Compositions and methods for mediating EPS

Abstract

The present disclosure relates to a method for inhibiting biofilm stability, comprising contacting a biofilm with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm. Also provided herein is a method for treating a biofilm in a subject, comprising administering to a subject infected with a biofilm an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm. Further described herein is a method for treating a biofilm in a patient with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 692,581, filed June 29, 2018, the contents of which are hereby incorporated by reference in their entirety.

[0002] The present disclosure relates generally to methods and compositions for removing or inhibiting bacterial extracellular polymeric substance (EPS). [Background technology]

[0003] Biofilms play a major role in medical, agricultural, and industrial environments. They are responsible for a significant portion of both animal and plant diseases and fouling of industrial facilities and are therefore the focus of intense research efforts. Eradication or treatment of biofilms is particularly difficult due to multiple factors, including the production of an extracellular matrix that forms a physical barrier against antimicrobial effectors, altered physiology that makes them less susceptible to environmental stressors, and cooperative interactions between biofilm components. The biofilm matrix is ​​variably composed of polysaccharides, proteins, and, perhaps universally, extracellular DNA (eDNA). The eDNA of microbial biofilms is a critical component of the extracellular matrix that provides protection. Compromising the biofilm eDNA structure through DNA degradation or removal of DNA-binding proteins that stabilize the structure can lead to catastrophic biofilm collapse and release of resident bacteria, leaving them in a more vulnerable state.

[0004] Bacteria are found in nature in two distinct states: planktonic bacteria are free-living, while bacteria that develop into community architectures called biofilms (either on surfaces or as aggregates). The CDC and NIH estimate that approximately 80% of all bacterial infections involve the necessary biofilm state. Dongari-Bagtzoglou et al. (2008) Expert Rev Anti Infect Ther. 6(2):201-8. These include, among others, otitis media (OM), chronic rhinosinusitis (CRS), chronic lung infections, chronic wound infections, periodontitis, cystitis, and infections of medical implants and indwelling catheters. In fact, one of the most common reasons for seeking medical care in children is OM [caused by Nontypeable Haemphilus influenzae (NTHI), Streptococcus pneumoniae, and Moraxella catarrhalis], and in adults, cystitis [e.g., uropathogenic E. coli (UPEC)]; therefore, antibiotic prescriptions are most common for these complaints. It is estimated that 500,000 deaths per year in the United States are the direct result of bacterial biofilm infections. The economic impact is staggering [$25 billion for chronic wounds, $14 billion for periodontitis, $5 billion for OM, and $1 billion for cystitis]. The global epidemic of biofilm-mediated diseases, the increasing prevalence of antibiotic-resistant bacterial infections, especially among high-priority ESKAPE pathogens (Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecium), and the significant financial burden create a critical need to develop novel approaches to treat refractory infections caused by organized bacterial communities. Therefore, there is a need to penetrate the protective barrier of biofilms to treat or kill associated bacterial infections and remove them from surfaces and water systems. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Dongari-Bagtzoglou et al. (2008) Expert Rev Anti Infect Ther. 6(2):201-8 Summary of the Invention [Means for solving the problem]

[0006] Because biofilms serve as refractory reservoirs for bacteria that cause chronic and recurrent infections, a self-produced extracellular matrix (or extracellular polymeric substance, EPS) that protects resident bacteria within the biofilm from immune clearance and antimicrobial drugs is essential for these disease-causing pathogenic biofilms. While EPS components are specific to individual bacterial species, they universally contain extracellular DNA (eDNA) derived from resident bacteria within the biofilm. Indeed, bacteria from various genera typically participate in the shared community architecture of multi-species biofilms, which requires that the EPS not only be structurally conducive to all component species but also contain EPS components derived from or usable by all resident bacteria. In this regard, the EPS of single- and multi-species biofilms contains a scaffold eDNA that appears to be the underlying universal EPS common structure. As disclosed herein, Applicants have discovered that this eDNA-dependent structure is stabilized by the ubiquitous DNABII family of bacterial DNA-binding proteins. Applicants have shown that exogenous DNA and DNABII proteins can direct free-living (planktonic) bacteria into a biofilm community architecture, but these two components are insufficient to recapitulate the signature eDNA scaffold.

[0007] Applicants herein disclose that polyamines are the third key component of the universal eDNA-DNABII-dependent EPS. Polyamines are short, positively charged organic molecules ubiquitously present both inside and outside cells that, upon binding to DNA, neutralize the polyanionic charge of nucleotide phosphates and enrich / aggregate DNA molecules. Importantly, Applicants herein disclose that polyamines can transform DNA from the most common right-handed B-form to nuclease-resistant left-handed Z-form DNA. Indeed, while nucleases can prevent bacterial biofilm formation, they cannot disrupt mature biofilms. As biofilms mature, they acquire nuclease resistance, concomitant with both (1) an increase in polyamines and (2) the emergence of Z-form DNA.

[0008] Described herein is a method for inhibiting biofilm stability, comprising, alternatively consisting essentially of, or alternatively consisting of, contacting a biofilm with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In one aspect, the method for inhibiting biofilm stability comprises, alternatively consisting essentially of, or alternatively consisting of, contacting a biofilm with an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm. The present disclosure also relates to a method for inhibiting biofilm stability, comprising, or alternatively consisting essentially of, or alternatively consisting of, contacting a biofilm in vitro with an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises, or alternatively consisting essentially of, or alternatively consisting of, coating a surface with an effective amount of an agent that depletes cations, and wherein the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In one aspect, the method for inhibiting biofilm stability may comprise, or alternatively consisting essentially of, or alternatively consisting of, contacting a biofilm in vitro with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises, or alternatively consisting essentially of, or alternatively consisting of, coating a surface with an effective amount of one or more agents that deplete cations. The contacting may be in vitro or in vivo.

[0009] In one embodiment, the agent interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment. In a second embodiment, the agent comprises, alternatively consists essentially of, or even consists of an anti-B-DNA antibody or a fragment or derivative thereof. In a third embodiment, the agent comprises, alternatively consists essentially of, or even consists of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or a derivative thereof. In a fourth embodiment, the agent comprises, alternatively consists essentially of, or even consists of chloroquine or a derivative thereof.

[0010] Further described herein is a method for inhibiting biofilm stability, comprising, or alternatively consisting essentially of, or alternatively consisting of, contacting a biofilm in vitro with an effective amount of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof, wherein the contacting comprises, or alternatively consisting essentially of, or alternatively consisting of, coating a surface with an effective amount of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof. The present disclosure also relates to a method for inhibiting biofilm stability, comprising, or alternatively consisting essentially of, or alternatively consisting of, contacting a biofilm in vitro with an effective amount of chloroquine and an anti-B-DNA antibody or a fragment or derivative thereof, wherein the contacting comprises, or alternatively consisting essentially of, or alternatively consisting of, coating a surface with an effective amount of chloroquine and an anti-B-DNA antibody or a fragment or derivative thereof. The contacting can be in vitro or in vivo.

[0011] Also provided herein is a method for treating a biofilm in a subject, comprising, or alternatively consisting essentially of, or alternatively consisting of, administering to a subject infected with a biofilm an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In one embodiment, the method for treating a biofilm in a subject comprises, or alternatively consisting essentially of, or alternatively consisting of, administering to a subject infected with a biofilm an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm.

[0012] The present disclosure also relates to a method for preventing biofilm formation in a biofilm-prone subject, the method comprising, or alternatively consisting essentially of, or alternatively consisting of, administering to the subject an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In one aspect, the method for preventing biofilm formation in a biofilm-prone subject comprises, or alternatively consisting essentially of, or alternatively consisting of, administering to the subject an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm.

[0013] The present disclosure further relates to a method for treating an infection caused by biofilm-producing bacteria in a subject in need thereof, comprising, or alternatively consisting essentially of, or alternatively consisting of, administering to the subject an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm and an agent that inhibits the replication of the organism, wherein the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In one aspect, a method for treating an infection caused by biofilm-producing bacteria in a subject in need thereof comprises, or alternatively consisting essentially of, or alternatively consisting of, administering to the subject an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm.

[0014] For any of the methods described above, the polyamine may be selected from the group consisting of putrescine, spermine, cadaverine, 1,3-diaminopropane, or spermidine. In one embodiment, for the method described above, the agent that interferes with the binding of polyamines to DNA in biofilms is tRNA. In another embodiment, the agent is an inhibitor of polyamine synthesis or an agent that inhibits the binding of polyamines to DNA. In a second embodiment, the agent comprises, alternatively consists essentially of, or even further consists of the polyamine analogs difluoromethylornithine, trans-4-methylcyclohexylamine, sardomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, dicyclohexylamine, any derivative thereof, or a salt thereof. In a third embodiment, the agent comprises, or alternatively consists essentially of, or further consists of, an agent that depletes cations from the biofilm, optionally a cation exchange resin, aminopolycarboxylic acid, crown ether, azacrown, or cryptand. In a fourth embodiment, the agent that depletes cations from the biofilm is selected from the group of sulfonates, sulfopropyl, phosphocellulose, P11 phosphocellulose, heparin sulfate, or a derivative or analog thereof. In a fifth embodiment, the agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment. In a sixth embodiment, the agent comprises, or alternatively consists essentially of, or further consists of an anti-B-DNA antibody, or a fragment or derivative thereof. In an eighth embodiment, the agent comprises, alternatively consists essentially of, or even further consists of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or a derivative thereof.In a ninth embodiment, the agent comprises, alternatively consists essentially of, or even consists of chloroquine or a derivative thereof. In one aspect, the agent that depletes cations from a biofilm has a net negative charge. In another aspect, the agent that depletes cations from a biofilm has a net neutral charge.

[0015] Also provided herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising, alternatively consisting essentially of, or alternatively consisting of, administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein the agent is not an HMGB1 protein, fragment thereof, or equivalent thereof.Disclosed herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB, comprising, alternatively consisting essentially of, or alternatively consisting of, administering an effective amount of one or more agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein the agent is not an HMGB1 protein, fragment thereof, or equivalent thereof. In one aspect, the agent is administered in the absence of DNase. In one embodiment, the agent interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment. In a second embodiment, the agent comprises, alternatively consists essentially of, or further consists of an anti-B-DNA antibody, or a fragment or derivative thereof. In a third embodiment, the agent comprises, alternatively consists essentially of, or further consists of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine, or a derivative thereof. In a fourth embodiment, the agent comprises, alternatively consists essentially of, or further consists of chloroquine, or a derivative thereof. In one aspect, the chloroquine derivative retains the ability to intercalate between DNA bases.

[0016] Also provided herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), the method comprising, or alternatively consisting essentially of, or further consisting of, administering effective amounts of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof. In one aspect, the method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or tuberculosis (TB) comprises, or alternatively consisting essentially of, or further consisting of, effective amounts of chloroquine and an anti-B-DNA antibody or a fragment or derivative thereof. The present disclosure also relates to a method for treating a biofilm-producing infection associated with chemotherapy in a patient undergoing or who has undergone platinum-based chemotherapy, comprising, or alternatively consisting essentially of, or alternatively consisting of, administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment, wherein the agent is not an HMGB1 protein, fragment thereof, or equivalent thereof. In one embodiment, the method comprises, or alternatively consists essentially of, or alternatively consists of, an effective amount of one or more agents that interfere with the conversion of B-DNA to Z-DNA in a biofilm or its local environment. In one embodiment, the agent is administered in the absence of DNase. In a further embodiment, the agent comprises, or alternatively consists essentially of, or alternatively consists of chloroquine or a derivative thereof. In a particular embodiment, the chloroquine derivative retains the ability to intercalate between DNA bases. In still further embodiments, the agent comprises, or alternatively consists essentially of, or even additionally consists of, an anti-B-DNA antibody or a fragment or derivative thereof.In one embodiment, the agent comprises, alternatively consists essentially of, or even further consists of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or a derivative thereof.

[0017] The present disclosure further relates to a method for treating a biofilm-producing infection associated with the administration of chemotherapy in a patient undergoing or who has undergone platinum-based chemotherapy, the method comprising, or alternatively consisting essentially of, or alternatively consisting of, administering an effective amount of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof. Also provided herein is a method for treating a biofilm-producing infection associated with the administration of chemotherapy in a patient undergoing or who has undergone platinum-based chemotherapy, the method comprising, or alternatively consisting essentially of, or alternatively consisting of, an effective amount of chloroquine and an anti-B-DNA antibody or a fragment or derivative thereof.

[0018] The methods described above may further comprise, alternatively consist essentially of, or even further consist of, contacting the biofilm with, or alternatively administering to the subject, an effective amount of an agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins, wherein the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In one aspect, the agent that interferes with the binding of eDNA to DNA-binding proteins comprises, alternatively consist essentially of, or even further consists of, one or more of an anti-DNABII antibody, an anti-IHF antibody, and / or an anti-HU antibody, or fragments thereof. In one embodiment, the agent that interferes with the binding of eDNA to DNA-binding proteins has a net negative charge. In a second embodiment, the agent that interferes with the binding of eDNA to DNA-binding proteins has a net neutral charge. In a third embodiment, the agent that interferes with the binding of eDNA to DNA-binding proteins has a net positive charge. In one aspect, the agent is administered in the absence of DNase. The methods described above can be carried out in the absence of administration of DNase enzyme. The present invention provides, for example, the following items. (Item 1) A method for inhibiting biofilm stability, comprising contacting the biofilm with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 2) A method for treating a biofilm in a subject, comprising administering to the subject infected with a biofilm an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 3) A method for preventing the formation of a biofilm in a subject prone to developing a biofilm, comprising administering to the subject an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, and optionally the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 4) A method for treating an infection caused by a biofilm-producing bacterium in a subject in need thereof, comprising administering to the subject an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm and an agent that inhibits the replication of the organism, wherein optionally the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 5) A method for inhibiting biofilm stability, comprising contacting the biofilm with an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm, wherein the agents are not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 6) A method for treating a biofilm in a subject, comprising administering to the subject infected with a biofilm an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm, wherein the agents are not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 7) A method for preventing the formation of a biofilm in a subject prone to developing a biofilm, comprising administering to the subject an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm, and optionally wherein the agents are not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 8) A method for treating an infection caused by a biofilm-producing bacterium in a subject in need thereof, comprising administering to the subject effective amounts of one or more agents that interfere with the binding of polyamines to DNA in the biofilm and an agent that inhibits the replication of the organism, wherein optionally the agents are not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 9) 6. The method of item 1 or 5, wherein the contacting is in vitro or in vivo. (Item 10) 10. The method of any one of items 1, 5 or 9, wherein the agent that interferes with the binding of polyamines to DNA in the biofilm is a tRNA. (Item 11) The method according to any preceding item, wherein the agent is an inhibitor of polyamine synthesis or an agent that inhibits the binding of the polyamine to the DNA, and the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 12) The method of any preceding item, wherein the polyamine is selected from the group consisting of putrescine, spermine, cadaverine, 1,3-diaminopropane, and spermidine. (Item 13) 12. The method of claim 11, wherein the agent comprises a polyamine analogue difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, dicyclohexylamine, any derivative thereof, or a salt thereof. (Item 14) 10. The method of any one of items 1 to 9, wherein the agent comprises an agent that depletes cations from the biofilm, optionally a cation exchange resin, an aminopolycarboxylic acid, a crown ether, an azacrown, or a cryptand. (Item 15) 15. The method of claim 14, wherein the agent that depletes cations from the biofilm is an agent from the group of sulfonates, sulfopropyl, phosphocellulose, P11 phosphocellulose, heparin sulfate or a derivative or analog thereof. (Item 16) A method for inhibiting biofilm stability, comprising contacting the biofilm in vitro with an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises coating a surface with an effective amount of an agent that depletes cations, and wherein the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 17) A method for inhibiting biofilm stability, comprising contacting the biofilm in vitro with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises coating a surface with an effective amount of one or more agents that deplete cations, and wherein the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 18) 18. The method of any one of items 1 to 9 or 16 or 17, wherein the agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment. (Item 19) 19. The method of any one of items 16 to 18, wherein the agent comprises an anti-B-DNA antibody or a fragment or derivative thereof. (Item 20) 19. The method of any one of items 16 to 18, wherein the agent comprises riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or a derivative thereof. (Item 21) 19. The method of any one of items 16 to 18, wherein the agent comprises chloroquine or a derivative thereof. (Item 22) A method for inhibiting biofilm stability, comprising contacting the biofilm in vitro with an effective amount of HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof, wherein the contacting comprises coating a surface with an effective amount of HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof. (Item 23) A method for inhibiting biofilm stability, comprising contacting the biofilm in vitro with an effective amount of chloroquine and an anti-B-DNA antibody, or a fragment or derivative thereof, wherein the contacting comprises coating a surface with the effective amount of chloroquine and an anti-B-DNA antibody, or a fragment or derivative thereof. (Item 24) A method for treating biofilms in patients suffering from systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 25) A method for treating biofilms in patients suffering from systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising administering an effective amount of one or more agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein the agents are not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 26) 26. The method of claim 22 or 25, wherein the agent comprises chloroquine or a derivative thereof. (Item 27) 26. The method of claim 22 or 25, wherein the agent comprises an anti-B-DNA antibody or a fragment or derivative thereof. (Item 28) 26. The method of claim 22 or 25, wherein the agent comprises riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or a derivative thereof. (Item 29) A method for treating biofilms in patients suffering from systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising administering an effective amount of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof. (Item 30) A method for treating biofilms in patients suffering from systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising administering an effective amount of chloroquine and an anti-B-DNA antibody or a fragment or derivative thereof. (Item 31) A method for treating a biofilm-producing infection associated with the administration of platinum-based chemotherapy in a patient receiving or who has received said chemotherapy, comprising administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in said biofilm or its local environment, wherein said agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 32) A method for treating a biofilm-producing infection associated with the administration of platinum-based chemotherapy in a patient receiving or who has received said chemotherapy, comprising administering an effective amount of one or more agents that interfere with the conversion of B-DNA to Z-DNA in said biofilm or its local environment, wherein said agents are not HMGB1 protein, a fragment thereof, or an equivalent thereof. (Item 33) 33. The method of claim 31 or 32, wherein the agent comprises chloroquine or a derivative thereof. (Item 34) 33. The method of claim 31 or 32, wherein the agent comprises an anti-B-DNA antibody or a fragment or derivative thereof. (Item 35) 33. The method of claim 31 or 32, wherein the agent comprises riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or a derivative thereof. (Item 36) A method for treating biofilm-producing infections associated with the administration of platinum-based chemotherapy in patients undergoing or who have received said chemotherapy, comprising administering an effective amount of HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof. (Item 37) A method for treating biofilm-producing infections associated with the administration of platinum-based chemotherapy in patients undergoing or who have received said chemotherapy, comprising administering an effective amount of chloroquine and an anti-B-DNA antibody or a fragment or derivative thereof. (Item 38) 6. The method of claim 1 or 5, further comprising contacting the biofilm with an effective amount of an agent that interferes with binding of eDNA to DNA-binding proteins and / or an antimicrobial agent. (Item 39) 39. The method of claim 38, wherein the agent that interferes with the binding of the eDNA to the DNA binding protein comprises one or more of an anti-DNABII antibody, an anti-IHF antibody, and / or an anti-HU antibody, or fragments of each. (Item 40) 40. The method of any of items 2 to 39, further comprising administering to the subject an effective amount of an agent that interferes with binding of the eDNA to a DNA-binding protein and / or an antibacterial agent. (Item 41) 41. The method of claim 40, wherein the agent that interferes with the binding of the eDNA to the DNA binding protein comprises one or more of an anti-DNABII antibody, an anti-IHF antibody, and / or an anti-HU antibody, or fragments of each. (Item 42) 16. The method of claim 14 or 15, wherein the agent that depletes cations from the biofilm has a net negative charge. (Item 43) 16. The method of claim 14 or 15, wherein the agent that depletes cations from the biofilm has a net neutral charge. (Item 44) 39. The method of claim 38, wherein the agent that interferes with the binding of the eDNA to a DNA binding protein has a net negative charge. (Item 45) 39. The method of claim 38, wherein the agent that interferes with the binding of the eDNA to a DNA binding protein has a net neutral charge. (Item 46) 39. The method of claim 38, wherein the agent that interferes with the binding of the eDNA to a DNA binding protein has a net positive charge. (Item 47) 47. The method of any one of items 1 to 46, wherein the method is carried out in the absence of administration of a DNase enzyme. (Item 48) A composition comprising one, two or three or more of an agent that interferes with the binding of polyamines to DNA in a biofilm, an agent that depletes cations from a biofilm, an agent that interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment, an agent that interferes with the binding of eDNA to DNA binding proteins, and / or an antibacterial agent. (Item 49) 49. The composition of claim 48, further comprising a pharmaceutically acceptable carrier. (Item 50) 50. The composition of claim 48 or 49, wherein the agent that interferes with the binding of polyamines to DNA in the biofilm comprises one or more of the polyamine analogs difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, and / or dicyclohexylamine, any derivatives thereof, or salts thereof. (Item 51) 50. The composition of claim 48 or 49, wherein the agent that depletes cations from the biofilm comprises one or more of a cation exchange resin, an aminopolycarboxylic acid, a crown ether, an azacrown or cryptand, a sulfonate, a sulfopropyl, a phosphocellulose, a P11 phosphocellulose, and / or a heparin sulfate or a derivative or analog thereof. (Item 52) 50. The composition of claim 48 or 49, wherein the agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment comprises one or more of an HMGB1 protein, a fragment thereof or its respective equivalent, an anti-B-DNA antibody or a fragment or derivative thereof, and / or chloroquine, or any derivative thereof. (Item 53) 50. The composition of claim 48 or 49, wherein the agent that interferes with the binding of the eDNA to a DNA-binding protein comprises one or more of an anti-DNABII antibody, an anti-IHF antibody, and / or an anti-HU antibody, or fragments of each. (Item 54) 54. A kit comprising the composition according to any one of items 48 to 53 and instructions for use, wherein the agents are optionally combined or packaged separately. (Item 55) 55. The kit according to Item 54, wherein the instructions for use provide a method for carrying out the method according to any one of Items 1 to 47. [Brief explanation of the drawings]

[0019] [Figure 1] Figures 1A-1C show that polyamines modulate DNA structure. (Figure 1A) Immunofluorescence microscopy image of mucosal biofilm EPS found in the middle ear of a chinchilla with acute OM due to NTHI (adapted from Goodman et al. (2011) Mucosal Immunol. 4(6):625-37). DNABII protein (gray beads) and eDNA (white regions) were probed. DNABII localizes to the apex of eDNA strands in biofilms in vivo. (Figure 1B) Chemical structure of a general polyamine (adapted from Di Martino et al. (2013) Int J Med Microbiol. 303(8):484-91). (Figure 1C) Atomic force microscopy image of DNA alone or after incubation with polyamines (adapted from Iacomino et al. (2011) Biomacromolecules. 12(4):1178-86). Polyamines induce the formation of thick fibers and structural complexity in eDNA.

[0020] [Figure 2] Figures 2A-2B show that polyamines induce eDNA scaffold structure. (Figure 2A) Immunofluorescence CLSM images of mucosal biofilm EPS found in the middle ear of a chinchilla with acute OM due to NTHI. The images were probed for polyamines (white dots) [putrescine (Put), cadaverine (Cad), spermidine (Spd)] and eDNA (white) and counterstained with DAPI (gray area). Polyamines localize to eDNA strands in biofilms formed in vivo; spermidine is the most abundant polyamine in biofilm EPS. (Figure 2B) Transmission electron microscopy images of complexes of DNA (5 μM) and spermidine (700 μM) (top) and DNA (5 μM), spermidine (700 μM), and HU (50 nM) (bottom) (adapted from Sarkar et al. (2007) Nucleic Acids Res. 35(3):951-61). A similar structure is formed by the DNA-polyamine-IHF complex. Sarkar et al. (2009) Biochemistry. 48(4):667-75. Polyamines induce DNA condensation and, in combination with DNA BII proteins, form thick fibers.

[0021] [Figure 3]Figures 3A-3C show that polyamine synthesis inhibitors reduce biofilm formation. (Figure 3A) COMSTAT quantification of LIVE / DEAD®-stained NTHI biofilms grown in the presence of dicyclohexylamine (DCHA, 50 μM), spermidine (Spd) synthase inhibitor Spd (1 mM), or both. Bars represent SEM. Statistical significance compared to control was assessed by unpaired t-test; *P<0.05. The average biofilm thickness was reduced by DCHA, whereas the simultaneous addition of exogenous Spd restored biofilm formation. (Figure 3B) Immunofluorescence microscopy images of eDNA scaffold structures in NTHI in vitro biofilms grown for 3 hours in the presence of DCHA (50 μM). The structures were probed for dsDNA (white area). eDNA scaffold structure production was significantly reduced by the inhibition of polyamine synthesis. (Figure 3C) Immunofluorescence CLSM image of an NTHI in vitro biofilm grown for 40 h in the presence of DCHA, probed for Spd (white dots in the bottom panel of Figure 3C) and counterstained with DAPI (gray area). DCHA inhibits polyamine incorporation into the biofilm EPS.

[0022] [Figure 4]Figures 4A-4B show that anti-DNABII disrupts DNABII-polyamine (PA)-dependent structures. (Figure 4A) DNA structures were formed by incubating genomic DNA (gDNA; 2 μg / ml) in a buffer containing spermidine (300 μM) and HU (1 μM) for 40 hours. Immunofluorescence CLSM images of DNABII-polyamine-dependent DNA structures. Probed for DNABII protein (white; indicated on the right side of the image) and counterstained with DAPI (white; indicated on the left side of the image). DNABII-polyamine-dependent DNA structures incorporate DNABII protein. (Figure 4B) EPS structures formed over 24 hours, as in Figure 4A, and then treated with a 1:50 dilution of DNABII antiserum for an additional 16 hours (indicated at the bottom of the image). Fluorescence CLSM images stained with DAPI (white). DNABII-polyamine-dependent DNA structures require DNABII protein.

[0023] [Figure 5] Figure 5 shows that the cation exchanger phosphocellulose (P11) disrupts NTHI biofilm formation. P11 (1%) was added to the apical chamber, while NTHI biofilm growth was initiated in the basolateral chamber of a transwell plate system. Spermidine (1 mM) and HU (1 μM) were added at the time of seeding and maintained for 16 h. Biofilms were visualized by CLSM and analyzed by COMSTAT. Mean thickness (not shown) showed the same trend. Bars represent SEM. Statistical significance compared to control was assessed by unpaired t-test; *P<0.05; **P<0.01. P11 prevents biofilm formation. Exogenous spermidine and HU together, but not either alone, restored biofilm development (not shown), suggesting that the P11 antibiofilm activity is the result of titration of structural components (polyamines and DNABII proteins) derived from the biofilm EPS.

[0024] [Figure 6]Figure 6 shows that mature biofilms are resistant to disruption by DNase. DNase (Pulmozyme; 5 units) was added to preformed in vitro NTHI and UPEC biofilms at the time of inoculation (prevention) or at 24 hours (disruption). After a total of 40 hours, biofilms were stained with LIVE / DEAD®, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. Statistical significance compared to control was assessed by unpaired t-test, *P<0.05; **P<0.01. DNase can prevent biofilms but does not disrupt existing biofilms.

[0025] [Figure 7]Figures 7A-7D show that DNABII proteins and polyamines (PAs) synergistically interact to induce DNase resistance. (Figure 7A) Immunofluorescence CLSM image of an NTHI in vitro biofilm grown for 40 hours. Probed for spermidine (dark gray beads), HU (light gray beads), and counterstained with DAPI (gray area). Polyamines and DNABII proteins colocalize in the biofilm EPS in vitro (white beads). (Figure 7B) Immunofluorescence CLSM image of a mucosal biofilm EPS found in the middle ear of a chinchilla with acute OM due to NTHI. Probed for putrescine (white beads) and HU (light gray beads) and counterstained with DAPI (dark gray area). Polyamines and DNABII proteins colocalize in the eDNA strands in the biofilm in vivo (white area). (Figure 7C) Increasing levels of spermidine (Spd) and HU, separately or together, were incubated with genomic DNA (2 μg / ml) for 1.5 hours at 37°C, followed by treatment with Pulmozyme® for 20 minutes. DNA degradation was assayed using agarose gel electrophoresis. Spd and HU synergistically protect genomic DNA from DNase digestion. (Figure 7D) Spd (300 μM) and HU (1 μM) were incubated with genomic DNA (2 μg / ml) for 40 hours, followed by treatment with Pulmozyme® for 20 minutes. Structures were stained with LIVE / DEAD® and imaged by CLSM (white). HU-Spd-dependent DNA structures are resistant to DNase treatment.

[0026] [Figure 8]Figures 8A-8B show that DNABII and polyamines combine to convert B-DNA to Z-DNA. (Figure 8A) DNABII-polyamine-dependent DNA structures were formed by incubating genomic DNA (2 μg / ml) with HU (1 μM) and spermidine (300 μM) for 16 hours. Immunofluorescence CLSM images of DNABII-polyamine-dependent DNA structures probed for Z-DNA (white) and stained with DAPI (dark gray). Polyamines and DNABII proteins act synergistically to induce the conversion of B-DNA to Z-DNA. (Figure 8B) Top: Circular dichroism spectra of B-DNA substrates converted to Z-DNA by increasing concentrations of Z-DNA catalyst (adapted from Jang et al. (2015) Sci Rep. 5:9943). Note the negative peak around 250 nm and the inversion of the positive peak around 280 nm. Bottom: Poly(dGdC) DNA (20 μg / ml) was incubated with HU (15 μM) for 2 hours and CD spectra were collected. HU shifts the CD spectrum of poly(dGdC) toward the Z-DNA signature.

[0027] [Figure 9] Figure 9 shows that Z-DNA is present in the biofilm EPS of multiple human pathogens. Top: Immunofluorescence CLSM images of 40-hour biofilms of the indicated bacteria probed with no primary antibody (1° no) or with Z-DNA antibody (white). Z-DNA is a component of the EPS of multibacterial biofilms at different steady-state levels. Bottom: Immunofluorescence CLSM images of the indicated biofilms probed with HU (white) and spermidine (dark gray) antibodies; colocalization is shown (white). DNABII and polyamine components colocalize in the EPS of multibacterial biofilms at steady-state levels, which correlates with Z-DNA abundance.

[0028] [Figure 10]Figure 10 shows that Z-DNA and polyamines increase in abundance as UPEC and NTHI biofilms mature. Immunofluorescence CLSM images of UTI89 and NTHI in vitro biofilms at various stages of formation probed with anti-Z-DNA (white) or anti-spermidine (dark gray). Mature biofilms incorporate increasing amounts of Z-DNA (white) and spermidine (dark gray) into the biofilm EPS over time.

[0029] [Figure 11] Figure 11 shows that HU is required for the conversion of B-DNA to Z-DNA and the incorporation of polyamines into biofilm EPS. Immunofluorescence CLSM images of 40-hour NTHI wild-type and ΔHU mutant in vitro biofilms probed with spermidine (dark gray) or anti-Z-DNA (white). In the absence of HU, polyamines and Z-DNA are reduced in abundance within the biofilm EPS.

[0030] [Figure 12] Figure 12 shows nontypeable Haemophilus influenzae biofilms grown for 40 hours in supplemented BHI medium in 8-well chambered coverglass slides at 37°C and 5% CO2. Biofilms were washed and probed with the indicated primary antibodies, fluorescent secondary antibodies (dark gray dots), and stained with DAPI (gray areas). Note: The minimal dark gray staining in the bottom left-most image represents background. Putrescine, spermidine, and spermine were all present throughout the biofilm matrix.

[0031] [Figure 13]Figure 13 shows that nontypeable Haemophilus influenzae was grown for 16 hours in supplemented BHI medium containing the indicated additives in 96-well plates at 37°C and 5% CO2. Growth was quantified by spectrophotometer absorbance at 490 nm (left) and by counting colony-forming units (right). Spermidine synthase inhibitors did not affect normal growth.

[0032] [Figure 14] Figure 14 shows nontypeable Haemophilus influenzae biofilms grown for 40 hours at 37°C and 5% CO2 in supplemented BHI medium containing the additives indicated under each bar on an 8-well chambered cover glass slide. Biofilms were washed, stained with LIVE / DEAD®, fixed, and imaged by CLSM. Biofilm parameters were quantified using COMSTAT software. Dicyclohexylamine inhibited biofilm biogenesis, whereas exogenous spermidine was able to rescue biofilm growth.

[0033] [Figure 15] Figure 15 shows nontypeable Haemophilus influenzae biofilms grown for 40 hours at 37°C and 5% CO2 in supplemented BHI medium containing the indicated additives on 8-well chambered coverglass slides. Biofilms were washed and probed with a primary antibody against spermidine, a fluorescent secondary antibody (dark gray dots), and stained with DAPI (gray area). Spermidine synthase inhibitors reduced the presence of spermidine in the biofilm matrix.

[0034] [Figure 16]Figure 16 shows that surface-attached nontypeable Haemophilus influenzae were grown for 3 hours at 37°C and 5% CO in supplemented BHI medium containing the additives indicated above on Fluorodish coverglass dishes. Biofilms were washed and probed with a primary antibody against double-stranded DNA and a fluorescent secondary antibody (white). Spermidine synthase inhibitors reduce both the presence and complexity of extracellular DNA structures within the biofilm matrix.

[0035] [Figure 17] Figures 17A-17D show that spermidine is present in biofilm EPS formed by multiple human pathogens. (Figure 17A) Immunofluorescence CLSM images of the indicated biofilms probed with HU (light gray) and spermidine (dark gray) antibodies; colocalization is white. DNABII and polyamine components colocalize in the EPS of multibacterial biofilms at steady-state levels. (Figure 17B) Dicyclohexylamine (DCHA) inhibition of spermidine biosynthesis reduces spermidine levels in NTHI and UPEC biofilms, as indicated by a decrease in IF signal, resulting in significantly decreased mean thickness compared to sBHI controls (Figure 17C). UPEC biofilms are expressed as a percent change in mean thickness compared to LB controls (Figure 17D).

[0036] [Figure 18]Figures 18A-B show that phosphocellulose has a dose-dependent negative effect on biofilm formation and preformed NTHI biofilm stability in vitro. (Figure 18A) Biofilm growth was initiated and then maintained for 24 hours, followed by treatment with 0 (SBHI control), 0.1%, 1%, and 5% (w / v) phosphocellulose (P11) for 16 hours. (Figure 18B) Biofilm growth was initiated and then maintained for 40 hours in the presence of 0 (sBHI control), 0.1%, 1%, and 5% (w / v) (P11). Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate average thickness and biomass. All images were captured using a 63x objective.

[0037] [Figure 19] Figure 19 shows that heparin sepharose has a negative effect on NTHI biofilm formation in vitro. Biofilm growth was initiated and then maintained for 40 hours in the presence of 0 (sBHI control) or 5% (w / v) heparin sepharose resin. Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate average thickness and biomass. All images were captured using a 63x objective.

[0038] [Figure 20]Figure 20 shows that exogenous addition of HU rescues the negative effect of phosphocellulose on NTHI biofilm stability in vitro. Biofilm growth was initiated and maintained for 24 hours, then treated for 16 hours as indicated. Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT, and mean thickness and biomass were calculated and compared with sBHI controls. All images were captured using a 63x objective. Bars represent SEM. Statistical significance compared to control was assessed by unpaired t-test, *P<0.05; **P<0.01.

[0039] [Figure 21] Figure 21 shows that exogenous addition of MgCl2 rescues the negative effect of phosphocellulose on NTHI biofilm stability in vitro. Biofilm growth was initiated and maintained for 24 hours, then treated as indicated for 16 hours. Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate average thickness and biomass. All images were captured using a 63x objective.

[0040] [Figure 22] Figure 22 shows that exogenous addition of spermidine rescues the negative effect of phosphocellulose on NTHI biofilm stability in vitro. Biofilm growth was initiated and then maintained for 24 hours, followed by 16 hours of treatment as indicated. Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate average thickness and biomass. All images were captured using a 63x objective.

[0041] [Figure 23]Figure 23 shows that the cation-depleting effect of P11 phosphocellulose does not require direct contact with biofilms. Biofilm growth was initiated in the basolateral chamber of a transwell plate system, while 0, 0.5, 1, or 1.5% (w / v) P11 phosphocellulose was added to the apical chamber and maintained for 16 hours. Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT, and mean thickness and biomass were calculated and compared with sBHI controls. All images were captured using a 63x objective. Bars represent SEM. Statistical significance compared to controls was assessed by unpaired t-test; *P<0.05; **P<0.01.

[0042] [Figure 24] Figure 24 shows that exogenous addition of spermidine reduces the cation-depleting effect of P11 phosphocellulose without requiring direct contact with the biofilm. Biofilm growth was initiated in the basal chamber of a transwell plate system, while 0 or 1.5% (w / v) P11 phosphocellulose was added to the apical chamber and maintained for 16 hours in the presence of 100, 500, or 1000 μM spermidine. Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT, and mean thickness and biomass were calculated and compared with sBHI controls. All images were captured using a 63x objective. Bars represent SEM. Statistical significance compared to the control was assessed by unpaired t-test; *P<0.05; **P<0.01. Brackets indicate statistical comparisons between conditions.

[0043] [Figure 25]Figure 25 shows that exogenous addition of spermidine and DNABII reduces the cation-depleting effect of P11 phosphocellulose without direct contact with the biofilm. Biofilm growth was initiated in the basolateral chamber of a transwell plate system, while 0 or 1.5% (w / v) P11 phosphocellulose was added to the apical chamber and maintained for 16 hours in the presence of 100 μM spermidine, 500 nM HU, or a combination thereof. Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT, and mean thickness and biomass were calculated and compared with the sBHI control. All images were captured using a 63x objective. Bars represent SEM. Statistical significance compared to the control was assessed by unpaired t-test; *P<0.05; **P<0.01. Brackets indicate statistical comparisons between conditions.

[0044] [Figure 26] Figures 26A-B show that coating abiotic surfaces with cation exchange resin prevents biofilm formation in a dose-dependent manner. Chamber slides were coated with P11 phosphocellulose (Figure 26A) or heparin Sepharose (Figure 26B) solution, as indicated. Biofilm growth was initiated and maintained on the coated slides for 40 hours. Biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate average thickness and biomass. All images were captured using a 63x objective. Bars represent SEM.

[0045] [Figure 27]Figure 27 shows that mature biofilms are resistant to disruption by DNase. DNase (Pulmozyme; 5 units) was added to preformed in vitro NTHI or UPEC biofilms at the time of inoculation (prevention) or at 24 hours (disruption). After a total of 40 hours, biofilms were stained with LIVE / DEAD®, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. A similar trend was observed for biomass. Statistical significance compared to the control was assessed by unpaired t-test, *P<0.05; **P<0.01. DNase can prevent biofilms but does not disrupt existing biofilms.

[0046] [Figure 28] Figure 28 shows that Z-DNA and polyamines increase in abundance as UPEC and NTHI biofilms mature. Immunofluorescence CLSM images of in vitro formed UPEC and NTHI biofilms at various stages of maturation probed with anti-Z-DNA (light gray) or anti-spermidine (dark gray). Mature biofilms incorporate increasing amounts of Z-DNA (light gray) and spermidine (dark gray) into the biofilm EPS over time.

[0047] [Figure 29] Figure 29 shows that Z-DNA is present in mature pathogenic fungal biofilms. Immunofluorescence CLSM images of biofilms formed by Candida albicans in vitro counterstained with DAPI and probed with anti-B-DNA, anti-Z-DNA, or no primary antibody (light gray). Mature fungal biofilms incorporate Z-DNA (light gray) within the biofilm EPS.

[0048] [Figure 30]Figure 30 shows that anti-Z-DNA antibodies stimulate biofilm biogenesis. Anti-Z-DNA antibodies (1 mg) were added to NTHI in vitro biofilms at the time of inoculation. After 16 hours, biofilms were stained with LIVE / DEAD®, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. A similar trend was observed for biomass. Statistical significance compared to control was assessed by paired t-test. Anti-Z-DNA antibodies stabilize the biofilm extracellular matrix and stimulate biofilm biogenesis, whereas antibodies against B-DNA (e.g., anti-dsDNA) do not stimulate biofilm biogenesis.

[0049] [Figure 31] Figure 31 shows that DNase degrades B-DNA but not Z-DNA within the biofilm extracellular matrix. DNase (Pulmozyme; 5 units) was added to preformed NTHI biofilms in vitro 24 hours after biofilm seeding. After a total of 40 hours, biofilms were probed with anti-Z-DNA, anti-B-DNA, or no primary antibody, which was revealed by the use of corresponding secondary fluorescent antibodies and visualized by CLSM. DNase treatment degrades B-DNA-form eDNA structures within the biofilm extracellular matrix but reveals large Z-DNA-form eDNA structures.

[0050] [Figure 32] Figure 32 shows that Z-DNA formation protects DNA from nuclease degradation. Poly(dG-dC) substrate was incubated with salt-activated nuclease (SAN) or DNase I in increasing concentrations of NaCl, spermine, or spermidine, as indicated above. Degradation products were visualized by gel electrophoresis. High salt and polyamines protect DNA from degradation by conversion to the Z-DNA form.

[0051] [Figure 33]Figure 33 shows that DNABII protein and polyamines colocalize within the biofilm extracellular matrix. Nontypeable Haemophilus influenzae biofilms were grown in supplemented BHI medium on 8-well chambered cover slides at 37°C and 5% CO2. Biofilms were washed, probed with primary antibodies against DNABII protein (light gray) or polyamines (dark gray), fluorescent secondary antibodies, stained with DAPI (gray area), and visualized by fluorescence microscopy. Polyamines colocalize with HU, but not IHF, in the biofilm matrix. NTHI strains unable to produce HU had reduced accumulation of polyamines in the biofilm matrix. DNABII protein and polyamines interact to stabilize the biofilm extracellular matrix.

[0052] [Figure 34] Figures 34A-B show that DNABII proteins protect DNA by shifting it to the Z-DNA form. (Figure 34A) Poly(dG-dC) substrates were incubated with DNase I at increasing concentrations of NTHi HU, as indicated above. Degradation products were visualized by gel electrophoresis. HU protected DNA from degradation. (Figure 34B) Top: Circular dichroism spectra of B-DNA substrates converted to Z-DNA by increasing concentrations of Z-DNA catalyst (adapted from Rahmouni (1992) Mol Microbiol. 6(5):569-72). Note the negative peak around 250 nm and the inversion of the positive peak around 280 nm. Bottom: Poly(dGdC) DNA (5 μg) was incubated with HU (15 μM) for 2 hours, and CD spectra were collected. HU shifts the CD spectrum of poly(dGdC) toward the Z-DNA signature.

[0053] [Figure 35]Figures 35A-35D show that DNABII proteins and polyamines (PAs) synergistically interact to induce DNase resistance. (Figure 35A) Increasing levels of spermidine (Spd) and HU, separately or together, were incubated with genomic DNA (gDNA; 2 μg / ml) for 1.5 hours at 37°C, followed by treatment with Pulmozyme® for 20 minutes. DNA degradation was assayed using agarose gel electrophoresis. Spd and HU synergistically protect genomic DNA from DNase digestion. (Figure 35B) Spd (300 μM) and HU (1 μM) were incubated with genomic DNA (2 μg / ml) for 40 hours, followed by treatment with Pulmozyme® for 20 minutes. Structures were stained with LIVE / DEAD® and imaged by CLSM (white). (Figure 35C) Immunofluorescence CLSM image of mucosal biofilm EPS found in the middle ear of a chinchilla with experimental OM caused by NTHI. Probed for putrescine (dark gray) and HU (light gray) and counterstained with DAPI (gray). HU-Spd-dependent DNA structures are resistant to DNase treatment. Polyamines and DNABII proteins colocalize on eDNA strands in biofilms in vivo (white). (Figure 35D): Increasing concentrations of DNase were added to NTHI or UPEC biofilms for 16 hours, either at the time of seeding (prevention) or at 24 hours (disruption). Biofilms were stained, fixed, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. Statistical significance compared to the control (no DNase) was assessed by unpaired t-test; **P<0.01. DNase can prevent biofilm formation but does not disrupt existing biofilms.

[0054] [Figure 36]Figure 36 shows that Z-DNA is present in the biofilm EPS of multiple human pathogens. Top: Immunofluorescence CLSM images of 40-hour biofilms formed by the indicated bacteria probed with either no primary antibody (1° no) or a Z-DNA-specific antibody (light gray). Z-DNA is a component of the EPS of multi-bacterial biofilms at different steady-state levels. Bottom: Immunofluorescence CLSM images of the indicated biofilms probed with HU (light gray) and spermidine (dark gray) antibodies; co-localization is shown (white). DNABII and polyamine components co-localize in the EPS of multi-bacterial biofilms at steady-state levels, which correlates with Z-DNA abundance.

[0055] [Figure 37] Figure 37 shows that HMGB1 destabilizes Z-DNA structures in the biofilm extracellular matrix. Nontypeable Haemophilus influenzae biofilms were grown in supplemented BHI medium on 8-well chamber cover slides at 37°C and 5% CO2. After 24 hours, the biofilms were treated as indicated above. After 40 hours, the biofilms were washed, probed with a primary antibody against Z-DNA (light gray dots), a fluorescent secondary antibody, stained with DAPI (gray areas), and visualized by fluorescence microscopy. HMGB1 treatment reduced Z-DNA structures in the biofilm matrix.

[0056] [Figure 38]Figures 38A-C show that HMGB1 displaces DNABII protein, thereby disrupting NTHI biofilms. Nontypeable Haemophilus influenzae biofilms were grown in supplemented BHI medium on 8-well chambered coverglass slides at 37°C and 5% CO2. After 24 hours, biofilms were treated as indicated. (Figure 38A) After 40 hours, biofilms were washed, probed with a primary antibody against DNABII protein (light gray dots), a fluorescent secondary antibody, stained with DAPI (gray areas), and visualized by fluorescence microscopy. (Figure 38B) Media was collected from biofilm cultures and analyzed by Western blot using a primary antibody that recognizes DNABII protein. HMGB1 treatment displaced DNABII protein from the biofilm matrix. (Figure 38C) COMSTAT-quantified NTHI biofilms stained with LIVE / DEAD® and visualized by confocal microscopy after treatment with 5 mg / mL HMGB1. HMGB1 disrupts biofilms by displacing DNABII proteins.

[0057] [Figure 39] Figures 39A-B show that HMGB1 promotes biofilm lysis in an experimental model of OM (chinchilla host). Diluent or 5 μg rHMGB1 or mHMGB1 was delivered directly to the middle ear of chinchillas on days 4 and 5 after NTHI infection. Animals were sacrificed 24 hours later, and the middle ears were imaged (Figure 39A) and blindly scored (Figure 39B) based on the criteria described below (Figure 39A). Bars represent SEM. ***P<0.001. The images and scoring demonstrate that HMGB1 promoted the clearance of pre-existing NTHI biofilms in situ.

[0058] [Figure 40]Figures 40A-C show that HMGB1 disrupts Burkholderia cenocepacia biofilms. (Figure 40A) B. cenocepacia biofilms were grown in LB medium on 8-well chambered cover slides at 37°C and 5% CO. After 24 hours, biofilms were treated with 5 mg / mL HMGB1. After a total of 40 hours, biofilms were stained with LIVE / DEAD®, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. (Figure 40B) C57BL / 6 mice were infected with 10 CFU it and simultaneously received 5 mg rHMGB1 or mHMGB1, the C45S non-inflammatory variant. B. cenocepacia aggregates were visible by fluorescence microscopy (gray) in sections probed with α-B. cenocepacia antibody. After 18 hours (FIG. 40C), CFU were quantified in the BAL. Bars represent SD. *P<0.05, ***P<0.001. HMGB1 treatment significantly disrupts B. cenocepacia biofilms in situ.

[0059] [Figure 41] Figure 41 shows that HMGB1 restores polyamine-induced Z-DNA to the nuclease-sensitive B-DNA state. Poly(dG-dC) substrates were incubated with spermidine and HMGB1. Degradation products were visualized by gel electrophoresis. HMGB1 treatment restored nuclease sensitivity to spermidine-induced Z-DNA substrates.

[0060] [Figure 42]Figures 42A-D show that both DNABII protein and polyamines are required to rescue cation exchanger (P11)-mediated biofilm prevention. (Figure 42A) NTHI biofilm growth was initiated at 37°C and 5% CO2 in the basolateral chamber of a transwell plate system, while P11 resin was added to the apical chamber and incubated for 16 hours. (Figure 42B) Biofilms were stained, fixed, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. Statistical significance compared to control (no P11) was assessed by unpaired t-test; ****P<0.0001. (Figure 42C) Spermidine (Spd, 300 μM) and HU (500 nM) were added at the time of seeding. Biofilms were quantified as in (Figure 42B). Compared to the control (no P11), **P<0.01, ***P<0.001, and ****P<0.0001. (Figure 42D) Representative images of biofilms formed under the conditions quantified in (Figure 42C), with the average biomass shown in the upper right. P11 addition prevents biofilm formation in a dose-dependent manner. Exogenous spermidine and HU together, but not either alone, restored biofilm development, suggesting that P11 antibiofilm activity is the result of titration of structural components (polyamines, DNABII proteins) away from the biofilm EPS without direct contact.

[0061] [Figure 43]Figures 43A-B show the specificity of B-DNA and Z-DNA antibodies. (Figure 43A) Brominated genomic DNA (2 μg / mL) and poly(dGdC) were incubated in buffer, and the absorbance values ​​at 260 nm and 295 nm were measured to calculate the A260 / 295 ratio. A ratio >8.6 indicates B-DNA (dark gray), whereas a value around 3.2 indicates Z-DNA (white). (Figure 43B) ELISA plates were coated with 1 μg of poly(dGdC) (B-DNA) or brominated poly(dGdC) (Z-DNA, Hindler et al. (2013) J Clin Microbiol. 51(6):1678-84.), followed by blocking with 0.5% BSA. Wells were then probed with mouse (ms) IgG1 (negative control), mouse anti-B DNA, or mouse anti-Z DNA and detected with secondary goat anti-mouse IgG-HRP. TMB (3,3',5,5'-tetramethylbenzidine) was the colorimetric substrate used for HRP detection (dark gray wells). Anti-B and anti-Z antibodies were specific for their respective DNA forms.

[0062] [Figure 44] Figure 44 shows that DNABII proteins, polyamines, and eDNA (B-DNA and Z-DNA) steadily accumulate within the EPS of NTHI biofilms over time. Immunofluorescence CLSM images of NTHI biofilms at various stages of formation probed with anti-B-DNA antibody (light gray; first from left), anti-anti-spermidine (light gray; second from left), anti-DNABII (gray; third from left), or anti-Z-DNA (white; fourth from left). Mature biofilms increase the incorporation of each TEDS component into their EPS over time.

[0063] [Figure 45]Figure 45 shows that polyamines and DNABII protein stimulate DNase-resistant Z-DNA. Immunofluorescence images of EPS scaffold mimics formed de novo by adding DNABII protein (HUNTHI, 500 nM) and spermidine (300 μM) to purified genomic DNA (2 μg / ml) and incubating at 37°C for 16 hours. The biofilm scaffold mimics were incubated with Pulmozyme® (DNase, 5 U / ml) for 1 hour and then probed for B-DNA (dark gray) and Z-DNA (white). Scale bar: 10 μm. B-DNA and Z-DNA were observed within the mimic structures; DNase addition selectively eliminated B-DNA, while Z-DNA remained intact. Thus, polyamines and DNABII protein can induce DNase-resistant status by converting eDNA from B-DNA to Z-DNA.

[0064] [Figure 46]Figure 46 shows that TEDS and Z-DNA are present in biofilm EPS formed by multiple human pathogens. (A) Top: Immunofluorescence CLSM images of the indicated biofilms, either untreated or probed with anti-HU (gray) and anti-spermidine (Spd, light gray) antibodies; colocalization is shown in white. Bottom: Immunofluorescence CLSM images of 40-hour-old biofilms of the indicated bacteria, either untreated or probed with anti-B-DNA (dark gray) and anti-Z-DNA antibodies (white). Z-DNA was detected using a well-characterized Z-DNA-specific monoclonal antibody (clone Z22, Heydorn et al. (2002) COMSTAT. Microbiology; 146; Yang et al. (2017) Paediatr Respir Rev. 21:65-7; Xu et al. (2016) Molecules; 21(8).). DNABII, polyamines, Z-DNA, and B-DNA components are all present in the EPS of polymicrobial biofilms. Z-DNA is an integral part of the EPS of polymicrobial biofilms at different steady-state levels.

[0065] [Figure 47] Figures 47A-B show that Z-DNA is present in in vivo and ex vivo samples. Left: (Figure 47A) Z-DNA (white) and B-DNA (dark gray) in NTHI biofilms in experimental NTHI-induced OM and (Figure 47B) sputum from a cystic fibrosis patient. Inset, negative control. Scale bar, 10 μm. Right: (Figure 47A) NTHI biofilms in experimental NTHI-induced OM and (Figure 47B) sputum from a cystic fibrosis patient. Inset, negative control. Scale bar, 10 μm. DNABII protein (gray) and spermidine (Spd, light gray) in NTHI biofilms in experimental NTHI-induced OM and (Figure 47B) sputum from a cystic fibrosis patient. Inset, negative control. Scale bar, 10 μm. DNABII, polyamines, Z-DNA, and B-DNA components are all present in sections of chinchilla middle ears infected with NTHI biofilms. Z-DNA is an integral part of the EPS of multibacterial biofilms at different steady-state levels.

[0066] [Figure 48] Figures 48A-B show that Z-DNA in biofilms forms nuclease-resistant scaffolds. (Figure 48A) Biofilms formed by the indicated bacteria (Kp = K. pneumoniae) for 24 hours were incubated with DNase (Pulmozyme®; 40 U / ml) for an additional 16 hours. Biofilms were then probed with anti-B-DNA (dark gray) and anti-Z-DNA (white) antibodies and counterstained with the bacterial cell membrane stain FM4-64™ (not shown). (Figure 48B) ImageJ was used to quantitate changes in B-DNA or Z-DNA abundance by calculating the ratio of the fluorescence intensity of B-DNA or Z-DNA after DNase addition divided by the fluorescence intensity in the absence of DNase (control). Fluorescence intensity was normalized to the fluorescence signal of FM4-64™-stained cells. Bars represent SEM. Statistical significance compared to the control (no DNase, dotted line) was assessed by paired t-test, *P<0.05; ****P<0.0001. B-DNA is degraded by DNase, whereas Z-DNA is resistant and functions to maintain the structural integrity of the biofilm EPS.

[0067] [Figure 49] Figure 49 shows that Z-DNA stabilization stimulates biofilm formation. NTHI and UPEC biofilm growth was initiated untreated or in the presence of anti-Z-DNA antibody (5 mg / ml) for 16 hours. Biofilms were stained, fixed, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. Statistical significance compared to control (no antibody) was assessed by paired t-test; *P<0.05, **P<0.01. Anti-Z-DNA antibody stimulates biofilm formation in a dose-dependent manner, whereas untreated antibody does not.

[0068] [Figure 50]Figures 50A-50B show that an equilibrium shift in B-DNA to Z-DNA conversion alters biofilm formation. NTHI biofilms formed for 24 h were incubated with cerium chloride (CeCl) or chloroquine for an additional 16 h and probed untreated or with anti-Z-DNA (white) by immunofluorescence CLSM. (Figure 50A) Representative images of NTHI biofilms indicated an increase in Z-DNA abundance after the addition of 1 mM CeCl. (Figure 50B) Representative images of NTHI biofilms indicated a decrease in Z-DNA abundance after the addition of 1 μM chloroquine.

[0069] [Figure 51] Figures 51A-B show that shifting the equilibrium of B-DNA to Z-DNA conversion alters biofilm formation. NTHI biofilms formed for 24 h were incubated with cerium chloride (CeCl) (Figure 51A) or chloroquine (Figure 51B) for an additional 16 h. Z-DNA was measured by IF and CLSM using anti-Z-DNA, and cells were stained with the membrane stain FM4-64. Bars represent SEM. Statistical significance compared to control was assessed by paired t-test; *P<0.05, **P<0.01. CeCl increased Z-DNA and biofilm formation, while chloroquine reduced Z-DNA and biofilm development.

[0070] [Figure 52]Figures 52A-B show that RNA homeostasis modulates bacterial biofilm development. (Figure 52A) NTHI biofilm growth was initiated in the presence of RNase A for 16 hours. Biofilms were stained, fixed, visualized by CLSM, and analyzed by COMSTAT. Addition of RNase A stimulated biofilm formation in a dose-dependent manner, likely through the release of polyamines, which are crucial catalysts for the extracellular conversion of B-DNA to Z-DNA. (Figure 52B) NTHI biofilms formed for 16 hours were incubated with tRNA and analyzed as in (Figure 52A). Guanosine monophosphate (GMP) served as a negative control. Bars represent SEM. Statistical significance compared to the control (no RNase A or tRNA / GMP) was assessed by paired t-test; *P<0.05, **P<0.01. Addition of tRNA (but not GMP) disrupted primary NTHI biofilms in a dose-dependent manner, presumably by sequestration of polyamines from the biofilm EPS.

[0071] [Figure 53]Figure 53 shows that DNABII and polyamines synergize to convert B-DNA to Z-DNA. In one embodiment, the agent is administered in the absence of DNase. Left: Genomic DNA (2 μg / mL) was incubated with spermidine (spd: 300 μM), HU (500 nM), or a combination of both spd and HU for 2 hours at 37°C. Incubation of gDNA with 3.6 M NaCl was used as a positive Z-DNA control. Center: gDNA (2 μg / mL) was incubated with increasing concentrations of cerium chloride (CeCl), which is known to induce Z-DNA. Right: Poly(dGdC) (1 μg) was incubated with either NaCl (3.6 M), chloroquine (100 μM), or a combination of both NaCl and chloroquine. Chloroquine prevents the transition from B-DNA to Z-DNA. Absorbance values ​​at 260 nm and 295 nm were measured, and the A260 / 295 ratio was calculated. A ratio >8.6 indicates B-DNA, while values ​​around 3.2 indicate Z-DNA. Consistent with well-established and validated spectrophotometric absorbance ratio assays, CeCl3, along with DNABII and polyamines, acted synergistically to induce Z-DNA, while chloroquine prevented Z-DNA conversion. DETAILED DESCRIPTION OF THE INVENTION

[0072] 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 disclosure belongs. All nucleotide sequences provided herein are shown in the 5' to 3' direction. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, certain non-limiting exemplary methods, devices, and materials are described herein. All technical and patent publications cited herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior invention.

[0073] The practice of the present disclosure 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 rd edition; Series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; Series Methods in Enzymology (Academic Press, Inc., NY); 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 thedition;Gait ed. (1984) Oligonucleotide Synthesis;U.S. Patent No. 4,683,195;Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization;Hames and Higgins eds. (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 eds. (2003) Gene Transfer and Expression in Mammalian Cells;Mayer and Walker eds. Press, London); and Herzenberg et al. eds (1996) See Weir's Handbook of Experimental Immunology.

[0074] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or by a variation of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always expressly indicated, that all numerical designations are preceded by the term "about." It is also to be understood, although not always expressly indicated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0075] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a polypeptide" includes multiple polypeptides, including mixtures thereof.

[0076] 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, means excluding any other elements that are essential to the combination for its intended use. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods, and pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, and the like. "Consisting of" means excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0077] The term "biofilm" refers to an organized community of microorganisms that may attach to structural surfaces, which may be organic or inorganic, along with macromolecules such as DNA secreted and / or released by the microorganisms. Biofilms are highly resistant to microbiotics and antimicrobial agents. They survive on gum tissue, teeth, and restorations, causing dental caries and periodontal disease, also known as periodontal plaque disease. They also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter systems, and contact lenses. They grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The Centers for Disease Control estimates that more than 65% of nosocomial (hospital-acquired) infections are caused by biofilms. They cause chronic vaginal infections and life-threatening systemic infections in people with compromised immune systems. Biofilms are also involved in many diseases. For example, cystic fibrosis patients often have Pseudomonas infections, which result in antibiotic-resistant biofilms.

[0078] The term "inhibiting, competing or titrating" intends the reduction in the formation of the DNA / protein matrix that is a component of a microbial biofilm.

[0079] "DNABII polypeptide or protein" refers to a DNA-binding protein or polypeptide that consists of a DNA-binding domain and therefore has specific or general affinity for microbial DNA. In one embodiment, they bind to the minor groove of DNA. Non-limiting examples of DNABII proteins are integration host factor (IHF) protein and histone-like protein from E. coli strain U93 (HU). Other DNA-binding proteins that may be associated with biofilms 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).

[0080] "Integration host factors," or "IHF" proteins, are bacterial proteins used by bacteriophages to integrate their DNA into host bacteria. They also bind to extracellular microbial DNA. The genes encoding IHF protein subunits in E. coli are the himA (GenBank Accession No. POA6X7.1) and himD (POA6Y1.1) genes. Homologs of these genes have been found in other organisms, and peptides corresponding to these genes from other organisms are disclosed in the art, for example, in Table 10 of U.S. Pat. No. 8,999,291.

[0081] "HMGB1" is a high mobility group box (HMGB) 1 protein that has been reported to bind to and distort the minor groove of DNA and is an example of an agent. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova. HMGB1 is a small 215 amino acid protein (approximately 30 Kda) consisting of three domains: two positively charged domains, A and B boxes, each consisting of 80 amino acids, and a negatively charged carboxyl-terminal acidic C tail consisting of approximately 30 consecutive aspartic acid and glutamic acid residues. A non-limiting example of the polypeptide sequence of wild-type HMGB1 is shown below. [ka] The bolded amino acids (amino acids 1-70) represent the A-box domain. The italicized amino acids (amino acids 88 to 164) represent the B-box domain. The underlined amino acids (amino acids 186-215) represent the C-tail domain. These are non-limiting examples of fragments, such as the A-box domain, the B-box domain, the A and B-box domains (AB-box domain), the C-tail domain, and the N-domain (amino acids 1-185). In one embodiment, the fragment consists essentially of a polypeptide comprising the C-terminal domain, or the B-box domain.

[0082] "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 to 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 against the HU protein are commercially available from Abeam.

[0083] The term "surface antigen" or "surface protein" refers to a protein or peptide on the surface of a cell, for example, a bacterial cell. Examples of surface antigens are outer membrane proteins, such as OMP P5 (GenBank accession number YP_004139079.1), OMP P2 (GenBank accession number ZZX87199.1), OMP P26 (GenBank accession number YP_665091.1), rsPilA or recombinant soluble PilA (GenBank accession number EFU96734.1), and type IV Pilin (GenBank accession number Yp_003864351.1).

[0084] The term "Haemophilus influenzae" refers to a pathogenic bacterium that can cause many different infections, such as ear infections, eye infections, and sinusitis. Many different strains of Haemophilus influenzae have been isolated and contain the IhfA gene or protein. Some non-limiting examples of different strains of Haemophilus influenzae include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0085] By "microbial DNA" is intended single-stranded or double-stranded DNA from biofilm-producing microorganisms.

[0086] By "inhibiting, preventing or disrupting" a biofilm is intended a prophylactic or therapeutic reduction in biofilm structure.

[0087] By "bent polynucleotide" is intended a double-stranded polynucleotide containing a small loop in one strand that does not pair with the other strand. In some embodiments, the loop is 1 to about 20 bases in length, or alternatively 2 to about 15 bases in length, or alternatively about 3 to about 12 bases in length, or alternatively about 4 to about 10 bases in length, or alternatively has about 4, 5, 6, 7, 8, 9, or 10 bases.

[0088] A "subject" of diagnosis or treatment is a cell or an animal, e.g., a mammal or a human. Non-human animals for diagnosis or treatment include non-human animals or animal models subject to infection, such as primates, murines, e.g., rats, mice, chinchillas, canines, e.g., dogs, lagomorphs, e.g., rabbits, livestock, sport animals, and pets. The terms "subject," "host," "individual," and "patient," when used interchangeably herein, refer to an animal, typically a mammal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), livestock animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal may be of any age or at any stage of development (e.g., an adult, adolescent, child, juvenile, or in utero mammal). The mammal may be male or female. In some embodiments, the subject is a human.

[0089] The terms "protein," "peptide," and "polypeptide" are used interchangeably and in their broadest sense to 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 alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids that may make up a protein's or peptide's sequence. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acids, amino acid analogs, and peptidomimetics, including glycine and both D and L optical isomers.

[0090] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment disclosed herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0091] A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) instead of thymine. Thus, the term "polynucleotide sequence" is an alphabetic representation of a polynucleotide molecule. This alphabetic representation can be input into a database on a computer with a central processing unit and used in bioinformatics applications, such as functional genomics and homology searching.

[0092] The terms "isolated" or "recombinant," as used herein with respect to nucleic acids, e.g., DNA or RNA, refer to molecules separated from other DNAs or RNAs and polypeptides, respectively, present in the natural source of the polymer. The term "isolated or recombinant nucleic acid" is intended to include nucleic acid fragments not naturally occurring as fragments, not found in the natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins isolated from other cellular proteins and is intended to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" refers to being separated from cellular components and other cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragment(s) thereof, with which it is otherwise normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a dissimilar phenotype or genotype. An isolated polynucleotide is separated from the 3' and 5' flanking nucleotides with which it is normally associated in its native or natural environment, e.g., on a chromosome. As will be apparent to one 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.

[0093] Where the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be assumed that equivalents or biological equivalents of such are intended within the scope of the present disclosure, without explicit recitation and unless otherwise intended. As used herein, the term "biological equivalent thereof" when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid is intended to be synonymous with "equivalent thereof," and is intended to be synonymous with "equivalent thereof," and to have minimal homology while still maintaining the desired structure or functionality. Unless otherwise recited herein, any polynucleotide, polypeptide, or protein listed herein is also intended to include its equivalent. In one aspect, an equivalent polynucleotide is a polynucleotide that hybridizes under stringent conditions to the polynucleotide or complement of the polynucleotide described herein for use in the described method. In another embodiment, an equivalent antibody or antigen-binding polypeptide contemplates an antibody or antigen-binding polypeptide that binds to a reference antibody or antigen-binding fragment with at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% affinity or higher. In another embodiment, the equivalent competes with the binding of the antibody or antigen-binding fragment to its antigen in a competitive ELISA assay. In another embodiment, equivalent contemplates at least 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.

[0094] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 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. Alignment and percent homology or sequence identity can be determined using software programs known in the art, such as the software programs described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for the alignment. A non-limiting exemplary alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: Genetic coding = standard; Filter = none; Strand = both; Cutoff = 60; Prediction = 10; Matrix = BLOSUM62; Description = 50 sequences; Classification = high score; Database = non-redundant GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity were determined by incorporating them into clustalW (available at the web address: align.genome.jp (last accessed March 7, 2011)).

[0095] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The 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 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0096] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0097] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can 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.

[0098] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C, a hybridization buffer concentration of about 6xSSC to about 10xSSC, a formamide concentration of about 0% to about 25%, and a wash solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C, a buffer concentration of about 9xSSC to about 2xSSC, a formamide concentration of about 30% to about 50%, and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C, a buffer concentration of about 1xSSC to about 0.1xSSC, a formamide concentration of about 55% to about 75%, and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps, with wash incubation times of about 1, 2, or 15 minutes. SSC is a 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0099] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0100] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when, in its native state, or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA and the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0101] As used herein, the terms "treat," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be therapeutic in terms of a partial or complete cure of the disorder and / or adverse effects resulting from the disorder. As used herein, "treating" or "treatment" of a disease in a subject can also refer to (1) preventing symptoms or disease from occurring in a subject who is susceptible to the disease or who does not yet exhibit symptoms of the disease; (2) inhibiting or arresting the development of the disease; or (3) ameliorating or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For the purpose of the present technology, beneficial or desired results may include, but are not limited to, one or more of: alleviation or improvement of one or more symptoms; attenuation of the severity of a condition (including a disease); stabilization (i.e., non-worsening) of a condition (including a disease); delay or slowing of a condition (including a disease); progress, improvement or alleviation of a condition (including a disease), condition and remission (whether partial or complete), whether detectable or undetectable.In one embodiment, treatment excludes prevention.When the disease is SLE (systemic lupus erythematosus) and / or cystic fibrosis (CF), evidence of treatment includes evidence of inflammation and / or reduction in the level of autoimmune activity or symptoms.

[0102] By prevent is intended preventing a disorder or effect in vitro or in vivo in a system or subject susceptible to the disorder or effect, one example of which is preventing biofilm formation in a system infected with a microorganism known to produce biofilms.

[0103] A "composition" is intended to mean a combination of an active agent with another compound or composition, inert (e.g., a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc., and includes a pharmaceutically acceptable carrier. Carriers also include pharmaceutical excipients and additives, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-oligosaccharides, and oligosaccharides; derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, including 1-99.99% by weight or volume of any combination. Exemplary protein excipients include serum albumins, e.g., human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / antibody components that may also function in a buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also contemplated within the scope of this technology, examples of which include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol.

[0104] A "pharmaceutical composition" is intended to include a combination of an active agent with a carrier, inert or active, that makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0105] "Pharmaceutically acceptable carrier" refers to any diluent, excipient or carrier that can be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphate, 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, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, 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 textbook in this field. They may be selected with regard to the intended dosage form, ie, oral tablet, capsule, elixir, syrup, etc., and consistent with conventional pharmaceutical practice.

[0106] Compositions used in accordance with the present disclosure may be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of composition calculated to produce the desired response in association with its administration, i.e., an appropriate route and regimen. The amount to be administered, both by number of treatments and unit dose, depends on the desired outcome and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors affecting dosage include the subject's physical and clinical condition, the route of administration, the intended purpose of treatment (relief of symptoms versus cure), and the efficacy, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the administration formulation and in an amount that is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, for example, the types of injectable solutions described herein.

[0107] The term "contacting" refers to a direct or indirect binding or interaction between two or more entities. A specific example of a direct interaction is binding. A specific example of an indirect interaction is when one entity acts on an intermediate molecule, which in turn acts on a second, referenced entity. Contacting, as used herein, includes contacting in solution, in solid phase, in vitro, ex vivo, in a cell, and in vivo. In vivo contacting can be referred to as administering or administration.

[0108] By "bioactive agent" or active agent as disclosed herein is intended one or more of an isolated or recombinant polypeptide, an isolated or recombinant polynucleotide, a vector, an isolated host cell, or an antibody, and compositions comprising one or more of these.

[0109] "Administration" can be achieved in one dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be performed at dose levels and patterns selected by the treating physician. Suitable administration formulations and methods for administering the active substance are known in the art. In addition, the route of administration can be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of treatment, the health condition or disease stage of the subject being treated, and the target cells or tissues being treated. Non-limiting examples of administration routes include oral administration, nasal administration, injection, and topical application.

[0110] The agents of the present disclosure may be administered for therapy by any suitable route of administration, it being understood that the most suitable route will vary with the condition and age of the recipient and the disease being treated.

[0111] The term "effective amount" refers to an amount sufficient to achieve a desired effect. For therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. For immunogenic compositions, in some embodiments, the effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is an amount sufficient to produce antibody production against an antigen. In some embodiments, the effective amount is the amount necessary to confer passive immunity to a subject in need thereof. For immunogenic compositions, in some embodiments, the effective amount depends on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system, in addition to the factors described above. Those skilled in the art will be able to determine the appropriate amount depending on these and other factors.

[0112] In the case of intravital application, in some embodiments, the effective amount depends on the size and nature of the application in question. It also depends on the nature and sensitivity of the intravital target and the method of use. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount may include one or more administrations of the composition depending on the embodiment.

[0113] A "peptide conjugate" refers to the association of one or more polypeptides with another chemical or biological compound, either covalently or non-covalently. In a non-limiting example, the "conjugation" of a polypeptide with a chemical compound results in improved stability or efficacy of the polypeptide for its intended purpose. In one embodiment, the peptide is conjugated to a carrier, and the carrier is a liposome, a micelle, or a pharmaceutically acceptable polymer.

[0114] "Liposomes" are microscopic vesicles composed of concentric lipid bilayers. Structurally, liposomes range in size and shape from elongated tubes to spheres, with dimensions ranging from hundreds of angstroms to fractions of a millimeter. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity in the final complex, providing the lipid composition of the outer layer. They can be neutral (cholesterol) or bipolar and include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), as well as other types of bipolar lipids, including, but not limited to, dioleoylphosphatidylethanolamine (DOPE), with hydrocarbon chain lengths ranging from 14 to 22 and either saturated or containing one or more double C=C bonds.Examples of lipids that can form stable liposomes, alone or in combination with other lipid components, include phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan-olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), and the like. These are palmitoyloteoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1-carboxylate (DOPE-mal). Additional non-phosphorus-containing lipids that can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfates, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids listed above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol).Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioteoylphosphatidylglycerol (DOPG), and dicetyl phosphate, which can form vesicles. Typically, liposomes can be divided into three categories based on their overall size and the nature of their lamellar structure. The three classifications developed by the December 1977 New York Academy of Sciences conference, "Liposomes and Their Use in Biology and Medicine," are multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar vesicles (LUVs). Bioactive agents can be encapsulated in such vesicles for administration according to the methods described herein.

[0115] "Micelles" are aggregates of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solution form aggregates with their hydrophilic "head" regions in contact with the surrounding solvent and their hydrophobic tail regions sequestered in the center of the micelle. This type of micelle is known as a normal-phase micelle (oil-in-water micelle). Reverse micelles have head groups in the center and tails pointing outward (water-in-oil micelle). Micelles can be used to bind polynucleotides, polypeptides, antibodies, or compositions described herein to facilitate efficient delivery to target cells or tissues.

[0116] The phrase "pharmaceutically acceptable polymer" refers to a group of compounds that can be conjugated to one or more polypeptides described herein. It is contemplated that conjugation of a polymer to a polypeptide can extend the half-life of the polypeptide in vivo and in vitro. Non-limiting examples include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols, and mixtures thereof. Bioactive agents can be conjugated to pharmaceutically acceptable polymers for administration according to the methods described herein.

[0117] " Gene delivery vehicle " is defined as any molecule that can carry inserted polynucleotide into host cell. Examples of gene delivery vehicles include liposomes, micelles, biocompatible polymers including natural and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial virus envelopes, metal particles, and bacteria or viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art, which have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy and simple protein expression.

[0118] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery," "gene transfer," "transduction," and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for transfer. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection or various other protein-based or lipid-based gene delivery complexes) and techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used to introduce polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contain a replication origin compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, several vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0119] As used herein, the term "eDNA" refers to extracellular DNA found as a component of pathogenic biofilms.

[0120] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of it. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically provide a selective advantage under given environmental conditions. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the proteins produced may act as toxins under similar conditions.

[0121] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which also contains a gene that makes the cell resistant to a particular antibiotic and a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites and allowing easy insertion of DNA fragments at this location. Another major use of plasmids is to produce large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces the protein that confers its antibiotic resistance, it can also be induced to produce large amounts of the protein from the inserted gene. This is an inexpensive and easy way to mass-produce a gene or the protein it encodes.

[0122] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100 kb, up to 3000 kb). It is an artificially constructed chromosome that contains telomere, centromere, and origin of replication sequences required for replication and storage in yeast cells. Once assembled using initial circular plasmids, they can be linearized using restriction enzymes, and then DNA ligase can be used to add sequences or genes of interest into the linear molecule using the cohesive ends. Yeast expression vectors, such as YACs, YIp (yeast integrating plasmid), and YEp (yeast episomal plasmid), are very useful because yeast is itself a eukaryotic cell, allowing for the production of eukaryotic protein products with post-translational modifications. However, YACs are known to be more unstable than BACs and produce chimeric effects.

[0123] A "viral vector" is defined as a recombinant virus or virus particle containing a polynucleotide to be delivered to a host cell, either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and alphavirus vectors. Infectious tobacco mosaic virus (TMV)-based vectors are sometimes used to manufacture proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In embodiments where gene transfer is mediated by a retroviral vector, vector construct refers to a polynucleotide comprising the retroviral genome or portion thereof and a therapeutic gene.

[0124] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process by which a gene or nucleic acid sequence is stably transferred into a host cell by a virus that enters the cell and integrates the viral genome into the host cell genome. The virus may enter the host cell through its normal infection mechanism, or the virus may be modified to bind to a different host cell surface receptor or ligand and enter the cell. As used herein, a retroviral vector refers to a viral particle that can introduce exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

[0125] Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse transcribed into a DNA form that integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0126] In embodiments in which gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or an adeno-associated virus (AAV), the term vector construct refers to a polynucleotide comprising the viral genome or a portion thereof and a transgene. Adenoviruses (Ad) are a relatively well-characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., PCT International Application Publication No. WO95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those with reduced potential for recombination and wild-type virus generation. See, e.g., PCT International Application Publication Nos. WO95 / 00655 and WO95 / 11984. Wild-type AAV has high infectivity and specificity for integration into the host cell genome. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0127] Vectors containing both a promoter and a cloning site to which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from suppliers such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate extra, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to improve expression.

[0128] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can also be used in the methods disclosed herein. In addition to delivery of polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be achieved by, but is not limited to, protein transfection techniques, or culture conditions that allow for enhanced expression and / or activity of the proteins disclosed herein, among other techniques.

[0129] As used herein, the terms "antibody," "antibodies," and "immunoglobulin" include whole antibodies and any antigen-binding fragments or single chains thereof. Accordingly, the term "antibody" includes any protein- or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule. The terms "antibody," "antibodies," and "immunoglobulin" also include immunoglobulins of any isotype; fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappabodies; antibody fragments and multispecific antibody fragments formed from one or more isolated fragments. Examples of such antibodies include, but are not limited to, the complementarity determining regions (CDRs) of heavy or light chains or their ligand-binding portions, heavy or light chain variable regions, heavy or light chain constant regions, framework (FR) regions, or any portion thereof, at least a portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissue. The term "anti-" when used before a protein name, e.g., anti-DNABII, anti-IHF, anti-HU, anti-OMP P5, refers to a monoclonal or polyclonal antibody that exhibits binding to and / or affinity for a specific protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody may have affinity for or bind to proteins other than the protein against which it was raised, for example, anti-IHF was raised specifically against the IHF protein but may also bind to other proteins related through sequence homology or through structural homology.

[0130] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific) and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, for example, murines, rats, sheep and canines.

[0131] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. Because each monoclonal antibody is directed against a single determinant on the antigen, monoclonal antibodies are highly specific. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. Antibodies can be further conjugated to other moieties, such as members of specific binding pairs, for example, biotin (a member of the biotin-avidin specific binding pair), etc. Antibodies can also be bound to solid supports, including, but not limited to, polystyrene plates or beads.

[0132] Monoclonal antibodies can be produced using hybridoma technology or recombinant DNA methods known in the art. Hybridomas are cells produced in a laboratory from the fusion of antibody-producing lymphocytes with non-antibody-producing cancer cells, usually myelomas or lymphomas. The hybridomas grow and produce continuous samples of specific monoclonal antibodies. Alternative techniques for producing or selecting antibodies include in vitro exposure of lymphocytes to the antigen of interest and screening antibody display libraries in cell, phage, or similar systems.

[0133] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to an antibody in which substantially any portion of the protein (e.g., CDRs, framework, C L , C H Domain (e.g., C H1 , C H2 , C H3 ), hinge, (VL, VH)) also refer to antibodies that are substantially non-immunogenic in humans and involve only minor sequence changes or variations. Similarly, primate (monkey, baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig, hamster, etc.), and other mammalian designations of antibodies refer to such species-, subgenus-, genus-, subfamily-, and family-specific antibodies. Furthermore, chimeric antibodies include any combination of the above. Such changes or variations optionally retain or reduce immunogenicity in humans or other species compared to unmodified antibodies. Thus, human antibodies are distinct from chimeric or humanized antibodies. It is noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Furthermore, when a human antibody is a single-chain antibody, it may contain a linker peptide not found in native human antibodies. For example, an Fv may include a linker peptide, e.g., two to about eight glycine or other amino acid residues, which connects the heavy chain variable region and the light chain variable region, and such a linker peptide may be of human origin.

[0134] As used herein, a human antibody is "derived" from a particular germline sequence if the antibody is obtained from a system that uses human immunoglobulin sequences, for example, by immunizing a transgenic mouse harboring human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody "derived" from a human germline immunoglobulin sequence can itself be identified by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin. Typically, the selected human antibody is at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as a human antibody when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a murine germline sequence). In certain cases, the human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence displays no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0135] "Human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The term also contemplates recombinant human antibodies. Methods for producing these antibodies are described herein.

[0136] The term "recombinant human antibody," as used herein, includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, e.g., antibodies isolated from animals (e.g., mice) transgenic or transchromosomic for human immunoglobulin genes or hybridomas prepared therefrom; antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas; antibodies isolated from recombinant combinatorial human antibody libraries; and antibodies prepared, expressed, generated, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibody, while derived from and related to human germline VH and VL sequences, may not naturally occur within the human antibody germline repertoire in vivo. Methods for producing these antibodies are described herein.

[0137] As used herein, a chimeric antibody is an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.

[0138] As used herein, the term "humanized antibody" or "humanized immunoglobulin" refers to a human / non-human chimeric antibody containing minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's variable region are replaced by residues from a variable region (donor antibody) of a non-human species, such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. Humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. Humanized antibodies may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin; a non-human antibody containing one or more amino acids in the framework region, constant region, or CDR substituted with correspondingly positioned amino acids from a human antibody. Generally, humanized antibodies are expected to elicit a reduced immune response in a human host compared to a non-humanized version of the same antibody. Humanized antibodies may have conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions. Conservative substitution classes include glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine.

[0139] The terms "polyclonal antibody" or "polyclonal antibody composition," as used herein, refer to a preparation of antibodies derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a particular antigen, each recognizing a different epitope.

[0140] As used herein, the term "antibody derivative" includes a full-length antibody or a fragment of an antibody in which one or more of the amino acids have been chemically modified, for example, by alkylation, pegylation, acylation, ester formation, or amide formation, to link the antibody to a second molecule, including, but not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof.

[0141] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition, e.g., an N-terminal histidine tag (N-His), a magnetically rotatory isotope, e.g., a nucleotide sequence, which is directly or indirectly conjugated to a composition to be detected to produce a "labeled" composition. 115 Sn, 117 Sn and 119 Sn, non-radioactive isotopes, e.g. 13 C and 15The term also contemplates a polynucleotide or protein, such as an antibody. The term also encompasses sequences conjugated to a polynucleotide that provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP). Labels may be detectable by themselves (e.g., radioisotope labels or fluorescent labels) or, in the case of enzymatic labels, may catalyze a chemical change in a detectable substrate compound or composition. Labels may be suitable for small-scale detection or may be more suitable for high-throughput screening. Suitable labels thus include, but are not limited to, magnetically rotatory isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels may be simply detected, and labels may be quantified. A simply detected response generally includes a response whose presence is simply confirmed, while a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can be generated directly using a luminophore or fluorophore associated with the assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. The detectable luminescent response generally involves a change in, or the generation of, a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0142] As used herein, the term "immunoconjugate" includes an antibody or antibody derivative associated with or linked to a second agent, e.g., a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semi-synthetic antibody, or a multispecific antibody.

[0143] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).

[0144] In another embodiment, the fluorescent label is functionalized to facilitate covalent binding to cellular components present in cells or tissues, or on the cell or tissue surface, such as cell surface markers.Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to bind the fluorescent label to a second molecule.The choice of functional group of the fluorescent label depends on the binding site to either a linker, an agent, a marker, or a second labeling agent.

[0145] "Eukaryotic cells" include all kingdoms of life except the kingdom Monera. They can be easily distinguished by a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes, or organisms in which the cell is organized into a complex structure by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless otherwise recited, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include primates, bovines, porcines, murines, rats, birds, reptiles, and humans.

[0146] "Prokaryotic cells" typically lack a nucleus or any other membrane-bound organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells may also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, E. coli, and Salmonella.

[0147] A "native" or "natural" antigen is an epitope-containing polypeptide, protein, or fragment that is isolated from a natural biological source and is capable of specifically binding to an antigen receptor, particularly a T cell antigen receptor (TCR), in a subject.

[0148] The terms "antigen" and "antigenic" refer to a molecule capable of being recognized by an antibody or otherwise acting as a member of an antibody-ligand pair. "Specific binding" refers to the interaction of an antigen with the variable regions of immunoglobulin heavy and light chains. Antibody-antigen binding can occur in vivo or in vitro. Those skilled in the art will understand that macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides, have the potential to act as antigens. Those skilled in the art will further understand that nucleic acids encoding proteins with the potential to act as antibody ligands necessarily encode antigens. Those skilled in the art will further understand that antigens are not limited to full-length molecules but can also include partial molecules. The term "antigenic" is an adjective that refers to a molecule having the properties of an antigen. This term encompasses substances that are immunogenic, i.e., immunogens, and substances that induce immunological unresponsiveness or anergy, i.e., anergens.

[0149] An "altered antigen" is an antigen having a primary sequence different from that of the corresponding wild-type antigen. Altered antigens can be produced by synthetic or recombinant methods and include, but are not limited to, antigenic peptides that are differentially modified during or after translation, for example, by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, or linkage to antibody molecules, membrane molecules, or other ligands. (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320) The synthetic or altered antigens disclosed herein are intended to bind to the same TCR as the natural epitope.

[0150] "Immune response" broadly refers to an antigen-specific response of lymphocytes to a foreign substance. The terms "immunogen" and "immunogenic" refer to a molecule capable of inducing an immune response. All immunogens are antigens, however, not all antigens are immunogenic. The immune responses disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response can occur in vivo or in vitro. Those skilled in the art will understand that various macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides, have the potential to be immunogenic. Those skilled in the art will further understand that a nucleic acid encoding a molecule capable of inducing an immune response necessarily encodes an immunogen. Those skilled in the art will further understand that an immunogen is not limited to a full-length molecule but can include a partial molecule.

[0151] The term "passive immunity" refers to the transfer of immunity from one subject to another through the transfer of antibodies. Passive immunity can occur naturally when maternal antibodies are transferred to a fetus. Passive immunity can also occur artificially when an antibody composition is administered to a non-immunized subject. Antibody donors and recipients can be human or non-human subjects. Depending on the embodiment, antibodies can be polyclonal or monoclonal, generated in vitro or in vivo, and purified, partially purified, or unpurified. In some embodiments described herein, passive immunity is provided to a subject in need thereof through the administration of an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen. In some embodiments, passive immunity is provided through the administration of an isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen.

[0152] In the context of the present disclosure, a "ligand" is a polypeptide. In one aspect, the term "ligand" as used herein refers to any molecule that binds to a specific site on another molecule. In other words, the ligand confers specificity to the protein in its reaction with immune effector cells or antibodies to the protein or DNA to the protein. In one aspect, it is the ligand site within the protein that directly combines with the complementary binding site on the immune effector cell.

[0153] As used herein, the term "inducing an immune response in a subject" is a term well understood in the art and contemplates detecting or measuring an increase of at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold or greater in the immune response to an antigen (or epitope) after introduction of the antigen (or epitope) into a subject, compared 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, the production of antigen-specific (or epitope-specific) antibodies and the production of immune cells that express molecules on their surface that specifically bind to the antigen (or epitope). Methods for determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art. For example, antigen-specific antibodies can be detected using any of a variety of immunoassays known in the art, including, but not limited to, e.g., ELISA, in which binding of antibodies in a sample to immobilized antigens (or epitopes) is detected with a detectably labeled secondary antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).

[0154] As used herein, the terms "solid phase support" or "solid support," which are used interchangeably, are not limited to a particular type of support. Rather, numerous supports are available and known to those skilled in the art. Solid phase supports include silica gel, resins, derivatized plastic films, glass beads, cotton, plastic beads, and alumina gel. As used herein, "solid support" also includes synthetic antigen-presenting matrices, cells, and liposomes. A suitable solid phase support can be selected based on the desired end use and suitability for various protocols. For example, for peptide synthesis, a solid support may refer to a resin such as polystyrene (e.g., PAM-resin available from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE® resin (available from Aminotech, Canada), polyamide resin (available from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tübingen, Germany) or polydimethylacrylamide resin (available from Milligen / Biosearch, Calif.).

[0155] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the support can be either soluble to some extent or insoluble. The support material can have virtually any possible structural configuration, so long as the attached molecule can bind to a polynucleotide, polypeptide, or antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, or polystyrene beads. Those skilled in the art will know many other suitable supports for binding antibodies or antigens, or can ascertain such by use of routine experimentation.

[0156] The term "modulating an immune response" includes inducing (increasing, eliciting) an immune response and reducing (suppressing) an immune response. An immunomodulatory method (or protocol) is a method of modulating an immune response in a subject. Modes for Carrying Out This Disclosure I. Biofilm Structure and Disease

[0157] The bacterial reservoir that sustains chronic and recurrent bacterial infections resides within biofilms, which are communities of bacteria that adhere to surfaces and, in this state, can resist clearance by the host immune system and antimicrobial drugs. Indeed, bacteria within biofilms are typically over 1,000 times more resistant to antibiotics than the same bacteria in a free-living or planktonic state. (Ceri et al. (1999) J Clin Microbiol. 37(6):1771-6) The ability of biofilm bacteria to resist clearance is largely due to their semipermeable, self-assembled matrix, or extracellular polymeric substances (EPS), which act as a physical barrier to environmental hazards and create conditions for altered physiology that restricts metabolism to enhance this resistant state. The components of the EPS are specific to each bacterium and include proteins, polysaccharides, lipids, and nucleic acids, but the nature of the EPS must be sufficiently conducive to the bacterial genus as a whole to interact productively (e.g., as metabolic partners). To this end, several recent discoveries have raised the possibility of an underlying universal EPS structure common to all eubacteria. Whitchurch and colleagues (Whitchurch et al. (2002) Science. 295(5559)) showed that extracellular DNA (eDNA) is a common EPS component and that treatment of bacteria with DNase is sufficient to prevent biofilm formation. While this result was replicated for multiple genera, the use of DNase was unable to treat biofilms existing for more than one or two days after biofilm seeding, despite the fact that eDNA is evident within biofilms throughout their life cycle. Separately, applicants previously identified DNABII proteins, a family of nucleoid-associated proteins (NAPs) common to all eubacteria, as essential components of the eDNA-dependent EPS.Indeed, antibodies directed against the DNABII protein titrate it out of the bulk medium, thereby shifting the equilibrium of the DNABII protein from a DNA-bound to a non-bound state, which leads to the catastrophic disruption of all bacterial biofilms tested to date, including mixed-species biofilms. Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; Novotny et al. (2013) PLoS One. 8(6):e67629; Devaraj et al. (2015) Mol Microbiol. 96(6):1119-35; Rocco et al. (2017) Mol Oral Microbiol. 32(2):118-30; Gustave et al. (2013) J Cyst Fibros. 12(4):384-9; Novotny et al. (2016) EBioMedicine. 10:33-44. Importantly, unlike DNases, treatment with antibodies directed against DNABII proteins was effective at all stages of biofilm development, demonstrating that the eDNA-dependent EPS is a critical structure regardless of biofilm stage. Brockson et al. (2014) Mol Microbiol. 93(6):1246-58. Despite the recognition that eDNA and members of the DNABII family are essential components of the EPS, understanding of the complete EPS structure has proven elusive, and DNABII proteins and DNA are insufficient to recapitulate functional EPS structure in vitro. Applicants demonstrate herein that for multiple human pathogens, as biofilms mature, the eDNA-dependent EPS depends on both DNABII proteins and polyamines, resulting in a shift in eDNA from a B-DNA to a Z-DNA conformation. This latter result is particularly intriguing because it may explain the inability of DNases to disrupt mature biofilms; nucleases only cleave the more classical B-type DNA. Intracellular bacterial nucleoids

[0158] DNA in bacteria is highly structured and facilitates the regulation of all types of nucleic acid processes, including DNA replication, repair, transcription, and recombination. Unlike eukaryotic cells, bacteria lack histones. Instead, bacterial DNA is structured, in part, by a class of proteins called nucleoid proteins (NAPs). NAPs collectively bind to DNA to create functional structures. Dillon et al. (2010) Nat Rev Microbiol. 8(3):185-95. Of the numerous NAP members present across genuses, only the DNABII family is ubiquitous among all eubacteria. Dey et al. (2017) Mol Phylogenet Evol. 107:356-66. Proteins in the DNABII family function as dimers (homodimers or heterodimers, depending on the species) and include the histone-like proteins HU and IHF. HU binds weakly and nonspecifically to and bends double-stranded DNA (dsDNA), but has a significantly higher affinity for pre-bent or structured dsDNA. IHF, like HU, binds to and bends DNA with a strong preference for pre-bent / structured DNA. Unlike HU, IHF is expressed only by proteobacteria and has a preference for specific DNA consensus sequences. Swinger et al. (2004) Curr Opin Struct Biol. 14(1):28-35. Extracellular bacterial nucleoids (nucleoi)

[0159] Extracellular DNA (eDNA) has been known to have a biological role since the discovery that the "transforming principle" is a consequence of DNA. Avery et al. (1944) J Exp Med. 79(2):137-58. Indeed, eDNA is also important for the extracellular matrix (extracellular polymeric substances, EPS) of bacterial biofilms. Gunn et al. (2016) J Biol Chem. 291(24):12538-46. However, biofilm eDNA structure and the importance of that structure for eDNA function have not yet been investigated. Chronic and recurrent infections are the result of bacterial biofilms

[0160] Biofilms are further distinguished from planktonic bacteria by their cell-to-cell communication and transport systems, but their most distinctive feature is their self-produced EPS, which protects resident biofilm bacteria by both acting as a semi-permeable barrier and creating an environment for altered / slowed metabolism; in fact, biofilm bacteria are over 1,000 times more resistant to antibiotics than their planktonic counterparts. Ceri et al. (1999) J Clin Microbiol. 37(6):1771-6. Interestingly, the EPS of each bacterium is distinctive and composed of various proteins, lipids, polysaccharides, and nucleic acids. Gunn et al. (2016) J Biol Chem. 291(24):12538-46. However, while biofilms can consist of a single species, in chronic infections in general and in the environment at all times, they consist of multiple genera and therefore must be able to interact productively (e.g., coaggregation with specific metabolic partners; Stacy et al. (2016) Nat Rev Microbiol. 14(2):93-105; Wolcott et al. (2013) Clin Microbiol Infect. 19(2):107-12). This community concept implies that, despite the varying EPS composition, each EPS must be sufficiently accommodating to allow diverse bacteria to interact within the biofilm, and further suggests that biofilm EPS likely have a universal underlying structure. eDNA-dependent EPSs have universal underlying architectural qualities

[0161] Numerous groups have examined eDNA associated with bacterial biofilms from both humans and ecological genera and observed scaffold structures (Figure 1A). Jurcisek et al. (2017) Proc Natl Acad Sci US A. 114(32):E6632-E41; Sena-Velez et al. (2016) PLoS One. 11(6):e0156695; Wang et al. (2015) Environ Microbiol Rep. 7(2):330-40. Whitchurch and colleagues first showed that P. aeruginosa biofilms could be disrupted by deoxynuclease I (DNase) treatment (Whitchurch et al. (2002) Science. 295(5559)), indicating that eDNA is a key structural component of the EPS. DNases can inhibit initial biofilm formation in many genera (Frederiksen et al. (2006) Acta Paediatr. 95(9):1070-4; Martins et al. (2012) Mycoses. 55(1):80-5; Hymes et al. (2013) J Infect Dis. 207(10):1491-7)), but as biofilms mature, they become refractory to DNases over time, despite the fact that eDNA apparently persists. Goodman et al. (2011) Mucosal Immunol. 4(6):625-37;Hall-Stoodley et al. (2008) BMC Microbiol. 8:173;Izano et al. (2009) Microb Pathog. 46(4):207-13;Kaplan et al. (2012) J Antibiot (Tokyo). 65(2):73-7; Novotny et al. (2013) PLoS One. 8(6):e67629; Tetz et al. (2010) DNA Cell Biol. 29(8):399-405.While this finding has often been interpreted as meaning that eDNA is no longer important for the structural integrity of the EPS, applicants have shown that eDNA not only persists but also becomes a major underlying EPS structure. Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; Novotny et al. (2013) PLoS One. 8(6):e67629; Devaraj et al. (2015) Mol Microbiol. 96(6):1119-35; Rocco et al. (2017) Mol Oral Microbiol. 32(2):118-30; Brockson et al. (2014) Mol Microbiol. 93(6):1246-58. DNABII family proteins are linchpins maintaining the structural integrity of biofilm eDNA-scaffolded EPS

[0162] Applicants have previously shown that the ubiquitous DNABII protein, and likely not other NAPs, are structural components of eDNA (Devaraj et al. (2017) Microbiologyopen.), and that its removal disrupts eDNA structure. Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; Novotny et al. (2013) PLoS One. 8(6):e67629; Devaraj et al. (2015) Mol Microbiol. 96(6):1119-35; Rocco et al. (2017) Mol Oral Microbiol. 32(2):118-30. Indeed, DNABII proteins were found to specifically bind to the vertices (pre-bent DNA) of the eDNA scaffolds of biofilms formed in vivo, while antibodies directed against DNABII proteins were sufficient to damage the structure of the eDNA-scaffolded EPS, resulting in the catastrophic collapse of both mono- and multi-species biofilms for every species we investigated, regardless of biofilm maturity (Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; Novotny et al. (2013) PLoS One. 8(6):e67629; Devaraj et al. (2015) Mol Microbiol. 96(6):1119-35; Rocco et al. (2017) Mol Oral Microbiol. 32(2):118-30). Brockson et al. (2014) Mol Microbiol. 93(6):1246-58.This disruption releases resident bacteria into planktonic and therefore antimicrobial- and immune-susceptible states (Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; Novotny et al. (2013) PLoS One. 8(6):e67629; Brockson et al. (2014) Mol Microbiol. 93(6):1246-58), demonstrating the importance and ubiquity of this family of proteins within biofilm structure. Given the known interactions of the DNABII family with DNA, it was unexpected that applicants were unable to create conditions using only DNA and DNABII proteins to recapitulate the three-dimensional scaffold-like structure observed in bacterial biofilms. Polyamines are ubiquitous inside and outside the cell and are required for the eDNA-scaffolded EPS structure of biofilms

[0163] Polyamines are short organic molecules that are typically positively charged (basic) at neutral pH and contain numerous primary amines, generally derived from the decarboxylation of amino acids (Figure 1B). Michael et al. (2016) Biochem J. 473(15):2315-29. Polyamines are naturally occurring and ubiquitous, found at high mM concentrations both intracellularly and extracellularly (Tabor et al. (1985) Microbiol Rev. 49(1):81-99), with spermidine, spermine, and putrescine being the most abundant. While polyamines are involved in numerous processes in bacterial physiology, they are perhaps most important for eDNA-scaffolded EPS for five reasons. First, they bind to DNA, neutralizing the negative charge of the phosphate backbone and consequently altering DNA structure. Bachrach et al. (2005) Curr Protein Pept Sci. 6(6):559-66; Pasini et al. (2014) Amino Acids. 46(3):595-603. Second, polyamines tend to promote protein-DNA interactions at low concentrations (mM), whereas they tend to interfere at higher concentrations (mM). The only exception is DNABII-DNA interactions, which cause DNA to form thick fibers. Sarkar et al. (2009) Biochemistry. 48(4):667-75; Sarkar et al. (2007) Nucleic Acids Res. 35(3):951-61. Third, polyamine synthesis is known to be induced during biofilm formation, and some of these effects occur extracellularly (although to date, this has only been investigated in bacterial membranes). Wilton et al. (2015) Antimicrob Agents Chemother. 60(1):544-53. Fourth, atomic force microscopy (AFM) experiments visualizing mixtures of polyamines and DNA revealed structures highly similar to biofilm eDNA scaffolds (Figure 1C).Finally, polyamines can induce the conversion of native B-form DNA to Z-form DNA in prone sequences at physiological concentrations (100 μM). Thomas et al. (1986) Nucleic Acids Res. 14(16):6721-33; Thomas et al. (1988) J Mol Biol. 201(2):463-7. Conversion of B-DNA to Z-DNA may be a novel means to render eDNA-scaffolded EPS nuclease-resistant and create stable structural materials.

[0164] B-DNA and Z-DNA are distinct conformations of dsDNA that exist in equilibrium, with B-DNA predominating under most physiological conditions. Alternating purines and pyrimidines (especially dGdC) tend to exist as Z-DNA in high salt (molar concentrations of monovalent or divalent cations) or under negative supercoiling. Pohl et al. (1983) Cold Spring Harb Symp Quant Biol. 47 Pt 1:113-7; Pohl et al. (1986) Proc Natl Acad Sci U S A. 83(14):4983-7. In the latter case, regions that tend to form Z-DNA can be temporarily juxtaposed to B-DNA during transcription if negative supercoiling is transiently induced. Rahmouni et al. (1992) Mol Microbiol. 6(5):569-72. B-DNA bases adopt a right-handed helix (10 bp / turn), while Z-DNA forms a left-handed helix (12 bp / turn). Jovin et al. (1987) Ann Rev Phys Chem. 38:521-60. B-DNA has two grooves (major and minor grooves), and most interacting proteins recognize / bind to the major groove due to their larger size, identifying the hydrogen bond donors and acceptors of each nucleotide base. In contrast, the major groove is absent in Z-DNA, and most of their binding contacts are found on the convex surface. Jovin et al. (1987) Ann Rev Phys Chem. 38:521-60. Z-DNA possesses a single groove corresponding to the minor groove of B-DNA. Interestingly, DNABII proteins are one of only a few DNA-binding proteins that bind to the minor groove (Kim et al. (2014) Acta Crystallogr D Biol Crystallogr. 70(Pt 12):3273-89), suggesting that they may bind to Z-DNA. The shift of eDNA from B-DNA to Z-DNA is consistent with four observations of eDNA-scaffolded EPS.First, the shift to Z-DNA occurs under conditions present in biofilm EPS; the tendency shifts to Z-DNA in the presence of physiological concentrations (100 mM) of some polyamines (spermidine and spermine). Second, Z-DNA tends to aggregate and form fibers. Chaires et al. (1988) J Biomol Struct Dyn. 5(6):1187-207. Because the phosphates in the Z-DNA backbone are tightly packed together more tightly than those in B-DNA, neutralization of the strong negative charges of the phosphate backbone (e.g., polyamines) favors Z-DNA but also tolerates DNA aggregation. Third, Z-DNA is more rigid than B-DNA and has an almost three-fold increase in persistence length, consistent with the straight fibers we observed in eDNA scaffolds (Thomas et al. (1983) Nucleic Acids Res. 11(6):1919-30) (Figure 1A, Figure 2, Figure 3). Finally, Z-DNA is nuclease-resistant. Unlike standard B-DNA binding proteins, only a few known proteins recognize Z-DNA (Athanasiadis et al. (2012) Semin Cell Dev Biol. 23(3):275-80) (however, antibodies that distinguish Z-DNA from B-DNA are available). Bergen et al. (1987) J Immunol. 139(3):743-8). Interestingly, Z-DNA binding proteins are homologous, bind Z-DNA with 1,000- to 10,000-fold higher affinity than B-DNA (Herbert et al. (1996) J Biol Chem. 271(20):11595-8), induce Z-DNA conformation at target sequences, and, at least in the case of ZBP1, are part of the innate immune system that detects the presence of microbial DNA. Athanasiadis et al. (2012) Semin Cell Dev Biol. 23(3):275-80. Importantly, despite the fact that Watson-Crick base pairing is conserved, proteins (e.g., nucleases) that use B-DNA as a substrate cannot recognize and therefore function on the same DNA sequence in the Z configuration. II. The three-part approach

[0165] Applicants have previously shown that eDNA-DNABII interactions serve to maintain the structural integrity of biofilm EPS (Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; Novotny et al. (2013) PLoS One. 8(6):e67629; Devaraj et al. (2015) Mol Microbiol. 96(6):1119-35; Rocco et al. (2017) Mol Oral Microbiol. 32(2):118-30; Devaraj et al. (2017) Microbiologyopen.; Brockson et al. (2014) Mol Microbiol. 93(6):1246-58), and that disrupting these interactions results in ex vivo (Gustave et al. (2013) J Cyst Fibros. 12(4):384-9) and in vivo (Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; Novotny et al. (2016) EBioMedicine. 10:33-44; Freire et al. (2017) Mol Oral Microbiol. 32(1):74-88). While both DNA and DNABII proteins were shown to be necessary, they are not sufficient to recapitulate the EPS scaffold architecture. Disclosed herein is a tripartite eDNA-dependent scaffold (TEDS) for eDNA-DNABII-dependent EPS that relies on the presence and relative location of (1) eDNA, (2) DNABII proteins, and the newly discovered EPS component (3) polyamines.

[0166] In one aspect, Applicants show that, in addition to eDNA and DNABII proteins, polyamines are essential components of the TEDS structure of bacterial biofilms. Second, Applicants show that, together, all three of these components promote the formation of a universal EPS that can foster productive interactions among bacterial genera in a protective biofilm state. Third, using these components, Applicants define and recapitulate this universal structure, provide evidence consistent with observations of thick double-stranded DNA fibers, the induction of a nuclease-resistant state, and demonstrate whether this state requires Z-DNA as a structural endpoint. Finally, this provides diagnostic and therapeutic interventions that focus on the TEDS structure itself as a target for intervention. A. Polyamines Polyamines function in concert with DNABII proteins to direct the assembly of eDNA scaffolds

[0167] The chinchilla model of acute otitis media caused by NTHI faithfully recapitulates the human disease process and pathophysiology (Bakaletz et al. (2009) Expert Rev Vaccines. 8(8):1063-82) and relies on a refractory biofilm in the middle ear. Using this model, Applicants previously showed that DNABII protein associates with eDNA, localizes to the apex of eDNA strands (Figure 1A) (Goodman et al. (2011) Mucosal Immunol. 4(6):625-37), and that these eDNA strands appear to be strikingly similar to polyamines visualized in DNA by AFM (Figure 1C). Iacomino et al. (2011) Biomacromolecules. 12(4):1178-86. DNABII protein binds to DNA in vitro in the presence of mM spermidine (Figure 2B), which led us to investigate the potential interaction between DNABII protein and polyamines that produce biofilm eDNA scaffold structures. We performed immunofluorescence confocal laser scanning microscopy (CLSM) on sections of fixed, embedded middle ear mucosal biofilms to visualize polyamines interacting with eDNA within the biofilm EPS, as we previously observed for DNABII protein. Immunofluorescence imaging shows that eDNA and polyamines colocalize along fibers present within mucosal biofilms (Figure 2A), visually similar to what Hud and colleagues observed for DNABII proteins and polyamines in vitro (Sarkar et al. (2009) Biochemistry. 48(4):667-75; Sarkar et al. (2007) Nucleic Acids Res. 35(3):951 61) (Figure 2B). Polyamine biosynthesis is required for biofilm structure

[0168] Applicants investigated whether dicyclohexylamine (DCHA), a broad-spectrum polyamine biosynthesis inhibitor, could alter NTHI biofilm biogenesis in vitro. DCHA inhibits spermidine synthase, the enzyme that catalyzes the conversion of putrescine to spermidine (Paulin et al. (1986) Antonie Van Leeuwenhoek. 52(6):483-90; Pegg et al. (1983) FEBS Lett. 155(2):192-6). Although DCHA did not affect NTHI growth (data not shown), it inhibited biofilm biogenesis in vitro, reducing average thickness and biomass, as determined by COMSTAT analysis of CLSM images of LIVE / DEAD®-stained NTHI biofilms (Heydorn et al. (2000) Microbiology. 146 (Pt 10):2395-407) (Figure 3A). Furthermore, we found that DCHA inhibited the production of eDNA scaffolds during early biofilm development (Figure 3B), suggesting that polyamine biosynthesis is a necessary step for biofilm formation. In support of these findings, we observed by immunofluorescence that DCHA reduced the incorporation of extracellular polyamines into biofilm EPS (Figure 3C) and that the biofilm biogenesis defect could be compensated for by the addition of exogenous spermidine (Figure 3A). Together, these results reveal that the incorporation of polyamines into EPS is important for the development and function of the eDNA scaffold in supporting robust biofilm growth. Anti-DNABII disrupts DNABII-polyamine-dependent DNA structure

[0169] We used immunofluorescence to determine whether DNABII protein is incorporated into EPS mimetic structures. Spermidine (300 μM) and HU (1 μM) were incubated with genomic DNA (2 μg / ml). The EPS mimetic structures were then probed with either naive (control) or anti-DNABII IgG, a fluorescent secondary antibody, stained with DAPI, and imaged by CLSM. DNABII protein was fully incorporated into the EPS structures (Figure 4A). EPS mimetic structures were then allowed to form, treated with anti-DNABII antibody, stained with DAPI, and imaged by CLSM. Sequestration of DNABII protein from the EPS mimetic by anti-DNABII antibody dramatically reduced the abundance and size of aggregated structures (Figure 4B), further confirming DNABII incorporation and its importance for DNA structural stability. NTHI biofilm disruption by the cation exchanger P11 can be prevented by the addition of exogenous DNABII (HU) and spermidine

[0170] Phosphocellulose (P11) is a negatively charged resin with a high affinity for positively charged molecules, such as polyamines and DNABII proteins. To determine the effect of P11 sequestration of these molecules on bacterial biofilm formation, we utilized a transwell system. NTHI growth was initiated in the basolateral chamber, while P11 (1% w / v) was added to the apical chamber at the time of seeding. At 16 hours, biofilms were washed, stained with LIVE / DEAD®, imaged using CLSM, and analyzed by COMSTAT. P11 significantly reduced average thickness and biomass (Figure 5). To determine whether cation depletion by P11 involved polyamines and / or DNABII proteins, NTHI growth was initiated in the basolateral chamber with the exogenous addition of 1 mM spermidine and 1 μM HU, while P11 was added to the apical chamber at 1% w / v. Both spermidine and HU are required for the structural components of the biofilm matrix and only when present together prevented biofilm disruption by P11 (Fig. 5); individually, HU and spermidine were insufficient (data not shown). DNases are unable to disrupt mature pathogenic bacterial biofilms

[0171] Applicants evaluated the anti-biofilm effects of Pulmozyme®, a recombinant human DNase used in conjunction with standard therapy for the management of cystic fibrosis (CF) patients to improve lung function. Yang et al. (2017) Paediatr Respir Rev. 21:65-7. Pulmozyme® was added either at the time of seeding (biofilm prevention) or to preformed biofilms (biofilm disruption) (Figure 6). The resulting biofilms were stained with LIVE / DEAD® and evaluated using CLSM and COMSTAT analysis. Addition of DNase at the time of seeding resulted in a significant reduction in the average thickness and biomass of NTHI and UPEC biofilms compared to untreated biofilms (Figure 6). However, DNase was unable to disrupt mature biofilms (Figure 6), suggesting that eDNA loses susceptibility to nuclease digestion as biofilms develop. Applicants propose that as the biofilm matures, the eDNA either becomes sterically protected from nucleases and / or the eDNA adopts a novel structure that makes it refractory to nuclease digestion. DNABII proteins and polyamines interact synergistically to confer nuclease resistance to DNA

[0172] Immunofluorescence of NTHI biofilms probed with anti-DNABII and anti-spermidine antibodies showed that polyamines colocalized with DNABII protein in vitro (Figure 7A) and in vivo (Figure 7B). Applicants hypothesized that DNABII and polyamines interact synergistically with eDNA to confer DNase resistance to mature biofilms. To test for synergy in vitro, Applicants incubated NTHI genomic DNA (gDNA) with spermidine (10, 20, 50, or 100 μM) in the presence or absence of HU (50 or 100 nM) and DNase (0.5 units). Degradation was assessed by agarose gel electrophoresis and UV irradiation. While spermidine (above 100 μM) and HU (1 μM) individually protected gDNA, combined lower levels of spermidine (below 50 μM) and HU (below 100 nM) synergistically inhibited gDNA digestion (Figure 7), suggesting that nuclease resistance was due to the combination of DNABII proteins and polyamines within the biofilm matrix. Next, we tested whether EPS mimetic structures were resistant to DNases. HU (1 μM) and spermidine (300 mM) were incubated with gDNA (2 μg / ml) for 40 hours to create EPS scaffold mimetics, which were then treated with DNase (5 units). As shown by CLSM, these DNABII-polyamine-dependent DNA structures were resistant to DNases, similar to mature biofilms (Figure 7D). B. DNABII, polyamines, and DNA form Z-DNA in vitro

[0173] Upon binding, polyamines shift the Z-DNA orientation from the BZ equilibrium to the Z-DNA configuration (Thomas et al. (1986) Nucleic Acids Res. 14(16):6721-33; Thomas et al. (1988) J Mol Biol. 201(2):463-7), while DNABII proteins bend and condense DNA. Due to the synergistic inhibition by HU and spermidine binding in our in vitro DNase degradation assay (Figure 7) and the ability of Z-DNA to resist DNase, we hypothesized that EPS mimetic structures might contain Z-DNA. EPS structures were allowed to form with gDNA for 16 hours, as in Figures 4 and 7. Immunofluorescence CLSM was performed, probing with an anti-Z-DNA antibody, which confirmed the presence of Z-DNA (note the white area; Figure 8A). To determine whether DNABII proteins affect the BZ equilibrium, we used circular dichroism (CD). Mixtures of HU and poly(dGdC) DNA substrates exhibit an inversion of the B-DNA ellipticity peaks (250 and 280 nm), similar to the characteristic Z-DNA spectrum (Jang et al. (2015) Sci Rep. 5:9943) (Figure 8B). EPS in bacterial biofilms contains Z-DNA and polyamines

[0174] To further characterize the association of Z-DNA with polyamines, the applicants performed immunofluorescence on 40-hour biofilms. When biofilms of the bacterial pathogens shown were probed with anti-DNABII and anti-spermidine, or anti-Z-DNA antibodies and then imaged by CLSM; Z-DNA was detected within the biofilm EPS of each of the bacterial pathogens (Figure 9), while there was clearly an interspecies hierarchy (UPEC = Staphylococcus epidermidis < K. pneumoniae < NTHI). In all cases, extensive co-localization between DNABII (HU) and spermidine was present (note in white), and corresponded to the abundance of Z-DNA. The applicants further examined NTHI and UPEC biofilms at various stages of biofilm formation (24, 40, and 90 hours) and observed that spermidine and Z-DNA increased together within the biofilm EPS with biofilm maturation (Figure 10). These data suggest that Z-DNA and spermidine are indeed essential parts of the biofilm EPS and appear to contribute to its structure and DNase-resistant state. HU-deficient NTHI is unable to form native biofilms, incorporate polyamines, and induce a shift from B-DNA to Z-DNA

[0175] Since polyamines co-localize with HU within NTHI biofilm EPS in vitro (Figure 7A and Figure 9) and in vivo (Figure 7B), the applicants evaluated the role of HU in the incorporation of polyamines and Z-DNA within NTHI biofilm EPS. By immunofluorescence, HU-deficient NTHI (hupA-deficient; ΔHU) was compared to WT NTHI biofilms for the presence of polyamines and Z-DNA. Deficiency of HU resulted in a significant reduction of polyamines and Z-DNA within the biofilm EPS compared to WT (Figure 11), suggesting that HU is required for the presence of polyamines and Z-DNA within NTHI biofilm EPS. III. Diagnostic and treatment methods

[0176] Provided herein is a method for treating a biofilm in a subject, the method comprising, or alternatively consisting essentially of, or alternatively consisting of, administering to a subject infected with a biofilm an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm. In one embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In another embodiment, the agent is provided in the absence of DNase. In a further embodiment, DNase is administered subsequent to administration of the agent. In one particular aspect, the administered DNase is Pulmozyme. In one embodiment, the method for treating a biofilm in a subject comprises, or alternatively consisting essentially of, or alternatively consisting of, administering to a subject infected with a biofilm an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm. In one embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In another embodiment, the agent is provided in the absence of DNase. In a further embodiment, the DNase is administered subsequent to administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme. In another embodiment, a method for treating a biofilm in a subject comprises, or alternatively consists essentially of, or even further consists of, administering to a subject infected with a biofilm effective amounts of two or more agents that interfere with the binding of polyamines to DNA in the biofilm, wherein in one embodiment the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, the DNase is administered subsequent to administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme.In a further embodiment, a method for treating a biofilm in a subject comprises, or alternatively consists essentially of, or further consists of, administering to a subject infected with a biofilm an effective amount of three or more agents that interfere with the binding of polyamines to DNA in the biofilm, and in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a specific embodiment, the administered DNase is Pulmozyme. In yet a further embodiment, a method for treating a biofilm in a subject comprises, or alternatively consists essentially of, or alternatively consists of, administering to a biofilm-infected subject an effective amount of four or more, or alternatively five or more, or alternatively six or more, or alternatively seven or more, or alternatively eight or more, or alternatively nine or more, or alternatively ten or more agents that interfere with the binding of polyamines to DNA in the biofilm; in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a specific embodiment, the administered DNase is Pulmozyme.

[0177] The present disclosure also relates to a method for preventing biofilm formation in a biofilm-prone subject, comprising, alternatively consisting essentially of, or further consisting of, administering to the subject an effective amount of an agent that interferes with the binding of polyamines to DNA in biofilms; in one embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof; and in another embodiment, the agent is administered in the absence of DNase. In a further embodiment, DNase is administered subsequent to administration of the agent. In a specific embodiment, the administered DNase is Pulmozyme. In one embodiment, a method for preventing biofilm formation in a subject prone to developing biofilms comprises, or alternatively consists essentially of, or further consists of, administering to the subject an effective amount of one or more agents that interfere with the binding of polyamines to DNA in biofilms, wherein the agents are not HMGB1 protein, a fragment thereof, or an equivalent thereof; in another embodiment, the agents are administered in the absence of DNase. In a further embodiment, DNase is administered subsequent to administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme. In another embodiment, a method for preventing biofilm formation in a subject prone to developing biofilms comprises, or alternatively consists essentially of, or further consists of, administering to the subject effective amounts of two or more agents that interfere with the binding of polyamines to DNA in biofilms, wherein in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, a DNase is administered subsequent to administration of the agent. In one particular embodiment, the administered DNase is Pulmozyme.In a further embodiment, a method for preventing biofilm formation in a biofilm-prone subject comprises, or alternatively consists essentially of, or further consists of, administering to the subject an effective amount of three or more agents that interfere with the binding of polyamines to DNA in biofilms; in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme. In yet a further embodiment, a method for preventing biofilm formation in a biofilm-prone subject comprises, or alternatively consists essentially of, or alternatively consists of, administering to the subject an effective amount of four or more, or alternatively five or more, or alternatively six or more, or alternatively seven or more, or alternatively eight or more, or alternatively nine or more, or alternatively ten or more agents that interfere with the binding of polyamines to DNA in biofilms; in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme.

[0178] The present disclosure further relates to a method for treating an infection caused by biofilm-producing bacteria in a subject in need thereof, comprising, or alternatively consisting essentially of, or further consisting of, administering to the subject an effective amount of an agent that interferes with the binding of polyamines to DNA in a biofilm and an agent that inhibits the replication of the organism; in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, a method for treating an infection caused by biofilm-producing bacteria in a subject in need thereof comprises, or alternatively consisting essentially of, or further consisting of, administering to the subject an effective amount of one or more agents that interfere with the binding of polyamines to DNA in a biofilm. In another embodiment, a method for treating an infection caused by a biofilm-producing bacterium in a subject in need thereof comprises, or alternatively consists essentially of, or alternatively consists of, administering to the subject effective amounts of two or more agents that interfere with the binding of polyamines to DNA in biofilms and an agent that inhibits the replication of the organism. In one embodiment, the agent is administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a specific embodiment, the administered DNase is Pulmozyme.In a further embodiment, a method for treating an infection caused by a biofilm-producing bacterium in a subject in need thereof comprises, or alternatively consists essentially of, or further consists of, administering to the subject effective amounts of three or more agents that interfere with the binding of polyamines to DNA in a biofilm and an agent that inhibits the replication of the organism, wherein in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme. In yet a further embodiment, a method for treating an infection caused by a biofilm-producing bacterium in a subject in need thereof comprises, or alternatively consists essentially of, or further consists of, administering to the subject an effective amount of four or more, or alternatively five or more, or alternatively six or more, or alternatively seven or more, or alternatively eight or more, or alternatively nine or more, or alternatively ten or more agents that interfere with the binding of polyamines to DNA in biofilms and an agent that inhibits the replication of the organism, wherein in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme.

[0179] For any of the methods described above, the polyamine may be selected from the group consisting of putrescine, spermine, cadaverine, 1,3-diaminopropane, or spermidine. In one embodiment, for the method described above, the agent that interferes with the binding of polyamines to DNA in biofilms is tRNA. In another embodiment, the agent is an inhibitor of polyamine synthesis or an agent that inhibits the binding of polyamines to DNA. In a second embodiment, the agent comprises, alternatively consists essentially of, or even further consists of the polyamine analogs difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, dicyclohexylamine, any derivative thereof, or a salt thereof. In one aspect, derivatives of these compounds maintain the same mass-to-charge ratio. In a third embodiment, the agent comprises, alternatively consists essentially of, or further consists of an agent that depletes cations from biofilms, optionally a cation exchange resin, aminopolycarboxylic acid, crown ether, azacrown, or cryptand. In a fourth embodiment, the agent that depletes cations from biofilms is selected from the group of sulfonates, sulfopropyl, phosphocellulose, P11 phosphocellulose, heparin sulfate, or a derivative or analog thereof. In one aspect, the derivative or analog of the agent that depletes cations from biofilms is a resin with a net negative charge. In a fifth embodiment, the agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment. In a sixth embodiment, the agent comprises, alternatively consists essentially of, or further consists of an anti-B-DNA antibody, or a fragment or derivative thereof. In one aspect, the polyclonal or monoclonal anti-B-DNA antibody, or a fragment or derivative thereof, recognizes B-DNA over Z-DNA with at least 10-fold greater affinity / avidity.In a seventh embodiment, the agent comprises, alternatively consists essentially of, or even consists of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine, or a derivative thereof. In an eighth embodiment, the agent comprises, alternatively consists essentially of, or even consists of chloroquine, or a derivative thereof. In one aspect, derivatives of the compound retain the ability to intercalate between DNA bases. In one aspect, the agent is not an HGMB1 protein or a fragment thereof. In one aspect, the agent that depletes cations from a biofilm has a net negative charge. In another aspect, the agent that depletes cations from a biofilm has a net neutral charge.

[0180] Also provided herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising, alternatively consisting essentially of, or even further consisting of, administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one embodiment, the agent is administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to administration of the agent. In a specific embodiment, the administered DNase is Pulmozyme. Disclosed herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB, comprising, alternatively consisting essentially of, or further consisting of, administering an effective amount of one or more agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one embodiment, the agent is administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to administration of the agent. In a specific embodiment, the administered DNase is Pulmozyme. In one embodiment, disclosed herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB, comprising, alternatively consisting essentially of, or further consisting of, administering effective amounts of two or more agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one embodiment, the agents are administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to administration of the agents.In one particular embodiment, the administered DNase is Pulmozyme. In another embodiment, disclosed herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB, comprising, alternatively consisting essentially of, or even consisting of, administering effective amounts of three or more agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment. In a further aspect, disclosed herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB, comprising, alternatively consisting essentially of, or alternatively consisting of, administering an effective amount of four or more, or alternatively five or more, or alternatively six or more, or alternatively seven or more, or alternatively eight or more, or alternatively nine or more, or alternatively ten or more agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one aspect, the agents are administered in the absence of DNase. In another aspect, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further aspect, DNase is administered subsequent to administration of the agents. In one particular aspect, the administered DNase is Pulmozyme. In one embodiment, the agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment. In a second embodiment, the agent comprises, alternatively consists essentially of, or even consists of an anti-B-DNA antibody, or a fragment or derivative thereof. In one aspect, the polyclonal or monoclonal anti-B-DNA antibody, or a fragment or derivative thereof, recognizes B-DNA over Z-DNA with at least 10-fold greater affinity / avidity.In a third embodiment, the agent comprises, or alternatively consists essentially of, or alternatively consists of, riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine or a derivative thereof. In a fourth embodiment, the agent comprises, or alternatively consists essentially of, or alternatively consists of chloroquine or a derivative thereof. In one aspect, the compound derivative retains the ability to intercalate between DNA bases. The agent is not an HMGB1 protein or a fragment thereof.

[0181] Also provided herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB, comprising, alternatively consisting essentially of, or further consisting of, administering an effective amount of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof, wherein in one embodiment, the protein and antibody are administered in the absence of DNase. In a further embodiment, the DNase is administered subsequent to administration of the agent. In a specific embodiment, the administered DNase is Pulmozyme. In one embodiment, the polyclonal or monoclonal anti-B-DNA antibody or a fragment or derivative thereof recognizes B-type DNA over Z-type DNA with at least 10-fold greater affinity / binding activity. The biologically active fragment of HMGB1 may comprise, alternatively consisting essentially of, or further consisting of one or more of the A box, B box, and / or AB box, a C-terminal fragment, or an N-terminal fragment. In certain embodiments, the biologically active fragment of HMGB1 may comprise, or alternatively consist essentially of, or even further consist of, a B-box domain capable of binding DNA. In one aspect, a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB comprises, or alternatively consists essentially of, or even further consists of, administering an effective amount of chloroquine and an anti-B-DNA antibody, or a fragment or derivative thereof, and in one aspect, the chloroquine and antibody are administered in the absence of DNase. In another aspect, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further aspect, DNase is administered subsequent to administration of the agent. In a specific aspect, the administered DNase is Pulmozyme.The present disclosure also relates to a method for treating a biofilm-producing infection associated with the administration of chemotherapy in a patient undergoing or who has undergone platinum-based chemotherapy, the method comprising, or alternatively consisting essentially of, or further consisting of, administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment, wherein in one embodiment, the agent is administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, the method comprises, or alternatively consists essentially of, or further consisting of, administering an effective amount of one or more agents that interfere with the conversion of B-DNA to Z-DNA in a biofilm or its local environment, wherein in one embodiment, the agent is administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, a DNase is administered subsequent to administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme. In a further embodiment, the agent comprises, or alternatively consists essentially of, or even further consists of chloroquine or a derivative thereof. In yet a further embodiment, the agent comprises, or alternatively consists essentially of, or even further consists of an anti-B-DNA antibody, or a fragment or derivative thereof. In one embodiment, the polyclonal or monoclonal anti-B-DNA antibody, or a fragment or derivative thereof, recognizes B-DNA over Z-DNA with at least 10-fold greater affinity / avidity. In one embodiment, the agent comprises, alternatively consists essentially of, or even further consists of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or a derivative thereof.In one particular embodiment, derivatives of the compounds retain the ability to intercalate between DNA bases.

[0182] The present disclosure further relates to a method for treating biofilm-producing infections associated with chemotherapy in patients undergoing or who have received platinum-based chemotherapy, comprising, alternatively consisting essentially of, or further consisting of, administering an effective amount of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof, wherein in one embodiment, the protein and antibody are administered in the absence of DNase. In a further embodiment, the DNase is administered subsequent to administration of the agent. In a specific embodiment, the administered DNase is Pulmozyme. In one embodiment, the polyclonal or monoclonal anti-B-DNA antibody or a fragment or derivative thereof recognizes B-type DNA over Z-type DNA with at least 10-fold greater affinity / binding activity. The biologically active fragment of HMGB1 may comprise, alternatively consisting essentially of, or further consisting of one or more of the A box, B box, and / or AB box, a C-terminal fragment, or an N-terminal fragment. In certain embodiments, the biologically active fragment of HMGB1 may comprise, or alternatively consist essentially of, or even further consist of, a B-box domain capable of binding DNA. Also provided herein is a method for treating biofilm-producing infections associated with chemotherapy in patients undergoing or who have received platinum-based chemotherapy, comprising, or alternatively consisting essentially of, or even further consisting of, administering an effective amount of chloroquine and an anti-B-DNA antibody, or a fragment or derivative thereof, wherein in one aspect, the chloroquine and antibody are administered in the absence of DNase. In a further aspect, the DNase is administered subsequent to administration of the agent. In a specific aspect, the administered DNase is Pulmozyme. In one aspect, the polyclonal or monoclonal anti-B-DNA antibody, or a fragment or derivative thereof, recognizes B-DNA over Z-DNA with at least 10-fold greater affinity / binding activity.

[0183] The methods described above may further comprise, or alternatively consist essentially of, or even further consist of, administering to the subject an effective amount of an agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins; in one embodiment, the agent is administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to administration of the agent. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, the method may further comprise, or alternatively consist essentially of, or even further consist of, administering to the subject an effective amount of an agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins; in one embodiment, the agent is administered in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further aspect, DNase is administered subsequent to administration of the agent. In one particular aspect, the administered DNase is Pulmozyme. In another aspect, the agent that interferes with the binding of eDNA to a DNA-binding protein comprises, alternatively consists essentially of, or even consists of, one or more of an anti-DNABII antibody, an anti-IHF antibody, and / or an anti-HU antibody, or fragments of each. In one embodiment, the agent that interferes with the binding of eDNA to a DNA-binding protein has a net negative charge. In a second embodiment, the agent that interferes with the binding of eDNA to a DNA-binding protein has a net neutral charge. In a third embodiment, the agent that interferes with the binding of eDNA to a DNA-binding protein has a net positive charge.

[0184] Described herein are methods for inhibiting biofilm stability, comprising, alternatively consisting essentially of, or further consisting of, contacting a biofilm with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the agent is contacted in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is contacted following contact with the agent. In a particular embodiment, the DNase is Pulmozyme. In one embodiment, the method for inhibiting biofilm stability comprises, alternatively consisting essentially of, or further consisting of, contacting a biofilm with an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the agent is contacted in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, the DNase is contacted subsequent to contact with the agent. In a particular embodiment, the DNase is Pulmozyme. In another embodiment, a method for inhibiting biofilm stability comprises, or alternatively consists essentially of, or even consists of, contacting a biofilm with an effective amount of two or more agents that interfere with the binding of polyamines to DNA in the biofilm, and in one embodiment, the agents are contacted in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, the DNase is contacted subsequent to contact with the agent. In a particular embodiment, the DNase is Pulmozyme. In a further aspect, a method for inhibiting biofilm stability comprises, or alternatively consists essentially of, or even further consists of, contacting the biofilm with an effective amount of three or more agents that interfere with the binding of polyamines to DNA in the biofilm, and in one aspect, the agents are contacted in the absence of DNase.In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, a DNase is contacted following contact with the agent. In a particular embodiment, the DNase is Pulmozyme. In yet a further embodiment, a method for inhibiting biofilm stability comprises, or alternatively consists essentially of, or alternatively consists of, contacting a biofilm with an effective amount of four or more, or alternatively five or more, or alternatively six or more, or alternatively seven or more, or alternatively eight or more, or alternatively nine or more, or alternatively ten or more agents that interfere with the binding of polyamines to DNA in the biofilm; in one embodiment, the agents are contacted in the absence of DNase. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, the DNase is contacted subsequent to contact with the agent. In one particular embodiment, the DNase is Pulmozyme. The contacting can be in vitro or in vivo.

[0185] The present disclosure also relates to a method for inhibiting biofilm stability, comprising, alternatively, consisting essentially of, or even further consisting of, contacting a biofilm in vitro with an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises, alternatively, consisting essentially of, or even further consisting of coating a surface with an effective amount of the agent that depletes cations; in one embodiment, the agent is contacted in the absence of DNase, while in another embodiment, DNase is contacted according to the method. In another embodiment, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is contacted following contact with the agent. In a specific embodiment, the DNase is Pulmozyme. In one embodiment, a method for inhibiting biofilm stability may comprise, or alternatively essentially consist of, or alternatively further consist of, contacting a biofilm in vitro with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises, or alternatively essentially consist of, or alternatively further consist of coating a surface with an effective amount of one or more agents that deplete cations. In one embodiment, the agent is contacted in the absence of DNase, while in another embodiment, DNase is contacted according to the method. In another embodiment, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is contacted following contact with the agent. In a specific embodiment, the DNase is Pulmozyme.

[0186] In another embodiment, a method for inhibiting biofilm stability can comprise, or alternatively essentially consist of, or alternatively further consist of, contacting a biofilm in vitro with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises, or alternatively essentially consist of, or alternatively further consist of coating a surface with an effective amount of two or more agents that deplete cations. In one embodiment, the agent is contacted in the absence of DNase, while in another embodiment, DNase is contacted according to the method. In another embodiment, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is contacted following contact with the agent. In a specific embodiment, the DNase is Pulmozyme.

[0187] In a further embodiment, a method for inhibiting biofilm stability may comprise, or alternatively essentially consist of, or alternatively further consist of, contacting a biofilm in vitro with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises, or alternatively essentially consist of, or alternatively further consist of coating a surface with an effective amount of three or more agents that deplete cations. In one embodiment, the agent is contacted in the absence of DNase, while in another embodiment, DNase is contacted according to the method. In another embodiment, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is contacted following contact with the agent. In a particular embodiment, the DNase is Pulmozyme. In yet a further aspect, a method for inhibiting biofilm stability may comprise, or alternatively consist essentially of, or alternatively consist of, contacting a biofilm in vitro with an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises, or alternatively consists essentially of, or alternatively consists of coating the surface with an effective cation-depleting amount of four or more, or alternatively five or more, or alternatively six or more, or alternatively seven or more, or alternatively eight or more, or alternatively nine or more, or alternatively ten or more agents. In one embodiment, the agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, and in one aspect, the agent is contacted in the absence of DNase, while in another aspect, DNase is contacted according to the method. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof, hi a further embodiment, the DNase is contacted subsequent to contact with the agent.In a particular aspect, the DNase is Pulmozyme. In a second embodiment, the agent comprises, alternatively consists essentially of, or even further consists of an anti-B-DNA antibody, or a fragment or derivative thereof. In one aspect, the polyclonal or monoclonal anti-B-DNA antibody, or a fragment or derivative thereof, recognizes B-DNA over Z-DNA with at least 10-fold greater affinity / binding activity. In a third embodiment, the agent comprises, alternatively consists essentially of, or even further consists of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, a quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine, or a derivative thereof. In a fifth embodiment, the agent comprises, alternatively consists essentially of, or even further consists of chloroquine, or a derivative thereof. In a particular aspect, derivatives of the compound retain the ability to intercalate between DNA bases. The agent is not an HMGB1 protein or a fragment thereof.

[0188] Further described herein is a method for inhibiting biofilm stability, which comprises, or alternatively essentially consists of, or alternatively further consists of contacting a biofilm in vitro with an effective amount of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof, wherein the contacting comprises, or alternatively essentially consists of, or alternatively further consists of coating a surface with an effective amount of an HMGB1 protein or a biologically active fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof, wherein in one embodiment, the protein and the antibody are contacted in the absence of DNase, while in another embodiment, DNase is contacted according to the method. In a further embodiment, DNase is contacted following contact with the agent. In a specific embodiment, the DNase is Pulmozyme.

[0189] In one aspect, the polyclonal or monoclonal anti-B-DNA antibody, or a fragment or derivative thereof, recognizes B-DNA with at least 10-fold greater affinity / binding activity than Z-DNA. The biologically active fragment of HMGB1 may comprise, or alternatively, essentially consist of, or even consist of, one or more of the A box, AB box, B box, C-terminal fragment, and / or N-terminal fragment. In certain embodiments, the biologically active fragment of HMGB1 may comprise, or alternatively, essentially consist of, or even consist of, the B box domain capable of binding DNA. The present disclosure also relates to a method for inhibiting biofilm stability, comprising, or alternatively consisting essentially of, or alternatively consisting of, contacting a biofilm in vitro with an effective amount of chloroquine and an anti-B-DNA antibody, or a fragment or derivative thereof, wherein the contacting comprises, or alternatively consisting essentially of, or alternatively consisting of coating a surface with an effective amount of chloroquine and an anti-B-DNA antibody, or a fragment or derivative thereof, and in one embodiment, the chloroquine and antibody are contacted in the absence of DNase. In a further embodiment, the DNase is contacted subsequent to contact with the agent. In a particular embodiment, the DNase is Pulmozyme. In one embodiment, the polyclonal or monoclonal anti-B-DNA antibody, or a fragment or derivative thereof, recognizes B-DNA over Z-DNA with at least 10-fold greater affinity / binding activity.

[0190] The methods described above may further comprise, or alternatively consist essentially of, or even further consist of, contacting the biofilm with an effective amount of an agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins; in one embodiment, the agent is contacted in the absence of DNase, while in another embodiment, DNase is contacted according to the method. In another embodiment, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is contacted subsequent to contact with the agent. In a particular embodiment, the DNase is Pulmozyme. In one embodiment, the agent that interferes with the binding of eDNA to DNA-binding proteins comprises, or alternatively consists essentially of, or even further consists of, one or more of an anti-DNABII antibody, an anti-IHF antibody, and / or an anti-HU antibody, or fragments thereof; in one embodiment, the agent is contacted in the absence of DNase. In a further aspect, the DNase is contacted subsequent to contact with the agent. In a particular aspect, the DNase is Pulmozyme. In another aspect, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In one embodiment, the agent that interferes with the binding of eDNA to the DNA-binding protein has a net negative charge, and in one aspect, the agent is contacted in the absence of DNase. In a further aspect, the DNase is contacted subsequent to contact with the agent. In a particular aspect, the DNase is Pulmozyme. In a second embodiment, the agent that interferes with the binding of eDNA to the DNA-binding protein has a net neutral charge, and in one aspect, the agent is contacted in the absence of DNase, while in another aspect, the DNase is contacted according to the method. In another aspect, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further aspect, the DNase is contacted subsequent to contact with the agent. In a particular aspect, the DNase is Pulmozyme. In a third embodiment, the agent that interferes with the binding of eDNA to a DNA-binding protein has a net positive charge.In a further embodiment, the DNase is contacted subsequent to contact with the agent. In one particular embodiment, the DNase is Pulmozyme.

[0191] Provided herein is a method for inhibiting biofilm stability, comprising contacting a biofilm with an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the agent is contacted in the absence of DNase, while in another embodiment, DNase is contacted according to the method. In another embodiment, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is contacted following contact with the agent. In a particular embodiment, the DNase is Pulmozyme. Contacting can be in vitro or in vivo.

[0192] Also provided is a method for treating a biofilm in a subject, comprising administering to a subject infected with a biofilm an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the agent is administered in the absence of DNase, while in another embodiment, DNase is administered. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to administration of the agent. In a specific embodiment, the DNase is Pulmozyme.

[0193] Further provided is a method for preventing biofilm formation in a subject prone to biofilm development, comprising administering to the subject an effective amount of an agent that interferes with the binding of polyamines to DNA in biofilms, wherein in one embodiment the agent is administered in the absence of DNase, while in another embodiment DNase is administered. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to administration of the agent. In a specific embodiment, the DNase is Pulmozyme.

[0194] Still further provided is a method for treating an infection caused by a biofilm-producing bacterium in a subject in need thereof, comprising administering to the subject an effective amount of an agent that interferes with the binding of polyamines to DNA in the biofilm and an agent that inhibits the replication of the organism, wherein in one embodiment, the agent is administered in the absence of DNase, while in another embodiment, DNase is administered. In another embodiment, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further embodiment, DNase is administered subsequent to the administration of the agent. In a specific embodiment, the DNase is Pulmozyme.

[0195] In one embodiment, the agent is an inhibitor of polyamine synthesis or an agent that inhibits the binding of polyamines to DNA. In another embodiment, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. Non-limiting examples of polyamines include putrescine, spermine, cadaverine, 1,3-diaminopropane, or spermidine. In another embodiment, the agent includes polyamine analogs difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide (Boc Sciences), methylglyoxal-bis[guanylhydrazone] (methyl-GAG), 1-aminooxy-3-aminopropane (AKos Consulting & Solutions), oxaliplatin, cisplatin, dicyclohexylamine, any derivative thereof, or a salt thereof (all agents in this paragraph are commercially available from Millipore Sigma unless otherwise indicated). In one embodiment, derivatives of these compounds maintain the same mass-to-charge ratio.

[0196] In a further aspect, the agent comprises an agent that depletes cations from a biofilm, optionally a cation exchange resin, aminopolycarboxylic acid, crown ether, azacrown, or cryptand (various representative compounds of each class of agent are available from Millipore Sigma). In another aspect, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. Non-limiting examples of cation exchange resins include resins containing sulfonate, sulfopropyl, phosphocellulose, P11 phosphocellulose, heparin sulfate, or derivatives or analogs thereof. In one embodiment, the agent that depletes cations from a biofilm has a net negative charge. In one embodiment, the agent that depletes cations from a biofilm has a net neutral charge.

[0197] In one embodiment, provided herein is a method for inhibiting biofilm stability, comprising contacting a biofilm in vitro with an agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contacting comprises coating a surface with an agent that depletes cations, and in one aspect, the agent is administered in the absence of DNase, while in another aspect, DNase is administered. In another aspect, the agent is not an HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further aspect, DNase is administered subsequent to administration of the agent. In a particular aspect, the DNase is Pulmozyme.

[0198] In one embodiment of the above method, the agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment; in one embodiment, the agent is administered in the absence of DNase, while in another embodiment, DNase is administered. In a further embodiment, DNase is administered subsequent to administration of the agent. In one particular embodiment, the DNase is Pulmozyme. Examples of such include anti-B-DNA antibodies or fragments or derivatives thereof. In a further embodiment, the agent comprises an agent from the group consisting of riboflavin, ethidium bromide, bis(methidium)spermine, (Dervan et al. (1978) 100(6):1968-1970) daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, (Rajecky et al. (2015); Le et al. (2004) 69(8):2768-2772) quinacrine, 9-aminoacridine or a derivative thereof (all agents in this further embodiment are commercially available from Millipore Sigma unless otherwise indicated). The agent is not an HMGB1 protein or a fragment thereof.

[0199] Further provided is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment; in one embodiment, the agent is administered in the absence of DNase, while in another embodiment, DNase is administered. In a further embodiment, DNase is administered subsequent to administration of the agent. In a particular embodiment, the DNase is Pulmozyme. Examples of such include anti-B-DNA antibodies, or fragments or derivatives thereof. In a further embodiment, the agent comprises an agent from the group of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine, or a derivative thereof. In one embodiment, the method is carried out in the absence of DNase, and in one embodiment, the treatment of CF is carried out in the absence of DNase. The agent is not an HMGB1 protein or a fragment thereof.

[0200] In another aspect, provided herein is a method for treating a biofilm-producing infection associated with chemotherapy in a patient undergoing or who has undergone platinum-based chemotherapy, comprising administering an effective amount of an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment; in one aspect, the agent is administered in the absence of DNase, while in another aspect, DNase is administered. In a further aspect, the DNase is administered subsequent to administration of the agent. In a particular aspect, the DNase is Pulmozyme. Examples of such include anti-B-DNA antibodies or fragments or derivatives thereof. In a further aspect, the agent comprises an agent from the group consisting of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine, or a derivative thereof. The agent is not an HMGB1 protein or a fragment thereof.

[0201] The methods set forth above may further include contacting the biofilm (if in vitro) or administering to the subject an effective amount of an agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins; in one aspect, the agent is administered in the absence of DNase, while in another aspect, DNase is administered. In another aspect, the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In a further aspect, DNase is administered subsequent to administration of the agent. In a particular aspect, the DNase is Pulmozyme. In one embodiment, the agent that interferes with the binding of eDNA to DNA-binding proteins has a net positive charge. In one embodiment, the agent that interferes with the binding of eDNA to DNA-binding proteins has a net negative charge. In one embodiment, the agent that interferes with the binding of eDNA to DNA-binding proteins has a net neutral charge.

[0202] When performed in vitro, the method is useful for screening or identifying agents with the same, similar, or opposite abilities as the polypeptides, polynucleotides, antibodies, host cells, small molecules, and compositions disclosed herein. Alternatively, they can be used to identify which agent is best suited to treat microbial infections, or whether the treatment was effective. For example, new agents or combination therapies can be screened by preparing two samples containing, for example, a DNABII polypeptide and microbial DNA, and the agent to be tested. The second sample contains a DNABII polypeptide and microbial DNA, and a known active agent, such as an anti-IHF antibody or small molecule, to serve as a positive control. In a further embodiment, several samples are provided, and the agent is added to the system in increasing dilutions to determine the optimal dose that may be effective in treating a subject in a clinical setting. As will be apparent to those skilled in the art, a negative control containing a DNABII polypeptide and microbial DNA may also be provided. In a further embodiment, the DNABII polypeptide and microbial DNA are detectably labeled, for example, with a luminescent molecule that emits a signal when they come into close contact with each other. The sample is contained under similar conditions for a time period during which the agent is effective to inhibit, compete with, or titrate the interaction between the DNABII polypeptide and the microbial DNA, and then the sample is assayed for the emission of a signal from the luminescent molecule. If the sample emits a signal, the agent is not effective in inhibiting binding.

[0203] In another embodiment, the in vitro method is carried out in a miniaturized chamber slide system, in which an isolate of a microorganism (e.g., bacteria) causing an infection is isolated from a human / animal and then cultured to allow it to grow as a biofilm in vitro. An agent (e.g., anti-DNABII or IHF antibody) or a test agent or potential agent is added to the culture alone or in combination with another agent, with or without increasing dilutions of the potential agent or agent, for example, anti-DNABII or IHF (or other antibodies, small molecules, agents, etc.), to find the optimal dose that, when delivered to a subject with an existing infection, can be effective in treating the patient. As will be clear to those skilled in the art, positive and negative controls can also be performed simultaneously.

[0204] In a further embodiment, the method is carried out on a high-throughput platform with an agent (e.g., anti-DNABII or IHF antibody) and / or potential agent (alone or in combination with another agent) in a flow cell. The agent (e.g., anti-DNABII or IHF antibody) or potential agent biofilm is added to the culture alone or in combination with another agent, with or without increasing dilutions of the potential agent or agent, for example, anti-DNABII or IHF (or other antibodies, small molecules, agents, etc.), to find the optimal dose that would be effective in treating the patient if delivered to a subject with an infection. The biofilm isolate is sonicated to separate the biofilm bacteria from the DNABII polypeptide, for example, IHF, bound to the microbial DNA. The DNABII polypeptide-DNA complex is separated by the anti-DNABII or IHF antibody on the platform. The microbial DNA is then released, for example, by a salt wash, and used to identify the added biofilm bacteria. The released DNA is then identified, for example, by PCR sequencing. If DNA is not released, the agent(s) successfully acted on or bound to the microbial DNA. If DNA is found in the sample, the agent did not interfere with the DNABII polypeptide-microbial DNA binding. As will be apparent to those skilled in the art, positive and / or negative controls may be performed simultaneously.

[0205] Also, because a given bacterial species may respond better to disruption of its biofilm by one agent than another, the above method can be used as a diagnostic test; this rapid, high-throughput assay system may allow one skilled in the art to assay a panel of potential anti-DNABII or IHF-like agents to identify the most effective of the group.

[0206] An advantage of these methods is that most in-hospital clinical microbiology laboratories are already equipped to perform these types of assays (i.e., determining MIC, MBC values) using bacteria growing in liquid culture (or planktonically). As will be apparent to those skilled in the art, bacteria generally do not grow planktonically when they are causing disease. Instead, they grow as stable biofilms, and these biofilms are significantly more resistant to treatment with antibiotics, antibodies, or other therapeutic agents. This resistance is the reason why most MIC / MBC values ​​cannot accurately predict in vivo efficacy. Therefore, by determining what "dose" of an agent can reverse bacterial biofilms in vitro (as described above), Applicants' preclinical assays may become a more reliable predictor of clinical efficacy, even in personalized medicine applications.

[0207] In addition to clinical settings, the methods can be used to identify infection-causing microorganisms and / or validate effective agents in industrial settings, where the agents can be used to treat, inhibit, or titer biofilms.

[0208] In a further embodiment of the above method, antibiotics or antimicrobial agents known to inhibit the growth of the underlying infection are added sequentially or in combination to determine whether the infection can be inhibited. It is also possible to add an agent to the microbial DNA or DNABII polypeptide, and then add the missing complex to assay for biofilm inhibition.

[0209] When performed in vivo in non-human animals, e.g., chinchillas, the method provides a preclinical screen to identify agents that can be used alone or in combination with other agents to disrupt biofilms.

[0210] In another aspect, provided herein are methods of inhibiting, preventing, or disrupting a biofilm in a subject by administering to the subject an effective amount of an agent, thereby inhibiting, preventing, or disrupting the microbial biofilm. Non-limiting examples of such subjects include mammals, e.g., pets, and human patients.

[0211] The agents and compositions disclosed herein can be administered in conjunction with or sequentially with other antimicrobial agents and / or surface antigens. In a specific embodiment, administration is local to the site of infection, for example, by direct injection or by inhalation. Other non-limiting examples of administration include administration by one or more methods, including transdermal, urethral, ​​sublingual, rectal, vaginal, ocular, subcutaneous, intramuscular, intraperitoneal, intranasal, by inhalation, or orally.

[0212] Microbial infections and diseases that can be treated by the methods disclosed herein include infections caused by the organisms Streptococcus agalactiae, Neisseria meningitidis, Treponemes denticola, Treponemes pallidum, Burkholderia cepacia, or Burkholderia pseudomallei. In one embodiment, the microbial infection is one or more of Haemophilus influenzae (nontypeable), Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, or Mycobacterium tuberculosis. These microbial infections can be present in the upper, middle, and lower respiratory tract (such as otitis, sinusitis, bronchitis, as well as exacerbations of chronic obstructive pulmonary disease (COPD), chronic cough, complications of and / or the primary cause of cystic fibrosis (CF), and community acquired pneumonia (CAP)). Thus, by practicing the in vivo methods disclosed herein, these diseases and complications from these infections can also be prevented or treated.

[0213] Infections can also occur in the oral cavity (tooth caries, periodontitis) and be caused by Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacter actinomvctemcomitans. Infections can also be localized to the skin (abscesses, staph infections, impetigo, secondary infections of burns, Lyme disease) and be caused by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Borrelia burdorferi. Urinary tract infections (UTIs) can also be treated and are typically caused by Escherichia coli. Gastrointestinal tract infections (GI) (diarrhea, cholera, gallstones, stomach ulcers) are typically caused by Salmonella enterica serovar, Vibrio cholerae, and Helicobacter pylori. Genital infections typically involve and are caused by Neisseria gonorrhoeae. Infections can be bladder infections or indwelling device infections caused by Enterococcus faecalis. Infections typically caused by various bacteria associated with implanted prosthetic devices, such as artificial hip or knee replacements, or dental implants, or medical devices, such as pumps, catheters, stents, or monitoring systems, can be treated by the methods disclosed herein. These devices can be coated or conjugated with the agents described herein. Therefore, by implementing the in vivo methods disclosed herein, these diseases and complications from these infections can also be prevented or treated.

[0214] Infections caused by Streptococcus agalactiae, which is a leading cause of bacterial sepsis in newborns, can also be treated by the methods disclosed herein. Infections caused by Neisseria meningitidis, which can cause meningitis, can also be treated.

[0215] Thus, administration routes applicable to the methods disclosed herein include intranasal, intramuscular, urethral, ​​intratracheal, subcutaneous, intradermal, transdermal, topical application, intravenous, rectal, nasal, oral, inhalation, and other enteral and parenteral routes of administration. Administration routes can be combined or adjusted as desired, depending on the active agent and / or the desired effect. Active agents can be administered in a single dose or in multiple doses. Suitable embodiments of these methods and routes of delivery include systemic or local routes. Generally, suitable administration routes for the methods disclosed herein include, but are not limited to, direct injection, enteral, parenteral, or inhalation routes.

[0216] Parenteral administration routes 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 digestive tract. Parenteral administration can be carried out to result in systemic or local delivery of the inhibiting agent. When systemic delivery is desired, administration typically involves topical or mucosal administration of the pharmaceutical preparation for invasive or systemic absorption.

[0217] The agents disclosed herein can also be delivered to a subject by enteral administration, including, but not limited to, oral and rectal (e.g., using a suppository) delivery.

[0218] Methods for administering active agents through the skin or mucosa include, but are not limited to, topical application of suitable pharmaceutical preparations, transdermal delivery, transdermal delivery, injection, and epidermal administration. For transdermal delivery, absorption enhancers or iontophoresis are suitable methods. Iontophoretic delivery can be achieved using commercially available "patches" that deliver the product continuously through intact skin via electrical pulses for several days or longer.

[0219] In various embodiments of the methods disclosed herein, the agent is administered by inhalation, injection, or orally on consecutive days at least once daily (QD), and in various embodiments, twice daily (BID), three times daily (TID), or even four times daily. Typically, a therapeutically effective daily amount is at least about 1 mg, or at least about 10 mg, or at least about 100 mg, or about 200 to about 500 mg, and sometimes up to about 1 g to about 2.5 g or so, depending on the compound.

[0220] Dosing of can be achieved according to the methods disclosed herein using capsules, tablets, oral suspensions, intramuscular injection suspensions, intravenous infusion suspensions, topical gels (gets) or creams, or intra-articular injection suspensions.

[0221] The dosage, toxicity, and therapeutic efficacy of the 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, which can be expressed as the ratio LD50 / ED50. In certain embodiments, the compositions exhibit a high therapeutic index. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the affected tissue site in order to minimize potential damage to non-infected cells, thereby reducing side effects.

[0222] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such compounds, in certain embodiments, lies within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the present methods, a therapeutically effective dose can be initially estimated from cell culture assays. Doses 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 that achieves half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0223] In some embodiments, an effective amount of the composition sufficient to achieve a therapeutic or prophylactic effect ranges from about 0.000001 mg per kilogram of body weight per dose to about 10,000 mg per kilogram of body weight per dose. Preferably, the dosage range is from about 0.0001 mg per kilogram of body weight per dose to about 100 mg per kilogram of body weight per dose. The dose may be provided as an initial dose, followed by one or more "booster" doses. Booster doses may be provided 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months after the initial dose. In some embodiments, the booster dose is administered after evaluation of the subject's response to the previous dose.

[0224] Those skilled in the art will understand that certain factors, 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, can affect the dosage and timing required to effectively treat a subject. 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. Antibodies and their derivatives

[0225] The present disclosure also provides antibodies that bind to and / or specifically recognize and bind to B DNA for use in the methods disclosed herein. The antibody can be any of the various antibodies described herein, non-limiting examples of which include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, veneered antibodies, diabodies, humanized antibodies, antibody derivatives, recombinant humanized antibodies, or derivatives or fragments of each thereof. In one aspect, the fragment comprises, or alternatively consists essentially of, or even consists of, the CDRs of the antibody. In one aspect, the antibody is detectably labeled or further comprises a detectable label conjugated thereto. Hybridoma cell lines that produce the monoclonal antibodies disclosed herein are also provided. Further provided herein are compositions comprising, or alternatively consisting essentially of, or even consisting of one or more of the above-described embodiments. Further provided are polynucleotides encoding the amino acid sequences of the antibodies and fragments, as well as methods for recombinantly producing or chemically synthesizing antibody polypeptides and fragments thereof. Antibody polypeptides can be produced in eukaryotic or prokaryotic cells, or by other methods known in the art and described herein.

[0226] Variations of this methodology include modifications of adjuvants, route and site of administration, injection volume per site, and number of sites per animal for optimal production and humane treatment of animals. For example, adjuvants are typically used to improve or enhance the immune response to antigens. Most adjuvants provide an injection site antigen depot, allowing stow release of antigens to the draining lymph nodes. Other adjuvants include surfactants that promote enrichment of protein antigen molecules over a large surface area and immunostimulatory molecules. Non-limiting examples of adjuvants for polyclonal antibody production include Freund's adjuvant, Ribi adjuvant system, and Titermax. Polyclonal antibodies can be produced using methods known in the art, some of which are described in U.S. Patent Nos. 7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746; 6,322,788; 5,686,073; and 5,670,153.

[0227] Monoclonal antibodies can be produced using conventional hybridoma technology, which is known in the art and well described in the literature. For example, suitable immortal cell lines (e.g., myeloma cell lines including, but not limited to, Sp2 / 0, Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2 / O, etc.) or heteromyeloma, its fusion products, or any cells or fusion cells derived therefrom, or any other suitable cell line known in the art (see the following web addresses, e.g., atcc.org, Hybridomas are produced by fusing the cell lines (see cell lines at lifetech.com, last accessed November 26, 2007) with antibody-producing cells, including but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil or other immune or B-cell containing cells, or any other cells, expressing heavy or light chain constant or variable or framework or CDR sequences as endogenous or heterologous nucleic acid, recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, caprine, ovine, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-, double- or triple-stranded, hybridized, etc., or any combination thereof. Antibody-producing cells can also be obtained from the peripheral blood, or in certain embodiments, from the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest, and then screened for the activity of interest. Any other suitable host cells can also be used to express heterologous or endogenous nucleic acid encoding an antibody, specified fragment, or variant thereof, of the present disclosure.The fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods and cloned by limiting dilution or cell sorting, or other known methods.

[0228] Other suitable methods for producing or isolating antibodies of the required specificity can be used, including, but not limited to, recombinant antibody selection from peptide or protein libraries (e.g., but not limited to, bacteriophage, ribosomal, oligonucleotide, cDNA, or other display libraries; e.g., as available from various commercial vendors, such as MorphoSys (Martinsreid / Planegg, Del.), BioInvent (Lund, Sweden), and Affitech (Oslo, Norway)) using methods known in the art. Methods known in the art are described in the patent literature, some of which include U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. An alternative method relies on immunization of transgenic animals (e.g., SCID mice, Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Mumma 93:154-161) capable of producing a repertoire of human antibodies, as known in the art and / or as described herein.Such techniques include ribosome display (Wanes et al. (1997) Proc. Natl. Acad. Sci. USA 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA 95:14130-14135); single-cell antibody production techniques (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987) J. Immunol 17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems (Cambridge, Mass.); Gray et al. (1995) J. Imm. Meth. 182:155-163; and Kenny et al. (1995) Bio. Technol. 13:787-790); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134).

[0229] Antibody derivatives of the present disclosure can also be prepared by delivering a polynucleotide encoding an antibody disclosed herein to a suitable host, e.g., providing a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces the antibody in its milk. Such methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0230] The term "antibody derivatives" includes post-translational modifications to the linear polypeptide sequence of an antibody or fragment. For example, U.S. Patent No. 6,602,684 (B1) describes methods for the production of modified glycoforms of antibodies, including whole antibody molecules, antibody fragments, or fusion proteins containing a region equivalent to the Fc region of an immunoglobulin, with enhanced Fe-mediated cytotoxicity, and the glycoproteins thus produced.

[0231] The antibodies disclosed herein also include derivatives that are modified by the covalent attachment of any type of molecule to the antibody, provided that the covalent attachment does not prevent the antibody from generating an anti-idiotypic response. Antibody derivatives include, but are not limited to, antibodies modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Moreover, derivatives can contain one or more non-classical amino acids.

[0232] Antibody derivatives can also be prepared by providing transgenic plants and cultured plant cells (e.g., but not limited to, tobacco, corn, and duckweed) that deliver the polynucleotides disclosed herein and produce the antibody, specified portion, or variant in plant parts or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118 and references cited therein describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins, for example, using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activity equivalent to that produced in other recombinant systems or purified from natural sources. See, for example, Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 and references cited therein. Antibody derivatives have also been produced in large quantities from transgenic plant seeds containing antibody fragments, such as single-chain antibodies (scFv), including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and the references cited therein. Thus, transgenic plants can also be used to produce antibodies according to known methods.

[0233] Antibody derivatives can also be produced, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions are replaced with variable or constant regions from human or other amino acids or other isotypes.

[0234] In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibody humanization or engineering can be performed using any known method, including, but not limited to, those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

[0235] The chimeric, humanized, or primatized antibodies of the present disclosure can be prepared based on the sequence of a reference monoclonal antibody prepared using standard molecular biology techniques. Using standard molecular biology techniques, DNA encoding heavy and light chain immunoglobulins can be obtained from the desired hybridoma and engineered to contain non-reference (e.g., human) immunoglobulin sequences. For example, to produce a chimeric antibody, a mouse variable region can be linked to a human constant region using methods known in the art (U.S. Patent No. 4,816,567). To produce a humanized antibody, a mouse CDR region can be inserted into a human framework using methods known in the art (U.S. Patent No. 5,225,539 and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370). Similarly, to create primatized antibodies, murine CDR regions can be inserted into a primate framework using methods known in the art (WO93 / 02108 and WO99 / 55369).

[0236] Techniques for producing partially to fully human antibodies are known in the art, and any such technique can be used. In one embodiment, fully human antibody sequences are produced in transgenic mice engineered to express human heavy and light chain antibody genes. Several strains of such transgenic mice have been generated that are capable of producing different classes of antibodies. B cells from transgenic mice producing the desired antibody can be fused to generate hybridoma cell lines for sustained production of the desired antibody (see, e.g., Russell et al. (2000) Infection and Immunity April 2000:1820-1826; Gallo et al. (2000) European J. of Immun. 30:534-540; Green (1999) J. of Immun. Methods 231:11-23; Yang et al. (1999A) J. of Leukocyte Biology 66:401-410; Yang (1999B) Cancer Research 59(6):1236-1243; Jakobovits (1998) Advanced Drug Reviews 31:33-42; Green and Jakobovits (1998) J. Exp. Med. 188(3):483-495;Jakobovits (1998) Exp. Opin. Invest. Drugs 7(4):607-614;Tsuda et al. (1997) Genomics 42:413-421;Sherman-Gold (1997) Genetic Engineering News 17(14);Mendez et al. (1997) Nature Genetics 15:146-156;Jakobovits (1996) Weir's Handbook of Experimental Immunology, The Integrated Immune System Vol. IV, 194.1-194.7; Jakobovits (1995) Current Opinion in Biotechnology 6:561-566; Mendez et al. (1995) Genomics 26:294-307; Jakobovits (1994) Current Biology 4(8):761-763; Arbones et al. (1994) Immunity 1(4):247-260; Jakobovits (1993) Nature 362(6417):255-258; Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555; and U.S. Patent No. 6,075,181.

[0237] The antibodies disclosed herein can also be modified to produce chimeric antibodies. Chimeric antibodies are antibodies in which various domains of the heavy and light chains of an antibody are encoded by DNA from two or more species. See, for example, U.S. Patent No. 4,816,567.

[0238] Alternatively, the antibodies disclosed herein can be modified to create veneered antibodies. Veneering antibodies are antibodies in which the outer surface amino acid residues of an antibody from one species have been judiciously replaced, or "veneered," with amino acid residues from a second species, such that the antibody from the first species is not immunogenic in the second species, thereby reducing the antibody's immunogenicity. Because the antigenicity of a protein depends primarily on the nature of its surface, replacing exposed residues that differ from those typically found in antibodies from another mammalian species can reduce the antibody's immunogenicity. Such judicious outer surface residue replacements should have little or no effect on internal domains or interdomain contacts. Thus, as a result of changes limited to variable region framework residues, ligand binding properties should be unaffected. This process is referred to as "veneering," because only the outer surface, or skin, of the antibody is altered, leaving supporting residues undisturbed.

[0239] The procedure for "veneering" utilizes available sequence data for human antibody variable domains compiled by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Non-limiting examples of methods used to generate veneered antibodies include EP 519596; U.S. Pat. No. 6,797,492; and are described in Padlan et al. (1991) Mol. Immunol. 28(4-5):489-498.

[0240] The term "antibody derivative" also includes "diabodies," which are small antibody fragments with two antigen-binding sites, comprising a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with complementary domains in another chain, creating two antigen-binding sites (see also U.S. Pat. No. 6,632,926, Chen et al., which discloses antibody variants with one or more amino acids inserted into the hypervariable region of the parent antibody and having binding affinity for a target antigen that is at least about two-fold stronger than the binding affinity of the parent antibody for the antigen).

[0241] The term "antibody derivatives" further includes engineered antibody molecules, fragments and single domains, such as scFvs, dAbs, nanobodies, minibodies, unibodies and affibodies (& Hudson (2005) Nature Biotech 23(9):1126-36; U.S. Patent Application Publication No. 2006 / 0211088; PCT International Application Publication No. WO2007 / 059782; U.S. Patent No. 5,831,012).

[0242] The term "antibody derivative" further includes "linear antibodies." Procedures for generating linear antibodies are known in the art and are described in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, such antibodies comprise a pair of tandem Ed segments (V) that form a pair of antigen-binding regions. H -C H 1-VH-C H 1) Linear antibodies can be bispecific or monospecific.

[0243] The antibodies disclosed herein can be recovered and purified from recombinant cell culture by known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0244] The antibodies of the present disclosure include naturally occurring purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells, or alternatively from prokaryotic hosts, as described above. Several antibody production systems are described in Birch & Radner (2006) Adv. Drug Delivery Rev. 58: 671-685.

[0245] If the antibody being tested binds to a protein or polypeptide, the antibody being tested and the antibody provided by the present disclosure are equivalent. It is also possible to determine, without undue experimentation, whether an antibody has the same specificity as an antibody disclosed herein by determining whether the antibody being tested prevents the antibody disclosed herein from binding to the protein or polypeptide with which it normally reacts. If the antibody being tested competes with the antibody disclosed herein, as indicated by reduced binding by the monoclonal antibody disclosed herein, the two antibodies may bind to the same or closely related epitopes. Alternatively, the antibody disclosed herein can be preincubated with the protein with which it normally reacts to determine whether the antibody being tested is inhibited in its ability to bind to the antigen. If the antibody being tested is inhibited, it almost certainly has the same or closely related epitope specificity as the antibody disclosed herein.

[0246] The term "antibody" is also intended to include antibodies of all immunoglobulin isotypes and subclasses. Monoclonal antibodies of a particular isotype can be prepared directly by selection from the initial fusion, or can be subsequently prepared from parent hybridomas secreting monoclonal antibodies of a different isotype using sib selection techniques to isolate class-switched variants using the procedures described in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J. Immunol. Methods 74:307. Alternatively, recombinant DNA techniques can be used.

[0247] Isolation of other monoclonal antibodies having the specificity of the monoclonal antibodies described herein can also be achieved by one skilled in the art by raising anti-idiotypic antibodies. Herlyn et al. (1986) Science 232:100. Anti-idiotypic antibodies are antibodies that recognize unique determinants present on the monoclonal antibody of interest.

[0248] In some embodiments disclosed herein, it is useful to detectably or therapeutically label an antibody.Suitable labels are described above.Methods for conjugating antibodies to these agents are known in the art.For illustrative purposes only, antibodies can be labeled with detectable moieties such as radioactive atoms, chromophores, fluorophores, etc.Such labeled antibodies can be used in diagnostic techniques in vivo or in isolated test samples.

[0249] Coupling of an antibody to a low molecular weight hapten can increase the sensitivity of the antibody in an assay. The hapten can then be specifically detected using a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See Harlow and Lane (1988) supra.

[0250] The variable regions of the antibodies of the present disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR1, CDR2, and / or CDR3 regions to improve one or more binding characteristics (e.g., affinity) of the antibody. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on desired antibody binding or other functional properties can be assessed in appropriate in vitro or in vivo assays. In certain embodiments, conservative modifications are introduced, typically altering no more than one, two, three, four, or five residues within the CDR regions. Mutations can be amino acid substitutions, additions, or deletions.

[0251] For example, framework modifications can be made to the antibody to reduce immunogenicity by "backmutating" one or more framework residues to the corresponding germline sequence.

[0252] In addition, the antibodies disclosed herein can be engineered to contain modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Such modifications include, but are not limited to, altering the number of cysteine ​​residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody (U.S. Patent No. 5,677,425), and mutating amino acids in the Fc hinge region to decrease the biological half-life of the antibody (U.S. Patent No. 6,165,745).

[0253] Furthermore, the antibodies disclosed herein can be chemically modified. The glycosylation of an antibody can be altered, for example, by modifying one or more glycosylation sites within the antibody sequence to increase the affinity of the antibody for an antigen (U.S. Patent Nos. 5,714,350 and 6,350,861). Alternatively, to increase antibody-dependent cell-mediated cytotoxicity, hypofucosylated antibodies with reduced amounts of fucosyl residues or increased bisected GlcNac structures can be obtained by expressing the antibody in a host cell with altered glycosylation machinery (Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-180).

[0254] The antibodies disclosed herein can be pegylated to increase biological half-life by reacting the antibody or fragment thereof with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Antibody PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the PEG forms that have been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated can be an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies disclosed herein (EP 0154316 and EP 0401384).

[0255] Moreover, antibodies can be chemically modified by conjugating or fusing the antigen-binding region of the antibody to a serum protein such as human serum albumin to increase the half-life of the resulting molecule. Such approaches are described, for example, in EP 0322094 and EP 0486525.

[0256] The antibodies or fragments thereof of the present disclosure can be conjugated to diagnostic agents and used diagnostically, for example, to monitor the onset or progression of disease and determine the effectiveness of a given treatment regimen. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. Detectable substances can be coupled or conjugated to antibodies or fragments thereof directly or indirectly via a linker using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. An example of a luminescent material includes luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131I, Indium-111, Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111, Gallium-67, Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166, Holmium-166 , rhenium-186, rhenium-188, rhenium-189, lead-212, radium-223, actinium-225, iron-59, selenium-75, arsenic-77, strontium-89, molybdenum-99, rhodium-1105, palladium-109, praseodymium-143, promethium-149, erbium-169, iridium-194, gold-198, gold-199, and lead-211. Monoclonal antibodies can be indirectly conjugated to radioactive metal ions by use of a bifunctional chelator covalently linked to the antibody. Chelating agents can be attached through amino groups (Meares et al. (1984) Anal. Biochem. 142:68-78); sulfhydral groups of amino acid residues (Koyama (1994) Chem. Abstr. 120:217-262); and carbohydrate groups (Rodwell et al. (1986) PNAS USA 83:2632-2636; Quadri et al. (1993) Nucl. Med. Biol. 20:559-570).

[0257] Additionally, the antibodies or fragments thereof of the present disclosure can be conjugated to a therapeutic agent. Suitable therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, aspartame, thiazolinone ... Laginase, gemcitabine, cladribine, etc.), alkylating agents (mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin), antibiotics (dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), etc.), diphtheria toxin and related molecules (diphtheria A chain and its active fragments, and hybrid molecules), ricin toxin (ricin A or deglycosylated Ricin A chain toxin, etc.), cholera toxin, Shiga toxin-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrietocin, phenomycin, enomycin toxin and mixed toxins.

[0258] Additional suitable conjugated molecules include ribonucleases (RNases), DNase I, antisense nucleic acids, inhibitory RNA molecules such as siRNA molecules, immunostimulatory nucleic acids, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Aptamers are small nucleic acids ranging from 15 to 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets, and can bind small molecules such as ATP (U.S. Patent No. 5,631,146) and theophylline (U.S. Patent No. 5,580,737), as well as large molecules such as reverse transcriptase (U.S. Patent No. 5,786,462) and thrombin (U.S. Patent No. 5,543,293). Ribozymes are nucleic acid molecules that can catalyze chemical reactions, either intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate followed by cleavage. Triplex-forming functional nucleic acid molecules can interact with double-stranded or single-stranded nucleic acids by forming triplexes, where three DNA strands form a complex that relies on both Watson-Crick and Hoogsteen base pairing. Triplex molecules can bind to target regions with high affinity and specificity.

[0259] Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or they can possess a de novo activity independent of any other molecule.

[0260] Therapeutic agents can be linked to antibodies directly or indirectly using any of a number of available methods. For example, an agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation using a cross-linking agent such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP) or via a carbohydrate moiety in the Fc region of the antibody (Yu et al. 1994 Int. J. Cancer 56: 244; Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal antibodies: principles and applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal antibodies: Production, engineering and clinical application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995)).

[0261] Techniques for conjugating therapeutic agents to antibodies are well known (Amon et al. "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy; Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al. "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.); Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospects Of The Therapeutic Use Of Radiolabeled Antibody in Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al. “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” (1982) Immunol. Rev. 62:119-58).

[0262] The antibodies or antigen-binding regions thereof disclosed herein can be linked to another functional molecule, such as another antibody or a receptor ligand, to generate bispecific or multispecific molecules that bind to at least two or more different binding sites or target molecules. Linkage of the antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, can be achieved, for example, by chemical coupling, genetic fusion, or noncovalent association. Multispecific molecules can further include a third binding specificity in addition to the first and second target epitopes.

[0263] Bispecific and multispecific molecules can be prepared using methods known in the art. For example, each binding unit of a bispecific molecule can be produced separately and then conjugated to each other. When the binding molecule is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (sulfo-SMCC) (Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). When the binding molecule is an antibody, it can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains.

[0264] The antibodies of the present disclosure or fragments thereof can be linked to a moiety that is toxic to the cells to which the antibody is bound to form a "depleting" antibody.

[0265] The antibodies disclosed herein may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0266] Antibodies can also be bound to many different carriers. Thus, the present disclosure also provides compositions containing antibodies and other active or inactive substances. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes disclosed herein. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or can ascertain such using routine experimentation.

[0267] In some embodiments of the antibodies provided herein, the antibody is a full-length antibody.

[0268] In some embodiments of the antibodies provided herein, the antibody is a monoclonal antibody.

[0269] In some embodiments of the antibodies provided herein, the antibodies are chimeric or humanized.

[0270] In some embodiments of the antibodies provided herein, the antibodies may be Fab, F(ab)'2, Fab', scF v and F v is selected from the group consisting of:

[0271] In some embodiments of the antibodies provided herein, the antibody comprises an Fc domain. In some embodiments of the antibodies provided herein, the antibody is a non-human animal, such as a rat, sheep, cow, dog, cat, or rabbit antibody. In some embodiments of the antibodies provided herein, the antibody is a human or humanized antibody, or is non-immunogenic in humans.

[0272] In some embodiments of the antibodies provided herein, the antibodies comprise a human antibody framework region.

[0273] In other embodiments, one or more amino acid residues in the CDRs of the antibodies provided herein are substituted with another amino acid. The substitutions can be "conservative" in the sense that they are substitutions within the same amino acid family. Naturally occurring amino acids can be divided into four families, and conservative substitutions are made within these families:

[0274] 1) Amino acids with basic side chains: lysine, arginine, histidine.

[0275] 2) Amino acids with acidic side chains: aspartic acid, glutamic acid

[0276] 3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine.

[0277] 4) Amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.

[0278] In another embodiment, one or more amino acid residues are added to or deleted from one or more CDRs of the antibody, such additions or deletions occurring at the N- or C-terminus of the CDR or at a position within the CDR.

[0279] By varying the amino acid sequence of the antibody CDR by adding, deleting, or substituting amino acids, various effects can be achieved, such as increasing the binding affinity to the target antigen.

[0280] It should be appreciated that antibodies of the present disclosure containing such altered CDR sequences will still bind to DNABII proteins with a specificity and sensitivity profile similar to the disclosed antibodies, which can be tested by binding assays.

[0281] In a further aspect, the antibodies are characterized by being both immunodominant and immunoprotective, as determined using appropriate assays and screens. Functional analysis using antibodies

[0282] The antibodies disclosed herein can be used to purify the polypeptides disclosed herein and identify biologically equivalent polypeptides and / or polynucleotides.The antibodies disclosed herein can also be used to identify agents that alter the function of the polypeptides disclosed herein.Such antibodies include polyclonal antisera, monoclonal antibodies, and various reagents derived from such preparations, which are familiar to those skilled in the art and described above.

[0283] Antibodies that neutralize the activity of the proteins encoded by the identified genes can also be used in vivo and in vitro to demonstrate function by adding such neutralizing antibodies to in vivo and in vitro test systems. Antibodies are also useful as pharmaceuticals for modulating the activity of the polypeptides disclosed herein.

[0284] Various antibody preparations can also be used in analytical methods such as ELISA assays or Western blots to demonstrate the expression of proteins encoded by the identified genes by test cells in vitro or in vivo. Using appropriate polyclonal antisera and samples derived from experimental samples, fragments of such proteins generated by protease degradation during metabolism can also be identified.

[0285] The antibodies disclosed herein can be used for vaccination or to boost vaccination, either alone or in combination with peptide or protein-based vaccines or dendritic cell-based vaccines. composition

[0286] The present disclosure further provides compositions comprising, or alternatively consisting essentially of, or even consisting of, one, two or more, three or more of an agent that interferes with the binding of polyamines to DNA in a biofilm, an agent that depletes cations from a biofilm, an agent that interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment, an agent that interferes with the binding of eDNA to a DNA-binding protein, and / or an antimicrobial agent. In one aspect, the composition does not comprise, essentially comprise, or even consist of HMB1 protein, fragments thereof, and equivalents thereof. In another aspect, the composition comprises, essentially consist, or even consist of DNase. In a further aspect, the composition does not comprise, essentially consist, or even consist of DNase. In one embodiment, the composition comprises, or alternatively consists essentially of, or even consist of, an agent that interferes with the binding of polyamines to DNA in a biofilm and an agent that interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment. In a second embodiment, the composition comprises, or alternatively consists essentially of, or even further consists of, an agent that depletes cations from the biofilm, an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, and an agent that interferes with the binding of eDNA to DNA-binding proteins. In a third embodiment, the composition comprises, or alternatively consists essentially of, or even further consists of, an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, and an agent that interferes with the binding of eDNA to DNA-binding proteins. In a fourth embodiment, the composition comprises, or alternatively consists essentially of, or even further consists of, an agent that interferes with the binding of polyamines to DNA in the biofilm, and an agent that interferes with the binding of eDNA to DNA-binding proteins.In a fifth embodiment, the composition comprises, or alternatively consists essentially of, or even further consists of, an agent that interferes with the binding of polyamines to DNA in a biofilm, and an agent that interferes with the binding of eDNA to DNA-binding proteins, and / or an antimicrobial agent. In a sixth embodiment, the composition comprises, or alternatively consists essentially of, or even further consists of, an agent that interferes with the binding of polyamines to DNA in a biofilm, an agent that depletes cations from the biofilm, and an agent that interferes with the binding of eDNA to DNA-binding proteins. In a seventh embodiment, the composition comprises, or alternatively consists essentially of, or even further consists of, an agent that interferes with the binding of polyamines to DNA in a biofilm, an agent that depletes cations from the biofilm, and an agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment.

[0287] The compositions of the present disclosure can further comprise, alternatively consist essentially of, or even consist of, a pharmaceutically acceptable carrier.

[0288] In one embodiment, the agent that interferes with the binding of polyamines to DNA in biofilms comprises one or more of the polyamine analogs difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, and / or dicyclohexylamine, any derivatives thereof, or salts thereof. In another embodiment, the agent that depletes cations from biofilms comprises one or more of a cation exchange resin, aminopolycarboxylic acid, crown ether, azacrown, or cryptand, sulfonate, sulfopropyl, phosphocellulose, P11 phosphocellulose, and / or heparin sulfate, or derivatives or analogs thereof. In yet another embodiment, the agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment comprises one or more of an HMGB1 protein, a fragment thereof or its respective equivalent, an anti-B-DNA antibody, or a fragment or derivative thereof, and / or chloroquine, or any derivative thereof. In one particular embodiment, the agent that interferes with the binding of eDNA to DNA-binding proteins comprises one or more of an anti-DNABII antibody, an anti-IHF antibody, and / or an anti-HU antibody, or fragments of each thereof.

[0289] Compositions are also provided. The compositions include a carrier and one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, the small molecules, or the antibodies disclosed herein. The carrier can be one or more of a solid support or a pharmaceutically acceptable carrier. The composition can further include an adjuvant or other components suitable for administration as a vaccine. In one aspect, the composition is formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants. In addition, embodiments of the compositions of the present disclosure include one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the small molecules disclosed herein, the isolated host cells, or the antibodies disclosed herein, formulated with one or more pharmaceutically acceptable substances.

[0290] For oral preparations, any one or more of the isolated or recombinant polypeptides described herein, isolated or recombinant polynucleotides described herein, vectors described herein, isolated host cells described herein, small molecules, or antibodies described herein can be used alone or in combination with suitable additives to produce tablets, powders, granules, or capsules, for example, in pharmaceutical formulations disclosed herein that comprise or consist essentially of the compounds in combination with conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if desired, diluents, buffers, humectants, preservatives, and flavoring agents. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring.

[0291] Pharmaceutical formulations and unit dosage forms suitable for oral administration are particularly useful in the treatment of chronic conditions, infectious diseases, and in therapies where the patient self-administers the drug. In one aspect, the formulation is specific for pediatric administration.

[0292] The present disclosure provides pharmaceutical formulations in which one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, or the antibodies disclosed herein can be formulated into an injectable preparation in accordance with the present disclosure by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent such as a vegetable oil or other similar oil, a synthetic aliphatic acid glyceride, an ester of a higher aliphatic acid, or propylene glycol, along with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives or other antimicrobial agents, if desired. Non-limiting examples of such are surface antigens, e.g., OMP P5, OMP 26, OMP P2, or other vaccine components such as type IV pilin proteins (see Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875 and Murphy, TF, Bakaletz, LO, and Smeesters, PR (2009) The Pediatric Infectious Disease Journal, 28:S121-S126), and antimicrobial agents such as antibacterial agents. For intravenous administration, suitable carriers include physiological bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, compositions for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists.

[0293] The aerosol formulations provided by the present disclosure can be administered by inhalation and can be propellant or non-propellant based.For example, the pharmaceutical formulations disclosed herein include the compounds disclosed herein formulated in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, etc.For administration by inhalation, the compounds can be delivered in the form of aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.A non-limiting example of a non-propellant is a pump spray that is expelled from a closed container using mechanical force (i.e., by pressing down the plunger with a finger, or by compressive force applied to the wall of the container or the wall itself, for example, by the elastic force exerted by an elastic sac).

[0294] The suppositories disclosed herein can be prepared by mixing the compounds disclosed herein with any of a variety of bases, such as emulsifying bases or water-soluble bases. Such pharmaceutical formulations of the compounds disclosed herein can be administered rectally via suppositories. Suppositories can include vehicles such as cocoa butter, carbowax, and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0295] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, can be provided, with each dosage unit, e.g., a teaspoonful, tablespoonful, tablet, or suppository, containing a predetermined amount of a composition containing one or more compounds disclosed herein. Similarly, unit dosage forms for injection or intravenous administration can include a compound disclosed herein in a composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.

[0296] The embodiments of the pharmaceutical preparations disclosed herein include those in which one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the small molecules for use in the present disclosure, the isolated host cells disclosed herein, or the antibodies disclosed herein are formulated into an injectable composition.The injectable pharmaceutical preparations disclosed herein are prepared as liquid solutions or suspensions; or as solid forms suitable for dissolving or suspending in liquid media prior to injection.According to other embodiments of the pharmaceutical preparations disclosed herein, the preparations can also be emulsified or the active ingredient can be encapsulated in liposome media.

[0297] In certain embodiments, one or more of the isolated polypeptides disclosed herein, isolated polynucleotides disclosed herein, vectors disclosed herein, isolated host cells disclosed herein, or antibodies disclosed herein are formulated for delivery by a continuous delivery system. The term "continuous delivery system" is used interchangeably herein with "controlled delivery system" and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

[0298] Mechanical or electromechanical infusion pumps may also be suitable for use in the present disclosure.Examples of such devices include, for example, those described in U.S. Patent Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; and 6,198,966.Generally, the delivery of the compounds disclosed herein can be achieved using any of a variety of refillable pump systems.The pump provides consistent controlled release over time.In some embodiments, the compounds disclosed herein are present in a liquid formulation in a drug-impermeable reservoir and are delivered to an individual in a sustained manner.

[0299] In one embodiment, the drug delivery system is at least partially an implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices known in the art. An implantation site is a site within a subject's body where the drug delivery device is introduced and placed. Implantation sites include, but are not necessarily limited to, subdermal, subcutaneous, intramuscular, or other suitable sites within a subject's body. Subcutaneous implantation sites are used in some embodiments for convenience in implanting and removing the drug delivery device.

[0300] Drug release devices suitable for use in the present disclosure can be based on any of a variety of operating mechanisms. For example, the drug release device can be based on a diffusion system, a convective system, or an erodible system (e.g., an erosion-based system). For example, the drug release device can be an electrochemical pump, an osmotic pump, an electroosmotic pump, a vapor pressure pump, or an osmotic burst matrix, where, for example, the drug is incorporated into a polymer that releases the drug formulation concomitantly with the degradation of the drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material). In other embodiments, the drug release device is based on an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, or the like.

[0301] Drug release devices based on mechanical or electromechanical infusion pumps may also be suitable for use according to the present disclosure. Examples of such devices include, for example, those described in U.S. Patent Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; and 4,725,852. Generally, the subject treatment method can be achieved using any of a variety of refillable and non-replaceable pump systems. Pumps and other convective systems can be utilized because they generally have more consistent controlled release over time. In some embodiments, osmotic pumps are used because they offer the combined advantages of more consistent controlled release and relatively small size (see, for example, PCT International Application Publication No. WO97 / 27840 and U.S. Patent Nos. 5,985,305 and 5,728,396). Exemplary osmotically powered devices suitable for use in the present disclosure are described in U.S. Patent Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; and 4,203,440. Nos. 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396, etc. A further exemplary device that may be adapted for use with the present disclosure is the Synchromed infusion pump (Medtronic).

[0302] In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices known in the art. As described herein, an implantation site is a site within a subject's body where the drug delivery device is introduced and placed. Implantation sites include, but are not necessarily limited to, subdermal, subcutaneous, intramuscular, or other suitable sites within a subject's body.

[0303] Suitable excipient vehicles for the compounds disclosed herein include, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In addition, if desired, the vehicle can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents or pH buffering agents. Methods for preparing such dosage forms are known or will become apparent to those skilled in the art in light of the present disclosure. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be administered will, in any case, contain an amount of compound appropriate to achieve the desired state in the subject being treated.

[0304] Compositions of the present disclosure include compositions comprising sustained-release or controlled-release matrices. Additionally, embodiments of the present disclosure can be used in conjunction with other treatments that utilize sustained-release formulations. As used herein, a sustained-release matrix is ​​a matrix made of a material, typically a polymer, that can be degraded by enzymatic or acid-based hydrolysis or dissolution. Once inserted into the body, the matrix is ​​subjected to the action of enzymes and bodily fluids. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide-co-glycolide (copolymer of lactic and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, and isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. Illustrative biodegradable matrices include polylactide matrices, polyglycolide matrices, and polylactide-co-glycolide (copolymers of lactic and glycolic acid) matrices.

[0305] In another embodiment, the active agent (and combination composition) is delivered in a controlled release system. For example, the compounds disclosed herein can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other administration mechanisms. In one embodiment, a pump can be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, a polymeric material is used. In yet another embodiment, the controlled release system is placed in close proximity to the therapeutic target, i.e., the liver, thereby requiring only a small fraction of the systemic dose. In yet another embodiment, the controlled release system is placed in close proximity to the therapeutic target, thereby requiring only a small fraction of the systemic dose. Other controlled release systems are discussed in the review by Langer (1990) Science 249:1527-1533.

[0306] In another embodiment, the compositions of the present disclosure (and the combined compositions, separately or together) include compositions formed by impregnation of absorbent materials, such as sutures, bandages, and gauze, with the inhibitory agents described herein, or compositions coated onto solid materials, such as surgical staples, zippers, and catheters, to deliver the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the present disclosure.

[0307] The present disclosure provides methods and compositions for the administration of one or more agents to a host (e.g., a human) for the treatment of a microbial infection. In various embodiments, such methods disclosed herein cover nearly all available methods and routes suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local administration routes. Screening assays

[0308] The present disclosure provides methods for screening for equivalent agents, such as monoclonal antibodies equivalent to the polyclonal antibodies described herein, and various agents that modulate the activity of active agents and the function of pharmaceutical compositions disclosed herein, or polypeptide or peptide products encoded by polynucleotides disclosed herein. For purposes of this disclosure, "agent" is intended to include, but is not limited to, biological or chemical compounds, such as simple or complex organic or inorganic molecules, peptides, proteins (e.g., antibodies), polynucleotides (antisense), or ribozymes. A wide range of compounds can be synthesized, including polymers such as polypeptides and polynucleotides, as well as synthetic organic compounds based on various core structures, and these are also included in the term "agent." In addition, various natural sources, such as plant or animal extracts, can provide compounds for screening. Although not always explicitly stated, it is understood that an agent may be used alone or in combination with another agent that has the same or different biological activity as the agent identified by the screens described herein.

[0309] As will be apparent to those skilled in the art, suitable cells can be cultured in microtiter plates and several agents can be assayed simultaneously by looking for genotypic changes, phenotypic changes or reduction in microbial titer.

[0310] When the agent is a composition other than DNA or RNA, such as a small molecule, as described above, the agent can be added directly to the cell culture or added to the culture medium for addition. As will be apparent to those skilled in the art, an "effective" amount (a mount) must be added, which can be determined empirically.

[0311] If the agent is an antibody or antigen-binding fragment, the agent can be contacted or incubated with the target antigen and the polyclonal antibody described herein under conditions for performing a competitive ELISA. Such methods are known to those skilled in the art.

[0312] The assay can also be performed in a subject. When the subject is an animal such as a rat, chinchilla, mouse, or monkey, this method provides a convenient animal model system that can be used prior to clinical testing of the agent in human patients. In this system, if the symptoms of disease or microbial infection are reduced or eliminated, respectively, compared with untreated animals with the same infection, the candidate agent is a potential drug. It can be useful to have a separate negative control group of healthy, untreated cells or animals to provide a basis for comparison.

[0313] The agents and compositions can be used in the manufacture of medicaments, such as active ingredients in pharmaceutical compositions, and for the treatment of humans and other animals by administration in accordance with conventional procedures. Combination therapy

[0314] The compositions and related methods of the present disclosure can be used in combination with the administration of other therapies, including, but not limited to, the administration of DNase enzymes, antibiotics, antimicrobial agents, or other antibodies. In one embodiment, the agent is administered in the absence of DNase enzymes.

[0315] 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 protozoa. Biofilms are generally resistant to the action of antibiotics, but the compositions and methods described herein can be used to make biofilm-associated infections susceptible to conventional therapeutic methods for treating the infection. In other embodiments, the use of antibiotics or antimicrobials in combination with the methods and compositions described herein allows for a reduction in the effective amount of antimicrobial and / or biofilm-reducing agent. Some non-limiting examples of antimicrobials and antibiotics useful in combination with the methods of the present disclosure include amoxicillin, amoxicillin-clavulanate, cefdinir, azithromycin, and sulfamethoxazole-trimethoprim. Therapeutically effective doses of antimicrobials and / or antibiotics in combination with biofilm-reducing agents can be readily determined by conventional methods. In some embodiments, the dose of the antimicrobial agent in combination with the biofilm reducing agent is the average effective dose shown to be effective in other bacterial infections, for example, bacterial infections where the etiology of the infection does not involve 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 agent can be added before, simultaneously with, or after the addition of the anti-DNABII antibody.

[0316] In other embodiments, the methods and compositions can be combined with antibodies to treat bacterial infections. One example of an antibody useful in combination with the methods and compositions described herein is an antibody against an unrelated outer membrane protein (i.e., OMP P5). Treatment with this antibody alone does not reduce biofilms in vitro. Combined therapy with this antibody and a biofilm-reducing agent produces a greater effect than can be achieved by either agent used alone at the same concentration. Other antibodies that can produce a synergistic effect when combined with a biofilm-reducing agent or method for reducing biofilms include anti-rsPilA, anti-OMP26, anti-OMP P2, and anti-OMP whole preparations.

[0317] The compositions and methods described herein can be used to susceptibly treat biofilm-mediated bacterial infections to common therapeutic modalities that are effective in treating non-biofilm-mediated bacterial infections but are otherwise ineffective in treating biofilm-mediated bacterial infections. In other embodiments, the compositions and methods described herein can be used in combination with therapeutic modalities that are effective in treating biofilm-mediated bacterial infections, but the combination of such additional therapy and biofilm-reducing agent or method creates 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 creates a synergistic effect such that treatment is enhanced. Enhanced treatment can be evidenced by a shorter amount of time required to treat the infection.

[0318] The additional therapeutic treatment can be added before, simultaneously with, or after the method or composition used to reduce the biofilm, and can be contained within the same formulation or as a separate formulation. kit

[0319] Provided herein are kits comprising, alternatively consisting essentially of, or even consisting of, the compositions disclosed herein and instructions for use. In one aspect, the instructions for use provide directions for performing any of the methods disclosed herein. In one aspect, one or more, two or more, or three or more of the agents for use in the disclosed methods are packaged separately or together in the kit.

[0320] Kits containing the agents and instructions necessary to carry out the in vitro and in vivo methods described herein are also claimed.Thus, the present disclosure provides kits for carrying out these methods, which kits can include the ones disclosed herein and instructions for carrying out the methods disclosed herein, such as tissue collection and / or screening and / or result analysis and / or administering an effective amount of the agents defined herein.These can be used alone or in combination with other suitable antimicrobial agents.

[0321] The following examples are intended to illustrate, but not limit, the embodiments disclosed herein. [Example]

[0322] Example 1 Experiment No. 1 Polyamines are ubiquitous, small, aliphatic polycations produced and utilized by almost all living organ...

Claims

1. 1. A composition comprising at least one agent that interferes with the binding of polyamines to DNA in the biofilm for use in a method for inhibiting the stability of or treating an established biofilm in a subject, the method comprising contacting the established biofilm with said agent, wherein said agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof, and wherein the agent that interferes with the binding of polyamines to DNA in the biofilm is selected from tRNA, difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, or dicyclohexylamine.

2. 2. The composition for use according to claim 1, wherein the method further comprises contacting the established biofilm with an agent that inhibits the replication of organisms capable of forming a biofilm.

3. The method of claim 1 , wherein the contacting is in vitro or in vivo.

4. The composition for use according to any one of claims 1 to 3, wherein the polyamine is selected from the group of putrescine, spermine, cadaverine, 1,3-diaminopropane or spermidine.

5. The composition for use according to claim 3, wherein said contacting comprises coating the surface with an effective amount of an agent that depletes cations, said agent being other than HMGB1 protein, a fragment thereof or an equivalent thereof.

6. 6. The composition for use according to claim 5, wherein the cation-depleting agent is selected from a cation exchange resin, an aminopolycarboxylic acid, a crown ether, an azacrown or a cryptand.

7. 6. The composition for use according to claim 5, wherein the agent that depletes cations from the biofilm has a net negative charge.

8. The composition for use according to any one of claims 1 to 6, wherein the method is carried out in the absence of administration of a DNase enzyme.

9. A composition for inhibiting the stability of or treating an established biofilm, comprising an agent that interferes with the binding of polyamines to DNA in the established biofilm, wherein the agent is administered in combination with an agent that inhibits the replication of organisms capable of forming biofilms, wherein the agent that interferes with the binding of polyamines to DNA in the established biofilm is selected from tRNA, difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, or dicyclohexylamine.

10. A composition for inhibiting the stability of or treating an established biofilm, comprising an agent that inhibits the replication in the established biofilm of organisms capable of forming a biofilm, said agent being administered in combination with an agent that interferes with the binding of polyamines to DNA in the established biofilm, wherein the agent that interferes with the binding of polyamines to DNA in the established biofilm is selected from tRNA, difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, or dicyclohexylamine.

11. A composition comprising chloroquine and an agent that interferes with the binding of polyamines to DNA in an established biofilm, wherein the agent is selected from tRNA, difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, or dicyclohexylamine.

12. A composition for inhibiting the stability of or treating an established biofilm, comprising chloroquine and an agent that interferes with the binding of polyamines to DNA in the established biofilm, wherein the agent is selected from tRNA, difluoromethylornithine, trans-4-methylcyclohexylamine, saldomozide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, or dicyclohexylamine.

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