COMPOSITIONS AND METHODS FOR THE REMOVAL OF BIOFILMS
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.
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This application claims the benefit under 35 U.S.C. §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 INVENTIONThis invention generally relates to the methods and compositions to lessen and/or cure clinical or industrial bacterial biofilms.
BACKGROUNDBacteria 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.
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.
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.
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.
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.
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.
SUMMARYIt 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.
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.
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.
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.
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.
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.
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.
In an aspect of any of the above embodiments, 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).
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.
In some aspects, the isolated or recombinant protein is a mammalian protein. In a particular aspect, the mammalian protein is a human protein.
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.
The polypeptide is administered by a method comprising topically, transdermally, sublingually, rectally, vaginally, ocularly, subcutaneous, intramuscularly, intraperitoneally, urethrally, intranasally, by inhalation or orally.
In some aspects, the subject is a pediatric patient and the polypeptide is administered in a formulation for the pediatric patient.
In any of the above embodiments, the biofilm can comprise microbial DNA from a microorganism identified in Table 1.
In one embodiment, the polypeptide is administered locally to the microbial infection.
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.
In one aspect, 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).
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.
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.
Also provided is a kit comprising any one or more agent of the group
(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) 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.
Yet another embodiment of the present disclosure provides use of a polypeptide of the group 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) in the manufacture of a medicament for breaking down a biofilm or inhibiting, preventing or treating a microbial infection that produces a biofilm.
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.
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, 3rd 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, 5th 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.
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.
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.
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.
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.
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—418813).
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.
“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.
“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.
“Microbial DNA” intends single or double stranded DNA from a microorganism that produces a biofilm.
“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
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.
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.
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.
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.
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.
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.
“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.
“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.
“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.
Examples of stringent hybridization conditions include: incubation temperatures of about 25° C. to about 37° C.; hybridization buffer concentrations of about 6×SSC to about 10×SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4×SSC to about 8×SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40° C. to about 50° C.; buffer concentrations of about 9×SSC to about 2×SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples of high stringency conditions include: incubation temperatures of about 55° C. to about 68° C.; buffer concentrations of about 1×SSC to about 0.1×SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1×SSC, 0.1×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.
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.
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.
“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.
“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.
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.
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.
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.
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.
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.
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.
The antibodies can be polyclonal or monoclonal and can be isolated from any suitable biological source, e.g., murine, rat, sheep or canine.
“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).
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).
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.
PolypeptidesAn “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).
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™ 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 (Labbé 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.
Exemplary sequences of polypeptides comprising an HMG-box domain include NP—002119 (human HMGB1), NP—001124160 (human HMGB2), NP—005333 (human HMGB3) and NP—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.
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.
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.
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.
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.
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.
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 MethodsOne 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.
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.
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.
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.
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.
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.
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:
(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;
(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;
(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;
(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
(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).
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.
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.
In some aspects, the isolated or recombinant protein is a mammalian protein. In a particular aspect, the mammalian protein is a human protein.
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.
The polypeptide is administered by a method comprising topically, transdermally, sublingually, rectally, vaginally, ocularly, subcutaneous, intramuscularly, intraperitoneally, urethrally, intranasally, by inhalation or orally.
In some aspects, the subject is a pediatric patient and the polypeptide is administered in a formulation for the pediatric patient.
In any of the above embodiments, the biofilm can comprise microbial DNA from a microorganism identified in Table 1.
In one embodiment, the polypeptide is administered locally to the microbial infection.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 TherapyThe 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.
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.
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.
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.
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.
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.
KitsKits 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.
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.
The following example is intended to illustrate, but not limit the invention.
EXPERIMENTAL Example 1 Preparation of HMGB1 Antibodies Methods and Materials for Western Blot1. Ran 1 μg/well of following purified proteins on SDS-Page gels (Bio-Rad mini PROTEAN™ TGX gels Catalogue #456-1093): IHF, HU, TBP and HMG1
2. Loaded 20 μl total to each well; ran at 125 V.
3. Transferred to nitrocellulose for 1 hour at 100V at 4° C.
4. Blocked nitrocellulose with 2% BSA in TTBS for 1 hour on rocking platform.
5. Incubated with primary antibody, for 1 hour on rocking platform in 1% BSA-TTBS primary antibodies used and dilution:
naïve rabbit serum (1:5000)
rabbit anti-IHF (1:10,000)
rabbit anti-TBP (1:10,000)
6. Washed 3 times with TTBS; 5 minutes each wash.
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).
8. Washed 3 times with TTBS for 5 minutes per wash. Developed with CN/DAB.
As shown in
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 DNAThis 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.
To test the effect of HMGB1 on biofilms, biofilms generated by Nontypable Haemophilus influenzae (NTHI) were treated with naïve serum alone, which contained HMGB1, or with serum containing anti-HMGB1 antibody. As shown in
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 μM of thickness at 40 hours, 0.075 μg/ml, 0.75 μg/ml and 7.5 μg/ml HMGB1 treatment at 24 hours reduced the biofilm thickness to 21.5 μm, 20.0 μm, and 16.5 μm, respectively (
The competitive binding between HMGB1 and IHF was further confirmed by dual labeling. As shown in
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 (
A further enlarged image in
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. (
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 3This 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 4A 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.
The surface of machined titanium dental implants (1.2×4.5 mm) can be modified by grit blasting with A103 (100 μm) and HCl etching (pH 7.8 for 20 min at 80° 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° 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® BacLight™. 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 5This 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.
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 6This 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 7Applicants 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 ˜45 million infected with HIV, estimates are that ˜⅓ 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 ˜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 8Multiple 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 9Methods 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 10Methods 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Type: Application
Filed: Sep 9, 2011
Publication Date: May 24, 2012
Applicants: ,
Inventors: Steven D. Goodman (Los Angeles, CA), Lauren O. Bakaletz (Columbus, OH)
Application Number: 13/229,575
International Classification: A61K 38/17 (20060101); A01P 1/00 (20060101); A61P 31/04 (20060101); A61K 39/00 (20060101); A01N 37/46 (20060101);
