that comprises at least a portion of an immunoglobulin molecule.
Examples of such include, but are not limited to a complementarity
determining region (CDR) of a heavy or light chain or a ligand
binding portion thereof, a heavy chain or light chain variable
region, a heavy chain or light chain constant region, a framework
(FR) region or any portion thereof or at least one portion of a
binding protein.
[0092] The antibodies can be polyclonal or monoclonal and can be
isolated from any suitable biological source, e.g., murine, rat,
sheep or canine.
[0093] "Immune response" broadly refers to the antigen-specific
responses of lymphocytes to foreign substances. Any substance that
can elicit an immune response is said to be "immunogenic" and is
referred to as an "immunogen". All immunogens are antigens,
however, not all antigens are immunogenic. An immune response of
this invention can be humoral (via antibody activity) or
cell-mediated (via T cell activation).
[0094] As used herein, the term "inducing an immune response in a
subject" is a term well understood in the art and intends that an
increase of at least about 2-fold, more preferably at least about
5-fold, more preferably at least about 10-fold, more preferably at
least about 100-fold, even more preferably at least about 500-fold,
even more preferably at least about 1000-fold or more in an immune
response to an antigen (or epitope) can be detected or measured,
after introducing the antigen (or epitope) into the subject,
relative to the immune response (if any) before introduction of the
antigen (or epitope) into the subject. An immune response to an
antigen (or epitope), includes, but is not limited to, production
of an antigen-specific (or epitope-specific) antibody and
production of an immune cell expressing on its surface a molecule
which specifically binds to an antigen (or epitope). Methods of
determining whether an immune response to a given antigen (or
epitope) has been induced are well known in the art. For example,
antigen-specific antibody can be detected using any of a variety of
immunoassays known in the art, including, but not limited to,
ELISA, wherein, for example, binding of an antibody in a sample to
an immobilized antigen (or epitope) is detected with a
detectably-labeled second antibody (e.g., enzyme-labeled mouse
anti-human Ig antibody).
[0095] The term "modulate an immune response" includes inducing
(increasing, eliciting) an immune response; and reducing
(suppressing) an immune response. An immunomodulatory method (or
protocol) is one that modulates an immune response in a
subject.
Polypeptides
[0096] An "HMG domain" or "high mobility group (HMG) box domain"
refers to an amino acid sequence that is involved in binding DNA
(Stros et al., Cell Mol Life Sci. 64(19-20):2590-606 (2007)). In
one embodiment, the structure of the HMG-box domain consists of
three helices in an irregular array. In another embodiment, an
HMG-box domain enables a protein to bind non-B-type DNA
conformations (kinked or unwound) with high affinity. HMG-box
domains can be found in high mobility group proteins, which are
involved in the regulation of DNA-dependent processes such as
transcription, replication and DNA repair, all of which require
changing the conformation of chromatin (Thomas (2001) Biochem. Soc.
Trans. 29(Pt 4):395-401).
[0097] A "polypeptide comprising an HMG-box domain" or
alternatively an "HMG-box protein", refers to a polypeptide or
protein that contains one or more HMG-box domains. Identification
of an HMG-box domain can be carried by the BLAST.TM. program or
comparing a sequence with known HMG-box domain sequences. HMG-box
proteins are found in a variety of eukaryotic organisms and can be
broadly divided into two groups, based on sequence-dependent and
sequence-independent DNA recognition; the former usually contain
one HMG-box motif, while the latter can contain multiple HMG-box
motifs. Non-limiting examples of polypeptides comprising an HMG-box
domain include HMG1(HMGB1), HMG2(HMGB2), HMGB3 and HMGB4
non-histone components of chromatin; SRY (sex determining region Y
protein) involved in differential gonadogenesis; the SOX family of
transcription factors (Harley et al. (2003) Endocr. Rev.
24(4):466-87); sequence-specific LEF1 (lymphoid enhancer binding
factor 1) and TCF-1 (T-cell factor 1) involved in regulation of
organogenesis and thymocyte differentiation (Labbe et al. (2000)
Proc. Natl. Acad. Sci. USA. 97(15):8358-63); structure-specific
recognition protein SSRP involved in transcription and replication;
MTF1 mitochondrial transcription factor; nucleolar transcription
factors UBF 1/2 (upstream binding factor) involved in transcription
by RNA polymerase I; Abf2 yeast ARS-binding factor (Cho et al.
(2001) Biochim. Biophys. Acta. 1522(3):175-86); yeast transcription
factors 1xr1, Rox1, Nhp6b and Spp41; mating type proteins (MAT)
involved in the sexual reproduction of fungi (Barve et al. (2003)
Fungal Genet. Biol. 39(2):151-67); and the YABBY plant-specific
transcription factors.
[0098] Exemplary sequences of polypeptides comprising an HMG-box
domain include NP.sub.--002119 (human HMGB1), NP.sub.--001124160
(human HMGB2), NP.sub.--005333 (human HMGB3) and NP.sub.--660206
(human HMGB4). Amino acid residues from about 9 to about 76 of the
human HMGB1, for example, form an HMG-box domain and amino acid
residues from about 90 to about 138 form another HMG-box domain. An
HMGB1 fragment that contains either of these two HMG-box domains,
for example, also constitutes a polypeptide comprising an HMG-box
domain, within the meaning of the present disclosure.
[0099] Accordingly, a polypeptide comprising an HMG-box domain, as
contemplated in the present disclosure, intends any of the above
described proteins, fragments of these proteins that contain one or
more of the HMG-box domain or equivalents of these proteins or
fragments. As used herein, an equivalent of a polypeptide refers to
a sequence that is at least about 70%, or alternatively at least
about 75%, or at least about 80%, or at least about 85%, or at
least about 90%, or at least about 95%, or at least about 98% or at
least about 99% identical to the reference polypeptide. In some
aspects, the equivalent of a polypeptide retains the intended
function and/or structural characteristics of the polypeptide,
e.g., containing an HMG-box domain. In one aspect, the equivalent
polypeptide includes a domain that is at least about 70%, or
alternatively at least about 80%, or at least about 85%, or at
least about 90%, or at least about 95%, or at least about 98% or at
least about 99% identical to the HMG-box domain. In some aspects,
such an equivalent domain retains the function and/or structural
characteristics of the HMB-box domain, e.g., binding to a HMB-box
binding target. In one aspect, the equivalent polypeptide can
hybridize with the polypeptide under stringent conditions.
[0100] The polypeptides comprising an HMG-box domain are intended
to include wildtype and recombinantly produced polypeptides and
proteins from prokaryotic and eukaryotic host cells, as well as
muteins, analogs and fragments thereof. In some embodiments, the
term also includes antibodies and anti-idiotypic antibodies. Such
polypeptides can be isolated or produced using the methods
identified below.
[0101] The proteins and polypeptides are obtainable by a number of
processes known to those of skill in the art, which include
purification, chemical synthesis and recombinant methods.
Polypeptides can be isolated from preparations such as host cell
systems. by methods such as immunoprecipitation with antibody and
standard techniques such as gel filtration, ion-exchange,
reversed-phase and affinity chromatography. For such methodology,
see for example Deutscher et al. (1999) Guide To Protein
Purification: Methods In Enzymology (Vol. 182, Academic Press).
Accordingly, this invention also provides the processes for
obtaining these polypeptides as well as the products obtainable and
obtained by these processes.
[0102] The polypeptides also can be obtained by chemical synthesis
using a commercially available automated peptide synthesizer such
as those manufactured by Perkin/Elmer/Applied Biosystems, Inc.,
Model 430A or 431A, Foster City, Calif., USA. The synthesized
polypeptide can be precipitated and further purified, for example
by high performance liquid chromatography (HPLC). Accordingly, this
invention also provides a process for chemically synthesizing the
proteins of this invention by providing the sequence of the protein
and reagents, such as amino acids and enzymes and linking together
the amino acids in the proper orientation and linear sequence.
[0103] Alternatively, the proteins and polypeptides can be obtained
by well-known recombinant methods as described, for example, in
Sambrook et al. (1989) supra, using the host cell and vector
systems described herein.
[0104] The polypeptides of this invention also can be combined with
various solid phase carriers, such as an implant, a stent, a paste,
a gel, a dental implant or a medical implant or liquid phase
carriers, such as beads, sterile or aqueous solutions,
pharmaceutically acceptable carriers, suspensions or emulsions.
Examples of non-aqueous solvents include propyl ethylene glycol,
polyethylene glycol and vegetable oils. When used to prepare
antibodies or induce an immune response in vivo, the carriers also
can include an adjuvant that is useful to non-specifically augment
a specific immune response. A skilled artisan can easily determine
whether an adjuvant is required and select one. However, for the
purpose of illustration only, suitable adjuvants include, but are
not limited to Freund's Complete and Incomplete, mineral salts and
polynucleotides. Other suitable adjuvants include monophosphoryl
lipid A (MPL), mutant derivatives of the heat labile enterotoxin of
E. coli, mutant derivatives of cholera toxin, CPG oligonucleotides
and adjuvants derived from squalene.
Therapeutic Methods
[0105] One embodiment of the present disclosure provides a method
for inhibiting, competing or titrating the binding of a DNABII
polypeptide or protein to a microbial DNA, comprising contacting
the DNABII polypeptide or protein or the microbial DNA with a
polypeptide comprising an HMG-box domain, thereby inhibiting,
competing or titrating the binding of the DNABII protein or
polypeptide to the microbial DNA.
[0106] Polypeptides having one or more HMG-box domains are known in
the art and further described above. One such example is HMGB1 from
eukaryotes, a non-specific DNA binding protein. It was known that
HMGB1 is released from cells during necrosis, but not apoptosis,
and is also released by macrophage stimulated with endotoxin and
proinflammatory cytokines. Released HMGB1 recruits neutrophils and
act as a cytokine to promote inflammation. HMGB1 aksi activates
dendritic cells and promotes their functional maturation and
response to lymph node chemokines.
[0107] HMGB1 binds in the minor groove of DNA, but is not
homologous to DNABII family proteins. The data presented in Example
2, however, shows that HMGB1 has a high affinity for bent DNA
structures and is functionally similar to DNABII.
[0108] Another embodiment of the present disclosure provides a
method for inhibiting, preventing or breaking down a microbial
biofilm, comprising contacting the biofilm with a polypeptide
comprising an HMG-box domain, thereby inhibiting, preventing or
breaking down the microbial biofilm. In some aspects, the
contacting is in vitro or in vivo.
[0109] Yet another embodiment of the present disclosure provides a
method of inhibiting, preventing or breaking down a biofilm in a
subject, comprising administering to the subject an effective
amount of a polypeptide comprising an HMG-box domain, thereby
inhibiting, preventing or breaking down the microbial biofilm.
[0110] Also provided, in another embodiment, is a method for
inhibiting, preventing or treating a microbial infection that
produces a biofilm in a subject, comprising administering to the
subject an effective amount of a polypeptide comprising an HMG-box
domain, thereby inhibiting, preventing or treating a microbial
infection that produces the biofilm in the subject.
[0111] In an aspect of any of the above embodiments, the
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of an HMG-box domain that also comprises
or alternatively consisting essentially of, or yet further
consisting of one or more of:
[0112] (a) an isolated or recombinant protein HMGB1 or a fragment
thereof that comprises or alternatively consists essentially of, or
yet further consists of one or more HMG-box domains;
[0113] (b) an isolated or recombinant protein HMGB2 or a fragment
thereof that comprises or alternatively consists essentially of, or
yet further consists of one or more HMG-box domains;
[0114] (c) an isolated or recombinant protein HMGB3 or a fragment
thereof that comprises or alternatively consists essentially of, or
yet further consists of one or more HMG-box domains;
[0115] (d) an isolated or recombinant protein HMGB4 or a fragment
thereof that comprises or alternatively consists essentially of, or
yet further consists of one or more HMG-box domains; or
[0116] (e) a polypeptide that is at least about 70%, or
alternatively at least about 75%, or at least about 80%, or at
least about 85%, or at least about 90%, or at least about 95%, or
at least about 98% or at least about 99% identical to any of (a),
(b), (c) or (d).
[0117] In another aspect, the polypeptide comprising an HMG-box
domain comprises or alternatively consists essentially of, or yet
further consists of an isolated or recombinant protein HMGB1, a
polypeptide that is at least about 70%, or at least about 75%, or
at least about 80%, or at least about 85%, or at least about 90%,
or at least about 95%, or at least about 98% or at least about 99%
identical to HMGB1, or a fragment thereof that comprises or
alternatively consists essentially of, or yet further consists of
one or more HMG-box domains.
[0118] In some aspect, the polypeptide comprising an HMG-box domain
comprises or alternatively consists essentially of, or yet further
consists of a biological equivalent to any polypeptide recited
above.
[0119] In some aspects, the isolated or recombinant protein is a
mammalian protein. In a particular aspect, the mammalian protein is
a human protein.
[0120] Any of the above method can further comprise or
alternatively consists essentially of, or yet further consists of
administering to the subject an effective amount of one or more of
an antimicrobial, an antigenic peptide or an adjuvant. The subject,
in one aspect, is a non-human animal or a human patient.
[0121] The polypeptide is administered by a method comprising
topically, transdermally, sublingually, rectally, vaginally,
ocularly, subcutaneous, intramuscularly, intraperitoneally,
urethrally, intranasally, by inhalation or orally.
[0122] In some aspects, the subject is a pediatric patient and the
polypeptide is administered in a formulation for the pediatric
patient.
[0123] In any of the above embodiments, the biofilm can comprise
microbial DNA from a microorganism identified in Table 1.
[0124] In one embodiment, the polypeptide is administered locally
to the microbial infection.
[0125] In one embodiment, the present disclosure provides a method
for inducing or providing an immune response in a subject in need
thereof, comprising or alternatively consisting essentially of, or
yet further consisting of administering to the subject an effective
amount of a polypeptide comprising an HMG-box domain. In another
embodiment, the administration is local to where the immune
response is desired. Examples of polypeptides comprising an HMG-box
domain are described above.
[0126] The isolated or recombinant protein can be a mammalian
protein or in a particular aspect, a human protein. The subject, in
some aspects, is a non-human animal or a human patient.
[0127] The agents and compositions of this invention can be
concurrently or sequentially administered with other antimicrobial
agents and/or surface antigens. In one particular aspect,
administration is locally to the site of the infection. Other
non-limiting examples of administration include by one or more
method comprising transdermally, sublingually, rectally, vaginally,
ocularly, subcutaneous, intramuscularly, intraperitoneally,
intranasally, by inhalation or orally.
[0128] Also provided, in one embodiment, is use of any of the above
described polypeptide comprising or alternatively consisting
essentially of, or yet further consisting of an HMG-box domain for
the manufacture of a medicament in breaking down a biofilm or
inhibiting, preventing or treating a microbial infection that
produces a biofilm.
[0129] For some of these methods the contacting can be performed in
vitro or in vivo. When the contacting is in vitro, the method
provides a means to determine efficacy of the agents of this
invention prior to animal or clinical studies and can be used to
determine if the agents of this invention work synergistically with
additional antimicrobials. When performed in vivo in an animal
model, the method provides a means to determine efficacy of the
agents of this invention prior to studies in human patients and can
be used to determine if the agents of this invention work
synergistically with additional antimicrobials.
[0130] Microbial infections and disease that can be treated by the
methods of this invention include infection by the organisms
identified in Table 1, e.g., Streptococcus agalactiae, Neisseria
meningitidis, Treponemes, denticola, pallidum, Burkholderia cepacia
or Burkholderia pseudomallei. In one aspect, the microbial
infection is one or more of Haemophilus influenzae (nontypeable),
Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus
pyogenes, Pseudomonas aeruginosa, Mycobacterium tuberculosis. These
microbial infections may be present in the upper, mid or lower
airway (otitis, sinusitis or bronchitis) but also exacerbations of
chronic obstructive pulmonary disease (COPD), chronic cough,
complications of and/or primary cause of cystic fibrosis (CF) and
community acquired pneumonia (CAP).
[0131] Infections might also occur in the oral cavity (caries,
periodontitis) and caused by Streptococcus mutans, Porphyromonas
gingivalis, Aggregatibacter actinomycetemcomitans. Infections might
also be localized to the skin (abscesses, `staph` infections,
impetigo, secondary infection of burns, Lyme disease) and caused by
Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas
aeruginosa and Borrelia burdorferi. Infections of the urinary tract
(UTI) can also be treated and are typically caused by Escherichia
coli. Infections of the gastrointestinal tract (GI) (diarrhea,
cholera, gall stones, gastric ulcers) are typically caused by
Salmonella enterica serovar, Vibrio cholerae and Helicobacter
pylori. Infections of the genital tract include and are typically
caused by Neisseria gonorrhoeae. Infections can be of the bladder
or of an indwelling device caused by Enterococcus faecalis.
Infections associated with implanted prosthetic devices, such as
artificial hip or knee replacements or dental implants or medical
devices such as pumps or monitoring systems, typically caused by a
variety of bacteria, can be treated by the methods of this
invention. These devices can be coated or conjugated to an agent as
described herein.
[0132] Infections caused by Streptococcus agalactiae are the major
cause of bacterial septicemia in newborns. Such infections can also
be treated by the methods of this invention. Likewise, infections
caused by Neisseria meningitidis which can cause meningitis can
also be treated.
[0133] Thus, routes of administration applicable to the methods of
the invention include intranasal, intramuscular, intratracheal,
subcutaneous, intradermal, topical application, intravenous,
rectal, nasal, oral and other enteral and parenteral routes of
administration. Routes of administration may be combined, if
desired, or adjusted depending upon the agent and/or the desired
effect. An active agent can be administered in a single dose or in
multiple doses. Embodiments of these methods and routes suitable
for delivery, include systemic or localized routes. In general,
routes of administration suitable for the methods of the invention
include, but are not limited to, enteral, parenteral or
inhalational routes.
[0134] Parenteral routes of administration other than inhalation
administration include, but are not limited to, topical,
transdermal, subcutaneous, intramuscular, intraorbital,
intracapsular, intraspinal, intrasternal and intravenous routes,
i.e., any route of administration other than through the alimentary
canal. Parenteral administration can be conducted to effect
systemic or local delivery of the inhibiting agent. Where systemic
delivery is desired, administration typically involves invasive or
systemically absorbed topical or mucosal administration of
pharmaceutical preparations.
[0135] The compounds of the invention can also be delivered to the
subject by enteral administration. Enteral routes of administration
include, but are not limited to, oral and rectal (e.g., using a
suppository) delivery.
[0136] Methods of administration of the active through the skin or
mucosa include, but are not limited to, topical application of a
suitable pharmaceutical preparation, transcutaneous transmission,
transdermal transmission, injection and epidermal administration.
For transdermal transmission, absorption promoters or iontophoresis
are suitable methods. Iontophoretic transmission may be
accomplished using commercially available "patches" that deliver
their product continuously via electric pulses through unbroken
skin for periods of several days or more.
[0137] In various embodiments of the methods of the invention, the
active will be administered orally on a continuous, daily basis, at
least once per day (QD) and in various embodiments two (BID), three
(TID) or even four times a day. Typically, the therapeutically
effective daily dose will be at least about 1 mg, or at least about
10 mg, or at least about 100 mg or about 200--about 500 mg and
sometimes, depending on the compound, up to as much as about 1 g to
about 2.5 g.
[0138] Dosing of can be accomplished in accordance with the methods
of the invention using capsules, tablets, oral suspension,
suspension for intra-muscular injection, suspension for intravenous
infusion, gel or cream for topical application or suspension for
intra-articular injection.
[0139] Dosage, toxicity and therapeutic efficacy of compositions
described herein can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, for example,
to determine the LD50 (the dose lethal to 50% of the population)
and the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Compositions which exhibit high therapeutic indices are
preferred. While compounds that exhibit toxic side effects may be
used, care should be taken to design a delivery system that targets
such compounds to the site of affected tissue in order to minimize
potential damage to uninfected cells and, thereby, reduce side
effects.
[0140] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the methods, the therapeutically effective
dose can be estimated initially from cell culture assays. A dose
can be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration
of the test compound which achieves a half-maximal inhibition of
symptoms) as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by high performance liquid
chromatography.
[0141] In some embodiments, an effective amount of a composition
sufficient for achieving a therapeutic or prophylactic effect,
ranges from about 0.000001 mg per kilogram body weight per
administration to about 10,000 mg per kilogram body weight per
administration. Suitably, the dosage ranges are from about 0.0001
mg per kilogram body weight per administration to about 100 mg per
kilogram body weight per administration. Administration can be
provided as an initial dose, followed by one or more "booster"
doses. Booster doses can be provided a day, two days, three days, a
week, two weeks, three weeks, one, two, three, six or twelve months
after an initial dose. In some embodiments, a booster dose is
administered after an evaluation of the subject's response to prior
administrations.
[0142] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to, the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of the therapeutic
compositions described herein can include a single treatment or a
series of treatments.
Combination Therapy
[0143] The compositions and related methods of the present
invention may be used in combination with the administration of
other therapies. These include, but are not limited to, the
administration of DNase enzymes, antibiotics, antimicrobials, or
other antibodies.
[0144] In some embodiments, the methods and compositions include a
deoxyribonuclease (DNase) enzyme that acts synergistically with a
composition of this disclosure, e.g., a DNase. A DNase is any
enzyme that catalyzes the cleavage of phosphodiester linkages in
the DNA backbone. Three non-limiting examples of DNase enzymes that
are known to target not only cruciform structures, but also a
variety of secondary structure of DNA include DNAse I, T4 EndoVII
and T7 Endo I. In certain embodiments, the effective amount of
anti-DNABII antibody needed to destabilize the biofilm is reduced
when combined with a DNase. When administered in vitro, the DNase
can be added directly to the assay or in a suitable buffer known to
stabilize the enzyme. The effective unit dose of DNase and the
assay conditions may vary, and can be optimized according to
procedures known in the art.
[0145] In other embodiments, the methods and compositions can be
combined with antibiotics and/or antimicrobials. Antimicrobials are
substances that kill or inhibit the growth of microorganisms such
as bacteria, fungi, or protozoans. Although biofilms are generally
resistant to the actions of antibiotics, compositions and methods
described herein can be used to sensitize the infection involving a
biofilm to traditional therapeutic methods for treating infections.
In other embodiments, the use of antibiotics or antimicrobials in
combination with methods and compositions described herein allow
for the reduction of the effective amount of the antimicrobial
and/or biofilm reducing agent. Some non-limiting examples of
antimicrobials and antibiotics useful in combination with methods
of the current invention include amoxicillin,
amoxicillin-clavulanate, cefdinir, azithromycin, and
sulfamethoxazole-trimethoprim. The therapeutically effective dose
of the antimicrobial and/or antibiotic in combination with the
biofilm reducing agent can be readily determined by traditional
methods. In some embodiments the dose of the antimicrobial agent in
combination with the biofilm reducing agent is the average
effective dose which has been shown to be effective in other
bacterial infections, for example, bacterial infections wherein the
etiology of the infection does not include a biofilm. In other
embodiments, the dose is 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40,
0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.8, 0.85, 0.9, 0.95,
1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0 or 5
times the average effective dose. The antibiotic or antimicrobial
can be added prior to, concurrent with, or subsequent to the
addition of the anti-DNABII antibody.
[0146] In other embodiments, the methods and compositions can be
combined with antibodies that treat the bacterial infection. One
example of an antibody useful in combination with the methods and
compositions described herein is an antibody directed against an
unrelated outer membrane protein (e.g., OMP P5). Treatment with
this antibody alone does not debulk a biofilm in vitro. Combined
therapy with this antibody and a biofilm reducing agent results in
a greater effect than that which could be achieved by either
reagent used alone at the same concentration. Other antibodies that
may produce a synergistic effect when combined with a biofilm
reducing agent or methods to reduce a biofilm include anti-rsPilA,
anti-OMP26, anti-OMP P2, and anti-whole OMP preparations.
[0147] The compositions and methods described herein can be used to
sensitize the bacterial infection involving a biofilm to common
therapeutic modalities effective in treating bacterial infections
without a biofilm but are otherwise ineffective in treating
bacterial infections involving a biofilm. In other embodiments, the
compositions and methods described herein can be used in
combination with therapeutic modalities that are effective in
treating bacterial infections involving a biofilm, but the
combination of such additional therapy and biofilm reducing agent
or method produces a synergistic effect such that the effective
dose of either the biofilm reducing agent or the additional
therapeutic agent can be reduced. In other instances the
combination of such additional therapy and biofilm reducing agent
or method produces a synergistic effect such that the treatment is
enhanced. An enhancement of treatment can be evidenced by a shorter
amount of time required to treat the infection.
[0148] The additional therapeutic treatment can be added prior to,
concurrent with, or subsequent to methods or compositions used to
reduce the biofilm, and can be contained within the same
formulation or as a separate formulation.
Kits
[0149] Kits containing the agents and instructions necessary to
perform the in vitro and in vivo methods as described herein also
are claimed. Accordingly, the invention provides kits for
performing these methods which may include a biological agent of
this invention as well as instructions for carrying out the methods
of this invention such as collecting tissue and/or performing the
screen and/or analyzing the results and/or administration of an
effective amount of biological agent as defined herein. These can
be used alone or in combination with other suitable antimicrobial
agents.
[0150] In one embodiment, the present disclosure provides a kit
comprising a polypeptide comprising an HMG-box domain and
instructions for use in breaking down a biofilm or inhibiting,
preventing or treating a microbial infection that produces a
biofilm. Examples of polypeptides comprising an HMG-box domain are
described above. In one embodiment, the kit further comprises one
or more of an adjuvant, an antigenic peptide or an antimicrobial.
In yet another embodiment, the kit further comprises a carrier
selected from the group of a liquid carrier, a pharmaceutically
acceptable carrier, a solid phase carrier, a pharmaceutically
acceptable carrier, an implant, a stent, a paste, a gel, a dental
implant or a medical implant.
[0151] The following example is intended to illustrate, but not
limit the invention.
EXPERIMENTAL
Example 1
Preparation of HMGB1 Antibodies
Methods and Materials for Western Blot
[0152] 1. Ran 1 .mu.g/well of following purified proteins on
SDS-Page gels (Bio-Rad mini PROTEAN.TM. TGX gels Catalogue
#456-1093): IHF, HU, TBP and HMG1
[0153] 2. Loaded 20 .mu.l total to each well; ran at 125 V.
[0154] 3. Transferred to nitrocellulose for 1 hour at 100V at
4.degree. C.
[0155] 4. Blocked nitrocellulose with 2% BSA in TTBS for 1 hour on
rocking platform.
[0156] 5. Incubated with primary antibody, for 1 hour on rocking
platform in 1% BSA-TTBS primary antibodies used and dilution:
[0157] naive rabbit serum (1:5000)
[0158] rabbit anti-IHF (1:10,000)
[0159] rabbit anti-TBP (1:10,000)
[0160] 6. Washed 3 times with TTBS; 5 minutes each wash.
[0161] 7. Incubated with secondary antibody for 1 hour on rocking
platform in 1% BSA-TTBS. Secondary Antibody: Goat anti-rabbit
IgG-HRP (1:10,000).
[0162] 8. Washed 3 times with TTBS for 5 minutes per wash.
Developed with CN/DAB.
[0163] As shown in FIG. 1, these antibodies are specific to the
corresponding proteins. FIG. 2 further shows that the goat
anti-human HMGB1 antibodies are specific to the human HMGB1 protein
and the binding is in a dose-dependent manner.
[0164] Therefore, these antibodies are suitable for testing the
binding of HMGB1 to DNA scaffold in microbial biofilms. Subsequent
experiments shows that HMGB1 protein binds to DNA scaffold in
microbial biofilms permitting immune response from the host leading
to destruction and removal of the biofilm.
Example 2
HMGB1 Competes with HU and IHF for Binding to Biofilm DNA
[0165] This example demonstrates that HMGB1 has high affinity for
bent DNA structures and competes with HU and IHF for binding to the
DNA in biofilm leading to reduction of biofilm growth.
[0166] To test the effect of HMGB1 on biofilms, biofilms generated
by Nontypable Haemophilus influenzae (NTHI) were treated with naive
serum alone, which contained HMGB1, or with serum containing
anti-HMGB1 antibody. As shown in FIG. 3, reduction of HMGB1 by the
antibody caused enhanced biofilm growth, shown as thicker biofilm
at lower right as compared to a thinner one at lower left.
Therefore, less competition from HMGB1 for HU and IHF binding sites
on the biofilm DNA strengthens the biofilm.
[0167] FIG. 4 confirms that HMGB1 protein exists in mammalian naive
serum and the HMGB1 protein from human, rabbit and goat can all be
recognized by the prepared goat anti-human HMGB1 antibodies. The
estimated concentrations of HMGB1 in each serum sample were about
0.8 .mu.g, 0.8 and 2.8 .mu.g per 80 .mu.g total protein,
respectively.
[0168] It was further discovered that HMGB1 dose-dependently
inhibits biofilm formation. For instance, compared to NTHI in
sterile medium sBHI (BHI with 2 mg heme/mL and 2 mg b-NAD/mL) that
grew up to 22.5 .mu.M of thickness at 40 hours, 0.075 .mu.g/ml,
0.75 .mu.g/ml and 7.5 .mu.g/ml HMGB1 treatment at 24 hours reduced
the biofilm thickness to 21.5 .mu.m, 20.0 .mu.m, and 16.5 .mu.m,
respectively (FIG. 5). This indicates that HMGB1 competes for the
same binding target as HU and IHF.
[0169] The competitive binding between HMGB1 and IHF was further
confirmed by dual labeling. As shown in FIG. 6, HMGB1 and IHF were
co-localized in an OCT (Optimal Cutting Temperature medium,
available commercially from Fisher Scientific Cat. No. 14-373-65)
embedded human bronchoalveolar lavage (BAL). The localization of
HMGB1 was detected with Alexafluor 488 conjugated antibodies (FIG.
6A); the localization of IHF was detected with Alexafluor 594
conjugated antibodies (FIG. 6B); and FIG. 6C, a merged image
between FIGS. 6A and 6B, shows the co-localization.
[0170] Likewise, the co-localization of HMGB1 and IHF on biofilm
was also observed in vivo on the NTHI biofilm formed in the middle
year of a chinchilla (FIG. 7). Serial sections of an OCT embedded
biomass were co-labeled for HMGB1 and IHF using goat anti-HMGB1
(diluted 1:25) and rabbit anti-IHF (diluted 1:200). Labeling was
detected using Donkey anti-Goat AlexaFluor 488 and Donkey
anti-rabbit AlexaFluor 594. dsDNA was stained with DAPI and
pseudocolored white. In all images of FIG. 7, co-localization of
HMGB1 and IHF on the biofilm DNA was observed.
[0171] A further enlarged image in FIG. 8 shows that HMGB1 was
periodically along the length of dsDNA strands and in close
proximity of IHF at junctions. Moreover, different z-plane images
of the same section of the slide (FIG. 9) show that HMGB1 and IHF
are both detected at the junction of strands of dsDNA and are in
close proximity.
[0172] Different from IHF, however, electromobility shift assay of
HMGB1 bound to synthetic DNA Holliday junctions show that HMGB1
fails to stabilize Holliday junction structural integrity with
increasing temperature. (FIG. 10).
[0173] The data of this example therefore shows that HMGB1 competes
with IHF and HU for binding to the same target on biofilm DNA.
Exemplified by HMGB1, therefore, proteins containing a HMG-box
domain is useful in inhibiting the formation and growth of biofilm
and thus useful in treating diseases and conditions characterized
by biofilms.
Example 3
[0174] This example described an animal model for the treatment of
middle ear infections. Middle ear infection (or otitis media, OM)
is a highly prevalent disease worldwide, afflicting 50-330 million
children globally each year. The socioeconomic burden of OM is also
great, with cost estimates between $5-6 billion in the United
States alone annually. All three of the predominant bacterial
pathogens of OM are known to form biofilms both in vitro and in
vivo and recently, clinicians have come to appreciate that the
chronicity and recurrence of OM is due, at least in part, to the
formation of bacterial biofilms within the middle ear cavity. In
one chinchilla model of OM, juvenile chinchillas are first given a
viral `cold`, followed a week later by their being challenged
intranasally with an inoculum of viable bacteria. Similar to the
human condition wherein "my child has a cold and a week later gets
an ear infection" chinchillas will also develop a bacterial OM
approximately one week after a challenge, and while experiencing
the viral upper respiratory tract infection. Once bacteria gain
access to the middle ear (either via ascension of the Eustachian
tube or following direct challenge to the middle ear space), they
will form a robust biofilm. Applicants thus contemplate and indeed
have already used chinchilla models to demonstrate the protective
efficacy of the compositions and methods as described herein, which
results in rapid resolution of existing biofilms. This model is
also useful for therapeutic approaches via either passive delivery
of anti-DNABII antibody or via delivery of a small molecule or
other agent known to bind to IHF or other DNABII family
members.
Example 4
[0175] A number of oral bacteria (e.g., Aggregatibacter
actinomycetemcomitans, Porphyromonas gingivalis) have been
implicated in the pathogenesis of inflammatory diseases such as
periodontitis and peri-implantitis, which destroy alveolar bone and
gingiva. Investigations of the pathogenesis of these bacteria are
hampered by lack of effective animal models. One of the challenges
of investigating the pathogenicity of specific bacteria is the
difficulty of establishing a biofilm when exogenous bacteria are
introduced into the oral cavity of animals. Though animal models of
periodontitis have been developed, cultivable bacteria are rarely
recovered from the oral cavity of inoculated animals. Developing an
effective animal model which can assess the pathogenicity of
specific bacteria will greatly aid in elucidating their pathogenic
mechanisms.
[0176] The surface of machined titanium dental implants
(1.2.times.4.5 mm) can be modified by grit blasting with A103 (100
.mu.m) and HCl etching (pH 7.8 for 20 min at 80.degree. C.).
Machined and nano-textured implants can be incubated in TSB medium
inoculated with D7S clinical strain of Aggregatibacter
actinomycetemcomitans (Aa) for 1 to 3 days at 37.degree. C. The
bacterial biofilm on the implants can be analyzed by SEM, as well
as by confocal laser scanning microscopy following staining with
LIVE/DEAD.RTM. BacLight.TM.. Implants with and without established
Aa biofilm are transmucosally placed into the alveolar bone of
female rats between premolar and incisor region of the maxillae. To
detect the presence of Aa biofilm on the implants placed in vivo,
bacterial samples are collected from saliva and the oral surfaces
of implants after 2 days. Aa was detected by culture, as well as by
PCR analysis.
Example 5
[0177] This experiment provides a mouse model for pre-clinical
testing of interfering agents to treat Lyme disease. See Dresser et
al. Pathogens 5(12)e1000680, Epub 2009 Dec. 4. Lyme disease is the
most common tick-borne disease in the United States. Reported cases
have more than doubled between 1992 and 2006, with approximately
29,000 new cases confirmed in 2008. Estimates are that the actual
number of cases of Lyme disease may exceed that reported by a
factor of 6-12 in endemic areas. By definition, these endemic areas
are expanding as populations continue to move from cities to
suburban and rural areas and whitetail deer (which carry the tick
species Ixodes) increasingly roam these areas. Lyme disease is
caused by the microorganism Borrelia burgdorferi, a spirochete. B.
burgdorferi is transmitted via the bite of the Ixodes tick and
subsequently disseminates, via the bloodstream, to other tissues
and organs.
[0178] In this animal model, C3H/HeN mice are injected with
spirochetes via dorsal subcutaneous and intraperitoneal injection,
or via intravenous injection. Blood and biopsy specimens are
recovered at approximately 7 days post infection for evaluation of
microbial burden and assessment of pathology in tissues and organs.
The methods and compositions of this invention are contemplated to
develop both therapeutic as well as preventative strategies for
reduction and/or elimination of the resulting B. burgdorferi
biofilms which form subsequent to challenge and are believed to
contribute to both the pathogenesis and chronic nature of the
disease.
Example 6
[0179] This experiment provides a porcine model for pre-clinical
testing of agents to treat cystic fibrosis. See Stoltz et al.
(2010) Science Translational Medicine 2(29): 29ra31. Cystic
fibrosis is an autosomal recessive disease due to mutations in a
gene that encodes the CF transmembrane conductance regulator
(called CFTR) anion channel. In this model, pigs which have been
specifically bred to carry a defect in the genes called "CFTR" and
called CF pigs spontaneously develop hallmark features of CF lung
disease that includes infection of the lower airway by multiple
bacterial species. The pigs can be immunized with the interfering
agents to either 1) immunize these CF pigs with a polypeptide or
other immunogenic agent thereby inducing the formation of
antibodies which will eradicate bacterial biofilms in the lungs, to
deliver anti-IHF (or other interfering agent) to the lungs of these
animals by nebulization to assess the amelioration of the signs of
disease and associated pathologies.
Example 7
[0180] Applicants also provide a pre-clinical model for
tuberculosis (TB). See Ordway et al. (2010) Anti. Agents and
Chemotherapy 54:1820. The microorganism Mycobacterium tuberculosis
is responsible for a growing global epidemic. Current figures
suggest that there are approximately 8 million new cases of TB and
about 2.7 million deaths due to TB annually. In addition to the
role of this microbe as a co-infection of individuals with HIV (of
the .about.45 million infected with HIV, estimates are that
.about.1/3 are also co-infected with M. tuberculosis), its
particularly troublesome that isolates have become highly resistant
to multiple drugs and no new drug for TB has been introduced in
over a quarter of a century. In this animal model, SPF guinea pigs
are maintained in a barrier colony and infected via aerosolized
spray to deliver .about.20 cfu of M. tuberculosis strain Erdman K01
bacilli into their lungs. Animals are sacrificed with determination
of bacterial load and recovery of tissues for histopathological
assessment on days 25, 50, 75, 100, 125 and 150 days
post-challenge. Unlike mice which do not develop classic signs of
TB, guinea pigs challenged in this manner develop well-organized
granulomas with central necrosis, a hallmark of human disease.
Further, like humans, guinea pigs develop severe pyogranulomatous
and necrotizing lymphadenitis of the draining lymph nodes as part
of the primary lesion complex. Use of this model will provide a
pre-clinical screen to confirm and identify therapeutic as well as
preventative strategies for reduction and/or elimination of the
resulting M. tuberculosis biofilms which have been observed to form
in the lungs of these animals subsequent to challenge and are
believed to contribute to both the pathogenesis and chronicity of
the disease.
Example 8
[0181] Multiple animal models of catheter/indwelling device biofilm
infections are known. See Otto (2009) Nature Reviews Microbiology,
7:555. While typically considered normal skin flora, the microbe
Staphylococcus epidermidis has become what many regard as a key
opportunistic pathogen, ranking first among causative agents of
nosocomial infections. Primarily, this bacterium is responsible for
the majority of infections that develop on indwelling medical
devices which are contaminated by this common skin colonizer during
device insertion. While not typically life-threatening, the
difficulty associated with treatment of these biofilm infections,
combined with their frequency, makes them a serious public health
burden. Current costs associated with treatment of vascular
catheter associated bloodstream infections alone that are due to S.
epidermidis amount to $2 billion annually in the United States. In
addition to S. epidermidis, E. faecalis and S. aureus are also
contaminations found on indwelling medical devices. There are
several animal models of catheter-associated S. epidermidis
infections including rabbits, mice, guinea pigs and rats all of
which are used to study the molecular mechanisms of pathogenesis
and which lend themselves to studies of prevention and/or
therapeutics. Rat jugular vein catheters have been used to evaluate
therapies that interfere with E. faecalis, S. aureus and S.
epidermidis biofilm formation. Biofilm reduction is often measured
three ways--(i) sonicate catheter and calculate CFUs, (ii) cut
slices of catheter or simply lay on a plate and score, or (iii) the
biofilm can be stained with crystal violet or another dye, eluted,
and OD measured as a proxy for CFUs.
Example 9
[0182] Methods described herein may be used to elicit immune
responses in humans and animals. Immunogenic compositions may be
administered to a human and animal subjects in the presence of
adjuvants such as but not limited to aluminum salts and liposomes.
Those skilled in the art will understand that any number of
pharmaceutically acceptable adjuvants can also be used. Immunogenic
compositions may be administered to a human or animal subjects
intramuscularly, subdermally, intranasally, or through any other
suitable route. Immunogenic compositions may be prepared in a
manner consistent with the selected mode of administration.
Immunogenic compositions may take the form of polypeptides, nucleic
acids, or a combination thereof, and may comprise full-length or
partial antigens. Additionally or alternatively, immunogenic
compositions may take the form of APCs pulsed with a particular
antigen, or APCs transfected with one or more polynucleotides
encoding a particular antigen. Administration may comprise a single
dose of an immunogenic composition, or an initial administration,
followed by one or more booster doses. Booster doses may be
provided a day, two days, three days, a week, two weeks, three
weeks, one, two, three, six or twelve months, or at any other time
point after an initial dose. A booster dose may be administered
after an evaluation of the subject's antibody titer.
Example 10
[0183] Methods described herein may be used to confer passive
immunity on a non-immune subject. Passive immunity against a given
antigen may be conferred through the transfer of antibodies or
antigen binding fragments that specifically recognize or bind to a
particular antigen. Antibody donors and recipients may be human or
non-human subjects. Additionally or alternatively, the antibody
composition may comprise an isolated or recombinant polynucleotide
encoding an antibody or antigen binding fragment that specifically
recognizes or binds to a particular antigen.
[0184] Passive immunity may be conferred in cases where the
administration of immunogenic compositions poses a risk for the
recipient subject, the recipient subject is immuno-compromised, or
the recipient subject requires immediate immunity. Immunogenic
compositions may be prepared in a manner consistent with the
selected mode of administration. Compositions may comprise whole
antibodies, antigen binding fragments, polyclonal antibodies,
monoclonal antibodies, antibodies generated in vivo, antibodies
generated in vitro, purified or partially purified antibodies, or
whole serum. Administration may comprise a single dose of an
antibody composition, or an initial administration followed by one
or more booster doses. Booster doses may be provided a day, two
days, three days, a week, two weeks, three weeks, one, two, three,
six or twelve months, or at any other time point after an initial
dose. A booster dose may be administered after an evaluation of the
subject's antibody titer.
[0185] It is to be understood that while the invention has been
described in conjunction with the above embodiments, that the
foregoing description and examples are intended to illustrate and
not limit the scope of the invention. Other aspects, advantages and
modifications within the scope of the invention will be apparent to
those skilled in the art to which the invention pertains.
[0186] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
nucleotide sequences provided herein are presented in the 5' to 3'
direction.
[0187] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including," containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed.
[0188] Thus, it should be understood that although the present
invention has been specifically disclosed by preferred embodiments
and optional features, modification, improvement and variation of
the inventions embodied therein herein disclosed may be resorted to
by those skilled in the art, and that such modifications,
improvements and variations are considered to be within the scope
of this invention. The materials, methods, and examples provided
here are representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the
invention.
[0189] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0190] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0191] All publications, patent applications, patents, and other
references mentioned herein are expressly incorporated by reference
in their entirety, to the same extent as if each were incorporated
by reference individually. In case of conflict, the present
specification, including definitions, will control.
[0192] It is to be understood that while the invention has been
described in conjunction with the above embodiments, that the
foregoing description and examples are intended to illustrate and
not limit the scope of the invention. Other aspects, advantages and
modifications within the scope of the invention will be apparent to
those skilled in the art to which the invention pertains.
* * * * *