Compositions and methods for mediating EPS

Abstract

To provide compositions and methods for mediating EPS. [Solution] This disclosure relates to a method for inhibiting the stability of a biofilm, comprising contacting the biofilm with an effective amount of an active substance 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 an effective amount of an active substance that interferes with the binding of polyamines to DNA in the biofilm to a subject infected with a 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 active substance that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 692,581, filed on 29 June 2018, under 35 U.S.C. § 119(e) , the contents of which said application are incorporated herein by reference in their entirety.

[0002] This disclosure generally relates to methods and compositions for removing or inhibiting extracellular polymeric substances (EPS) of bacteria. [Background technology]

[0003] Biofilms play a major role in medical, agricultural, and industrial environments. They are responsible for a significant portion of diseases in both animals and plants, as well as for the contamination of industrial equipment, and are therefore the subject of intense research efforts. Eradicating or treating biofilms is particularly difficult due to several factors, including the production of an extracellular matrix that forms a physical barrier against antimicrobial effectors, changes in physiological function that make the biofilm less susceptible to environmental stressors, and cooperative interactions between biofilm components. The biofilm matrix consists invariably of polysaccharides, proteins, and, perhaps universally, extracellular DNA (eDNA). The eDNA of microbial biofilms is a crucial component of the extracellular matrix that provides protection. Damaging the biofilm eDNA structure through DNA degradation or removal of DNA-binding proteins that stabilize the structure leads to the catastrophic collapse of the biofilm and the release of commensal bacteria, making it more vulnerable.

[0004] Bacteria are found in nature in two distinct states: planktonic bacteria are free-living, while bacteria that develop into community architectures are called biofilms (either on a surface 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 many 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 attention in children is omniferous venereal disease (OM) [caused by Nontypeable Haemphilus influenzae (NTHI), Streptococcus pneumoniae, and Moraxella catarrhalis], and for adults, cystitis [e.g., urinary tract pathogenic E. coli (UPEC)]; therefore, antibiotic prescriptions are the most common for these complaints. In the United States, it is estimated that 500,000 deaths annually are attributable to the direct consequences 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-borne diseases, particularly the increasing rate of antibiotic-resistant bacterial infections 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 overcome the protective barrier of biofilms in order 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 [Overview of the project] [Means for solving the problem]

[0006] Because biofilms act as refractory reservoirs for bacteria that cause chronic and recurrent infections, a self-producing extracellular matrix (or extracellular macromolecule, EPS) that protects the commensal bacteria within the biofilm from immune clearance and antimicrobial agents is essential for pathogenic biofilms that cause these diseases. EPS components are specific to individual bacterial species but universally contain extracellular DNA (eDNA) derived from the commensal bacteria within the biofilm. In fact, bacteria of various genera typically enter the shared community architecture of multi-species biofilms, which requires that the EPS not only be structurally helpful to all component species but also contain EPS components derived from or usable by all commensal bacteria. In this regard, the EPS of single-species and multi-species biofilms contains scaffold eDNA that appears to be a common structure of the underlying universal EPS. As disclosed herein, the applicant has found that this eDNA-dependent structure is stabilized by the ubiquitous DNABII family of bacterial DNA-binding proteins. The applicant has shown that exogenous DNA and DNABII proteins can lead free-living (planktonic) bacteria to the community architecture of a biofilm, but these two components are insufficient to replicate the signature eDNA scaffold.

[0007] The applicant hereby discloses that polyamines are a third important component of universal eDNA-DNABII-dependent EPS. Polyamines are ubiquitous, positively charged short organic molecules found both intracellularly and extracellularly that, upon binding to DNA, neutralize the polyanionic charge of nucleotide phosphates, enriching / aggregating DNA molecules. Importantly, the applicant hereby discloses that polyamines can transform DNA from the most common right-handed B-type to nuclease-resistant left-handed Z-type DNA. Indeed, while nucleases can prevent bacterial biofilm formation, they cannot disrupt mature biofilms. As biofilms mature, accompanied by both (1) an increase in polyamines and (2) the appearance of Z-type DNA, they acquire nuclease resistance.

[0008] Methods for inhibiting the stability of a biofilm are described herein, comprising, or essentially thereof, or further comprising, a method for inhibiting the stability of a biofilm, comprising contacting the biofilm with an effective amount of an active substance that interferes with the binding of a polyamine to DNA in the biofilm, wherein the active substance is not the HMGB1 protein, a fragment thereof, or any of its equivalents. In one embodiment, a method for inhibiting the stability of a biofilm comprises, or essentially thereof, or further comprising, contacting the biofilm with an effective amount of one or more active substances that interfere with the binding of a polyamine to DNA in the biofilm. Furthermore, the present disclosure relates to a method for inhibiting the stability of a biofilm, comprising, or essentially, or further comprising, in vitro contacting a biofilm with an active substance that interferes with the binding of polyamines to DNA in the biofilm, wherein the contact comprises, or essentially, or further comprising, coating the surface with an effective amount of the active substance that depletes cations, and wherein the active substance is not the HMGB1 protein, a fragment thereof, or each of its equivalents. In one embodiment, the method for inhibiting the stability of a biofilm comprises, or essentially, or further comprising, in vitro contacting a biofilm with an effective amount of an active substance that interferes with the binding of polyamines to DNA in the biofilm, wherein the contact comprises, or essentially, or further comprises, coating the surface with an effective amount of one or more active substances that depletes cations. The contact may be in vitro or in vivo.

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

[0010] A method for inhibiting the stability of a biofilm is further described herein, comprising, or essentially thereof, or further comprising, contacting the biofilm in vitro with an effective amount of HMGB1 protein or its bioactive fragment and an anti-B-DNA antibody or its fragment or derivative, and the contact comprising, or essentially thereof, or further comprising, coating the surface with an effective amount of HMGB1 protein or its bioactive fragment and an anti-B-DNA antibody or its fragment or derivative. The disclosure also relates to a method for inhibiting the stability of a biofilm, comprising, or essentially thereof, or further comprising, contacting the biofilm in vitro with an effective amount of chloroquine and an anti-B-DNA antibody or its fragment or derivative, and the contact comprising, or essentially thereof, or further comprising, coating the surface with an effective amount of chloroquine and an anti-B-DNA antibody or its fragment or derivative. The contact may be in vitro or in vivo.

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

[0012] Also provided herein is 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, or consisting essentially of, or consisting of, such an agent, wherein the agent is not HMGB1 protein, a fragment thereof, or an equivalent thereof. In one aspect, a method for preventing the formation of a biofilm in a subject prone to developing a biofilm comprises administering to the subject an effective amount of one or more agents that interfere with the binding of polyamines to DNA in the biofilm, or consisting essentially of, or consisting of, such agents.

[0013] This disclosure further relates to a method for treating an infection caused by biofilm-forming bacteria in a subject requiring its use, comprising, or essentially, or further comprising, an effective amount of an active substance that interferes with the binding of polyamines to DNA in a biofilm and an active substance that inhibits the replication of the organism, wherein the active substances are not the HMGB1 protein, fragments thereof, or their respective equivalents. In one embodiment, a method for treating an infection caused by biofilm-forming bacteria in a subject requiring its use comprises, or essentially, or further comprising, an effective amount of one or more active substances that interfere with the binding of polyamines to DNA in a 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 active agent that interferes with the binding of the polyamine to DNA in the biofilm is tRNA. In another embodiment, the active agent is an inhibitor of polyamine synthesis or an active agent that inhibits the binding of the polyamine to DNA. In a second embodiment, the active agent includes, or is essentially, or 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 the third embodiment, the active agent comprises, or is essentially thereof, or further comprises an active agent that depletes cations from the biofilm, optionally containing a cation exchange resin, aminopolycarboxylic acid, crown ether, azacrown, or cryptand. In the fourth embodiment, the active agent that depletes cations from the biofilm is selected from the group consisting of sulfonates, sulfopropyls, phosphocellulose, P11 phosphocellulose, heparin sulfate, or derivatives or analogs thereof. In the fifth embodiment, the active agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment. In the sixth embodiment, the active agent comprises, or is essentially thereof, or further comprises an anti-B-DNA antibody or a fragment or derivative thereof. In the eighth embodiment, the active substance includes, or is essentially, or further comprises, riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine or its derivatives.In the ninth embodiment, the agent comprises, consists essentially of, or consists of chloroquine or a derivative thereof. In one aspect, the agent that depletes cations from the biofilm has a net negative charge. In another aspect, the agent that depletes cations from the biofilm has a net neutral charge.

[0015] Also disclosed herein is a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising, or essentially therein, or further comprising therein, wherein the active substance is neither HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In one embodiment, the active agent is administered in the absence of DNase. In one embodiment, the active agent interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment. In a second embodiment, the active agent includes, or is essentially, an anti-B-DNA antibody or a fragment or derivative thereof. In a third embodiment, the active agent includes, or is essentially, a derivative thereof, riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or a derivative thereof. In a fourth embodiment, the active agent includes, or is essentially, a derivative thereof, chloroquine or a derivative thereof. In one embodiment, the chloroquine derivative retains the ability to intervene between DNA bases.

[0016] Furthermore, methods for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF), comprising, or essentially thereof, or further comprising, the administration of an effective amount of HMGB1 protein or its bioactive fragment and an anti-B-DNA antibody or its fragment or derivative. In one embodiment, a method for treating biofilms in patients with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or tuberculosis (TB) comprises, or essentially thereof, or further comprising, the administration of an effective amount of chloroquine and an anti-B-DNA antibody or its fragment or derivative. Furthermore, the Disclosure relates to a method for treating a biofilm-producing infection associated with the administration of chemotherapy in a patient receiving or having received platinum-based chemotherapy, comprising, or essentially thereof, or further comprising thereof, the activator being neither the HMGB1 protein nor a fragment thereof nor any of its equivalents. In one embodiment, the method comprises, or essentially thereof, or further comprising thereof, the activator being neither the HMGB1 protein nor a fragment thereof nor any of its equivalents. In further embodiments, the active substance includes, or is essentially composed of, an anti-B-DNA antibody or a fragment or derivative thereof.In one embodiment, the active substance includes, or is essentially, or further comprises, riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine or its derivatives.

[0017] This disclosure relates to a method for treating a biofilm-producing infection associated with the administration of platinum-based chemotherapy in a patient receiving or having received platinum-based chemotherapy, comprising, or essentially thereof, or further comprising, a method comprising, an effective amount of HMGB1 protein or a bioactive 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 platinum-based chemotherapy in a patient receiving or having received platinum-based chemotherapy, comprising, or essentially thereof, or further comprising, a method comprising, an effective amount of chloroquine and an anti-B-DNA antibody or a fragment or derivative thereof.

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

[0019] [Figure 1] Figures 1A-1C demonstrate 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 induced by NTHI (adapted from Goodman et al. (2011) Mucosal Immunol. 4(6):625-37). DNABII protein (gray dots) and eDNA (white regions) were probed. DNABII is localized to the vertices of eDNA strands in biofilms in vivo. (Figure 1B) Chemical structure of a typical 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 polyamine (adapted from Iacomino et al. (2011) Biomacromolecules. 12(4):1178-86). Polyamines induce the formation of thick filaments and structural complexity in eDNA.

[0020] [Figure 2]Figures 2A and 2B illustrate how polyamines induce eDNA scaffold structures. (Figure 2A) Immunofluorescence CLSM image of mucosal biofilm EPS found in the middle ear of a chinchilla with acute OM induced by NTHI. Polyamines (white dots) [putrescine (Put), cadaverine (Cad), spermidine (Spd)] and eDNA (white) were probed and counterstained with DAPI (gray areas). Polyamines are localized to eDNA strands in biofilms formed in vivo; spermidine is the most abundant polyamine in biofilm EPS. (Figure 2B) Transmission electron microscope 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). Similar structures are formed by the DNA-polyamine-IHF complex. Sarkar et al. (2009) Biochemistry. 48(4):667-75. Polyamines induce DNA condensation and, in combination with DNABII 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-tests, *P<0.05. The mean biofilm thickness was reduced by DCHA, but co-addition of exogenous Spd restored biofilm formation. (Figure 3B) Immunofluorescence microscopy image of eDNA scaffold structures in NTHI in vitro biofilms grown for 3 hours in the presence of DCHA (50 μM). dsDNA was probed (white area). eDNA scaffold structure production was significantly reduced by inhibition of polyamine synthesis. (Figure 3C) Immunofluorescence CLSM image of NTHI in vitro biofilm grown for 40 hours in the presence of DCHA. Spd was probed (white dots in the lower panel of Figure 3C) and counterstained with DAPI (gray areas). DCHA inhibits polyamine uptake into the biofilm EPS.

[0022] [Figure 4]Figures 4A and 4B show that anti-DNABII disrupts the DNABII-polyamine (PA)-dependent structure. (Figure 4A) The DNA structure was formed by incubating spermidine (300 μM) and HU (1 μM) in a buffer containing genomic DNA (gDNA; 2 μg / ml) for 40 hours. Immunofluorescence CLSM image of the DNABII-polyamine-dependent DNA structure. DNABII protein was probed (white; indicated on the right side of the image) and counterstained with DAPI (white; indicated on the left side of the image). The DNABII-polyamine-dependent DNA structure incorporates the DNABII protein. (Figure 4B) The EPS structure was formed in 24 hours, similar to (Figure 4A), and treated for a further 16 hours with a 1:50 dilution of DNABII antiserum (indicated at the bottom of the image). Fluorescence CLSM image stained with DAPI (white). The DNABII-polyamine-dependent DNA structure requires the DNABII protein.

[0023] [Figure 5] Figure 5 shows that cation exchanger phosphocellulose (P11) disrupts NTHI biofilm formation. P11 (1%) was added to the apical chamber, while NTHI biofilm growth was initiated in the basal chamber of the Transwell plate system. Spermidine (1 mM) and HU (1 μM) were added at seeding and maintained for 16 hours. Biofilms were visualized by CLSM and analyzed by COMSTAT. Average thickness (not shown) showed a similar trend. Bars represent SEM. Statistical significance compared to controls was assessed by unpaired t-tests: *P<0.05; **P<0.01. P11 prevents biofilm formation. Exogenous spermidine and HU together restore biofilm development, but neither alone restores it (not shown). This result suggests that the P11 antibiofilm activity is a 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 pre-formed NTHI and UPEC biofilms in vitro at the time of sowing (prevention) or at 24 hours (disruption). After a total of 40 hours, the biofilms were stained with LIVE / DEAD®, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. Statistical significance compared to the control was assessed by an unpaired t-test: *P<0.05; **P<0.01. DNase can prevent biofilm formation but does not disrupt existing biofilms.

[0025] [Figure 7]Figures 7A to 7D show that DNABII proteins and polyamines (PA) synergistically interact to induce DNase resistance. (Figure 7A) Immunofluorescence CLSM image of an NTHI in vitro biofilm grown for 40 hours. Spermidine (dark gray dots) and HU (light grayish-white dots) were probed and counterstained with DAPI (gray areas). Polyamines and DNABII proteins colocalize in vitro in biofilm EPS (white dots). (Figure 7B) Immunofluorescence CLSM image of a mucosal biofilm EPS found in the middle ear of a chinchilla with acute OM due to NTHI. Putrescine (white dots) and HU (light grayish-white dots) were probed and counterstained with DAPI (dark gray areas). Polyamines and DNABII proteins colocalize in vivo in eDNA strands within the biofilm (white areas). (Figure 7C) Sequentially increasing levels of spermidine (Spd) and HU were incubated separately or together with genomic DNA (2 μg / ml) at 37°C for 1.5 hours, 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 and 8B demonstrate that DNABII and polyamines work together to convert B-DNA morphology to Z-DNA morphology. (Figure 8A) The DNABII-polyamine-dependent DNA structure was formed by incubating genomic DNA (2 μg / ml) with HU (1 μM) and spermidine (300 μM) for 16 hours. Immunofluorescence CLSM image of the DNABII-polyamine-dependent DNA structure. Z-DNA was probed (white) and stained with DAPI (dark gray). Polyamines and DNABII proteins synergistically induce the conversion of B-DNA to Z-DNA. (Figure 8B) Top: Circular dichroism spectrum of B-DNA substrate converted to Z-DNA by a Z-DNA catalyst with increasing concentration (adapted from Jang et al. (2015) Sci Rep. 5:9943). Note the inversion of the negative peak around 250 nm and the positive peak around 280 nm. Bottom: Poly(dGdC)DNA (20 μg / ml) was incubated with HU (15 μM) for 2 hours, and the CD spectrum was 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 EPS of multiple human pathogen biofilms. Top: Immunofluorescence CLSM image of the indicated bacterial biofilm at 40 hours, probed with no primary antibody (1°) or with Z-DNA antibody (white). Z-DNA is a component of the EPS of multiple bacterial biofilms at different steady-state levels. Bottom: Immunofluorescence CLSM image of the indicated biofilm, probed with HU (white) and spermidine (dark gray) antibodies, co-localization is (white). DNABII and polyamine components co-localize in the EPS of multiple bacterial biofilms at steady-state levels, and this correlates with Z-DNA abundance.

[0028] [Figure 10]Figure 10 shows that the abundance of Z-DNA and polyamines increases 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 show increased uptake of Z-DNA (white) and spermidine (dark gray) within 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 for 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, the abundance of polyamines and Z-DNA in the biofilm EPS is reduced.

[0030] [Figure 12] Figure 12 shows the growth of an unclassifiable Haemophilus influenzae biofilm for 40 hours in supplemented BHI medium on an 8-well chamber coverslip slide at 37°C and 5% CO2. The biofilm was washed, probed with the indicated primary antibody and fluorescent secondary antibody (dark gray dots), and stained with DAPI (gray areas). Note: The smallest dark gray stain in the bottom left image represents the background. Putrescine, spermidine, and spermine were all present throughout the biofilm matrix.

[0031] [Figure 13]Figure 13 shows the growth of unclassifiable Haemophilus influenzae for 16 hours at 37°C and 5% CO2 in BHI medium supplemented with the additives indicated above in a 96-well plate. Growth was quantified by spectrophotometric 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 the growth of unclassifiable Haemophilus influenzae biofilms for 40 hours at 37°C and 5% CO2 in supplemented BHI medium containing the additives indicated below each bar on an 8-well chamber coverslip slide. The biofilms were washed, stained with LIVE / DEAD®, fixed, and imaged by CLSM. Biofilm parameters were quantified using COMSTAT software. Dicyclohexylamine inhibited biofilm development, while exogenous spermidine was able to rescue biofilm growth.

[0033] [Figure 15] Figure 15 shows the growth of an unclassifiable Haemophilus influenzae biofilm for 40 hours at 37°C and 5% CO2 in supplemented BHI medium containing the additives indicated above, on an 8-well chamber coverslip slide. The biofilm was washed, probed with primary and secondary fluorescent antibodies against spermidine (dark gray dots), and stained with DAPI (gray areas). Spermidine synthase inhibitors reduced the presence of spermidine in the biofilm matrix.

[0034] [Figure 16]Figure 16 shows the growth of surface-adhering, unclassifiable Haemophilus influenzae for 3 hours at 37°C and 5% CO2 in supplemented BHI medium containing the additives indicated above, in a Fluorodish coverslip dish. The biofilm was washed and probed with primary and secondary fluorescent antibodies against double-stranded DNA (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 the presence of spermidine within the EPS of biofilms formed by multiple human pathogens. (Figure 17A) Immunofluorescence CLSM images of the indicated biofilms probed with HU antibody (light gray) and spermidine (dark gray) antibodies; co-localization is shown in white. DNABII and polyamine components co-localize in the EPS of multiple bacterial biofilms at steady-state levels. (Figure 17B) Dicyclohexylamine (DCHA) inhibition of spermidine biosynthesis reduces spermidine levels in NTHI and UPEC biofilms, indicated by a decrease in IF signaling, resulting in a significantly reduced mean thickness compared to the sBHI control (Figure 17C). UPEC is shown as a percentage change in mean thickness compared to the LB control (Figure 17D).

[0036] [Figure 18]Figures 18A and 18B demonstrate that phosphocellulose has a dose-dependent negative effect on biofilm formation and the stability of pre-formed NTHI biofilms in vitro. (Figure 18A) Biofilm growth was initiated and maintained for 24 hours, then treated with 0 (SBHI control), 0.1%, 1%, and 5% (w / v) phosphocellulose (P11) for 16 hours. (Figure 18B) Biofilm growth was initiated and 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 the average thickness and biomass. All images were captured using a 63× objective lens.

[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. The biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate the average thickness and biomass. All images were captured using a 63× objective lens.

[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 to calculate mean thickness and biomass and compared with sBHI controls. All images were captured using a 63× objective lens. Bars represent SEM. Statistical significance compared to controls was assessed by unpaired t-tests, *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, followed by 16 hours of treatment as indicated. The biofilms were washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate the average thickness and biomass. All images were captured using a 63× objective lens.

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

[0041] [Figure 23]Figure 23 shows that the cation depletion effect of P11 phosphocellulose does not require direct contact with the biofilm. Biofilm growth was initiated in the basal 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. The biofilm was washed with saline solution and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate the mean thickness and biomass and compared with the sBHI control. All images were captured using a 63× objective lens. Bars represent SEM. Statistical significance compared to the control was assessed by unpaired t-tests, *P<0.05; **P<0.01.

[0042] [Figure 24] Figure 24 shows that exogenous spermidine addition reduces the cation depletion effect of P11 phosphocellulose without requiring direct contact with the biofilm. Biofilm growth was initiated in the basal chamber of the 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. The biofilm was washed with saline solution and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate mean thickness and biomass and compared with the sBHI control. All images were captured using a 63× objective lens. Bars represent SEM. Statistical significance compared to the control was assessed by unpaired t-tests, *P<0.05; **P<0.01. Square brackets indicate statistical comparisons between conditions.

[0043] [Figure 25]Figure 25 shows that exogenous addition of spermidine and DNABII reduces the cation depletion effect of P11 phosphocellulose without 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 μM spermidine or 500 nM HU or a combination thereof. The biofilm was washed with saline and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate mean thickness and biomass and compared with the sBHI control. All images were captured using a 63× objective lens. Bars represent SEM. Statistical significance compared to the control was assessed by unpaired t-tests, *P<0.05; **P<0.01. Square brackets indicate statistical comparisons between conditions.

[0044] [Figure 26] Figures 26A and 26B demonstrate that coating abiotic surfaces with cation exchange resins prevents biofilm formation in a dose-dependent manner. Chamber slides were coated with P11 phosphocellulose (Figure 26A) or heparin-sepharose (Figure 26B) solutions as indicated. Biofilm growth was initiated and maintained on the coated slides for 40 hours. The biofilms were washed with saline solution and stained with LIVE / DEAD® stain. Images were analyzed by COMSTAT to calculate the average thickness and biomass. All images were captured using a 63× objective lens. 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 pre-formed NTHI or UPEC biofilms in vitro at seeding time (prevention) or at 24 hours (disruption). After a total of 40 hours, the 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 an unpaired t-test: *P<0.05; **P<0.01. DNase can prevent biofilm formation but does not disrupt existing biofilms.

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

[0047] [Figure 29] Figure 29 shows the presence of Z-DNA 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 (pale gray). Mature fungal biofilms incorporate Z-DNA (pale gray) within the biofilm EPS.

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

[0049] [Figure 31] Figure 31 shows that DNase degrades B-DNA within the biofilm extracellular matrix but not Z-DNA. DNase (pulmozyme; 5 units) was added to pre-formed NTHI biofilms in vitro 24 hours after biofilm seeding. After a total of 40 hours, the biofilms were probed with anti-Z-DNA, anti-B-DNA, or without primary antibody, which were revealed using the corresponding secondary fluorescent antibody and visualized by CLSM. DNase treatment degrades eDNA structures in B-DNA morphology within the biofilm extracellular matrix, but reveals eDNA in large Z-DNA morphology.

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

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

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

[0053] [Figure 35]Figures 35A–35D demonstrate that DNABII proteins and polyamines (PAs) synergistically interact to induce DNase resistance. (Figure 35A) Gradual levels of spermidine (Spd) and HU were incubated separately or together with genomic DNA (gDNA; 2 μg / ml) at 37°C for 1.5 hours, 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 chinchillas with experimental OM by NTHI. Probes were used for putrescine (dark gray) and HU (light grayish white), and counterstained with DAPI (gray). The HU-Spd-dependent DNA structure is resistant to DNase treatment. Polyamines and DNABII proteins co-localize on eDNA strands in biofilm in vivo (white). (Figure 35D): Gradually increasing concentrations of DNase were added to NTHI or UPEC biofilms for 16 hours at seeding (prevention) or at 24 hours (destruction). Biofilms were stained, fixed, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. Statistical significance compared to control (no DNase) was assessed by unpaired t-tests, **P<0.01.** DNase can prevent biofilm formation but does not destroy existing biofilms.

[0054] [Figure 36]Figure 36 shows that Z-DNA is present in the EPS of multiple human pathogen biofilms. Top: Immunofluorescence CLSM image of a 40-hour biofilm formed by the indicated bacteria, probed with either no primary antibody (1°) or Z-DNA-specific antibody (light gray). Z-DNA is a component of the EPS of multiple bacterial biofilms at different steady-state levels. Bottom: Immunofluorescence CLSM image of the indicated biofilm, probed with HU (light gray) and spermidine (dark gray) antibodies, co-localization is (white). DNABII and polyamine components co-localize in the EPS of multiple 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 extracellular matrix of biofilms. Unclassified Haemophilus influenzae biofilms were grown in supplemented BHI medium on 8-well chamber coverslip 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 primary antibody against Z-DNA (pale gray dots) and fluorescent secondary antibody, stained with DAPI (gray areas), and visualized by fluorescence microscopy. HMGB1 treatment reduced the Z-DNA structure in the biofilm matrix.

[0056] [Figure 38]Figures 38A-38C demonstrate that HMGB1 displaces the DNABII protein, thereby disrupting the NTHI biofilm. Unclassifiable Haemophilus influenzae biofilms were grown in supplemented BHI medium on 8-well chamber coverslip slides at 37°C and 5% CO2. After 24 hours, the biofilms were treated as indicated. (Figure 38A) After 40 hours, the biofilms were washed, probed with primary antibody against DNABII protein (pale gray dots) and fluorescent secondary antibody, stained with DAPI (gray areas), and visualized by fluorescence microscopy. (Figure 38B) Medium was collected from the biofilm cultures and analyzed by Western blotting using a primary antibody that recognizes DNABII protein. HMGB1 treatment displaced the 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 and 39B demonstrate that HMGB1 promotes biofilm lysis in an experimental model of OM (chinchilla host). On days 4 and 5 post-NTHI infection, diluents or 5 μg rHMGB1 or mHMGB1 were directly delivered to the middle ear of chinchillas. Animals were sacrificed 24 hours later, and their middle ears were imaged (Figure 39A) and scored blindly based on the criteria described below (Figure 39A) (Figure 39B). Bars represent SEM. ***P<0.001. The imaging and scoring demonstrate that HMGB1 promoted the clearance of existing NTHI biofilms in situ.

[0058] [Figure 40]Figures 40A-40C show that HMGB1 disrupts Burkholderia cenocepacia biofilms. (Figure 40A) B. cenocepacia biofilms were grown in LB medium on 8-well chamber coverslip slides at 37°C and 5% CO2. After 24 hours, the biofilms were treated with 5 mg / mL HMGB1. After a total of 40 hours, the biofilms were stained with LIVE / DEAD®, visualized by CLSM, and analyzed by COMSTAT. Bars represent SEM. (Figure 40B) C57BL / 6 mice were infected with 107 CFU it and simultaneously given 5 mg rHMGB1 or mHMGB1, a C45S non-inflammatory variant. B. cenocepacia aggregates were visible by fluorescence microscopy in sections probed with α-B. cenocepacia antibody (gray). After 18 hours, CFU was quantified in BAL (Figure 40C). Bars represent SD. *P<0.05, ***P<0.001. HMGB1 treatment significantly disrupts B. cenocepacia biofilm in situ.

[0059] [Figure 41] Figure 41 shows that HMGB1 reverts polyamine-inducible Z-DNA to a 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-inducible Z-DNA substrates.

[0060] [Figure 42]Figures 42A–42D show that both DNABII proteins 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 basal 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 the control (without P11) was assessed by an unpaired t-test, ****P<0.0001. (Figure 42C) Spermidine (Spd, 300 μM) and HU (500 nM) were added at seeding. Biofilms were quantified in the same manner as in (Figure 42B). Compared to the control (without P11), **P<0.01, ***P<0.001, and ****P<0.0001. Representative images of biofilms formed under the conditions quantified in (Figure 42D)(Figure 42C) are shown in the upper right, with mean biomass. P11 addition prevents biofilm formation in a dose-dependent manner. Exogenous spermidine and HU together restore biofilm development, but neither alone does, suggesting that the P11 anti-biofilm activity is the result of titration of structural components (polyamines, DNABII proteins) detached from the biofilm EPS without direct contact.

[0061] [Figure 43]Figures 43A and 43B 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 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), while values ​​around 3.2 indicate 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. Next, the wells were 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). The anti-B and anti-Z antibodies were specific to their respective DNA morphologies.

[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 (pale gray; 1st from left), anti-anti-spermidine (pale gray; 2nd from left), anti-DNABII (gray; 3rd from left), or anti-Z-DNA (white; 4th from left (elft)). Mature biofilms increase the uptake of each TEDS component into the EPS over time.

[0063] [Figure 45]Figure 45 shows that polyamines and DNABII proteins stimulate DNase-resistant Z-DNA. Immunofluorescence images of EPS scaffold mimes formed de novo by adding DNABII protein (HUNTHI, 500 nM) and spermidine (300 μM) to purified genomic DNA (2 μg / ml) and incubation at 37°C for 16 hours. Biofilm scaffold mimes 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 mime structure, and DNase addition selectively excluded B-DNA while Z-DNA remained intact. Thus, polyamines and DNABII proteins can induce a DNase-resistant state by converting eDNA from B-DNA to Z-DNA.

[0064] [Figure 46]Figure 46 shows the presence of TEDS and Z-DNA within biofilm EPS formed by multiple human pathogens. (A) Top: Immunofluorescence CLSM image of the pointed biofilm, either naive or probed with anti-HU (gray) and anti-spermidine (Spd, light grayish white) antibodies; co-localization is white. Bottom: Immunofluorescence CLSM image of the biofilm formed over 40 hours of the pointed bacterium, either naive 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 multibacterial biofilms. Z-DNA is an essential part of the EPS of multibacterial biofilms at different steady-state levels.

[0065] [Figure 47] Figures 47A and 47B show the presence of Z-DNA in in vivo and ex vivo samples. Left: (Figure 47A) NTHI biofilm in experimentally NTHI-induced OM and (Figure 47B) Z-DNA (white) and B-DNA (dark gray) in sputum from a cystic fibrosis patient. Inset, negative control. Scale bar 10 μm. Right: (Figure 47A) NTHI biofilm in experimentally NTHI-induced OM and (Figure 47B) DNABII protein (gray) and spermidine (Spd, light grayish-white) in 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 ear infected with NTHI biofilm. Z-DNA is an integral part of EPS in multiple bacterial biofilms at different steady-state levels.

[0066] [Figure 48] Figures 48A and 48B show that Z-DNA in biofilms forms nuclease-resistant scaffolds. (Figure 48A) A biofilm formed by the indicated bacterium (Kp = K. pneumoniae) for 24 hours was incubated with DNase (Pulmozyme®; 40 U / ml) for a further 16 hours. The biofilm was 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) The change in the abundance of B-DNA or Z-DNA was quantified using ImageJ 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 against the fluorescence signal of FM4-64® stained cells. Bars represent SEM. Statistical significance compared to the control (without DNase, dotted line) was assessed by paired t-tests: *P<0.05;****P<0.0001. B-DNA is degraded by DNase, but Z-DNA is resistant and functions to maintain the structural integrity of biofilm EPS.

[0067] [Figure 49] Figure 49 shows that Z-DNA stabilization stimulates biofilm formation. NTHI and UPEC biofilm growth was initiated in 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 the control (no antibody) was assessed by paired t-tests, *P<0.05, **P<0.01. Anti-Z-DNA antibody stimulated biofilm formation in a dose-dependent manner, while untreated antibody did not.

[0068] [Figure 50]Figures 50A and 50B demonstrate that an equilibrium shift in the conversion of B-DNA to Z-DNA alters biofilm formation. NTHI biofilms formed over 24 hours were incubated with cerium chloride (CeCl3) or chloroquine for a further 16 hours and probed untreated or with anti-Z-DNA (white) by immunofluorescence CLSM. (Figure 50A) A representative image of the NTHI biofilm indicates an increase in Z-DNA abundance after the addition of 1 mM CeCl3. (Figure 50B) A representative image of the NTHI biofilm indicates a decrease in Z-DNA abundance after the addition of 1 μM chloroquine.

[0069] [Figure 51] Figures 51A and 51B show that an equilibrium shift in the conversion of B-DNA to Z-DNA alters biofilm formation. NTHI biofilms formed over 24 hours were incubated for an additional 16 hours with cerium chloride (CeCl3) (Figure 51A) or chloroquine (Figure 51B). 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 controls was assessed by paired t-tests, *P<0.05, **P<0.01. CeCl3 increased Z-DNA and biofilm formation, while chloroquine decreased Z-DNA and biofilm development.

[0070] [Figure 52]Figures 52A and 52B demonstrate 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. The addition of RNase A stimulated biofilm formation in a dose-dependent manner, likely by the release of polyamines, which are probably the decisive 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 similarly to (Figure 52A). Guanosine monophosphate (GMP) was used as a negative control. Bars represent SEM. Statistical significance compared to controls (without RNase A or tRNA / GMP) was assessed by paired t-tests, *P<0.05, **P<0.01. The addition of tRNA (not GMP) likely disrupted the initial NTHI biofilm in a dose-dependent manner, presumably by sequestration of polyamines from the biofilm EPS.

[0071] [Figure 53]Figure 53 shows that DNABII and polyamines synergistically convert (covert) B-DNA to Z-DNA. In one embodiment, the active ingredient is administered in the absence of DNase. Left: Genomic DNA (2 μg / mL) was incubated at 37°C for 2 hours with spermidine (spd: 300 μM), HU (500 nM), or a combination of both spd and HU. Incubation with 3.6 M NaCl and gDNA was used as a positive Z-DNA control. Center: gDNA (2 μg / mL) was incubated with gradually increasing concentrations of cerium chloride (CeCl3), 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 conversion from B-DNA to Z-DNA. Absorbance values ​​were measured at 260 nm and 295 nm, 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 spectroscopic absorbance ratio assays, CeCl3, along with DNABII and polyamines, synergistically induced Z-DNA, while chloroquine prevented Z-DNA conversion. [Modes for carrying out the invention]

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the field to which this disclosure belongs. All nucleotide sequences provided herein are shown in the 5'-3' direction. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but certain non-limiting exemplary methods, devices and materials are described herein. All technical and patent publications referenced herein are incorporated herein by reference in their entirety. Nothing herein should be construed as an acknowledgment that no prior invention grants this disclosure the right to precede such disclosure.

[0073] The implementation of this disclosure will, unless otherwise indicated, utilize prior art in tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which is within the scope of the art in that field. For example, 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 th 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. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London; and Herzenberg et al. eds. See (1996) Weir's Handbook of Experimental Immunology.

[0074] All numerical values, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) in increments of 1.0 or 0.1 as appropriate, or by variations of + / - 15%, or 10%, or 5%, or 2%. It should be understood that, although not always explicitly stated, all numerical values ​​are preceded by the term "approximately." Furthermore, although not always explicitly stated, it should be understood that the reagents described herein are merely illustrative, and equivalents of such reagents are known in the art.

[0075] As used herein and in the claims, the singular forms “a,” “an,” and “the” include plural nouns unless the context clearly specifies otherwise. For example, the term “polypeptide (a polypeptide)” includes plural polypeptides, including mixtures thereof.

[0076] Where used herein, the term “comprising” is intended to mean that a composition and method includes the listed elements but does not exclude others. “Essentially consisting of” means, when used to define a composition and method, to exclude any other elements that are essentially important to the combination for the intended use. Thus, a composition essentially consisting of the elements defined herein does not exclude trace impurities from isolation and purification methods, and pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, etc. “Consists of” means to exclude more than other components and trace elements of 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] A "biofilm" refers to an organized community of microorganisms that may adhere to structural surfaces, whether organic or inorganic, along with macromolecules such as DNA secreted and / or released by microorganisms. Biofilms are highly resistant to microbiotics and antimicrobial agents. They survive on gingival 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. Centers for Disease Control estimate that more than 65% of hospital-acquired infections are caused by biofilms. They cause chronic vaginal infections and lead to life-threatening systemic infections in people with compromised immune systems. Biofilms are also involved in many diseases. For example, patients with cystic fibrosis often have Pseudomonas infections, which result in antibiotic-resistant biofilms.

[0078] The terms "inhibit, compete, or titrate" refer to reducing the formation of the DNA / protein matrix, which is a component of microbial biofilms.

[0079] "DNABII polypeptides or proteins" are intended to be DNA-binding proteins or polypeptides consisting of a DNA-binding domain and therefore having 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) proteins and histone-like proteins (HU) from E. coli strain U93. 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] "Integrated 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. In E. coli, the genes encoding IHF protein subunits are himA (Genbank accession number POA6X7.1) and himD (POA6Y1.1). 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. Patent 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 active agent. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova. HMGB1 is a small protein of 215 amino acids (approximately 30 kda) consisting of three domains: two positively charged domains A and B boxes, each composed of 80 amino acids, and a loaded electrocarbosyl-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 amino acids in bold (amino acids 1-70) represent A-box domains. The italicized amino acids (amino acids 88-164) 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, e.g., A-box domain, B-box domain, A and B-box domain (AB-box domain), C-tail domain, and N-domain (amino acids 1-185). In one embodiment, the fragment essentially consists of a polypeptide containing a C-terminal domain or a B-box domain.

[0082] "HU" or "histone-like protein from E. coli strain U93" typically refers to a class of heterodimeric proteins 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 HU proteins are commercially available from Abeam.

[0083] The terms "surface antigen" or "surface protein" refer to proteins or peptides on the surface of cells, such as bacterial cells. Examples of surface antigens include 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 pathogenic bacteria that can cause a wide variety of infections, such as ear infections, eye infections, and sinusitis. Many different strains of Haemophilus influenzae have been isolated and possess the IhfA gene or protein. Some non-exclusive examples of various strains of Haemophilus influenzae include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0085] "Microbial DNA" refers to single-stranded or double-stranded DNA from microorganisms that produce biofilms.

[0086] To “inhibit, prevent, or disrupt” a biofilm means to intend a preventive or therapeutic reduction of the biofilm’s structure.

[0087] "Bent polynucleotide" refers to a double-stranded polynucleotide containing small loops on one strand that do not pair with the other strand. In some embodiments, the loops are 1 to about 20 nucleotides long, or 2 to about 15 nucleotides long, or 3 to about 12 nucleotides long, or 4 to about 10 nucleotides long, or have about 4, 5 or 6 or 7 or 8 or 9 or 10 nucleotides.

[0088] The “subject” of a diagnosis or treatment is a cell or animal, e.g., a mammal or a human. Non-human animals used for diagnosis or treatment are non-human animals or animal models used for infection, e.g., primates, murids, e.g., rats, mice, chinchillas; canids, e.g., dogs; rabbits, e.g., rabbits; livestock, sports animals, and pets. The terms “subject,” “host,” “individual,” and “patient,” where used interchangeably herein, refer to an animal, typically a mammal. Non-exclusive examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domesticated animals (e.g., dogs and cats), livestock animals (e.g., horses, cattle, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, mammal is human. The mammal may be of any age or developmental stage (e.g., adult, adolescent, juvenile, nymph, or in utero). The mammal may be male or female. In some embodiments, the subject is human.

[0089] The terms “protein,” “peptide,” and “polypeptide” are interchangeable and used in their broadest sense to refer to compounds of two or more subunit amino acids, amino acid analogs, or peptidomimetics. Subunits may be linked by peptide bonds. In other embodiments, subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may constitute a protein sequence or peptide 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 polymeric forms of any nucleotide or analogue of a deoxyribonucleotide or ribonucleotide of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include: genes or gene fragments (e.g., probes, primers, EST 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 may include modified nucleotides, e.g., methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure, where present, may be given before or after the assembly of the polynucleotide. The sequence of a nucleotide may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeled components. Furthermore, this term 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 two complementary single-stranded forms that are known or expected 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. Therefore, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database on a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.

[0092] The terms “isolated” or “recombinant,” as used herein for nucleic acids, e.g., DNA or RNA, refer to molecules isolated from other DNA or RNA and polypeptides, respectively, that are present in their natural sources as macromolecules. The term “isolated or recombinant nucleic acid” means that it includes nucleic acid fragments that do not exist naturally as fragments and are not found in nature. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins isolated from other cellular proteins and means that it includes both purified polypeptides and recombinant polypeptides. In other embodiments, the term “isolated or recombinant” means that it is isolated from cellular components and, otherwise, cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof that normally associate in nature. For example, an isolated cell is a cell isolated from a tissue or cell of a dissimilar phenotype or genotype. An isolated polynucleotide is one that is isolated from the 3' and 5' adjacent nucleotides with which it normally associates in its native or natural environment, e.g., on a chromosome. As will be apparent to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies or their fragments(s) that do not exist in nature do not require "isolation" to distinguish them from their naturally occurring counterparts.

[0093] Where this disclosure relates to polypeptides, proteins, polynucleotides, or antibodies, it should be inferred, without express enumeration and unless otherwise intended, that equivalents or bioequivalents of such are intended within the scope of this disclosure. Where used herein, the term “its bioequivalent” is intended to be synonymous with “its equivalent” when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid, and is intended to have minimal homology while still maintaining the desired structure or functionality. Unless specifically enumerated herein, any polynucleotide, polypeptide, or protein mentioned herein is also intended to include its equivalent. In one embodiment, an equivalent polynucleotide is a polynucleotide that, under stringent conditions, hybridizes to the polynucleotide or polynucleotide complement described herein for use in the manner described. In another embodiment, the equivalent antibody or antigen-binding polypeptide is intended to bind to a reference antibody or antigen-binding fragment with an affinity of at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or higher. In another embodiment, the equivalent competes for the binding of the antibody or antigen-binding fragment to its antigen under a competitive ELISA assay. In another embodiment, the equivalent is intended to exhibit substantially equivalent biological activity to a reference protein, polypeptide, or nucleic acid with at least about 80% homology or identity, and at least about 85%, or at least about 90%, or at least about 95%, or at least 98% percent homology or identity.

[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” with respect to another sequence means that, when aligned, that percentage of bases (or amino acids) is the same in comparison to the two sequences. Alignment and percentage homology or sequence identity can be determined using software programs known in the art, e.g., 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 alignment. A non-limiting exemplary alignment program that uses default parameters is BLAST. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: gene code=standard; filter=none; strand=both; cutoff=60; prediction=10; matrix=BLOSUM62; description=50 sequences; classification=high score; database=non-duplicate 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 web address:align.genome.jp (last accessed March 7, 2011)).

[0095] "Homologie," "identity," or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homologie can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. Molecules are homologous at a position if the positions in the sequences being compared are occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of aligned or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity or less than 25% identity with any of the sequences in this disclosure.

[0096] Furthermore, "homology," "identity," or "similarity" can 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 that is stabilized via hydrogen bonds between the bases of nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific mode. The complex may consist of two strands forming a double-stranded structure, three or more strands forming a polychain complex, a single self-hybridized strand, or any combination thereof. A hybridization reaction may constitute a step in a broader process, such as the initiation of a PCR reaction or enzymatic cleavage of a polynucleotide by a ribozyme.

[0098] Examples of stringent hybridization conditions include: incubation temperature of approximately 25°C to 37°C; hybridization buffer concentration of approximately 6×SSC to 10×SSC; formamide concentration of approximately 0% to 25%; and washing solution of approximately 4×SSC to 8×SSC. Examples of moderate hybridization conditions include: incubation temperature of approximately 40°C to 50°C; buffer concentration of approximately 9×SSC to 2×SSC; formamide concentration of approximately 30% to 50%; and washing solution of approximately 5×SSC to 2×SSC. Examples of highly stringent conditions include: incubation temperature of approximately 55°C to 68°C; buffer concentration of approximately 1×SSC to 0.1×SSC; formamide concentration of approximately 55% to 75%; and washing solution of approximately 1×SSC, 0.1×SSC, or deionized water. Generally, the hybridization incubation time is 5 minutes to 24 hours, accompanied by one, two or more washing steps, with a washing incubation time of approximately 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems may 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 subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0100] The term “coding” refers to a polynucleotide that, when applied to a polynucleotide, is said to “code” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce mRNA and polypeptides and / or fragments thereof. The antisense strand is the complement of such nucleic acid, and the coding sequence can be inferred from it.

[0101] As used herein, terms such as “to treat” and “treatment” are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in that it is a partial or complete cure of a disorder and / or adverse effects resulting from the disorder. As used herein, “to treat” or “treatment” of a disease in a subject may mean (1) preventing the occurrence of symptoms or disease in a subject that is susceptible to the disease or has not yet shown symptoms of the disease; (2) inhibiting or preventing the onset of the disease; or (3) improving or reducing the symptoms of the disease or disease. As understood in the art, “treatment” is a method for obtaining a beneficial or desired result, including a clinical outcome. For the purposes of this technology, beneficial or desired outcomes may include, but are not limited to, one or more: reduction or improvement of one or more symptoms; attenuation of the degree of a condition (including disease); a stabilization (i.e., non-worsening) of a condition (including disease); delay or slowing of a condition (including disease); or progression, improvement or mitigation of a condition (including disease), condition, and remission (whether partial or total), whether detectable or undetectable. In one embodiment, the treatment excludes prevention. If the disease is SLE (systemic lupus erythematosus) and / or cystic fibrosis (CF), the evidence of the treatment includes evidence of inflammation and / or a reduction in the level of autoimmune activity or symptoms.

[0102] Prevention aims to prevent damage or effect in vitro or in vivo in systems or subjects susceptible to such damage or effect. One example of this is preventing biofilm formation in systems infected with microorganisms known to produce biofilms.

[0103] "Composition" is intended to mean a combination of an activator and another compound or composition, either inert (e.g., a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, etc., and includes a pharmaceutically acceptable carrier. The carrier also includes pharmaceutical excipients and additives, proteins, peptides, amino acids, lipids and carbohydrates (e.g., monosaccharides, di, tri, tetra-oligosaccharides and oligosaccharides; derivatized sugars, such as alditol, aldonic acid, esterified sugars, etc.; and polysaccharides or sugar polymers), which may exist alone or in combinations of 1 to 99.99% by weight or volume. Exemplary protein excipients include serum albumin, e.g., human serum albumin (HSA). This includes albumin, recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acids / antibody components that can also function in buffering capacity include alanine, arginine, glycine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Furthermore, carbohydrate excipients are intended within the scope of this technology, and examples include monosaccharides, e.g., fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides, e.g., lactose, sucrose, trehalose, cellobiose; polysaccharides, e.g., raffinose, melegitose, maltodextrin, dextran, starch; and algitols, e.g., mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myo-inositol.

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

[0105] "Pharmacologically acceptable carrier" means any diluent, excipient, or carrier that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, e.g., human serum albumin, buffers, e.g., phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. These can be selected in accordance with the intended dosage form, namely oral tablets, capsules, elixirs, syrups, etc., and in accordance with conventional pharmaceutical practices.

[0106] The compositions used in accordance with this disclosure may be packaged in dosing unit form for ease of administration and uniformity of dosage. The terms “unit dose” or “dosage” refer to physically distinct units suitable for use in the subject, each unit containing a predetermined amount of the composition calculated to produce a desired response in conjunction with its administration, i.e., an appropriate route and regimen. The amount to be administered, both by the number of treatments and the unit dose, depends on the desired outcome and / or protection. The exact amount of the composition is also subject to the judgment of the practitioner and is specific to each individual. Factors influencing the dosage include the physical and clinical condition of the subject, the route of administration, the purpose of the intended treatment (symptom relief versus cure), and the potency, stability, and toxicity of the particular composition. In formulation, the liquid formulation is administered in a manner compatible with the administration formulation and in a therapeutically or prophylactically effective amount. The formulation is readily administered in various dosage forms, e.g., the types of injectable liquid formulations described herein.

[0107] The term “contact” means direct or indirect binding or interaction between two or more things. 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 then acts on a second reference entity. Contact, as used herein, includes contact in solution, in a solid phase, in vitro, ex vivo, in cells, and iv vivo. Contact in iv vivo may be called administration or dosing.

[0108] "Bioactive agent" or the activators disclosed herein means one or more isolated or recombinant polypeptides, isolated or recombinant polynucleotides, vectors, isolated host cells, or antibodies, and compositions comprising one or more of these.

[0109] "Administration" can be delivered in a single dose, continuously or intermittently throughout the course of treatment. The most effective means of administration and methods for determining the dosage are known to those skilled in the art and vary depending on the composition used in treatment, the purpose of treatment, the target cells being treated, and the subject being treated. Single or multiple administrations may be given at dose levels and patterns selected by the treating physician. Suitable formulations for administration and methods for administering their active ingredients are known in the art. The route of administration can also 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 in treatment, the purpose of treatment, the health status or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, intranasal administration, injection, and topical application.

[0110] The active ingredients of this disclosure may be administered for therapeutic purposes by any preferred route of administration. It is understood that the optimal route will vary depending on the recipient's condition and age, as well as the disease being treated.

[0111] The term "effective dose" refers to an amount sufficient to achieve the desired effect. With respect to therapeutic or preventive applications, the effective dose depends on the type and severity of the condition, as well as the characteristics of the individual subject, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. With respect to immunogenic compositions, in some embodiments, the effective dose is sufficient to produce a protective response to a pathogen. In other embodiments, the effective dose of an immunogenic composition is sufficient to produce antibodies against an antigen. In some embodiments, the effective dose is the amount necessary to provide passive immunity to a subject requiring it. With respect to immunogenic compositions, in some embodiments, the effective dose depends, in addition to the factors described above, on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system. Those skilled in the art can determine an appropriate dose depending on these and other factors.

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

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

[0114] A "liposome" is a microscopic vesicle consisting of a concentric lipid bilayer. Structurally, liposomes range in size and shape from tubular to spherical, with dimensions ranging from several hundred angstroms to a fraction of a millimeter. The vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex, which provides the lipid composition of the outer layer. These are 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 dioleoylphosphatidylethanolamine (DOPE), which have hydrocarbon chain lengths ranging from 14 to 22 and are saturated or have one or more double C=C bonds. Examples of lipids that can produce stable liposomes, either alone or in combination with other lipid components, include phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC). Soy phosphatidylcholine), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC) These are palmitoyloteoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1-carboxylate (DOPE-mal). Additional phosphorus-free lipids that can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, 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).Loaded 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 layered structure. The three classifications developed by the December 1977 New York Academy Scientific Conference, “Liposomes and Their Use in Biology and Medicine,” are multi-lamellar vesicles (MLV), small uni-lamellar vesicles (SUV), and large uni-lamellar vesicles (LUV). Bioactive agents may be encapsulated in such for administration according to the methods described herein.

[0115] A "micelle" is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with a hydrophilic "head" region in contact with the surrounding solvent and a hydrophobic tail region isolated at the micelle center. This type of micelle is known as a normal-phase micelle (oil-in-water micelle). An inverse micelle has a head group at the center and a tail facing outward (water-in-oil micelle). Micelles can be used to conjugate polynucleotides, polypeptides, antibodies, or compositions described herein to facilitate their efficient delivery to target cells or tissues.

[0116] The phrase "pharmaceutically acceptable polymers" refers to a group of compounds that can be conjugated to one or more polypeptides as described herein. The conjugation of polymers to polypeptides is intended to extend the half-life of the polypeptides 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] A “gene delivery medium” is defined as any molecule capable of delivering an inserted polynucleotide to a host cell. Examples of gene delivery mediums include liposomes; micelles; biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metallic particles; as well as bacteria or viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant mediums typically used in the art for expression in various eukaryotic and prokaryotic hosts, and for gene therapy and simple protein expression.

[0118] The polynucleotides disclosed herein may be delivered to cells or tissues using gene delivery media. “Genetic delivery,” “gene transfer,” “transduction,” etc., as used herein, are terms referring to the introduction of exogenous polynucleotides (sometimes called “transgenes”) into host cells, regardless of the method used for introduction. Such methods include a variety of well-known techniques, e.g., vector-mediated gene transfer (e.g., viral infection / transfection, or by 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 for the introduction of polynucleotides). The introduced polynucleotides may be maintained stably or transiently in host cells. Stable maintenance typically requires that the introduced polynucleotides either contain a replication origin compatible with the host cell or integrate into a replicon of the host cell, e.g., an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As is known in the art and as described herein, some vectors can mediate 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 chromosomal DNA. Often, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population and typically offer selective benefits under given environmental conditions. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins they produce may act as toxins under similar conditions.

[0121] The "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 a plasmid that contains the gene that makes cells resistant to a particular antibiotic, and a multiple cloning site (MCS: or polylinker), which is a short region containing several commonly used restriction sites that allow for the easy insertion of DNA fragments at this location. Another major use of plasmids is to produce large quantities of protein. In this case, researchers grow bacteria containing a plasmid that harbors the gene of interest. Just as the bacteria produce the protein that gives them their antibiotic resistance, it can also be induced to produce large quantities of protein from the inserted gene. This is an inexpensive and easy way to produce large quantities of a gene or the protein that the gene encodes.

[0122] A "yeast artificial chromosome" or "YAC (yeast artificial chromosome)" refers to a vector used to clone large DNA fragments (larger than 100kb, up to 3000kb). It is an artificially constructed chromosome containing telomere sequences, centromere sequences, and origin of replication sequences necessary for replication and preservation in yeast cells. When assembled using an initial circular plasmid, they can be made linear using restriction enzymes, and then the desired sequence or gene can be added to the linear molecule using DNA ligase and adherent ends. Yeast expression vectors, such as YAC, 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 less stable than BACs and can produce chimeric effects.

[0123] A "viral vector" is defined as a recombinant virus or viral particle containing polynucleotides to be delivered to a host cell in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and alphavirus vectors. Infectious tobacco mosaic virus (TMV)-based vectors may be used as protein manufacturers 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 Semlik Forest virus-based vectors and Sindbis virus-based vectors, are also being 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, the vector construct refers to a polynucleotide containing the retroviral genome or a portion thereof and the therapeutic gene.

[0124] As used herein, “retroviral gene transfer” or “retroviral transduction” are synonymous 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 it may be modified to enter the cell by binding to a different host cell surface receptor or ligand. As used herein, a retroviral vector refers to a viral particle that can introduce exogenous nucleic acids into a cell through a virus or a virus-like entry mechanism.

[0125] Retroviruses carry their genetic information in the form of RNA; however, when a virus infects a cell, the RNA is reverse-transcribed into DNA, which is then incorporated into the infected cell's genomic DNA. This incorporated DNA form is called a provirus.

[0126] In embodiments where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), the vector construct refers to a polynucleotide containing the viral genome or a portion thereof and the introduced gene. Adenoviruses (Ad) are a relatively well-characterized group of viral homologs, including more than 50 serotypes. See, for example, PCT International Publication No. WO95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors, particularly recombinant Ad-derived vectors with reduced potential for the generation of recombinant and wild-type viruses, have also been constructed. See PCT International Publication Nos. WO95 / 00655 and WO95 / 11984, where 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 on which polynucleotides can be operably ligated are well known in the art. Such vectors can transcribe 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 remove extra, potentially inappropriate, selective translation start codons, or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site may be inserted immediately 5' of the start codon to improve expression.

[0128] Furthermore, gene delivery media include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can also be used in the manner disclosed herein. In addition to the delivery of polynucleotides to cells or cell populations, the direct introduction of proteins described herein into cells or cell populations may be carried out by non-limiting techniques of protein transfection, or by other non-limiting techniques such as culture conditions that enable enhanced expression and / or promotion of the activity of the proteins disclosed herein.

[0129] As used herein, the terms “antibody,” “multiple antibodies,” and “immunoglobulin” include the entire antibody and any antigen-binding fragment or a single chain thereof. Therefore, the term “antibody” includes any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule. Furthermore, the terms “antibody,” “multiple antibodies,” and “immunoglobulin” include any isotype of immunoglobulin; non-limitingly, antibody fragments that retain specific binding to antigens, including Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragment, 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 ones. Examples of such entities include, but are not limited to, the complementarity determining region (CDR) of the heavy or light chain or its ligand-binding portion, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the framework (FR) region, or any part 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 the antigen. 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, such as anti-DNABII, anti-IHF, anti-HU, anti-OMP P5, refers to a monoclonal or polyclonal antibody that indicates binding to and / or possession of affinity to a particular protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody is a protein that has affinity for or can bind to proteins other than the protein against which the antibody was produced. For example, an anti-IHF antibody is specifically produced against the IHF protein, but can also bind to other related proteins through sequence homology or structural homology.

[0130] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific antibodies), or antibody fragments, insofar as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, such as rodents, rats, sheep, and canines.

[0131] As used herein, “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population. Since each monoclonal antibody is directed to a single determinant on an antigen, monoclonal antibodies are highly specific. Antibodies can be detected by labeling with, for example, radioisotopes, enzymes that produce detectable products, fluorescent proteins, etc. Antibodies can be further conjugated with other parts, such as members of specific binding pairs, e.g., biotin (a member of the biotin-avidin specific binding pair). Antibodies can also be conjugated to solid supports, including, but not limited to, polystyrene plates or beads.

[0132] Monoclonal antibodies can be produced using hybridoma techniques or recombinant DNA methods known in the art. Hybridomas are cells produced in the laboratory from the fusion of antibody-producing lymphocytes with non-antibody-producing cancer cells, typically myeloma or lymphoma. Hybridomas proliferate and produce serial samples of specific monoclonal antibodies. Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to antigens of interest and screening of antibody display libraries in cells, phages, or similar systems.

[0133] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, e.g., mouse, is grafted onto a human framework sequence. Therefore, as used herein, the term "human antibody" refers to substantially any portion of the protein (e.g., CDR, framework, C). L , C H Domain (for example, 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, antibodies designated for primates (monkeys, baboons, chimpanzees, etc.), rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals designate such species, subgenus, genus, subfamily, and family-specific antibodies. Furthermore, chimeric antibodies include any combination of the above. Such changes or variations, as necessary, retain or reduce immunogenicity in humans or other species compared to unmodified antibodies. Thus, human antibodies are distinct from chimeric antibodies or humanized antibodies. It should be 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 chain and / or light chain) genes. Furthermore, if a human antibody is a single-chain antibody, it may contain linker peptides not found in native human antibodies. For example, Fv may contain a linker peptide, such as 2 to about 8 glycine or other amino acid residues, which connects the variable region of the heavy chain to the variable region of the light chain. Such linker peptides are thought to be of human origin.

[0134] As used herein, a human antibody is “derived” from a specific germline sequence if it is obtained from a system using a human immunoglobulin sequence, for example, by immunizing transgenic mice having a human immunoglobulin gene, or by screening a human immunoglobulin gene library. A human antibody “derived” from a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin. Typically, a 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, when compared with the germline immunoglobulin amino acid sequence of another species (e.g., murine germline sequence), identify the human antibody as a human antibody. In certain cases, a human antibody may be at least 95%, or even more, at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, human antibodies derived from specific human germline sequences exhibit differences of 10 or fewer amino acids from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, human antibodies may exhibit differences of 5 or fewer amino acids from the amino acid sequence encoded by the germline immunoglobulin gene, or even 4, 3, 2, or 1 or fewer amino acids.

[0135] A "human monoclonal antibody" refers to a single antibody exhibiting binding specificity, possessing a variable region and a constant region derived from a human germline immunoglobulin sequence. This term also refers to 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, produced or isolated by recombinant means, e.g., antibodies isolated from transgenic or chromosome-transformed animals (e.g., mice) or hybridomas prepared therefrom for human immunoglobulin genes; antibodies isolated from host cells transformed to express antibodies, e.g., from transfectomas; antibodies isolated from recombinant combinatorial human antibody libraries; and antibodies prepared, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences to 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 be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, if transgenic animals for human Ig sequences are used), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody may be derived from and related to human germline VH and VL sequences, while they may not be naturally present in 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 in which light and heavy chain genes are constructed, typically by genetic engineering, from antibody variable region and constant region genes belonging to different species.

[0138] As used herein, the terms “humanized antibody” or “humanized immunoglobulin” refer to a human / non-human chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the variable region of the recipient are replaced by residues from the variable region (donor antibody) of a non-human species, e.g., mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capability. A humanized antibody may also contain residues not found in the recipient antibody or the donor antibody. Furthermore, a humanized antibody may optionally contain a non-human antibody containing one or more amino acids in the immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region of human immunoglobulin; or in the framework region, constant region, or CDR, replaced with correspondingly positioned amino acids from the human antibody. Generally, humanized antibodies are expected to result in a reduced immune response in the human host compared to the non-humanized version of the same antibody. Humanized antibodies may have conserved amino acid substitutions that do not substantially affect antigen binding or other antibody functions. Conservative substitutions 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 preparations of antibodies derived from various B cell lines. They are mixtures of immunoglobulin molecules secreted in response to specific antigens, each recognizing a different epitope.

[0140] As used herein, the term “antibody derivative” includes full-length antibodies or fragments of antibodies in which one or more amino acids are chemically modified, for example, by alkylation, pegylation, acylation, esterification, or amide formation, in order to link the antibody to a second molecule. This includes, but is not limited to, pegylated antibodies, cysteine-pegylated antibodies, and their variants.

[0141] As used herein, the term "label" refers to a direct or indirect detectable compound or composition conjugated directly or indirectly to a composition to be detected in order to generate a "labeled" composition, e.g., an N-terminal histidine tag (N-His), a magnetic optically active isotope, e.g., 115 Sn, 117 Sn and 119 Sn, a non-radioactive isotope, e.g., 13 C and 15The term refers to N, polynucleotides, or proteins, such as antibodies. It also includes sequences conjugated to polynucleotides that provide a signal during the expression of an inserted sequence, such as green fluorescent protein (GFP). The label may be detectable on its own (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The label may be suitable for small-scale detection or for high-throughput screening. Therefore, suitable labels include, but are not limited to, magnetic optical isotopes, non-radioactive isotopes, radioactive isotopes, fluorescent dyes, chemiluminescent compounds, dyes, and enzymes in proteins. The label may be simply detected or quantified. Simply detected responses generally include responses where the presence is simply confirmed, while quantified responses generally include responses with quantifiable (e.g., numerically reportable) values, such as intensity, polarization, and / or other properties. In luminescence or fluorescence assays, a detectable response may be generated directly using a luminophore or fluorescent phore associated with the assay component actually involved in binding, or indirectly using a luminophore or fluorescent phore associated with another component (e.g., a reporter or indicator). Examples of luminescence labeling that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescence response generally involves a change in the luminescence signal or the generation of a luminescence signal. Suitable methods and luminophores for luminescent labeling assay components are known in the art, e.g., Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th This is described in (ed). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0142] As used herein, the term “immunoconjugate” includes antibodies or antibody derivatives associated with or conjugated with a second active substance, such as 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 listed in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th It is explained in the ed.

[0144] In another embodiment, the fluorescent label is functionalized to promote covalent binding to cellular components present within or on the cell or tissue surface, such as a cell surface marker. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides; all of these can be used to bind the fluorescent label to a second molecule. The choice of functional groups for the fluorescent label depends on the binding site to the linker, active substance, marker, or second labeling agent.

[0145] "Eukaryotic cells" include all living things except the Monera kingdom. They can be easily distinguished by their membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes, or organisms in which cells are organized into complex structures by an internal membrane and cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" includes eukaryotic hosts, such as yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include primates, cattle, pigs, mures, rats, birds, reptiles, and humans.

[0146] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelle 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 (about 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and helical. Instead of undergoing the elaborate replication process of eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus bacteria, E. coli bacteria, and Salmonella bacteria.

[0147] "Native" or "natural" antigens are isolated from natural biological sources and, in the subject, have antigen receptors, particularly T cell antigen receptors (TCRs). A polypeptide, protein, or fragment containing an epitope that can specifically bind to a receptor.

[0148] The terms “antigen” and “antigenicity” refer to molecules that have the ability to be recognized by an antibody, or otherwise, the ability to act as a member of an antibody-ligand pair. “Specific binding” refers to the interaction between an antigen and the variable regions of the heavy and light chains of immunoglobulins. 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 may also include partial molecules. The term “antigenicity” is an adjective reference to a molecule that possesses 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] A “modified antigen” is an antigen having a primary sequence different from the primary sequence of the corresponding wild-type antigen. Modified antigens can be produced by synthesis or recombinant methods and include, but are not limited to, antigenic peptides that are differentially modified during or after translation by, for example, phosphorylation; glycosylation; crosslinking; 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 modified antigens disclosed herein are intended to bind to the same TCR as the natural epitope.

[0150] "Immune response" broadly refers to the antigen-specific response of lymphocytes to exogenous substances. The terms "immunogen" and "immunogenicity" refer to molecules that have the ability to induce an immune response. All immunogens are antigens; however, not all antigens are immunogenic. The immune responses disclosed herein may be humoral (mediated by antibody activity) or cell-mediated (mediated by T cell activation). The response may occur in vivo or in vitro. Those skilled in the art will understand that a variety of 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 nucleic acids that encode molecules capable of inducing an immune response necessarily encode immunogens. Those skilled in the art will further understand that immunogens may include partial molecules, not just full-length molecules.

[0151] The term "passive immunity" refers to the transfer of immunity from one target 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-immune target. Antibody donors and recipients can be human or non-human targets. Depending on the embodiment, antibodies may be polyclonal or monoclonal, may be produced in vitro or in vivo, and may be purified, partially purified or unpurified. In some embodiments described herein, passive immunity is administered to a target in need 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 administered through the administration of isolated or recombinant polynucleotides encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen.

[0152] In the context of this disclosure, “ligand” means polypeptide. In one embodiment, the term “ligand” as used herein refers to any molecule that binds to a specific site on another molecule. In other words, a ligand confers the specificity of an immunoeffector cell or protein to an antibody or to a protein in a reaction with DNA. In one embodiment, the ligand site within the protein is what directly combines with the complementary binding site on the immunoeffector cell.

[0153] As used herein, the term “inducing an immune response in a subject” is a term well understood in the art and is intended to mean that an increase of at least approximately 2, at least approximately 5, at least approximately 10, at least approximately 100, at least approximately 500, or at least approximately 1000, or greater, in the immune response to an antigen (or epitope) after introduction of an antigen (or epitope) to a subject, compared to the immune response before introduction of the antigen (or epitope) to the subject (if any), can be detected or measured. 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, without limitation, using any of the various immunoassays known in the art, including, for example, ELISAs in which the binding of an antibody in a sample to an immobilized antigen (or epitope) is detected with a detectably labeled secondary antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).

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

[0155] Examples of solid-phase supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The properties of the support can be either soluble or insoluble to some extent. The support material can have virtually any possible structural arrangement, as long as the bound molecule can bind to a polynucleotide, polypeptide, or antibody. Thus, the support arrangement can be spherical, as in beads, or cylindrical, as on the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be planar, e.g., a sheet, test paper, or polystyrene beads. Those skilled in the art know of many other suitable supports for binding antibodies or antigens, or can confirm this by using them in routine experiments.

[0156] The term "modulate an immune response" includes inducing (increasing, triggering) an immune response and reducing (suppressing) an immune response. An immunomodulatory method (or protocol) is a method for modulating an immune response in a subject. Form for making this disclosure I. Biofilm Structure and Diseases

[0157] The bacterial reservoirs that maintain chronic and recurrent bacterial infections reside within biofilms, which are communities of bacteria that adhere to surfaces and, in this state, are resistant to clearance by the host immune system and antimicrobial agents. In fact, bacteria in biofilms are typically more than 1000 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 a semi-permeable, self-forming matrix or extracellular macromolecule (EPS) that acts as a physical barrier to environmental hazards and creates conditions for altered physiological functions that restrict metabolism to enhance this resistant state. The components of EPS are specific to each bacterium and include proteins, polysaccharides, lipids, and nucleic acids, but the properties of the EPS must be sufficiently beneficial to the bacterial genus as a whole in order to interact productively (e.g., as metabolic partners). For this purpose, several recent discoveries have suggested the possibility of a universal EPS structure that underlies all eubacteria. Whitchurch et al. (2002) Science. 295(5559) showed that extracellular DNA (eDNA) is a common EPS component and that treatment with bacterial DNases is sufficient to inhibit biofilm formation. While this result has been replicated for several genera, the use of DNases was unable to treat biofilms that were present for longer than one or two days after biofilm seeding, despite the fact that eDNA is evident within biofilms throughout their entire life cycle. Separately, the applicants previously identified DNABII proteins, a family of nucleoid-associated proteins (NAPs) common to all eubacteria, as essential components of eDNA-dependent EPS.Indeed, antibodies directed against DNABII proteins titrate them from bulk medium, thereby shifting the equilibrium of DNABII proteins from an eDNA-bound state to an unbound state, which results in the catastrophic collapse 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 is effective at all stages of biofilm development, demonstrating that eDNA-dependent EPS is an important structure regardless of the biofilm stage. Brockson et al. (2014) Mol Microbiol. 93(6):1246-58. Despite the knowledge that eDNA and members of the DNABII family are essential components of EPS, the complete understanding of EPS structure remains unclear, and DNABII proteins and DNA are insufficient to reproduce functional EPS structure in vitro. The applicants hereby describe that for several human pathogens, as biofilms mature, eDNA-dependent EPS becomes dependent on both DNABII proteins and polyamines, and therefore eDNA shifts from B-DNA to Z-DNA conformation. This latter result is particularly interesting because it may explain why DNases were unable to disrupt mature biofilms, as nucleases only cleave the more classical B-type DNA. Intracellular bacterial nucleoid

[0158] DNA within 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). Collectively, NAPs bind to DNA to create functional structures. Dillon et al. (2010) Nat Rev Microbiol. 8(3):185-95. Of the numerous NAP members present throughout the genus, only the DNABII family is ubiquitous in all eubacteria. Dey et al. (2017) Mol Phylogenet Evol. 107:356-66. Proteins of the DNABII family function as dimers (homodimers or heterodimers depending on the species) and include histone-like proteins HU and IHF. HU binds weakly and nonspecifically to double-stranded DNA (dsDNA) and bends it, but has a fairly high affinity for pre-curved or structured dsDNA3. IHF, like HU, binds to and bends pre-curved / structured DNA with strong preference. Unlike HU, IHF is expressed only by proteobacteria and has preference for specific DNA consensus sequences. Swinger et al. (2004) Curr Opin Struct Biol. 14(1):28-35. Extracellular bacterial nucleoid (nucleoi)

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

[0160] Biofilms are further distinguished from planktonic bacteria by their intercellular communication and transport systems, but their most distinctive feature is their self-produced EPS, which protects commensal biofilm bacteria both by acting as a semi-osmotic barrier and by creating an environment for altered / slowed metabolism; in fact, biofilm bacteria are more than 1000 times more resistant to antibiotics than their planktonic relatives. Ceri et al. (1999) J Clin Microbiol. 37(6):1771-6. Interestingly, the EPS of each bacterium is characteristic and consists 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 general, in chronic infections and always in the environment, they consist of multiple genera and therefore need to be able to interact productively (e.g., co-aggregation 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 means that, despite the diverse EPS composition, each EPS must be sufficiently accommodating to allow diverse bacteria to interact within the biofilm, and further, it suggests that biofilm EPS appear to have a universal underlying structure. eDNA-dependent EPS possesses the qualities of a universally underlying architecture.

[0161] Numerous groups examined eDNA associated with bacterial biofilms from both human 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 collaborators were the first to show that P. aeruginosa biofilms can be inhibited by deoxynuclease I (DNase) treatment (Whitchurch et al. (2002) Science. 295(5559)), indicating that eDNA is an important structural component of EPS. DNases can inhibit early 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 the biofilm matures, it becomes refractory to DNases over time, despite the fact that eDNA is clearly present. Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; Hall-Stoodley et al. 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 result was often interpreted as meaning that eDNA is no longer important for the structural integrity of EPS, the applicants showed that eDNA not only persists but also forms the primary 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 essential for maintaining the structural integrity of biofilm eDNA-scaffolded EPS.

[0162] The applicants have previously shown that the ubiquitous DNABII protein is a structural component of eDNA, and that other NAPs are not (Devaraj et al. (2017) Microbiologyopen.), and that once removed, the eDNA structure is disrupted. Goodman et al. (2011) Mucosal Immunol. 4(6):625-37; 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. In fact, DNABII proteins were found to specifically bind to the vertices (pre-bent DNA) of eDNA scaffolds in biofilms formed in vivo. On the other hand, antibodies directed against DNABII proteins were sufficient to damage the structure of eDNA-scaffolded EPS, resulting in catastrophic collapse of both single-species and multi-species biofilms in all species investigated by the applicants, 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 commensal bacteria, making them detached and thus susceptible to antimicrobial agents and immunosuppressants (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), this demonstrates the importance and universality of this family of proteins within biofilm structures. Assuming the known interactions of the DNABII family with DNA, it was unexpected that the applicants could not create the conditions to reproduce the three-dimensional scaffold-like structures observed in bacterial biofilms using only DNA and DNABII proteins. Polyamines are ubiquitous both inside and outside cells and are required for the eDNA-scaffolded EPS structure of biofilms.

[0163] Polyamines are typically short organic molecules that are positively charged (basic) at neutral pH and generally contain numerous primary amines derived from the decarboxylation of amino acids (Figure 1B). Michael et al. (2016) Biochem J. 473(15):2315-29. Polyamines are naturally ubiquitous and found at high mM concentrations both intracellularly and extracellularly (Tabor et al. (1985) Microbiol (Rev. 49(1):81-99), spermidine, spermine, and putrescine are the most abundant. Polyamines are involved in numerous processes in bacterial physiological function, but they are perhaps most important for eDNA-scaffolded EPS for five reasons. Firstly, they bind to DNA and neutralize the negative charge of the phosphate backbone, thereby altering the DNA structure. Bachrach et al. (2005) Curr Protein Pept Sci. 6(6):559-66; Pasini et al. (2014) Amino Acids. 46(3):595-603. Secondly, while polyamines tend to promote protein-DNA interactions at low concentrations (mM), they tend to interfere at higher concentrations (mM), with the sole exception being the DNABII-DNA interaction, which causes DNA to form thicker fibers. Sarkar et al. (2009) Biochemistry. 48(4):667-75; Sarkar et al. (2007) Nucleic Acids Res. 35(3):951 61. Thirdly, it is known that polyamine synthesis is induced during biofilm formation, and that some of these effects occur extracellularly (although this has so far only been studied in bacterial membranes). Wilton et al. (2015) Antimicrob Agents Chemother. 60(1):544-53. Fourthly, atomic force microscopy (AFM) experiments visualizing polyamine-DNA mixtures have revealed structures highly similar to biofilm eDNA scaffolds (Figure 1C). Finally, polyamines can induce the conversion of native B-type DNA to Z-type 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. The conversion of B-DNA to Z-DNA may be a novel method for making eDNA-scaffolded EPS nuclease-resistant and creating stable structural materials.

[0164] B-DNA and Z-DNA are distinct conformations of dsDNA that exist in equilibrium, with B-DNA being dominant under most physiological conditions. Alternating purines and pyrimidines (particularly dGdC) tend to exist more as Z-DNA at high salt concentrations (monovalent or divalent cations) or under negative superhelics. Pohl et al. (1983) Cold Spring Harb Symp Quant Biol. 47 Pt 1:113-7; Pohl et al. (1986) Proc Natl Acad Sci US A. 83(14):4983-7. In the latter case, the region that tends to form Z-DNA may be temporarily juxtaposed with B-DNA during transcription if negative superhelions are 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 its larger size, identifying hydrogen bond donors and acceptors for each nucleotide base. In contrast, the major groove is absent in Z-DNA, and most of their binding contacts are found on the convex side. 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 be able to 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 sequence shifts to Z-DNA in the presence of certain polyamines (spermidine and spermine) at physiological concentrations (100 mM). Second, Z-DNA tends to aggregate and form fibers. Chaires et al. (1988) J Biomol Struct Dyn. 5(6):1187-207. Since phosphates in the Z-DNA backbone are more tightly bound together than phosphates in B-DNA, neutralization of the strong negative charge of the phosphate backbone (e.g., polyamines) is favorable to Z-DNA but also tolerant of DNA aggregation. Thirdly, Z-DNA is stiffer than B-DNA and has nearly three times the persistence length, consistent with the linear fibers observed by the applicants in the eDNA scaffold (Thomas et al. (1983) Nucleic Acids). Res. 11(6):1919-30) (Figures 1A, 2, and 3). And finally, Z-DNA is nuclease-resistant. Unlike standard B-DNA binding proteins, there are only a few known proteins that recognize Z-DNA (Athanasiadis et al. (2012) Semin Cell Dev Biol. 23(3):275-80) (however, Z-DNA versus B-DNA discriminant antibodies are available). Bergen et al. (1987) J Immunol. 139(3):743-8. Interestingly, Z-DNA binding proteins are homologous and bind to Z-DNA with 1,000–10,000 times higher affinity than B-DNA (Herbert et al. (1996) J Biol Chem. 271(20):11595-8), induce Z-DNA conformation in the predispositional sequence, and are part of the innate immune system that detects the presence of microbial DNA, at least in the case of ZBP1. Athanasiadis et al. (2012) Semin Cell Dev Biol. 23(3):275-80. Importantly, despite the fact that Watson-Crick base pairing is conserved, proteins that use B-DNA as a substrate (e.g., nucleases) cannot recognize the same DNA sequence in the Z configuration and therefore cannot function against it. II. A three-part approach

[0165] The applicants previously showed that eDNA-DNABII interactions work 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 disrupting these interactions ex vivo (Gustave et al. (2013) It has been shown to yield positive results in (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). Although both DNA and DNABII proteins have been shown to be necessary, they are not sufficient to reproduce the EPS scaffold architecture. A tripartite eDNA-dependent scaffold (TEDS) of eDNA-DNABII-dependent EPS, which relies on the presence and relative location of (1) eDNA, (2) DNABII proteins and newly discovered EPS components, and (3) polyamines, is disclosed herein.

[0166] In one embodiment, the applicants demonstrate that polyamines, in addition to eDNA and DNABII proteins, are essential components of the TEDS structure in bacterial biofilms. Secondly, the applicants demonstrate that all three of these components together promote the formation of a universal EPS that can foster productive interactions within the bacterial genus in a protective biofilm state. Thirdly, using these components, the applicants define and reproduce this universal structure, providing evidence consistent with the observation of thick double-stranded DNA fibers, induction of a nuclease-resistant state, and demonstrating 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 work in conjunction with DNABII proteins to direct the assembly of eDNA scaffolds.

[0167] A chinchilla model of acute otitis media induced by NTHI faithfully reproduces the disease process and pathophysiology in humans (Bakaletz et al. (2009) Expert Rev Vaccines. 8(8):1063-82) and relies on refractory biofilms in the middle ear. Using this model, the applicants previously showed that DNABII proteins associate with eDNA and localize to the vertices of eDNA strands (Figure 1A) (Goodman et al. (2011) Mucosal Immunol. 4(6):625-37), and that these eDNA strands appear remarkably similar to polyamines visualized in DNA by AFM (Figure 1C). Iacomino et al. (2011) Biomacromolecules. 12(4):1178-86. The DNABII protein binds to DNA in vitro in the presence of mM spermidine (Figure 2B), leading to an investigation into the potential interaction between the DNABII protein and polyamines in producing biofilm eDNA scaffold structures. The applicants 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 they had previously observed with the DNABII protein. Immunofluorescence imaging shows that eDNA and polyamines co-localize along fibers present in mucosal biofilms (Figure 2A), visually similar to what was observed in vitro for DNABII proteins and polyamines by Hud and collaborators (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] The applicants investigated whether dicyclohexylamine (DCHA), a broad-acting polyamine biosynthesis inhibitor, can alter NTHI biofilm biogenesis in vitro. DCHA inhibits spermidine synthase, an 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). DCHA did not affect NTHI growth (data not shown), but as determined by COMSTAT analysis of CLSM images of LIVE / DEAD®-stained NTHI biofilms (Heydorn et al. (2000) Microbiology. 146 (Pt 10):2395-407), DCHA inhibited biofilm biogenesis in vitro and reduced average thickness and biomass (Figure 3A). Furthermore, the applicants found that DCHA inhibits eDNA scaffold production in early biofilm development (Figure 3B), suggesting that polyamine biosynthesis is a necessary step in biofilm formation. Supporting these findings, the applicants observed by immunofluorescence that DCHA reduces the incorporation of extracellular polyamines into the biofilm EPS (Figure 3C), and that the defect in biofilm biogenesis can be compensated for by the addition of exogenous spermidine (Figure 3A). Furthermore, these results reveal that the incorporation of polyamines into EPS is important for the development and function of eDNA scaffolds in supporting robust biofilm growth. Anti-DNABII disrupts DNABII-polyamine-dependent DNA structure.

[0169] Immunofluorescence was used to determine whether DNABII proteins were incorporated into EPS mimetic structures. Spermidine (300 μM) and HU (1 μM) were incubated with genomic DNA (2 μg / ml). Subsequently, the EPS mimetic structures were probed with naive (control) or anti-DNABII IgG, a fluorescent secondary antibody, stained with DAPI, and imaged by CLSM. DNABII proteins were fully incorporated into the EPS structures (Figure 4A). Next, EPS mimetic structures were allowed to form, treated with anti-DNABII antibody, stained with DAPI, and imaged by CLSM. Sequestering of DNABII proteins from EPS mimetic structures by anti-DNABII antibody dramatically reduced the abundance and size of aggregated structures (Figure 4B), further confirming their importance for DNABII integration and DNA structure stability. NTHI biofilm disruption by cation exchanger P11 can be inhibited by the addition of exogenous DNABII(HU) and spermidine.

[0170] Phosphocellulose (P11) is a charged resin with high affinity for positively charged molecules, such as polyamines and DNABII proteins. To determine the effect of P11 on the sequestering of these molecules on bacterial biofilm formation, the applicants utilized a Transwell system. NTHI growth was initiated in the basal chamber, while P11 (1% w / v) was added to the apical chamber at seeding. After 16 hours, the biofilms were washed, stained with LIVE / DEAD®, imaged using CLSM, and analyzed by COMSTAT. P11 significantly reduced the average thickness and biomass (Figure 5). To determine whether cation depletion by P11 included polyamines and / or DNABII proteins, NTHI growth was initiated in the basal chamber with 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 structural components of the biofilm matrix and inhibit P11-mediated biofilm disruption only when they are present together (Figure 5); individually, HU and spermidine were insufficient (data not shown). DNases cannot destroy mature pathogenic bacterial biofilms.

[0171] The applicants evaluated the antibiofilm effect 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 seeding (biofilm prevention) or to pre-formed biofilms (biofilm disruption) (Figure 6). The resulting biofilms were stained with LIVE / DEAD® and evaluated using CLSM and COMSTAT analysis. Addition of the DNase at seeding resulted in a significant reduction in the mean thickness and biomass of NTHI and UPEC biofilms compared to untreated biofilms (Figure 6). However, the DNase was unable to disrupt mature biofilms (Figure 6), suggesting that eDNA loses sensitivity to nuclease digestion as the biofilm develops. The applicants propose that as the biofilm matures, the eDNA is either sterically protected from nucleases, or / or adopts a novel structure that makes the eDNA 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 co-localize with DNABII proteins in vitro (Figure 7A) and in vivo (Figure 7B). The applicants hypothesized that DNABII and polyamines synergistically interact with eDNA to confer DNase resistance to mature biofilms. To test the synergistic effect in vitro, the applicants incubated NTHI genomic DNA (gDNA) with spermidine (10, 20, 50, or 100 μM) in or without HU (50 or 100 nM) and DNase (0.5 units). Degradation was evaluated by agarose gel electrophoresis and UV irradiation. While spermidine (≥100 μM) and HU (1 μM) individually protected gDNA, mixed, lower levels of spermidine (≤50 μM) and HU (≤100 nM) synergistically inhibited gDNA digestion (Figure 7), suggesting that nuclease resistance is due to the combination of DNABII protein and polyamine within the biofilm matrix. Next, the applicants decided to test whether EPS mimetic structures were resistant to DNase. EPS scaffold mimetic structures were prepared by incubating HU (1 μM) and spermidine (300 mM) with gDNA (2 μg / ml) for 40 hours and then treated with DNase (5 units). As shown by CLSM, these DNABII-polyamine-dependent DNA structures were resistant to DNase, as were mature biofilms (Figure 7D). B.DNABII, polyamines, and DNA form Z-DNA in vitro.

[0173] Upon binding, polyamines shift the Z-DNA-like sequence from BZ equilibrium to 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 effect of inhibition by HU and spermidine binding in the applicants' in vitro DNase degradation assay (Figure 7), and the Z-DNA's ability to resist DNase, the applicants hypothesized that EPS mimetic structures could contain Z-DNA. EPS structures were formed with gDNA for 16 hours as shown in Figures 4 and 7. Immunofluorescence CLSM was performed using an anti-Z-DNA antibody to confirm the presence of Z-DNA (note the white areas; Figure 8A). To determine whether the DNABII protein affects the BZ equilibrium, the applicants used circular dichroism (CD). A mixture of HU and poly(dGdC)DNA substrates showed inversion of the B-DNA single elliptic 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 indicated bacterial pathogens 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 a hierarchy among species (UPEC = Staphylococcus epidermidis < K. pneumoniae < NTHI). In all cases, extensive co-localization existed between DNABII (HU) and spermidine (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 actually 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 (Figures 7A and 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 are methods for treating a biofilm in a subject, comprising, or essentially thereof, or further comprising, administering to a subject infected with a biofilm an effective amount of an active agent that interferes with the binding of polyamines to DNA in the biofilm. In one embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any equivalent thereof. In another embodiment, the active agent is provided in the absence of a DNase. In a further embodiment, the DNase is administered following the administration of the active agent. In one particular embodiment, the administered DNase is Pulmozyme. In one embodiment, the method for treating a biofilm in a subject comprises, or essentially thereof, or further comprising, administering to a subject infected with a biofilm an effective amount of one or more active agents that interfere with the binding of polyamines to DNA in the biofilm. In one embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any equivalent thereof. In another embodiment, the active agent is provided in the absence of a DNase. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme. In another embodiment, a method for treating a biofilm in a subject comprises, or is essentially thereof, or further comprises thereof, administering to a biofilm-infected subject two or more species of active agents that interfere with the binding of polyamines to DNA in the biofilm, and in one embodiment, the active agent is administered in the absence of a DNase. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is administered following the administration of the active 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 is essentially, or further comprises, administering to a biofilm-infected subject an effective amount of three or more species of active agents that interfere with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the active agents are administered in the absence of DNases. In another embodiment, the active agents are neither HMGB1 protein, nor fragments thereof, nor any of their equivalents. In a further embodiment, DNases are administered following the administration of the active agents. In a particular embodiment, the administered DNase is Pulmozyme. In further embodiments, a method for treating a biofilm in a subject comprises, or is essentially, or further comprises, administering to a biofilm-infected subject an effective amount of four or more species, or or five or more species, or or six or more species, or or seven or more species, or or eight or more species, or or nine or more species, or or ten or more species of active agents that interfere with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the active agents are administered in the absence of DNase. In another embodiment, the active agents are neither HMGB1 protein, nor fragments thereof, nor any of their equivalents. In further embodiments, DNase is administered following the administration of the active agents. In a particular embodiment, the administered DNase is Pulmozyme.

[0177] Furthermore, the present disclosure relates to a method for preventing biofilm formation in a biofilm-prone subject, comprising, or essentially therein, or further comprising therein, administering to the subject an effective amount of an active agent that interferes with the binding of polyamines to DNA in the biofilm, wherein in one embodiment the active agent is not the HMGB1 protein, a fragment thereof, or any of its equivalents, and in another embodiment the active agent is administered in the absence of a DNase. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, a method for preventing biofilm formation in a biofilm-prone subject comprises, or is essentially thereof, or further comprises thereof, the subject, wherein the active substance is neither HMGB1 protein nor a fragment thereof nor any equivalent thereof, and in another embodiment, the active substance is administered in the absence of DNase. In a further embodiment, the DNase is administered following the administration of the active substance. In a particular embodiment, the administered DNase is Pulmozyme. In another embodiment, a method for preventing biofilm formation in a biofilm-prone subject comprises, or is essentially thereof, or further comprises thereof, the subject, wherein the active substance is administered in the absence of DNase. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In yet another embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme.In a further embodiment, a method for preventing biofilm formation in a biofilm-prone subject comprises, or is essentially thereof, or further comprises thereof, administering to the subject an effective amount of three or more species of active agents that interfere with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the active agents are administered in the absence of DNases. In another embodiment, the active agents are neither HMGB1 protein, nor fragments thereof, nor any of their equivalents. In a further embodiment, DNases are administered following the administration of the active agents. In a particular embodiment, the administered DNase is Pulmozyme. In a further embodiment, a method for preventing biofilm formation in a biofilm-prone subject comprises, or is essentially, or further comprises, administering to the subject an effective amount of four or more species of active agents, or or five or more species, or or six or more species, or or seven or more species, or or eight or more species, or or nine or more species, or or ten or more species, that interfere with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the active agents are administered in the absence of DNase. In another embodiment, the active agents are neither HMGB1 protein, nor fragments thereof, nor any of their equivalents. In a further embodiment, DNase is administered following the administration of the active agents. In a particular embodiment, the administered DNase is Pulmozyme.

[0178] This disclosure relates to a method for treating an infection caused by biofilm-forming bacteria in a subject where it is needed, comprising, or essentially therein, or further comprising therein, the method comprising administering to the subject an effective amount of an active agent that interferes with the binding of polyamines to DNA in a biofilm and an active agent that inhibits the replication of the organism, and in one embodiment, the method further relating to the method in which the active agents are administered in the absence of a DNase. In another embodiment, the active agent is not the HMGB1 protein, a fragment thereof, or any of its equivalents. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, a method for treating an infection caused by biofilm-forming bacteria in a subject where it is needed comprises, or essentially therein, or further comprising therein, the method comprising administering to the subject an effective amount of one or more active agents that interfere with the binding of polyamines to DNA in a biofilm. In another embodiment, a method for treating an infection caused by biofilm-forming bacteria in an object requiring it comprises, or is essentially therein, or further comprises therein, administering to the object an effective amount of two or more species of active agents that interfere with the binding of polyamines to DNA in the biofilm, and an active agent that inhibits the replication of the organism. In one embodiment, the active agents are administered in the absence of a DNase. In another embodiment, the active agents are neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is administered following the administration of the active agents. In a particular embodiment, the administered DNase is Pulmozyme.In a further embodiment, a method for treating an infection caused by biofilm-forming bacteria in an object requiring it comprises, or is essentially thereof, or further comprises thereof, administering to the object an effective amount of three or more species of active agents that interfere with the binding of polyamines to DNA in the biofilm, and an active agent that inhibits the replication of the organism, wherein in one embodiment, the active agents are administered in the absence of DNases. In another embodiment, the active agents are neither the HMGB1 protein, nor fragments thereof, nor any of their equivalents. In a further embodiment, DNases are administered following the administration of the active agents. In a particular embodiment, the administered DNase is Pulmozyme. In further embodiments, a method for treating an infection caused by biofilm-forming bacteria in an object requiring it comprises, or is essentially thereof, or further comprises thereof, administering to the object an effective amount of four or more species of active agents that interfere with the binding of polyamines to DNA in the biofilm, or five or more species, or six or more species, or seven or more species, or eight or more species, or nine or more species, or ten or more species, along with an active agent that inhibits the replication of the organism, wherein in one embodiment, the active agents are administered in the absence of DNase. In another embodiment, the active agents are neither HMGB1 protein, nor fragments thereof, nor any of their equivalents. In further embodiments, DNase is administered following the administration of the active agents. 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 active agent that interferes with the binding of the polyamine to DNA in the biofilm is tRNA. In another embodiment, the active agent is an inhibitor of polyamine synthesis or an active agent that inhibits the binding of the polyamine to DNA. In a second embodiment, the active agent includes, or is essentially, or further consists of, the polyamine analogs difluoromethylornithine, trans-4-methylcyclohexylamine, saldomodide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin, dicyclohexylamine, any derivative thereof, or a salt thereof. In one embodiment, derivatives of these compounds maintain the same mass-to-charge ratio. In the third embodiment, the active agent comprises, or is essentially thereof, or further comprises, an active agent that depletes cations from the biofilm, optionally comprising a cation exchange resin, aminopolycarboxylic acid, crown ether, azacrown, or cryptand. In the fourth embodiment, the active agent that depletes cations from the biofilm is selected from the group consisting of sulfonates, sulfopropyls, phosphocellulose, P11 phosphocellulose, heparin sulfate, or derivatives or analogs thereof. In one embodiment, the derivative or analog of the active agent that depletes cations from the biofilm is a resin having a net negative charge. In the fifth embodiment, the active agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment. In the sixth embodiment, the active agent comprises, or is essentially thereof, or further comprises, an anti-B-DNA antibody or a fragment or derivative thereof. In one embodiment, a polyclonal or monoclonal anti-B-DNA antibody or a fragment or derivative thereof recognizes B-DNA with at least 10-fold affinity / binding activity compared to Z-DNA.In the seventh embodiment, the active substance includes, or is essentially thereof, or further comprises riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine or its derivatives. In the eighth embodiment, the active substance includes, or is essentially thereof, or further comprises. In one embodiment, the derivative of the compound retains the ability to intervene between DNA bases. In one embodiment, the active substance is neither the HGMB1 protein nor a fragment thereof. In one embodiment, the active substance that depletes cations from the biofilm has a net negative charge. In another embodiment, the active substance that depletes cations from the biofilm has a net neutral charge.

[0180] Furthermore, the Specified Method for Treating Biofilms in Patients with Systemic Lupus Erythematosus (SLE) and / or Cystic Fibrosis (CF) comprises, or is essentially, or further comprises, administering an effective amount of an active agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one embodiment, the active agent is administered in the absence of a DNase. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme. A method for treating a biofilm in a patient with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB is disclosed herein, comprising, or essentially therein, or further comprising therein, the method wherein in one embodiment, the activator is administered in the absence of a DNase. In another embodiment, the activator is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is administered following the administration of the activator. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, a method for treating a biofilm in a patient with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB is disclosed herein, comprising, or essentially therein, or further comprising therein, an effective amount of two or more species of active agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one embodiment, the active agents are administered in the absence of DNase. In another embodiment, the active agents are neither HMGB1 protein, nor fragments thereof, nor any of their equivalents. In a further embodiment, DNase is administered following the administration of the active agents.In one particular embodiment, the administered DNase is Pulmozyme. In another embodiment, a method for treating a biofilm in a patient with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB is disclosed herein, comprising, or essentially therein, or further comprising, administering an effective amount of three or more species of active agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment. In a further embodiment, a method for treating a biofilm in a patient with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB is disclosed herein, comprising, or essentially therein, or further comprising thereof, an effective amount of four or more species, or or five or more species, or or six or more species, or or seven or more species, or or eight or more species, or or nine or more species, or or ten or more species of active agents that interfere with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one embodiment, the active agents are administered in the absence of DNases. In another embodiment, the active agents are neither HMGB1 protein, nor fragments thereof, nor any of their equivalents. In a further embodiment, DNases are administered following the administration of the active agents. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, the active ingredient interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment. In a second embodiment, the active ingredient includes, or is essentially thereof, or further consists of, an anti-B-DNA antibody or a fragment or derivative thereof. In one embodiment, a polyclonal or monoclonal anti-B-DNA antibody or a fragment or derivative thereof recognizes B-DNA with at least 10-fold affinity / binding activity compared to Z-DNA.In the third embodiment, the active substance includes, or is essentially composed of, riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine or its derivatives. In the fourth embodiment, the active substance includes, or is essentially composed of, chloroquine or its derivatives. In one embodiment, the derivatives of the compound retain the ability to intervene between DNA bases. The active substance is neither the HMGB1 protein nor a fragment thereof.

[0181] Furthermore, the Specified Method for Treating Biofilms in Patients with Systemic Lupus Erythematosus (SLE) and / or Cystic Fibrosis (CF) and / or TB comprises, or is essentially thereof, or further comprises thereof, a method in which, in one embodiment, the protein and antibody are administered in the absence of a DNase. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, the polyclonal or monoclonal anti-B-DNA antibody or its fragment or derivative recognizes B-type DNA with at least 10-fold affinity / binding activity compared to Z-type DNA. The bioactive fragment of HMGB1 may comprise, or be essentially thereof, or further comprise thereof, one or more of an A-box, a B-box and / or an AB-box, a C-terminal fragment, or an N-terminal fragment. In certain embodiments, the bioactive fragment of HMGB1 may contain, or be essentially thereof, or further consist of, a B-box domain capable of binding DNA. In one embodiment, a method for treating a biofilm in a patient with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) and / or TB comprises administering an effective amount of chloroquine and an anti-B-DNA antibody or its fragment or derivative, or, essentially thereof, or further consists of, in one embodiment, the chloroquine and antibody are administered in the absence of a DNase. In another embodiment, the active agent is neither the HMGB1 protein, nor its fragment, nor any of its equivalents. In a further embodiment, the DNase is administered following the administration of the active agent. In a certain embodiment, the administered DNase is Pulmozyme.Furthermore, the present disclosure relates to a method for treating a biofilm-producing infection associated with the administration of chemotherapy in a patient receiving or having received platinum-based chemotherapy, comprising, or essentially therein, or further comprising therein, the method wherein the activator is administered in the absence of a DNase. In another embodiment, the activator is not the HMGB1 protein, a fragment thereof, or any of its equivalents. In a further embodiment, the DNase is administered following the administration of the activator. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, the method comprises, or essentially therein, or further comprising therein, the method wherein the activator is administered in the absence of a DNase. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any equivalent thereof. In yet another embodiment, a DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme. In yet another embodiment, the active agent comprises, or is essentially thereof, or consists of thereof. In yet another embodiment, the active agent comprises, or is essentially thereof, or consists of an anti-B-DNA antibody or a fragment or derivative thereof. In one embodiment, a polyclonal or monoclonal anti-B-DNA antibody or a fragment or derivative thereof recognizes B-type DNA with at least 10-fold affinity / binding activity compared to Z-type DNA. In one embodiment, the active substance includes, or is essentially, or further comprises, riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine or its derivatives.In a particular embodiment, derivatives of the compound retain the ability to intervene between DNA bases.

[0182] This disclosure relates to a method for treating a biofilm-producing infection associated with the administration of chemotherapy in a patient receiving or having received platinum-based chemotherapy, comprising, or essentially thereof, or further comprising thereof, an effective amount of HMGB1 protein or a bioactive fragment thereof and an anti-B-DNA antibody or a fragment or derivative thereof, and further relating in one embodiment to a method in which the protein and antibody are administered in the absence of a DNase. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular 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 with at least 10-fold affinity / binding activity compared to Z-type DNA. The bioactive fragment of HMGB1 may comprise, or essentially thereof, or further comprise thereof, one or more of an A-box, a B-box and / or an AB-box, a C-terminal fragment, or an N-terminal fragment. In certain embodiments, the bioactive fragment of HMGB1 may include, or be essentially thereof, or further consist of, a B-box domain capable of binding DNA. Also provided herein is a method for treating a biofilm-producing infection associated with the administration of chemotherapy in a patient receiving or having received platinum-based chemotherapy, comprising, or being essentially thereof, or further consisting of, an effective amount of chloroquine and an anti-B-DNA antibody or a fragment or derivative thereof, wherein in one embodiment, the chloroquine and antibody are administered in the absence of a DNase. In a further embodiment, the DNase is administered following the administration of the active agent. In a certain 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 with at least 10-fold affinity / binding activity compared to Z-type DNA.

[0183] The method described above further comprises, or is essentially thereof, or further consists of, administering to a subject an effective amount of an active agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins, in one embodiment, the active agent is administered in the absence of DNase. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any equivalent thereof. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme. In one embodiment, the method further comprises, or is essentially thereof, or further consists of, administering to a subject an effective amount of an active agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins, in one embodiment, the active agent is administered in the absence of DNase. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any equivalent thereof. In a further embodiment, a DNase is administered following the administration of the active agent. In a particular embodiment, the administered DNase is Pulmozyme. In another embodiment, the active agent that interferes with the binding of eDNA to DNA-binding proteins comprises, or is essentially thereof, or further comprises thereof, one or more of the anti-DNABII antibody, anti-IHF antibody and / or anti-HU antibody or fragments thereof. In one embodiment, the active agent that interferes with the binding of eDNA to DNA-binding proteins has a net negative charge. In a second embodiment, the active agent that interferes with the binding of eDNA to DNA-binding proteins has a net neutral charge. In a third embodiment, the active agent that interferes with the binding of eDNA to DNA-binding proteins has a net positive charge.

[0184] A method for inhibiting the stability of a biofilm is described herein, comprising, or essentially, or further comprising, contacting the biofilm with an effective amount of an active agent that interferes with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the active agent is contacted in the absence of a DNase. In another embodiment, the active agent is not the HMGB1 protein, its fragments, or any of its equivalents. In a further embodiment, the DNase is contacted following contact with the active agent. In a particular embodiment, the DNase is a Pulmozyme. In one embodiment, a method for inhibiting the stability of a biofilm comprises, or essentially, or further comprising, contacting the biofilm with an effective amount of one or more active agents that interfere with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the active agent is contacted in the absence of a DNase. In another embodiment, the active agent is not the HMGB1 protein, its fragments, or any of its equivalents. In a further embodiment, the DNase is contacted following contact with the active substance. In a particular embodiment, the DNase is a Pulmozyme. In another embodiment, a method for inhibiting the stability of a biofilm comprises, or is essentially thereof, or further comprises thereof, contacting the biofilm with an effective amount of two or more species of active substances that interfere with the binding of polyamines to DNA in the biofilm, and in one embodiment, the active substances are contacted in the absence of the DNase. In another embodiment, the active substance is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is contacted following contact with the active substance. In a particular embodiment, the DNase is a Pulmozyme. In a further embodiment, a method for inhibiting the stability of a biofilm comprises, or is essentially, or further comprises, contacting the biofilm with an effective amount of three or more species of active agents that interfere with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the active agents are contacted in the absence of DNase.In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In yet another embodiment, a DNase is brought into contact with the active agent. In one particular embodiment, the DNase is a Pulmozyme. In yet another embodiment, a method for inhibiting the stability of a biofilm involves, or is essentially, or further consists of, bringing the biofilm into contact with four or more species of active agents, or or five or more species, or or six or more species, or seven or more species, or eight or more species, or nine or more species, or ten or more species, in an effective amount that interferes with the binding of polyamines to DNA in the biofilm, and in one embodiment, the active agent is brought into contact in the absence of a DNase. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is brought into contact with the active substance. In a particular embodiment, the DNase is a Pulmozyme. The contact may be in vitro or in vivo.

[0185] Furthermore, the present disclosure relates to a method for inhibiting the stability of a biofilm, comprising, or essentially therein, or further comprising, in vitro contacting a biofilm with an active agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contact comprises, or essentially therein, or further comprising, coating the surface with an effective amount of the active agent that depletes the cations, wherein in one embodiment, the active agent is contacted in the absence of a DNase, while in another embodiment, the DNase is contacted according to the present method. In another embodiment, the active agent is not the HMGB1 protein, a fragment thereof, or any of its equivalents. In a further embodiment, the DNase is contacted following contact with the active agent. In a particular embodiment, the DNase is a Pulmozyme. In one embodiment, a method for inhibiting the stability of a biofilm comprises, or is essentially thereof, or further comprises thereof, in vitro contacting the biofilm with an effective amount of an active agent that interferes with the binding of polyamines to DNA in the biofilm, the contact comprising, or is essentially thereof, or further comprises thereof, coating the surface with an effective amount of one or more active agents that depletes cations, in one embodiment, the active agent is contacted in the absence of a DNase, while in another embodiment, the DNase is contacted according to the method. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is contacted following contact with the active agent. In a particular embodiment, the DNase is a Pulmozyme.

[0186] In another embodiment, a method for inhibiting the stability of a biofilm comprises, or may be essentially thereof, or further comprised thereof, in vitro contacting the biofilm with an effective amount of an active agent that interferes with the binding of polyamines to DNA in the biofilm, the contact comprising, or may be essentially thereof, or further comprised thereof, coating the surface with an effective amount of two or more species of active agents that deplete the cations, in one embodiment, the active agent is contacted in the absence of a DNase, while in another embodiment, the DNase is contacted according to the method. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is contacted following contact with the active agent. In a particular embodiment, the DNase is a Pulmozyme.

[0187] In a further embodiment, a method for inhibiting the stability of a biofilm involves a biofilm being subjected to an effective amount of an active substance that interferes with the binding of polyamines to DNA in the biofilm. The method may include, or be essentially thereof, or further consist thereof, and the contact may include, or be essentially thereof, or further consist thereof, coating a surface with an effective amount of three or more species of active agents that deplete the cations, in one embodiment, the active agents are contacted in the absence of the DNase, while in another embodiment, the DNase is contacted according to the method. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is contacted following contact with the active agent. In a particular embodiment, the DNase is a Pulmozyme. In further embodiments, a method for inhibiting the stability of a biofilm may include, or be essentially thereof, or further consist thereof, contacting the biofilm in vitro with an effective amount of an active agent that interferes with the binding of polyamines to DNA in the biofilm, and the contact may include, or be essentially thereof, or further consist thereof, coating the surface with an effective amount of four or more species of the active agent, or five or more species, or six or more species, or seven or more species, or eight or more species, or nine or more species, or ten or more species, to deplete the cations. In one embodiment, the active agent interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, in one embodiment the active agent is contacted in the absence of a DNase, while in another embodiment the DNase is contacted according to the method. In another embodiment, the active substance is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In yet another embodiment, the DNase is brought into contact with the active substance. In a particular embodiment, the DNase is a Pulmozyme.In the second embodiment, the active substance includes, or is essentially thereof, or further comprises 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-type DNA with at least 10 times the affinity / binding activity compared to Z-type DNA. In the third embodiment, the active substance includes, or is essentially thereof, or further comprises riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloid, quinacrine, 9-aminoacridine, or a derivative thereof. In the fifth embodiment, the active substance includes, or is essentially thereof, or further comprises chloroquine or a derivative thereof. In a particular embodiment, a derivative of the compound retains the ability to intervene between DNA bases. The active substance is neither the HMGB1 protein nor a fragment thereof.

[0188] A method for inhibiting the stability of a biofilm is described herein, comprising, or essentially, or further comprising, contacting the biofilm in vitro with an effective amount of HMGB1 protein or its bioactive fragment and an anti-B-DNA antibody or its fragment or derivative, wherein the contact comprises, or essentially, or further comprising, coating the surface with an effective amount of HMGB1 protein or its bioactive fragment and an anti-B-DNA antibody or its fragment or derivative, wherein in one embodiment, the protein and antibody are contacted in the absence of a DNase, while in another embodiment, the DNase is contacted according to the present method, and such method is described herein further. In a further embodiment, the DNase is contacted following contact with the active substance. In a particular embodiment, the DNase is a Pulmozyme.

[0189] In one embodiment, a polyclonal or monoclonal anti-B-DNA antibody or a fragment or derivative thereof recognizes B-type DNA with at least 10-fold affinity / binding activity compared to Z-type DNA. The bioactive fragment of HMGB1 may contain, or be essentially thereof, or further 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 bioactive fragment of HMGB1 may contain, or be essentially thereof, or further consist of, a B-box domain capable of binding DNA. Furthermore, the Disclosure relates to a method for inhibiting the stability of a biofilm, comprising, or essentially therein, or further 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 contact comprises, or essentially therein, or further comprising, coating the 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 a DNase. In a further embodiment, the DNase is contacted following contact with the active substance. In a particular embodiment, the DNase is a Pulmozyme. In one embodiment, the polyclonal or monoclonal anti-B-DNA antibody or a fragment or derivative thereof recognizes B-type DNA with at least 10-fold affinity / binding activity compared to Z-type DNA.

[0190] The method described above further comprises, or is essentially thereof, or further consists of, contacting a biofilm with an effective amount of an active agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins, in one embodiment, the active agent is contacted in the absence of a DNase, while in another embodiment, the DNase is contacted according to the method. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is contacted following contact with the active agent. In a particular embodiment, the DNase is Pulmozyme. In one embodiment, the active agent that interferes with the binding of eDNA to DNA-binding proteins comprises, or is essentially thereof, or further consists of, one or more of anti-DNABII antibodies, anti-IHF antibodies and / or anti-HU antibodies or fragments thereof, and in one embodiment, the active agent is contacted in the absence of a DNase. In a further embodiment, the DNase is brought into contact with the active agent. In a particular embodiment, the DNase is a Pulmozyme. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any equivalent thereof. In one embodiment, the active agent that interferes with the binding of eDNA to the DNA-binding protein has a net negative charge, and in one embodiment, the active agent is brought into contact in the absence of the DNase. In a further embodiment, the DNase is brought into contact with the active agent. In a particular embodiment, the DNase is a Pulmozyme. In a second embodiment, the active agent that interferes with the binding of eDNA to the DNA-binding protein has a net neutral charge, and in one embodiment, the active agent is brought into contact in the absence of the DNase, while in another embodiment, the DNase is brought into contact according to the present method. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any equivalent thereof. In a further embodiment, the DNase is brought into contact with the active substance. In a particular embodiment, the DNase is a Pulmozyme. In a third embodiment, the active substance that interferes with the binding of eDNA to the DNA-binding protein has a net positive charge.In a further aspect, the DNase is contacted subsequent to contact with the agent. In a particular aspect, the DNase is Pulmozyme.

[0191] A method for inhibiting the stability of a biofilm, the method comprising contacting the biofilm with an agent that interferes with the binding of polyamine to DNA in the biofilm, wherein in one aspect, the agent is contacted in the absence of DNase, while in another aspect, DNase is contacted according to the method, is provided herein. In another aspect, the agent is not the HMGB1 protein, nor a fragment thereof, nor 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. The contacting can be in vitro or in vivo.

[0192] Also provided is a method for treating a biofilm in a subject, the method comprising administering to a subject infected with the biofilm an effective amount of an agent that interferes with the binding of polyamine to DNA in the biofilm, wherein 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 the HMGB1 protein, nor a fragment thereof, nor an equivalent thereof. In a further aspect, the DNase is administered subsequent to administration of the agent. In a particular aspect, the DNase is Pulmozyme.

[0193] A method for preventing biofilm formation in a biofilm-prone subject is provided, comprising administering to the subject an effective amount of an active substance that interferes with the binding of polyamines to DNA in the biofilm, wherein in one embodiment, the active substance is administered in the absence of a DNase, while in another embodiment, a DNase is administered. In another embodiment, the active substance is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is administered following the administration of the active substance. In a particular embodiment, the DNase is a Pulmozyme.

[0194] A method for treating an infection caused by biofilm-forming bacteria in a subject requiring such treatment is also provided, comprising administering to the subject an effective amount of an active agent that interferes with the binding of polyamines to DNA in the biofilm, and an active agent that inhibits the replication of the organism, wherein in one embodiment, the active agent is administered in the absence of a DNase, while in another embodiment, a DNase is administered. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the DNase is a Pulmozyme.

[0195] In one embodiment, the active substance is an inhibitor of polyamine synthesis or an active substance that inhibits the binding of polyamines to DNA. In another embodiment, the active substance is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. Non-limiting examples of polyamines include putrescine, spermine, cadaverine, 1,3-diaminopropane, or spermidine. In another embodiment, the active substance includes polyamine analogs such as 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 salts thereof (all active substances 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 further embodiments, the active agent comprises an active agent that depletes cations from the biofilm, optionally a cation exchange resin, aminopolycarboxylic acid, crown ether, azacrown, or cryptand (various representative compounds of each class of active agents are available from Millipore Sigma). In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. Non-limiting examples of cation exchange resins include resins containing sulfonates, sulfopropyls, phosphocellulose, P11 phosphocellulose, heparin sulfate, or derivatives or analogs thereof. In one embodiment, the active agent that depletes cations from the biofilm has a net negative charge. In one embodiment, the active agent that depletes cations from the biofilm has a net neutral charge.

[0197] In one embodiment, a method for inhibiting the stability of a biofilm is provided herein, comprising contacting the biofilm in vitro with an active agent that interferes with the binding of polyamines to DNA in the biofilm, wherein the contact comprises coating the surface with a cation-depleting active agent, wherein in one embodiment the active agent is administered in the absence of a DNase, while in another embodiment the DNase is administered. In another embodiment, the active agent is neither the HMGB1 protein, nor a fragment thereof, nor any of its equivalents. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the DNase is a Pulmozyme.

[0198] In one aspect of the above method, the active agent interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment, and in one aspect, the active agent is administered in the absence of DNase, while in another aspect, DNase is administered. In a further aspect, DNase is administered following the administration of the active agent. In a particular aspect, DNase is a pulsemozyme. Examples of such include anti-B-DNA antibodies or fragments or derivatives thereof. In a further embodiment, the active substance includes active substances 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 its derivatives (all active substances in this further embodiment are commercially available from Millipore Sigma unless otherwise indicated). The active substance is neither the HMGB1 protein nor a fragment thereof.

[0199] A method for treating a biofilm in a patient with systemic lupus erythematosus (SLE) and / or cystic fibrosis (CF) is provided, comprising administering an effective amount of an active agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one embodiment the active agent is administered in the absence of a DNase, while in another embodiment the DNase is administered. In a further embodiment the DNase is administered following the administration of the active agent. In a particular embodiment the DNase is a Pulmozyme. Examples of such include anti-B-DNA antibodies or fragments or derivatives thereof. In a further embodiment the active agent includes active agents from the group of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine or derivatives thereof. In one embodiment, the method is carried out in the absence of DNase, and in another embodiment, the treatment of CF is carried out in the absence of DNase. The active substance is neither the HMGB1 protein nor a fragment thereof.

[0200] In another embodiment, a method is provided herein for treating a biofilm-producing infection associated with the administration of chemotherapy in a patient receiving or having received platinum-based chemotherapy, comprising administering an effective amount of an active agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, wherein in one embodiment, the active agent is administered in the absence of a DNase, while in another embodiment, a DNase is administered. In a further embodiment, the DNase is administered following the administration of the active agent. In a particular embodiment, the DNase is a Pulmozyme. Examples of such include anti-B-DNA antibodies or fragments or derivatives thereof. In a further embodiment, the active agent (the gent) includes active agents from the group of riboflavin, ethidium bromide, bis(methidium)spermine, daunorubicin, TMPyP4, quaternary benzo[c]phenanthridine alkaloids, quinacrine, 9-aminoacridine or derivatives thereof. The active substance is neither the HMGB1 protein nor a fragment of it.

[0201] The methods described above may further include contacting (in vitro) or administering to a subject an effective amount of an active agent and / or antimicrobial agent that interferes with the binding of eDNA to DNA-binding proteins, in one embodiment, the active agent being administered in the absence of DNase, while in another embodiment, DNase being administered. In another embodiment, the active agent is neither the HMGB1 protein, its fragments, nor any of its equivalents. In a further embodiment, DNase is administered following the administration of the active agent. In a particular embodiment, DNase is Pulmozyme. In one embodiment, the active agent that interferes with the binding of eDNA to DNA-binding proteins has a net positive charge. In one embodiment, the active agent that interferes with the binding of eDNA to DNA-binding proteins has a net negative charge. In one embodiment, the active agent that interferes with the binding of eDNA to DNA-binding proteins has a net neutral charge.

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

[0203] In another embodiment, the in vitro method is performed in a miniaturized chamber-slide system that allows the isolation of infection-causing microorganisms (e.g., bacteria) from humans / animals, followed by cultivation, and growth as a biofilm in vitro. The active agent (e.g., anti-DNABII or IHF antibody) or test active agent or potential active agent is added to the culture alone or in combination with another active agent, with or without increasing the dilution of the potential active agent or active agent, e.g., anti-DNABII or IHF (or other antibodies, small molecules, active agents, etc.), to find an optimal dose that may be effective in treating a patient if delivered to a subject with an infection. As will be apparent to those skilled in the art, positive and negative controls may be performed simultaneously.

[0204] In a further embodiment, the method is carried out on a high-throughput platform with an active agent (e.g., anti-DNABII or IHF antibody) and / or a potential active agent (alone or in combination with another active agent) in a flow cell. The active agent (e.g., anti-DNABII or IHF antibody) or potential active agent biofilm is added to the culture, either alone or in combination with another active agent, with increasing or no increase in the dilution of the potential active agent or active agent, e.g., anti-DNABII or IHF (or other antibodies, small molecules, active agents, etc.), to find an optimal dose that may be effective in treating a patient if delivered to an infected subject. Biofilm isolates are sonicated to separate the biofilm bacteria from the DNABII polypeptide, e.g., IHF, bound to 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, e.g., by salt washing, and used to identify the added biofilm bacteria. The released DNA is then identified, e.g., by PCR sequencing. If DNA is not released, the active agent(s) acted successfully on or bound to the microbial DNA. If DNA is present in the sample, the active agent did not interfere with 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] Furthermore, since a given bacterial species may respond better to the destruction of its biofilm by a certain agent than to another, the above method can be used as a diagnostic test, and this rapid, high-throughput assay system may enable those skilled in the art to assay a population of potential anti-DNABII or IHF-like agents to identify the most effective one among them.

[0206] The advantage of these methods is that most hospital clinical microbiology laboratories are already prepared to perform these types of assays (i.e., determining MIC and MBC values) using bacteria growing in liquid culture (or suspension). As is evident to those skilled in the art, bacteria generally do not grow in suspension if 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 why most MIC / MBC values ​​cannot accurately predict efficacy in vivo. Therefore, by determining which “dose” of the active ingredient (as described above) can reverse bacterial biofilms in vitro, our preclinical assays can be more reliable predictors of clinical efficacy, even as an application of personalized medicine.

[0207] In addition to clinical settings, this method can be used to identify infection-causing microorganisms and / or to confirm effective active agents in industrial settings. Therefore, in industrial settings, active agents can be used to treat, inhibit, or titrate 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. Alternatively, the active agent can be added to microbial DNA or DNABII polypeptide, followed by the addition of a deficient complex to assay for biofilm inhibition.

[0209] When performed in vivo in non-human animals, such as chinchillas, this method provides preclinical screening to identify active ingredients that can be used alone or in combination with other active ingredients to disrupt biofilms.

[0210] In another embodiment, a method for inhibiting, preventing, or destroying biofilms in a subject is provided herein, by administering an effective amount of an active substance to the subject, thereby inhibiting, preventing, or destroying the microbial biofilm. Non-limiting examples of such subjects include mammals, e.g., pets and human patients.

[0211] The active ingredients and compositions disclosed herein may be administered in combination with or sequentially with other antimicrobial agents and / or surface antigens. In a particular embodiment, administration is topical to the site of infection, for example, by direct injection or inhalation. Other non-limiting examples of administration include administration by one or more methods, including percutaneous, 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 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 (unclassified), Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, or Mycobacterium tuberculosis. These microbial infections can be present in the upper, middle, and lower respiratory tracts (otitis, sinusitis, bronchitis, as well as exacerbations of chronic obstructive pulmonary disease (COPD), chronic cough, complications of cystic fibrosis (CF) and / or the primary cause of CF, and community-acquired pneumonia (CAP)). Therefore, by implementing the in vivo methods disclosed herein, these diseases and complications from these infections can also be prevented or treated.

[0213] In addition, infectious diseases can occur in the oral cavity (caries, periodontitis) and be caused by Streptococcus mutans, Porphyromonas gingivalis, Aggregatibacter actinomvctemcomitans. Also, infectious diseases can be localized on the skin (abscess, "staphylococcal" infection, impetigo, secondary infection 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, gastric ulcers) are typically caused by Salmonella enterica serovar, Vibrio cholerae, and Helicobacter pylori. Genital infections typically include Neisseria gonorrhoeae and are caused by it. Infectious diseases can be bladder infections or infections of indwelling devices caused by Enterococcus faecalis. Typically, infections associated with implanted prosthetic devices, such as artificial hips or knee replacements, or dental implants, or medical devices, such as pumps, catheters, stents, or monitoring systems, which are caused by various bacteria, can be treated by the methods disclosed herein. These devices can be coated or conjugated with the agents described herein. Thus, by performing the in vivo methods disclosed herein, complications from these diseases and these infectious diseases can also be prevented or treated.

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

[0215] Accordingly, applicable routes of administration to the methods disclosed herein include intranasal, intramuscular, urethral, ​​intratracheal, subcutaneous, intradermal, transdermal, topical, intravenous, rectal, nasal, oral, inhalation, and other enteral and parenteral routes of administration. The routes of administration may be combined or modified, if desired, depending on the active ingredient and / or the desired effect. The activator may be administered in single or multiple doses. Embodiments of these methods and preferred routes of delivery include systemic or local routes. Generally, preferred routes of administration for the methods disclosed herein include, but are not limited to, direct injection, enteral, parenteral, or inhalation routes.

[0216] Other parenteral administration routes besides inhalation include, but are not limited to, local, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any administration route other than those passing through the gastrointestinal tract. Parenteral administration is used to inhibit the active ingredient (inhibiting The administration may be carried out to result in systemic or local delivery of the agent. If systemic delivery is desired, the administration typically involves invasive or systemically absorbable local or mucosal administration of the pharmacomodulator.

[0217] Furthermore, the active ingredients disclosed herein may be delivered to subjects by enteral administration. Enteral administration routes include, but are not limited to, oral and rectal (e.g., using suppositories) delivery.

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

[0219] In various embodiments of the methods disclosed herein, the active ingredient is administered by inhalation, injection, or orally at least once daily (QD) and, in various embodiments, twice daily (BID), three times daily (TID), or four times daily, such as daily. Typically, the therapeutically effective daily dose may be 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, depending on the compound, up to about 1 g to about 2.5 g.

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

[0221] The dosage, toxicity, and therapeutic efficacy of the compositions described herein can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine the LD50 (lethal dose in 50% of the population) and ED50 (therapeutably effective dose in 50% of the population). The dose ratio between the toxic effect and the therapeutic effect 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 exhibiting toxic side effects may be used, care should be taken to design delivery systems that target affected tissue sites to minimize potential damage to non-infected cells and thereby reduce side effects.

[0222] Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for human use. In certain embodiments, the dose of such compounds falls within a circulating concentration range that includes an ED50 with little to no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in this method, the therapeutically effective dose can first be estimated from a cell culture assay. The dose can then be formulated in an animal model to achieve the circulating plasma concentration range including the IC50 (i.e., the concentration of the test compound that achieves the maximum half of the inhibition of symptoms) determined in the cell culture. Such information can be used to more accurately determine a useful dose in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0223] In some embodiments, the effective amount of composition sufficient to achieve a therapeutic or preventive 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 dose range is about 0.0001 mg per kilogram of body weight per dose to about 100 mg per kilogram of body weight per dose. The administration is 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, booster doses are administered after evaluation of the subject’s response to the previous dose.

[0224] Those skilled in the art will understand, without limitation, that certain factors, including the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing conditions, may influence the dosage and timing adjustments required to effectively treat the subject. Furthermore, treatment of the subject with a therapeutically effective amount of the therapeutic composition described herein may include a single treatment or a series of treatments. Antibodies and their derivatives

[0225] This disclosure also provides antibodies for use in the methods disclosed herein that bind to and / or specifically recognize and bind to B DNA. The antibodies can be any of the various antibodies described herein, non-limiting examples of such antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, veneered antibodies, diabodies, humanized antibodies, antibody derivatives, recombinant humanized antibodies, or derivatives or fragments thereof. In one embodiment, the fragment comprises, or is essentially derived from, or further comprises from, an antibody CDR. In one embodiment, the antibody is detectably labeled, or further comprises a detectable label conjugated thereto. Hybridoma cell systems for producing monoclonal antibodies disclosed herein are also provided. Compositions comprising, or being essentially derived from, or further comprising, one or more of the embodiments described herein are further provided. Polynucleotides encoding the amino acid sequences of antibodies and fragments, as well as methods for producing antibody polypeptides and fragments therefrom by recombination or chemical synthesis are further provided. 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 to adjuvants, routes and sites of administration, injection volume per site, and the 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 an antigen. Most adjuvants provide injection-site antigen depots that allow for stow release of the antigen to the influx region lymph nodes. Other adjuvants include surfactants that promote enrichment of protein antigen molecules across a large surface area and immunostimulant molecules. Non-limiting examples of adjuvants for polyclonal antibody production include Freund's adjuvant, the 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 generated using conventional hybridoma techniques that are well known in the art and have been adequately described in the literature. For example, suitable immortal cell lines (e.g., 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., myeloma cell lines) 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 address, e.g., atcc.org, Hybridomas are produced by fusing antibody-producing cells, such as but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil or other immune or B cell-containing cells, or any other cell type, expressing heavy or light chain constant or variable or framework or CDR sequences, as endogenous or heterologous nucleic acids (see cell lines, last accessed November 26, 2007), including recombinant or endogenous viral, bacterial, algal, prokaryotic, amphibian, insect, reptile, fish, mammalian, rodent, horse, sheep, goat, lamb, primate, or eukaryotic genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, or any combination thereof, with antibody-producing cells, such as, but not limited to, those expressing heavy or light chain constant or variable or framework or CDR sequences, including those of viruses, bacteria, algae, prokaryotes, amphibians, insects, reptiles, fish, mammals, rodents, horses, sheep, goats, lambs, primates, or eukaryotes. Antibody-producing cells may also be obtained from the peripheral blood of a human or other suitable animal immunized with the antigen of interest, or, in certain embodiments, from the spleen or lymph nodes, and then screened for the activity of interest. Any other suitable host cell may also be used to express heterologous or endogenous nucleic acids encoding the antibodies of this disclosure, their designated fragments or variants.Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and then cloned by limiting dilution, cell sorting, or other known methods.

[0228] Other suitable methods can be used to produce or isolate antibodies of the required specificity, including, but not limited to, methods for selecting recombinant antibodies from peptide or protein libraries (e.g., bacteriophages, ribosomes, oligonucleotides, cDNA, or other display libraries; for example, those available from various commercial vendors such as MorphoSys (Martinsreid / Planegg, Del.), BioInvent (Lund, Sweden), and Affitech (Oslo, Norway)) using methods known in the art. These known methods are described in the patent documents, some of which include U.S. Patents 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. Alternative methods include transgenic animals (e.g., SCID mice) capable of producing a repertoire of human antibodies, as is known in the art and / or as described herein. (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 This technique relies on immunization (93:154-161). 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., selected lymphocyte antibody methods). 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 microdroplet 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 This includes, but is not limited to, 19:125-134.

[0229] The antibody derivatives of this disclosure can also be prepared by delivering a polynucleotide encoding the antibody disclosed herein to a suitable host to prepare a transgenic animal or mammal, such as a goat, cattle, horse, or sheep, which produces such antibody in its milk. Such methods are known in the art and are described, for example, in U.S. Patents 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 derivative” includes post-translational modifications of a linear polypeptide sequence of an antibody or fragment. For example, U.S. Patent No. 6,602,684(B1) describes a method for producing a modified glycol form of antibody, comprising a whole antibody molecule, an antibody fragment, or a fusion protein containing a region equivalent to the Fc region of an immunoglobulin, having enhanced Fe-mediated cytotoxicity, and the glycoprotein thus produced.

[0231] The antibodies disclosed herein also include derivatives modified by covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from generating an anti-idiotype 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 cell ligands or other proteins, etc. Furthermore, derivatives may contain one or more non-classical amino acids.

[0232] Antibody derivatives can also be prepared by delivering the polynucleotides disclosed herein to prepare transgenic plants and cultured plant cells (e.g., tobacco, maize, and duckweed) that produce such antibodies, specified moieties, or variants in plant moieties or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118 and the references cited therein describe, for example, the production of transgenic tobacco leaves expressing large quantities of recombinant proteins using inducible promoters. Transgenic maize has been used to express mammalian proteins with bioactivity equivalent to those produced in other recombinant systems or purified from natural sources at commercially viable levels. For example, see Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 and the 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, for example, Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and the references cited therein. Therefore, antibodies can also be produced using transgenic plants according to known methods.

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

[0234] Generally, CDR residues are directly and most substantially involved in the influence of antigen binding. The humanization or manipulation of antibodies can be carried out using any known method, including but not limited to the methods 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 this 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 a hybridoma of interest and manipulated to contain a non-reference (e.g., human) immunoglobulin sequence. For example, to produce a chimeric antibody, a mouse variable region can be ligated to a human constant region using a method 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 a method known in the art (U.S. Patents No. 5,225,539, 5,530,101, 5,585,089, 5,693,762, and 6,180,370). Similarly, to produce primate-like antibodies, mouse CDR regions can be inserted into primate frameworks 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. Multiple lines of such transgenic mice capable of producing different classes of antibodies have been produced. By fusing B cells derived from transgenic mice that produce the desired antibody, a hybridoma cell line for sustained antibody production can be created (e.g., Russel 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 see U.S. Patent Nos. 6,075,181).

[0237] Chimeric antibodies can also be produced by modifying the antibodies disclosed herein. A chimeric antibody is an antibody in which various domains of the heavy and light chains of the 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 produce veneering antibodies. Veneering antibodies are antibodies in which the outer amino acid residues of one species of antibody have been intelligently replaced or "veneering" with amino acid residues of a second species, so that the antibody of the first species is not immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein depends primarily on the properties of its surface, the immunogenicity of an antibody can be reduced by replacing exposed residues that differ from those normally found in antibodies of another mammalian species. Such intelligent replacement of outer residues should have little to no effect on the inner domain or interdomain contacts. Therefore, as a result of changes limited to variable region framework residues, the ligand binding properties should remain unaffected. Because only the outer surface or skin of the antibody is modified and the supporting residues remain undisturbed, this process is called "veneering."

[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 US and foreign databases (both nucleic acid and protein). Non-limiting examples of methods used to produce veneered antibodies include EP519596; US Patent No. 6,797,492; Padlan et al. This is described in al. (1991) Mol. Immunol. 28(4-5):489-498.

[0240] The term “antibody derivative” also includes “diabody,” which is a small antibody fragment having two antigen-binding sites, the fragment containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (see, e.g., EP404,097;WO93 / 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 on the same chain, the domains are forced to pair with a complementary domain on another chain, creating two antigen-binding sites (see also Chen et al., U.S. Patent No. 6,632,926, disclosing an antibody variant having one or more amino acids inserted into the hypervariable region of the parent antibody and having a binding affinity to a target antigen that is at least about twice as strong as the binding affinity of the parent antibody to the antigen).

[0241] The term “antibody derivative” further includes engineered antibody molecules, fragments, and single domains such as scFv, dAb, nanobody, minibody, unibody, and affibody (& 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 antibody." Procedures for producing linear antibodies are well known in the art and are described in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, such antibodies consist of a pair of tandem Ed segments (V) that form a pair of antigen-binding regions. H -C H 1-VH-C H 1) is included. Linear antibodies can be bispecific or monospecific.

[0243] The antibodies disclosed herein can be recovered and purified from recombinant cell cultures 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, hydroxyl apatite chromatography, and lectin chromatography. High-performance liquid chromatography ("HPLC") may also be used for purification.

[0244] The antibodies disclosed herein include naturally occurring and purified products, products of chemical synthesis procedures, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells, or 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, then the antibody being tested and the antibody provided herein are equivalent. It is also possible to determine, without excessive experimental procedures, whether an antibody has the same specificity as the antibody disclosed herein by determining whether the antibody being tested prevents the antibody disclosed herein from binding to the protein or polypeptide to which this antibody normally reacts. If the antibody being tested competes with the antibody disclosed herein, as indicated by the reduced binding by the monoclonal antibody disclosed herein, then these two antibodies may bind to the same or closely related epitopes. Alternatively, the antibody disclosed herein can be pre-incubated with the protein to 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, then 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 any immunoglobulin isotype and subclass. Monoclonal antibodies of a specific isotype can be prepared directly by selection from the initial fusion, or secondarily by using sib selection techniques to prepare from parent hybridomas secreting monoclonal antibodies of different isotypes, and then class-switched variants can be isolated 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] The isolation of other monoclonal antibodies having the specificity of the monoclonal antibodies described herein can also be achieved by those skilled in the art by producing anti-idiotype antibodies. Herlyn et al. (1986) Science 232:100. Anti-idiotype antibodies are antibodies that recognize specific determinants present in the monoclonal antibody of interest.

[0248] In some embodiments disclosed herein, it is useful to label antibodies in a detectable or therapeutic manner. Suitable labeling methods are described above. Methods for conjugating antibodies with these active ingredients are known in the art. For illustrative purposes only, antibodies can be labeled with detectable moieties such as radioactive atoms, chromophores, or fluorophores. Such labeled antibodies can be used in diagnostic techniques in vivo or in isolated test samples.

[0249] Coupling antibodies to low molecular weight haptens can increase the sensitivity of the antibodies in the assay. The haptens 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) above.

[0250] The variable regions of the antibodies of this 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 of the antibody's binding properties (e.g., affinity). Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and their effects on the desired antibody binding or other functional properties can be evaluated in appropriate in vitro or in vivo assays. In certain embodiments, conservative modifications are introduced, typically involving changes to one, two, three, four, or five or fewer residues within the CDR region. Mutations may be amino acid substitutions, additions, or deletions.

[0251] For example, by "backmutating" one or more framework residues to the corresponding germline sequence, the framework of an antibody can be modified, thereby reducing its immunogenicity.

[0252] In addition, the antibodies disclosed herein can be manipulated to include 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 cell-mediated cytotoxicity. Such modifications include, but are not limited to, alterations in the number of cysteine ​​residues in the hinge region to facilitate light- and heavy-chain assembly or to increase or decrease antibody stability (U.S. Patent No. 5,677,425), and amino acid mutations in the Fc hinge region to reduce the biological half-life of the antibody (U.S. Patent No. 6,165,745).

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

[0254] Antibodies disclosed herein can be pegylated to increase their biological half-life by reacting an antibody or antibody fragment with PEG or a reactive ester or aldehyde derivative of PEG under conditions that cause one or more polyethylene glycol (PEG) groups to bind to the antibody or antibody fragment. Antibody pegylation can be carried out by acylation or alkylation reactions with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any PEG form that has 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 may be an aglycosylated antibody. Methods for pegyrating proteins are known in the art and can be applied to the antibodies disclosed herein (EP0154316 and EP0401384).

[0255] Furthermore, antibodies can be chemically modified by conjugating or fusing their antigen-binding domain to serum proteins such as human serum albumin, thereby increasing the half-life of the resulting molecule. Such approaches are described, for example, in EP0322094 and EP0486525.

[0256] The antibodies or fragments thereof of this disclosure can be conjugated into diagnostic agents and used diagnostically, for example, to monitor the onset or progression of a disease and to 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 used in various positron emission tomography (PET) scans, and non-radioactive paramagnetic metal ions. Detectable substances can be coupled or conjugated directly or indirectly via linkers to the antibodies or fragments thereof 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. Suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. 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 This includes 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 with radioactive metal ions by the use of a bifunctional chelating agent covalently linked to the antibody. Chelating agents can be attached via 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] Furthermore, the antibodies or fragments thereof of this disclosure can be conjugated into therapeutic agents. Suitable therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, antimetabolites (methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparagus) Laginase, gemcitabine, cladribine, etc.; alkylating agents (mechloretamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin, etc.); antibiotics (dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mitramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), etc.); diphtheria toxin and related molecules (diphtheria A chain and its active fragments, as well as hybrid molecules, etc.); lysine toxin (lysine A or deglycosylated Lysine 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 bark protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alpha-sarcin, AleuritesIt contains fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, geronin, 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, immunostimulant nucleic acids, aptamers, ribozymes, triple-helix-forming molecules, and external guide sequences. Aptamers are small nucleic acids ranging from 15 to 50 nucleotides in length that fold into defined secondary and tertiary structures such as stem-loops or G-quartets, and can bind to small molecules such as ATP (US Patent No. 5,631,146) and theophiline (US Patent No. 5,580,737), as well as large molecules such as reverse transcriptase (US Patent No. 5,786,462) and thrombin (US 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 target substrates and subsequent cleavage. Triplican-forming nucleic acid molecules can interact with double-stranded or single-stranded nucleic acids by forming a triple helix, where three DNA strands form a complex dependent on both Watson-Crick and Hoogsteen base pairing. Triplican molecules can bind to target regions with high affinity and specificity.

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

[0260] Therapeutic agents can be linked to antibodies directly or indirectly using one of several available methods. For example, the active ingredient can be linked to the hinge region of the reduced antibody component via disulfide bond formation using a crosslinking agent such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP), or via the 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 into 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 Prospective 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)およびThorpe et al. ”The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” (1982) Immunol. Rev. 62:119-58)。

[0262] The antibodies or their antigen-binding regions disclosed herein can be ligated to other functional molecules, such as ligands of other antibodies or receptors, to produce bispecific or polyspecific molecules that bind to at least two or more different binding sites or target molecules. The ligation of an 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 non-covalent association. The polyspecific molecules may further include a third binding specificity in addition to the first and second target epitopes.

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

[0264] The antibodies or fragments thereof can be linked to a portion of the cell to which the antibody is bound to form a “depletion” antibody.

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

[0266] Antibodies can also be conjugated to many different carriers. Therefore, this 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 properties of the carriers may be either soluble or insoluble for the purposes disclosed herein. Those skilled in the art will know of other suitable carriers for conjugating monoclonal antibodies, or can verify such carriers using routine experimental methods.

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

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

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

[0270] In some embodiments of the antibodies provided herein, the antibodies are Fab, F(ab)'2, Fab', scF v and F v It is selected from the group consisting of the following.

[0271] In some embodiments of antibodies provided herein, the antibody includes an Fc domain. In some embodiments of antibodies provided herein, the antibody is an antibody from a non-human animal, such as a rat, sheep, cattle, dog, cat, or rabbit. In some embodiments of 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 antibody includes a human antibody framework region.

[0273] In other embodiments, one or more amino acid residues in the CDR of the antibodies provided herein are substituted with other amino acids. 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 occur 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 addition or deletion occurs at the N-terminus or C-terminus of the CDR or at a position within the CDR.

[0279] By altering the amino acid sequence of the antibody's CDR through the addition, deletion, or substitution of amino acids, various effects can be obtained, such as increased binding affinity to the target antigen.

[0280] It should be fully understood that the antibodies of this disclosure, containing such modified CDR sequences, still bind to DNABII proteins with similar specificity and sensitivity profiles to those of the disclosed antibodies. This can be verified by binding assays.

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

[0282] The antibodies disclosed herein can be used to purify the polypeptides disclosed herein and identify bioequivalent polypeptides and / or polynucleotides. The antibodies disclosed herein can also be used to identify active agents that modify 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] The function can also be demonstrated by using antibodies that neutralize the activity of the protein encoded by the identified gene in vivo and in vitro, and by adding such neutralizing antibodies to in vivo and in vitro test systems. The antibodies are also useful as pharmaceuticals for modulating the activity of the polypeptides disclosed herein.

[0284] Using various antibody preparations in analytical methods such as ELISA assays or Western blotting, the expression of proteins encoded by identified genes in test cells can also be demonstrated in vitro or in vivo. By using appropriate polyclonal antiserum and samples derived from experimental specimens, fragments of such proteins produced by protease degradation in metabolism can also be identified.

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

[0286] This disclosure further provides compositions comprising, or essentially, or further comprising, one, two or more, three or more, of the following: an agent that interferes with the binding of polyamines to DNA in a biofilm; 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; an agent that interferes with the binding of eDNA to DNA-binding proteins; and / or antimicrobial agents. In one embodiment, the composition is not essentially, nor further comprising, HMB1 protein, fragments thereof, or equivalents. In another embodiment, the composition is not essentially, nor further comprising, a DNase. In a further embodiment, the composition is not essentially, nor further comprising, a DNase. In one embodiment, the composition is not essentially, nor further comprising, 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 the biofilm or its local environment. In the second embodiment, the composition comprises, or essentially derived from, or further derived from, an active agent that depletes cations from the biofilm, an active agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, and an active agent that interferes with the binding of eDNA to DNA-binding proteins. In the third embodiment, the composition comprises, or essentially derived from, or further derived from, an active agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment, and an active agent that interferes with the binding of eDNA to DNA-binding proteins. In the fourth embodiment, the composition comprises, or essentially derived from, or further derived from, an active agent that interferes with the binding of polyamines to DNA in the biofilm, and an active agent that interferes with the binding of eDNA to DNA-binding proteins.In the fifth embodiment, the composition comprises, or essentially derived from, or further derived from, an active agent that interferes with the binding of polyamines to DNA in a biofilm, an active agent that interferes with the binding of eDNA to DNA-binding proteins, and / or an antimicrobial agent. In the sixth embodiment, the composition comprises, or essentially derived from, or further derived from, an active agent that interferes with the binding of polyamines to DNA in a biofilm, an active agent that depletes cations from the biofilm, and an active agent that interferes with the binding of eDNA to DNA-binding proteins. In the seventh embodiment, the composition comprises, or essentially derived from, or further derived from, an active agent that interferes with the binding of polyamines to DNA in a biofilm, an active agent that depletes cations from the biofilm, and an active agent that interferes with the conversion of B-DNA to Z-DNA in the biofilm or its local environment.

[0287] The compositions of this disclosure further comprise, or are essentially derived from, or may further comprise from, pharmaceutically acceptable carriers.

[0288] In one embodiment, the active agent that interferes with the binding of polyamines to DNA in a biofilm includes one or more of the polyamine analogs difluoromethylornithine, trans-4-methylcyclohexylamine, saldomodide, methylglyoxal-bis[guanylhydrazone] (MGBG), 1-aminooxy-3-aminopropane, oxaliplatin, cisplatin and / or dicyclohexylamine, any derivative thereof, or salts thereof. In another embodiment, the active agent that depletes cations from the biofilm includes one or more of the cation exchange resins, aminopolycarboxylic acids, crown ethers, azacrowns, or cryptands, sulfonates, sulfopropyls, phosphocellulose, P11 phosphocellulose and / or heparin sulfate, or derivatives or analogs thereof. In yet another embodiment, the active agent that interferes with the conversion of B-DNA to Z-DNA in a biofilm or its local environment includes one or more of the HMGB1 protein, its fragments or equivalents thereof, an anti-B-DNA antibody, its fragments or derivatives, and / or chloroquine, or any derivative thereof. In a particular embodiment, the active agent that interferes with the binding of eDNA to a DNA-binding protein includes one or more of the anti-DNABII antibody, an anti-IHF antibody, and / or an anti-HU antibody, or fragments thereof.

[0289] Further compositions are provided. A composition comprises a carrier and one or more of the isolated polypeptides, isolated polynucleotides, vectors, isolated host cells, small molecules, or antibodies disclosed herein. The carrier may be one or more solid supports or pharmaceutically acceptable carriers. The composition may further comprise other components suitable for administration as an adjuvant or vaccine. In one embodiment, the composition is formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants. In addition, embodiments of the compositions of this disclosure comprise one or more of the isolated polypeptides, isolated polynucleotides, vectors, small molecules, isolated host cells, or antibodies disclosed herein, formulated with one or more pharmaceutically acceptable substances.

[0290] For oral preparations, one or more of the isolated or recombinant polypeptides, isolated or recombinant polynucleotides, vectors, isolated host cells, small molecules, or antibodies described herein may be used alone or in combination with appropriate additives for making tablets, powders, granules, or capsules, for example, with conventional additives such as lactose, mannitol, corn starch, or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; with disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and optionally with diluents, buffers, humectants, preservatives, and flavorings, in pharmaceutical formulations disclosed herein that contain or are essentially derived from the compounds. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavoring.

[0291] Pharmaceutical formulations and unit dose forms suitable for oral administration are particularly useful in the treatment of chronic conditions, infections, and in therapies where patients self-administer drugs. In one embodiment, the formulation is specific for pediatric administration.

[0292] This disclosure provides pharmaceutical formulations in which one or more of the isolated polypeptides, isolated polynucleotides, vectors, isolated host cells, or antibodies disclosed herein may be formulated in accordance with this disclosure by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol, together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives or other antimicrobial agents, as desired. Non-limiting examples of such substances include surface antigens, such as other vaccine components like OMP P5, OMP 26, OMP P2, or type IV pyrin protein (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. Suitable carriers for intravenous administration 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 fluid enough to be easily injected.

[0293] The aerosol formulations provided herein can be administered by inhalation and may be propellant or non-propellant based. For example, embodiments of the pharmaceutical formulations disclosed herein include compounds disclosed herein formulated in a pressurized, acceptable propellant such as dichlorodifluoromethane, propane, or nitrogen. For administration by inhalation, the compounds may be delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant, such as a gas such as carbon dioxide. Non-limiting examples of non-propellants are pump sprays ejected from a closed container using mechanical force (i.e., by pushing down a piston with a finger, or by compression of the container, such as by a compressive force applied to the container wall or by the wall itself, such as an elastic force exerted by an elastic sac).

[0294] The suppositories disclosed herein can be prepared by mixing the compounds disclosed herein with any of various bases, such as emulsifying agents or water-soluble bases. Such pharmaceutical formulations of the compounds disclosed herein can be administered rectally by suppository. The suppositories may contain media that melt at body temperature but solidify at room temperature, such as cocoa butter, carbowax, and polyethylene glycol.

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

[0296] Embodiments of the pharmaceutical formulations disclosed herein include embodiments in which one or more of the isolated polypeptides, isolated polynucleotides, vectors, small molecules for use in this disclosure, isolated host cells, or antibodies disclosed herein are formulated into an injectable composition. The injectable pharmaceutical formulations disclosed herein are prepared as liquid solutions or suspensions; or as solid forms suitable for dissolution or suspension in a liquid medium prior to injection. According to other embodiments of the pharmaceutical formulations disclosed herein, the preparations may also be emulsified or as active ingredients encapsulated in a liposome medium.

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

[0298] Mechanical or electromechanical injection pumps may also be suitable for use according to this disclosure. Examples of such devices include, for example, those described in U.S. Patents No. 4,692,147; No. 4,360,019; No. 4,487,603; No. 4,360,019; No. 4,725,852; No. 5,820,589; No. 5,643,207; No. 6,198,966, etc. In general, delivery of the compounds disclosed herein can be achieved using any of a variety of refillable pump systems. The pumps provide a consistent and controlled release over time. In some embodiments, the compounds disclosed herein are present in a liquid formulation in a drug-impermeable reservoir and delivered to a solid 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 in any suitable implantation site using methods and devices known in the art. The implantation site is a site within the subject's body where the drug delivery device is introduced and placed. The implantation site includes, but is not limited to, subdermal, subcutaneous, intramuscular, or other suitable sites within the subject's body. Subcutaneous implantation sites are used in some embodiments for convenience in the implantation and removal of the drug delivery device.

[0300] Drug release devices suitable for use in this disclosure can be based on any of a variety of operating mechanisms. For example, a drug release device can be based on a diffusion system, a convection system, or an erosive system (e.g., an erosion-based system). For example, a drug release device can be an electrochemical pump, an osmotic pump, an electroosmotic pump, a vapor pressure pump, or an osmotic burst matrix, in which case, for example, the drug is incorporated into a polymer, and the polymer releases the drug formulation in conjunction with the decomposition of the drug-impregnated polymer material (e.g., a biodegradable, drug-impregnated polymer material). In other embodiments, a drug release device can be based on an electrodiffusion system, an electrolytic pump, a foaming pump, a piezoelectric pump, a hydrolysis system, etc.

[0301] Drug release devices based on mechanical or electromechanical infusion pumps may also be suitable for use according to this disclosure. Examples of such devices include, for example, those described in U.S. Patents 4,692,147; 4,360,019; 4,487,603; 4,360,019; and 4,725,852. Generally, the treatment methods in question can be achieved using any of a variety of refillable and non-replaceable pump systems. Pumps and other convection systems can be used because they generally offer more consistent controlled release over time. Osmotic pumps are used in some embodiments 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. Patents 5,985,305 and 5,728,396). Examples of osmotic pressure-driven devices suitable for use in this disclosure are 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; 4,203,440. This includes, but is not limited to, the devices described in Patent 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, and others. A further exemplary device to which this disclosure may apply is a Synchromed injection pump (Medtronic).

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

[0303] Suitable excipient media for the compounds disclosed herein include, for example, water, saline solution, dextrose, glycerol, ethanol, and combinations thereof. In addition, if desired, the media may contain small amounts of auxiliary substances such as wetting agents, emulsifiers, or pH buffers. Methods for preparing such dosage forms are known or will become apparent to those skilled in the art by considering this disclosure. For example, Remington's Pharmaceutical Sciences, Mack Publishing Company, See Easton, Pa., 17th edition, 1985. In any case, the composition or preparation to be administered shall contain a compound content appropriate for achieving the desired state in the subject being treated.

[0304] The compositions of this disclosure include compositions comprising a sustained-release matrix or a controlled-release matrix. In addition, embodiments of this disclosure can be used in conjunction with other treatments using sustained-release formulations. As used herein, a sustained-release matrix is ​​a matrix made from materials, usually polymers, that are degradable by enzymatic or acid-based hydrolysis or dissolution. When inserted into the body, the matrix is ​​subjected to the action of enzymes and body fluids. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolides (polymers of glycolic acid), polylactide co-glycolides (copolymers of lactic acid and glycolic acid), polyacid anhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids (carboxcylic acid), fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, and isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. The biodegradable matrices described include polylactide matrices, polyglycolide matrices, and polylactide co-glycolide (a copolymer of lactic acid and glycolic acid) matrices.

[0305] In another embodiment, the active substance (and combination composition) is delivered in a controlled-release system. For example, the compounds disclosed herein can be administered by intravenous injection, implantable osmotic pump, transdermal patch, liposome or other administration mechanism. 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, polymer materials are used. In yet another embodiment, the controlled-release system is positioned close to the therapeutic target, i.e., the liver, and therefore requires only a small fraction of the systemic dose. In yet another embodiment, the controlled-release system is positioned close to the therapeutic target and therefore requires only a small fraction of the systemic dose. Other controlled-release systems are discussed in the overview by Langer (1990) Science 249:1527-1533.

[0306] In another embodiment, the compositions of the present disclosure (and separate or combined compositions) include compositions formed by impregnating absorbable materials such as sutures, bandages, and gauze with the inhibitory substances described herein, or compositions coated on the surface of solid-phase materials such as surgical staples, zippers, and catheters, for the purpose of delivering the compositions. Other delivery systems of this kind will be readily apparent to those skilled in the art in consideration of the present disclosure.

[0307] This disclosure provides methods and compositions for the administration of one or more active substances to a host (e.g., human) for the treatment of microbial infections. In various embodiments, such methods disclosed herein encompass virtually all available methods and routes suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local administration routes. Screening assay

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

[0309] As will be apparent to those skilled in the art, suitable cells can be cultured in microtiter plates, and several active substances can be simultaneously assayed by focusing on genotypic changes, phenotypic changes, or reductions in microbial titer.

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

[0311] If the active agent is an antibody or an antigen-binding fragment, the active agent can be contacted with 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 on subjects. When the subjects are animals such as rats, chinchillas, mice, or monkeys, this method provides a simple animal model system that can be used prior to clinical testing of active ingredients in human patients. In this system, if the symptoms of a disease or microbial infection are reduced or eliminated compared to untreated animals with the same infection, the candidate active ingredient is a potential drug. Having separate negative control groups of healthy, untreated cells or animals to provide a basis for comparison may be useful.

[0313] The active substances and compositions can be used in the manufacture of pharmaceuticals, such as as active ingredients in pharmaceutical compositions, and for the treatment of humans and other animals by administration according to conventional procedures. Combination therapy

[0314] The compositions and related methods of this 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 active ingredient is administered in the absence of a DNase enzyme.

[0315] In other embodiments, the methods and compositions may be combined with antibiotics and / or antimicrobial agents. Antimicrobial agents 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-related infections susceptible to conventional treatments for treating the infection. In other embodiments, the use of antibiotics or antimicrobial agents in combination with the methods and compositions described herein allows for a reduction in the effective amount of antimicrobial agent and / or biofilm reducer. Some non-limiting examples of antimicrobial agents and antibiotics useful in combination with the methods of this disclosure include amoxicillin, amoxicillin clavulanate, cefdinir, azithromycin, and sulfamethoxazole trimethoprim. The therapeutically effective dose of antimicrobial agents and / or antibiotics in combination with biofilm reducers 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 that has been shown to be effective in other bacterial infections, e.g., bacterial infections in which the etiology of infection is not biofilm-dependent. 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. Antibiotics or antimicrobial agents may be added before, simultaneously with, or after the addition of anti-DNABII antibodies.

[0316] In other embodiments, the methods and compositions can be combined with antibodies that treat bacterial infections. An 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 biofilm in vitro. The combined treatment with this antibody and a biofilm reducing agent yields a greater effect than that which can be achieved by using either reagent alone at the same concentration. Other antibodies that can produce a synergistic effect when combined with a biofilm reducing agent or a method for reducing biofilm include anti-rsPilA, anti-OMP26, anti-OMP P2, and whole anti-OMP preparations.

[0317] The compositions and methods described herein can be used to make biofilm-mediated bacterial infections susceptible to common therapeutic modalities that are effective in treating non-biofilm-mediated bacterial infections but 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, such combination of additional therapeutic agents and biofilm reducers or methods may produce a synergistic effect that may reduce the effective dose of either the biofilm reducer or the additional therapeutic agent. In other examples, such combination of additional therapeutic agents and biofilm reducers or methods may produce a synergistic effect that enhances the treatment. This enhancement of treatment can be demonstrated by the fact that a shorter time dose is required to treat the infection.

[0318] Additional therapeutic measures may be added before, simultaneously with, or after the method or composition used to reduce the biofilm, and may be contained within the same formulation or as separate formulations. kit

[0319] Provided herein are kits comprising, or essentially derived from, or further derived from, the compositions disclosed herein and instructions for use. In one embodiment, the instructions for use provide a method of use for performing any of the methods disclosed herein. In one embodiment, one or more, two or more, or three or more of the active ingredients for use in the methods di...

Claims

[Claim 1] The invention as shown in the drawings.

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