Composition and method for treatment of burkholderia infection

Abstract

To provide a composition and method for treatment of burkholderia infection.SOLUTION: According to the present invention, there is provided a method for breaking down a biofilm in a cystic fibrosis patient. According to the present invention, there is also provided an anti-IHF antibody for use in treating or preventing the recurrence of a Burkholderia infection in a Cystic Fibrosis (CF) patient in need of treating or preventing the recurrence of a Burkholderia infection. Here, the anti-IHF antibody may be administered to the patient in the absence of a DNase treatment. According to the present invention, there is further provided a hybridoma cell line producing monoclonal antibodies of the invention.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Citation of Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61 / 834,846, filed June 13, 2013, the contents of which are hereby incorporated by reference into this application. [Background technology]

[0002] background Throughout this disclosure, references, such as technical publications, scientific publications, patent publications, etc., are cited within parentheses. This information in its entirety is incorporated by reference into this disclosure to more fully describe the state of the art and to support the claimed subject matter.

[0003] Cystic fibrosis (CF) is the most common genetic fatal disorder affecting Caucasians (Campana et al. (2004) J. Cyst. Fibros. 3:159-163). The greatest problem with this genetic disorder is the inability of CF patients to eradicate bacterial pathogens from the lungs. As a result, bacterial proliferation is followed by the formation of adherent extracellular biota. These bacteria persist within the film and / or locally invade host cells, creating a vicious cycle of chronic infection. Each acute reproductive cycle elicits a strong host response, and to clear these infections, an exaggerated inflammatory response damages lung tissue, resulting in scarring, impaired lung function, and often death. Even today, at least 90% of CF patients die from respiratory failure. Current treatment strategies rely heavily on antibiotics, but certain pathogens retain a high degree of resistance to antibiotic treatment, a phenotype largely attributed to their ability to reside within biofilms. Once biofilms form and infection is established in the CF lung, bacterial eradication is rare (George et al. (2009) FEMS Microbiol. Lett. 300:153-164).

[0004] While the predominant pathogen in adult CF patients is Pseudomonas aeruginosa, the most harmful CF-associated pathogens are members of the Burkholderia cepacia complex (Bcc), with Burkholderia cenocepacia (a Gram-negative opportunistic pathogen) being perhaps the most virulent member. Of the 17 officially named species within the Bcc complex, B. multivorans and B. cenocepacia predominate in CF (Simpson et al. (1994) J. Antimicrob. Chemother. 34:353-361; Butler et al. (1995) J. Clin. Microbiol. 33:1001-1004; Castellani et al. (1995) Arch Dis Child 73:276; LiPuma et al. (1995) N. Engl. J. Med. 332:820-821), accounting for approximately 85-97% of all Burkholderia infections. While CF patients infected with any B. cerevisiae species often have a poor prognosis, B. cenocepacia infection is more severe, resulting in decreased survival and a higher risk of developing the fatal "cepacia syndrome" (Simpson et al. (1994) J. Antimicrob. Chemother. 34:353-361; Butler et al. (1995) J. Clin. Microbiol. 33:1001-1004; Castellani et al. (1995) Arch. Dis. Child. 73:276; LiPuma et al. (1995) N. Engl. J. Med. 332:820-821; Burns et al. (1999) Pediatr. Infect. Dis. J. 18:155-156; Hopkins et al. (2009) Am. J. Respir. Crit. Care Med. 179:257-258; De Soyza et al. (2010) J. Heart. Lung Transplant 29:1395-1404; Nash et al. (2010) Transpl. Infect. Dis. 12:551-554).B. cenocepacia is intrinsically resistant to polymyxins, aminoglycosides, and most β-lactams and can develop resistance to essentially all antibiotic classes (Aaron et al. (2000) Am. J. Respir. Crit. Care Med. 161:1206-1212; Golini et al. (2006) Eur. J. Clin. Microbiol. Infect. Dis. 25:175-180; Dubarry et al. (2010) Appl. Environ. Microbiol. 76:1095-1102).

[0005] B. cenocepacia has gained notoriety as a pathogen in CF because it is difficult to identify and treat and can be easily transmitted between CF individuals. Even outside of CF, highly contagious epidemic strains of B. cenocepacia have been shown to contaminate healthcare settings and spread within hospitals (Graindorge et al. (2010) Diagn. Microbiol. Infect. Dis. 66:29-40). It is also likely that multidrug-resistant B. cenocepacia can transfer resistance mechanisms to other microorganisms present in the human airway, particularly those that co-colonize the CF lung. This hypothesis is supported by the finding that 25-45% of adult CF patients are chronically infected with multiple multidrug-resistant bacteria (Lechtzin et al. (2006) Respiration 73:27-33).

[0006] The pathogenesis of CF airway disease is multifactorial and includes defects in the antibacterial activity of airway secretions, altered mucociliary clearance, abnormal submucosal gland function, and excessive production of reactive oxygen species (ROS). Chronic inflammation is central to CF pathogenesis as a result of pulmonary infection, leading to lung damage and 85% mortality. Excessive cytokine-secreting alveolar macrophages further contribute to the lung pathology observed in CF patients. Proinflammatory mediators, such as IL-1β, IL-8, TNF-α, and anti-inflammatory IL-10, are detected in CF patients, even in young patients without culture-positive infections. Furthermore, whereas alveolar macrophages typically phagocytose microorganisms that access the lung, sequestering them within vacuoles and fusing with lysosomes, where their contents are degraded and removed through a process called autophagy, CF macrophages are defective in this regard. This inherent autophagy vulnerability (Luciani et al. (2010) Nat. Cell. Biol. 12:863-875; Luciani et al. (2011) Autophagy 7:104-106; Luciani et al. (2012) Autophagy 8) is further exacerbated by B. cenocepacia's ability to downregulate essential autophagy molecules (Abdulrahman et al. (2011) Autophagy 7:1359-1370). B. cenocepacia infection in CF patients is considered a virtual death sentence because it is extremely difficult to treat and often precludes the patient from receiving a lung transplant, a last resort for lifesaving care. Therefore, the need to develop novel, highly effective approaches to eradicate B. cenocepacia from the lungs of CF patients as well as from the medical environment cannot be underestimated. The present invention fulfills this need and provides related advantages as well. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Campana et al. (2004) J.Cyst.Fibros.3:159-163 [Non-patent document 2] George et al. (2009) FEMS Microbiol. Lett. 300:153-164 [Non-patent document 3] Simpson et al. (1994) J. Antimicrob. Chemother. 34:353-361 [Non-patent document 4] Butler et al. (1995) J.Clin.Microbiol.33:1001-1004 [Non-patent document 5] Castellani et al. (1995) Arch. Dis Child 73:276 [Non-patent document 6] LiPuma et al. (1995) N. Engl. J. Med. 332:820-821 [Non-Patent Document 7] Burns et al. (1999) Pediatr. Infect. Dis. J. 18:155-156 [Non-patent document 8] Hopkins et al. (2009) Am. J. Respir. Crit. Care Med. 179:257-258 [Non-Patent Document 9] De Soyza et al. (2010) J. Heart. Lung Transplant 29:1395-1404 [Non-Patent Document 10] Nash et al. (2010) Transpl. Infect. Dis. 12:551-554 [Non-Patent Document 11] Aaron et al. (2000) Am.J.Respir.Crit.Care Med.161:1206-1212 [Non-Patent Document 12] Golini et al. (2006) Eur.J.Clin.Microbiol.Infect.Dis.25:175-180 [Non-Patent Document 13] Dubarry et al. (2010) Appl.Environ.Microbiol.76:1095-1102 [Non-Patent Document 14] Graindorge et al. (2010) Diagn. Microbiol. Infect. Dis. 66:29-40 [Non-Patent Document 15] Lechtzin et al. (2006) Respiration 73:27-33 [Non-Patent Document 16] Luciani et al. (2010) Nat.Cell.Biol.12:863-875 [Non-Patent Document 17] Luciani et al. (2011) Autophagy 7:104-106 [Non-Patent Document 18] Abdulrahman et al. (2011) Autophagy 7:1359-1370 Summary of the Invention [Means for solving the problem]

[0008] Disclosure Overview The present inventors have identified a protein target for the development of a novel approach to treating CF infection. This protein target (a member of the DNABII family of DNA-binding proteins) is essential for biofilm formation and stability by multiple human pathogens (Goodman et al. (2011) Mucosal Immunol. 4:625-637; Justice et al. (2012) PLoS One 7:e48349; Gustave et al. (2012) J. Cyst. Fibros.) due to its contribution to the structural lattice of extracellular DNA (eDNA) within these bacterial populations. The DNABII family is a member of a protein class called nucleoid-associated proteins (NAPs), bacterial proteins that partially shape the intracellular bacterial nucleoid (Browning et al. (2010) Curr. Opin. Microbiol. 13:773-780). Furthermore, this family is ubiquitous and expressed by virtually all eubacteria. All family members characterized to date function as either homodimers or heterodimers of subunits. This family is divided into two types: HU (histone-like protein) and IHF (integration host factor), both of which can be expressed in B. cenocepacia (J2315 genetic strains: BCAL3530, hupA; BCAL1585, hupB; BCAL1487, ihfA, and BCAL2949, ihfb). The main difference between these family members is that HU binds to DNA in a sequence-independent manner, while IHF binds to a consensus sequence (WATCAANNNNTTR (where W is A or T and R is purine), SEQ ID NO: 36) that is conserved across genera (Swinger et al. (2004) Curr. Opin. Struct. Biol. 14:28-35). All DNA BII proteins bind to and bend DNA significantly (e.g., E. coli IHF can bend DNA into a substantial U-turn (Rice et al. (1996) Cell 87:1295-1306)). Furthermore, all family members prefer pre-bent or curved DNA structures (e.g., Holliday junctions, the cruciform structures central to DNA recombination).Indeed, DNABII proteins function as cofactors that facilitate all intracellular DNA functions, including gene expression, recombination, repair, and replication (Swinger et al. (2004) Curr. Opin. Struct. Biol. 14:28-35).

[0009] Over the past 20 years, several researchers have discovered that these proteins also exist extracellularly (Gao (2000) Los Angeles: University of Southern California; Winters et al. (1993) Infect. Immun. 61:3259-3264; Lunsford et al. (1996) Curr. Microbiol. 32:95-100; Boleij et al. (2009) Infect. Immun. 77:5519-5527). To date, three extracellular functions have been described. First, streptococcal HU has been shown to induce a potent inflammatory innate immune response by inducing the release of TNFα and interleukin-1 (Zhang et al. (1999) Infect. Immun. 67:6473-6477). Interestingly, B. cenocepacia induces peripheral damage by exacerbating IL-1β production via the host receptor Pyrin through a still unknown mechanism (Gavrilin et al. (2012) Immunol. 188:3469-3477; Kotrange et al. (2011) J. Leukoc. Biol. 89:481-488). In this regard, the extracellular role of DNABII family members remains to be tested for B. cenocepacia. Second, extracellular DNABII proteins are thought to bind to laminin and induce direct contact with host cells (Winters et al. (1993) Infect. Immun. 61:3259-3264). Third, DNABII proteins are known to stabilize the structural integrity of eDNA within the extracellular polymeric matrix or polymeric substances (EPS) of biofilms of several pathogens (Goodman et al. (2011) Mucosal Immunol. 4:625-637). Antisera directed against these proteins are sufficient to destabilize biofilms, thereby exposing / releasing resident bacteria and thus sensitizing them to the action of both antimicrobial agents and immune system effectors (Goodman et al. (2011) Mucosal Immunol. 4:625-637).Recently, it has been shown that both IHF subunits are required for effective colonization of the bladder by uropathogenic E. coli and that both IHF subunits also influence the population structure of intracellular bacterial populations (Justice et al. (2012) PLoS One 7:e48349). Without being bound by theory, and given the large amount of eDNA in B. cenocepacia-induced biofilms, we investigated whether there is a role for the DNABII protein family in stabilizing these biofilms as well and, therefore, whether this protein family could serve as a target for therapeutic intervention. Furthermore, the presence of extracellular DNABII proteins, possibly associated with bacterial cells, may influence B. cenocepacia interactions with host cells, particularly with its primary reservoir (macrophages).

[0010] Therefore, after research and investigation, the present inventors disclose herein an immunotherapeutic approach targeting the DNABII protein, which has been found to be highly effective in both reducing the biofilm formed by B. cenocepacia and sensitizing bacteria newly released from the biofilm EPS to antibiotic action, especially when combined with existing conventional treatments. This method can be used to reduce and eradicate B. cenocepacia reservoirs from the lungs of CF patients. Furthermore, this method has been shown to prevent CF disease relapse by inhibiting the ability of B. cenocepacia to multiply in macrophages isolated from CF mice (an important animal model of CF in humans).

[0011] Thus, in one aspect, there is provided a method of inhibiting, competing with, or determining the titer of a biofilm (e.g., a B. cenocepacia-induced biofilm) present in or contributing to CF, comprising, consisting essentially of, or consisting of contacting the biofilm with an interfering agent, thereby inhibiting, competing with, or determining the titer of the biofilm. The contacting can be performed in vitro or in vivo.

[0012] In another aspect, provided herein is a method for inhibiting, preventing, or determining the titer of bacterial cells in a biofilm in a CF patient or a patient carrying an infection that contributes to CF, comprising, consisting essentially of, or consisting of contacting the bacterial cells with an interfering agent, thereby inhibiting, preventing, or determining the titer of the bacterial cells and infection. The contacting can be in vitro or in It can be performed in vivo.

[0013] Also provided are methods for treating or preventing recurrence of bacterial infection in a CF patient or a patient at risk of developing a bacterial infection, comprising, consisting essentially of, or consisting of administering to the subject an effective amount of an interfering agent, thereby treating or preventing recurrence of bacterial infection in the CF patient.

[0014] To further complement the treatment, the interfering agent can be combined with an antibacterial agent (e.g., ceftazidime, ciprofloxacin, imipenem, and minocycline). Thus, any of the above methods can further comprise, consist essentially of, or further consist of administering or contacting with an effective amount of an antibacterial agent (e.g., ceftazidime, ciprofloxacin, imipenem, and minocycline). In another embodiment, the administration or contacting of the interfering agent is performed without the use of DNase treatment. In one embodiment, the DNase treatment excluded from the treatment includes an enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes known to target not only cruciform structures but also various secondary structures of DNA include DNase I, T4 Endo VII, and T7 Endo I. In one embodiment, the DNase treatment excluded from therapy comprises, consists essentially of, or further consists of Pulmozyme® (dornase alfa; Genentech, Inc.).

[0015] For the methods described herein, any agent that occludes critical surfaces necessary for intracellular persistence or that interferes with or prevents the binding of microbial DNA to DNABII proteins or DNABII polypeptides is intended to be included within the scope of the invention. Non-limiting examples of interfering agents include: (a) an isolated or recombinant integration host factor (IHF) polypeptide, or its respective fragment or equivalent; (b) an isolated or recombinant protein or polypeptide identified in Table 1, Table 2, an Arm fragment identified in Table 2, Table 3, or a DNA-binding peptide identified in Figure 9, or a fragment or equivalent thereof; (c) an isolated or recombinant polypeptide of SEQ ID NO: 1-33 or its equivalent, or a fragment or equivalent thereof; (d) an isolated or recombinant C-terminal polypeptide of SEQ ID NOs: 5-11, 28, 29, or identified in Table 1, or a fragment or equivalent thereof; (e) a polypeptide that occludes a critical surface without competing with or displacing integration host factors in binding to microbial DNA; (f) four-way junction polynucleotides that resemble Holliday junctions, three-way junction polynucleotides that resemble replication forks, polynucleotides with inherent flexibility, or bent polynucleotides; (g) an isolated or recombinant polynucleotide encoding any one of (a) through (e); (h) an antibody or antigen-binding fragment that specifically recognizes or specifically binds to any one of (a) through (e), or their respective equivalents or fragments; (i) an isolated or recombinant polynucleotide encoding the antibody or antigen-binding fragment of (h); and (j) a small molecule that competes with the binding of a DNABII protein or DNABII polypeptide to microbial DNA; Examples include:

[0016] In one aspect, an isolated or recombinant polypeptide is provided that consists essentially of an amino acid sequence selected from SEQ ID NOs: 1-5, 12-27, or 30-33, or a DNA-binding peptide identified in Figure 9.

[0017] In another embodiment, the method is practiced using an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NO: 1 or 2, but not any of SEQ ID NOs: 6-11, 28, or 29.

[0018] In another embodiment, the method is practiced using an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NO: 3, 4, or 5, but not any of SEQ ID NOs: 6-11, 28, or 29.

[0019] In another embodiment, the method is practiced using an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 30, or 32, but not any of SEQ ID NOs: 6-11, 28, or 29.

[0020] In one aspect, the method is practiced using an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 31, or 33, but not any of SEQ ID NOs: 6-11, 28, or 29.

[0021] In another embodiment, the method is practiced using an isolated or recombinant polypeptide comprising, consisting essentially of, or consisting of SEQ ID NOs: 12 and 13, or 14 and 15, or 16 and 17, or 18 and 19, or 20 and 21, or 22 and 23, or 24 and 25, or 26 and 27, or 30 and 31, or 32 and 33, but not any of SEQ ID NOs: 6-11, 28, or 29.

[0022] In another aspect, the method is practiced using an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of the C-terminal region or peptide of a DNABII polypeptide, an IHF polypeptide, a polypeptide of SEQ ID NOs: 6-11, 28, 29, or the group of polypeptides identified in Table 1, or a fragment or equivalent thereof.

[0023] Further non-limiting examples of agents that can be used in the methods of the present invention include: (a) an isolated or recombinant integration host factor (IHF) polypeptide, or a fragment or equivalent thereof; (b) an isolated or recombinant histone-like protein (HU) polypeptide from E. coli strain U93, or a fragment or equivalent thereof; (c) an isolated or recombinant protein or polypeptide identified in Table 1, Table 2, an Arm fragment identified in Table 2, Table 3, Table 4, or a DNA-binding peptide identified in Figure 9, or a fragment or equivalent thereof; (d) an isolated or recombinant polypeptide of SEQ ID NO: 1-348, or a fragment or equivalent thereof; (e) an isolated or recombinant C-terminal polypeptide of SEQ ID NOs: 6-11, 28, 29, 42-100, Table 1, or a C-terminal polypeptide identified in Table 4, or a fragment or equivalent thereof; (f) a polypeptide or polynucleotide that competes with an integration host factor for binding to microbial DNA; (g) four-way junction polynucleotides that resemble Holliday junctions, three-way junction polynucleotides that resemble replication forks, polynucleotides that have inherent flexibility or are bent; (h) an isolated or recombinant polynucleotide encoding any one of (a) to (f), or an isolated or recombinant polynucleotide of SEQ ID NO: 36, or their respective equivalents, or a polynucleotide that hybridizes under stringent conditions to the polynucleotide or its equivalent or its complement; (i) an antibody or antigen-binding fragment that specifically recognizes or specifically binds to any one of (a) to (f), or an equivalent or fragment of each antibody or antigen-binding fragment thereof; (j) an isolated or recombinant polynucleotide encoding the antibody or antigen-binding fragment of (i), or its complement; or (k) a small molecule that blocks or competes with the binding of a DNABII protein or DNABII polypeptide to microbial DNA Examples include:

[0024] Provided herein is a method for inducing an immune response in a CF subject in need thereof or conferring passive immunity to a subject in need thereof, comprising administering to the CF subject a medicament selected from the group consisting of: (a) an isolated or recombinant integration host factor (IHF) polypeptide, or a fragment or equivalent thereof; (b) an isolated or recombinant histone-like protein (HU) polypeptide from E. coli strain U93, or a fragment or equivalent thereof; (c) an isolated or recombinant protein polypeptide identified in Table 1, Table 2, an Arm fragment identified in Table 2, Table 3, Table 4, or a DNA-binding peptide identified in Figure 9, or a fragment or equivalent thereof; (d) an isolated or recombinant polypeptide of SEQ ID NO: 1-348, or a fragment or equivalent thereof; (e) an isolated or recombinant C-terminal polypeptide of SEQ ID NOs: 6-11, 28, 29, 42-100, Table 1, or a C-terminal polypeptide identified in Table 4, or a fragment or equivalent thereof; (f) an isolated or recombinant polynucleotide encoding any one of (a) through (e), or an isolated or recombinant polynucleotide of SEQ ID NO: 36, or their respective equivalents, or a polynucleotide that hybridizes under stringent conditions to the polynucleotide, its equivalent, or its complement; (g) an antibody or antigen-binding fragment that specifically recognizes or specifically binds to any one of (a) to (e), or their respective equivalents or fragments; (h) an isolated or recombinant polynucleotide encoding the antibody or antigen-binding fragment of (g); (i) antigen-presenting cells pulsed with any one of (a) to (e); and (j) An antigen-presenting cell transfected with one or more polynucleotides encoding any one of (a) to (e). Also provided are methods comprising, consisting essentially of, or consisting of administering an effective amount of one or more agents of

[0025] Subjects in need of such an immune response or support include those at risk for or suffering from an infection in which a microbial biofilm occurs.

[0026] Also provided herein are polynucleotides, polypeptides and antibodies, antigen-binding fragments and compositions for use in the above methods, non-limiting examples of which are discussed below.

[0027] In one aspect, an isolated or recombinant polypeptide is provided comprising, or consisting essentially of, an amino acid sequence selected from SEQ ID NOs: 1-5, or 12-27, 30-35, 101-348, or a DNA-binding peptide identified in Figure 9.

[0028] In another aspect, an isolated or recombinant polypeptide is provided that comprises, consists essentially of, or further consists of SEQ ID NO: 1 or 2, but is not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0029] In one aspect, an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NO: 3, 4, or 5, but not any of SEQ ID NOs: 6-11, 28, 29, or 42-100, is also provided.

[0030] In one aspect, there is provided an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 30, or 32, but not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0031] In one aspect, there is provided an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 31, or 33, but not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0032] In one aspect, there is provided an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NO: 337, 338, 339, or 340, but not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0033] In one aspect there is provided an isolated or recombinant polypeptide comprising, consisting essentially of, or further consisting of SEQ ID NOs: 12 and 13, or 14 and 15, or 16 and 17, or 18 and 19, or 20 and 21, or 22 and 23, or 24 and 25, or 26 and 27, or 30 and 31, or 32 and 33, but not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0034] In one aspect, there is provided an isolated or recombinant polypeptide comprising, or consisting essentially of, or further consisting of a C-terminal region comprising at least 10, alternatively at least 15, alternatively at least 20, alternatively at least 25, alternatively at least 30, C-terminal amino acids of a DNABII polypeptide, an IHF polypeptide, an HU polypeptide, a polypeptide of SEQ ID NOs: 6-11, 28, 29, or the group of polypeptides identified in Table 1, Table 2, an Arm fragment identified in Table 2, Table 4, or a respective fragment or equivalent thereof, or a polypeptide of SEQ ID NOs: 337-340, or an equivalent thereof.

[0035] In one aspect, A polypeptide comprising SEQ ID NOs: 12 and 13; a polypeptide comprising SEQ ID NOs: 14 and 15; a polypeptide comprising SEQ ID NOs: 16 and 17; a polypeptide comprising SEQ ID NOs: 18 and 19; A polypeptide comprising SEQ ID NOs: 20 and 21; A polypeptide comprising SEQ ID NOs: 23 and 24, a polypeptide comprising SEQ ID NOs: 25 and 26; A polypeptide comprising SEQ ID NOs: 30 and 31; a polypeptide comprising SEQ ID NOs: 32 and 33; a polypeptide comprising SEQ ID NOs: 34 and 35; a polypeptide comprising SEQ ID NOs: 337 and 338, or a polypeptide comprising SEQ ID NOs: 339 and 340; Polynucleotides or polypeptides comprising SEQ ID NOs: 341 to 348 and wherein the polypeptide is not the wild type of any one of IHF alpha, IHF beta, or any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0036] In one aspect, A polypeptide consisting essentially of SEQ ID NOs: 12 and 13; A polypeptide consisting essentially of SEQ ID NOs: 14 and 15; A polypeptide consisting essentially of SEQ ID NOs: 16 and 17; A polypeptide consisting essentially of SEQ ID NOs: 18 and 19; A polypeptide consisting essentially of SEQ ID NOs: 20 and 21; A polypeptide consisting essentially of SEQ ID NOs: 23 and 24; A polypeptide consisting essentially of SEQ ID NOs: 25 and 26; A polypeptide consisting essentially of SEQ ID NOs: 30 and 31; A polypeptide consisting essentially of SEQ ID NOs: 32 and 33; A polypeptide consisting essentially of SEQ ID NOs: 34 and 35; a polypeptide consisting essentially of SEQ ID NOs: 337 and 338, or A polypeptide consisting essentially of SEQ ID NOs: 339 and 340; A polynucleotide or polypeptide consisting essentially of any one of SEQ ID NOs: 341 to 348;

[0013] Provided are isolated or recombinant polynucleotides or polypeptides of the group: wherein the polypeptide is not the wild type of any one of IHF alpha, IHF beta, or any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0037] Also provided are isolated or recombinant polypeptides comprising, consisting essentially of, or consisting of two or more, three or more, four or more, or a number of the above-identified isolated polypeptides, including fragments and equivalents thereof, examples of which include isolated polypeptides comprising SEQ ID NOs: 1-4 and / or 12-29, and / or 30-33, and / or 30-35, e.g., SEQ ID NOs: 1 and 2, or 1 and 3, or 1 and 4, or 2 and 3, or SEQ ID NOs: 1, 2 and 3, or 2, 3 and 4, or 1, 3 and 4, or equivalent polypeptides, examples of which are set forth in Table 2, and in particular, the Arm fragments identified in Table 2 or equivalents thereof, or the polypeptides of SEQ ID NOs: 337-340, or equivalents thereof. The polypeptides can be in any orientation, for example, SEQ ID NOs: 1, 2, and 3, or SEQ ID NOs: 3, 2 and 1, or SEQ ID NOs: 2, 1 and 3, or 3, 1 and 2, or 11 and 12, or 1 and 12, or 2 and 12, or 1 and 12, or 2 and 13, or 12, 16 and 1, or 1, 16 and 12.

[0038] In another aspect, the present invention provides an isolated or recombinant polypeptide comprising SEQ ID NO: 1 or 2 and 3 or 4, or a fragment of a DNABII protein, e.g., a polypeptide of Haemophilus influenzae IHFα organism or Haemophilus The present invention provides a polypeptide or recombinant polypeptide comprising, consisting essentially of, or further consisting of amino acids corresponding to, for example, the beta-3 and / or alpha-3 fragments of an IHFα or IHFβ microorganism, non-limiting examples of which include SEQ ID NOs: 12-27, or the respective fragments or equivalents of the polypeptides, examples of which are set forth in Tables 2 and 3. In one aspect, isolated wild-type polypeptides are specifically excluded, e.g., the polypeptide is not any of SEQ ID NOs: 6-11 or the wild-type sequences identified in Table 1. In this embodiment, the polypeptide comprising, consisting essentially of, or further consisting of amino acids corresponding to SEQ ID NO: 1 or 2, or the beta-3 and / or alpha-3 fragments of an IHFα or IHFβ microorganism, non-limiting examples of which include SEQ ID NOs: 12-27 and 30-33, or their respective equivalents, is located upstream or amino-terminal to SEQ ID NO: 3 or 4, or a fragment or equivalent thereof. In another aspect, the isolated polypeptide comprises, consists essentially of, or further consists of amino acids corresponding to SEQ ID NO: 3 or 4, or the beta-3 fragment and / or alpha-3 fragment of an IHFα microorganism or an IHFβ microorganism, including, but not limited to, SEQ ID NOs: 12-27, or an equivalent thereof located upstream or amino terminal to SEQ ID NO: 1 or 2, or an equivalent thereof.

[0039] In any of the above embodiments, a peptide linker can be added to the N- or C-terminus of the polypeptide, fragment, or equivalent. In one aspect, the linker connects a polypeptide of the invention, e.g., a polypeptide comprising, consisting essentially of, or consisting of amino acids corresponding to SEQ ID NOS: 1-4, 28, 29, 34, or 35, or 30-33, 34, or 35, or the beta-3 fragment and / or alpha-3 fragment of a Haemophilus influenzae IHFα microorganism or a Haemophilus influenzae IHFβ microorganism, non-limiting examples of which include SEQ ID NOS: 12-27, or their respective equivalents. A "linker" or "peptide linker" refers to a peptide sequence linked to either the N- or C-terminus of a polypeptide sequence. In one aspect, the linker is from about 1 to about 20 amino acid residues in length, alternatively from 2 to about 10 amino acid residues, or from about 3 to about 5 amino acid residues in length. An example of a peptide linker is Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO: 37).

[0040] Further provided are isolated or recombinant polypeptides of any one of the above-identified polypeptides, as well as isolated or recombinant polypeptide fragments or equivalents comprising, consisting essentially of, or even consisting of two or more of the above-identified isolated or recombinant polypeptides.

[0041] Further provided are isolated or recombinant polynucleotides encoding the above polypeptides or antibodies or fragments thereof, which can be operably linked to regulatory elements necessary for expression and / or replication of the polynucleotide, such as polynucleotides that interfere with the binding of microbial DNA to a polypeptide or a fragment thereof or equivalent thereof, for example, SEQ ID NO: 36, or a four-way junction polynucleotide resembling a Holliday junction, a three-way junction polynucleotide resembling a replication fork, a polynucleotide with inherent flexibility, or a curved polynucleotide;The polynucleotide may be contained within a vector.

[0042] Also provided are isolated host cells comprising, consisting essentially of, or further consisting of the above-mentioned isolated or recombinant polypeptides, four-way junction polynucleotides resembling Holliday junctions, three-way junction polynucleotides resembling replication forks, polynucleotides with inherent flexibility or bent polynucleotides; the above-mentioned isolated or recombinant polynucleotides, or the above-mentioned vectors.

[0043] Further provided are isolated or recombinant polypeptides of any one of the above-identified polypeptides, as well as isolated or recombinant polypeptide fragments or equivalents comprising, consisting essentially of, or even consisting of two or more of the above-identified isolated or recombinant polypeptides.

[0044] In another aspect, the method is carried out using an antibody or antigen-binding fragment that specifically recognizes and binds to the isolated or recombinant polypeptide, including a fragment or equivalent of the polypeptide. Non-limiting examples of antibodies include polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, antibody derivatives, veneered antibodies, diabodies, chimeric antibodies, antibody derivatives, recombinant human antibodies, or antibody fragments. In certain aspects, the antibody is a monoclonal antibody. Hybridoma cell lines that produce monoclonal antibodies are further provided.

[0045] The present invention also provides isolated or recombinant polynucleotides encoding one or more of the above-identified isolated or recombinant polypeptides or antibodies, or fragments thereof.Vectors containing the isolated polynucleotides are further provided.In one embodiment of the two or more isolated polypeptides of the present invention, the isolated polynucleotides may be contained within a polycistronic vector.

[0046] Further provided is an isolated host cell, or an isolated or recombinant polynucleotide, or a vector, comprising one or more isolated or recombinant polypeptides described herein. In one aspect, the isolated host cell is a eukaryotic cell, such as an antigen-presenting cell, such as a dendritic cell.

[0047] The antibody, polynucleotide, polypeptide, vector or host cell may further comprise a detectable label or carrier (eg, a pharmaceutically acceptable carrier).

[0048] Also provided are compositions comprising a carrier and one or more of the isolated or recombinant polypeptides of the invention, the isolated or recombinant polynucleotides of the invention, the vectors of the invention, the isolated host cells of the invention, or the antibodies of the invention. The carrier may be one or more of a solid support, a medical device such as a stent or dental implant, or a liquid such as a pharmaceutically acceptable carrier. The composition may further comprise an adjuvant, an antimicrobial agent, or an antigenic peptide.

[0049] The composition may further comprise additional biologically active agents, non-limiting examples of which are an antimicrobial agent, such as another vaccine component (i.e., an antigenic peptide), such as a surface antigen, such as a type IV Pilin protein (see Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875).

[0050] The present invention also provides a method for producing an antigenic peptide by growing or culturing a host cell containing the isolated polynucleotide described above under conditions favorable for expression of the polynucleotide. The polypeptide produced by this method can be isolated for further in vitro or in vivo use.

[0051] Kits for use in diagnosis or treatment are also provided, which contain the above compositions and instructions for use. Kits for conducting screens for new drugs and / or combination therapies provided herein are also provided. Sequence Listing SEQ ID NO: 1 A1-A2-A3-A4-A5-A6-A7-A8-A9 where: A1 is V or I; A2 is any one of K, Q, E, A, V or Y; A3 is any one of K, L, I, V or F; A4 is any one of S, I, R or V; A5 is any one of G or S; A6 is F; A7 is G; A8 is any one of N or S or T or K; A9 is F. SEQ ID NO: 2 is VKKSGFGNF. SEQ ID NO: 3 is B1-B2-B3-B4-B5-B5-B6-B7, B1 is absent or any one of G or K; B2 is absent or any one of R, I or K; B3 is N or V; B4 is P or I; B5 is any one of K, Q, S or G; B6 is any one of T, K or S; B7 is any one of G, K, Q or D. SEQ ID NO: 4 is NP(K / Q)TG. SEQ ID NO: 5 GRNP(K / Q)TG SEQ ID NO: 6 Full length wild type (wt) 86-028NP Haemophilus influenzae IhfA; Genbank accession number: AAX88425.1, last accessed March 21, 2011: MATITKLDIIEYLSDKYHLSK QDTKNVVENFLEEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARRVVTFKPGQKLRARVEKTK. SEQ ID NO: 7 Full length wt 86-028NP Haemophilus influenzae HU, Genbank accession number: YP_248142.1, last accessed March 21, 2011: MRFVTIFINHAFNSSQVRLSFAQFLR QIRKDTFKESNFLFNRRYKFMNKTDLIDAIANAAELNKKQAKAALEATLDAITASLKEGEPVQLIGFGTFKVNERAARTGRNPQTGAEIQIAASKVPAFVSGKALKDAIK. SEQ ID NO: 8 Full length wt R2846 Haemophilus influenzae IhfA, Genbank accession number: ADO96375, last accessed March 21, 2011: MATITKLDIIEYLSDKYHLSKQDTKNVVENFL EEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARRVVTFKPGQKLRARVEKTK. SEQ ID NO: 9 Full length wt Rd Haemophilus influenzae IhfA, Genbank accession number: AAC22959.1, last accessed on March 21, 2011: MATITKLDIIEYLSDKYHLSKQDTK NVVENFLEEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARRVVTFKPGQKLRARVEKTK; SEQ ID NO: 10 Full length wt E. coli K12 IhfA; Genbank accession number: AAC74782.1, last accessed March 21, 2011: MALTKAEMSEYLFDKLGLSKRDAKELVELFFE EIRRALENGEQVKLSGFGNFDLRDKNQRPGRNPKTGEDIPITARRVVT FRPGQKLKSRVENASPKDE; DNA Genbank number NC_000913. SEQ ID NO: 11 Full length wt P. aeruginosa PA 01 IhfA; Genbank accession number: AAG06126.1, last accessed March 21, 2011: MGALTKAEIAERLYEELGLNKREA KELVELFFEEIRQALEHNEQVKLSGFGNFDLRDKRQRPGRNPKTGEEIPITARRVVTFRPGQKLKARVEAYAGTKS SEQ ID NOs: 12 and 13: the β-3 and α-3 portions of (IHFβ), SEQ ID NO: 12: TFRPGQ and SEQ ID NO: 13: KLKSRVENASPKDE SEQ ID NOs: 14 and 15: the β-3 and α-3 portions of (IHFβ), SEQ ID NO: 14: HFKPGK and SEQ ID NO: 15: ELRDRANIYG SEQ ID NOs: 16 and 17: the β-3 and α-3 portions of SEQ ID NO: 6, SEQ ID NO: 16: TFKPGQ and SEQ ID NO: 17: KLRARVEKTK SEQ ID NOs: 18 and 19:2019 Haemophilus influenzae The β-3 and α-3 portions of IhfA, SEQ ID NO: 18: TFKPGQ and SEQ ID NO: 19: KLRARVENTK SEQ ID NOs: 20 and 21: the β-3 and α-3 portions of SEQ ID NO: 8, SEQ ID NO: 20: TFKPGQ and SEQ ID NO: 21: KLRARVEKTK SEQ ID NOs: 22 and 23: the β-3 and α-3 portions of SEQ ID NO: 9, SEQ ID NO: 22: TFKPGQ and SEQ ID NO: 23: KLRARVEKTK SEQ ID NOs: 24 and 25: the β-3 and α-3 portions of SEQ ID NO: 10, SEQ ID NO: 24: TFRPGQ and SEQ ID NO: 25: KLKSRVENASPKDE SEQ ID NOs: 26 and 27: the β-3 and α-3 portions of SEQ ID NO: 11, SEQ ID NO: 26: TFRPGQ and SEQ ID NO: 27: KLKARVEAYAGTKS SEQ ID NO: 28: E. coli hupA, Genbank accession number: AP_003818, last accessed March 21, 2011: MNKTQLIDVIAEKAELSKTQAKAALESTLAAITESLKEGDAVQLVGFGTFK VNHRAERTGRNPQTGKEIKIAAANVPAFVSSGKALKDAVK SEQ ID NO: 29: E. coli hupB, Genbank accession number: AP_001090.1, last accessed March 21, 2011: MNKSQLIDKIAAGADISKAAAGRALDAIIASVTESLKEGDDVALVGFG TFAVKERAARTGRNPQTGKEITIAAAKVPSFRAGKALKDAVN SEQ ID NOs: 30 and 31: the β-3 and α-3 portions of SEQ ID NO: 28, SEQ ID NO: 30: AFVSGK and SEQ ID NO: 31: ALKDAVK SEQ ID NOs: 32 and 33: the β-3 and α-3 portions of SEQ ID NO: 29, SEQ ID NO: 32: SFRAGK and SEQ ID NO: 33: ALKDAVN SEQ ID NO: 34: C-terminal 20 amino acids of IHF alpha: TFRPGQKLKSRVENASPKDE SEQ ID NO: 35: C-terminal 20 amino acids of IHF β: KYVPHFKPGKELRDRANIYG SEQ ID NO: 36: DNABII binding consensus sequence: WATCAANNNNTTR, where W is A or T, N is any base, and R is a purine SEQ ID NO: 337: E. coli IHF alpha: GRNPKTGEDIPI SEQ ID NO: 338: E. coli IHF beta: GRNPKTGDKVEL SEQ ID NO: 339: E. coli HU alpha: GRNPQTGKEIKI SEQ ID NO: 340: E. coli HU beta: GRNPQTGKEITI.

[0052] Table Description Table 1 is a non-limiting summary of DNA-binding proteins produced by Gram (+) and Gram (-) bacteria that can be used in the methods provided herein.

[0053] Table 2 is a sequence alignment of relevant portions of DNA-binding proteins of various embodiments of the present invention. Bold text indicates exact matches to the consensus, light gray text indicates conserved amino acid changes, and lightly or darkly shaded sequences are highly conserved across species. Gray-shaded undefined sequences at the amino and / or carboxy termini are undefined amino acids not shared with the consensus sequence. Table 2 is based on information previously published in Obeto et al. (1994) Biochimie 76:901-908. Fragments "ARM" are listed at the bottom of the table; polypeptide fragments comprising, consisting essentially of, or further consisting of these fragments, or equivalents thereof, possess the biological activity described herein.

[0054] Table 3 is a comparison of the 16 amino acid peptide motif to Liu et al. (2008) Cell Microbiol. 10(1):262-276.

[0055] Table 4 lists the α, β, and C-terminal portions of DNABII proteins from the indicated organisms. In certain embodiments, for example, the following are provided: (Item 1) An anti-IHF antibody for use in treating or preventing recurrence of Burkholderia infection in a cystic fibrosis (CF) patient in need thereof. (Item 2) 2. The antibody of item 1, wherein the anti-IHF antibody is administered to the patient without DNase treatment. (Item 3) The antibody according to item 1 or 2 and an antibacterial agent that inhibits the growth of Burkholderia. (Item 4) 4. The antibody according to any one of items 1 to 3, wherein the Burkholderia is Burkholderia cenocepacia or B. multivorans. (Item 5) 5. The antibody according to any one of items 1 to 4, wherein the anti-IHF antibody is an anti-IHFα antibody or an anti-IHFβ antibody. (Item 6) The antibody of any preceding item, wherein the antibody is an IgG antibody. (Item 7) The antibody of any preceding item, wherein the antibody is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof. (Item 8) The antibody of any preceding item, wherein the antibody specifically recognizes and binds to a polynucleotide of the group TCTCAACGATTTA or WATCAANNNNTTR (where W is A or T, N is any nucleotide, and R is A or G) or their respective equivalents, or a polypeptide selected from the group of MATITKLDIIEYLSDKYHLS;KYHLSKQDTKNVVENFLEEI;FLEEIRLSLESGQDVKLSGF;KLSGFGNFELRDKSSRPGRN;RPGRNPKTGDVVPVSARRVV; or ARRVVTFKPGQKLRARVEKTK or their equivalents, or a polypeptide identified in Table 2, or an Arm fragment of a polypeptide identified in Table 2, or their respective equivalents, or a polypeptide of SEQ ID NOs: 337 to 340 or their equivalents. (Item 9) An anti-IHF antibody for use in treating an infection or disease caused by Burkholderia in a subject infected with Burkholderia. (Item 10) 10. The antibody of item 9, wherein the anti-IHF antibody is administered to the subject without DNase treatment. (Item 11) 11. The antibody according to item 9 or 10, and an antibacterial agent that inhibits the growth of Burkholderia. (Item 12) 12. The antibody according to any one of items 9 to 11, wherein the Burkholderia is Burkholderia cenocepacia or B. multivorans. (Item 13) 13. The antibody according to any one of items 9 to 12, wherein the anti-IHF antibody is an anti-IHFα antibody or an anti-IHFβ antibody. (Item 14) 14. The antibody according to any one of items 9 to 13, wherein the antibody is an IgG antibody. (Item 15) 15. The antibody according to any one of items 9 to 14, wherein the antibody is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof. (Item 16) 16. The antibody of any one of items 9 to 15, wherein the antibody specifically recognizes and binds to a polynucleotide of the group TCTCAACGATTTA or WATCAANNNNTTR (where W is A or T, N is any nucleotide, and R is A or G) or their respective equivalents, or a polypeptide selected from the group of MATITKLDIIEYLSDKYHLS;KYHLSKQDTKNVVENFLEEI;FLEEIRLSLESGQDVKLSGF;KLSGFGNFELRDKSSRPGRN;RPGRNPKTGDVVPVSARRVV;or ARRVVTFKPGQKLRARVEKTK or their respective equivalents, or a polypeptide identified in Table 2, or an Arm fragment identified in Table 2, or an equivalent thereof, or a polypeptide of SEQ ID NO: 337 to 340, or an equivalent thereof. (Item 17) 17. The antibody according to any one of items 1 to 16, wherein the subject is a mammal. (Item 18) 18. The antibody of item 17, wherein the mammal is a human patient. (Item 19) 19. The antibody of item 17 or 18, wherein the mammal or human patient is an immature mammal or a pediatric patient. (Item 20) 20. The antibody of any one of items 9 to 19, wherein the subject infected with Burkholderia is a cystic fibrosis subject. (Item 21) Antibodies for use in inhibiting, competing with, or titrating biofilms produced by Burkholderia. (Item 22) 22. The antibody of item 21, wherein the antibody is contacted with a biofilm in vitro or in vivo. (Item 23) 23. The antibody of item 21 or 22, wherein the contacting of the antibody is in the absence of DNase treatment. (Item 24) 24. The antibody of any one of items 21 to 23, further comprising contacting the biofilm or Burkholderia with an effective amount of an antibacterial agent that inhibits the growth of the Burkholderia that produces the biofilm. (Item 25) An isolated polynucleotide comprising the sequence TCTCAACGATTTA or WATCAANNNNTTR (where W is A or T, N is any nucleotide, and R is A or G) or an equivalent thereof; or an isolated polypeptide comprising the sequence of the group MATITKLDIIEYLSDKYHLS;KYHLSKQDTKNVVENFLEEI;FLEEIRLSLESGQDVKLSGF;KLSGFGNFELRDKSSRPGRN;RPGRNPKTGDVVPVSARRVV;ARRVVTFKPGQKLRARVEKTK, or an equivalent thereof, or a polypeptide identified in Table 2, or an Arm fragment of a polypeptide identified in Table 2, or an equivalent thereof, or a polypeptide of SEQ ID NO: 337 to 340, or an equivalent thereof. (Item 26) 26. The isolated polynucleotide or polypeptide of item 25, further comprising an adjuvant or a detectable label. (Item 27) 26. An isolated antibody that specifically recognizes and binds to the isolated polynucleotide or polypeptide of item 25. (Item 28) 28. The antibody of item 27, wherein the antibody is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof. (Item 29) 29. A hybridoma cell line producing the monoclonal antibody according to item 28. (Item 30) 29. The antibody of item 27 or 28, and a carrier. (Item 31) 31. The antibody of item 30, wherein the carrier is a pharmaceutically acceptable carrier. (Item 32) 28. An isolated polynucleotide encoding the isolated polypeptide of Item 25 or the antibody of Item 27. [Brief explanation of the drawings]

[0056] [Figure 1] Figures 1A-1C show the presence of both eDNA and IHF within biofilms formed by B. cenocepacia. Figure 1A - A 24-hour unfixed biofilm formed by B. cenocepacia stained with FilmTracerFM1-43. Figure 1B - A 24-hour unfixed biofilm formed by B. cenocepacia immunolabeled with a monoclonal antiserum directed against dsDNA (white in the image). Figure 1C - A 24-hour unfixed biofilm formed by B. cenocepacia labeled with both an antiserum directed against dsDNA and a rabbit anti-IHF serum to demonstrate the presence of IHF and dsDNA (white) within the biofilm. Note the robust biofilm formed by B. cenocepacia in Figure 1A. A particularly high density of eDNA can be seen in Figure 1B, particularly at the bottom of the biofilm. As indicated by the abundance in Figure 1C, the 24-hour biofilm formed by B. cenocepacia is also abundant in IHF. All images were captured using a 63X objective.

[0057] [Figure 2]Figure 2 shows labeling of IHF and eDNA in sputum collected from cultures of B. cenocepacia-positive CF patients. Immunolabeled light micrographs show strong labeling of both eDNA (white strands) and the DNABII protein IHF (punctate labeling; see arrows) in sputum samples collected from B. cenocepacia-infected CF patients. As previously shown using nontypeable Haemophilus influenzae-formed biofilms, in biofilms formed by B. cenocepacia, cross-strand junctions of bacterial eDNA strongly label the presence of IHF, suggesting its role in maintaining the structural scaffolding of these biofilms. Scale bar indicates 5 μm.

[0058] [Figure 3A-C]Figures 3A-3H show the disruption of preformed B. cenocepacia biofilms by incubation with antisera directed against IHF. B. cenocepacia biofilms were grown in chamber slides for 24 hours and then treated for 16 hours with the following: Figure 3A—sterilized medium; Figure 3B—naive rabbit serum; Figure 3C—rabbit antisera directed against isolated IHF; Figure 3D—IgG-enriched anti-IHF; Figure 3E—serum eluate from an enrichment column; Figure 3F—Western blots showing the recognition of antibodies directed against IHF and IgG-enriched anti-IHF against purified IHF and IHF in B. cenocepacia whole-cell lysate. Arrows indicate recognition of the monomeric form of IHF in each blot. Furthermore, each serum fraction recognized the dimeric and trimeric forms of IHF in whole-cell lysate of B. cenocepacia strain K56-2. Figure 3G—Plot of the change in mean biofilm thickness ± SEM after each indicated treatment. Figure 3H—Plot of change in biofilm biomass ± SEM after each indicated treatment. Note that statistically significant disruption of pre-formed B. cenocepacia biofilms was mediated only by rabbit anti-IHF serum and the IgG-enriched IHF serum fraction when compared to treatments with sterile medium, naive rabbit serum, or serum eluate from an IgG-enriched column. Asterisks indicate statistical significance (p<0.05) compared to sterile medium, naive serum, and enriched column eluate. [Figure 3D-H]Figures 3A-3H show the disruption of preformed B. cenocepacia biofilms by incubation with antisera directed against IHF. B. cenocepacia biofilms were grown in chamber slides for 24 hours and then treated for 16 hours with the following: Figure 3A—sterilized medium; Figure 3B—naive rabbit serum; Figure 3C—rabbit antisera directed against isolated IHF; Figure 3D—IgG-enriched anti-IHF; Figure 3E—serum eluate from an enrichment column; Figure 3F—Western blots showing the recognition of antibodies directed against IHF and IgG-enriched anti-IHF against purified IHF and IHF in B. cenocepacia whole-cell lysate. Arrows indicate recognition of the monomeric form of IHF in each blot. Furthermore, each serum fraction recognized the dimeric and trimeric forms of IHF in whole-cell lysate of B. cenocepacia strain K56-2. Figure 3G—Plot of the change in mean biofilm thickness ± SEM after each indicated treatment. Figure 3H—Plot of change in biofilm biomass ± SEM after each indicated treatment. Note that statistically significant disruption of pre-formed B. cenocepacia biofilms was mediated only by rabbit anti-IHF serum and the IgG-enriched IHF serum fraction when compared to treatments with sterile medium, naive rabbit serum, or serum eluate from an IgG-enriched column. Asterisks indicate statistical significance (p<0.05) compared to sterile medium, naive serum, and enriched column eluate.

[0059] [Figure 4A-L]Figures 4A-4N show the synergistic behavior of antibodies directed against IHF in combination with antimicrobial agents or antibiotics. Figures 4A-4C—Untreated B. cenocepacia biofilms. Figures 4D-4F—B. cenocepacia biofilms after treatment with anti-IHF at a 50-fold dilution. Figures 4G-4I—B. cenocepacia biofilms after treatment with ceftazidime (16 μg / ml) at the indicated MIC. Figures 4J-4L—B. cenocepacia biofilms after treatment with the combination of anti-IHF and ceftazidime at the indicated MIC. Note that treatment with both anti-IHF and ceftazidime significantly reduced B. cenocepacia biofilm height and significantly increased B. cenocepacia killing compared to treatment with antibiotics alone (shown in the second row of images; compare panels H and K). [Figure 4M-N] Figures 4A-4N show the synergistic behavior of antibodies directed against IHF in combination with antimicrobial agents or antibiotics. Figure 4M—Mean biofilm thickness ± SEM after treatment with antibiotics or antibiotics + anti-IHF. Figure 4N—Biofilm biomass ± SEM after incubation with antibiotics or antibiotics + anti-IHF. Asterisks indicate statistical significance between the indicated pairs (p<0.05). Note the significant reduction in both mean biofilm thickness and biomass mediated by the combination of anti-IHF serum + ceftazidime, ciprofloxacin, imipenem, and minocycline.

[0060] [Figure 5A-C]Figures 5A-5E show the induction of more robust B. cenocepacia biofilms after exposure to Pulmozyme (DNase). Figure 5A - Treatment of B. cenocepacia biofilms for 24 hours with saline diluent alone. Figure 5B - Treatment of B. cenocepacia biofilms for 24 hours with Pulmozyme (DNase) induced the formation of significantly denser and thicker biofilms than treatment with saline diluent alone (compare panels A and B). Figure 5C - Treatment of B. cenocepacia biofilms for 24 hours with both Pulmozyme and anti-IHF. Biofilms were stained for viability and pseudocolored white (live cells) and dark (dead cells), indicating minimal bacterial death upon any treatment. [Figure 5D-E] Figures 5A-5E show the induction of more robust B. cenocepacia biofilms after exposure to Pulmozyme (DNase). Figure 5A - Treatment of B. cenocepacia biofilms for 24 hours with saline diluent alone. Figure 5B - Treatment of B. cenocepacia biofilms for 24 hours with Pulmozyme (DNase) induced the formation of significantly denser and thicker biofilms than treatment with saline diluent alone (compare panels A and B). Figure 5C - Treatment of B. cenocepacia biofilms for 24 hours with both Pulmozyme and anti-IHF. Biofilms were stained for viability and pseudocolored white (live cells) and dark (dead cells) to indicate minimal bacterial death upon any treatment. Mean relative biofilm thickness ± SD and biomass ± SEM are shown graphically in Figures 5D and 5E. Asterisks indicate significantly thicker biofilms after exposure to Pulmozyme compared to treatment with saline diluent (p<0.05).

[0061] [Figure 6]Figure 6 shows that pretreatment of B. cenocepacia with anti-IHF induced a significant decrease in viability in murine CF macrophages. B. cenocepacia was significantly reduced 6 hours after bacterial pretreatment with anti-IHF compared to naive serum (*p<0.05). Bacterial CFU / ml ± SD for each time point is also shown.

[0062] [Figure 7] Figures 7A-7E show that the development of robust B. cenocepacia biofilms incorporating IHF depended on an active T6SS. Figure 7A—Biofilm formed by the parent isolate (B. cenocepacia strain K56-2). Figure 7B—Biofilm formed by the type III secretion system mutant (strain JRL2) stained with propidium iodide. Figure 7C—Biofilm formed by the type VI secretion system mutant (strain DFA2) stained with propidium iodide. All biofilms were labeled for the presence of the DNABII protein (IHF). Note that biofilms formed by either secretion system mutant were significantly less robust than those formed by the parent isolate, whereas biofilms formed by the T6SS mutants showed significantly reduced labeling (see Figure 7C). This suggests that the T6SS mutants had impaired ability to incorporate IHF into biofilms formed under these conditions. Figure 7D - IHF-bound DNase footprint of the intergenic gap (386 bp) between BCAL0339 and BCAL0340 (part of the T6SS gene cluster of B. cenocepacia). The IHF footprint targeted the region 25 bp to 52 bp upstream of the BACL0340 start codon, while a putative promoter was found 75 bp to 104 bp upstream of the start codon (Figure 7E). HSS: Highly sensitive site indicating DNA bending. This finding suggested that IHF can self-regulate its own release and possibly the release of eDNA incorporated into the biofilm matrix.

[0063] [Figure 8]Figures 8A-8F show the relative eDNA content of biofilms formed in chamber slides by either the parent isolate K56-2, its T3SS mutants, or its T6SS mutants. Biofilms formed by either the parent B. cenocepacia strain or its T3SS and T6SS mutants and subsequently stained with FilmTracerFM1-43 are seen in Figures 8A, 8C, and 8E, respectively. The relative eDNA content of each unfixed biofilm can be confirmed by immunolabeling each biofilm with a monoclonal antibody directed against dsDNA, as seen in Figures 8B, 8D, and 8F.

[0064] [Figure 9] Figure 9A is a map showing the amino acid residues of an IHF that interact with or bind to another IHF in an IHF-IHF dimer (indicated by triangles in the upper column) or to DNA (indicated by triangles in the lower column). Peptides are divided into regions containing three amino acids by short vertical bars. Figure 9B graphically illustrates the interaction of microbial DNA with an IHF.

[0065] [Figure 10] Figures 10A-10B show the disruption of NTHI biofilms by anti-IHFE. coli. (A) Representative images of NTHI biofilms and (B) calculations of average biomass. a, Medium; b, Naive serum; c, Anti-IHFE. coli. Anti-IHFE. coli significantly disrupted established NTHI biofilms from 16 hours to 2 weeks compared with naive serum or medium. Data are shown as the mean ± SEM of three independent assays. *p<0.05; **p<0.01 compared with each naive serum treatment (one-way ANOVA).

[0066] [Figure 11A]Figures 11A-11C show the rate of biofilm disruption by anti-IHFE. coli. (A) Representative images of NTHI biofilms and (B) calculations of average biomass. a, medium; b, naive serum; c, anti-IHFE. coli. Anti-IHFE. coli-mediated biomass reduction was maximal at 6 h and sustained for 24 h. (C) Biofilms treated with medium, naive serum, or a 5-fold dilution of anti-IHFE. coli for 24 h. Treatment with higher concentrations of anti-IHFE. coli eradicated the biofilm, leaving a bacterial monolayer. Data are shown as the mean ± SEM of three independent assays. *p<0.01 compared to each naive serum treatment (one-way ANOVA). [Figure 11B] Figures 11A-11C show the rate of biofilm disruption by anti-IHFE. coli. (A) Representative images of NTHI biofilms and (B) calculations of average biomass. a, medium; b, naive serum; c, anti-IHFE. coli. Anti-IHFE. coli-mediated biomass reduction was maximal at 6 h and sustained for 24 h. (C) Biofilms treated with medium, naive serum, or a 5-fold dilution of anti-IHFE. coli for 24 h. Treatment with higher concentrations of anti-IHFE. coli eradicated the biofilm, leaving a bacterial monolayer. Data are shown as the mean ± SEM of three independent assays. *p<0.01 compared to each naive serum treatment (one-way ANOVA). [Figure 11C] Figures 11A-11C show the rate of biofilm disruption by anti-IHFE. coli. (A) Representative images of NTHI biofilms and (B) calculations of average biomass. a, medium; b, naive serum; c, anti-IHFE. coli. Anti-IHFE. coli-mediated biomass reduction was maximal at 6 h and sustained for 24 h. (C) Biofilms treated with medium, naive serum, or a 5-fold dilution of anti-IHFE. coli for 24 h. Treatment with higher concentrations of anti-IHFE. coli eradicated the biofilm, leaving a bacterial monolayer. Data are shown as the mean ± SEM of three independent assays. *p<0.01 compared to each naive serum treatment (one-way ANOVA).

[0067] [Figure 12] Figures 12A-12F show that direct contact between anti-IHFE. coli and biofilms was not required to mediate disruption. (A) Representative images of biofilms treated on the basolateral side with medium or serum. (B-C) Biofilms treated on the apical side by placement of antibodies coupled to agarose beads within a transwell. (D) NTHI biofilms after layering bare beads beneath antibody-coupled beads on the apical side. (E) Biofilms after mixing bare beads and antibody-coupled beads. (F) Biomass values after incubation with: a, medium; b, naive serum; c, anti-IHFE.coli; d, coupled IgG-enriched naive serum; e, coupled IgG-enriched anti-IHFE.coli; f, bare beads layered under coupled IgG-enriched naive serum; g, bare beads layered under coupled IgG-enriched anti-IHFE.coli; h, mixture of naked beads and coupled IgG-enriched naive serum; i, mixture of naked beads and coupled IgG-enriched anti-IHFE.coli. Data are shown as the mean ± SEM of three independent assays. *p<0.05 (one-way ANOVA) compared with representative naive serum or IgG-enriched naive serum conjugated to agarose bead treatments.

[0068] [Figure 13A] Figures 13A-13B show the adsorption of anti-IHF-specific antibodies. (A) Representative images of biofilms incubated with IHF-adsorbed serum. (B) Changes in biofilm biomass and average thickness. a, naive serum; b, anti-IHFE.coli. Anti-IHFE.coli (4.4 μg) adsorbed with the following: c, 0 μg IHFE.coli; d, 2.2 μg IHFE.coli; e, 4.4 μg IHFE.coli; f, 4.4 μg rsPilA. Adsorption of IHF-specific antibodies prevented biofilm disruption. Data are shown as the mean ± SEM of three independent assays. *p<0.05 compared to naive serum (one-way ANOVA). [Figure 13B] Figures 13A-13B show the adsorption of anti-IHF-specific antibodies. (A) Representative images of biofilms incubated with IHF-adsorbed serum. (B) Changes in biofilm biomass and average thickness. a, naive serum; b, anti-IHFE.coli. Anti-IHFE.coli (4.4 μg) adsorbed with the following: c, 0 μg IHFE.coli; d, 2.2 μg IHFE.coli; e, 4.4 μg IHFE.coli; f, 4.4 μg rsPilA. Adsorption of IHF-specific antibodies prevented biofilm disruption. Data are shown as the mean ± SEM of three independent assays. *p<0.05 compared to naive serum (one-way ANOVA).

[0069] [Figure 14] Figures 14A-14E show that anti-IHFE. coli acted synergistically with antibiotics. (A-D) Representative images of biofilms treated with medium, antibiotics at MIC90, or antiserum. (E) Biomass and average thickness of treated biofilms. a, medium; b, naive serum; c, anti-IHFE. coli. Incubation of NTHI biofilms with anti-IHFE. coli plus antibiotics significantly altered biofilm structure, significantly reduced biofilm biomass and average thickness, and negatively affected viability (note the yellow color of the biofilms). Data are shown as mean ± SEM of three independent assays. Bars indicate p<0.05 (one-way ANOVA).

[0070] [Figure 15]Figures 15A-15F show that treatment with anti-IHFE. coli enhanced the antibiotic susceptibility of bacteria newly released from biofilms. Biofilms were incubated with ampicillin (A and D), amoxicillin-clavulanate (B and E), or cefdinir (C and F) in the absence or presence of 50-fold dilutions of anti-IHFE. coli or naive serum. (A-C) Adherent CFU NTHI within biofilms. (D-F) The sum of planktonic and adherent NTHI. a, medium only; b, naive serum; c, anti-IHFE. coli. Data are shown as the mean ± SEM of three independent assays. Bars indicate p < 0.05 (one-way ANOVA).

[0071] [Figures 16A-C] Figures 16A-16F show epitope mapping of IHFNTHI and the design of a minimal IHFNTHI targeting peptide. (A) Three-dimensional model showing the reactivity of chinchilla anti-IHFE.coli and (B) chinchilla anti-IHFE.coli complexed to DNA to a synthetic peptide representing IHFNTHI. Reactive regions are shown in gray, and non-reactive regions are shown in black. (C) Three-dimensional model of DNA bound to IHF to show blockage of the tip-binding region. (D) Localization of IhfA-3NTHI (yellow) and IhfA-5NTHI (green) within the IHF model. (E) Representative image and (F) average biomass of biofilms after incubation with chinchilla serum. Data are shown as the mean ± SEM of three independent assays. Bars indicate p<0.05 (one-way ANOVA). a, naive serum; b, anti-IHFE.coli; c, anti-IHFE.coli complexed with DNA; d, anti-IhfA-3NTHI; e, anti-IhfA-5NTHI. [Figure 16D-F]Figures 16A-16F show epitope mapping of IHFNTHI and the design of a minimal IHFNTHI targeting peptide. (A) Three-dimensional model showing the reactivity of chinchilla anti-IHFE.coli and (B) chinchilla anti-IHFE.coli complexed to DNA to a synthetic peptide representing IHFNTHI. Reactive regions are shown in gray, and non-reactive regions are shown in black. (C) Three-dimensional model of DNA bound to IHF to show blockage of the tip-binding region. (D) Localization of IhfA-3NTHI (yellow) and IhfA-5NTHI (green) within the IHF model. (E) Representative image and (F) average biomass of biofilms after incubation with chinchilla serum. Data are shown as the mean ± SEM of three independent assays. Bars indicate p<0.05 (one-way ANOVA). a, naive serum; b, anti-IHFE.coli; c, anti-IHFE.coli complexed with DNA; d, anti-IhfA-3NTHI; e, anti-IhfA-5NTHI.

[0072] [Figure 17] Figure 17 shows that IgG-enriched anti-IHFE. coli bound to agarose beads and placed in the apical chamber of a transwell did not diffuse into the basolateral chamber, as judged by Western blot against purified IHFE. coli. Media from the basolateral chamber was collected 24 hours after treatment with: a, naive serum added to the basolateral side; b, anti-IHFE. coli added to the basolateral side; c, IgG-enriched naive serum tethered to agarose beads added to the apical side; d, IgG-enriched anti-IHFE. coli tethered to agarose beads added to the apical side.

[0073] [Figure 18]Figures 18A-18B show the adsorption of anti-IHFE.coli antibodies. (A) Incubation of anti-IHFE.coli with purified IHFE.coli reduced band intensity as shown by slot blot, and this intensity was quantified by densitometry (B). a, naive serum; b, anti-IHFE.coli; c, anti-IHFE.coli adsorbed with 0 μg IHFE.coli; d, anti-IHFE.coli adsorbed with 2.2 μg IHFE.coli; e, anti-IHFE.coli adsorbed with 4.4 μg IHFE.coli; f, anti-IHFE.coli adsorbed with 4.4 μg rsPilA.

[0074] [Figure 19] Figures 19A-19C show that treatment with anti-IHFE. coli did not increase the susceptibility of planktonic cultures of NTHI to antibiotics. (A-C) Broth cultures of NTHI were incubated with ampicillin, amoxicillin-clavulanate, or cefdinir in the absence or presence of anti-IHFE. coli or naive serum. a, culture only; b, naive serum; c, anti-IHFE. coli. Data represent the mean ± SEM of three independent assays. Bars indicate p<0.05 (one-way ANOVA). DETAILED DESCRIPTION OF THE INVENTION

[0075] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to the methods and materials described herein can be used in the practice or testing of this invention, preferred methods, devices, and materials are described herein.All technical publications and patent publications cited herein are incorporated herein by reference in their entirety.Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0076] The practice of the present invention employs, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, within the skill of the art. See, e.g., Sambrook and Russell (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition; Ausubel et al. (eds.) (2007) Current Protocols in Molecular Biology series; Methods in Enzymology (Academic Press, Inc., NY) series; 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. Manual;Freshney(2005)Culture of Animal Cells: A Manual of Basic Technique, 5th ed.; 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 See Mammalian Cells; Mayer and Walker (eds.) (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. (eds.) (1996) Weir's Handbook of Experimental Immunology.

[0077] All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximate and may vary (+) or (-) by 1.0 or 0.1 increments, as appropriate, or alternatively, by a variance of + / - 15%, or 10%, or 5%, or 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and that equivalents thereof are known in the art, although not always explicitly stated.

[0078] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "polypeptide" includes multiple polypeptides, including mixtures thereof.

[0079] As used herein, the term "comprising" shall mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, shall mean excluding any other elements of essential importance for the combination for the intended use. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, etc. "Consisting of" shall mean excluding trace elements of other components and more than substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0080] "Biofilm" refers to a thin layer of microorganisms that adhere to the surface of a structure. It can be organic or inorganic, along with polymers such as DNA that they secrete. Biofilms are highly resistant to microbiotics and antimicrobial agents. Biofilms inhabit gum tissue, teeth, and restorations, causing dental caries and periodontal disease, also known as periodontal plaque disease. Biofilms also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter lines, and contact lenses. Biofilms grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The Centers for Disease Control estimate that more than 65% of nosocomial infections (hospital-acquired infections) are caused by biofilms. Fungal biofilms also frequently contaminate medical devices. Biofilms 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, cystic fibrosis patients have Pseudomonas and B. cenocepacia infections that often result in antibiotic-resistant biofilms.

[0081] The term "inhibiting, competing with, or titrating" means reducing or preventing the formation of a DNA / protein matrix that is a component of a microbial biofilm.

[0082] "DNA BII polypeptide or DNA BII protein" refers to a DNA-binding protein or polypeptide that is composed of a DNA-binding domain and thus has specific or general affinity for microbial DNA. In one embodiment, a DNA BII polypeptide or DNA BII protein binds to DNA in the minor groove. A non-limiting example of a DNA BII protein is the integration host factor (IHF) protein. 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).

[0083] "Integration host factor" proteins are bacterial proteins that bacteriophages use to integrate their DNA into host bacteria. They also bind to extracellular microbial DNA.

[0084] "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 interfering agent. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova.

[0085] "HU" refers to a class of heterodimeric proteins commonly associated with E. coli. HU proteins are known to bind to DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU was reported by Laine et al. (1980) Eur. J. Biochem. 103(3):447-481. Antibodies against the HU protein are commercially available from Abcam.

[0086] "HU" or "histone-like protein from E. coli strain U93" refers to a class of heterodimeric proteins generally associated with E. coli. HU proteins are known to bind to DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU was reported by Laine et al. (1980) Eur. J. Biochem, 103(3):447-481. Antibodies against the HU protein are commercially available from Abeam. The genes encoding the HU protein subunits in E. coli are hupA and hupB, which correspond to SEQ ID NOs:28 and 29, respectively. Homologs to these genes are found in other organisms, and the corresponding peptides from these genes from other organisms can be found in Table 4.

[0087] "Microbial DNA" means single-stranded or double-stranded DNA derived from a biofilm-producing microorganism.

[0088] "Inhibiting, preventing, or disrupting" a biofilm means prophylactically or therapeutically reducing the structure of a biofilm.

[0089] "Interfering agent" refers to an agent that competes with, inhibits, prevents, titrates, or occludes a DNA BII polypeptide, such as IHF, for microbial DNA, or that also disrupts microbial biofilms. An interfering agent can be any one or more chemical or biological molecules. For example, IHF can specifically bind to, bend, or distort DNA structures, such as DNA containing four-way junctions, cis-platinum adducts, DNA loops, or base bulges. Examples of such agents include, but are not limited to, (1) small molecules that inhibit the DNA-binding activity of IHF, (2) small molecules such as polyamines and spermine that compete with IHF for binding to DNA, (3) polypeptides, such as peptide fragments of IHF, that compete with IHF for binding to DNA, (4) antibodies or fragments thereof directed against IHF, or (5) four-way or bent polynucleotides or other types of polynucleotides containing bent or distorted DNA structures that compete for IHF-binding. "Small molecules that inhibit the binding of IHF to nucleic acids" refers to (1) or (2) above and includes small molecules that bind to DNA in the minor groove, i.e., minor groove binding molecules. "Four-way polynucleotide" refers to a polynucleotide that contains a four-way junction, also known as a Holliday junction, between the four strands of DNA.

[0090] "Bent polynucleotide" refers to a double-stranded polynucleotide that contains a small loop on one strand that does not pair with the other strand. In some embodiments, the loop is from 1 to about 20 bases in length, or from 2 to about 15 bases in length, or from about 3 to about 12 bases in length, or from about 4 to about 10 bases in length, or about 4, 5, or 6, or 7, or 8, or 9, or 10 bases in length.

[0091] "Polypeptides that compete with IHF for binding to DNA" refers to proteins or peptides that occlude bent or distorted DNA structures or compete with IHF for binding to the DNA, but do not form biofilms with the DNA. Examples include, but are not limited to, fragments of IHF containing one or more DNA-binding domains of IHF, or biological equivalents thereof. The DNA-binding domains are shown in Figure 9.

[0092] A "subject" of diagnosis or treatment is a cell or an animal, such as a mammal or a human. Non-human animal subjects for diagnosis or treatment, infection or animal models are, for example, monkeys, murines, e.g., rats, mice, chinchillas, canines, e.g., dogs, lagomorphs, e.g., rabbits, farm animals, sport animals, and pets.

[0093] The terms "protein," "peptide," and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as ester bonds, ether bonds, etc. A protein or peptide must contain at least two amino acids, there is no limit on the maximum number of amino acids, and may include a protein sequence or a peptide sequence. As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0094] By "C-terminal polypeptide" is intended the C-terminal half of a polypeptide. As an example, for a polypeptide containing 90 amino acids, the C-terminal polypeptide includes amino acids 46-90. In another embodiment, the term refers to the 20 C-terminal amino acids from the carboxy terminus.

[0095] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST tags, 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 contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms that are known or predicted to be in double-stranded form.

[0096] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) is substituted for thymine. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications, such as functional genomics and homology searching.

[0097] The terms "isolated" or "recombinant," when used herein with reference to nucleic acids, such as DNA or RNA, refer to molecules separated from other DNA or RNA, respectively, present in the macromolecule and the natural source of the polypeptide. The term "isolated or recombinant nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in nature. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins that have been isolated from other cellular proteins and is intended to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" refers to a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof, separated from constituents, cells, and other entities with which it is normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a different phenotype or genotype. An isolated polynucleotide is separated from its native or natural environment, e.g., the 3' and 5' contiguous nucleotides with which it is normally associated on the chromosome. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require "isolation" to be distinguished from its naturally occurring counterpart.

[0098] Unless explicitly recited or otherwise intended, when the present invention relates to a polypeptide, protein, polynucleotide, or antibody, it should be presumed that its equivalent or biological equivalent is intended within the scope of the present invention. As used herein, "biological equivalent thereof" when referring to a reference protein, antibody, polypeptide, or nucleic acid is intended to be synonymous with the term "equivalent thereof," meaning one that has minimal homology or sequence identity while still maintaining the desired structure or functionality. Unless specifically recited herein, any polynucleotide, polypeptide, or protein referred to herein is also considered to encompass its equivalent. For example, in another embodiment, equivalent refers to a percentage of homology or sequence identity of at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95%, or alternatively at least about 98%, and exhibits substantially the same biological activity as the reference protein, reference polypeptide, or reference nucleic acid. Examples of biologically equivalent polypeptides are provided in Table 2, and Arm fragments are identified in Table 2, where conservative amino acid substitutions to the preferred amino acid sequences are identified.

[0099] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a particular percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, the percentage of bases (or amino acids) in the two sequences being compared are the same. Alignment and percentages of homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Appendix 30, section 7.7.18, table 7.7.1. Default parameters are preferably used for alignment. A preferred alignment program is BLAST, using default parameters. Specifically, the preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH. SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0100] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions within each sequence, which can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.

[0101] "Homology" or "identity" or "similarity" may also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0102] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a larger process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0103] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6xSSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1xSSC to about 0.1xSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps, with wash incubation times of approximately 1, 2, or 15 minutes. SSC is a 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0104] 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 splicing of the mRNA in a eukaryotic cell.

[0105] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that, in its native state or when manipulated by methods well known to those of skill in the art, is considered to "encode" a polypeptide that can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0106] As used herein, the terms "treating," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disorder or its signs or symptoms, and / or it may be therapeutic, in that it partially or completely cures a disorder and / or adverse effects resulting from the disorder.

[0107] By prevent is intended preventing a disorder or effect in vitro or in vivo in a system or subject predisposed to the disorder or effect, an example of which would be preventing biofilm formation in a system infected with a microorganism known to produce biofilms.

[0108] A "composition" is intended to mean a combination of an active agent and another compound or composition, either inert (e.g., a detectable agent or label) or active, such as an adjuvant.

[0109] A "pharmaceutical composition" is intended to include a combination of an active agent with a carrier, inert or active, forming a composition suitable for use in in vitro, in vivo or ex vivo diagnosis or treatment.

[0110] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, polymer, micelle, liposome, vector, plasmid or the like that can be used in the compositions of the present invention. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. Suitable pharmaceutical carriers are preferably selected based on the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., and in accordance with conventional pharmaceutical practice.

[0111] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where the components are water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, compositions for parenteral administration must be sterile and fluid to the extent that easy syringeability exists. Pharmaceutical compositions should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0112] Oral compositions generally contain an inert diluent or edible carrier.For therapeutic oral administration, active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules (for example, gelatin capsules).Oral compositions can also be prepared using a liquid carrier to be used as a mouthwash.Pharmaceutically compatible binders and / or adjuvants can be incorporated as part of the composition.Tablets, pills, capsules, troches, etc. can contain any of the following ingredients or compounds of similar nature: 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 flavor.

[0113] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0114] By "biologically active agent" or active agent of the present invention is meant one or more of an isolated or recombinant polypeptide, an isolated or recombinant polynucleotide, a vector, an isolated host cell, or an antibody, as well as compositions containing one or more of them.

[0115] "Administration" can be carried out in a single dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosages are known to those skilled in the art and vary depending on the composition used in the therapy, the purpose of the therapy, the target cells being treated, and the subject being treated. Single or multiple administrations can be carried out, with the dosage level and pattern selected by the treating physician. Appropriate dosage formulations and methods for administering active agents are known in the art. The route of administration can be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary depending on the composition used in the therapy, the purpose of the therapy, the health condition or stage of disease of the subject being treated, and the target cells or tissues. Non-limiting examples of administration routes include oral administration, nasal administration, injection, and topical application.

[0116] The agents of the present invention can be administered for therapy by any suitable route of administration, it being understood that the preferred route will vary with the condition and age of the recipient, and the disease being treated.

[0117] The term "effective amount" refers to a quantity sufficient to achieve a desired effect. For therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in question and the characteristics of the individual subject, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. For immunogenic compositions, in some embodiments, the effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is an amount sufficient to produce antibody production against an antigen. In some embodiments, the effective amount is the amount necessary to confer passive immunity in a subject in need thereof. For immunogenic compositions, in some embodiments, the effective amount depends on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system, in addition to the factors described above. Those skilled in the art can determine the appropriate amount depending on these and other factors.

[0118] In the case of in vitro applications, in some embodiments, the effective amount depends on the size and nature of the application in question. The effective amount also depends on the in vitro target and the nature and sensitivity of the method used. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount may include one or more administrations of the composition, depending on the embodiment.

[0119] The term "conjugated moiety" refers to a moiety that can be added to an isolated chimeric polypeptide by forming a covalent bond with a residue of the chimeric polypeptide. The moiety can be directly attached to the residue of the chimeric polypeptide or can form a covalent bond with a linker, which in turn forms a covalent bond between the linker and a residue of the chimeric polypeptide.

[0120] A "peptide conjugate" refers to a covalent or non-covalent association of one or more polypeptides with another chemical or biological compound. In a non-limiting example, the "conjugation" of a polypeptide with a chemical compound improves the stability or efficacy of the polypeptide for its intended purpose. In one embodiment, the peptide is conjugated to a carrier, where the carrier is a liposome, a micelle, or a pharmaceutically acceptable polymer.

[0121] Liposomes are microscopic vesicles composed of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes with dimensions ranging from hundreds of angstroms to fractions of a millimeter to spheres. Vesicle-forming lipids are selected to achieve a particular degree of fluidity or rigidity in the final complex, resulting in the lipid composition of the outer layer. They can be neutral (cholesterol) or bipolar, and include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), as well as other types of bipolar lipids, including, but not limited to, dioleoylphosphatidylethanolamine (DOPE), with hydrocarbon chain lengths ranging from 14 to 22, saturated or containing one or more C=C double bonds. Examples of lipids that can be used alone or in combination with other lipid components to produce stable liposomes include phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan-olamine (DSPE), dioleo ... These include dipalmitoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal).Additional non-phosphite containing lipids that can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine lauryl sulfate, alkyl-aryl sulfates, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate. Liposomes include steroids, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the above-mentioned cationic lipids (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, and DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol (DOPG), and dicetylphosphate, which can form vesicles. Generally, liposomes can be divided into three categories based on their overall size and the nature of their lamellar structure. The three classifications developed by the New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Medicine," December 1977, are multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar vesicles (LUVs). Biologically active agents can be encapsulated in such for administration according to the methods described herein.

[0122] "Micelles" are aggregates of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solution have hydrophilic "head" regions in contact with the surrounding solvent, forming aggregates with sequestered hydrophobic tail regions in the center of the micelle. This type of micelle is known as a normal-phase micelle (oil-in-water micelle). Reverse micelles have a central head group with protruding tails (water-in-oil micelle). Micelles can be used to attach polynucleotides, polypeptides, antibodies, or compositions described herein to facilitate efficient delivery to target cells or tissues.

[0123] The phrase "pharmaceutically acceptable polymer" refers to a group of compounds that can be conjugated to one or more polypeptides described herein.Conjugating a polymer to a polypeptide is believed to extend the half-life of the polypeptide in vivo and in vitro.Non-limiting examples include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols, and mixtures thereof.Biologically active agents can be conjugated to pharmaceutically acceptable polymers for administration according to the methods described herein.

[0124] " Gene delivery vehicle " is defined as any molecule that can carry inserted polynucleotide into host cell. Examples of gene delivery vehicle include biocompatible polymers, including liposomes, micelles, natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors, and other recombinant vehicles commonly used in the art, which are described for expression in various eukaryotic and prokaryotic hosts, and can be used for gene therapy and for the expression of simple proteins.

[0125] The polynucleotide of the present invention can be delivered to cells or tissues using a gene delivery vehicle. As used herein, "gene delivery," "gene transfer," "transduction," and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used to introduce polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotide contain a replication origin compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, a number of vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0126] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of it. Plasmids are often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and generally confer a selective advantage under given environmental conditions. Plasmids may carry genes that confer resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may function as toxins under similar circumstances.

[0127] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids for such use are commercially available. The gene to be replicated is inserted into a copy of the plasmid, which contains a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites that allow genes that make cells resistant to a particular antibiotic and DNA fragments to be easily inserted into this location. Another major use of plasmids is to produce large amounts of proteins. In this case, researchers grow bacteria containing a plasmid with the gene of interest. Just as bacteria produce proteins to confer antibiotic resistance, they can also be induced to produce large amounts of proteins from the inserted gene. This is an inexpensive and easy way to mass-produce a gene, or in turn, the protein it encodes.

[0128] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100 kb, up to 3000 kb). It is an artificially constructed chromosome that contains telomere, centromere, and origin of replication sequences necessary for replication and storage in yeast cells. It is constructed using an initial circular plasmid, which is linearized using restriction enzymes. The target sequence or gene can then be added to the linear molecule using DNA ligase and overhanging 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 post-translationally modified eukaryotic protein products to be obtained. However, YACs are more unstable than BACs and have been found to produce chimeric effects.

[0129] A "viral vector" is an in vivo vector containing a polynucleotide to be delivered to a host cell. Viral vectors are defined as viruses or viral particles produced by recombinant means, either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and alphavirus vectors. Infectious tobacco mosaic virus (TMV)-based vectors can be used to produce proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have been developed for use in gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In aspects where gene transfer is mediated by a retroviral vector, vector construct refers to a polynucleotide comprising the retroviral genome or a portion thereof and a therapeutic gene.

[0130] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process by which a virus enters a cell and integrates its genome into the host cell genome, thereby stably transferring a gene or nucleic acid sequence into the host cell. The virus can enter the host cell through its normal infection mechanism or can be modified to bind to a different host cell surface receptor or ligand to enter the cell. As used herein, a retroviral vector refers to a viral particle that can introduce exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

[0131] Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse transcribed into a DNA form and integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0132] In embodiments in which gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or an adeno-associated virus (AAV), the vector construct refers to a polynucleotide comprising the viral genome or a portion thereof and a transgene. Adenoviruses (Ad) are a relatively well-characterized, homogeneous group of viruses, including more than 50 serotypes. See, for example, International PCT Publication No. WO95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the potential for recombination and generation of wild-type viruses. See International PCT Publication Nos. WO95 / 00655 and WO95 / 11984. Wild-type AAV has high infectivity and specificity for integration into the host cell genome. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0133] Vectors containing both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate additional, potentially inappropriate, alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' to the initiation codon to enhance expression.

[0134] Gene delivery vehicles also include DNA / liposome complexes, micelles, and target viral protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can be used in the methods of the present invention. In addition to delivering polynucleotides to cells or cell populations by non-limiting techniques of protein transfection, the proteins described herein can be directly introduced into cells or cell populations, or culturing conditions that enhance expression and / or promote the activity of the proteins of the present invention are other non-limiting techniques.

[0135] As used herein, the term "eDNA" refers to extracellular DNA found as a component to pathogenic (e.g., bacterial) biofilms.

[0136] As used herein, the terms "antibody" and "immunoglobulin" encompass antibodies or immunoglobulins of any isotype, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains, and fragments of antibodies that retain specific binding to an antigen; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies; multispecific antibody fragments formed from antibody fragments and isolated fragments; CDRs or functional paratopes; chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins and non-antibody proteins containing the antigen-binding portion of an antibody. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissue.

[0137] The term "antibody" is used herein in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, human monoclonal antibodies, recombinant human antibodies, chimeric antibodies, antibody derivatives, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and antigen-binding fragments, so long as they exhibit the desired biological activity.

[0138] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies are highly specific because each antibody is directed against a single determinant on an antigen. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, or the like. Antibodies can also be conjugated to other moieties, such as members of specific binding pairs, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies can also be bound to solid supports, including, but not limited to, polystyrene plates or beads.

[0139] Monoclonal antibodies can be produced using hybridoma or recombinant DNA methods known in the art. Alternative techniques for producing or selecting antibodies include exposing lymphocytes in vitro to the antigen of interest and screening antibody-displayed libraries in cell, phage, or similar systems.

[0140] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention include antibodies that have amino acid residues not encoded by human germline immunoglobulin sequences (e.g., modified by random or site-specific mutagenesis in vitro, or in vitro). However, the term "human antibody," as used herein, does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to an antibody in which substantially all portions of a protein (e.g., CDRs, framework, Cs) have been grafted onto human framework sequences. L Domain, C H Domain (e.g., C H1 , C H2 , C H3(VL, VH) refers to antibodies in which the VL, VH) are substantially non-immunogenic in humans and involve only minor sequence changes or mutations. Similarly, antibodies designated for primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals refer to antibodies specific for such species, subgenus, genus, subfamily, or family. Furthermore, chimeric antibodies include any combination of the above. Such changes or mutations preferably retain or are less immunogenic, as the case may be, compared to unmodified antibodies in humans or other species. Thus, human antibodies differ from chimeric or humanized antibodies. It is noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Furthermore, when a human antibody is a single-chain antibody, the human antibody may include a linker peptide not found in native human antibodies. For example, an Fv may include a linker peptide, such as two to about eight glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region, and such a linker peptide is considered to be of human origin.

[0141] As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, for example, by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin. The selected human antibody generally has at least 90% amino acid sequence identity to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human compared to germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some cases, the human antibody has at least 95%, or even at least 96%, 97%, 98%, or even 99% amino acid sequence identity to the amino acid sequence encoded by the germline immunoglobulin gene. Generally, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a human antibody may display no more than 5, or even no more than 4, no more than 3, no more than 2, or no more than 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0142] "Human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. This term also refers to recombinant human antibodies. Methods for producing these antibodies are described herein.

[0143] The term "recombinant human antibody," as used herein, encompasses all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, antibodies isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the recombinant antibody VH and VL regions are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur in the human antibody germline repertoire in vivo. Methods for generating these antibodies are described herein.

[0144] As used herein, a chimeric antibody is an antibody whose light and heavy chain genes have been constructed, generally by genetic engineering, from antibody variable and constant region genes belonging to different species.

[0145] As used herein, the term "humanized antibody" or "humanized immunoglobulin" refers to a human / non-human chimeric antibody that contains minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's variable region are replaced with residues from the variable region of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. Humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. Humanized antibodies may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The non-human antibody contains one or more amino acids in the framework, constant, or CDR regions that are substituted for similarly positioned amino acids from a human antibody. Generally, humanized antibodies are expected to generate a reduced immune response in a human host compared to non-humanized versions of the same antibody. Humanized antibodies may have conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions. Conservative substitution groupings include glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine.

[0146] As used herein, the term "antibody derivative" includes a full-length antibody or a fragment of an antibody in which one or more of the amino acids have been chemically modified, for example, by alkylation, pegylation, acylation, ester formation, or amide formation, to link the antibody to a second molecule. Antibody derivatives include, but are not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof.

[0147] As used herein, the term "immunoconjugate" includes an antibody or antibody derivative bound or linked to a second agent, such as a cytotoxic agent, a detectable agent, a fluorescent label, 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.

[0148] As used herein, the term "detectable label" refers to a directly or indirectly detectable compound or composition, e.g., an N-terminal histidine tag (N-His), a magnetically active isotope, e.g., a nucleotide sequence, which is directly or indirectly conjugated to a composition to be detected to produce a "labeled" composition. 115 Sn, 117 Sn and 119 Sn, non-radioactive isotopes, e.g. 13 C and 15The term "label" refers to a polynucleotide or protein, such as an antibody, such as N. The term also encompasses sequences conjugated to a polynucleotide that provide a signal when the inserted sequence is expressed, such as green fluorescent protein (GFP). The label may be detectable alone (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a detectable chemical alteration of a substrate compound or composition. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. As such, suitable labels include, but are not limited to, magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, luminescent compounds, dyes, and proteins, including enzymes. A label may be simply detectable or quantifiable. A reaction that is simply detected generally involves a reaction whose presence is simply confirmed, while a reaction that is quantified generally involves a reaction that has a value that can be quantified (e.g., reported numerically), such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can be generated directly using a luminophore or fluorophore attached to the component of the assay that actually participates in the binding, or indirectly using a luminophore or fluorophore attached to another (e.g., reporter or indicator) component.

[0149] Examples of luminescent labels that produce signals include, but are not limited to, bioluminescence and chemiluminescence. Detectable luminescent reactions generally involve the change or appearance of a luminescent signal. Suitable methods and luminophores for luminescent labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th Edition). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0150] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0151] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present within or on the surface of cells or tissues, such as cell surface markers. Suitable functional groups, including but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, can all be used to attach the fluorescent label to a second molecule. The choice of functional group on the fluorescent label depends on the site of attachment, either to a linker, agent, marker, or second labeling agent.

[0152] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue®, and Texas Red®. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and It is described in Research Chemicals (6th edition).

[0153] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present within or on the surface of cells or tissues, such as cell surface markers. Suitable functional groups, including but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, can all be used to attach the fluorescent label to a second molecule. The choice of functional group on the fluorescent label depends on the site of attachment, either to a linker, agent, marker, or second labeling agent.

[0154] "Eukaryotic cells" include all kingdoms of life except Monera. Eukaryotes can be easily distinguished by their membrane-bound nuclei. Animals, plants, fungi, and protists are eukaryotic organisms or organisms whose cells are organized into complex structures by internal membranes and cytoskeleton. The most distinctive membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" encompasses eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include monkeys, cows, pigs, mice, rats, birds, reptiles, and humans.

[0155] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelles and are divided into two kingdoms: bacteria and archaea. Furthermore, instead of possessing chromosomal DNA, the genetic information of these cells resides within circular loops called plasmids. Bacterial cells are very small, roughly the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, E. coli, and Salmonella.

[0156] A "native" or "natural" antigen is a polypeptide, protein, or fragment that contains an epitope, has been isolated from a natural biological source, and is capable of specifically binding to an antigen receptor, specifically a T cell antigen receptor (TCR), in a subject.

[0157] The terms "antigen" and "antigenic" refer to a molecule capable of being recognized by an antibody or otherwise functioning as a member of an antibody-ligand pair. "Specific binding" refers to the interaction of an antigen with the variable regions of the heavy and light chains of an immunoglobulin. 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 function as antigens. Those skilled in the art will further understand that nucleic acids encoding proteins with the potential to function 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 "antigenic" is an adjective reference to a molecule having the properties of an antigen. This term encompasses substances that are immunogenic, i.e., immunogens, as well as substances that induce immunological unresponsiveness or anergy, i.e., anergens.

[0158] An "altered antigen" is an antigen having a primary sequence that differs from that of the corresponding wild-type antigen. Altered antigens can be produced by synthetic or recombinant methods and include, but are not limited to, antigenic peptides that have been differentially modified during or after translation by, for example, phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, or linkage to an antibody molecule, membrane molecule, or other ligand (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320). The synthetic or altered antigens of the present invention will bind to the same TCR as the natural epitope.

[0159] An "autoantigen," also referred to herein as a native antigen or wild-type antigen, is an antigenic peptide that induces little or no immune response in a subject due to autoimmune tolerance to the antigen. An example of an autoantigen is the melanoma-specific antigen gp100.

[0160] The term "major histocompatibility complex" or "MHC" refers to a complex of genes that encodes cell surface molecules necessary for antigen presentation to T cells and for rapid graft rejection. In humans, the MHC is also known as the "human leukocyte antigen" or "HLA" complex. The proteins encoded by the MHC are known as "MHC molecules" and are classified into class I and class II MHC molecules. Class I MHC comprises a membrane heterodimeric protein made of an α chain encoded in the MHC noncovalently linked to β2-microglobulin. Class I MHC molecules are expressed by nearly all nucleated cells and are expressed by CD8 + They have been shown to function in the presentation of antigens to T cells. Class I molecules include HLA-A, HLA-B, and HLA-C in humans. Class II MHC molecules also include membrane heterodimeric proteins consisting of noncovalently associated α and β chains. Class II MHC molecules are expressed by CD4 + HLA-like molecules are known to function in T cells, and in humans, these include HLA-DP, HLA-DQ, and HLA-DR. In preferred embodiments, the compositions and ligands of the present invention can form complexes with MHC molecules of any HLA type. Those skilled in the art are familiar with HLA serotypes and genotypes. See the bimas.dcrt.nih.gov / cgi-bin / molbio / hla coefficient viewing page; Rammensee HG, Bachmann J., and Stevanovic S. MHC Ligands and Peptide Motifs (1997) Chapman & Hall Publishers; Schreuder GM Th. et al. The HLA dictionary (1999) Tissue Antigens 54:409-437.

[0161] "Immune response" broadly refers to an antigen-specific response of lymphocytes to a foreign substance. The terms "immunogen" and "immunogenic" refer to a molecule capable of eliciting an immune response. This response may include antibody production or immune cell activation. The response may occur in vivo or in vitro. Those skilled in the art will appreciate that a variety of macromolecules have the potential to be immunogenic, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides. Those skilled in the art will further appreciate that a nucleic acid encoding a molecule capable of eliciting an immune response necessarily encodes an immunogen. Those skilled in the art will further appreciate that an immunogen is not limited to a full-length molecule, but may include a partial molecule.

[0162] The term "passive immunity" refers to the transfer of immunity from one subject to another through the transfer of antibodies. Passive immunity can occur naturally, such as when a mother's antibodies are transferred to her fetus. Passive immunity can also occur artificially, such as when an antibody composition is administered to a non-immune subject. The donor and recipient of the antibody can be a human or non-human subject. The antibody can be polyclonal or monoclonal, can be generated in vitro or in vivo, and can be purified, partially purified, or unpurified, depending on the embodiment. In some embodiments described herein, passive immunity is conferred by administering to a subject in need thereof an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen. In some embodiments, passive immunity is conferred by administering an isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen.

[0163] In the context of the present invention, a "ligand" is a polypeptide. In one aspect, the term "ligand" as used herein refers to any molecule that binds to a specific site on another molecule. In other words, the ligand confers specificity to the protein in response to immune effector cells or antibodies against the protein or DNA against the protein. In one aspect, the ligand site in the protein directly combines with a complementary binding site on immune effector cells.

[0164] As used herein, the terms "solid support" and "solid support" are used interchangeably and are not limited to a particular 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. As used herein, "solid support" also encompasses synthetic antigen-presenting matrices, cells, and liposomes. An appropriate solid support can be selected based on the desired end use and suitability for various protocols. For example, with respect to peptide synthesis, a solid support may refer to a resin such as polystyrene (e.g., PAM-resin available from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE.RTM. resin (available from Aminotech, Canada), polyamide resin (available from Peninsula Laboratories), polyethylene glycol-grafted polystyrene resin (TentaGel.RTM., Rapp Polymere, Tübingen, Germany) or polydimethylacrylamide resin (available from Milligen / Biosearch, Calif.).

[0165] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural cellulose, modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the carrier may be soluble to some extent or insoluble. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polynucleotide, polypeptide, or antibody. Thus, the configuration of the support may be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface may be flat, e.g., a sheet, test strip, or polystyrene beads. Those of skill in the art will know many other suitable carriers for binding antibodies or antigens, or will be able to ascertain such by routine experimentation.

[0166] MODES FOR CARRYING OUT THE DISCLOSURE Cystic fibrosis (CF) is the most common fatal genetic disorder affecting Caucasians. Despite medical advances, CF is short-lived, with patients typically only surviving to age 38. Patients with CF lung infections caused by Burkholderia cenocepacia (B. cenocepacia) are exceptionally difficult to manage because this organism is clinically resistant to virtually all antibiotics, highly contagious, and often precludes CF patients from lung transplantation, a last-ditch, life-saving option. For immune intervention, we targeted two abundant components of B. cenocepacia biofilms: extracellular DNA and DNABII proteins (the latter being bacterial nucleic acid-binding proteins). Treatment of B. cenocepacia biofilms with antisera directed against one of these DNABII proteins (integration host factor, or IHF) significantly disrupted the biofilms. Furthermore, when anti-IHF-mediated destabilization of B. cenocepacia biofilms was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm, which are typically highly resistant to antibiotic action, were rapidly killed. Preincubation of B. cenocepacia with anti-IHF serum before exposure to murine CF macrophages, which normally cannot effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytosed B. cenocepacia. Collectively, these findings indicate that targeting DNABII proteins represents a novel approach for the treatment of CF patients, particularly those with lung infections caused by B. cenocepacia.

[0167] Diagnostic and Therapeutic Methods The present disclosure provides a method for inhibiting, competing with, or determining the titer of a biofilm produced by Burkholderia, comprising, consisting essentially of, or further consisting of contacting the biofilm with an effective amount of an anti-integration host factor (anti-IHF) antibody, thereby inhibiting, competing with, or determining the titer of the biofilm. The method can be performed in vitro or in vivo. In one embodiment, the anti-IHF antibody contact is performed without the use of DNase treatment. In one embodiment, the DNase treatment excluded from treatment comprises an enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes excluded from treatment and known to target not only cruciform structures but also various secondary structures in DNA include DNase I, T4 Endo VII, and T7 Endo I. In one embodiment, the DNase treatment excluded from treatment comprises, consists essentially of, or further consists of Pulmozyme® (dornase alfa; Genentech, Inc.).

[0168] In another embodiment, the method further comprises, consists essentially of, or consists of contacting the biofilm or Burkholderia with an effective amount of an antimicrobial agent that inhibits the growth of biofilm-producing Burkholderia. Non-limiting examples of such antimicrobial agents include ampicillin, amoxicillin-clavulanate, ceftazidime, ciprofloxacin, imipenem, minocycline, and cefdinir. Contacting can be performed in vitro or in vivo.

[0169] Also provided herein are methods for treating an infection or disease caused by Burkholderia infection in a subject, comprising, consisting essentially of, or further consisting of administering to the subject an effective amount of an anti-IHF antibody, thereby treating the infection or disease caused by Burkholderia infection.

[0170] The present disclosure also provides a method of treating or preventing recurrence of infection in a CF patient in need thereof, comprising, consisting essentially of, or further consisting of administering to the patient an effective amount of an anti-IHF antibody, thereby treating or preventing recurrence of infection in the CF patient.

[0171] In one embodiment, the anti-IHF antibody is administered without DNase treatment. In one embodiment, the DNase treatment excluded from treatment includes an enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes that are excluded from treatment and are known to target not only cruciform structures but also various secondary structures of DNA include DNase I, T4 Endo VII, and T7 Endo I. In one embodiment, the DNase treatment excluded from treatment includes, consists essentially of, or further consists of Pulmozyme® (Dornase alfa; Genentech, Inc.). In yet a further embodiment, the method further includes, or even more essentially consists of, or further consists of administering to the subject an effective amount of an antimicrobial agent that inhibits the growth of biofilm-forming Burkholderia. Non-limiting examples of such antibacterial agents include ampicillin, amoxicillin-clavulanate, ceftazidine, ciprofloxacin, imipenem, minocycline, and cefdinir.

[0172] In each of the above methods, non-limiting examples of Burkholderia are Burkholderia cenocepacia (B. cenocepacia), B. multivorans, B. mallei, B. cepaci, or B. pseudomallei.

[0173] The anti-IHF antibody used in the above method is one or more anti-IHFα or anti-IHFβ antibodies. In a further embodiment, the anti-IHF antibody is an IgG antibody. The antibody can be any of the various antibodies described herein, and non-limiting examples of such antibodies include full-length monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, human monoclonal antibodies, recombinant human antibodies, chimeric antibodies, veneered antibodies, bispecific antibodies, humanized antibodies, antibody derivatives, recombinant humanized antibodies, fragments, or antigen-binding fragments thereof, so long as they exhibit the desired biological activity. In one embodiment, the fragment comprises, consists essentially of, or further consists of the CDRs of the antibody. In one embodiment, the antibody is detectably labeled or further comprises a detectable label conjugated to the antibody.

[0174] Non-limiting examples of anti-IHF antibodies are described herein and in one embodiment are one or more antibodies that specifically recognize and bind to a polypeptide identified in Table 2 or an Arm fragment identified in Table 2, or an equivalent of such a polypeptide, or a polynucleotide or polypeptide comprising one or more of the following sequences:

number

[0175] For these methods, the infection occurs in vivo and in a mammalian host (such as a human patient), for example, in an immature mammalian host or a pediatric patient.

[0176] The present disclosure also provides methods for inhibiting, competing with, or determining the titer of a biofilm (e.g., a B. cenocepacia-induced biofilm) present in or contributing to CF, comprising, consisting essentially of, or consisting of contacting the biofilm with an interfering agent, thereby inhibiting, competing with, or determining the titer of the biofilm. Contacting can be performed in vitro or in vivo.

[0177] In another aspect, there is provided a method of inhibiting, preventing, or disrupting a bacterial biofilm in a CF patient or a patient carrying an infection that contributes to CF, comprising, consisting essentially of, or consisting of contacting the biofilm with an interfering agent, thereby inhibiting, preventing, disrupting, contacting the bacterial biofilm. The patient may be an animal, mammal, or human patient.

[0178] When performed in vitro, the method is useful for screening or identifying interfering agents with the same, similar, or opposite potency as the polypeptides, polynucleotides, antibodies, host cells, small molecules, and compositions of the present invention. Alternatively, the method can be used to identify which interfering agents are optimal for treating microbial infections. For example, new agents or combination therapies can be screened by, for example, preparing two samples containing a DNA BII polypeptide and microbial DNA or biofilm and the agent being tested. The second sample contains a DNA BII polypeptide and microbial DNA or biofilm and a known active agent, such as an anti-IHF antibody or a small molecule that serves as a positive control. In another embodiment, several samples are prepared and the interfering agent is added to the system in increasing dilutions to determine the optimal dose that will be effective in treating subjects in a clinical setting. As will be apparent to one of skill in the art, a negative control containing a DNA BII polypeptide and microbial DNA or biofilm can be prepared. In another embodiment, the DNA BII polypeptide and microbial DNA or biofilm are detectably labeled, for example, with luminescent molecules that emit a signal when brought into close contact with each other. A sample is contained under similar conditions for a time effective to determine the ability of the agent to inhibit, compete with, or titer the interaction between the DNA BII polypeptide and the microbial DNA or biofilm, and the sample is then assayed for emission of a signal from the luminescent molecule. If a signal is emitted from the sample, the agent is not effective to inhibit binding.

[0179] In another embodiment, an in vitro method is performed in a miniaturized chamber slide system in which an isolate of a microorganism (e.g., bacteria) causing a CF infection is isolated from a human / animal and then cultured and grown as a biofilm in vitro. An interfering agent (e.g., an anti-IHF antibody) or potential interfering agent biofilm is added to the culture, alone or in combination with another agent, with or without increasing dilutions of the potential interfering agent or interfering agent, e.g., anti-IHF (or other antibody, small molecule, agent, etc.), to find the optimal dose that will be effective in treating the patient when delivered to the site of the target infection. As will be apparent to one skilled in the art, positive and negative controls can be performed simultaneously.

[0180] In another embodiment, the method is performed on a high-throughput platform using an interfering agent (e.g., an anti-IHF antibody) and / or a potential interfering agent (alone or in combination with another agent) in a flow cell. The interfering agent (e.g., an anti-IHF antibody) or potential interfering agent biofilm, alone or in combination with another agent, is added to the culture with or without increasing dilutions of the potential interfering agent or interfering agent, such as anti-IHF (or other antibodies, small molecules, agents, etc.), to find an optimal dose that will be effective in treating the patient when delivered to the site of the target infection. The biofilm isolate is sonicated to separate the biofilm bacteria from the DNA BII polypeptide, such as IHF, bound to the microbial DNA. The DNA BII polypeptide-DNA complex is isolated by the anti-IHF antibody on the platform. The microbial DNA is then released, for example, using a salt wash, for use in identifying the attached biofilm bacteria. The released DNA is then identified, for example, by sequencing by PCR. If no DNA is released, the interfering agent(s) successfully functioned or bound to the microbial DNA. If DNA is found in the sample, the agent did not interfere with the DNA BII polypeptide-microbial DNA binding. As will be apparent to one skilled in the art, positive and / or negative controls can be performed simultaneously.

[0181] The above method can also be used as a diagnostic test, as a given bacterial species may respond better to reversal of its biofilm by one agent than another, and this rapid, high-throughput assay system allows one skilled in the art to assay a panel of potential anti-IHF-like agents to identify the most effective group.

[0182] An advantage of these methods is that most hospital clinical microbiology laboratories are already equipped to perform these types of assays (i.e., determining MIC and MBC values) using bacteria growing in liquid media (or planktonically). As will be apparent to those skilled in the art, bacteria generally do not grow in planktonic form when causing disease. Instead, they grow as stable biofilms, and these biofilms are significantly more resistant to treatment with antibiotics, antibodies, or other treatments. This resistance is the reason why most MIC / MBC values do not accurately predict in vivo efficacy. Therefore, by determining which "dose" of an agent can reverse bacterial biofilms in vitro (as described above), Applicants' preclinical assays become more reliable predictors of clinical efficacy, even for personalized medicine applications.

[0183] In addition to clinical settings, this method can be used to identify and / or validate effective interfering agents in industrial settings.

[0184] In another embodiment of the above method, an antibiotic or antimicrobial agent known to inhibit the growth of the underlying infection is added sequentially or simultaneously to determine whether the infection can be inhibited. To assay for biofilm inhibition, an interfering agent can be added to the microbial DNA or DNA BII polypeptide before adding the missing complex.

[0185] When performed in vivo in non-human animals, the method provides a preclinical screen to identify interfering agents that can be used alone or in combination with other agents to degrade biofilms.

[0186] In another aspect, there is provided a method of inhibiting, preventing, disrupting, and / or eliminating a bacterial biofilm and / or a bacterial infection in a CF patient or a patient carrying an infection that contributes to CF, comprising, consisting essentially of, or consisting of contacting the biofilm with an interfering agent, thereby inhibiting, preventing, disrupting, and / or eliminating the bacterial infection or bacterial biofilm. The patient may be an animal, mammal, or human patient.

[0187] Also provided is a method for treating or preventing the recurrence of biofilms in CF patients or patients at risk of developing infections that contribute to biofilm formation, comprising, consisting essentially of, or consisting of administering to the patient an effective amount of an interfering agent, thereby treating or preventing the recurrence of bacterial infections in CF patients. The CF patient can be an animal, mammal, or human patient.

[0188] Also provided is a method of treating or preventing recurrent infections in CF in a patient in need thereof, comprising, consisting essentially of, or consisting of administering to the patient an effective amount of an interfering agent.

[0189] To further aid in treatment, the interfering agent can be combined with an antimicrobial agent. Thus, any of the above methods can further comprise, consist essentially of, or further consist of administering or contacting with an effective amount of an antimicrobial agent. Non-limiting examples of antimicrobial agents include...

[0190] Interfering agents and compositions for the above in vitro and in vivo methods are described in U.S. Patent Application Publication No. 2011 / 0236306, particularly paragraphs 238-263 and 277-329, which are incorporated herein by reference.

[0191] In another embodiment, the method further comprises, consists essentially of, or consists of administering to the subject an effective amount of one or more of an antimicrobial agent, an antigenic peptide, or an adjuvant.

[0192] A non-limiting example of an antibacterial agent is another vaccine component, such as a surface antigen, such as a type IV Pilin protein (Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875).

[0193] The agents and compositions of the present invention can be administered simultaneously or sequentially with each other, or with other antibacterial agents and / or surface antigens. In a particular embodiment, administration is local to the site of infection, for example, by direct injection or inhalation. Other non-limiting examples of administration include by one or more methods including transdermally, urethrally, sublingually, rectally, vaginally, ocularly, subcutaneously, intramuscularly, intraperitoneally, intranasally, by inhalation, or orally.

[0194] Microbial infections and diseases that can be treated by the methods of the present invention include infections that result in or are associated with CF infection. 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 and / or primary causes of cystic fibrosis (CF), and community-acquired pneumonia (CAP). Thus, by practicing the in vivo methods of the present invention, these diseases and complications resulting from these infections can also be prevented or treated.

[0195] Therefore, administration routes applicable to the methods of the present invention include intranasal, intramuscular, urethral, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, inhalation, and other enteral and parenteral routes of administration. Administration routes can be combined if desired or adjusted depending on the agent and / or the desired effect. The active agent can be administered in a single dose or multiple doses. Suitable embodiments of these methods and routes for delivery include systemic or localized routes. In general, administration routes suitable for the methods of the present invention include, but are not limited to, direct injection, enteral, parenteral, or inhalation routes.

[0196] Parenteral administration routes other than administration by inhalation include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the digestive tract. Parenteral administration can be carried out to result in systemic or local delivery of the inhibitor. When systemic delivery is desired, administration generally involves invasive or systemically absorbed topical or mucosal administration of the pharmaceutical preparation.

[0197] Interfering agents of the present invention can also be delivered to a subject by enteral administration, including, but not limited to, oral, urethral, and rectal (e.g., using a suppository).

[0198] Methods for administering active substances through the skin or mucosa include, but are not limited to, topical application of appropriate pharmaceutical preparations, transdermal delivery, injection, and epidermal administration. For transdermal delivery, absorption enhancers or iontophoresis are suitable methods. Iontophoretic delivery can be achieved using commercially available "patches" that deliver the product continuously through intact skin via electrical pulses for periods of several days or longer.

[0199] In various embodiments of the methods of the invention, the interfering agent is administered by inhalation, injection, or orally continuously, daily, at least once per day (QD), and in various embodiments, twice per day (BID), three times per day (TID), or even four times per day. Generally, a therapeutically effective daily dose is at least about 1 mg, or at least about 10 mg, or at least about 100 mg, or about 200 mg to about 500 mg, and sometimes as much as about 1 g to about 2.5 g, depending on the compound.

[0200] Dosing can be achieved in accordance with the methods of the present invention using capsules, tablets, oral suspensions, intramuscular suspensions, intravenous suspensions, topical gels or creams, or intra-articular suspensions.

[0201] The dosage, toxicity, and therapeutic effect of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as procedures for determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compositions that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects can be used, but care should be taken to design a delivery system that targets such compounds to the affected tissue site in order to minimize potential damage to uninfected cells and thereby reduce side effects.

[0202] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a circulating concentration range that includes the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the method, a therapeutically effective amount can be initially estimated from cell culture assays. Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0203] In some embodiments, an effective amount of the composition sufficient to achieve a therapeutic or prophylactic effect ranges from about 0.000001 mg per kilogram of body weight per dose to about 10,000 mg per kilogram of body weight per dose. A suitable dosage range is from about 0.0001 mg per kilogram of body weight per dose to about 100 mg per kilogram of body weight per dose. Administration can be provided as an initial administration, followed by one or more "booster" administrations. Booster administrations can 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 administration. In some embodiments, booster administrations are administered after assessing the subject's response to the previous administration.

[0204] Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[0205] The compositions and related methods of the present invention 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.

[0206] In some embodiments, the methods and compositions include a deoxyribonuclease (DNase) enzyme that acts synergistically with an anti-DNABII antibody. DNase is any enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes that are known to target not only cruciform structures but also various secondary structures of DNA include DNase I, T4 Endo VII, and T7 Endo I. In certain embodiments, when combined with DNase, the effective amount of anti-DNABII antibody required to destabilize biofilms is reduced. When administered in vitro, DNase can be added directly to the assay or in an appropriate buffer known to stabilize the enzyme. The effective unit dose of DNase and assay conditions can vary and can be optimized according to procedures known in the art.

[0207] In other embodiments, the methods and compositions can 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 sensitize biofilm-associated infections to conventional therapies for treating infections. 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 the antimicrobial and / or biofilm-reducing agent. Some non-limiting examples of antimicrobial agents and antimicrobial agents useful in combination with the methods of the present invention include amoxicillin, amoxicillin-clavulanate, cefdinir, azithromycin, and sulfamethoxazole-trimethoprim. The treatment-effective amount of the antimicrobial and / or antimicrobial agent combined with the biofilm-reducing agent can be readily determined by conventional methods. In some embodiments, the dose of the antimicrobial agent combined with the biofilm reducing agent is the average effective dose that has been shown to be effective in other bacterial infections, for example, bacterial infections whose etiology does not involve biofilms. In other embodiments, the dose is 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.8, 0.85, 0.9, 0.95, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, or 5 times the average effective dose. The antibiotic or antimicrobial agent can be added before, simultaneously with, or after the addition of the anti-DNABII antibody.

[0208] In other embodiments, the methods and compositions can be combined with antibodies to treat bacterial infections. One example of an antibody useful in combination with the methods and compositions described herein is an antibody directed against an unrelated outer membrane protein (i.e., OMP P5 protein). Treatment with this antibody alone does not debulk biofilms in vitro. Combination of this antibody with biofilm-reducing agents and / or antimicrobial agents can also be used to treat bacterial infections. Combination therapy results in a greater effect than can be achieved by using the same concentration of either agent alone. Other antibodies that may provide a synergistic effect when used in combination with biofilm-reducing agents or methods to reduce biofilm include anti-rsPilA. Anti-OMP26, anti-OMP P2, and anti-total OMP antibody preparations are included.

[0209] The compositions and methods described herein can be used to sensitize biofilm-associated bacterial infections to common treatment modalities that are effective in treating non-biofilm-associated bacterial infections but are otherwise ineffective in treating biofilm-associated bacterial infections. In other embodiments, the compositions and methods described herein can be used in combination with treatment modalities that are effective in treating biofilm-associated bacterial infections, where the combination of such additional therapy with a biofilm-reducing agent or method results in a synergistic effect that may reduce the effective amount of the biofilm-reducing agent or additional therapeutic agent. In other cases, the combination of such additional therapy with a biofilm-reducing agent or method results in a synergistic effect that enhances treatment. Enhanced treatment can be evidenced by a reduction in the amount of time required to treat the infection.

[0210] The additional therapeutic treatment may be added prior to, concurrently with, or after the method or composition used to reduce biofilm, and may be included in the same formulation or as a separate formulation. It can also be done.

[0211] kit Kits containing the necessary agents and instructions for carrying out the in vitro and in vivo methods described herein are also claimed. Thus, the present invention provides kits for carrying out the methods of the present invention, which may involve interfering with the present invention, as well as instructions for carrying out the methods of the present invention, such as collecting tissue, and / or carrying out screening, and / or analyzing the results, and / or administering an effective amount of the interfering agent defined herein. These can be used alone or in combination with other suitable antibacterial agents.

[0212] For example, the kit may include an isolated or recombinant integration host factor (IHF) polypeptide, polynucleotide, or a fragment or equivalent thereof; an isolated or recombinant protein polypeptide identified in Table 1 or Table 2, an Arm fragment identified in Table 2, Table 3, or Table 4, a DNA-binding peptide identified in Figure 9, or a fragment or equivalent thereof; an isolated or recombinant polypeptide, polynucleotide, or a fragment or equivalent thereof of SEQ ID NOs: 1-33 or 341-348; an isolated or recombinant C-terminal polypeptide of SEQ ID NOs: 6-11, 28, 29, or an isolated or recombinant C-terminal polypeptide identified in Table 1 or Table 4, or a fragment or equivalent thereof; a peptide for binding to microbial DNA; The kit may comprise, consist essentially of, or even consist of one or more of the following agents: a polypeptide that competes with integration host factors; a four-way junction polynucleotide resembling a Holliday junction, a three-way junction polynucleotide resembling a replication fork, a polynucleotide with inherent flexibility or a curved polynucleotide; an isolated or recombinant polynucleotide encoding any one of the above-mentioned polypeptides; an antibody, or an equivalent or fragment thereof, that specifically recognizes or specifically binds to any one of the above-mentioned polypeptides; or a small molecule that competes with the binding of DNABII protein or DNABII polypeptide to microbial DNA, together with instructions for use. The kit may further comprise one or more of an adjuvant, an antigenic peptide, or an antimicrobial agent. Examples of carriers include liquid carriers, pharmaceutically acceptable carriers, solid-phase carriers, pharmaceutically acceptable polymers, liposomes, micelles, implants, stents, pastes, gels, dental implants, or medical implants.

[0213] Polypeptides Also provided herein are polypeptide interfering agents and compositions for use in the methods described herein, said interfering agents comprising: (a) an isolated or recombinant integration host factor (IHF) polypeptide, or a fragment or equivalent thereof; (b) an isolated or recombinant histone-like protein (HU) polypeptide from E. coli strain U93, or a fragment or equivalent thereof; (c) an isolated or recombinant protein polypeptide identified in Table 1, Table 2, a polypeptide comprising or consisting of an Arm fragment identified in Table 2, Table 3, Table 4, or a DNA-binding peptide identified in Figure 9, or a fragment or equivalent thereof; (d) an isolated or recombinant polypeptide of SEQ ID NO: 1-348, or a fragment or equivalent thereof; (e) an isolated or recombinant C-terminal polypeptide of SEQ ID NOs: 6-11, 28, 29, 42-100, Table 1, or a C-terminal polypeptide identified in Table 4, or a fragment or equivalent thereof, respectively; or (f) a polypeptide or polynucleotide that competes with integration host factors for binding to microbial DNA It is from the group of.

[0214] In a particular embodiment, the interfering agent is an isolated or recombinant DNABII polypeptide, or its respective fragment or equivalent, such as, but not limited to, IHF or HU alpha or beta polypeptides; IHF alpha polypeptide; Moraxella catarrhalis HU; E. coli HupA, HupB, himA, himD; E. faecalis HU (e.g., V583), HMGB1, and those identified in Table 1.

[0215] In another embodiment, the interfering agent is an isolated or recombinant polypeptide consisting essentially of an amino acid sequence selected from SEQ ID NOs: 1-5, or 12-27, 30-35, 101-340, or a DNA-binding peptide identified in FIG. 9.

[0216] In another embodiment, the isolated or recombinant polypeptide comprises, consists essentially of, or further consists of SEQ ID NO: 1 or 2, but is not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0217] In another embodiment, the isolated or recombinant polypeptide comprises, consists essentially of, or further consists of SEQ ID NO: 3, 4, or 5, but is not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0218] In another embodiment, the isolated or recombinant polypeptide comprises, consists essentially of, or further consists of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 30, or 32, but is not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0219] In another embodiment, the isolated or recombinant polypeptide comprises, consists essentially of, or further consists of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 31, 33, 34, or 35, but is not any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0220] In another aspect, the isolated or recombinant polypeptide is A polypeptide comprising SEQ ID NOs: 12 and 13; a polypeptide comprising SEQ ID NOs: 14 and 15; a polypeptide comprising SEQ ID NOs: 16 and 17; a polypeptide comprising SEQ ID NOs: 18 and 19; A polypeptide comprising SEQ ID NOs: 20 and 21; A polypeptide comprising SEQ ID NOs: 23 and 24, a polypeptide comprising SEQ ID NOs: 25 and 26; A polypeptide comprising SEQ ID NOs: 30 and 31; a polypeptide comprising SEQ ID NOs: 32 and 33; a polypeptide comprising SEQ ID NOs: 34 and 35; a polypeptide comprising SEQ ID NOs: 337 and 338, or Polypeptides comprising SEQ ID NOs: 339 and 340 and wherein the polypeptide is not the wild type of any one of IHF alpha, IHF beta or any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0221] In another aspect, the isolated or recombinant polypeptide is A polypeptide consisting essentially of SEQ ID NOs: 12 and 13; A polypeptide consisting essentially of SEQ ID NOs: 14 and 15; A polypeptide consisting essentially of SEQ ID NOs: 16 and 17; A polypeptide consisting essentially of SEQ ID NOs: 18 and 19; A polypeptide consisting essentially of SEQ ID NOs: 20 and 21; A polypeptide consisting essentially of SEQ ID NOs: 23 and 24; A polypeptide consisting essentially of SEQ ID NOs: 25 and 26; A polypeptide consisting essentially of SEQ ID NOs: 30 and 31; A polypeptide consisting essentially of SEQ ID NOs: 32 and 33; A polypeptide consisting essentially of SEQ ID NOs: 34 and 35; a polypeptide consisting essentially of SEQ ID NOs: 337 and 338, or A polypeptide consisting essentially of SEQ ID NOs: 339 and 340 and wherein the isolated or recombinant polypeptide is not the wild type of any one of IHF alpha, IHF beta, or any of SEQ ID NOs: 6-11, 28, 29, or 42-100.

[0222] Also provided herein is an isolated polynucleotide or polypeptide comprising one or more of the following sequences:

number

[0223] Fragments or equivalents of the above-mentioned isolated or recombinant polypeptides are also provided as agents for use in the methods of the present invention. An example of a fragment is the C-terminal polypeptide. In another embodiment, the isolated or recombinant polypeptide comprises, consists essentially of, or further consists of two or more of the above-mentioned isolated or recombinant polypeptides.

[0224] For example, the isolated or recombinant polypeptide comprises, consists essentially of, or further consists of any one of SEQ ID NOs: 1-5, 12-27, or 30-33, or fragments or equivalent polypeptides, examples of which are identified in Table 1 or set forth in Table 2, or Arm fragments identified in Table 2, Table 3, or Table 4. In one aspect, the isolated wild-type polypeptide is excluded, i.e., the polypeptide is not any of SEQ ID NOs: 6-11, 28, 29, or the wild-type sequences identified in Table 1 or set forth in Table 2.

[0225] In one aspect, the present invention provides an isolated or recombinant polypeptide consisting essentially of an amino acid sequence of the group of SEQ ID NOs: 1-5, 12-27 or 30-35, 1-6 and 13-35, or a polypeptide comprising, consisting essentially of, or further consisting of amino acids corresponding to the beta-3 fragment and / or alpha-3 fragment of Haemophilus influenzae IHFα or Haemophilus influenzae IHFβ, non-limiting examples of which include SEQ ID NOs: 12-27, or respective fragments or equivalents thereof. In another aspect, the present invention provides isolated or recombinant polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence of the group of SEQ ID NOs: 1-4, or their respective fragments or equivalents, or polypeptides comprising, consisting essentially of, or consisting of amino acids corresponding to the β-3 and / or α-3 fragments of Haemophilus influenzae IHFα or Haemophilus influenzae IHFβ, non-limiting examples of which include SEQ ID NOs: 12-27, or fragments or biological equivalents thereof, each independently further comprising at least 2, alternatively at least 3, alternatively at least 4, alternatively at least 5, alternatively at least 6, alternatively at least 7, alternatively at least 8, alternatively at least 9, alternatively at least 10 amino acids at the amino and / or carboxyl termini of the polypeptide. In one aspect, isolated wild-type DNA-binding polypeptides are excluded, i.e., the polypeptide is not any of SEQ ID NOs: 6-11, 28, 29, or 42-100, or an isolated wild-type polypeptide sequence listed in Table 1 or shown in Table 2.

[0226] In another aspect, the present invention provides isolated or recombinant polypeptides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 or 2, or in combination with polypeptides comprising, consisting essentially of, or consisting of amino acids corresponding to the β-3 and / or α-3 fragments of Haemophilus influenzae IHF-α or IHFβ, non-limiting examples of which include SEQ ID NOs: 12-27, or their respective fragments or biological equivalents. In one aspect, isolated wild-type DNA-binding polypeptides are excluded. That is, the polypeptide is not SEQ ID NO: 6-11, 28, 29, 42-100, an isolated polypeptide sequence listed in Table 1, or set forth in Table 2.

[0227] In still further aspects, the present invention provides isolated or recombinant polypeptides comprising, consisting essentially of, or consisting of SEQ ID NO: 3 or 4, or their respective fragments or equivalents, in combination with polypeptides comprising, consisting essentially of, or consisting of amino acids corresponding to the β-3 and / or α-3 fragments of Haemophilus influenzae IHF-α or IHFβ, non-limiting examples of which include SEQ ID NOs: 12-27 and 34-35, or their respective biological equivalents. In one aspect, isolated wild-type DNA-binding polypeptides are excluded. That is, the polypeptide is not SEQ ID NO: 6-11, 28, 29, 42-100, an isolated wild-type polypeptide sequence listed in Table 1, or set forth in Table 2.

[0228] The present invention also provides isolated or recombinant polypeptides comprising, consisting essentially of, or consisting of two or more, or three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more of the total 14 isolated polypeptides, or their respective fragments or equivalents. Examples include isolated or recombinant polypeptides comprising SEQ ID NOS: 1-4, e.g., SEQ ID NOS: 1 and 2, or 1 and 3, or 1 and 4, or 2 and 3, or SEQ ID NOS: 1, 2 and 3, or 2, 3 and 4, or 1, 3 and 4. The polypeptides can be in any orientation, e.g., SEQ ID NOS: 1, 2, and 3, or SEQ ID NOS: 3, 2 and 1, or 2, 1 and 3, or 3, 1 and 2. Biological equivalents of these polypeptides are also encompassed by the present invention, but the sequences do not include isolated wild-type sequences, such as those identified in Tables 1, 2, and 3.

[0229] In another aspect, the present invention provides an isolated or recombinant polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 1 or 2 and 3 or 4, or their respective fragments or equivalents, but which is not any of SEQ ID NOs: 6-11, and may further comprise any one or more of SEQ ID NOs: 11-26, e.g., 11 and 12, or 1 and 11, or 2 and 11, or 1 and 12, or 2 and 12, or 11, 12 and 1, or 2, 11 and 12. In this embodiment, SEQ ID NO: 1 or 2 is located upstream or amino terminal to SEQ ID NO: 3 or 4, but the amino acid sequence is not the isolated wild-type polypeptide, e.g., is not any of SEQ ID NOs: 6-11, 28, or 29. In another aspect, the isolated polypeptide comprises SEQ ID NO: 3 or 4 located upstream or amino terminal to SEQ ID NO: 1 or 2. Biological equivalents of these polypeptides are also included in the present invention, but the sequences do not include the isolated wild-type polypeptide.

[0230] In one embodiment, the wild-type polypeptide or the polypeptide of Pedulla et al. (1996) PNAS 93:15411-15416. Any polypeptides or proteins having sequence identity to those disclosed in are excluded from the present invention.

[0231] In any of the above embodiments, a peptide linker can be added to the N-terminus or C-terminus of the polypeptide. "Linker" or "peptide linker" refers to a peptide sequence linked to either the N-terminus or C-terminus of a polypeptide sequence. In one aspect, the linker is from about 1 to about 20 amino acid residues in length, or alternatively, from 2 to about 10 amino acid residues, or from about 3 to about 5 amino acid residues in length. An example of a peptide linker is Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO: 37). Other examples include Gly-Gly-Gly; Gly-Pro-Ser-Leu (SEQ ID NO: 38); Gly-Pro-Ser; Pro-Ser-Leu-Lys (SEQ ID NO: 39); Gly-Pro-Ser-Leu-Lys (SEQ ID NO: 40), and Ser-Leu-Lys-Leu (SEQ ID NO: 41).

[0232] Isolated polypeptides of the present invention are intended to include wild-type and recombinantly produced polypeptides and proteins isolated from prokaryotic and eukaryotic host cells, as well as mutant proteins, analogs, and fragments thereof; examples of such cells are described above. In some embodiments, the term also encompasses the antibodies and anti-idiotypic antibodies described herein. Such polypeptides are known in the art and can be isolated or produced using methods briefly described herein.

[0233] It is understood that functional equivalents or variants of wild-type polypeptides or proteins, e.g., those with conservative amino acid substitutions, are also within the scope of the present invention. See, e.g., Table 2. Other analogs include fusion proteins comprising the proteins or polypeptides of the present invention, which may include the polypeptide linked to an antigen-presenting matrix.

[0234] In another aspect, the polypeptide is conjugated or linked to a detectable label. Suitable labels are known in the art and described herein.

[0235] In a further embodiment, the polypeptide, with or without a detectable label, can be contained on or expressed on the surface of a host prokaryotic or eukaryotic host cell, such as a dendritic cell.

[0236] Proteins and polypeptides can be obtained by a number of processes known to those skilled in the art, including purification, chemical synthesis, and recombinant methods. Polypeptides can be isolated from preparations, such as host cell lines, by methods such as immunoprecipitation with antibodies, and standard techniques such as gel filtration, ion exchange, reverse-phase chromatography, and affinity chromatography. Such methodologies are described, for example, in Deutscher et al. (1999) Guide To Protein See Purification: Methods In Enzymology (Vol. 182, Academic Press). Accordingly, the present invention also provides processes for obtaining these polypeptides, as well as products obtainable by these processes and the products obtained.

[0237] Polypeptides can also be obtained by chemical synthesis using commercially available automated peptide synthesizers, such as those manufactured by Perkin / Elmer / Applied Biosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. Synthesized polypeptides can be precipitated and further purified, for example, by high performance liquid chromatography (HPLC). Thus, the present invention also provides a process for chemically synthesizing the proteins of the present invention by providing the protein sequence and reagents, such as amino acids and enzymes, and linking the amino acids together in the appropriate direction and linear sequence.

[0238] Alternatively, proteins and polypeptides can be obtained by well-known recombinant methods, such as those described in Sambrook et al. (1989) supra, using the host cell and vector systems described herein.

[0239] The present application also provides the polypeptides described herein conjugated to a detectable agent for use in diagnostic methods. For example, detectably labeled polypeptides can be bound to a column and used to detect and purify antibodies. Detectably labeled polypeptides are also useful as immunogens for producing antibodies, as described below. The polypeptides of the present invention are useful in in vitro assay systems for screening agents or drugs that modulate cellular processes.

[0240] It is well known to those skilled in the art that the peptide of the present invention can be modified to change its properties.As used herein, the term "amino acid" refers to either natural amino acid and / or unnatural amino acid or synthetic amino acid, including glycine and both D optical isomer and L optical isomer, and amino acid analogs and peptidomimetics.A peptide of three or more amino acids is generally called an oligopeptide if the peptide chain is short.If the peptide chain is long, the peptide is generally called a polypeptide or protein.

[0241] The peptides of the present invention can be modified to include unnatural amino acids. Thus, peptides may contain D-amino acids, combinations of D- and L-amino acids, and various "designer" amino acids (e.g., beta-methyl amino acids, C-alpha-methyl amino acids, and N-alpha-methyl amino acids) to impart special properties to the peptides. Furthermore, by assigning specific amino acids to specific coupling steps, peptides with alpha helices, beta turns, beta sheets, gamma turns, and cyclic peptides can be generated. Generally, alpha-helical or random secondary structures are believed to be preferred.

[0242] The polypeptides of the present invention can also be combined with various solid-phase carriers, such as implants, stents, pastes, gels, dental implants, or medical implants, or liquid-phase carriers, such as beads, sterile or aqueous solutions, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, suspensions, and emulsions. Examples of non-aqueous solvents include propylethylene glycol, polyethylene glycol, and vegetable oils. When used to prepare antibodies or induce immune responses in vivo, the carrier may also contain an adjuvant useful for nonspecifically enhancing a specific immune response. Those skilled in the art can easily determine whether an adjuvant is necessary and select one accordingly. However, for illustrative purposes only, suitable adjuvants include, but are not limited to, Freund's complete and incomplete adjuvants, inorganic salts, and polynucleotides. Other suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of E. coli heat-labile enterotoxin, mutant derivatives of cholera toxin, CPG oligonucleotides, and squalene-derived adjuvants.

[0243] The present invention also provides pharmaceutical compositions comprising, consisting essentially of, or further consisting of any of the polypeptides, analogs, muteins, or fragments of the invention, alone or in combination with each other or with other agents, such as antibiotics and acceptable carriers or solid supports. These compositions are useful for the various diagnostic and treatment methods described herein.

[0244] Polynucleotides The present invention also provides isolated or recombinant polynucleotides encoding one or more of the above-identified isolated or recombinant polypeptides and their respective complementary strands.Vector containing isolated or recombinant polynucleotides is further provided, examples of which are known in the art and are briefly described herein.In one embodiment, where two or more isolated or recombinant polynucleotides are to be expressed as a single unit, the isolated or recombinant polynucleotides can be contained in a polycistronic vector.Polynucleotides can be DNA, RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA.

[0245] In another aspect, the present invention provides interfering agents that are four-way junction polynucleotides resembling Holliday junctions, three-way junction polynucleotides resembling replication forks, polynucleotides with inherent flexibility, or curved polynucleotides, which can treat or inhibit the binding of DNA BII polynucleotides to microbial DNA and treat, prevent, or inhibit biofilm formation and related infections and disorders. Those skilled in the art can create such polynucleotides using the information provided herein and their own knowledge. See Goodman and Kay (1999) J. Biological Chem. 274(52):37004-37011 and Kamashev and Rouviere-Yaniv (2000) EMBO J. 19(23):6527-6535.

[0246] The present invention further provides isolated or recombinant polynucleotides operably linked to a promoter of RNA transcription and other regulatory sequences for DNA or RNA replication and / or transient or stable expression. As used herein, the term "operably linked" means that the promoter is positioned to direct transcription of RNA from the DNA molecule. Examples of such promoters are SP6, T4, and T7. In certain embodiments, cell-specific promoters are used to achieve cell-specific expression of an inserted polynucleotide. Vectors containing a promoter or promoter / enhancer, a termination codon and a selectable marker sequence that can be operably linked to the promoter, and a cloning site into which a piece of DNA can be inserted, are known in the art and commercially available. For general methodologies and cloning strategies, see Gene Expression Technology (Goeddel, ed., Academic Press, Inc. (1991)) and the references cited therein, as well as Vectors: Essential Data Series (Gacesa and Ramji, eds., John Wiley & Sons, NY (1994)), which contains maps, functional properties, commercial suppliers, and GenEMBL accession number references for a variety of suitable vectors.

[0247] In one embodiment, polynucleotides derived from the polynucleotides of the present invention encode polypeptides or proteins having diagnostic and therapeutic utility as described herein, as well as probes for identifying protein transcripts that may or may not be present. These nucleic acid fragments can be prepared, for example, by restriction enzyme digestion of larger polynucleotides and then labeled with a detectable marker. Alternatively, nick translation of molecules can be used to generate random fragments. For methodologies for preparing and labeling such fragments, see Sambrook et al. (1989) supra.

[0248] Expression vectors containing these nucleic acids are useful for creating host-vector systems for producing proteins and polypeptides. It has been shown that these expression vectors must be replicable in the host organism, either as episomes or as an integral part of the chromosomal DNA. Non-limiting examples of suitable expression vectors include plasmids, yeast vectors, viral vectors, and liposomes. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo, as they efficiently transform cells with high levels of expression both in vitro and in vivo. When the nucleic acid is inserted into a suitable host cell, such as a prokaryotic or eukaryotic cell, and the host cell replicates, the protein can be produced recombinantly. The appropriate host cell depends on the vector and can include mammalian, animal, human, simian, insect, yeast, and bacterial cells constructed using known methods. See Sambrook et al. (1989) supra. In addition to using viral vectors to insert exogenous nucleic acids into cells, nucleic acids can be inserted into host cells by methods known in the art, such as transformation of bacterial cells; transfection of mammalian cells using calcium phosphate precipitation; or DEAE-dextran; electroporation; or microinjection. See Sambrook et al. (1989) supra for methodology. Thus, the present invention provides host cells, e.g., mammalian cells, animal cells (rat or mouse), human cells, or prokaryotic cells, e.g., bacterial cells, containing a polynucleotide encoding a protein or polypeptide or antibody.

[0249] When a vector is used for in vivo or ex vivo gene therapy, a pharmaceutically acceptable vector, such as a replication-incompetent retroviral or adenoviral vector, is preferred. A pharmaceutically acceptable vector containing a nucleic acid of the present invention can be further modified to transiently or stably express the inserted polynucleotide. As used herein, the term "pharmaceutically acceptable vector" includes, but is not limited to, a vector or delivery vehicle capable of selectively targeting and introducing a nucleic acid into dividing cells. An example of such a vector is a "replication-incompetent" vector, defined by its inability to produce viral proteins and its prevention of vector propagation in infected host cells. An example of a replication-incompetent retroviral vector is LNL6 (Miller et al. (1989) BioTechniques 7:980-990). Methodologies using replication-incompetent retroviruses for retroviral-mediated gene transfer of genetic markers have been established (Bordignon (1989) PNAS USA 86:8912-8952; Culver (1991) PNAS USA 88:3155; and Rill (1991) Blood 79(10):2694-2700).

[0250] The present invention also provides genetically modified cells that contain and / or express the polynucleotides of the present invention. Genetically modified cells can be made by inserting upstream regulatory sequences, such as promoters or gene activators (see U.S. Patent No. 5,733,761).

[0251] Polynucleotides can be conjugated with detectable markers, such as enzyme labels or radioisotopes, to detect the expression of nucleic acids and / or genes in cells. A wide variety of suitable detectable markers are known in the art, including fluorescent, radioactive, enzymatic, or other ligands capable of producing detectable signals, such as avidin / biotin. In one embodiment, it may be desirable to use fluorescent labels or enzyme tags, such as urease, alkaline phosphatase, or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates can be used to provide a visible means, either by the human eye or spectrophotometrically, for identifying specific hybridization with complementary nucleic acid-containing samples. Therefore, the present invention further provides a method for detecting a single-stranded polynucleotide or its complement by contacting the target single-stranded polynucleotide with a labeled single-stranded polynucleotide (probe) that is part of the polynucleotide of the present invention under conditions that allow hybridization of complementary single-stranded polynucleotides (preferably moderately stringent hybridization conditions), or more preferably, under highly stringent hybridization conditions. The hybridized polynucleotide pair is separated from unhybridized single-stranded polynucleotides. The hybridized polynucleotide pair is detected using methods known to those skilled in the art, for example, as described in Sambrook et al. (1989) supra.

[0252] The polynucleotide of the present invention can be obtained by chemical synthesis, recombinant cloning method, PCR, or any combination thereof.Methods for chemical polynucleotide synthesis are known in the art and do not need to be described in detail herein.Those skilled in the art can use the sequence data provided herein to obtain desired polynucleotides by using a DNA synthesizer or ordering commercial services.

[0253] The polynucleotides of the present invention can be isolated or replicated using PCR. PCR technology is the subject of U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, and is also described in PCR: The Polymerase Chain Reaction (Mullis et al., eds., Birkhauser Press, Boston (1994)) or MacPherson et al. (1991) and (1995) supra, and the references cited therein. Alternatively, those skilled in the art can replicate DNA using the sequences provided herein and a commercial DNA synthesizer. Thus, the present invention also provides a process for obtaining the polynucleotides of the present invention by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes, and instructions for replicating them, and chemically replicating or linking the nucleotides in the appropriate direction to obtain a polynucleotide. In another embodiment, these polynucleotides are further isolated. Furthermore, those skilled in the art can insert the polynucleotide into a suitable replicable vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA thus amplified can be isolated from the cell by methods known to those skilled in the art. The process for obtaining polynucleotides by this method, as well as the polynucleotides thus obtained, are further provided herein.

[0254] RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be delivered by any suitable method, for example, by using a suitable gene delivery vehicle (e.g., liposome, plasmid, or vector) or by electroporation. After the cells have replicated and the DNA has been transcribed into RNA, the RNA can then be isolated using methods known to those skilled in the art, for example, the method described in Sambrook et al. (1989) supra. For example, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures described in Sambrook et al. (1989) supra, or extracted with a nucleic acid-binding resin according to the accompanying instructions provided by the manufacturer.

[0255] Polynucleotides exhibiting sequence complementarity or homology to the polynucleotides of the invention are useful as hybridization probes. Because the entire coding sequence of the transcript is known, any portion of this sequence or a homologous sequence can be used in the methods of the invention.

[0256] It is known in the art that a "perfect match" probe is not required for specific hybridization. Minor variations in the probe sequence can be achieved by substitution, deletion, or insertion of a small number of bases that do not affect the specificity of hybridization. Generally, a base pair mismatch of about 20% (when optimally aligned) can be tolerated. Probes useful for detecting the above-mentioned mRNAs are preferably at least about 80% identical to the homologous region. More preferably, the probe is 85% identical to the corresponding gene sequence after alignment with the homologous region; even more preferably, the probe exhibits 90% identity.

[0257] These probes can be used in radioassays (e.g., Southern blot analysis and Northern blot analysis) to detect, prognose, diagnose, or monitor various cells or tissues containing these cells. The probes can also be attached to a solid support or array, such as a chip, for use in high-throughput screening assays to detect expression of genes corresponding to the polynucleotides of the invention. Thus, the present invention also provides probes comprising or corresponding to the polynucleotides of the invention, or their equivalents, or their complements, or fragments, attached to a solid support for use in high-throughput screening.

[0258] The overall size of the fragment, as well as the size of the complementary stretch, will depend on the intended use or application of the particular nucleic acid segment. Smaller fragments are generally used in hybridization embodiments, and the length of the complementary region can vary, for example, from at least 5-10 nucleotides to about 100 nucleotides, or even the full length, depending on the complementary sequence desired to be detected.

[0259] Nucleotide probes having a stretch of complementary sequence greater than 5-10 nucleotides in length are generally preferred to increase hybrid stability and selectivity, thereby improving the specificity of the resulting hybrid molecules. More preferably, gene-complementary stretches of polynucleotides longer than 10 nucleotides, or even longer if desired, can be designed. Such fragments can be readily prepared, for example, by directly synthesizing the fragment by chemical means, by applying nucleic acid reproduction techniques such as the PCR technique using two primer oligonucleotides described in U.S. Pat. No. 4,603,102, or by introducing selected sequences into a recombinant vector for recombinant production. In one embodiment, the probe is about 50-75 nucleotides or more, or 50-100 nucleotides in length.

[0260] The polynucleotide of the present invention can serve as a primer for detecting genes or gene transcripts expressed in the cells described herein.In this case, amplification refers to any method using a primer-dependent polymerase that can replicate the target sequence with reasonable fidelity.Amplification can be performed by natural DNA polymerase or recombinant DNA polymerase, such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase.For illustrative purposes only, the primer has the same length as that shown for the probe.

[0261] One method for amplifying polynucleotides is PCR, and PCR amplification kits are commercially available. After amplification, the resulting DNA fragments can be detected by any suitable method known in the art, for example, by agarose gel electrophoresis followed by visualization using ethidium bromide staining and ultraviolet illumination.

[0262] Methods for administering an effective amount of a gene delivery vector or vehicle to cells have been developed, and are known to those skilled in the art and are described herein.Methods for detecting gene expression in cells are known in the art, and include techniques such as hybridization to DNA microarrays, in situ hybridization, PCR, RNase protection assays, and Northern blot analysis.Such methods are useful for detecting and quantifying the expression of genes in cells.Alternatively, the expression of encoded polypeptides can be detected by various methods.Specifically, it is useful to prepare polyclonal or monoclonal antibodies that specifically react with target polypeptides.Such antibodies are useful for visualizing cells expressing polypeptides using techniques such as immunohistochemistry, ELISA, and Western blotting.These techniques can be used to determine the expression level of expressed polynucleotides.

[0263] Antibodies and their derivatives The present invention also provides antibodies that specifically recognize and / or bind to the isolated polypeptides used in the methods of the present invention. The antibodies can be any of the various antibodies described herein, and 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 antigen-binding fragments thereof. In one aspect, the fragment comprises, consists essentially of, or further consists of the CDRs of the antibody. In one aspect, the antibody is detectably labeled or further comprises a detectable label conjugated thereto. Also provided are hybridoma cell lines that produce the monoclonal antibodies of the present invention. Further provided herein are compositions comprising, consisting essentially of, or further consisting of one or more of the above embodiments. Further provided are polynucleotides encoding the amino acid sequences of the antibodies and fragments, as well as methods for recombinantly producing antibody polypeptides and fragments thereof. Antibody polypeptides can be produced in eukaryotic cells, prokaryotic cells, or through other methods known in the art and described herein.

[0264] Antibodies can be produced using conventional techniques known in the art and fully described in the literature. There are several methods for producing polyclonal antibodies. For example, polyclonal antibodies are typically produced by immunizing suitable mammals, such as, but not limited to, chickens, goats, guinea pigs, hamsters, horses, mice, rats, and rabbits. An antigen is injected into a mammal, which induces B lymphocytes to produce immunoglobulins specific to the antigen. The immunoglobulins can be purified from the serum of the mammal. Antibodies specific to IHFα and IHFβ can be produced by injecting polypeptides corresponding to different epitopes of IHFα and IHFβ. For example, antibodies can be generated using the 20 amino acids of each subunit, such as TFRPGQKLKSRVENASPKDE (SEQ ID NO: 34) for IHFα and KYVPHFKPGKELRDRANIYG (SEQ ID NO: 35) for IHFβ, or antibodies that specifically recognize and bind to polynucleotides or peptides containing one or more of the following sequences:

number

[0265] Monoclonal antibodies can be produced using conventional hybridoma methods, which are known in the art and fully described in the literature. For example, hybridomas can be produced using a suitable immortal cell line (e.g., Sp2 / 0 cells, Sp2 / 0-AG14 cells, NSO cells, NS1 cells, NS2 cells, AE-1 cells, L.5 cells, P3X63Ag8.653 cells, Sp2 SA3 cells, Sp2 MAI cells, Sp2 SS1 cells, Sp2 SA5 cells, U397 cells, MLA 144 cells, ACT IV cells, MOLT4 cells, DA-1 cells, JURKAT cells, WEHI cells, K-562 cells, COS cells, RAJI cells, NIH 3T3 cells, HL-60 cells, MLA 144 cells, NAMAIWA cells, NEURO cells, or the like). Myeloma cell lines, such as, but not limited to, myeloma cell lines, such as, but not limited to, 2A cells, CHO cells, PerC.6 cells, YB2 / 0 cells, or heteromyelomas, fusion products thereof, or any cells or fusion cells derived therefrom, or any other suitable cell line known in the art (see cell lines at the following web addresses, e.g., atcc.org, lifetech.com, last accessed November 26, 2007), isolated or cloned spleen cells, peripheral blood cells, lymphocytes, tonsil cells, or other immune or B-cell containing cells, or recombinant or endogenous viruses, bacteria, algae, prokaryotes, amphibians, insects, reptiles, fish, mammals, rodents, horses, sheep, etc. The antibodies are produced by fusing with antibody-producing cells such as, but not limited to, any other cell that expresses heavy chain constant sequences or heavy chain variable sequences or heavy chain framework sequences or heavy chain CDR sequences or light chain constant sequences or light chain variable sequences or light chain framework sequences or light chain CDR sequences as endogenous or heterologous nucleic acids, such as genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded, or triple-stranded nucleic acids, hybridized nucleic acids, or any combination thereof, of eukaryotes.Antibody-producing cells can also be obtained from the peripheral blood, or preferably the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. Any other suitable host cells can also be used to express heterologous or endogenous nucleic acid encoding the antibodies, specified fragments, or variants thereof of the present invention. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other appropriate known methods and cloned via limiting dilution, or cell sorting, or other known methods.

[0266] Other suitable methods for producing or isolating antibodies with the required specificity can be used, including, but not limited to, recombinant antibody selection from peptide or protein libraries (e.g., but not limited to, bacteriophage display libraries, ribosome display libraries, oligonucleotide display libraries, RNA display libraries, cDNA display libraries, etc.; for example, commercially available libraries from commercial sources such as MorphoSys (Martinsreid / Planegg, Del.), BioInvent (Lund, Sweden), and Affitech (Oslo, Norway) using methods known in the art). Methods known in the art are described in the patent literature, some of which include U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. An alternative method relies on immunization of transgenic animals (e.g., SCID mice; Nguyen et al. (1977), Microbiol. Immunol. 41:901-907 (1997); Sandhu et al. (1996), Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998), Immunol. 93:154-161) that are capable of producing a repertoire of human antibodies as known in the art and / or described herein.Such techniques include ribosome display (Hanes 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 (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987), J. Immunol. 17:887-892; Babcook et al. (1996), Proc. Natl. Acad. Sci. USA 93:7843-7848), gel 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 methods (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134).

[0267] Antibody derivatives of the invention can also be prepared by delivering a polynucleotide encoding an antibody of the invention to a suitable host to provide a transgenic animal or mammal that produces such an antibody in its milk, such as a goat, cow, horse, sheep, etc. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0268] The term "antibody derivative" includes post-translational modifications to the linear polypeptide sequence of an antibody or fragment. For example, U.S. Patent No. 6,602,684 B1 describes modifications of antibodies, including whole antibody molecules, antibody fragments, or fusion proteins, that include a region equivalent to the Fc region of an immunoglobulin and have enhanced Fc-mediated cytotoxicity. Methods for producing glycoforms and glycoproteins so produced are described.

[0269] The antibodies of the present invention include derivatives that have been modified by covalently attaching any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from eliciting an anti-idiotypic response. Antibody derivatives include, but are not limited to, antibodies that have been modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization through known protecting / blocking groups, proteolytic cleavage, conjugation to intracellular ligands or other proteins, etc. In addition, derivatives may contain one or more non-classical amino acids.

[0270] Antibody derivatives can also be prepared by generating transgenic plants and cultured plant cells (e.g., but not limited to, tobacco, corn, and duckweed) that deliver polynucleotides of the present invention and produce such antibodies, specified portions, or variants in plant parts or cultured cells derived therefrom. For example, Cramer et al. (1999), Curr. Top. Microbol. Immunol., 240:95-118, and references cited therein, describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins, for example, using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activity equivalent to mammalian proteins produced in other recombinant systems or purified from natural sources. See, for example, Hood et al. (1999), Adv. Exp. Med. Biol., 464:127-147, and references cited therein. Antibody derivatives have also been produced in large quantities from transgenic plant seeds, including tobacco seeds and potato tubers, that contain antibody fragments such as single-chain antibodies (scFv). See, e.g., Conrad et al. (1998), Plant Mol. Biol., 38:101-109, and references cited therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

[0271] Antibody derivatives can also be produced, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, some or all of the non-human or human CDR sequences are maintained while substituting human or other amino acids for the non-human sequences of the variable and constant regions.

[0272] In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Humanization or engineering of antibodies can be carried out using any known method, such as, but not limited to, those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

[0273] Chimeric, humanized, or primatized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared using standard molecular biology techniques. DNA encoding heavy and light chain immunoglobulins can be obtained from the subject murine hybridoma and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (U.S. Patent No. 4,816,567). To create a humanized antibody, murine CDR regions can be inserted into a human framework using methods known in the art (U.S. Patent No. 5,225,539, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370). Similarly, to create primatized antibodies, murine CDR regions can be inserted into a primate framework using methods known in the art (WO93 / 02108 and WO99 / 55369).

[0274] The antibodies of the present invention can also be modified to create chimeric antibodies, which are antibodies in which the various domains of the antibody's heavy and light chains are encoded by DNA from multiple species. See, e.g., U.S. Patent No. 4,816,567.

[0275] Alternatively, the antibodies of the present invention can also be modified to create veneered antibodies. A veneered antibody is an antibody in which the exterior amino acid residues of an antibody from one species have been carefully replaced, or "veneered," with exterior amino acid residues from a second species, so that the antibody from the first species is not immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Because the immunogenicity of a protein depends primarily on the nature of its surface, substituting exposed residues that differ from those typically found in antibodies from another mammalian species may reduce the immunogenicity of the antibody. Such careful substitution of exterior residues should have little or no effect on the internal domains or interdomain junctions. Therefore, changes limited to framework residues in the variable region should not affect the binding properties of the ligand. This process is referred to as "veneering," because only the outer surface or skin of the antibody is altered, leaving the supporting residues undisturbed.

[0276] The "veneering" procedure utilizes searchable 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 searchable databases (both nucleic acid and protein databases) in the United States and abroad. Non-limiting examples of methods used to produce veneered antibodies include EP 519596, U.S. Pat. No. 6,797,492, and are also described in Padlan et al. (1991), Mol. Immunol. 28(4-5):489-498.

[0277] The term "antibody derivative" also encompasses "diabodies," which are small antibody fragments with two antigen-binding sites, wherein the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain, creating two antigen-binding sites. (See also U.S. Pat. No. 6,632,926 by Chen et al., which discloses antibody mutants in which one or more amino acids are inserted into the hypervariable region of the parent antibody, resulting in antibody mutants with binding affinity for a target antigen that is at least about twice as strong as the parent antibody's binding affinity for that antigen.)

[0278] The term "antibody derivative" further encompasses engineered antibody molecules, engineered antibody fragments, and engineered antibody single domains such as scFvs, dAbs, nanobodies, minibodies, unibodies, and affibodies (Holliger and Hudson (2005) Nature Biotech 23(9):1126-36; U.S. Patent Publication No. US2006 / 0211088; PCT Publication No. WO2007 / 059782; U.S. Patent No. 5,831,012).

[0279] The term "antibody derivative" further encompasses "linear antibodies." Procedures for producing linear antibodies are known in the art and are also described in Zapata et al. (1995), Protein Eng., 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (V) that form a pair of antigen-binding regions. H -C H 1-VH-C H 1) Linear antibodies can be bispecific or monospecific.

[0280] Antibodies of the invention can be recovered and purified from recombinant cell culture by known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0281] Antibodies of the present invention include naturally occurring purified products, products of chemical synthetic procedures, and products produced via recombinant methods from eukaryotic hosts, including, for example, yeast, higher plants, insect, and mammalian cells, or alternatively, from prokaryotic hosts as described above. Birch and Radner (2006), Adv. Drug Delivery Rev., 58:671-685, describe many antibody production systems.

[0282] The term "antibody" is also intended to encompass antibodies of all immunoglobulin isotypes and immunoglobulin subclasses. A particular isotype of monoclonal antibody can be prepared directly by selection from the initial fusion, or can be subsequently prepared from parent hybridomas secreting monoclonal antibodies of a different isotype by using sib selection methods to isolate class-switched variants using 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 methods can be used.

[0283] Isolation of other monoclonal antibodies with the specificity of the monoclonal antibodies described herein can also be achieved by one skilled in the art through the generation of anti-idiotypic antibodies (Herlyn et al. (1986) Science 232:100), which are antibodies that recognize unique determinants present in the monoclonal antibody of interest.

[0284] In some aspects of the present invention, it may be useful to detectably or therapeutically label the antibody. Suitable labels have been described above. Methods for conjugating antibodies with these agents are known in the art. For illustrative purposes only, antibodies can be labeled with detectable moieties such as radioactive atoms, chromophores, fluorophores, etc. Such labeled antibodies can be used in diagnostic methods in vivo or in isolated test samples.

[0285] The sensitivity of an antibody in an assay can be increased by linking it to a low molecular weight hapten. The hapten can then be specifically detected by a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See Harlow and Lane (1988), supra.

[0286] The variable regions of the antibodies of the present invention can be modified by mutating amino acid residues within the VH CDR1 and / or VL CDR1 regions, VH CDR2 and / or VL CDR2 regions, and / or VH CDR3 and / or VL CDR3 regions to improve one or more binding characteristics (e.g., affinity) of the antibody. Mutations can be introduced via site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding or other functional properties of interest can be assessed in appropriate in vitro or in vivo assays. Conservative modifications are preferably introduced, typically altering no more than one, two, three, four, or five residues within the CDR regions. Mutations can be amino acid substitutions, additions, or deletions.

[0287] For example, antibodies can be modified with framework modifications to reduce immunogenicity by "backmutating" one or more framework residues to the corresponding germline sequence.

[0288] In addition, antibodies of the invention can be engineered to include modifications in the Fc region that alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Such modifications include, but are not limited to, altering the number of cysteine residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease antibody stability (U.S. Patent No. 5,677,425), and mutating amino acids in the Fc hinge region that decrease the biological half-life of the antibody (U.S. Patent No. 6,165,745).

[0289] In addition, antibodies of the present invention can be chemically modified. For example, the glycosylation of an antibody can be altered by modifying one or more glycosylation sites within the antibody sequence to increase the affinity of the antibody for the antigen (U.S. Patent Nos. 5,714,350 and 6,350,861). Alternatively, to increase antibody-dependent cell-mediated cytotoxicity, the antibody can be expressed in a host cell with an altered glycosylation machinery to produce a hypofucosylated antibody with reduced amounts of fucosyl residues or an antibody with increased bisecting GlcNac structures (Shields et al., 2002, J. Biol. Chem., 277:26733-26740; Umana et al., 1999, Nat. Biotech., 17:176-180).

[0290] The antibodies of the present invention, or fragments thereof, can be pegylated to extend their biological half-life by reacting them with polyethylene glycol (PEG), a reactive ester of PEG, or an aldehyde derivative of PEG under conditions that result in attachment of one or more PEG groups to the antibody or antibody fragment. Pegylation of the antibody can be carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG used to derivatize other proteins, such as mono(C1-C10) alkoxypolyethylene glycol or aryloxypolyethylene glycol, or polyethylene glycol maleimide. The antibody to be pegylated can be an aglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the present invention (EP 0154316 and EP 0401384).

[0291] Additionally, antibodies can be chemically modified by conjugating or fusing the antigen-binding region of the antibody to a serum protein, such as human serum albumin, to extend the half-life of the resulting molecule. Such techniques are described, for example, in EP0322094 and EP0486525.

[0292] The antibodies of the present invention or fragments thereof can be conjugated with diagnostic agents and used for diagnosis, e.g., to monitor the onset or progression of disease and determine the effectiveness of a given treatment regimen. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, various positron-emitting metals used in positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substance can be directly linked or conjugated to the antibody or fragment thereof, or indirectly linked or conjugated to the antibody or fragment thereof via a linker using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131I, Indium-111, Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111, Gallium-67, Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166, Holmium-166 Radioactive metal ions include rhenium-186, rhenium-188, rhenium-189, lead-212, radium-223, actinium-225, iron-59, selenium-75, arsenic-77, strontium-89, molybdenum-99, rhodium-105, palladium-109, praseodymium-143, promethium-149, erbium-169, iridium-194, gold-198, gold-199, and lead-211. Monoclonal antibodies can be indirectly conjugated to radioactive metal ions by using bifunctional chelating agents that are covalently attached to the antibodies. Chelating agents can be attached through amines (Meares et al., 1984, Anal. Biochem., 142:68-78), sulfhydral groups of amino acid residues (Koyama, 1994, Chem. Abstr., 120:217-262t), and carbohydrate groups (Rodwell et al., 1986, PNAS USA, 83:2632-2636; Quadri et al., 1993, Nucl. Med. Biol., 20:559-570).

[0293] Additionally, the antibodies or fragments thereof of the present invention can be conjugated to a therapeutic agent, such as an antibacterial agent that will treat or prevent recurrence of Burklederia infection. Suitable therapeutic agents include ceftazidime, ciprofloxacin, imipenem, and minocycline.

[0294] Additional suitable conjugated molecules include ribonucleases (RNases), DNase I, antisense nucleic acids, inhibitory RNA molecules such as siRNA molecules, immunostimulatory nucleic acids, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Aptamers are small nucleic acids ranging from 15 to 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets, and can bind to small molecules such as ATP (U.S. Pat. No. 5,631,146) and theophylline (U.S. Pat. No. 5,580,737), as well as macromolecules such as reverse transcriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Ribozymes are nucleic acid molecules capable of catalyzing chemical reactions intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate, which is then cleaved. Nucleic acid molecules with triplex-forming function can interact with double-stranded nucleic acids by forming triplexes, and can also interact with single-stranded nucleic acids, in which the triplex of DNA forms a complex by relying on both Watson-Crick and Hoogsteen base pairing. The triplex molecule can bind to a target region with high affinity and specificity.

[0295] Functional nucleic acid molecules may act as effectors, inhibitors, regulators, and stimulators of specific activities possessed by target molecules, or may possess novel activities that are independent of other molecules.

[0296] Therapeutic agents can be linked to antibodies, either directly or indirectly, using any of a number of available methods. For example, agents can be attached to the hinge region of a reduced antibody component via disulfide bond formation using a cross-linking agent such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP), or via carbohydrate moieties 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.), pp. 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.), pp. 60-84 (Cambridge University Press, 1995)).

[0297] Techniques for conjugating therapeutic agents to antibodies are well known (see, for example, 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, "Antibodies for Drug Delivery," in Monoclonal Antibodies '84: Biological And Clinical Applications "Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospect of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy," in "Monoclonal Antibodies For Cancer Detection And Therapy," Baldwin et al. (eds.), pp. 303-16 (Academic Press, 1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," 1982, Immunol. Rev. 62:119-58).

[0298] The antibodies of the present invention or their antigen-binding regions can be linked to another functional molecule, such as another antibody or a receptor ligand, to produce bispecific or multispecific molecules that bind to at least two or more different binding sites or target molecules. Linking an antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimic, can be achieved, for example, via chemical linkage, genetic fusion, or noncovalent association. Multispecific molecules can further include a third binding specificity in addition to the first and second target epitopes.

[0299] Bispecific and multispecific molecules can be prepared using methods known in the art.For example, the binding units of bispecific molecules can be produced separately and then conjugated with each other.When binding molecules are proteins or peptides, various linking or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (sulfo-SMCC) (Karpovsky et al., 1984, J. Exp. Med., 160:1686; Liu et al., 1985, Proc. Natl. Acad. Sci. USA, 82:8648). When the binding molecule is an antibody, it can be conjugated by sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains.

[0300] The antibodies of the present invention or fragments thereof can be linked to a moiety that is toxic to the cells to which the antibody binds to form a "depleting" antibody. These antibodies are particularly useful in applications in which it is desired to deplete NK cells.

[0301] The antibodies of the present invention can also be attached to a solid support, which is particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0302] Antibodies can also be bound to many different carriers (e.g., pharmaceutically acceptable carriers). Thus, the present invention also provides compositions containing antibodies and another active or inactive substance. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be soluble or insoluble, depending on the purpose of the present invention. Those skilled in the art will know of other suitable carriers for antibodies, or will be able to ascertain such carriers using routine experimentation. [Example]

[0303] The following examples are intended to illustrate, but not limit, the present invention.

[0304] Experimental procedure Experiment No. 1 Materials and Methods Ethics Statement: Written informed consent and permission was obtained from parents or legally authorized representatives prior to collection of sputum from pediatric patients at Nationwide Children's Hospital. The procedure was performed in accordance with all institutional and federal guidelines and in accordance with a protocol approved by the Institutional Review Board of Nationwide Children's Hospital, Columbus, Ohio.

[0305] In vitro biofilm formation. B. cenocepacia strains (including strains K56-2, DFA2, and JRL2) were inoculated from frozen stocks into LB broth (BBL) and grown overnight at 37°C with shaking at 200 rpm. Cultures were then diluted 100-fold with fresh broth, the optical density at 600 nm was read, and a final concentration of 10 CFU B. cenocepacia / ml of medium was adjusted. Bacteria were further diluted 2500-fold with LB broth, and 200 μl of the bacterial suspension was added to each well of a LabTek II 8-well chambered cover glass (LabTek). Slides were incubated stationary at 37°C in a humidified atmosphere for 16 hours, at which point the medium was aspirated and replaced with fresh LB broth. After an additional 8 hours (total incubation time 24 hours), biofilms were stained or treated with antisera or antibiotics. Assays were performed a minimum of three times.

[0306] Distribution of IHF and DNA within B. cenocepacia biofilms. To examine the relative distribution of IHF within biofilms formed by B. cenocepacia strains, 24-hour unfixed biofilms were incubated with naive rabbit serum or rabbit anti-E. coli IHF (Goodman et al. (2011) Mucosal Immunol. 4:625-637) and revealed using goat anti-rabbit IgG conjugated to AlexaFluor 594 (Invitrogen). DNA within the biofilms was stained with an anti-dsDNA antibody (Abcam, Inc.) and revealed using goat anti-mouse IgG conjugated to AlexaFluor 647 (Molecular Probes). Intact bacterial cells were stained using the membrane dye FilmTracer™ FM® 1-43 Green Biofilm Stain (Molecular Probes) according to the manufacturer's instructions. Images were collected using a Zeiss 510 meta-laser scanning confocal microscope (Carl Zeiss) with a 63X objective. Three-dimensional images were reconstructed using AxioVision Rel. 4.8 (Carl Zeiss).

[0307] To determine the relative abundance of eDNA in 24-h biofilms formed by B. cenocepacia, the DNA-to-bacteria ratio was determined using Zeiss image acquisition software, which compares the relative fluorescence intensity of the anti-dsDNA label to that of the FilmTracer™. Biofilms formed by nontypeable Haemophilus influenzae were also established (Goodman et al. (2011) Mucosal Immunol. 4:625-637; Jurcisek et al. (2011) J. Vis. Exp.) and stained similarly.

[0308] Distribution of IHF in human sputum. To visualize IHF labeling using human sputum, sputum samples were collected from three pediatric CF patients who were also culture-positive for B. cenocepacia under an approved IRB protocol. Sputum was embedded in OCT compound (Fisher Scientific) and flash-frozen in the vapor phase of liquid nitrogen. 10-micron serial sections were cut and adhered to glass slides. Slides were air-dried, fixed in cold acetone, equilibrated in a buffer containing 0.05 M Tris-HCl, 0.15 M NaCl, and 0.05% Tween 20 (pH 7.4), and then blocked using image-iT FX signal enhancer (Molecular Probes) and Background Sniper (BioCare Medical). Sections were incubated with polyclonal anti-IHF antibody or naive rabbit serum and revealed using goat anti-rabbit IgG-Alexafluor 594 (Invitrogen). DNA was counterstained using DAPI-containing Prolong Gold antifade mounting medium (Molecular Probes). Images were viewed as described above.

[0309] Degradation of established B. cenocepacia biofilms. To evaluate the ability of antisera to degrade biofilms formed in vitro, 24-hour biofilms were first established using strain K56-2 as described and then exposed to either naive rabbit serum or rabbit anti-E. coli IHF diluted 50-fold in LB broth, or LB broth alone for 16 hours. Biofilms were then stained using the LIVE / DEAD® BacLight™ Bacterial Viability Kit (Molecular Probes) for microscopy as previously described (Jurcisek et al. (2011) J. Vis. Exp.), and then fixed in a solution of 16% paraformaldehyde, 2.5% glutaraldehyde, and 4% acetic acid in 0.2 M phosphate buffer (pH 7.4) and immediately imaged by confocal microscopy as previously described. Average thickness and average biomass were quantified by COMSTAT2 analysis (Heydorn et al. (2000) Microbiology 146(Pt 10):2409-2415). As an additional measure to confirm that application of naive and immune rabbit serum did not induce cell death, both planktonic and biofilm-adherent B. cenocepacia were harvested 16 hours after treatment, and unfixed cells were stained using the LIVE / DEAD® BacLight™ Bacterial Viability and Counting Kit for flow cytometry (Molecular Probes). Live and dead bacteria were distinguished using a C6 compensated flow cytometer (Accuri). Assays were performed a minimum of three times.

[0310] Enrichment of IgG from whole serum. To confirm that biofilm degradation was mediated by the antibody and not other serum components, IgG was purified from rabbit anti-IHF serum using a HiTrap Protein G HP column (GE Healthcare) according to the manufacturer's instructions. After serum application, both the eluate and the IgG-enriched fraction that passed through the column were collected. SDS-PAGE and Western blotting were performed to demonstrate that anti-IHF and IgG-enriched anti-IHF recognized purified IHF in addition to the native IHF expressed by B. cenocepacia strain K56-2. Briefly, 0.5 μg purified IHF (

[25] ; gift from Howard Nash, NIH) and 5 μg of B. cenocepacia whole-cell lysate were separated on a 4-15% Mini-PROTEAN TGX gel (BioRad) containing Tris / glycine / SDS buffer (BioRad) and then transferred to a nitrocellulose membrane (Invitrogen). Membranes were blocked overnight at 4°C in 3% skim milk in Tris-buffered saline containing 0.5% Tween 20 (Fisher Scientific) and then incubated with rabbit anti-IHF or IgG-enriched anti-IHF, followed by goat anti-rabbit IgG-HRP (Invitrogen). Blots were developed using a CN / DAB substrate kit (Pierce), and images were captured using a BioRad GS800 densitometer.

[0311] Synergistic effect of anti-IHF and antibiotics on biofilm degradation. To test the synergistic effects of antibiotics in combination with antisera directed against IHF when applied to biofilms formed by B. cenocepacia strain K56-2, it was necessary to determine the minimum inhibitory concentrations (MICs) of several antibiotics for planktonic cultures of B. cenocepacia strain K56-2. To do so, 100 CFU of B. cenocepacia were inoculated into LB broth containing two-fold serial dilutions of the following antibiotics: ceftazidime, ciprofloxacin, imipenem, meropenem, minocycline, sulfamethoxazole-trimethoprim, or tobramycin (Sigma). The cultures were incubated statically for 24 hours, and the turbidity of the cultures was assessed. The MICs for planktonic B. cenocepacia were determined as the antibiotic concentrations at which no bacterial growth was observed. Using this information, established B. cenocepacia biofilms were treated for 16 hours with 50-fold dilutions of anti-IHF, antibiotics at determined MICs, anti-IHF plus antibiotics, or medium alone, followed by viability staining using the LIVE / DEAD® BacLight™ Bacterial Viability Kit and visualization by confocal microscopy as previously described. The following antibiotic concentrations were used: ceftazidime 16 μg / ml, ciprofloxacin 2 μg / ml, imipenem 32 μg / ml, meropenem 16 μg / ml, minocycline 4 μg / ml, sulfamethoxazole-trimethoprim 16 μg / ml, and tobramycin 512 μg / ml. Mean thickness and mean biomass were quantified by COMSTAT2 analysis. Assays were performed a minimum of three times.

[0312] Treatment of established biofilms with Pulmozyme®. To test the outcome of treatment of established B. cenocepacia strain K56-2 biofilms with Pulmozyme® (dornase alfa; Genentech, Inc.), established biofilms were treated with a 50-fold dilution of anti-IHF, 0.25 mg of Pulmozyme®, anti-IHF + Pulmozyme®, or medium alone for 16 hours, followed by viability staining and visualization by confocal microscopy. Average thickness and average biomass were quantified by COMSTAT2 analysis. Assays were performed a minimum of three times.

[0313] Effect of anti-IHF antibodies on B. cenocepacia survival within macrophages. The B. cenocepacia strain MHK1, which harbors a mutation in an antibiotic efflux pump that confers gentamicin sensitivity but does not alter the transport of the mutant in macrophages (Hamad et al. (2010) Appl. Environ. Microbiol. 76:3170-3176), was used for this assay. The MHK1 strain was cultured as described above and then treated with naive serum or anti-IHF serum at a 1:1000 dilution for 15 minutes. The treated B. cenocepacia were then added to murine bone marrow-derived macrophages for 1 hour. To kill extracellular bacteria, Iscove's medium (GIBCO) containing 10% heat-inactivated FBS (GIBCO) and 50 μg gentamicin / ml (GIBCO) was added for 30 minutes. Macrophages were lysed at 2, 4, and 6 hours postinfection, and the lysates were plated to determine CFU B. cenocepacia / ml. Assays were performed a minimum of three times.

[0314] DNase footprinting experiments. 386 bp of upstream DNA from BCAL0339 and BCAL0340 was amplified by PCR. The isotopically end-labeled (P) amplicon was used in a binding reaction with crystallographically pure E. coli IHF (Rice et al. (1996) Cell 87:1295-1306; a gift from Howard Nash, NIH) and subsequently used in DNase I footprinting assays (Hung et al. (2011) J. Bacteriol. 193:3642-3652).

[0315] Statistical Methods. To determine significant differences in mean biofilm thickness and mean biomass and macrophage binding assays, paired two-sample t-tests were performed using GraphPad Prism software, version 6.00.

[0316] result Evidence for the presence of abundant extracellular DNA (eDNA) in biofilms formed by B. cenocepacia in vitro. To initially characterize the basic structure of B. cenocepacia biofilms formed in vitro, B. cenocepacia strain K56-2 was grown statically in chamber slides for 24 hours and then labeled with FilmTracer FM 1-43. As shown in Figure 1A, B. cenocepacia formed robust biofilms with a characteristic tower-like structure approximately 26 μm in height. To determine whether B. cenocepacia incorporated DNA into its biofilm matrix, unfixed biofilms were labeled with a monoclonal antibody to detect the presence of dsDNA (white). The formed biofilms contained large amounts of eDNA, which was particularly dense at the base of the biofilm (Figure 1B), suggesting that this matrix component may play an essential role in bacterial adhesion and anchoring during the early stages of biofilm development. Interestingly, although visually apparent, further COMSTAT analysis of both B. cenocepacia biofilms and biofilms formed by nontypeable Haemophilus influenzae (NTHI) (data not shown), followed by identical labeling and the use of separate laser channels (to detect bacteria and DNA), confirmed in a more objective manner that B. cenocepacia incorporated approximately 30% more eDNA per bacterial cell into its biofilms than this additional important human respiratory tract pathogen (Jurcisek et al. (2007) J. Bacteriol. 189:3868-3875).

[0317] Demonstration of DNABII proteins in biofilms produced by B. cenocepacia. To determine whether biofilms formed by B. cenocepacia K56-2 contained members of the DNABII protein family, biofilms formed in vitro by B. cenocepacia K56-2 were incubated with rabbit anti-IHF antibody (antiserum against E. coli IHF, which cross-reacts with multiple DNABII family members (Goodman et al. (2011) Mucosal Immunol. 4:625-637)) and observed labeling throughout the biofilm (Figure 1C).

[0318] Here, to assess whether our in vitro findings correlated with clinical symptoms in CF patients and to determine whether IHF was located at the intersections of eDNA strands present within biofilms formed by B. cenocepacia, as observed in NTHI (20), we obtained sputum samples from CF patients with known B. cenocepacia infection. Samples were flash-frozen and immunolabeled using the same anti-IHF antibody. We observed strong labeling in three-thirds (100%) of the sputum samples collected to date. Notably, specific labeling was observed at virtually 100% of the intersections formed by overlapping dsDNA strands present within these samples (Figure 2). Given the large amount of eDNA present within the EPS matrix of B. cenocepacia-producing biofilms and the fact that IHF appears to reside critically within this matrix, it was argued that biofilm structure could be disrupted by targeting this protein for intervention. It was further reasoned that if this disruption occurred, it could greatly facilitate access of immune effectors, antibiotics, or other therapeutic agents to bacteria previously protected by the EPS, thus facilitating their eradication.

[0319] Demonstration of the ability of anti-IHF to disrupt biofilms formed by B. cenocepacia in vitro. To test one of these hypotheses, 24-h biofilms of B. cenocepacia strain K56-2 were treated with either medium (Figure 3A), naive rabbit serum (50x dilution) (Figure 3B), or rabbit anti-IHF serum (arbitrarily selected 50x dilution) (Figure 3C). After 24 h of incubation, these biofilms were analyzed using COMSTAT software, and it was found that treatment with naive rabbit serum resulted in small and statistically insignificant changes in biofilm thickness, but had no effect on the mean biomass above that observed when sterile medium was used (Figures 3G and 3H). This effect is attributed to nonspecific antibodies in whole rabbit serum that cross-react with outer membrane proteins of multiple Gram-negative bacteria. Conversely, treatment with antisera directed against IHF resulted in a 44% reduction in thickness, a 56% reduction in biomass, and a 52% reduction in height compared to naive serum. These latter effects were statistically significant compared to the use of either sterile medium or naive serum (Figures 3G and 3H). To explain why treatment with antisera directed against IHF did not kill resident bacteria and thus disrupt the biofilm, we harvested both the planktonic subpopulation of bacteria contained within the culture medium and the subpopulation of bacteria remaining within the biofilm after treatment and subjected both preparations to flow cytometric analysis to determine the relative ratio of live to dead bacteria. Regardless of the treatment used, we found that over 80% of the bacteria in the planktonic subpopulation and over 93% of the bacteria remaining within the biofilm remained viable. This result suggested that treatment with anti-IHF mediated the release of live bacteria into the planktonic phase.

[0320] To demonstrate that the biofilm-disrupting activity observed upon treatment with rabbit anti-IHF serum was primarily due to IgG antibodies directed against IHF in this polyclonal, yet hyperimmune, serum, we enriched the serum for IgG as described in Methods (above) and then assayed both the IgG-enriched fraction (Fig. 3D) and the serum fraction that passed through the column (Fig. 3E) for their relative abilities to disrupt preformed B. cenocepacia biofilms. As seen in Figs. 3D and 3E and depicted in 3G and 3H, the IgG-enriched antiserum preparation retained the activity of the whole rabbit anti-IHF, whereas the eluate from the enrichment column was similarly incapable of disrupting biofilms. Western blots demonstrated that antibodies contained in both the total anti-IHF serum and the IgG-enriched fraction recognized the purified IHF protein as well as monomeric, dimeric, and trimeric forms of this protein in whole-cell lysates of B. cenocepacia strain K56-2 (Fig. 3F); the DNABII family member PG0121 (HUβ from Porphyromonas gingivalis) also exhibits the ability to maintain multimers during SDS-PAGE (S. Goodman, personal communication).

[0321] Demonstration of synergistic interactions between anti-IHF and traditional antibiotics. To determine whether anti-IHF-mediated biofilm disruption could render resident bacteria more susceptible to treatment with other existing potential therapeutic agents, we exposed preformed B. cenocepacia biofilms to each of seven antibiotics (using the respective MICs of planktonic B. cenocepacia strain K56-2 as described in the Experimental Procedures section) alone or in combination with anti-IHF serum (1:50 dilution). As shown in Figure 4 (using incubation with ceftazidime (16 μg / ml) as an example), treatment with anti-IHF (Figure 4D) reduced biofilm height, thickness, and biomass compared to untreated biofilms (Figure 4A), whereas these treatments did not induce significant bacterial cell death (Figures 4B and 4E). Furthermore, antibiotic treatment alone had little observable effect on B. cenocepacia-induced biofilms (Figure 4G), and while bacterial cell death was increased relative to that observed after treatment with either sterile medium or anti-IHF alone, this effect was minimal (Figure 4H). However, when used together, a 43% reduction in height (Figure 4J) and a significant and significant reduction in bacterial cell death (indicated by red / orange) (Figure 4K) was observed compared to antibiotic use alone (compare Figures 4H and K), suggesting a synergistic interaction between anti-IHF and ceftazidime. Significant decreases in mean biofilm height (Figure 4M) and biomass (Figure 4N) between anti-IHF + antibiotic treatment and antibiotic treatment alone were similarly obtained after treatment of B. cenocepacia biofilms with ciprofloxacin + anti-IHF, imipenem + anti-IHF, and minocycline + anti-IHF (p<0.05).

[0322] Evidence that the use of Pulmozyme (DNase) may be contraindicated in CF patients infected with B. cenocepacia. Given that DNase treatment is used as the standard of care for CF patients and that B. cenocepacia incorporates large amounts of eDNA into its biofilms, we hypothesized that the use of anti-IHF in combination with DNase might also have a potentially synergistic effect on bacterial weight reduction and eradication within B. cenocepacia-induced biofilms in vitro, as we previously demonstrated with NTHI (Goodman et al. (2011) Mucosal Immunol. 4:625-637). However, this was not true. Indeed, in multiple replicate assays, exposure of B. cenocepacia biofilms to DNase induced significantly more robust biofilms in terms of average thickness (Figures 5B and 5D) than treatment with dilution alone (Figure 5A). These DNase-treated biofilms showed only a slight increase in maximum height (compare 32 μm with 28 μm), but the average increase in surface-to-volume ratio was 12%. The average increase in biomass was 174%, and when compared for average thickness, biofilms formed by DNase-treated B. cenocepacia were 204% thicker than those formed by diluent-treated B. cenocepacia alone. While antisera directed against IHF were still able to effectively reduce these enhanced biofilms (Fig. 5C, D, and E), pursuing this line of investigation into potential synergistic use became counterintuitive given the unexpected results obtained with DNase alone. Importantly, this result has significant promising clinical implications in B. cenocepacia-infected CF patients, as repeated use of DNase may actually worsen the disease and contribute to the very poor clinical outcomes suffered by these patients.

[0323] We demonstrated that incubation of B. cenocepacia with antibodies directed against the DNABII protein, but not with naive serum, reduced bacterial recovery from macrophages. B. cenocepacia can persist and replicate in murine and human CF macrophages. However, wild-type murine macrophages or macrophages from non-CF patients limit B. cenocepacia infection by transporting the organism to lysosomes for degradation. Given the recent evidence that IHF associated with uropathogenic E. coli affects the bacterium's ability to efficiently colonize the bladder (Justice et al. (2012) PLoS One 7:e48349), and the importance of this host cell in bacterial clearance from CF lungs, we hypothesized that incubation of B. cenocepacia with antisera directed against IHF might affect its interaction with macrophages. Using a variant of strain K56-2 (strain MHK1) that was conferred gentamicin susceptibility, we determined the intracellular amplification counts of B. cenocepacia (following the killing of extracellular bacteria by gentamicin treatment). The macrophage-associated B. cenocepacia counts at 2 h postinfection were not significantly different between pretreatment with naive serum and pretreatment with anti-IHF serum (1:1000 dilution), suggesting equivalent uptake by macrophages at this time point. However, by 6 h postinfection, pretreatment with anti-IHF serum significantly prevented B. cenocepacia from multiplying within CF macrophages (p<0.05) (Fig. 6). Thus, pretreatment of B. cenocepacia with anti-IHF serum promoted its clearance by CF macrophages, but the mechanism underlying this finding remains to be determined and is the subject of ongoing research.

[0324] Demonstration of the association of the T3SS or T6SS with the integration of DNABII proteins into the B. cenocepacia biofilm matrix. To begin to elucidate the molecular mechanism by which B. cenocepacia integrates both eDNA and DNABII proteins into its biofilms, we incubated biofilms formed at 24 h by either B. cenocepacia strain K56-2 (parental isolate), B. cenocepacia strain JRL2 (ΔbcsV; type III secretion system mutant - T3SS), or B. cenocepacia strain DFA2 (ΔBcsK; type VI secretion system mutant - T6SS) with anti-IHF antibodies (Aubert et al. (2010) J. Biol. Chem. 285:35988-35998). In biofilms formed by the parental isolate, positive labeling was distributed throughout the biofilm, as previously described (Figure 7A). Biofilms formed by the T3SS mutants were generally less robust than those formed by the parental isolate (biomass: 4.5 μm / μm compared with 18 μm / μm for the parental isolate; mean thickness: 5.5 μm compared with 25.2 μm for the parental isolate). Labeling with anti-IHF serum was also present throughout the biofilm, although, as observed with the parental isolate, labeling appeared to be much stronger at the bottom of the biofilm (Figure 7B). Conversely, biofilms formed by the T6SS mutants showed significantly less labeling (Figure 7C), and these biofilms were also significantly less robust than those formed by the parental isolate (biomass: 2.9 μm / μm; thickness: 3.9 μm). This latter finding was unexpected, as the T6SS of B. cenocepacia is known to be involved in biofilm formation (Aubert et al. (2008) Infect. Immun. 76:1979-1991). However, taken together, these data suggested that export of DNABII proteins (and possibly eDNA as well) is dependent on the T6SS of B. cenocepacia .

[0325] Given the effect of T6SS mutants on the relative amounts of IHF and eDNA present in in vitro established biofilms (Figure 8), we wondered whether IHF might act intracellularly to affect transcription of the T6SS gene cluster (BCAL0340–BCAL0348; the BcsK gene is equivalent to BCAL0342) in B. cenocepacia. As a first step in this regard, we examined the upstream region of this gene cluster and identified the sequence TCTCAACGATTTA, which was a near-perfect match with the IHF-binding consensus sequence WATCAANNNNTTR (where W is A or T, N is any nucleotide, and R is A or G). In the presence of 50 nM E. coli IHF, a strong DNase footprint was visible (Figure 7D), targeting a region 25–52 bp upstream of the coding sequence of BCAL0340 and overlapping with the match with this consensus sequence. This result may indicate that IHF itself autoregulates its release and possibly the release of eDNA that is incorporated into the biofilm matrix.

[0326] Consideration Cystic fibrosis (CF) is a characteristic example of a chronic, persistent disease that is refractory to current treatment methods. Biofilms that reside within the lungs of CF patients contribute significantly to both pathogenesis and chronicity. Biofilms are highly organized multicellular communities coated with an extracellular polymeric matrix or substance (EPS) attached to inert or biological surfaces and are the preferred lifestyle of virtually all bacteria. Bacterial populations within biofilms, in contrast to their planktonic or free-living counterparts, have slower growth rates (due to nutrient limitation), distinct transcriptomes (Post et al. (2007) Curr. Opin. Otolaryngol. Head Neck Surg. 15:347-351; Post et al. (2004) Curr. Opin. Otolaryngol. Head Neck Surg. 12:185-190), and are substantially more resistant to antibiotic action as well as to effectors of innate and acquired immunity (Slinger et al. (2006) Diagn. Microbiol. Infect. Dis. 56:247-253). Furthermore, EPS provides a formidable physical barrier to phagocytes and other bacterial clearance mechanisms (both physical and physiological), making biofilms extremely difficult to eradicate (Flemming et al. (2010) Nat. Rev. Microbiol. 8:623-633). Therefore, diseases in which biofilms play a key role in pathogenesis and chronicity, such as CF, require novel treatment and prevention strategies. While the composition of biofilm EPS is highly variable among genera and influenced by the environment in which they are formed, a very common and important component is the incorporation of extracellular DNA (eDNA). While the mechanisms by which eDNA is released by microorganisms and / or incorporated into the biofilm matrix remain unclear for many pathogens, eDNA is nonetheless of great interest for both its biological functionality and its role as a structural component of biofilms.

[0327] We disclose herein that Burkholderia cenocepacia incorporates large amounts of eDNA into the biofilm it forms. The presence of this large amount of eDNA likely provides exceptional protection to resident B. cenocepacia as a physical barrier, and as we have recently shown, eDNA within biofilms can also bind to effectors of innate immunity (Jones et al. (2012) J. Innate Immun), thereby limiting or preventing access to bacterial cells within the biofilm. Specifically, with regard to B. cenocepacia, Peeters et al. (Peeters et al. (2008) J. Hosp. Infect. 70:361-368) showed that sessile B. cenocepacia exhibited high resistance to chlorhexidine, hydrogen peroxide, and 5% bleach, even after 5 minutes of treatment. Furthermore, an analysis of FDA product recall data for non-sterile pharmaceutical products from 1998 to 2006 showed that 48% of recalls were due to contamination with either B. cepacia, Pseudomonas species, or Ralstonia picketti (Jimenez (2007) PDA J. Pharm. Sci. Technol. 61:383-399). For both non-sterile and sterile products, B. cenocepacia was the most frequently isolated species. Collectively, these data indicate that B. cenocepacia contamination of surfaces, equipment, and pharmaceutical devices, likely in the form of biofilms, serves as a source of infection not only in CF patients but also in any hospitalized, mechanically ventilated, or immunocompromised patient (Graindorge et al. (2010) Diagn. Microbiol. Infect. Dis. 66:29-40; Lucero et al. (2011) Am. J. Infect. Control. 39:775-778), and / or immunocompromised patients (Vandamme et al. (1997) Int. J. Syst. Bacteriol. 47:1188-1200). These findings led us to develop novel immunotherapeutic strategies for CF patients, particularly those infected with B. cenocepacia.To do this, we focused on eDNA and a family of proteins known to bind to this extracellular DNA (DNABII proteins).

[0328] We disclose herein that B. cenocepacia incorporates large amounts of eDNA into its biofilms. This eDNA is associated with the DNABII protein, which interacts with antisera directed against isolated, native IHF produced by E. coli. When B. cenocepacia biofilms were examined to determine the spatial distribution of this DNABII protein, positive labeling was found to be associated with each intersection of eDNA strands present within the biofilm, as observed for NTHI (Goodman et al. (2011) Mucosal Immunol. 4:625-637). Biofilms produced by B. cenocepacia were sensitive to disruption by anti-IHF but not naive serum in an in vitro assay system. Furthermore, this disruptive effect of antisera directed against IHF rendered bacteria within B. cenocepacia-induced biofilms susceptible to the traditional, but usually ineffective, killing effects of several antibiotics used to treat CF patients. These antibiotics were ineffective or significantly less effective at killing B. cenocepacia in biofilms in vitro without treatment with anti-IHF serum. Anti-IHF serum disrupted B. cenocepacia biofilms by targeting the lynchpin protein responsible for bending and stabilizing eDNA into the lattice structure observed in these biofilms, whereas DNase was not found to be effective when incubated with B. cenocepacia biofilms in vitro. Indeed, unlike the reduction observed using biofilms formed by either NTHI (Gustave et al. (2012) J. Cyst. Fibros.) or P. aeruginosa (Whitchurch et al. (2002) Science 295:1487), treatment of B. cenocepacia biofilms with DNase induced the formation of significantly more robust biofilms, suggesting that DNase is contraindicated for the treatment of CF patients infected with B. cenocepacia.We also demonstrated that pretreatment of B. cenocepacia with antiserum directed against IHF likely facilitates the routing of ingested B. cenocepacia to more efficient degradation pathways within murine CF macrophages, as the killing of ingested B. cenocepacia treated with anti-IHF at 6 h postinfection was statistically significantly increased compared with that of ingested B. cenocepacia preincubated with naive serum. While the mechanism of this observation remains unknown, we hypothesize that binding of anti-IHF to bacterial cell-associated extracellular IHF may have played a role in this. Finally, to begin to elucidate the molecular mechanism by which B. cenocepacia incorporates both eDNA and DNABII proteins into its biofilms, we examined biofilms assembled by both T3SS and T6SS mutants of the K56-2 isolate used herein. While biofilms of both mutants were overall impaired in terms of relative biofilm robustness, biofilms assembled by the T6SS mutant lacked labeling with antisera directed against IHF, suggesting that secretion of this protein and robust biofilm formation depend on an active T6SS. Furthermore, we identified a putative IHF binding site immediately upstream of the T6SS gene cluster, suggesting that IHF may regulate the excretion of IHF itself. Indeed, formal transcriptional analysis of IHF-deficient mutants could further delineate the role of IHF in B. cenocepacia pathogenesis; IHF is known to be both part of the extracellular matrix and to regulate virulence factor expression in uropathogenic E. coli (Justice et al., 2012). The mechanisms underlying these findings are under further investigation.

[0329] Despite significant recent advances that allow for better management of CF patients, these patients can still only expect to survive into their mid-30s. At least 90% of CF patients die from respiratory failure after years of chronic, recurrent, and persistent bacterial lung infections. Bacterial residence within biofilms in the lungs of CF patients poses a formidable challenge. Therefore, to design novel and effective strategies to better treat and / or prevent the long-term bacterial infections characteristic of CF, it is necessary to understand both the unique biological properties of biofilms and determine how these structures can be weakened to mediate therapeutic or preventative "heals." Herein, we demonstrate that targeting bacterial proteins that stabilize eDNA present within biofilms is highly effective in reducing or eradicating biofilm structures in vitro. Furthermore, treatment of B. cenocepacia-induced biofilms with anti-IHF acted synergistically with multiple standard antibiotics, resulting in the susceptibility and death of resident bacterial cells. Finally, we discovered that pretreatment with antisera directed against B. cenocepacia IHF significantly inhibited its survival when ingested by murine CF macrophages. Based on the data obtained to date, the approach of targeting the DNABII protein associated with eDNA within B. cenocepacia biofilms promises to provide a promising novel approach for the treatment of CF patients, particularly those colonized with B. cenocepacia. Most importantly, given that multiple human pathogens appear to use a similar strategy in which eDNA within biofilms associates with members of the DNABII family of nucleic acid-binding proteins, the approach developed herein is likely to be useful for other airway pathogens whose proliferation often precedes B. cenocepacia infection of CF lungs (George et al. (2009) FEMS Microbiol. Lett. 300:153-164).

[0330] Experiment No. 2 Bacterial strains, biofilm formation, IHF, and serum NTHI 86-028NP is a minimally passaged clinical isolate cultured from the nasopharynx of a child undergoing tympanostomy tube placement for chronic otitis media. NTHI biofilm formation in 8-well chambered coverglass slides has been described (Jurcisek et al. (2011) J. Vis. Esp). For all biofilm assays, duplicate wells were observed with a Zeiss 510 Meta laser scanning confocal microscope. Images were compiled with Zeiss Zen software, and biomass and / or mean biofilm thickness values were calculated using COMSTAT2 software (Heydorn et al. (2000) Microbiology 146(Pt10):2395-2407). All biofilm assays were repeated a minimum of three times on separate days. Data are presented as mean ± SEM.

[0331] Purified E. coli IHF and rabbit antiserum directed against purified E. coli IHF ("anti-IHF") E.coli ") was kindly provided by Howard Nash (Granston et al. (1993) J. Mol. Biol. 234:45-59; Rice et al. (1996) Cell 87:1295-1306). Naive rabbit serum was purchased from Spring Valley Laboratories.

[0332] Quantification of IHF-specific IgG IHF-specific IgG was purified from polyclonal serum using a HiTrap Protein G HP column (GE Healthcare). Polyclonal and IgG-enriched anti-IHF E.coli IHF-specific IgG in both purified IHF vs. slot blots E.coli A standard curve was generated using rabbit reference serum versus purified rabbit IgG (Bethyl Laboratories, Inc.), and band intensities were analyzed using AlphaView software (ProteinSimple).

[0333] Degradation of mature biofilms Biofilms were allowed to establish for 24, 48, and 96 hours, or for 1 and 2 weeks. To maintain bacterial survival, the medium (2 μg / ml -1 The medium (brain heart exudate broth supplemented with β-NAD and heme) was changed twice daily. E.coli (50-fold dilution (4.4 μg IHF-specific IgG / well for biofilms ≤48 h) -1 equivalent to 10.0 μg IHF-specific IgG / well for biofilms over 96 hours -1 After 16 hours, biofilms were stained with the BacLight™ Bacterial Viability Kit (Molecular Probes), fixed in a solution of 1.6% paraformaldehyde, 2.5% glutaraldehyde, and 4.0% acetic acid in 0.1 M phosphate buffer, and observed as described.

[0334] Biofilm degradation kinetics Biofilms established for 24 h were incubated with medium or anti-IHF. E.coli After incubation with either 1:50 dilution of naive serum or 1:50 dilution of naive serum for 0, 6, 12, 16, or 24 hours, viability staining and fixation were performed as described. For 0 hours, treatment was applied and then immediately removed. To maintain bacterial viability of the 24-hour-treated biofilms, the treatment was replaced after 16 hours and incubated for an additional 8 hours. To completely eradicate the 24-hour biofilms, anti-IHF was used. E.coli Alternatively, an equal volume of a 5-fold dilution of naive serum was applied.

[0335] Inhibition of direct contact between anti-IHF antibodies and NTHI biofilms IgG enriched anti-IHF E.coli was covalently coupled to agarose beads (diameter >45 μm) using the AminoLink Plus kit (Thermo Scientific). E.coliTo determine whether direct contact of antibodies with biofilms is necessary to induce degradation, biofilms were established in optical-bottom 96-well plates for 24 hours and then incubated with medium, anti-IHF, or IgG. E.coli or incubated with an equal volume of naive serum and applied directly to the biofilms (total volume 80 μl) or after insertion into 96-well plates of 5 μm-pore-sized HTS Transwell (Corning) plates, medium, 0.5 μg, 5.0 μg, or 50.0 μg of IgG-enriched anti-IHF covalently bound to agarose beads. E.coli An equal volume of IgG-enriched naive serum bound to agarose beads was placed in the apical chamber (total volume 80 μl). Biofilms were incubated for 16 h and then processed as described. To confirm that IHF-specific antibodies had not diffused into the basolateral chamber, supernatants from the basolateral chamber were collected and analyzed by Western blotting to identify purified IHF. E.coli The antibodies were assayed for reactivity against

[0336] anti-IHF E.coli To determine whether biofilm degradation by IHF could be prevented by steric hindrance, 80 μl of bare agarose beads was added to the apical chamber, and after 1 h, 50 μg of IgG-enriched anti-IHF bound to beads was added. E.coli Or an equivalent volume of IgG enriched from naive serum coupled to beads was overlaid. Plates were incubated for an additional 16 hours, and then biofilms were stained, observed, and analyzed as described.

[0337] To determine whether the ability of IHF-specific antibodies to sequester free IHF is limited by relative accessibility, 50.0 μg of IgG-enriched anti-IHF conjugated to beads was used. E.coliAlternatively, an equal volume of IgG-enriched naive serum bound to beads was applied to the apical chamber of a transwell whose basolateral chamber contained a 24-h NTHI biofilm. After 6 h, the contents of the apical chamber were mixed by vortexing and incubated for an additional 10 h. Biofilms were then stained, observed, and analyzed as described.

[0338] anti-IHF E.coli Adsorption of anti-IHF E.coli To abolish the IHF-mediated biofilm weight loss, IHF-specific antibodies were used to treat purified IHF. E.coli Anti-IHF was adsorbed from serum by incubation with E.coli An aliquot of 4.4 μg of IHF-specific IgG was added to either 2.2 μg or 4.4 μg of purified IHF. E.coli , saline dilutions, or a recombinant protein of equivalent molecular mass called "rsPilA" (Novotny et al. (2009) PLoS One 8:e67629) for 1 hour. Western blots were performed with anti-IHF. E.coli The adsorption of anti-IHF was confirmed. E.coli To assess the functional significance of adsorption, the adsorbed serum was then applied to NTHI biofilms for 24 hours following standard treatment and processing protocols.

[0339] anti-IHF E.coli and synergistic effects of antibiotics on NTHI biofilms To visualize changes in NTHI biofilm viability upon exposure to antibiotics typically used to treat NTHI infection, we established biofilms for 24 h and then treated them with anti-IHF. E.coli or a 50-fold dilution of naive serum, ampicillin (32.0 μg / ml -1 ), cefdinir (0.25μg / ml -1 ), or amoxicillin (1.0 μg / ml -1 ) + clavulanate-lithium (0.5 μg / ml -1) for 16 hours. Each antibiotic was tested to determine the MIC of planktonic NTHI as determined by standard broth microdilution method. 90 (Biedenbach et al. (2003) Diagn. Microbiol. Infect. Dis. 46:55-61; Tristam et al. (2007) Clin. Microl. Rev. 20:368-389).

[0340] To quantify NTHI adherence within biofilms and newly released bacteria into planktonic forms after treatment, 24-hour biofilms were analyzed by MIC. 90 The biofilms were incubated with each antibiotic or its 4-fold or 8-fold dilutions with or without antisera. To culture the newly released NTHI, the supernatant was collected by aspiration, the biofilms were gently washed twice with sterile saline to remove loosely adherent bacteria, and NTHI within the biofilms was collected by repeated vigorous pipetting. Planktonic and adherent bacteria were plated separately to determine CFU NTHI / ml. -1 were determined and these values were combined to establish total CFU bacteria as shown. Data are shown as the mean ± SEM of three independent assays.

[0341] Anti-IHF against planktonic NTHI E.coli and antibiotic synergy NTHI was prepared as described (Jurcisek et al. (2011) J. Vis. Exp.) and 10 6 CFU NTHI was inoculated into the wells of a 96-well plate, and the MIC 90 antibiotics or their 4-fold or 8-fold dilutions and anti-IHF E.coli Or incubated with or without an equal volume of 1:50 dilution of naive serum. After 16 hours, cultures were serially diluted and plated onto chocolate agar to semiquantitative CFU NTHI / well. Data are shown as the mean ± SEM of three independent assays.

[0342] Epitope mapping of NTHI IHF To identify immunodominant regions within IHF, a series of 12 five-residue overlapping 20-mer synthetic peptides were synthesized from IHF predicted to be expressed by the NTHI strain 86-028NP ("IHF"). NTHI The peptides were synthesized to mimic the N- to C-terminus of the α-subunit of the β-lactamase inhibitor β-lactamase (IHF). Synthesis, purification, and sequence verification of all synthetic peptides were performed by Ohio Peptide, LLC. E.coli or IHF pre-bound to excess double-stranded DNA E.coli An archived sample of polyclonal sera collected from chinchillas immunized with either IHF or IHF (Goodman et al. (2011) Mucosal. Immunol. 4:625-637) was used to map the immunodominant epitopes of IHF. NTHI Interactions between the synthetic peptides and antibodies present in chinchilla serum were analyzed using a Biacore 3000 (GE) as described. Analysis was performed using the IHF (IHF Healthcare) (Novotny et al. (2000) Infect. Immun. 68:2119-2128; Novotny et al. (2009) Vaccine 28:279-289). NTHI The reactivity of chinchilla serum to the peptides was depicted using PyMol software (Schroedinger) to generate a 3D model image.

[0343] IHF for disrupting NTHI biofilms NTHI Evaluation of epitope-specific antisera Based on the results from the epitope mapping study, IHF NTHI Two regions within the α-subunit were selected to generate the following polyclonal chinchilla antisera: IhfA-3 NTHI (non-reactive region) and IhfA-5 NTHI (IHF E.coli It reacts with antibodies against IHF, but not with anti-IHF pre-bound to DNA. E.coli NTHI biofilms established for 24 hours were treated with a 1:50 dilution of the following chinchilla sera: anti-IHF; E.coli, anti-IHF pre-bound to DNA E.coli , naive serum, anti-IhfA-3 NTHI , or anti-IhfA-5 NTHI Experiments on animals were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and under protocols approved by the Animal Care and Use Committee at Nationwide Children's Hospital.

[0344] statistical analysis Statistical analysis was performed using GraphPad Prism software. Biofilm biomass and thickness were compared using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test set at 5%. Significant differences in CFU NTHI after treatment were determined by one-way ANOVA followed by a Holm-Sidak test for multiple comparisons; a p value of 0.05 or less was considered significant.

[0345] result Anti-IHF-induced disassembly of NTHI biofilms In a previous study, polyclonal rabbit antiserum against E. coli IHF (i.e., “anti-IHF”) was used at a 1:50 dilution. E.coli ") (Used at a 50-fold dilution, 4.4 μg IHF-specific IgG / ml -1 (equivalent to Mucosal ) has been shown to disrupt NTHI biofilms in vitro for 24 hours (Goodman et al. (2011) Mucosal Immunol. 4:625-637). Next, we investigated the effect of anti-IHF on both early-formed and mature NTHI biofilms. E.coli Biofilms formed 16, 24, 48, or 96 hours and 1 or 2 weeks before treatment were shown to be anti-IHF. E.coli While the residual biofilm was significantly reduced after incubation with IHF, the biofilms exposed to naive serum were comparable to those maintained in culture (Figure 10A). E.coliThe biomass of 16-, 24-, and 48-h biofilms exposed to IHF was significantly reduced by 94%, 86%, and 74%, respectively, compared to naive serum (p<0.05, Figure 10B). To achieve a similar effect on more mature biofilms, anti-IHF was added. E.coli was diluted 10-fold. As a result, the biomass of 96-hour, 1-week, and 2-week biofilms was significantly reduced by 74%, 43%, and 57%, respectively, compared to naive serum (p<0.01 or 0.05). As NTHI biofilms mature in vitro, the relative amount of eDNA increases (Jones et al. (2013) J. Innate Immun. 5:24-38); therefore, it was not expected that higher concentrations of IHF-specific antisera would be required for similar reductions in biomass of these "older" and denser biofilms. Taken together, these data support the anti-IHF E.coli was significantly effective and demonstrated the ability to destroy both early-forming and mature NTHI biofilms.

[0346] The kinetics of anti-IHF-mediated disruption was then examined. Biofilms were allowed to establish for 24 hours and then treated with either medium or a 50-fold dilution of anti-IHF for 0, 6, 12, 16, or 24 hours. E.coli Treatment with either IFN-γ or naïve serum did not induce a change in biofilm biomass compared to the medium (Figures 11A and 11B). E.coli Biofilms exposed to 100% IHF-1000 for 6, 12, 16, or 24 hours showed a 76%, 43%, 65%, and 67% reduction in biomass compared to naive serum (p<0.01), with the greatest reduction observed at 6 hours. The moderate effect of naive serum from non-SPF animals compared to biofilms maintained in culture has previously been reported (Goodman et al. (2011) Mucosal Immunol. 4:625-637; Novotny et al. (2013) PLoS One 8:e67629). We next compared the biofilms exposed to higher concentrations of anti-IHF-1000. E.coliWe also tested whether prolonged exposure to NTHI biofilms could further reduce or eradicate pre-formed biofilms. E.coli Incubation with anti-IHF resulted in an 86% reduction in biomass compared to naive serum (p<0.01; Figures 11A and 11B). E.coli This result appears to be maximal, as extending the treatment time to 24 hours or 30 days did not completely eradicate all viable bacteria. In both cases, monolayers of bacteria remained after treatment, and anti-IHF antibodies were present in these monolayers. E.coli This suggests that there was no target for

[0347] Direct contact with anti-IHF was not required to disrupt NTHI biofilms. So far, anti-IHF was applied directly to NTHI biofilms. E.coli To determine here whether direct contact between the NTHI and the biofilm is necessary, NTHI biofilms were established in the basolateral chamber of a transwell. IgG-enriched anti-IHF antibodies covalently bound to agarose beads were used. E.coli The antibody was placed in the apical chamber, thus physically separating the antibody from the biofilm due to the presence of the membrane. We demonstrated the antibody-IHF coupled to agarose beads by Western blot analysis, collecting the medium in the basolateral chamber 24 hours after placing the antibody-coupled beads in the apical chamber of the transwell. E.coli We first confirmed that IHF did not diffuse into the basolateral chamber (Figure 18). E.coli Compared to direct application of IHF to the biofilm (Figure 12A), anti-IHF tethered to agarose beads in the apical chamber of the transwell E.coliBiofilm reduction in the presence of IgG was equivalent (Figure 12C), with a significant reduction in biomass (p<0.05) observed compared to tethered IgG from naive serum (Figures 12B and 12F). Three concentrations of antibody coupled to agarose beads were assayed; all were effective, and no dose-dependent biofilm disruption was observed. These data suggested saturation of available antibody binding sites at the interface between the biofilm medium and the transwell membrane. To test this theory, a layer of bare agarose beads was placed in IHF. E.coli The antibody was placed under the bound layer, thus sterically blocking its ability to bind to "free" IHF. As expected, no biofilm disruption was observed (Figures 12D and 12F). However, biofilm disruption was restored upon mixing of the naked beads and the anti-IHF-bound antibody (Figure 12E). Taken together, these data support the role of anti-IHF in the biofilm destruction. E.coli demonstrated that direct contact with the NTHI biofilm was not necessary to mediate disruption, and furthermore, this disruption was likely mediated by a forced equilibrium shift, as free IHF was captured by the antibody when the protein was naturally dissociated from eDNA within the biofilm (i.e., in the “off” state).

[0348] To demonstrate that the observed biofilm disruption was specifically mediated by antibodies directed against IHF and not due to the influence of other components within the hyperimmune rabbit serum (since this serum was not heat-inactivated before use), anti-IHF antibodies were used. E.coliAliquots of the samples were adsorbed using either purified IHF (i.e., as a negative control) or an NTHI protein of similar molecular mass called rsPilA (Novotny et al. (2009) Vaccine 28:279-289). We first confirmed that the arbitrarily selected 50-fold dilutions of antisera utilized in the biofilm assays were equivalent to applying 4.4 μg of IHF-specific IgG to each biofilm. Western blotting revealed a decrease in IHF-specific antibody reactivity after incubation with increasing concentrations of purified IHF (Figure 19). This result was specific, as no decrease in reactivity was observed after incubation with an unrelated recombinant NTHI protein. Furthermore, incubation of biofilms with IHF-adsorbed serum reduced their ability to effectively disrupt preformed biofilms compared with whole serum (Figures 13A and 13B). These data demonstrated that the observed NTHI biofilm disruption was mediated by IHF-specific antibodies within the polyclonal rabbit serum.

[0349] Synergistic effects of anti-IHF antibiotics We next investigated the anti-IHF E.coli We tested whether biofilm disruption by α-glucan increases the susceptibility of biofilm-resident bacteria to killing by antibiotics commonly used to treat NTHI infections. The minimum inhibitory concentration (MIC) required to inhibit 90% of planktonic NTHI growth was calculated. 90 ) was determined as previously described (Tristram et al. (2007) Clin. Microbiol. Rev. 20:368-389) and the following concentrations were used: ampicillin (32 μg / ml -1 ), amoxicillin-clavulanate (each 1 μg / ml -1 and 0.5 μg / ml -1 ), and cefdinir (0.25 μg / ml -1 24-hour NTHI biofilms were then incubated with 50-fold diluted anti-IHF E.coli , antibiotics, or anti-IHF E.coli+ exposure to a combination of antibiotics. As expected, anti-IHF E.coli mediated significant biofilm disruption (Fig. 14A), but the MIC of planktonic NTHI 90 Incubation with any of the three antibiotics at 100°C did not kill the bacteria as determined by viability staining (Figures 14B-14D), and the mean biofilm thickness and mean biomass were similar between medium and antibiotics alone (Figure 14E). Notably, anti-IHF activity of established NTHI biofilms was significantly reduced. E.coli Treatment with anti-IHF in combination with any of the three antibiotics induced significant changes in biofilm structure and a statistically significant decrease in mean biomass (p ≤ 0.05) compared with treatment with antibiotics alone. E.coli + Cell death was observed within biofilms treated with any of the three antibiotics. These data suggested that anti-IHF-mediated disruption of NTHI biofilms rendered resident bacteria more susceptible to the action of previously ineffective antimicrobial agents.

[0350] To expand this line of investigation beyond the descriptive data of physical disruption of preformed biofilms, anti-IHF E.coli We tested whether exposure to planktonic NTHI increased antibiotic-mediated killing of newly released bacteria. To do so, we administered three targeted antibiotics at MICs of 1000 to 1000 of planktonic NTHI. 90 (Biedenbach et al. (2003) Diagn. Microbiol. Infect. Dis. 46:291-294; Tristram et al. (2007) Clin. Microbiol. Rev. 20:368-389) and assayed at both 4-fold and 8-fold dilutions. 90 Adherent colony forming units (CFU) bacteria / ml compared to medium when incubated with any of the three antibiotics -1 No significant difference was observed between the anti-IHF and anti-IHF groups (Figs. 15A to 15C). E.coliThe addition of anti-IHF significantly reduced the number of NTHI cells that remained attached to the chamber slides compared to medium (p<0.05) (Goodman et al. (2011) Mucosal Immunol. 4:625-637). E.coli MIC combined with 90 The use of any antibiotic induced an even further significant decrease in the number of adherent bacteria compared to medium alone (p<0.05). This decrease was observed at 1 / 8 the ampicillin concentration (4 μg / ml -1 ) and amoxicillin-clavulante at 1 / 8 the concentration (0.125 μg / ml each) -1 and 0.0625 μg / ml -1 ) and 1 / 4 of the cefdinir concentration (0.0625 μg / ml -1 ) was maintained.

[0351] Bacterial anti-IHF in both adherent and planktonic populations E.coli To demonstrate biofilm destruction through enhanced mediated killing, anti-IHF E.coli The total viable bacteria / well after treatment with each antibiotic delivered with or without anti-IHF (Figures 16D-16F) was tested. E.coli MIC in combination with a 50-fold dilution of 90 The use of either antibiotics or their 4-fold or 8-fold dilutions significantly reduced the total viable CFU NTHI / chamber slide well. In all cases, these differences were consistent with the use of antibiotics alone, anti-IHF, or anti-IHF. E.coli This was significant compared to treatment with antibiotics alone or antibiotics plus naive serum (p<0.05).

[0352] To determine whether newly released NTHI, or NTHI presumably associated with disrupted biofilms at low stringency, would normally be susceptible to these antibiotics, NTHI broth cultures were treated with anti-IHF antibodies delivered alone or in the presence of ampicillin, amoxicillin / clavulanate, or cefdinir. E.coliAntibiotics were administered at MICs of planktonic NTHI. 90 and its 4-fold and 8-fold dilutions. E.coli Antibiotic-mediated killing was not enhanced upon exposure to anti-IHF alone (Figures 20A-20C). E.coli These results suggest that NTHI newly released from biofilms by this action are phenotypically distinct from either their biofilm or planktonic counterparts.

[0353] Identification of immunodominant regions within IHF In previous studies, native IHF E.coli Immunization of chinchillas with IHF induced antibody formation and demonstrated rapid degradation of established NTHI biofilms in the chinchilla middle ear during experimental OM. However, IHF pre-complexed with DNA was not shown to be effective. E.coli Immunization with IHF (a form likely naturally occurring during disease) did not reverse the disease (Goodman et al. (2011) Mucosal Immunol. 4:625-637). Collectively, the data to date imply that there is a conserved domain (IHF and HU) within the DNABII proteins of many bacteria that can be targeted for effective disruption of biofilm structure, and that this domain is masked or occluded when IHF / HU associate with DNA. To determine the location of this effective / masked domain, we investigated the IHF ("IHF") from NTHI. NTHI The DNABII protein was epitope mapped using a series of overlapping 20-mer peptides designed to mimic the predicted N- to C-termini of the α-subunit of the DNABII protein. These peptides were then subjected to epitope mapping using native IHF. E.coli or IHF complexed with excess DNA E.coli Screening was performed using antisera collected from chinchillas immunized with either native IHF or denatured IHF (Goodman et al. (2011) Mucosal Immunol. 4:625-637). E.coliThe antiserum against IHF showed reactivity to a peptide predicted to represent the DNA-binding front region (Fig. 16A). E.coli IHF with antiserum against the -DNA complex NTHI The highest reactivity was obtained with a peptide representing the N-terminal tail of IHF (Figure 16B). E.coli This result was logical because the DNA-binding tip region is likely to be occluded when the ribosomal protein binds to DNA, and therefore the tip-binding region is expected to be immunologically inaccessible, whereas the tail region is exposed.

[0354] Having revealed specific regions within the Ihf molecule to which serum antibodies reacted through our epitope mapping studies, we next sought to determine whether antibodies directed against this targeted epitope would be as effective as antibodies directed against the native protein. To this end, we selected the following two peptides for generating immune sera in chinchillas: peptide IhfA-5 NTHI (showing a reactive DNA tip-binding region within the α-subunit of IHF) (Figure 16D) and as a negative control, IhfA-3 NTHI (showing a peptide of the same size but shown to be non-reactive by epitope mapping) (Figure 16D). Purified IHF E.coli and IHF precomplexed with DNA E.coli was used as a comparative immunogen. As expected, the anti-IHF complexed with DNA was significantly higher than that of the native chinchilla serum. E.coli NTHI biofilms incubated with either IhfA-3 or IhfA-3 NTHI did not change biofilm morphology or biomass (Figures 16E and 16F). E.coli Similar to biofilms observed using anti-IhfA-5 NTHI Incubation with sera was equally effective in inducing a significant reduction (p<0.01) in biofilm biomass compared to naive serum.

[0355] Consideration Bacterial biofilms contribute significantly to most recurrent and chronic bacterial diseases, including those of the respiratory tract, genitourinary tract, and oral cavity. Biofilms are refractory to the host immune system and antimicrobial agents, making it necessary to develop novel treatment methods for diseases with biofilm components. eDNA is a common component of the biofilm EPS of many microorganisms, and we have previously demonstrated that members of the DNABII family of proteins play an important role in stabilizing biofilm structure, as exposure of biofilms to antibodies directed against IHF mediates significant disruption (Goodman et al. (2011) Mucosal Immunol. 4:625-637).

[0356] As NTHI biofilms mature, the concentration of eDNA within the EPS increases (Jones et al. (2013) J. Innate Immun. 5:24-38), and by corollary, the concentration of the correspondingly associated DNABII protein increases relative to it. Thus, we demonstrate herein that older biofilms require higher concentrations of anti-IHF to mediate their destruction. E.coli Anti-IHF antibodies were shown to be necessary. E.coli The ability of anti-IHF to disrupt established 24-hour NTHI biofilms was rapid, with maximal effects within 6 hours of exposure (76% reduction in biomass and 71% reduction in mean thickness compared to naive serum), with no further disruption occurring after an additional 24 hours of incubation using a single treatment. E.coli Regardless of the relative increase in antibody concentration or exposure time, complete eradication of NTHI could not be achieved. Instead, a monolayer of viable bacteria persisted, suggesting that in the absence of EPS containing eDNA and IHF, there is no target for anti-IHF-directed therapy.

[0357] Therefore, a combination approach would be ideal, likely allowing existing antibiotics or other therapeutic agents to be used to reverse these diseases.

[0358] To better define the mechanism of action, biofilm and anti-IHF E.coli We investigated whether direct contact between antibodies is required for biofilm disruption, since it has previously been assumed that such a requirement is not true (Goodman et al. (2011) Mucosal Immunol. 4:625-637). As expected, separation of agarose bead-anchored antibodies from biofilms by microporous membranes did not inhibit biofilm disruption, suggesting that direct contact is not required. Instead, taken together, the data suggest that free IHF dissociated from eDNA within biofilms as part of the normal equilibrium between IHF and DNA, resulting in IHF E.coli showed that antibodies directed against IHF captured free IHF. Indeed, epitope mapping experiments pointed to competitive inhibition of the DNA-binding domain of IHF as the site of antibody action. Thus, the presence of excess antibodies against IHF shifts this equilibrium and mediates the disruption or destruction of the biofilm structure.

[0359] Because treatment of biofilms with anti-IHF mediates the release of bacteria into the planktonic phase (Goodman et al. (2011) Mucosal Immunol. 4:625-637), anti-IHF E.coli We tested whether IFN-γ-α can act in combination with traditional antibiotics to enhance their killing ability. We determined that this was indeed true for three antibiotics traditionally used to treat treatment-resistant airway infections caused by NTHI. We also investigated the anti-IFN-γ-α (IFN-γ) activity of established biofilms. E.coli Treatment with antibiotics made the resident bacteria susceptible to killing. Furthermore, these newly released bacteria were also susceptible to killing, and this killing was due to the anti-IHF E.coli All three tested antibiotics were found to be over MICs of planktonic cells. 90These compounds were able to mediate killing when used at concentrations ¼ to ⅛ times lower than the original concentration, thus demonstrating true synergy. These findings also suggested the possibility of a unique phenotype for NTHI newly released from biofilm growth compared with either NTHI living within biofilms or NTHI growing planktonically. Importantly, similar findings were obtained with S. pneumoniae in a pioneering study by Anders Hakansson, who found that pneumococci released from biofilms, when mediated by several treatments, had a unique transcriptome and increased virulence compared with both bacteria growing as biofilms and planktonic bacteria grown in nutrient-rich media (Marks et al. (2013) MBio.4).

[0360] Taken together, these data support a model to explain the mechanism by which antisera to IHF induce the disruption of established biofilms. Exposure of biofilms to anti-IHF induces an equilibrium shift between IHF molecules bound to eDNA within (or "on") the biofilm and IHF molecules in an "off" state. Free IHF molecules are removed into the surrounding aqueous environment, forcing the bound IHF to dissociate from the biofilm eDNA, thus mediating the breakdown of the biofilm structure. These findings demonstrate the feasibility of targeting molecules critical to biofilm integrity for the treatment of multiple diseases with a biofilm component.

[0361] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All nucleotide sequences presented herein are presented in the 5' to 3' orientation.

[0362] The present invention illustratively described herein can suitably be practiced in the absence of any element or elements, or any limitation or limitations, not specifically disclosed herein. Thus, for example, terms such as "comprises," "includes," and "contains" are intended to be read expansively and without limitation. In addition, the terms and expressions used herein are used for descriptive purposes, not for limiting purposes, and no attempt is made to use such terms and expressions to exclude any equivalents of the shown and described features or portions thereof, recognizing that various modifications are possible within the scope of the invention as claimed.

[0363] Thus, while the present invention has been specifically disclosed in terms of preferred embodiments, it is to be understood that the optional features, modifications, improvements, and variations disclosed herein may be recovered by those skilled in the art, and that such modifications, improvements, and variations are considered to be within the scope of the present invention. The materials, methods, and examples presented herein are indicative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention.

[0364] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within this generic disclosure also form part of the invention. This includes generic descriptions of the invention with a qualification or negative limitation excluding any subject matter from the genus, regardless of whether the excluded material is specifically recited herein.

[0365] Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member of the Markush group or subgroup of members of the Markush group.

[0366] While the present invention has been described in conjunction with the above embodiments, it should be understood that the foregoing descriptions and examples are intended to be illustrative of the invention and not to limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. [Table 1-1] [Table 1-2] [Table 1-3] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 3-1] [Table 3-2] [Table 3-3] [Table 4-1] [Table 4-2]

Claims

1. An antibody that specifically recognizes and binds to the amino acid sequence RPGRNPKTGDVVPVSARRVV (sequence number 347).

2. The antibody described in claim 1, wherein the antibody is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof.

3. An antibody described in claim 1 or 2 conjugated to a detectable label.

4. A hybridoma cell line that produces the monoclonal antibody described in claim 2.

5. An isolated polynucleotide encoding an antibody described in any one of claims 1 to 3.

6. A composition comprising one or more of the antibodies described in any one of claims 1 to 3, the hybridoma cell line described in claim 4, or the isolated polynucleotide described in claim 5.

7. The composition described in claim 6, further comprising a carrier.

8. The composition described in claim 7, wherein the carrier is a pharmaceutically acceptable carrier.

9. A composition described in any one of claims 6 to 8, further comprising a detectable label.

10. A composition for interfering with or preventing the binding of microbial DNA to DNABII protein, comprising one or more of the antibodies described in any one of claims 1 to 3, a hybridoma cell line described in claim 4, an isolated polynucleotide described in claim 5, or a composition described in any one of claims 6 to 9.

11. The composition of claim 10 for use in vitro, in vivo or ex vivo.

12. A kit for diagnostic or therapeutic use, comprising one or more of the antibodies of any one of claims 1 to 3, the hybridoma cell line of claim 4, the isolated polynucleotide of claim 5, or the composition of any one of claims 6 to 9.

13. A kit according to claim 12 for use in therapy, comprising an antibody that specifically recognizes and binds to an amino acid sequence consisting of RPGRNPKTGDVVPVSARRVV (sequence number 347).

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