Peptides and antibodies for removing biofilms

Abstract

To provide peptides and antibodies for removing biofilm.SOLUTION: The disclosure provides an isolated or recombinant polypeptide that is useful to vaccinate individuals suffering from a chronic / recurrent biofilm disease or as a therapeutic drug for those with an existing infection. The individual's immune system will then naturally generate an antibody which will prevent or clear these bacteria from the host by interfering with the construction and / or maintenance of a functioning and protective biofilm. Alternatively, an antibody against the polypeptide can be administered to treat or prevent infection. Bacteria released from the biofilm by the technology made by the present inventors are more readily cleared by the remainder of the host's immune system.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under U.S.C. §119(e) to U.S. Provisional Patent Application Nos. 62 / 199,957, filed July 31, 2015, 62 / 387,562, filed December 23, 2015, and 62 / 361,400, filed July 12, 2016, the contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates generally to methods and compositions for reducing and / or curing bacterial biofilms and treating biofilm-associated disorders or diseases.

[0003] Statement of Government Support This invention was made with government support under Contract No. R01DC011818 awarded by the National Institute of Deafness and Communication Disorders (NIDCD) of the National Institutes of Health. The government has certain rights in this invention.

[0004] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on July 28, 2016, is named 106887-0360_SL.txt and is 73,105 bytes in size. [Background technology]

[0005] At least one protein from the DNABII family of proteins is found in all known eubacteria and is naturally found on the outside of bacterial cells. These elicit a strong innate immune response, but host subjects are unable to naturally produce specific protective antibodies against family members as a result of infection. DNABII proteins and extracellular DNA (eDNA) contribute to the lattice structure of a "biofilm." A major problem with bacterial biofilms is the inability of the host immune system and / or antibiotics and other antimicrobial agents to access the bacteria protected within the biofilm.

[0006] Biofilms also exist in industrial settings. For example, biofilms are associated with a wide range of petroleum processing problems, from production sites to gas station storage tanks. In the field, sulfate-reducing biofilm bacteria produce hydrogen sulfide (soured oil). In processing pipelines, biofilm activity creates slime that clogs filters and openings. Biofilms and biofilm organisms also cause corrosion in pipelines and petroleum processing equipment. These problems can manifest throughout oil or gas production facilities, to the point where fouling and corrosive biofilm organisms are found on the surface of the end product in storage tanks.

[0007] In homes, biofilms are found in or on any surface that supports microbial growth, such as in drains, on countertops, in toilets, and in swimming pools and spas. Biofilms are implicated in a wide range of water processing applications, both domestic and industrial. Biofilms can grow on the surfaces of processing equipment and impair its performance, such as reducing heat transfer efficiency or clogging filters and membranes. Biofilms growing on cooling tower fill can add enough weight to cause the fill to collapse. Biofilms can corrode even highly specialized stainless steel. Biofilms in water processing, such as biofilm contamination in paper processing or the attachment of even single cells to silicon chips, can degrade the value of the final product. Biofilms growing in drinking water distribution systems can harbor potential pathogenic, corrosive, or bacterial organisms that degrade the aesthetic quality of the water.

[0008] partial sequence table SEQ ID NO: 1: Full length wild type (wt) 86-028NP Haemophilus influenzae IhfA; Genbank accession number: AAX88425.1 (last accessed March 21, 2011): [ka]

[0009] SEQ ID NO: 2: Full length wild-type (wt) 86-028NP Haemophilus influenzae IhfB; Genbank accession number: AAX88699.1 (last accessed May 13, 2015): [ka]

[0010] SEQ ID NO: 3: Full length wt86-028NP Haemophilus influenzae HU; Genbank accession number: YP_248142.1 (last accessed March 21, 2011): [ka]

[0011] SEQ ID NO: 4: Full length wtR2846 Haemophilus influenzae IhfA; Genbank accession number: ADO96375 (last accessed March 21, 2011): [ka]

[0012] SEQ ID NO: 5: Full length wtRd Haemophilus influenzae IhfA; Genbank accession number: AAC22959.1 (last accessed March 21, 2011): [ka]

[0013] SEQ ID NO: 6: Full length wt E. coli K12 IhfA; Genbank accession number: AAC74782.1 (last accessed March 21, 2011): [ka]

[0014] SEQ ID NO: 7: Full length wt E. coli K12 IhfB; Genbank accession number: BAA35656 (last accessed May 19, 2015): [ka]

[0015] SEQ ID NO: 8: E. coli hupA; Genbank accession number: AP_003818 (last accessed March 21, 2011): [ka]

[0016] SEQ ID NO: 9: E. coli hupB; Genbank accession number: AP_001090.1 (last accessed March 21, 2011): [ka]

[0017] SEQ ID NO: 10: Full length wtP. aeruginosa PA 01 IhfA; Genbank accession number: AAG06126.1 (last accessed March 21, 2011): [ka]

[0018] SEQ ID NO: 11: Full length wtP. aeruginosa PA 01 IhfB; Genbank accession number: AAF72950.1 (last accessed May 19, 2015): [ka]

[0019] SEQ ID NO: 12: Haemophilus influenzae IhfA, A-3 fragment: [ka]

[0020] SEQ ID NO: 13: Haemophilus influenzae IhfA, A5 fragment: [ka]

[0021] SEQ ID NO: 14: Haemophilus influenzae HU, A5 fragment: [ka]

[0022] SEQ ID NO: 15: Haemophilus influenzae IhfB, B2 fragment: [ka]

[0023] SEQ ID NO: 16: Haemophilus influenzae IhfB, B4 fragment: [ka]

[0024] SEQ ID NO: 17: Haemophilus influenzae IhfB, modified B4 (mB4) fragment: [ka]

[0025] SEQ ID NO: 18: Haemophilus influenzae IhfA, A-1 fragment: [ka]

[0026] SEQ ID NO: 19: Haemophilus influenzae IhfA, A2 fragment: [ka]

[0027] SEQ ID NO: 20: Haemophilus influenzae IhfA, A4 fragment [ka]

[0028] SEQ ID NO: 21: Haemophilus influenzae IhfA, A6 fragment: [ka]

[0029] SEQ ID NO: 22: Haemophilus influenzae IhfB, B1 fragment: [ka]

[0030] SEQ ID NO: 23: Haemophilus influenzae IhfB, B3 fragment: [ka]

[0031] SEQ ID NO: 24: Haemophilus influenzae IhfB, B5 fragment: [ka]

[0032] SEQ ID NO: 25: Haemophilus influenzae IhfB, B6 fragment: [ka]

[0033] SEQ ID NO: 26: Haemophilus influenzae IhfA, A distal fragment: [ka]

[0034] SEQ ID NO: 27: Haemophilus influenzae IhfB, B tip fragment: [ka]

[0035] SEQ ID NO: 28: Haemophilus influenzae HU fragment: [ka]

[0036] SEQ ID NO: 29: Haemophilus influenzae HU fragment: [ka]

[0037] SEQ ID NO: 30: Haemophilus influenzae HU fragment: [ka]

[0038] SEQ ID NO: 31: Haemophilus influenzae HU fragment: [ka]

[0039] SEQ ID NO: 32: Haemophilus influenzae HU fragment: [ka]

[0040] SEQ ID NO: 33: Haemophilus influenzae HU, A3 fragment: [ka]

[0041] SEQ ID NO: 34: Human IgD constant region, Uniprot: P01880: [ka]

[0042] SEQ ID NO: 35: Human IgG1 constant region, Uniprot: P01857: [ka]

[0043] SEQ ID NO: 36: Human IgG2 constant region, Uniprot: P01859: [ka]

[0044] SEQ ID NO: 37: Human IgG3 constant region, Uniprot: P01860: [ka]

[0045] SEQ ID NO: 38: Human IgM constant region, Uniprot: P01871: [ka]

[0046] SEQ ID NO: 39: Human IgG4 constant region, Uniprot: P01861: [ka]

[0047] SEQ ID NO: 40: Human IgA1 constant region, Uniprot: P01876: [ka]

[0048] SEQ ID NO: 41: Human IgA2 constant region, Uniprot: P01877: [ka]

[0049] SEQ ID NO: 42: Human Ig kappa constant region, Uniprot: P01834: [ka]

[0050] SEQ ID NO: 43: Non-limiting exemplary linker: GPSLKL.

[0051] SEQ ID NO: 44: Non-limiting exemplary linker: GGG.

[0052] SEQ ID NO: 45: Non-limiting exemplary linker: GPSL.

[0053] SEQ ID NO: 46: Non-limiting exemplary linker: GPS.

[0054] SEQ ID NO: 47: Non-limiting exemplary linker: PSLK.

[0055] SEQ ID NO: 48: Non-limiting exemplary linker: GPSLK.

[0056] SEQ ID NO: 49: Non-limiting exemplary linker: SLKL.

[0057] SEQ ID NO: 50: Non-limiting exemplary heavy chain variable region nucleotide sequence, IhfA5 fragment: [ka] [ka]

[0058] SEQ ID NO: 51: Non-limiting exemplary heavy chain variable region amino acid sequence, IhfA5 fragment: [ka]

[0059] SEQ ID NO: 52: Non-limiting exemplary heavy chain variable region nucleotide sequence, IhfmB4 fragment: [ka]

[0060] SEQ ID NO: 53: Non-limiting exemplary heavy chain variable region amino acid sequence, IhfmB4 fragment: [ka]

[0061] SEQ ID NO: 54: Non-limiting exemplary light chain variable region nucleotide sequence, IhfA5 fragment: [ka]

[0062] SEQ ID NO: 55: Non-limiting exemplary light chain variable region amino acid sequence, IhfA5 fragment: [ka]

[0063] SEQ ID NO: 56: Non-limiting exemplary light chain variable region nucleotide sequence, IhfmB4 fragment: [ka]

[0064] SEQ ID NO: 57: Non-limiting exemplary light chain variable region amino acid sequence, IhfmB4 fragment: [ka]

[0065] SEQ ID NO: 58: Non-limiting exemplary partial CDRH1 sequence; IhfA5 fragment: FSLTSYS

[0066] SEQ ID NO: 59: Non-limiting exemplary partial CDRH1 sequence; IhfA5 fragment: FSLTSYSV.

[0067] SEQ ID NO: 60: Non-limiting exemplary partial CDRH1 sequence; IhfA5 fragment: FSLTSYSVH.

[0068] SEQ ID NO: 61: Non-limiting exemplary partial CDRH1 sequence; IhfA5 fragment: GFSLTSYS.

[0069] SEQ ID NO: 62: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: IWAGGST.

[0070] SEQ ID NO: 63: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: VIWAGGST.

[0071] SEQ ID NO: 64: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: GVIWAGGST.

[0072] SEQ ID NO: 65: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: LGVIWAGGST.

[0073] SEQ ID NO: 66: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: WLGVIWAGGST.

[0074] SEQ ID NO: 67: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: IWAGGSTN.

[0075] SEQ ID NO: 68: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: VIWAGGSTN.

[0076] SEQ ID NO: 69: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: GVIWAGGSTN.

[0077] SEQ ID NO: 70: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: LGVIWAGGSTN.

[0078] SEQ ID NO: 71: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: WLGVIWAGGSTN.

[0079] SEQ ID NO: 72: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: IWAGGSTNY.

[0080] SEQ ID NO: 73: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: VIWAGGSTNY.

[0081] SEQ ID NO: 74: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: GVIWAGGSTNY.

[0082] SEQ ID NO: 75: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: LGVIWAGGSTNY.

[0083] SEQ ID NO: 76: Non-limiting exemplary partial CDRH2 sequence; IhfA5 fragment: WLGVIWAGGSTNY.

[0084] SEQ ID NO: 77: Non-limiting exemplary partial CDRH3 sequence; IhfA5 fragment: REDS.

[0085] SEQ ID NO: 78: Non-limiting exemplary partial CDRH3 sequence; IhfA5 fragment: AREDS.

[0086] SEQ ID NO: 79: Non-limiting exemplary partial CDRL1 sequence; IhfA5 fragment: QNVGTN.

[0087] SEQ ID NO: 80: Non-limiting exemplary partial CDRL1 sequence; IhfA5 fragment: QNVGTNV.

[0088] SEQ ID NO: 81: Non-limiting exemplary partial CDRL1 sequence; IhfA5 fragment: QNVGTNVA.

[0089] SEQ ID NO: 82: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: SAS.

[0090] SEQ ID NO: 83: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: YSAS.

[0091] SEQ ID NO: 84: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: IYSAS

[0092] SEQ ID NO: 85: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: LIYSAS.

[0093] SEQ ID NO: 86: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: ALIYSAS.

[0094] SEQ ID NO: 87: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: SASY.

[0095] SEQ ID NO: 88: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: YSASY.

[0096] SEQ ID NO: 89: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: IYSASY.

[0097] SEQ ID NO: 90: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: LIYSASY.

[0098] SEQ ID NO: 91: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: ALIYSASY.

[0099] SEQ ID NO: 92: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: SASYR.

[0100] SEQ ID NO: 93: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: YSASYR.

[0101] SEQ ID NO: 94: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: IYSASYR.

[0102] SEQ ID NO: 95: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: LIYSASYR.

[0103] SEQ ID NO: 96: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: ALIYSASYR.

[0104] SEQ ID NO: 97: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: SASYRY.

[0105] SEQ ID NO: 98: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: YSASYRY.

[0106] SEQ ID NO: 99: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: IYSASYRY.

[0107] SEQ ID NO: 100: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: LIYSASYRY.

[0108] SEQ ID NO: 101: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: ALIYSASYRY.

[0109] SEQ ID NO: 102: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: SASYRYS.

[0110] SEQ ID NO: 103: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: YSASYRYS.

[0111] SEQ ID NO: 104: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: IYSASYRYS.

[0112] SEQ ID NO: 105: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: LIYSASYRYS.

[0113] SEQ ID NO: 106: Non-limiting exemplary partial CDRL2 sequence; IhfA5 fragment: ALIYSASYRYS.

[0114] SEQ ID NO: 107: Non-limiting exemplary linker: GGSGGS.

[0115] SEQ ID NO: 108: Non-limiting exemplary partial CDRL3 sequence; IhfA5 fragment: QQYNSYP.

[0116] SEQ ID NO: 109: Non-limiting exemplary partial CDRL3 sequence; IhfA5 fragment: QQYNSYPT.

[0117] SEQ ID NO: 110: Non-limiting exemplary partial CDRH1 sequence; IhfmB4 fragment: FNIKDYY.

[0118] SEQ ID NO: 111: Non-limiting exemplary partial CDRH1 sequence; IhfmB4 fragment: FNIKDYYM.

[0119] SEQ ID NO: 112: Non-limiting exemplary partial CDRH1 sequence; IhfmB4 fragment: FNIKDYYMH.

[0120] SEQ ID NO: 113: Non-limiting exemplary partial CDRH1 sequence; IhfmB4 fragment: GFNIKDYY.

[0121] SEQ ID NO: 114: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: IDPENDDT.

[0122] SEQ ID NO: 115: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: WIDPENDDT.

[0123] SEQ ID NO: 116: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: GWIDPENDDT.

[0124] SEQ ID NO: 117: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: IGWIDPENDDT.

[0125] SEQ ID NO: 118: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: WIGWIDPENDDT.

[0126] SEQ ID NO: 119: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: IDPENDDTE.

[0127] SEQ ID NO: 120: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: WIDPENDDTE.

[0128] SEQ ID NO: 121: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: GWIDPENDDTE.

[0129] SEQ ID NO: 122: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: IGWIDPENDDTE.

[0130] SEQ ID NO: 123: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: WIGWIDPENDDTE.

[0131] SEQ ID NO: 124: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: IDPENDDTEY.

[0132] SEQ ID NO: 125: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: WIDPENDDTEY.

[0133] SEQ ID NO: 126: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: GWIDPENDDTEY.

[0134] SEQ ID NO: 127: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: IGWIDPENDDTEY.

[0135] SEQ ID NO: 128: Non-limiting exemplary partial CDRH2 sequence; IhfmB4 fragment: WIGWIDPENDDTEY.

[0136] SEQ ID NO: 129: Non-limiting exemplary partial CDRH3 sequence; IhfmB4 fragment: TELGAY.

[0137] SEQ ID NO: 130: Non-limiting exemplary partial CDRL1 sequence; IhfmB4 fragment: QSLLDSNGKTY.

[0138] SEQ ID NO: 131: Non-limiting exemplary partial CDRL1 sequence; IhfmB4 fragment: QSLLDSNGKTYL.

[0139] SEQ ID NO: 132: Non-limiting exemplary partial CDRL1 sequence; IhfmB4 fragment: QSLLDSNGKTYLN.

[0140] SEQ ID NO: 133: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LVS.

[0141] SEQ ID NO: 134: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: YLVS.

[0142] SEQ ID NO: 135: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: IYLVS.

[0143] SEQ ID NO: 136: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LIYLVS.

[0144] SEQ ID NO: 137: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: RLIYLVS.

[0145] SEQ ID NO: 138: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LVSK.

[0146] SEQ ID NO: 139: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: YLVSK.

[0147] SEQ ID NO: 140: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: IYLVSK.

[0148] SEQ ID NO: 141: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LIYLVSK.

[0149] SEQ ID NO: 142: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: RLIYLVSK.

[0150] SEQ ID NO: 143: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LVSKL.

[0151] SEQ ID NO: 144: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: YLVSKL.

[0152] SEQ ID NO: 145: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: IYLVSKL.

[0153] SEQ ID NO: 146: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LIYLVSKL.

[0154] SEQ ID NO: 147: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: RLIYLVSKL.

[0155] SEQ ID NO: 148: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LVSKLD.

[0156] SEQ ID NO: 149: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: YLVSKLD.

[0157] SEQ ID NO: 150: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: IYLVSKLD.

[0158] SEQ ID NO: 151: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LIYLVSKLD.

[0159] SEQ ID NO: 152: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: RLIYLVSKLD.

[0160] SEQ ID NO: 153: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LVSKLDS

[0161] SEQ ID NO: 154: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: YLVSKLDS.

[0162] SEQ ID NO: 155: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: IYLVSKLDS.

[0163] SEQ ID NO: 156: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: LIYLVSKLDS.

[0164] SEQ ID NO: 157: Non-limiting exemplary partial CDRL2 sequence; IhfmB4 fragment: RLIYLVSKLDS.

[0165] SEQ ID NO: 158: Non-limiting exemplary partial CDRL3 sequence; IhfmB4 fragment: WQSTHFPH.

[0166] SEQ ID NO: 159: Non-limiting exemplary partial CDRL3 sequence; IhfmB4 fragment: WQSTHFPHT.

[0167] Table Description Table 1 lists non-limiting exemplary antibody-producing hybridomas.

[0168] Table 2 is a non-limiting list of antibodies and their combinations used in Example 3.

[0169] Table 3 is a non-limiting list of antibodies used in Example 4.

[0170] Table 4 outlines the scoring method used in the otitis media model of Example 4.

[0171] Table 5 describes the cohort studied in Example 7.

[0172] Table 6 is a non-limiting list of exemplary antibodies and their corresponding CDRs.

[0173] Table 7 is a non-limiting list of exemplary antibodies and their corresponding heavy and light chain variable regions. Summary of the Invention [Problem to be solved by the invention]

[0174] Within bacterial cells, DNABII proteins are DNA-binding proteins that, upon binding, naturally bend the DNA substrate. Similarly, DNA already in a bent conformation is an exemplary substrate, as it eliminates the energy required for bending.

[0175] The DNABII family is a member of a class of proteins called nuclear inclusion-associated proteins (NAPs), which are bacterial proteins that partially form bacterial nuclear inclusions within cells (Browning et al. (2010) Curr. Opin. Microbiol. 13:773-780). In addition, this family is ubiquitous and expressed by virtually all eubacteria. All family members characterized to date function as homodimers or heterodimers of subunits. The family is divided into two classes: HU (histone-like proteins) and IHF (integration host factors). The primary distinction between these family members is that HU binds to DNA in a sequence-independent manner, whereas IHF binds to a consensus sequence (WATCAANNNNTTR [where W is A or T and R is a purine] (SEQ ID NO: 160)) that is conserved across genuses (Swinger et al. (2004) Curr. Opin. Microbiol. 13:773-780). Opin. Struct. Biol., 14:28-35). All DNABII proteins bind to DNA and bend it significantly; for example, E. coli IHF bends DNA into a virtual U-shaped turn (Rice et al. (1996), Cell, 87:1295-1306). In addition, all family members are likely to adopt pre-bent or curved DNA structures, such as Holliday junctions, cruciform-like structures that are central to DNA recombination. Indeed, DNABII proteins function as accessory factors that facilitate all DNA functions within the cell, including gene expression, recombination, repair, and replication (Swinger et al. (2004), Curr. Opin. Struct. Biol., vol. 14:28-35).

[0176] The DNABII family of proteins is found on the outside of bacterial cells in biofilms. Applicants have shown that these proteins indeed bind to extracellular DNA at critical branched junctions. In one aspect, Applicants have shown that delivering DNABII-specific antibodies to a host allows the host to significantly reduce or eradicate pre-formed biofilms. [Means for solving the problem]

[0177] Embodiments of the present disclosure relate to one or more antibodies or antigen-binding fragments specific for a DNABII polypeptide. In certain embodiments, the polypeptide is derived from the IHF or HU sequence of Haemophilus influenzae. In some embodiments, the antibody or antigen-binding fragment may have affinity for a specific region of the alpha or beta subunit of IHF, such as, but not limited to, a polypeptide that comprises, alternatively consists essentially of, or further comprises a proline-containing loop; in some embodiments, the antibody or antigen-binding fragment may have affinity for a specific region of HU, such as, but not limited to, the flexible arm of its 3D structure.

[0178] In certain aspects, the present disclosure relates to an antibody or antigen-binding fragment that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of an amino acid sequence selected from SEQ ID NOs: 12 to 17, SEQ ID NO: 31, SEQ ID NO: 33, a polypeptide comprising the amino acid sequence NPXT, or their respective equivalents. In further aspects, the isolated or recombinant polypeptide comprises a total of at least 15, or alternatively at least 18, or alternatively at least 20 amino acids. In further aspects, the NPXT sequence is not the terminal amino acid of the polypeptide. In some embodiments, the antibody or antigen-binding fragment is not a polyclonal antibody.

[0179] In alternative embodiments, the present disclosure provides antibodies or antigen-binding fragments that can be administered alone or in combination with each other and with a pharmaceutically acceptable carrier. Some embodiments of the present disclosure also relate to compositions that comprise, or alternatively consist essentially of, and / or further consist of, these combinations. Further embodiments relate to the use of the antibodies or antigen-binding fragments, for example, as disclosed herein, in diagnostic, therapeutic, industrial, veterinary, and functional assays, including, but not limited to, the use of the antibodies or antigen-binding fragments in the treatment and / or visualization and / or detection of biofilms. They are also useful for treating microbial infections caused by biofilms.

[0180] The present disclosure also provides isolated or recombinant polypeptides comprising, alternatively consisting essentially of, and / or further consisting of, an amino acid sequence selected from SEQ ID NOs: 12 to 17, SEQ ID NO: 31, SEQ ID NO: 33, a polypeptide comprising the amino acid sequence NPXT, or their respective equivalents. The use of isolated or recombinant polypeptides as immunogens is disclosed herein. In some method embodiments, such isolated or recombinant polypeptides are used to generate antibodies that specifically recognize or specifically bind to these amino acid sequences. In addition, isolated or recombinant polypeptides can be used in vaccine formulations and immunization methods. They are also useful for treating microbial infections caused by biofilms.

[0181] The present disclosure also provides isolated or recombinant polynucleotides encoding one or more of the above-identified isolated or recombinant polypeptides or antibodies, or their respective fragments. Also provided are vectors comprising the isolated polynucleotides, which in one embodiment also contain regulatory sequences operably linked to the polynucleotide for its replication and / or expression. In embodiments disclosed herein involving more than one isolated polypeptide, the isolated polynucleotides may be contained within a polycistronic vector.

[0182] Further provided is an isolated host cell comprising one or more of the isolated or recombinant polypeptides, or isolated or recombinant polynucleotides or vectors described herein.

[0183] The polynucleotide, polypeptide, antibody, antigen-binding fragment, or vector, or host cell may further comprise a detectable label and / or a purification label. In one embodiment, the detectable label is not a naturally occurring detectable compound, such as a fluorescent polynucleotide or an amino acid. In a further embodiment, the detectable label is a naturally occurring molecule located at a non-naturally occurring position on the polypeptide or polynucleotide.

[0184] Also provided are compositions comprising, alternatively consisting essentially of, and / or further consisting of a carrier and one or more of the isolated or recombinant polypeptides disclosed herein, the isolated or recombinant polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, or the antibodies or fragments thereof disclosed herein. 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, or an antimicrobial agent other than the disclosed antibodies or fragments thereof.

[0185] The composition may further comprise an additional biologically active agent.

[0186] Non-limiting examples of such compositions include antimicrobial agents, one or more antibodies against vaccine antigens, or antimicrobial peptides. In some embodiments, these exemplary biologically active agents are used in combination with one or more antibodies disclosed herein. In further embodiments, such combinations can be used as therapeutic compositions in any of the methods disclosed herein.

[0187] Further non-limiting examples include vaccine components (i.e., antigenic peptides) such as surface antigens, e.g., OMP P5, rsPilA, OMP 26, OMP P2, or type IV Pilin proteins (see Jurcisek and Bakaletz (2007), J. of Bacteriology, 189(10):3868-3875; Murphy et al. (2009), The Pediatric Infectious Disease Journal, 28:S121-S126; and Novotny et al. (2015), Mol. Microbiol. 96(2):276-92), and antibacterial agents. In some embodiments, these exemplary biologically active agents are used in combination with one or more isolated or recombinant polypeptides disclosed herein. In further embodiments, such combinations can be used as vaccine compositions in any of the methods disclosed herein.

[0188] Additional non-limiting agents include additional antibodies, fragments, derivatives, or polypeptides disclosed herein or disclosed in U.S. Patent Publication No. 2011 / 0236306 and PCT International Patent Publication No. WO2014 / 201305. As will be apparent to one of skill in the art, the choice and amount of agent will vary with the purpose of treatment, general health, sex, gender, and species (e.g., human or animal).

[0189] The present disclosure also provides a method for producing an antigenic peptide by growing or culturing a host cell containing an isolated polynucleotide encoding the antigenic peptide described above under conditions suitable for expression of the polynucleotide. Also provided is a vector containing a polynucleotide encoding an antigenic polypeptide, which in one embodiment also contains regulatory sequences operably linked to the polynucleotide for its replication and / or expression. The polypeptide produced by this method can be further isolated for in vitro or in vivo use. Alternatively, the polypeptide can be produced using chemical synthesis methods known to those skilled in the art.

[0190] Also provided are kits for diagnostic or therapeutic use that include the compositions described herein and instructions for use. Also provided are kits for conducting screens for new drugs and / or combination therapies provided herein. [Brief explanation of the drawings]

[0191] [Figure 1] FIG. 1 depicts the reduction of biofilms formed by Haemophilus influenzae upon incubation with polyclonal rabbit anti-IHF or polyclonal rabbit anti-HU compared to incubation with naive rabbit serum.

[0192] [Figure 2-1]Figure 2A shows the culture results and purification of monoclonal antibodies generated against IHF and HU of Haemophilus influenzae. Figure 2B shows the ELISA results confirming the specificity of each antibody for its corresponding epitope. Figure 2C depicts the specificity of IHFNTHI monoclonal antibodies for the IHF fragments (e.g., A3, A5, B2, B4) to which they were directed and the IHFNTHI protein subunits (i.e., IHF alpha subunit or IHF beta subunit) from which they were derived. Dark wells in the scanned image of a representative ELISA plate indicated the reactivity of monoclonal antibodies to their respective peptides and protein subunits. [Figure 2-2] Same as above [Figure 2-3] Same as above

[0193] [Figure 3-1] Figure 3A depicts the disruption of biofilms formed by Haemophilus influenzae during incubation with monoclonal anti-IHF antibodies and their combinations. Specifically, anti-IHF A5 and anti-IHF B4 or anti-IHF mB4, either alone or in combination with each other, demonstrated robust disruption of biofilms. Figure 3B depicts COMSTAT plots of biofilm layer thickness after administration of each antibody or their combination. Again, anti-IHF A5 and anti-IHF B4 or anti-IHF mB4, either alone or in combination with each other, demonstrated robust reductions in biofilm layer thickness. Figure 3C depicts the mean biofilm biomass remaining after treatment, as determined by COMSTAT2 analysis. ***p≦0.001, ****p≦0.0001. [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 3-4] Same as above [Figure 3-5] Same as above

[0194] [Figure 4-1] Figure 4A shows the concentration of nontypeable Haemophilus influenzae (NTHI) colony-forming units per ml of fluid extracted from the chinchilla middle ear by treatment group. Figure 4B shows the concentration of nontypeable Haemophilus influenzae (NTHI) colony-forming units per mg of biofilm attached to the middle ear mucosa by treatment group. Figure 4C depicts the mucosal biofilm score by treatment group. Figure 4D presents stained images of middle ear biofilm (or lack thereof) in dissected bullae collected from chinchillas by treatment group. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 4-4] Same as above

[0195] [Figure 5] Figure 5A depicts antibody labeling of biofilms in situ using IhfA3 confirms detection of DNABII protein in 2-week-old NTHI. Figure 5B depicts antibody labeling of biofilms in vitro using IhfA3.

[0196] [Figure 6-1] Figure 6A shows the concentration of Pseudomonas aeruginosa colony-forming units per mouse lung by treatment group, and Figure 6B shows the concentration of Pseudomonas aeruginosa colony-forming units per mouse lung by treatment group (with and without tobramycin). [Figure 6-2] Same as above

[0197] [Figure 7-1]Figure 7A depicts the reduction of biofilms formed by B. cenocepacia K56 upon incubation with monoclonal anti-IHF and its combination, and polyclonal anti-IHF. Specifically, anti-IHF A-5 and anti-IHF mB-4, either alone or in combination with each other, demonstrated robust biofilm disruption. Figure 7B depicts similar results for biofilms formed by Pseudomonas aeruginosa 28753. Figure 7C depicts similar results for biofilms formed by Staphylococcus aureus 29213. Figure 7D depicts similar results for biofilms formed by Moraxella catarrhalis 7169. [Figure 7-2] Same as above [Figure 7-3] Same as above [Figure 7-4] Same as above

[0198] [Figure 8-1] Figures 8A-8B depict a computer model of the three-dimensional (3D) structure of the DNABII protein, highlighting predicted amino acid positions within peptides used to generate antibodies. Figure 8A depicts a top-down view and a front view of the specific IHFB epitope, and Figure 8B depicts the 3D structure of the IHFA, HU, and IHFB epitopes, highlighting the epitopes (arrows indicate the highlighted regions). The figures disclose SEQ ID NOs: 12-17, respectively, in order of appearance. [Figure 8-2] Same as above

[0199] [Figure 9-1]Figure 9A depicts the disruption of biofilms formed by Haemophilus influenzae NTHI strain no. 1885 upon incubation with monoclonal anti-IHF and combinations thereof. Figure 9B depicts the disruption of biofilms formed by Haemophilus influenzae NTHI strain no. 1714 upon incubation with monoclonal anti-IHF and combinations thereof. Figure 9C depicts the disruption of biofilms formed by Haemophilus influenzae NTHI strain no. 214 upon incubation with monoclonal anti-IHF and combinations thereof. [Figure 9-2] Same as above

[0200] [Figure 10] Figure 10A depicts the mean biofilm biomass of Haemophilus influenzae NTHI 1885 remaining after treatment as determined by COMSTAT2 analysis. Figure 10B depicts the mean biofilm biomass of Haemophilus influenzae NTHI 1714 remaining after treatment as determined by COMSTAT2 analysis. Figure 10C depicts the mean biofilm biomass of Haemophilus influenzae NTHI 214 remaining after treatment as determined by COMSTAT2 analysis.

[0201] [Figure 11-1]Figure 11A depicts the mean biofilm biomass of B. cenocepacia K56 remaining after treatment as determined by COMSTAT2 analysis. Figure 11B depicts the mean biofilm biomass of Pseudomonas aeruginosa 28753 remaining after treatment as determined by COMSTAT2 analysis. Figure 11C depicts the mean biofilm biomass of Staphylococcus aureus 29213 remaining after treatment as determined by COMSTAT2 analysis. Figure 11D depicts the mean biofilm biomass of Moraxella catarrhalis 7169 remaining after treatment as determined by COMSTAT2 analysis. [Figure 11-2] Same as above

[0202] [Figure 12-1]Figures 12A-12C depict a reduction in both the number and size of P. aeruginosa aggregates in lungs treated with mAbs directed to the IhfNTHI tip in an experimental mouse model of pulmonary infection. Before treatment (Figure 12A), after administration of nonspecific mouse IgG1 + IgG2a (Figure 12B), or a cocktail of tail-directed mAb IhfA3NTHI + mAb IhfB2NTHI (Figure 12C), multiple bacterial aggregates were observed throughout the lungs of mice (yellow, aggregates circled). In contrast, after delivery of apical-directed mAb IhfA5NTHI + mAb IhfmB4NTHI, P. aeruginosa was barely detectable, confirming the efficacy of this treatment strategy (Figure 12D). Counterstaining with DAPI (blue); scale bar: 50 μm. Figures 12E-12H show high-magnification images of representative aggregates, indicating the relative size of the aggregates or the presence of single bacteria for each cohort [Figure 12E: before treatment; Figure 12F: treatment with nonspecific mouse IgG1 + IgG2a; Figure 12G: treatment with tail-directed mAb IhfA3NTHI + mAb IhfB2NTHI; and Figure 12H: treatment with tip-directed mAb IhfA5NTHI + mAb IhfmB4NTHI (P. aeruginosa aggregates were labeled with a fluorescently tagged rabbit antibody against whole P. aeruginosa, an antibody that labels both the bacteria themselves as well as bacterial proteins contained within the biofilm matrix, so that individual bacterial cells cannot be easily resolved)]. Note that although the aggregates in Figures 12A, 12B, and 12C and those in 12E, 12F, and 12G are similar in size, aggregates imaged in lungs treated with the apically directed mAb cocktail appear as single bacterial cells (Figures 12D and 12H). Scale bar: 5 μm. Representative TEM images of P. aeruginosa aggregates shown in Figures 12A, 12B, and 12C or 12E, 12F, and 12G are presented in Figure 12I. To aid visualization, bacterial cells are pseudocolored green, and mouse lung tissue is pseudocolored blue.Bacterial aggregates in the lungs from mice treated with mouse IgG or tail-directed mAb typically contained 50-100 bacteria. The relatively limited number of very small clusters or the presence of only single bacteria inhabiting the lungs from mice treated with apical-directed mAb precluded visualization by TEM. Scale bar: 2 μm. Legend: FOV: field of view. [Figure 12-2] Same as above [Figure 12-3] Same as above

[0203] [Figure 13-1] 13A-13B show in vitro disruption of B. cenocepacia biofilms by monoclonal antibodies against the DNA-binding "tip" region of IHF (A5 and mB4) and synergism with antibiotics. [Figure 13-2] Same as above [Figure 13-3] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0204] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in a 5' to 3' orientation. Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing the present disclosure, specific, non-limiting, exemplary methods, devices, and materials are described herein. All technical publications and patent disclosures cited herein are incorporated by reference in their entirety. Nothing herein shall be construed as an admission that the present disclosure is not entitled to antedate such disclosure by way of prior invention.

[0205] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, within the skill of the art. See, e.g., Green and Sambrook (eds.) (2012), Molecular Cloning: A Laboratory "PCR Manual," 4th edition; Ausubel et al. (eds.), (2015), "Current Protocols in Molecular Biology"; "Methods in Enzymology" series (Academic Press, Inc., NY); MacPherson et al. (2015), "PCR 1: A Practical Approach" (Oxford University Press, IRL Press); MacPherson et al. (1995), "PCR 2: A Practical Approach"; McPherson et al. (2006), "PCR: The Basics," (Garland Science); Harlow and Lane (eds.), (1999), "Antibodies, A Laboratory Manual"; Greenfield (ed.) (2014) "Antibodies, A Laboratory Manual"; Freshney (ed.) (2010) "Culture of Animal Cells: A Manual of Basic Technique", 6th 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"; Herdewijn (eds.) (2005) "Oligonucleotide Synthesis: Methods and Applications"; Hames and Higgins (eds.) (1984) "Transcription and Translation"; Buzdin and Lukyanov (eds.) (2007) "Nucleic Acids Hybridization: Modern Applications"; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi (ed.) (2007) "In Vitro Transcription and Translation (2006), "Immobilization of Enzymes and Cells," Perbal (1988), "A Practical Guide to Molecular Cloning," 2nd ed.; Miller and Calos (1987), "Gene Transfer Vectors for Mammalian Cells" (Cold Spring Harbor Laboratory); Makrides (2003), "Gene Transfer and Expression in Mammalian Cells"; Mayer and Walker (1987), "Immunochemical Methods in Cell and Molecular Biology" (Academic Press, London); Lundblad and Macdonald (2010), "Handbook of Biochemistry and Molecular Biology," 4th ed.; and Herzenberg et al. (1996), "Weir's Handbook of Experimental Immunology," 5th ed.

[0206] 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.

[0207] 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.

[0208] 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 saline, preservatives, etc. "Consisting of" shall mean excluding more than trace elements of other components and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0209] The term "biofilm" refers to an organized community of microorganisms, sometimes attached to the surface of a structure, which may be organic or inorganic, along with polymers such as DNA secreted and / or released by the microorganisms. 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 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. Biofilms cause vaginal infections and lead to life-threatening systemic infections in people with compromised immune systems. Biofilms may also be used to support the growth of bacteria such as Aggregatibacter actinomycetemcomitans, Borrelia burgdorferi (e.g., B31), Bordetella pertussis (e.g., Tohama I), Burkholderia pseudomallei (e.g., 668), Burkholderia cenocepacia (e.g., HI2424), Escherichia coli (e.g., K12 MG1655), Enterococcus faecalis (e.g., V583), Haemophilus influenzae (e.g., Rd KW20), Helicobacter pylori (e.g., 26695), Klebsiella pneumoniae, Moraxella catarrhalis (e.g., RH4), Mycobacterium smegmatis (e.g., MC2), Mycobacterium tuberculosis (e.g., CDC1551), Neisseria gonorrhoeae, and the like. gonorrhoeae (e.g., FA1090), Neisseriameningitidis (e.g., MC58), Pseudomonas aeruginosa, Porphyromonas gingivalis (e.g., W83), Prevotella intermedia (e.g., 17), Prevotella melaninogenica (e.g., ATCC (registered trademark) 25845), Staphylococcus aureus (e.g., MW2), Staphylococcus epidermidis (e.g., RP62A), Streptococcus agalactiae (e.g., 2603V / R), Streptococcus bovis, Streptococcus gallolyticus (e.g., UCN34), Streptococcus gordonii (e.g., NCTC 7868 (Challis)), Streptococcus mutans (e.g., UA159), Streptococcus pneumoniae (e.g., R6), Streptococcus Biofilms are also implicated in numerous diseases, including, but not limited to, those caused by S. pyogenes (e.g., MGAS10270), Streptococcus sobrinus (e.g., 6715), Salmonella enterica (e.g., typhi, CT18), Treponema denticola (e.g., ATCC® 35405), Treponema palladum (e.g., Nichols), and Vibrio cholera (e.g., El Tor, N16961). Additional organisms known to be associated with and / or form biofilms include, but are not limited to, Campylobacter species, Candida species, Legionella pneumophila, and Listeria monocytogenes. For example, cystic fibrosis patients have Pseudomonas infections, which often result in antibiotic-resistant biofilms. Other diseases associated with biofilms include lung infections in cystic fibrosis patients, otitis media, post-tympanostomy tube ottorhea, chronic suppurative otitis media, and native valve infective endocarditis. These include, but are not limited to, bacterial infections caused by the organisms listed above (e.g., Listeria monocytogenes, Escherichia coli, Salmonella typhimurium ... Foodborne pathogens, such as, but not limited to, some strains of Escherichia coli (S. enterica), can also form biofilms on the foods they contaminate. Biofilms that cause disease in animals (e.g., Escherichia coli, Salmonella species, and Shigella species) can also cause food contamination and / or disease in downstream human hosts. Furthermore, biofilms need not be a homogenous population of microorganisms but may incorporate other pathogens, and even host cells. In addition to being associated with disease (both hospital-based and other) and food contamination, biofilms are often a source of industrial contamination, particularly associated with process water and surfaces that come into contact with it. Problems involving biofilm-forming organisms as industrial contaminants include, but are not limited to, biocorrosion, biofouling, and equipment damage as a result of biofilm formation. Non-limiting exemplary biofilm-associated organisms in industrial settings include those disclosed in Ferrera et al. (2015), Biofouling, 31(2):173-180, and Desulfovibrio species. Further details regarding biofilms can be found, for example, in Donlan (2002), Emerging Infectious Diseases, 8(9):881-890.

[0210] The term "inhibiting, competing with, or diminishing" intends to prevent, reduce, or disrupt the formation or structure of the DNA / protein matrix that is a component of a microbial biofilm.

[0211] As used herein, the term "nuclear inclusion-associated protein" or "NAP" refers to a class of proteins that perform dynamic spatial organization of nucleic acids within the nuclear inclusions of prokaryotic cells. These proteins organize the genome through DNA bending, binding, and aggregation. Certain NAPs are DNA-binding proteins that may be associated with biofilms, including DNABII proteins, 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). NAPs generally share strong sequence identity with the alpha-helix dimerization domain of DNABII proteins and often comprise antiparallel beta ribbons with NPXT-containing tips that bind to and insert into the minor groove of DNA, causing the DNA to twist. A functional promoter is a dimer of identical or homologous subunits.

[0212] "DNABII polypeptide or DNABII protein" refers to a DNA-binding protein or polypeptide that is composed of a DNA-binding domain and therefore has a specific or general affinity for microbial DNA. In one embodiment, the DNABII polypeptide or DNABII protein binds to DNA in the minor groove. Non-limiting examples of DNABII proteins are integration host factor (IHF) protein and histone-like protein (HU).

[0213] "Integration host factor" or "IHF" proteins are bacterial proteins used by bacteriophages to integrate their DNA into host bacteria. They also bind to extracellular microbial DNA. The genes encoding the IHF protein subunits in E. coli are the himA gene (Genbank accession number: POA6X7.1) and the himD gene (POA6Y1.1). Homologs to these genes are found in other organisms, and peptides corresponding to these genes from other organisms can be found in Table 10 of U.S. Pat. No. 8,999,291, incorporated herein by reference.

[0214] "HMGB1" is the high mobility group box (HMGB) 1 protein, which 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.

[0215] "HU" or "histone-like protein" 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. The genes encoding the HU protein subunits in E. coli are hupA and hupB, which correspond to SEQ ID NOs:8 and 9, respectively. The Haemophilus influenzae homologs from nontypeable Haemophilus influenzae (NTHI) correspond to SEQ ID NO:3. Homologs of these genes are also found in other organisms, and peptides corresponding to these genes from other organisms can be found in Table 10 of U.S. Pat. No. 8,999,291, incorporated herein by reference.

[0216] The term "surface antigen" or "surface protein" refers to a protein or peptide on the surface of a cell, such as a bacterial cell. Examples of surface antigens are outer membrane proteins, such as OMP P5 (Genbank accession number: YP_004139079.1), OMP P2 (Genbank accession number: ZZX87199.1), OMP P26 (Genbank accession number: YP_665091.1), rsPilA or recombinant soluble PilA (Genbank accession number: EFU96734.1), and type IV Pilin (Genbank accession number: Yp_003864351.1).

[0217] The term "Haemophilus influenzae" refers to a pathogenic bacterium that can cause many different infections, such as ear infections, eye infections, and sinusitis. Many different strains of Haemophilus influenzae have been isolated that contain the IhfA gene or protein. Some non-limiting examples of different strains of Haemophilus influenzae include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0218] "Microbial DNA" means single-stranded or double-stranded DNA derived from a biofilm-producing microorganism.

[0219] "Inhibiting, preventing, or disrupting" a biofilm means prophylactically or therapeutically reducing the structure of the biofilm. An example of biofilm disruption or reduction is shown in Figure 1. Methods for determining whether a biofilm has been inhibited, prevented, or disrupted are known in the art and include in vivo and ex vivo assays, as well as the reduction of clinical symptoms of an infection or disease associated with the biofilm.

[0220] "Treating an infection" refers to reducing the number of microorganisms, for example, bacteria associated with the formation of a biofilm. Methods for determining whether the number of microorganisms has been reduced are known in the art, including in vivo and ex vivo assays, and the alleviation of clinical symptoms of the infection. Because bacteria are protected by biofilms, they become resistant to the use of antibacterial drugs. By destroying biofilms, bacterial resistance to antibacterial drugs and other agents can be reduced or inhibited.

[0221] "Interfering agent" refers to an agent that competes with, inhibits, prevents, or reduces the activity of a DNABII polypeptide, such as IHF or HU, against microbial DNA, or disrupts a microbial biofilm (e.g., by disrupting the structural integrity of the eDNA-based extracellular matrix). Interfering agents can be any one or more of 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 or HU for DNA binding, (3) polypeptides such as peptide fragments of IHF or HU that compete with IHF or HU for DNA binding, (4) antibodies or fragments thereof directed against IHF or HU, or (5) four-way or bent polynucleotides, or other types of polynucleotides containing bent or distorted DNA structures that compete for IHF or HU-binding. "Small molecules that inhibit the binding of IHF or HU 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 containing a four-way junction, also known as a Holliday junction, between the four strands of DNA.

[0222] "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.

[0223] "Polypeptides that compete with DNABII proteins for DNA binding" refers to proteins or peptides that compete with IHF or HU for binding to bent or distorted DNA structures but do not form biofilms with DNA. Examples include, but are not limited to, fragments of IHF containing one or more DNA-binding domains of IHF, or biological equivalents thereof.

[0224] As used herein, the term "specifically recognize or specifically bind to" intends that binding of an agent, e.g., a monoclonal antibody, to its intended target or binding partner is more likely than non-binding.

[0225] A "subject" of diagnosis or treatment is a cell or an animal, such as a mammal or a human. Non-human animals for diagnosis or treatment, infection, or animal models include, for example, monkeys, murines, such as rats, mice, chinchillas, canines, such as dogs, lagomorphs, such as rabbits, farm animals, sport animals, and pets.

[0226] 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 the D and L optical isomers, amino acid analogs, and peptidomimetics.

[0227] By "C-terminal polypeptide" is meant at least 10, alternatively at least 15, alternatively at least 20, or alternatively at least 25 C-terminal amino acids, or alternatively half of the polypeptide. In another embodiment, for a polypeptide containing 90 amino acids, the C-terminal polypeptide includes amino acids 46-90. In one embodiment, the term refers to the 20 C-terminal amino acids from the carboxy terminus.

[0228] 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 disclosed herein 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.

[0229] 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.

[0230] 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 components, cells, and other items 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.

[0231] Without explicit recitation or unless otherwise intended, when the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be assumed that its equivalent or biological equivalent is intended within the scope of the present disclosure. As used herein, "biological equivalent thereof" is intended to be synonymous with the term "equivalent thereof" when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid, and means one that has minimal homology 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. In one aspect, an equivalent polynucleotide is a polynucleotide that hybridizes under stringent conditions with the polynucleotide or complement of the polynucleotide described herein for use in the described method. In another embodiment, an equivalent antibody or antigen-binding polypeptide refers to an antibody or antigen-binding polypeptide that binds with at least 70%, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95% of the affinity or greater of the reference antibody or reference antigen-binding fragment. In another embodiment, the equivalent is an antibody or antigen-binding polypeptide that binds with at least 70%, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95% of the affinity or greater of the reference antibody or reference antigen-binding fragment. (e.g., a nucleotide sequence of a polypeptide that competes with the binding of an antibody or antigen-binding fragment to its antigen) In another embodiment, equivalent refers to a percentage of homology or identity of at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or 98%, that exhibits substantially the same biological activity as the reference protein, polypeptide, or nucleic acid. Examples of biologically equivalent polypeptides are provided in Table 9 of U.S. Pat. No. 8,999,291, incorporated herein by reference, where conservative amino acid substitutions to the disclosed amino acid sequences are identified.

[0232] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in the two sequences being compared. 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. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST using default parameters. Specifically, exemplary programs include BLASTN and BLASTP, which used 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. Sequence identities and percentage identities were determined by importing them into clustalW (available at the web address: align.genome.jp, last accessed March 7, 2011).

[0233] "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 the sequences share. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0234] "Homology" or "identity" or "similarity" may also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0235] "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.

[0236] 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 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalent SSCs using other buffer systems can be used.

[0237] 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.

[0238] 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.

[0239] 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 preventative, in that a disorder or its signs or symptoms are completely or partially prevented, and / or may be therapeutic, in that a disorder and / or adverse effects resulting from the disorder are partially or completely cured. Methods for determining whether treatment has occurred are known in the art and are outlined herein.

[0240] By prevent is meant preventing a disorder or effect in vitro or in vivo in a system or subject predisposed to the disorder or effect, such as preventing biofilm formation in a system infected with a microorganism known to produce biofilms.

[0241] 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.

[0242] 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 diagnosis or therapy in vitro, in vivo or ex vivo.

[0243] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, salts or electrolytes such as sodium chloride and zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, microspheres, microparticles, or nanoparticles (including, for example, biodegradable polymers such as Poly(lactic acid-co-glycolic acid)), 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 can be selected with respect to the intended form of administration, ie, oral tablet, capsule, elixir, syrup, etc., and consistent with conventional pharmaceutical practice.

[0244] As disclosed herein, a "biologically active agent" or active agent refers to 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.

[0245] "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 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, inhalation, injection, and topical application. Administration can be for use in industrial and therapeutic applications.

[0246] The agent (antibody or fragment thereof, polypeptide or polynucleotide, or cell) of the present disclosure can be administered by any suitable route for treatment. It will also be recognized that the optimal route will vary depending on the condition and age of the recipient and the disease being treated. The agent can be used in an industrial setting and can be used for the treatment of animals. When used in an industrial setting, a biofilm is contacted with the agent, for example, an antibody.

[0247] The term "effective amount" refers to a quantity sufficient to achieve a desired effect. With respect to 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, species, and tolerance to the pharmaceutical composition. In some embodiments, in the context of an immunogenic composition, an effective amount is an amount sufficient to result in the disruption of a biofilm. In other aspects, the amount is effective to treat a bacterial infection associated with a biofilm in a subject. In other embodiments, an effective amount of an agent or immunogenic composition is an amount sufficient to result in antibody production against an antigen. In some embodiments, an effective amount is the amount necessary to confer passive immunity to a subject in need thereof. With respect to 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.

[0248] 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 nature and sensitivity of the target in vitro and the method used. Those skilled in the art can determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may include one or more administrations of the composition.

[0249] 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.

[0250] 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.

[0251] 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 create 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), dioleoyl These include dipalmitoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloteoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1-carboxylate (DOPE-mal).Additional non-phosphorus-containing lipids that can be incorporated into the liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine lauryl sulfate, alkyl-aryl sulfates, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate. Liposomes include stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the above-mentioned cationic lipids (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioteoylphosphatidylglycerol (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. [New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Three classifications developed by the Journal of Clinical Immunology, "Medicine," December 1977, are multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar vesicles (LUVs). Bioactive agents can be encapsulated in such for administration according to the methods described herein.

[0252] "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.

[0253] The phrase "pharmaceutically acceptable polymer" refers to a group of compounds that can be conjugated to one or more polypeptides or antibodies described herein. Conjugating a polymer to a polypeptide or antibody may 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.

[0254] " 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.

[0255] The polynucleotides disclosed herein 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.

[0256] As used herein, the term "eDNA" refers to extracellular DNA found as a component to pathogenic biofilms.

[0257] 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.

[0258] "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.

[0259] "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 YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), 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.

[0260] 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 Semulki 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 embodiments where gene transfer is mediated by a retroviral vector, the vector construct refers to a polynucleotide comprising the retroviral genome or a portion thereof and a therapeutic gene. Further details about current vector methods for use in gene transfer can be found, for example, in Kotterman et al. (2015), "Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering," p. 17.

[0261] 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.

[0262] 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.

[0263] 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, PCT International Patent Application Publication No. WO95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the potential for recombination and generation of wild-type viruses. See, PCT International Patent Application Publication Nos. WO95 / 00655 and WO95 / 11984. Wild-type AAV has high infectivity and specificity for integration into the host cell genome. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0264] 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, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate 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' of the initiation codon to enhance expression.

[0265] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can be used in the methods disclosed herein. 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 culture conditions that enhance the expression and / or activity of the proteins disclosed herein are other non-limiting techniques.

[0266] As used herein, the terms "antibody," "antibody," and "immunoglobulin" include whole antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" encompasses any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule. The terms "antibody," "antibody," and "immunoglobulin" also encompass immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies; multispecific antibody fragments formed from antibody fragments, and isolated fragments of one or more antibodies. Examples of such antibodies include, but are not limited to, a heavy or light chain complementarity-determining region (CDR) or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, at least a portion of a binding protein, a chimeric antibody, a humanized antibody, a species-specific antibody, a single-chain antibody, and fusion proteins and non-antibody proteins comprising 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 tissues. When the term "anti-" is used before the name of a protein, e.g., anti-IHF, anti-HU, anti-OMP P5, refers to a monoclonal or polyclonal antibody that binds to and / or has affinity for a specific protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody may have affinity for or bind to proteins other than the protein against which it was raised. For example, anti-IHF may be raised specifically against the IHF protein but may also bind to other proteins related by sequence or structural homology.

[0267] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific), diabodies, and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, for example, human, mouse, rat, sheep, and dog.

[0268] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies are highly specific because each is directed against a single determinant on an antigen. Antibodies can be detectably labeled, for example, with radioisotopes, enzymes that generate detectable products, fluorescent proteins, etc. 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.

[0269] Monoclonal antibodies can be produced using hybridoma or recombinant DNA methods known in the art. Hybridomas are cells produced in laboratories by fusing antibody-producing lymphocytes with non-antibody-producing cancer cells, usually myeloma or lymphoma. Hybridomas grow and produce continuous samples of specific monoclonal antibodies. Alternative techniques for producing or selecting antibodies include exposing lymphocytes to the antigen of interest in vitro and screening antibody display libraries in cell, phage, or similar systems.

[0270] 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 disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, 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 the protein (e.g., CDRs, framework, CL domain, CH domain (e.g., CH1, CH2, CH3), hinge, (VL, VH)) are substantially non-immunogenic in humans and contain only minor sequence changes or mutations. Similarly, antibodies designated for primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rat, rabbit, guinea pig, hamster, etc.), and other mammals refer to antibodies specific to that species, subgenus, genus, subfamily, or family. Furthermore, chimeric antibodies include any combination of the above. Such alterations or mutations may retain or be less immunogenic than the unmodified antibody in humans or other species, as the case may be. Thus, human antibodies differ from chimeric or humanized antibodies. It has been noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Furthermore, when a human antibody is a single-chain antibody, it may contain a linker peptide not found in native human antibodies. For example, an Fv may contain 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. Such linker peptides are considered to be of human origin.

[0271] 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., mouse 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.

[0272] "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.

[0273] 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 VH and VL regions of the recombinant antibodies 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.

[0274] 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.

[0275] 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. For the most part, 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 regions, constant region, or CDRs that are substituted for similarly positioned amino acids from a human antibody. In general, 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. The term "speciation" refers to antibodies that have been modified in the same or similar manner for non-human species.

[0276] The terms "polyclonal antibody" or "polyclonal antibody composition," as used herein, refer to a preparation of antibodies derived from different B-cell lines. These are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope. In some embodiments, the antibody or antigen-binding fragment is not a polyclonal antibody.

[0277] 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, such as by alkylation, pegylation, acylation, ester formation, or amide formation, for example, to link the antibody to a second molecule. Antibody derivatives include, but are not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof.

[0278] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition, e.g., an N-terminal histidine tag (N-His), a magnetically 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). While the term "label" generally contemplates a composition covalently attached to the composition to be detected, in one embodiment, the term "label" specifically excludes naturally occurring nucleosides and amino acids that are known to fluoresce under certain conditions (e.g., temperature, pH, etc.) when placed within a polynucleotide or protein in their natural environment, and generally excludes any natural fluorescence that may be present in the composition to be detected. Labels may be detectable themselves (e.g., radioisotope labels or fluorescent labels) or, in the case of enzymatic labels, may catalyze chemical alteration of a substrate compound or composition that is detectable. Labels 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, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels can be simply detected or quantified. A simply detected reaction generally involves a reaction whose presence is simply confirmed, while a quantified reaction generally involves a reaction that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other properties. In luminescent or fluorescent assays, the detectable reaction can be generated directly using a luminophore or fluorophore attached to the assay component actually involved in the binding, or indirectly using a luminophore or fluorophore attached to another component (e.g., a reporter or indicator). Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent reaction generally involves a change or appearance of a luminescent signal.Suitable methods and luminophores for luminescently labeling assay components are known in the art, see, eg, 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.

[0279] 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 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.

[0280] 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.).

[0281] 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.

[0282] "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.

[0283] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells may also contain genetic information within circular loops called epitopes. 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.

[0284] 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.

[0285] The terms "antigen" and "antigenic" refer to a molecule capable of being recognized by an antibody or otherwise serving 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 serve as antigens. Those skilled in the art will further understand that nucleic acids encoding proteins with the potential to serve 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.

[0286] 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, for example, by 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 disclosed herein are intended to bind to the same TCR as the natural epitope.

[0287] "Autoantigens," also referred to herein as native antigens or wild-type antigens, are antigenic peptides that induce little or no immune response in a subject due to self-tolerance to the antigen. An example of an autoantigen is the melanoma-specific antigen gp100.

[0288] "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. All immunogens are antigens, but not all antigens are immunogenic. The immune responses disclosed herein can be humoral (through antibody activity) or cell-mediated (through T cell activation). The response can occur in vivo or in vitro. Those skilled in the art will understand 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 understand that a nucleic acid encoding a molecule capable of eliciting an immune response necessarily encodes an immunogen. Those skilled in the art will further understand that an immunogen is not limited to a full-length molecule but can include a partial molecule.

[0289] 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.

[0290] As used herein, the term "inducing an immune response in a subject" is a term well understood in the art and means being able to detect or measure an increase in the immune response to an antigen (or epitope) of at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold or more after introducing the antigen (or epitope) into a subject, compared to the immune response (if any) before introducing the antigen (or epitope) into the subject. An immune response to an antigen (or epitope) includes, but is not limited to, the production of antigen-specific (or epitope-specific) antibodies and the production of immune cells expressing on their surface a molecule that specifically binds to the antigen (or epitope). Methods for determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art. For example, antigen-specific antibodies can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, in which binding of antibodies in a sample to an immobilized antigen (or epitope) is detected using a detectably labeled second antibody (e.g., an enzyme-labeled mouse anti-human Ig antibody).

[0291] 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® resin (available from Aminotech, Canada), polyamide resin (available from Peninsula Laboratories), polyethylene glycol-grafted polystyrene resin (TentaGel®, Rapp Polymere, Tübingen, Germany) or polydimethylacrylamide resin (available from Milligen / Biosearch, Calif.).

[0292] 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 can be soluble to some extent or insoluble. The support material can 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 can 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 can 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 use of routine experimentation.

[0293] Antibodies, their fragments, and derivatives The present disclosure provides antibodies that bind to and / or specifically recognize and bind to isolated polypeptides for use in the methods disclosed herein. The antibodies can be any of the various antibodies described herein, non-limiting examples of which include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, veneered antibodies, diabodies, humanized antibodies, antibody derivatives, recombinant humanized antibodies, or their respective derivatives or fragments. In one aspect, the fragment comprises, consists essentially of, or further consists of the CDRs of the antibody, examples of which are provided herein. In one aspect, the antibody is detectably labeled or further comprises a detectable and / or purification label conjugated thereto. Also provided are hybridoma cell lines that produce the monoclonal antibodies disclosed herein. Further provided herein are compositions comprising, consisting essentially of, or further consisting of one or more of the above embodiments. Further provided herein are polynucleotides encoding the amino acid sequences of the antibodies and fragments, as well as methods for recombinantly producing or chemically synthesizing antibody polypeptides and fragments thereof. Antibody polypeptides can be produced in eukaryotic or prokaryotic cells, or by other methods known in the art and outlined herein.The antibodies disclosed herein can be selected so that they have a high level of epitope binding specificity and high binding affinity to biofilms.Generally, the greater the binding affinity of the antibody, the more stringent the washing conditions can be implemented in immunoassays to remove non-specifically bound materials without removing the target.Therefore, the antibodies of the present technology useful in the disclosed methods are usually at least 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , or 10 -12In certain embodiments, the antibody has a binding affinity of less than or equal to about 1000 picomolar (pM), less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, or less than about 10 pM, or about 9 pM, or about 8 pM, or alternatively, less than about 4 pM, or alternatively, less than about 2 pM.

[0294] Antibodies such as equivalent antibodies can also 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. When an antigen is injected into a mammal, it induces B lymphocytes to produce immunoglobulins specific to the antigen. Immunoglobulins can be purified from the serum of the mammal. Antibodies specific to the IHFα subunit and / or IHFβ subunit can be produced by injecting polypeptides corresponding to different epitopes of IHFα and IHFβ. For example, antibodies can be produced using 20 amino acid subunits such as SEQ ID NOs: 12 and 13 (fragments A3 and A5 of IHF, respectively) for IHFα, SEQ ID NO: 14 (fragment A5 of HU) for HU, and SEQ ID NOs: 15 to 17 (fragments B2, B4, and mB4 of IHF) for IHFβ.

[0295] Additionally, antibodies can be generated against the "tip" region of the DNABII protein, which in one aspect contains or has been altered to contain an antiparallel beta ribbon turn and / or the sequence NPXT, where "X" refers to any amino acid. In some embodiments, X is selected from the amino acids Q, R, K, S, or T. Such antibodies can be generated using fragments of the DNABII protein that contain the NPXT sequence, optionally flanked by between about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids on either or both sides of the sequence. Non-limiting examples of such antibodies are provided herein, including those based on the consensus amino acid sequence [ka] In another embodiment, antibodies can be raised against the apical region of the DNABII protein, which leads to the consensus sequence NPXT, where "X" is any amino acid, or alternatively, X is selected from the amino acids Q, R, K, S, or T. Those skilled in the art will recognize that in any one of these sequences, the residue at position X (marked with a double underline in the examples below) can be substituted with any amino acid (e.g., Q, R, K, S, or T). Examples are:

[0296] SEQ ID NO: 13: Haemophilus influenzae IhfA; A5 fragment: [ka]

[0297] SEQ ID NO: 14: Haemophilus influenzae HU; A5 fragment: [ka]

[0298] SEQ ID NO: 17: Haemophilus influenzae IhfB; modified B4 (mB4) fragment: [ka]

[0299] SEQ ID NO: 26: Haemophilus influenzae IhfA; A tip fragment: [ka]

[0300] SEQ ID NO: 27: Haemophilus influenzae IhfB; B tip fragment: [ka]

[0301] SEQ ID NO: 31: Haemophilus influenzae HU; fragment: [ka] Includes.

[0302] In some embodiments, the polypeptide comprising NPXT is at least about 20 amino acids long, and NPXT is centrally located within the sequence.Non-limiting examples of such sequences include SEQ ID NOs: 13, 14, and 31.Alternatively, polypeptides with NPXT motifs (for example, as mentioned above) can be modified to remove NPXT motifs by substituting or deleting one or more amino acids, and monoclonal antibodies can be raised against these polypeptides.Applicants have determined that antibodies raised against DNABII polypeptides that lack NPXT motifs are useful in diagnostic methods for imaging and monitoring biofilm formation and / or destruction.In one aspect, a kit is also provided that includes an antibody raised against or binding to DNABII with NPXT motifs and an antibody raised against or binding to DNABII (for example, modified polypeptide or naturally occurring polypeptide) that lacks NPXT motifs. For example, the kit may include antibodies that recognize and bind to polypeptides A5, B4, or mB4 (therapeutic), or may include antibodies that recognize and bind to polypeptides A3 or B2 (diagnostic). The kits are useful for diagnosis, treatment, and monitoring of biofilm treatment.

[0303] Variations of this method include modifying the adjuvant, route and site of administration, injection volume per site, and number of sites per animal to optimize production and ensure humane animal handling. For example, adjuvants are typically used to improve or enhance the immune response to an antigen. Most adjuvants cause antigen retention at the injection site, allowing for stowed release of the antigen into the draining lymph nodes. Other adjuvants include surfactants, which promote the concentration of protein antigen molecules over a large surface area, and immunostimulatory molecules. Non-limiting examples of adjuvants for generating polyclonal antibodies include Freund's adjuvant, the Ribi adjuvant system, adjuvants derived from MPL, adjuvants derived from E. coli heat-labile enterotoxin (e.g., dmLT = double mutant heat-labile toxin), and Titermax. Polyclonal antibodies can be produced using methods known in the art, some of which are described in U.S. Patent Nos. 7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746; 6,322,788; 5,686,073; and 5,670,153.

[0304] 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, MIA 144 cells, ACT IV cells, MOLT4 cells, DA-1 cells, JURKAT cells, WEHI cells, K-562 cells, COS cells, RAJI cells, NIH 313 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 / O 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, peripheral blood, lymph, tonsil, or other immune or B-cell containing cells, or recombinant or endogenous viruses, bacteria, algae, prokaryotes, amphibians, insects, reptiles, fish, mammals, rodents, horses, sheep (o 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 as nucleic acids of eukaryotic 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, etc., or any combination thereof, of a mammalian animal.Antibody-producing cells can also be obtained from the peripheral blood, or in certain embodiments, 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 disclosure. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other appropriate known methods and cloned via limiting dilution, cell sorting, or other known methods. Some embodiments disclosed herein relate to specific hybridomas producing monoclonal antibodies against Ihf fragments, non-limiting examples include IhfA5 NTHI 14G8.F5.G6 (ATCC No.: PTA-122334), IhfB4 NTHI 4E11.E5.G2 (ATCC No.: PTA-122336), and mIhfmB4 NTHI 12E6.F8.D12.D5 (ATCC No.: PTA-122335).

[0305] Other suitable methods for producing or isolating antibodies with the required specificity can be used, including, but not limited to, selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, bacteriophage display libraries, ribosome display libraries, oligonucleotide 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), Mumma 93:154-161) to produce a repertoire of human antibodies, as known in the art and / or described herein. Such techniques include ribosome display (e.g., Wanes et al. (1997), Proc. Natl. Acad. Sci. USA 94:4937-4942; Hanes et al. (1998), Proc. Natl. Acad. Sci. USA 95:14130-14135), single-cell antibody production methods (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987), J. Immunol. 17:887-892; Babcook et al. (1996), Proc. Natl. Acad. Sci. USA 93:7843-7848), gel microdroplets and flow cytometry (Powell et al. (1990), Biotechnol. 8:333-337; One Cell Systems (Cambridge, Mass); Gray et al. (1995), J. Imm. Meth., 182:155-163; and Kenny et al. (1995), Bio. Technol., 13:787-790), B cell selection (Steenbakkers et al. (1994), Molec. Biol. Reports, 19:125-134).

[0306] Antibodies can also be produced by inducing lymphocyte populations in vivo or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents (Orlandi et al. (1989) PNAS 86:3833-3837; Winter et al. (1991) Nature 349:293-299).

[0307] Alternatively, techniques for producing single chain antibodies can be used. Single chain antibodies (scF v ) comprises a heavy chain variable region and a light chain variable region connected by a linker peptide (typically about 5 to 25 amino acids in length). v Within an scF, the heavy and light chain variable regions may be derived from the same antibody or from different antibodies. v have used recombinant techniques to express scF in a host organism such as E. coli.v scF can be synthesized by expression of a vector encoding v The DNA encoding scF can be obtained by using a DNA selected from the DNA encoding the heavy chain or the heavy chain variable region of the above-mentioned antibody and the DNA encoding the light chain or the light chain variable region thereof as a template, and amplifying the entire amino acid sequence or a partial DNA encoding a desired amino acid sequence using a primer pair that defines both ends of the template, and further amplifying the DNA encoding the polypeptide linker portion with a primer pair that defines both ends of the template so that both ends of the linker are ligated to the heavy chain and the light chain, respectively. v An expression vector containing DNA encoding the above and a host transformed with the expression vector can be obtained according to conventional methods known in the art.

[0308] Antigen-binding fragments can also be produced, such as F(ab')2 fragments, which can be produced by pepsin digestion of antibody molecules, and Fab fragments, which can be generated by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed, which allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 256:1275-1281).

[0309] Antibody derivatives of the present disclosure can also be prepared by delivering a polynucleotide encoding an antibody or fragment thereof disclosed herein to a suitable host to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces such an antibody in its milk. 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.

[0310] 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 methods for producing modified glycoforms of antibodies, including whole antibody molecules, antibody fragments, or fusion proteins, that contain a region equivalent to the Fc region of an immunoglobulin and have enhanced Fe-mediated cytotoxicity, and the glycoproteins thus produced.

[0311] The antibodies disclosed herein also encompass derivatives 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 modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization via known protecting / blocking groups, proteolytic cleavage, conjugation to intracellular ligands or other proteins, and the like. Non-limiting examples of modified antibodies contemplated herein are unglycosylated whole antibody molecules, antibody fragments, or fusion proteins containing a region equivalent to the Fc region of an immunoglobulin. Such unglycosylated forms can be produced, for example, in host cells lacking the ability to modify proteins with N-linked glycans, or by mutating N-linked consensus sites on the antibody of interest. In addition, derivatives can contain one or more non-classical amino acids.

[0312] 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 the polynucleotides disclosed herein 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.

[0313] 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, the non-human sequences of the variable and constant regions are substituted with human or other amino acids, while maintaining some or all of the non-human or human CDR sequences.

[0314] 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.

[0315] The general structure of an antibody is known in the art, and will only be briefly summarized here. An immunoglobulin monomer contains two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is paired with one of the light chains to which it is directly linked via a disulfide bond. Each heavy chain contains a constant region (which varies depending on the antibody isotype) and a variable region. The variable region contains three hypervariable regions (or complementarity-determining regions), designated CDRH1, CDRH2, and CDRH3, supported within framework regions. Each light chain contains a constant region and a variable region, and the variable region, like the heavy chain variable region, contains three hypervariable regions (designated CDRL1, CDRL2, and CDRL3), supported by framework regions.

[0316] The hypervariable regions of each pair of heavy and light chains cooperate with each other to form an antigen-binding site capable of binding to a target antigen. The binding specificity of a pair of heavy and light chains is defined by the sequences of CDR1, CDR2, and CDR3 of the heavy and light chains. Therefore, once a set of CDR sequences (i.e., the sequences of CDR1, CDR2, and CDR3 of the heavy and light chains) that confer a particular binding specificity is determined, the set of CDR sequences can, in principle, be inserted into an appropriate position in any other antibody framework region linked to the constant region of any antibody to create a different antibody with the same antigen-binding specificity.

[0317] In certain aspects, the present disclosure relates to antibodies or antigen-binding fragments that specifically recognize or specifically bind to an isolated or recombinant polypeptide comprising or consisting essentially of an amino acid sequence selected from SEQ ID NOs: 12-17, SEQ ID NO: 31, SEQ ID NO: 33, a polypeptide comprising the amino acid sequence NPXT, or their respective equivalents. In some embodiments, the antibody or antigen-binding fragment is not a polyclonal antibody. Non-limiting exemplary antibodies produced by the disclosed hybridomas are disclosed in Table 1. Hybridoma cell lines producing monoclonal antibodies that specifically recognize and bind to Haemophilus influenzae IhfA fragment A5 (SEQ ID NO: 13), IhfB fragment B4 (SEQ ID NO: 16), and IhfB fragment mB4 (SEQ ID NO: 17) were deposited with the American Type Culture Collection (ATCC) pursuant to the terms of the Budapest Treaty on July 30, 2015, under the accession numbers listed in Table 1, and each hybridoma cell line is listed in Table 1. Further non-limiting exemplary antibodies include antibodies that specifically recognize and specifically bind to the Haemophilus influenzae IhfA fragment, A3 (SEQ ID NO: 12), or the IhfB fragment, B2 (SEQ ID NO: 15), produced by hybridoma cell lines IhfA3 NTHI 9B10.F2.H3, IhfB2 NTHI 7A4.E4.G4, and IhfB2 NTHI 7A4.E4.G11 (these hybridomas were deposited with the American Type Culture Collection (ATCC) under the terms of the Budapest Treaty on August 1, 2016, under the accession numbers listed in Table 1); and antibodies that specifically recognize or specifically bind to a polypeptide comprising SEQ ID NO: 31, SEQ ID NO: 33, the amino acid sequence NPXT, or their respective equivalents. [Table 1-1]

[0318] In one aspect, the disclosure provides an isolated antibody that is at least 85% identical to an antibody selected from the group consisting of: (i) an antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) an antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0319] In one aspect, the disclosure provides an isolated antibody comprising a CDR of (i) an antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5. In one aspect, the disclosure provides an isolated antibody having a CDR that is at least 85% identical to (i) an antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0320] In some embodiments of the antibodies provided herein, the HC variable domain sequence comprises the HC variable domain sequence of (i) an antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line MIhfB4 NTHI 12E6.F8.D12.D5; and / or the LC variable domain sequence comprises the HC variable domain sequence of (i) an antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0321] In some embodiments of the antibodies provided herein, the HC variable domain sequence comprises an HC variable domain sequence at least 85% identical to the HC variable domain sequence of (i) an antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) an antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5; and / or the LC variable domain sequence comprises an HC variable domain sequence at least 85% identical to the HC variable domain sequence of (i) an antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) an antibody produced by hybridoma cell line mIhfmB4 NTHI It comprises an LC variable domain sequence that is at least 85% identical to the LC variable domain sequence of the antibody produced by 12E6.F8.D12.D5.

[0322] In one aspect, the disclosure provides an isolated antibody comprising a heavy chain (HC) variable domain sequence and a light chain (LC) variable domain sequence, wherein the heavy chain immunoglobulin variable domain sequence and the light chain immunoglobulin variable domain sequence form an antigen binding site that binds to an epitope of a DNABII protein.

[0323] In some embodiments, the heavy chain variable region comprises a CDRH1 sequence comprising, alternatively consisting essentially of, and / or further consisting of, an amino acid sequence comprising the CDRH1 of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0324] In some embodiments, the heavy chain variable region comprises a CDRH2 sequence comprising, alternatively consisting essentially of, and / or further consisting of, an amino acid sequence comprising the CDRH2 of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line IhfmB4 NTHI 12E6.F8.D12.D5.

[0325] In some embodiments, the heavy chain variable region comprises a CDRH3 sequence that comprises, alternatively consists essentially of, and / or further consists of an amino acid sequence comprising the CDRH3 of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0326] In some embodiments, the heavy chain variable region comprises, or alternatively consists essentially of, and / or further consists of, an amino acid sequence comprising the heavy chain variable region sequence of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0327] In some embodiments, the light chain variable region comprises a CDRL1 sequence that comprises, alternatively consists essentially of, and / or further consists of an amino acid sequence comprising the CDRL1 of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0328] In some embodiments, the light chain variable region comprises a CDRL2 sequence comprising, alternatively consisting essentially of, and / or further consisting of, an amino acid sequence comprising the CDRL2 of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0329] In some embodiments, the light chain variable region comprises a CDRL3 sequence that comprises, alternatively consists essentially of, and / or further consists of an amino acid sequence comprising the CDRL3 of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0330] In some embodiments, the light chain variable region comprises, or alternatively consists essentially of, and / or further consists of, a polypeptide encoded by a polynucleotide sequence comprising the light chain variable region sequence of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0331] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH1 sequence comprising, alternatively consisting essentially of, and / or further consisting of an amino acid sequence comprising FSLTSYS (SEQ ID NO: 58), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of FSLTSYSV (SEQ ID NO: 59), FSLTSYSVH (SEQ ID NO: 60), GFSLTSYS (SEQ ID NO: 61), or their respective biological equivalents.

[0332] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH1 sequence comprising, alternatively consisting essentially of, and / or further consisting of an amino acid sequence comprising FNIKDYY (SEQ ID NO: 110), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of FNIKDYYM (SEQ ID NO: 111), FNIKDYYMH (SEQ ID NO: 112), GFNIKDYY (SEQ ID NO: 113), or their respective biological equivalents.

[0333] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH2 sequence comprising, alternatively consisting essentially of, and / or further consisting of an amino acid sequence comprising IWAGGST (SEQ ID NO:62), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of VIWAGGST (SEQ ID NO:63), GVIWAGGST (SEQ ID NO:64), LGVIWAGGST (SEQ ID NO:65), WLGVIWAGGST (SEQ ID NO:66), IWAGGSTN (SEQ ID NO:67), VIWAGGSTN (SEQ ID NO:68), GVIWAGGSTN (SEQ ID NO:69), LGVIWAGGSTN (SEQ ID NO:70), WLGVIWAGGSTN (SEQ ID NO:71), IWAGGSTNY (SEQ ID NO:72), VIWAGGSTNY (SEQ ID NO:73), GVIWAGGSTNY (SEQ ID NO:74), LGVIWAGGSTNY (SEQ ID NO:75), WLGVIWAGGSTNY (SEQ ID NO:76), or their respective biological equivalents.

[0334] In some embodiments, the heavy chain variable region of the antibody or fragment thereof has an amino acid sequence comprising IDPENDDT (SEQ ID NO: 114), and is selected from the group consisting of WIDPENDDT (SEQ ID NO: 115), GWIDPENDDT (SEQ ID NO: 116), IGWIDPENDDT (SEQ ID NO: 117), WIGWIDPENDDT (SEQ ID NO: 118), IDPENDDTE (SEQ ID NO: 119), WIDPENDDTE (SEQ ID NO: 120), GWIDPENDDTE (SEQ ID NO: 121), IGWIDPENDDTE (SEQ ID NO: 122), WIGWIDPENDDTE (SEQ ID NO: 123), and CDRH2 sequences including, alternatively consisting essentially of, and / or further consisting of, an amino acid sequence such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of SEQ ID NO: 123), IDPENDDTEY (SEQ ID NO: 124), WIDPENDDTEY (SEQ ID NO: 125), GWIDPENDDTEY (SEQ ID NO: 126), IGWIDPENDDTEY (SEQ ID NO: 127), WIGWIDPENDDTEY (SEQ ID NO: 128), or their respective biological equivalents.

[0335] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH3 sequence that comprises, alternatively consists essentially of, and / or further consists of an amino acid sequence comprising AREDS (SEQ ID NO: 77), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of AREDS (SEQ ID NO: 78) or a biological equivalent thereof.

[0336] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH3 sequence comprising, alternatively consisting essentially of, and / or further consisting of, an amino acid sequence comprising TELGAY (SEQ ID NO: 129) or a biological equivalent thereof.

[0337] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises the polynucleotide sequence referred to below: [ka] or a biological equivalent thereof.

[0338] In some embodiments, the heavy chain variable region of the antibody or fragment thereof has the amino acid sequence: [ka] or a biological equivalent thereof, or alternatively consisting essentially of, and / or further consisting of.

[0339] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises the polynucleotide sequence referred to below: [ka] [ka] or a biological equivalent thereof.

[0340] In some embodiments, the heavy chain variable region of the antibody or fragment thereof has the amino acid sequence: [ka] or a biological equivalent thereof, or alternatively consisting essentially of, and / or further consisting of.

[0341] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL1 sequence comprising, alternatively consisting essentially of, and / or further consisting of an amino acid sequence comprising QNVGTN (SEQ ID NO: 79), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of QNVGTNV (SEQ ID NO: 80), QNVGTNVA (SEQ ID NO: 81), or their respective biological equivalents.

[0342] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL1 sequence comprising, alternatively consisting essentially of, and / or further consisting of an amino acid sequence comprising QSLLDSNGKTY (SEQ ID NO: 130), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of QSLLDSNGKTYL (SEQ ID NO: 131), QSLLDSNGKTYLN (SEQ ID NO: 132), or their respective biological equivalents.

[0343] In some embodiments, the light chain variable region of the antibody or fragment thereof has an amino acid sequence comprising SAS (SEQ ID NO: 82), and is selected from the group consisting of YSAS (SEQ ID NO: 83), IYSAS (SEQ ID NO: 84), LIYSAS (SEQ ID NO: 85), ALIYSAS (SEQ ID NO: 86), SASY (SEQ ID NO: 87), YSASY (SEQ ID NO: 88), IYSASY (SEQ ID NO: 89), LIYSASY (SEQ ID NO: 90), ALIYSASY (SEQ ID NO: 91), SASYR (SEQ ID NO: 92), YSASYR (SEQ ID NO: 93), IYSASYR (SEQ ID NO: 94), LIYSASYR (SEQ ID NO: 95), ALIYSASYR (SEQ ID NO: 96), SASYRY (SEQ ID NO: 97), YS CDRL2 sequences include, or alternatively consist essentially of, and / or further consist of, an amino acid sequence such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of ASYRY (SEQ ID NO: 98), IYSASYRY (SEQ ID NO: 99), LIYSASYRY (SEQ ID NO: 100), ALIYSASYRY (SEQ ID NO: 101), SASYRYS (SEQ ID NO: 102), YSASYRYS (SEQ ID NO: 103), IYSASYRYS (SEQ ID NO: 104), LIYSASYRYS (SEQ ID NO: 105), ALIYSASYRYS (SEQ ID NO: 106), or their respective biological equivalents.

[0344] In some embodiments, the light chain variable region of the antibody or fragment thereof has an amino acid sequence comprising LVS (SEQ ID NO: 133), and is selected from the group consisting of YLVS (SEQ ID NO: 134), IYLVS (SEQ ID NO: 135), LIYLVS (SEQ ID NO: 136), RLIYLVS (SEQ ID NO: 137), LVSK (SEQ ID NO: 138), YLVSK (SEQ ID NO: 139), IYLVSK (SEQ ID NO: 140), LIYLVSK (SEQ ID NO: 141), RLIYLVSK (SEQ ID NO: 142), LVSKL (SEQ ID NO: 143), YLVSKL (SEQ ID NO: 144), IYLVSKL (SEQ ID NO: 145), LIYLVSKL (SEQ ID NO: 146), RLIYLVSKL (SEQ ID NO: 147), LVSKLD (SEQ ID NO: 148), and CDRL2 sequences comprising, alternatively consisting essentially of, and / or further consisting of, an amino acid sequence such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of: YLVSKLD (SEQ ID NO: 148), YLVSKLD (SEQ ID NO: 149), IYLVSKLD (SEQ ID NO: 150), LIYLVSKLD (SEQ ID NO: 151), RLIYLVSKLD (SEQ ID NO: 152), LVSKLDS (SEQ ID NO: 153), YLVSKLDS (SEQ ID NO: 154), IYLVSKLDS (SEQ ID NO: 155), LIYLVSKLDS (SEQ ID NO: 156), RLIYLVSKLDS (SEQ ID NO: 157), or their respective biological equivalents.

[0345] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL3 sequence that comprises, alternatively consists essentially of, and / or further consists of an amino acid sequence comprising QQYNSYP (SEQ ID NO: 108), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of QQYNSYPT (SEQ ID NO: 109), or a biological equivalent thereof.

[0346] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL3 sequence that comprises, alternatively consists essentially of, and / or further consists of an amino acid sequence comprising WQSTHFPH (SEQ ID NO: 158), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of WQSTHFPHT (SEQ ID NO: 159) or a biological equivalent thereof.

[0347] In some embodiments, the light chain variable region of the antibody or fragment thereof has the polynucleotide sequence: [ka] [ka] or a biological equivalent thereof.

[0348] In some embodiments, the light chain variable region of the antibody or fragment thereof has the amino acid sequence: [ka] or a biological equivalent thereof, or alternatively consisting essentially of, and / or further consisting of.

[0349] In some embodiments, the light chain variable region comprises the polynucleotide sequence: [ka] or a biological equivalent thereof.

[0350] In some embodiments, the light chain variable region has the amino acid sequence: [ka] or a biological equivalent thereof, or alternatively consisting essentially of, and / or further consisting of.

[0351] Exemplary antibodies comprising the disclosed CDR sequences and heavy and light chain variable sequences are disclosed in Tables 6 and 7, respectively. Alternative CDR predictions can be made based on the heavy and / or light chain sequences (e.g., based on the Kabat, Cothia, AbM, or contact definitions of CDR specificity; details of these CDR prediction methods are known in the art (see, e.g., bioinf.org.uk / abs / #cdrid) and / or are commercially available). The results disclosed in Table 6 were generated using the CDR prediction algorithm provided by Ofran Lab (Paratome, available at ofranservices.biu.ac.il / site / services / paratome / index.html) and Green This is the result of utilizing Mountain Antibodies' CDR prediction program. [Table 6] [Table 7]

[0352] In one aspect, the present disclosure provides an isolated antibody that is at least 85%, or alternatively, at least 90%, or alternatively, at least 95% identical to an antibody selected from the group consisting of Ihf A5, Ihf mB4, or their respective biological equivalents.

[0353] In one aspect, the present disclosure provides an isolated antibody comprising the CDRs of Ihf A5. In one aspect, the present disclosure provides an isolated antibody that is at least 85%, or alternatively, at least 90%, or alternatively, at least 95% identical to Ihf A5 or a biological equivalent thereof.

[0354] In one aspect, the present disclosure provides an isolated antibody comprising the CDRs of Ihf mB4. In one aspect, the present disclosure provides an isolated antibody that is at least 85%, or alternatively, at least 90%, or alternatively, at least 95% identical to Ihf mB4 or a biological equivalent thereof.

[0355] In some embodiments of the antibodies provided herein, the HC variable domain sequence comprises the variable domain sequence of Ihf A5, and the LC variable domain sequence comprises the variable domain sequence of Ihf A5.

[0356] In some embodiments of the antibodies provided herein, the HC variable domain sequence comprises the variable domain sequence of Ihf mB4 and the LC variable domain sequence comprises the variable domain sequence of Ihf mB4.

[0357] Further provided herein are isolated polynucleotides encoding the above-referenced polypeptides, vectors and host cells containing the same, and methods for recombinantly producing the polypeptides using recombinant cell lines known in the art and described herein.

[0358] In another aspect of the present technology, the isolated antibody has the following characteristics: (a) the light chain immunoglobulin variable domain sequence comprises one or more CDRs at least 85%, or alternatively at least 90%, or alternatively at least 95% identical to the CDRs of the light chain variable domain of any of the disclosed light chain sequences; (b) the heavy chain immunoglobulin variable domain sequence comprises one or more CDRs at least 85%, or alternatively at least 90%, or alternatively at least 95% identical to the CDRs of the heavy chain variable domain of any of the disclosed heavy chain sequences; (c) the light chain immunoglobulin variable domain sequence is at least 85%, or alternatively, at least 90%, or alternatively, at least 95% identical to the light chain variable domain of any of the disclosed light chain sequences; (d) the HC immunoglobulin variable domain sequence is at least 85%, or alternatively, at least 90%, or alternatively, at least 95% identical to the heavy chain variable domain of any of the disclosed light chain sequences; and (e) the antibody binds to an epitope that overlaps with the epitope bound by any of the disclosed sequences Contains one or more of:

[0359] In some embodiments of the antibodies provided herein, the antibodies bind to the DNABII protein. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 A dissociation constant (K D ) In some embodiments of the antibodies provided herein, the antigen-binding site specifically binds to a DNABII protein. In other embodiments, the affinity of the antibody or antigen-binding fragment is less than or about 1000 picomolar (pM), 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, about 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, and 8 pM.

[0360] In some embodiments of the antibodies provided herein, the antibody is a soluble Fab.

[0361] In some embodiments of the antibodies provided herein, the HC variable domain sequence and the LC variable domain sequence are components of the same polypeptide chain. In some embodiments of the antibodies provided herein, the HC variable domain sequence and the LC variable domain sequence are components of different polypeptide chains.

[0362] In some embodiments of the antibodies provided herein, the antibody is a full-length antibody.

[0363] In some embodiments of the antibodies provided herein, the antibody is a monoclonal antibody.

[0364] In some embodiments of the antibodies provided herein, the antibody is a chimeric antibody or a humanized antibody.

[0365] In other aspects, the antibodies provided herein are diabodies.

[0366] In some embodiments of the antibodies provided herein, the antibody is a Fab, F(ab)'2, Fab', scF v , and F v is selected from the group consisting of:

[0367] In some embodiments of the antibodies provided herein, the antibody comprises an Fc domain. In some embodiments of the antibodies provided herein, the antibody is a rabbit antibody. In some embodiments of the antibodies provided herein, the antibody is a human antibody or a humanized antibody, or is non-immunogenic in humans.

[0368] In some embodiments of the antibodies provided herein, the antibody comprises a framework region of a human antibody (e.g., a humanized antibody). In some embodiments of the antibodies provided herein, the antibody comprises an antibody framework region derived from a non-human species, such as monkey, horse, cat, dog, cow, pig, goat, or sheep. In some embodiments of the antibodies provided herein, the antibody is a chimeric antibody. In any one or more of these embodiments, the antibody may further comprise one or more of the CDR regions and / or heavy and / or light chains disclosed herein.

[0369] In other embodiments, one or more residues within the CDRs of the antibodies provided herein are substituted with another amino acid. The substitutions may be "conservative," meaning that they are within the same amino acid family. Naturally occurring amino acids fall into four families:

[0370] 1) Amino acids with basic side chains: lysine, arginine, histidine;

[0371] 2) Amino acids with acidic side chains: aspartic acid, glutamic acid;

[0372] 3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine;

[0373] 4) Amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine and conservative substitutions will occur within these families.

[0374] In another embodiment, one or more residues are added to or deleted from one or more CDRs of the antibody, either at the N-terminus or C-terminus of the CDR or at a position within the CDR.

[0375] By varying the amino acid sequence of the antibody CDR by adding, deleting, or substituting amino acids, a variety of effects can be achieved, such as increasing the binding affinity for the target antigen.

[0376] It will be appreciated that antibodies of the present disclosure containing such variations in the CDR sequences will still bind to DNABII proteins with a specificity and sensitivity profile similar to the disclosed antibodies, which can be determined by binding assays.

[0377] The constant region of the antibody can also be varied. For example, antibodies can be provided with the Fc region of any isotype: IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), or IgM. Furthermore, the constant region of the antibody can be adapted to a specific therapeutic indication, for example, following the teachings of Irani et al. (2015), Molecular Immunol., 67:171-182.

[0378] As mentioned in Irani (2015), Fc engineering is important for developing therapeutic antibodies with potent and specific activity, thus reducing both dosage and potential side effects. Fc engineering has used an understanding of the differences across IgG subclasses, and studies have shown that introducing specific residues from one subclass into specific residues from another subclass can convert certain effector functions while retaining others (Armour et al. (1999), Eur. J. Immunol., 29:2613-2624; Armour et al. (2003), Mol. Immunol., 40:585-593; Hessell et al. (2007), Nature, 449:101-104; Redpath et al. (1998), Hum. Immunol., 59:720-727; Vafa et al. (2014), Methods (San Diego, CA), 65:114-126). These Fc engineering methods are relevant in the context of infectious disease because the effector functions of specific antibodies are often crucial in the efficient clearance of pathogens.

[0379] The structural and functional properties of IgG subclasses vary, as do their response profiles to different infectious diseases, and these differences can be exploited in the development of effective therapeutic antibodies (Carter (2006) Nat. Rev. Immunol. 6:343-357; Jefferis (2012) Arch. Biochem. Biophys. 526:159-166). Heavy chains share greater than 90% sequence identity across IgG subclasses (Rispens and Vidarsson (2014) Nimmerjahn, MEA (ed.), Chapter 9, "Human IgG Subclasses," Academic Press, Boston, pp. 159-177), but there are differences in surface-exposed residues in the constant (CH1, CH2, and CH3) domains, as well as substantial variation within the hinge region. It is the hinge structure that confers many of the unique properties of each IgG subclass, such as stability, flexibility, and the distance spanned by the two Fabs and associated Fc (Liu and May (2012), mAbs, 4:17-23; Roux et al. (1997), J. Immunol. (Baltimore, MD: 1950), 159:3372-3382; Tian et al. (2014), Pharm. Sci., 103:1701-1710). Importantly, some regions of the Fc and hinge that differ among IgG subclasses clearly overlap with residues known to be involved in binding to both activating and inhibitory Fcγ receptors (FcγRs), the neonatal IgG receptor (FcRn), and the complement component C1q. The occurrence of key amino acid differences within the binding sites of these effector molecules helps explain the observed differences in the effector properties of IgG subclasses. This structural and molecular information is important when selecting subclass frameworks for therapeutic antibodies or when introducing key amino acid changes to tailor antibodies for specific purposes.

[0380] Of the approved antibodies, the majority are IgG1, likely due to their potent effector functions. When lack of specific cellular activity is desired, IgG2 or IgG4 have been used. The lack of approved IgG3 therapeutic antibodies suggests that this is due to (i) the increased likelihood of proteolysis due to the extended hinge region (Carter, 2006), (ii) the prevalence of IgG3 allotypes among the population, (iii) the inability of IgG3 to be purified with protein A, or (iv) the short serum half-life of IgG3 compared to other subclasses, despite its high antibody-dependent cellular cytotoxicity (ADCC). IgG1 is the predominant class of antibody among therapeutic antibodies approved or under review in Europe or the United States, and in particular, therapeutic antibodies generated against infectious diseases caused by Gram-positive and Gram-negative bacteria.

[0381] Non-limiting examples of constant region sequences include:

[0382] Human IgD constant region; Uniprot:P01880 (SEQ ID NO: 34): [ka]

[0383] Human IgG1 constant region; Uniprot:P01857 (SEQ ID NO: 35): [ka]

[0384] Human IgG2 constant region; Uniprot:P01859 (SEQ ID NO: 36): [ka]

[0385] Human IgG3 constant region; Uniprot:P01860 (SEQ ID NO: 37): [ka]

[0386] Human IgM constant region; Uniprot:P01871 (SEQ ID NO: 38): [ka] [ka]

[0387] Human IgG4 constant region; Uniprot:P01861 (SEQ ID NO: 39): [ka]

[0388] Human IgA1 constant region; Uniprot:P01876 (SEQ ID NO: 40): [ka]

[0389] Human IgA2 constant region; Uniprot:P01877 (SEQ ID NO: 41): [ka]

[0390] Human Ig kappa constant region; Uniprot:P01834 (SEQ ID NO: 42): [ka]

[0391] In some embodiments, the antibody is specific for IhfA fragment A5 (SEQ ID NO: 13) and comprises a constant region derived from IgM or IgG, such as, but not limited to, IgG2 (in certain embodiments, IgG2a). When the antibody is a monoclonal antibody, exemplary isotypes include, but are not limited to, IgG, such as IgG1, IgG2, IgG3, and IgG4; IgM; IgA, such as IgA1 and IgA2; IgD; and IgE, and may preferably include IgG and IgM. The isotype and subclass of a monoclonal antibody can be determined, for example, by Ouchterlony test, ELISA, or radioimmunoassay (hereinafter referred to as "RIA"). Commercially available kits for identification (e.g., Mouse Typer Kit; Bio-Rad Laboratories, Inc.; and Rat Monoclonal Antibody Isotyping Test Kit: AbD Serotec) can be used.

[0392] In some embodiments, the antibody is specific for the IhfB fragment mB4 (SEQ ID NO: 17) and comprises a constant region derived from an IgG, such as, but not limited to, IgG1.

[0393] In some embodiments, the antibody comprises a heavy chain constant region that is at least 80% identical to the heavy chain constant region sequence of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0394] In some embodiments, the antibody comprises a light chain constant region that is at least 80% identical to the light chain constant region sequence of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5.

[0395] In some embodiments of the antibodies provided herein, the antibody binds to an epitope that is bound by a DNABII antibody.

[0396] In some embodiments of the antibodies provided herein, the antibody contains structural modifications that facilitate rapid binding and cellular uptake and / or slow release from cells. In some embodiments, the DNABII antibody contains a deletion in the antibody heavy chain CH2 constant region that facilitates rapid binding and cellular uptake and / or slow release from cells. In some embodiments, a Fab fragment is used to facilitate rapid binding and cellular uptake and / or slow release from cells. In some embodiments, a F(ab)'2 fragment is used to facilitate rapid binding and cellular uptake and / or slow release from cells.

[0397] The antibodies, fragments thereof, and equivalents thereof can be combined with a carrier, eg, a pharmaceutically acceptable carrier or other agent, to provide a formulation for use and / or storage.

[0398] The chimeric, humanized, or primatized antibodies of the present disclosure can be prepared based on the sequence of a reference monoclonal antibody prepared using standard molecular biology techniques. DNA encoding heavy and light chain immunoglobulins can be obtained from the desired hybridoma and engineered to contain non-reference (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, a mouse variable region can be linked to a human constant region using methods known in the art (U.S. Patent No. 4,816,567). To create a humanized antibody, a mouse CDR region can be inserted into a human framework using methods known in the art (U.S. Patent No. 5,225,539, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370). Similarly, to create primatized antibodies, murine CDR regions can be inserted into a primate framework using methods known in the art (PCT International Patent Application Publication Nos. WO93 / 02108 and WO99 / 55369).

[0399] Techniques for producing partially to fully human antibodies are known in the art, and any such technique can be used. According to one embodiment, fully human antibody sequences are produced in transgenic mice engineered to express human heavy and light chain antibody genes. Several such transgenic mouse strains have been generated that are capable of producing different classes of antibodies. B cells from transgenic mice producing the desired antibody can be fused to generate hybridoma cell lines for continuous production of the desired antibody (see, e.g., Russell et al. (2000), Infection and Immunity, April 2000:1820-1826; Gallo et al. (2000), European J. of Immun., 30:534-540; Green (1999), J. of Immun. Methods, 231:11-23; Yang et al. (1999A), J. of Leukocyte Biology, 66:401-410; Yang (1999B), Cancer Research, 59(6):1236-1243; Jakobovits (1998), Advanced Drug Reviews, 31:33-42; Green and Jakobovits (1998), J. Exp. Med., 188(3):483-495; Jakobovits (1998), Exp. Opin. Invest. Drugs, 7(4):607-614; Tsuda et al. (1997), Genomics, 42:413-421; Sherman-Gold (1997), Genetic Engineering News, 17(14); Mendez et al. (1997), Nature Genetics, 15:146-156; Jakobovits (1996), "Weir's Handbook of Experimental Immunology, The Integrated Immune System," IV, 194.1-194.7; Jakobovits (1995), Current Opinion in Biotechnology, 6:561-566; Mendez et al. (1995), Genomics, 26:294-307; Jakobovits (1994), Current Biology, 4(8):761-763; Arbones et al. (1994), Immunity, 1(4):247-260; Jakobovits (1993), Nature, 362(6417):255-258; Jakobovits et al. (1993), Proc. Natl. Acad. Sci. USA, 90(6):2551-2555; and U.S. Patent No. 6,075,181).

[0400] The antibodies disclosed herein 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.

[0401] Alternatively, the antibodies disclosed herein 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 internal domains or interdomain contacts. Therefore, changes limited to framework residues in the variable regions 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, while supporting residues remain unperturbed.

[0402] The "veneering" procedure utilizes available sequence data for human antibody variable domains compiled by Kabat et al. (1987), "Sequences of Proteins of Immunological Interest," 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, and other accessible 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.

[0403] The term "antibody derivative" also includes "diabodies", which are small antibody fragments with two antigen-binding sites, which 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, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. (See also U.S. Pat. No. 6,632,926 by Chen et al., which discloses antibody variants in which one or more amino acids are inserted into the hypervariable region of a parent antibody such that the binding affinity for a target antigen is at least about twice as strong as that of the parent antibody for that antigen.) A non-limiting exemplary method for producing diabodies utilizes an expression vector encoding the diabody and transfecting it into a bacterial host. Diabodies can be isolated from the resulting bacterial periplasm using methods such as immobilized metal ion affinity chromatography, culture, and ELISA, as well as other known purification methods. These isolated diabodies can then be characterized, for example, by immunoblotting or gel filtration. Additional methods for producing diabodies are known in the art, including, but not limited to, those disclosed in Takemura et al. (2000), Protein Eng., 13(8):583-588; and Methods in Molecular Biology, (2012), 907:713-727.

[0404] In some embodiments, the antibody derivative is a diabody, in which the heavy chain variable domain (VH) and light chain variable domain (VL) are the same as any one of the antibodies disclosed hereinabove. In some embodiments, the diabody is specific for IhfA fragment A5 (SEQ ID NO: 13). In some embodiments, the diabody is specific for IhfB fragment mB4 (SEQ ID NO: 17).

[0405] The term "antibody derivatives" further encompasses engineered antibody molecules, fragments, and single domains, e.g., scFv, dAb, nanobodies, minibodies, unibodies, and affibodies (Hudson (2005) Nature Biotech 23(9):1126-36; U.S. Patent Application Publication No. 2006 / 0211088; PCT International Patent Application Publication No. WO2007 / 059782; U.S. Patent No. 5,831,012).

[0406] 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 tandem pair of Ed segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0407] The antibodies disclosed herein can be recovered and purified from recombinant cell culture by known methods, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0408] Antibodies of the present disclosure include naturally occurring purified products, products of chemical synthetic procedures, and products produced via recombinant methods from eukaryotic hosts, including, for example, yeast, higher plant, 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.

[0409] If the test antibody binds to the protein or polypeptide, the test antibody and the antibody provided by the present disclosure are equivalent. It is also possible to determine, without undue experimentation, whether an antibody has the same specificity as an antibody disclosed herein by determining whether the test antibody prevents the antibody of the present disclosure from binding to the protein or polypeptide with which the antibody disclosed herein is normally reactive. If the test antibody competes with the antibody disclosed herein, as indicated by reduced binding by the monoclonal antibody disclosed herein, it is likely that the two antibodies bind to the same epitope or a closely related epitope. Alternatively, the antibody disclosed herein can be preincubated with the protein with which it is normally reactive to determine whether the test antibody's ability to bind to the antigen is inhibited. If the test antibody is inhibited, it is highly likely that the antibody has the same epitope specificity as the antibody disclosed herein or a closely related epitope specificity.

[0410] 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.

[0411] 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.

[0412] In some embodiments disclosed herein, 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.

[0413] The sensitivity of an antibody in an assay can be increased by coupling 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.

[0414] The variable regions of the antibodies of the present disclosure 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. In certain embodiments, conservative modifications are introduced, typically changing no more than one, two, three, four, or five residues within the CDR regions. Mutations can be amino acid substitutions, additions, or deletions.

[0415] For example, antibodies can be modified with framework modifications to reduce immunogenicity by "backmutating" one or more framework residues to the corresponding germline sequence.

[0416] In addition, the antibodies disclosed herein 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).

[0417] In addition, the antibodies disclosed herein can also 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 an 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).

[0418] The antibodies or fragments thereof disclosed herein 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 an 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). The term "polyethylene glycol" as used herein is intended to encompass any of the forms of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxypolyethyleneglycol or aryloxypolyethyleneglycol, or polyethyleneglycolmaleimide. 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 disclosed herein (EP 0154316 and EP 0401384).

[0419] 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.

[0420] The antibodies or fragments thereof of the present disclosure can be conjugated with diagnostic agents and used for diagnosis, for example, to monitor the onset or progression of disease and determine the effectiveness of a given treatment regimen. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, various positron-emitting metals used in positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substance can be directly coupled or conjugated to the antibody or its fragment, or indirectly linked or conjugated to the antibody or its fragment 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).

[0421] Additionally, the antibodies or fragments thereof of the present disclosure can be conjugated to a therapeutic agent, including taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin. , antimetabolites (methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decaffeinated, hydroxyurea, asparaginase, gemcitabine, cladribine, etc.), alkylating agents (mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, etc.) toxin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin), antibiotics (dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (diphtheria toxin A chain, and and their active fragments, and hybrid molecules), ricin toxin (such as ricin toxin A or deglycosylated ricin toxin A chain), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin toxins, mixed toxins, and antimicrobial agents or host defense peptides (e.g., specifically targeted antimicrobial peptides (STAMPs)).

[0422] 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 larger molecules 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 case the triplex of DNA relies on both Watson-Crick and Hoogsteen base pairing to form a complex. Triplex molecules can bind to target regions with high affinity and specificity. Suitable conjugate molecules can also include any protein that binds to DNA, provided that it does not create or stabilize biofilm architecture, and at least a subset of such proteins promotes the binding kinetics of the interfering agents disclosed herein.

[0423] 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.

[0424] Therapeutic agents can be linked to antibodies, 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, "Monoclonal antibodies: Production, Engineering and Clinical "Production and Characterization of Synthetic Peptide-Derived Antibodies," in "Applications," Ritter et al. (eds.), pp. 60-84 (Cambridge University Press, 1995).

[0425] 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. (1982) Immunol. Rev. 62:119-58).

[0426] The antibodies or their antigen-binding regions disclosed herein 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 coupling, genetic fusion, or noncovalent association. Multispecific molecules can further include a third binding specificity in addition to the first and second target epitopes.

[0427] Bispecific and multispecific molecules can be prepared using methods known in the art.For example, each binding unit of a bispecific molecule (hi-specific) can be produced separately, and then they can be conjugated with each other.When the binding molecule is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (sulfo-SMCC) (Karpovsky et al. (1984) J. Exp. Med., 160:1686; Liu et al. (1985) Proc. Natl. Acad. Sci. USA, 82:8648). When the binding molecule is an antibody, it can be conjugated by sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains.

[0428] The antibodies disclosed herein 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.

[0429] Antibodies can also be bound to many different carriers. Thus, the present disclosure also provides compositions containing antibodies and other active or inactive substances. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be soluble or insoluble, depending on the purpose disclosed herein. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such carriers using routine experimentation.

[0430] Provided herein are compositions and methods of use involving the antibodies disclosed above as interfering agents. Also disclosed are other interfering agents and compositions for use in the methods described herein. In some embodiments, the interfering agents disclosed herein are: (a) an isolated or recombinant integration host factor (IHF) polypeptide, or their respective fragments or equivalents; (b) an isolated or recombinant histone-like protein (HU) polypeptide or their respective fragments or equivalents; (c) an isolated or recombinant polypeptide comprising or consisting essentially of SEQ ID NOs: 12 to 17, SEQ ID NO: 31, SEQ ID NO: 33, a polypeptide comprising the amino acid sequence NPXT, or a fragment or equivalent thereof; (d) a polypeptide that competes with integration host factors for binding to microbial DNA; (e) an isolated or recombinant polynucleotide encoding any one of (a) through (d), or a polynucleotide that hybridizes under stringent conditions to the above polynucleotide, its equivalent, or its complement; (f) an antibody or antigen-binding fragment, or an equivalent or fragment of each antibody or antigen-binding fragment thereof, that specifically recognizes or specifically binds to any one of (a) to (e); (g) an isolated or recombinant polynucleotide encoding the antibody or antigen-binding fragment of (f), or its complement; (h) a small molecule that competes with the binding of a DNABII protein or DNABII polypeptide to microbial DNA; (i) an antibody from the group of (i) antibodies produced by hybridoma cell line IhfA5 nTHI 14G8.F5.G6, (ii) antibodies produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) antibodies produced by hybridoma cell line mIhfmB4 NTHI 12E6.F8.D12.D5; (j) an antibody or antigen-binding fragment comprising one or more of the CDR sequences disclosed in SEQ ID NOs: 58-106, 108, and 109, the heavy chain sequence disclosed in SEQ ID NO: 51, and / or the light chain sequence disclosed in SEQ ID NO: 55, or their respective equivalents; or (k) Interfering agents of the group of antibodies or antigen-binding fragments comprising one or more of the CDR sequences disclosed in SEQ ID NOs: 110-159, the heavy chain sequence disclosed in SEQ ID NO: 53, and / or the light chain sequence disclosed in SEQ ID NO: 57, or their respective equivalents.

[0431] In some embodiments, the antibody or antigen-binding fragment of (f), (i), (j), and / or (k) is not a polyclonal antibody.

[0432] Polypeptides Aspects of the present disclosure relate to isolated or recombinant DNABII polypeptides or fragments thereof that include at least the turn or "tip" portion of the protein, which in one aspect includes an antiparallel beta ribbon turn and / or the consensus sequence NPXT, where "X" is any amino acid, or alternatively, X is selected from the amino acids Q, R, K, S, or T. Non-limiting examples of such antibodies are provided herein, including those that bind to the consensus amino acid sequence [ka] In another aspect, methods are provided for obtaining immunogens by mutating or selecting the apical region of a DNABII family member that contains the consensus sequence NPXT, where "X" is any amino acid, or alternatively, X is selected from the amino acids Q, R, K, S, or T. Those skilled in the art will recognize that in any one of these sequences, the residue at position X (marked with a double underline in the examples below) can be substituted with any amino acid (e.g., Q, R, K, S, or T). Examples include:

[0433] SEQ ID NO: 13: Haemophilus influenzae IhfA; A5 fragment: [ka]

[0434] SEQ ID NO: 14: Haemophilus influenzae HU; A5 fragment: [ka]

[0435] SEQ ID NO: 17: Haemophilus influenzae IhfB; modified B4 (mB4) fragment: [ka]

[0436] SEQ ID NO: 26: Haemophilus influenzae IhfA; A tip fragment: [ka]

[0437] SEQ ID NO: 27: Haemophilus influenzae IhfB; B tip fragment: [ka]

[0438] SEQ ID NO: 31: Haemophilus influenzae HU; fragment: [ka] Includes.

[0439] In some embodiments, the polypeptide comprising NPXT is at least about 20 amino acids in length, and NPXT is centrally located within the sequence. Non-limiting examples of such sequences include SEQ ID NOs: 13, 14, and 31.

[0440] Embodiments of the present disclosure relate to isolated or recombinant integration host factor (IHF) polypeptides, or their respective fragments or equivalents, and isolated or recombinant histone-like protein (HU) polypeptides, or their respective fragments or equivalents, as interfering agents. In some embodiments, the interfering agent is an isolated or recombinant polypeptide comprising or consisting essentially of an amino acid sequence selected from SEQ ID NOs: 12 to 17, or SEQ ID NO: 33, a polypeptide comprising the amino acid sequence NPXT, or their respective equivalents.

[0441] The present disclosure also provides isolated or recombinant polypeptides comprising, alternatively consisting essentially of, and / or additionally consisting of, two or more, 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 all fourteen isolated polypeptides or their respective fragments or equivalents. Examples of such polypeptides include isolated or recombinant polypeptides comprising SEQ ID NOs: 1, 2, 3, 12-17, and 33, polypeptides comprising the amino acid sequence NPXT, and their respective equivalents. Biological equivalents of these polypeptides are also included in the present disclosure, provided that the sequences do not include the isolated complete wild-type sequence.

[0442] In one embodiment, the present disclosure excludes any polypeptide or protein having sequence identity to the wild-type polypeptide or to the polypeptides disclosed in Pedulla et al. (1996), PNAS 93:15411-15416.

[0443] In any of the above embodiments, a peptide linker can be added to the N-terminus or C-terminus of the polypeptide. A "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, 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: 43). Other examples include Gly-Gly-Gly (SEQ ID NO: 44); Gly-Pro-Ser-Leu (SEQ ID NO: 45); Gly-Pro-Ser (SEQ ID NO: 46); Pro-Ser-Leu-Lys (SEQ ID NO: 47); Gly-Pro-Ser-Leu-Lys (SEQ ID NO: 48), and Ser-Leu-Lys-Leu (SEQ ID NO: 49).

[0444] As disclosed herein, isolated polypeptides are intended to include wild-type and recombinantly produced polypeptides and proteins isolated from prokaryotic and eukaryotic host cells, examples of which are described above, as well as muteins, analogs, and fragments thereof. In some embodiments, the term also includes the antibodies and anti-idiotypic antibodies described herein. Such polypeptides can be isolated or produced using methods known in the art and outlined herein.

[0445] It is understood that functional equivalents or variants of the wild-type polypeptides or proteins, e.g., functional equivalents or variants having conservative amino acid substitutions, are also within the scope of the present disclosure. Other analogs include fusion proteins comprising the proteins or polypeptides disclosed herein, which may include polypeptides linked to antigen-presenting matrices.

[0446] In a further aspect, the polypeptide is conjugated or linked to a detectable label. Suitable labels are known in the art and described herein.

[0447] In still further aspects, the polypeptide with or without a detectable label may be contained on or expressed on the surface of a prokaryotic or eukaryotic host cell, such as a dendritic cell.

[0448] 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 standard techniques such as immunoprecipitation with antibodies, as well as gel filtration, ion exchange, reverse-phase, and affinity chromatography. For such methods, see, for example, Deutscher et al. (1999), "Guide to Protein Purification: Methods in Enzymology" (Vol. 182, Academic Press). Thus, the present disclosure also provides processes for obtaining these polypeptides, as well as products that can be obtained by these processes and the products obtained.

[0449] Polypeptides can also be obtained by chemical synthesis using a commercially available automated peptide synthesizer, such as an automated peptide synthesizer manufactured by Perkin / Elmer / Applied Biosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. The synthesized polypeptides can be precipitated and further purified, for example, by high performance liquid chromatography (HPLC). Thus, the present disclosure also provides a process for chemically synthesizing the proteins disclosed herein by providing the protein sequences and reagents, such as amino acids and enzymes, and linking the amino acids together in the proper orientation and linear sequence.

[0450] Alternatively, proteins and polypeptides can be obtained by well-known recombinant methods using the host cell and vector systems described herein, for example as described in Sambrook et al. (1989), supra.

[0451] The present application also provides a polypeptide described herein conjugated with a detectable agent for use in diagnostic methods. For example, a detectably labeled polypeptide 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 disclosed herein are useful in in vitro assay systems for screening agents or drugs that regulate cellular processes.

[0452] Those skilled in the art are well aware that the peptides disclosed herein can be modified to change their 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 either D optical isomer or L optical isomer, as well as amino acid analogs and peptidomimetics.The peptide of three or more amino acids is generally called oligopeptide if the peptide chain is short.If the peptide chain is long, the peptide is generally called polypeptide or protein.

[0453] The peptides disclosed herein can be modified to include unnatural amino acids. Thus, the peptides can include D-amino acids, combinations of D-amino acids 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 unique properties to the peptides. In addition, by assigning specific amino acids in specific coupling steps, peptides with alpha helices, beta turns, beta sheets, gamma turns, and cyclic peptides can also be generated. It is generally believed that alpha-helical or random secondary structures can be particularly useful.

[0454] The polypeptides disclosed herein can be combined with various solid-phase carriers, such as implants, stents, pastes, gels, dental implants, or medical implants, or with 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 propyl ethylene glycol, polyethylene glycol, and vegetable oils. When used to prepare antibodies or induce an immune response in vivo, the carrier can also include an adjuvant useful for non-specifically enhancing a specific immune response. Those skilled in the art can easily determine whether an adjuvant is necessary and select one. However, by way of example 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 adjuvants derived from squalene.

[0455] The present disclosure also provides pharmaceutical compositions comprising, or alternatively consisting essentially of, and / or further consisting of, any of the polypeptides, analogs, muteins, or fragments disclosed herein, alone or in combination with each other or with other agents, such antibiotics, and an acceptable carrier or solid support.

[0456] These compositions are useful in a variety of diagnostic and therapeutic methods described herein. In specific embodiments, it is contemplated that the polypeptides disclosed herein may be used in the generation of antibodies or in more generalized immune responses. For example, the use of the polypeptides as immunogens may be useful in the generation of antibodies according to the methods disclosed above, as well as in the diagnostic and therapeutic methods disclosed below. Embodiments contemplated according to the present disclosure include, but are not limited to, the use of isolated or recombinant polynucleotides to characterize, identify, purify, or otherwise assay for the antibodies disclosed herein; the use of isolated or recombinant polynucleotides to generate the antibodies disclosed herein; or the use of isolated or recombinant polynucleotides, alone or in combination with the antibodies disclosed herein, in contemplated vaccine or therapeutic formulations.

[0457] Aspects of the present disclosure relate to two or more of the polypeptides disclosed herein being used as immunogens. For example, (a) SEQ ID NO: 12 [ka] (b) SEQ ID NO: 13 [ka] (c) SEQ ID NO: 14 [ka] (d) SEQ ID NO: 15 [ka] (e) SEQ ID NO: 16 [ka] (f) SEQ ID NO: 17 [ka] (g) SEQ ID NO: 33 [ka] (h) SEQ ID NO: 31 [ka] (i) a polypeptide comprising the amino acid sequence NPXT; or (j) Equivalents of (a) through (i), wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complement, wherein the high stringency conditions comprise an incubation temperature of from about 55° C. to about 68° C.; a buffer concentration of from about 1×SSC to about 0.1×SSC; a formamide concentration of from about 55% to about 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. The present invention relates to polypeptides including, but not limited to:

[0458] In some embodiments, the polypeptides disclosed herein can also be used to determine the binding affinity of antibodies and / or to screen for equivalent antibodies based on the predicted affinity of antibodies raised against the peptides.

[0459] Polynucleotides The present disclosure also provides isolated or recombinant polynucleotides encoding one or more of the above-identified isolated or recombinant polypeptides and their respective complementary strands. The present disclosure further provides isolated or recombinant polynucleotides encoding one or more of the above-identified antibodies. Vectors comprising isolated or recombinant polynucleotides or antibodies, examples of which are known in the art and outlined herein, are also provided. In one embodiment, where more than one isolated or recombinant polynucleotide is to be expressed as a single unit, the isolated or recombinant polynucleotides can be contained within a polycistronic vector. The polynucleotide can be DNA, RNA, mRNA, or an interfering RNA such as siRNA, miRNA, or dsRNA.

[0460] In another aspect, the present disclosure provides an interference agent that is a polynucleotide that interferes with the binding of DNA to polypeptides or proteins in microbial biofilms, or a four-way junction polynucleotide similar to a Holliday junction, a three-way junction polynucleotide similar to a replication fork, a polynucleotide with inherent flexibility, or a curved polynucleotide, and can treat DNABII polynucleotides or inhibit DNABII polynucleotides from binding to microbial DNA, and can treat, prevent, or inhibit the formation of biofilms and related infections and disorders.Those skilled in the art can use the information provided herein and their knowledge to produce such polynucleotides.Goodman and Kay (1999), J. Biological Chem., vol. 274(52): pp. 37004-37011 and Kamashev and Rouviere-Yaniv (2000), EMBO J., vol. 19(23): pp. 6527-6535.

[0461] The present disclosure also 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 direct cell-specific expression of an inserted polynucleotide. Vectors containing a promoter or promoter / enhancer, along with a termination codon and selectable marker sequence, and a cloning site into which a small piece of DNA can be operably linked to the promoter, are known in the art and commercially available. For general methods 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 references to maps, functional characteristics, commercial sources, and GenEMBL accession numbers for a variety of suitable vectors.

[0462] In one embodiment, polynucleotides derived from the polynucleotides disclosed herein encode polypeptides or proteins having diagnostic and therapeutic utility as described herein, as well as probes for identifying protein transcripts, which 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 methods of preparing and labeling such fragments, see Sambrook et al. (1989), supra.

[0463] 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 because 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 recombinantly produced. Suitable host cells depend 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 for bacterial cells; transfection using calcium phosphate precipitation or DEAE-dextran for mammalian cells; electroporation; or microinjection. For methods, see Sambrook et al. (1989), supra. Thus, the present disclosure also provides host cells, e.g., prokaryotic cells such as mammalian cells, animal cells (rat or mouse), human cells, or bacterial cells, containing a polynucleotide encoding a protein or polypeptide or antibody.

[0464] Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be added before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can also be further modified after polymerization, such as by conjugation with a labeled component. This term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment disclosed herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms that are known or predicted to be in a double-stranded form.

[0465] When vectors are used for in vivo or ex vivo gene therapy, pharmaceutically acceptable vectors, such as replication-incompetent retroviral or adenoviral vectors, are exemplary (but not limited to) and particularly useful. Pharmaceutically acceptable vectors containing the nucleic acids disclosed herein 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, vectors or delivery vehicles capable of selectively targeting and introducing nucleic acids into dividing cells. An example of such a vector is a "replication-incompetent" vector, defined by its inability to produce viral proteins and its inability to spread within infected host cells. An example of a replication-incompetent retroviral vector is LNL6 (Miller et al. (1989) BioTechniques 7:980-990). Methods 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).

[0466] The present disclosure also provides genetically modified cells that contain and / or express the polynucleotides disclosed herein. Genetically modified cells can be produced by inserting upstream regulatory sequences, such as promoters or gene activators (see U.S. Patent No. 5,733,761).

[0467] Polynucleotides can be conjugated with detectable markers and / or purification 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 ligands, radioactive ligands, enzyme ligands, or other ligands such as avidin / biotin, which can produce detectable signals.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 visual or spectrophotometrically detectable means for identifying specific hybridization with complementary nucleic acid-containing samples. Therefore, the present disclosure also 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 disclosed herein under conditions that allow hybridization of complementary single-stranded polynucleotides (optionally, moderately stringent hybridization conditions), or optionally, under highly stringent hybridization conditions. Hybridized polynucleotide pairs are separated from unhybridized single-stranded polynucleotides. Hybridized polynucleotide pairs are detected using methods known to those skilled in the art, for example, the methods described in Sambrook et al. (1989), supra.

[0468] The polynucleotides embodied in the present disclosure can be obtained by chemical synthesis, recombinant cloning, PCR, or any combination thereof.Chemical polynucleotide synthesis methods 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 by requesting commercial services.

[0469] The polynucleotides disclosed herein can be isolated or replicated using PCR. PCR technology is the subject of U.S. Pat. Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, and is 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, one skilled in the art can replicate DNA using the sequences provided herein and a commercially available DNA synthesizer. Thus, the present disclosure also provides a process for obtaining the polynucleotides disclosed herein 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 orientation to obtain a polynucleotide. In another embodiment, these polynucleotides are further isolated. Furthermore, those skilled in the art can insert the polynucleotide into an appropriate replicable vector and then insert the vector into an appropriate host cell (prokaryotic or eukaryotic cell) for replication and amplification. The DNA thus amplified can be isolated from the cell by methods known to those skilled in the art. Also provided herein is a process for obtaining a polynucleotide by this method, as well as the polynucleotide thus obtained.

[0470] 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 an appropriate 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 methods set forth in Sambrook et al. (1989), supra. For example, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989), supra, or extracted with nucleic acid-binding resins according to the accompanying instructions provided by the manufacturer.

[0471] Polynucleotides exhibiting sequence complementarity or sequence homology to the polynucleotides disclosed herein are useful as hybridization probes or as equivalents of the specific polynucleotides identified herein. Because the complete coding sequence of the transcript is known, any portion of this sequence or a homologous sequence can be used in the methods disclosed herein.

[0472] It is known in the art that a "perfect match" probe is not required for specific hybridization. Minor changes in the probe sequence, achieved by substitution, deletion, or insertion of a small number of bases, do not affect the specificity of hybridization. Generally, a base pair mismatch of around 20% (when optimally aligned) can be tolerated. In some embodiments, probes useful for detecting the aforementioned mRNAs are at least about 80% identical to the homologous region. In some embodiments, the probe is 85% identical to the corresponding gene sequence after alignment with the homologous region, and in some embodiments, the probe exhibits 90% identity.

[0473] These probes can be used in radioassays (for example, Southern blot analysis and Northern blot analysis) to detect, prognose, diagnose, or monitor various cells or tissues containing these cells.Probes can also be attached to solid supports or arrays such as chips for use in high-throughput screening assays to detect the expression of genes corresponding to the polynucleotides disclosed herein.Therefore, the present disclosure also provides probes that comprise or correspond to the polynucleotides disclosed herein, or their equivalents, or their complements, or their fragments, attached to solid supports for use in high-throughput screening.

[0474] 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, such as between at least 5-10 nucleotides and about 100 nucleotides, or even full length, depending on the complementary sequence desired to be detected.

[0475] Nucleotide probes with complementary sequences spanning a stretch greater than 5 to 10 nucleotides in length are generally preferred to increase hybrid stability and selectivity, thereby improving the specificity of the resulting hybrid molecules. In certain embodiments, polynucleotides can be designed with gene-complementary stretches of 10 nucleotides or more, or even longer if desired. Such fragments can be readily prepared, for example, by direct synthesis of the fragments via chemical means, by applying nucleic acid reproduction techniques such as the PCR technique involving two priming oligonucleotides described in U.S. Pat. No. 4,603,102, or by introducing selected sequences into a recombinant vector for recombinant production. In one aspect, the probes are about 50 to 75 nucleotides or more in length, or alternatively, 50 to 100 nucleotides in length.

[0476] The polynucleotides of the present disclosure can be used as primers for detecting genes or gene transcripts expressed in the cells described herein. In this context, amplification refers to any method that utilizes a primer-dependent polymerase that can replicate the target sequence with reasonable fidelity. Amplification can be performed with natural or recombinant DNA polymerases, such as T7 DNA polymerase, the Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. For illustrative purposes only, the primers are the same length as those identified for the probe.

[0477] 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, such as agarose gel electrophoresis, followed by visualization with ethidium bromide staining and ultraviolet illumination.

[0478] Methods for administering an effective amount of gene delivery vector or vehicle to cells have been developed and are known to those skilled in the art, and are also 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 also be detected by various methods.In particular, it is useful to prepare polyclonal or monoclonal antibodies that are specifically reactive with target polypeptides.Such antibodies are useful for visualizing cells that express polypeptides, for example, using techniques such as immunohistochemistry, ELISA, and Western blotting.These techniques can be used to determine the expression level of expressed polynucleotides.

[0479] composition Compositions are also provided. The compositions include a carrier and one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, the small molecules, or the antibodies disclosed herein. The carrier may be one or more of a solid support or a pharmaceutically acceptable carrier. The composition may further include an adjuvant or other components suitable for administration as a vaccine. In one aspect, the composition is formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants. In addition, embodiments of the compositions of the present disclosure also include one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the small molecules, the isolated host cells disclosed herein, or the antibodies disclosed herein, formulated with one or more pharmaceutically acceptable substances.

[0480] For oral preparations, any one or more of the isolated or recombinant polypeptides described herein, the isolated or recombinant polynucleotides described herein, the vectors described herein, the isolated host cells described herein, the small molecules described herein, or the antibodies described herein can be used alone or in combination with suitable additives to produce tablets, powders, granules, or capsules, such as conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as microcrystalline cellulose, cellulose derivatives, acacia gum, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if desired, diluents, buffers, humectants, preservatives, and flavoring agents. Pharmaceutically compatible binders and / or adjuvant materials can be incorporated as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0481] Pharmaceutical formulations and unit dosage forms suitable for oral administration are particularly useful in the treatment of chronic conditions, infectious diseases, and therapies where the patient self-administers the drug. In one embodiment, the formulation is specific for pediatric administration.

[0482] The present disclosure can be formulated into preparations for injection in accordance with the present disclosure by dissolving, suspending, or emulsifying one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, or the antibodies disclosed herein in an aqueous solvent or a non-aqueous solvent such as a vegetable oil or other similar oil, a synthetic aliphatic acid glyceride, an ester of a higher aliphatic acid, or propylene glycol, and, if desired, with conventional additives such as solubilizing agents, isotonicity agents, suspending agents, emulsifying agents, stabilizers and preservatives, or other antimicrobial agents. Non-limiting examples of such additives are surface antigens, e.g., OMP P5, OMP 26, OMP P2, or vaccine components, e.g., type IV pilin proteins (see Jurcisek and Bakaletz (2007), J. of Bacteriology, 189(10):3868-3875; and Murphy, T. F. et al. (2009), The Pediatric Infectious Disease Journal, 28:S121-S126), and other therapeutic agents that are antibodies against other vaccine components, such as antimicrobial agents. For intravenous administration, suitable carriers include physiological, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In any case, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists.

[0483] The aerosol formulations provided by the present disclosure can be administered via inhalation, and can be propellant-based or non-propellant-based.For example, the embodiments of the pharmaceutical formulations disclosed herein include the compounds disclosed herein that are formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. For inhalation administration, the compounds can be delivered in the form of aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.A non-limiting example of a non-propellant is a pump spray that is propelled from a closed container by mechanical force (i.e., by pressing down the plunger with a finger, or by compressing the container through the compressive force applied to the wall of the container, or the elastic force exerted by the wall itself (for example, through an elastic bladder)).

[0484] The suppositories disclosed herein can be prepared by mixing the compounds disclosed herein with any of a variety of bases, such as emulsifying bases or water-soluble bases.The pharmaceutical formulations of the compounds disclosed herein can be administered rectally via suppositories.Suppositories can contain vehicles such as cocoa butter, carbowax, and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0485] It is possible to provide a unit dosage form for oral or rectal administration, such as syrup, elixir, and suspension, and each dosage unit, for example, a teaspoonful, a spoonful, a tablet, or a suppository, contains a predetermined amount of the composition containing one or more compounds disclosed herein.Similarly, a unit dosage form for injection or intravenous administration can contain the compound disclosed herein in a composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.

[0486] The embodiments of the pharmaceutical formulations disclosed herein include pharmaceutical formulations in which one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the small molecules used in this disclosure, the isolated host cells disclosed herein, or the antibodies disclosed herein are formulated into an injectable composition.The injectable pharmaceutical formulations disclosed herein are prepared as solutions or suspensions, or as solid forms suitable for dissolving or suspending in a liquid vehicle before injection.The preparations can also be emulsified, or the active ingredient can be encapsulated in a liposome vehicle, according to other embodiments of the pharmaceutical formulations disclosed herein.

[0487] In certain embodiments, one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, or the antibodies disclosed herein are formulated for delivery via a sustained delivery system. As used herein, the term "sustained delivery system" is used interchangeably with "controlled delivery system" and encompasses sustained (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

[0488] Mechanical infusion pump or electromechanical infusion pump may also be suitable for use with the present disclosure.Examples of such devices include, for example, the devices described in U.S. Patent No. 4,692,147; U.S. Patent No. 4,360,019; U.S. Patent No. 4,487,603; U.S. Patent No. 4,360,019; U.S. Patent No. 4,725,852; U.S. Patent No. 5,820,589; U.S. Patent No. 5,643,207; U.S. Patent No. 6,198,966, etc. Generally, the delivery of the compounds disclosed herein can be achieved using any of a variety of rechargeable pump systems.The pump provides a constant controlled release over a period of time.In some embodiments, the compounds disclosed herein are in a liquid formulation in a drug-impermeable reservoir and are continuously delivered to individuals.

[0489] In one embodiment, the drug delivery system is at least partially an implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices known in the art. An implantation site is a site within a subject's body where the drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, subdermal, intramuscular, or other suitable sites within a subject's body. In some embodiments, a subcutaneous implantation site is used because it allows for convenient implantation and removal of the drug delivery device.

[0490] Drug release devices suitable for use in the present disclosure may be based on any of a variety of actuation modes. For example, the drug release device may be based on a diffusion system, a convection system, or an erosion system (e.g., an erosion-based system). For example, the drug release device may be an electrochemical pump, an osmotic pump, an electroosmotic pump, a vapor pressure pump, or an osmotic bursting matrix, e.g., a pump in which a drug is incorporated into a polymer that releases a drug formulation concurrently with the degradation of a drug-impregnated polymeric material (e.g., a biodegradable drug-impregnated polymeric material). In other embodiments, the drug release device is based on an electrodiffusion system, an electrolytic pump, a bubble pump, a piezoelectric pump, a hydrolysis system, or the like.

[0491] Drug release devices based on mechanical infusion pumps or electromechanical infusion pumps can also be suitable for use with the present disclosure.Examples of such devices include, for example, the devices described in United States Patent No. 4,692,147; United States Patent No. 4,360,019; United States Patent No. 4,487,603; United States Patent No. 4,360,019; United States Patent No. 4,725,852, etc. Generally, the treatment of the subject can be achieved using any of a variety of rechargeable non-replaceable pump systems.Pumps and other convective systems can be utilized because of their generally constant and highly controlled release over a period of time.In some embodiments, osmotic pumps are used because of the advantages of combining constant and highly controlled release with relatively small size (see, for example, PCT International Patent Application Publication No. WO 97 / 27840; and United States Patent No. 5,985,305 and 5,728,396). Exemplary osmotically driven devices suitable for use in the present disclosure include those described in U.S. Patent Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; Such devices include, but are not necessarily limited to, those described in: 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396, etc. A further exemplary device that may be adapted for use with the present disclosure is the Synchromed infusion pump (Medtronic).

[0492] In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices known in the art. As referred to herein, an implantation site is a site within a subject's body where the drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, subdermal (subcutaneous) sites, intramuscular sites, or other suitable sites within a subject's body.

[0493] Suitable excipient vehicles for the compounds disclosed herein are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof.In addition, if desired, vehicle can also contain minor amounts of auxiliary substances, such as wetting agents, emulsifying agents, or pH buffering agents.In view of the present disclosure, those skilled in the art will know or will be able to understand how to prepare such dosage forms.See, for example, "Remington's Pharmaceutical Sciences", Mack Publishing Company, Easton, Pa., 17th Edition, 1985.In any case, the composition or formulation to be administered contains a sufficient amount of compound to achieve the desired state in the treated subject.

[0494] The compositions of the present disclosure include compositions comprising sustained-release or controlled-release matrices. Additionally, embodiments of the present disclosure can be used in conjunction with other treatments that utilize sustained-release formulations. As used herein, a sustained-release matrix is ​​a matrix made of a material, usually a polymer, that is degradable through enzymatic or acid-based hydrolysis or dissolution. Once inserted into the body, the matrix is ​​acted upon by enzymes and bodily fluids. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide-co-glycolide (copolymer of lactic and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, and isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicone. Exemplary biodegradable matrices include polylactide matrices, polyglycolide matrices, and polylactide-co-glycolide (copolymers of lactic and glycolic acid) matrices.

[0495] In another embodiment, the interfering agent or antibody (as well as the combination composition) is delivered by a controlled release system. For example, the compounds disclosed herein can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump is used (Sefton (1987), CRC Crit. Ref. Biomed. Eng., 14:201; Buchwald et al. (1980), Surgery, 88:507; Saudek et al. (1989), N. Engl. J. Med., 321:574). In another embodiment, a polymeric material is used. In yet another embodiment, the controlled release system is placed in the vicinity of the therapeutic target, i.e., the liver, thereby requiring only a fraction of the systemic dose. In yet another embodiment, the controlled release system is placed in the vicinity of the therapeutic target, thereby requiring only a fraction of the systemic dose. Other controlled release systems are reviewed by Langer (1990) Science 249:1527-1533.

[0496] In another embodiment, the compositions of the present disclosure (as well as the individual or combined compositions) include compositions formed by impregnating the inhibitors described herein into absorbent materials such as sutures, bandages, and gauze, or compositions coated onto the surface of solid materials such as surgical staples, zippers, and catheters for delivery of the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art with the present disclosure in mind.

[0497] The present disclosure provides methods and compositions for administering one or more interfering agents to a host (e.g., a human) to treat a microbial infection. In various embodiments, the methods disclosed herein span almost any available method and route suitable for delivering a drug, including in vivo and ex vivo methods, as well as systemic and local administration routes.

[0498] Formulations and co-formulations More specifically, the disclosure provided herein contemplates specific formulations and combination formulations of the disclosed interfering agents with pharmaceutically acceptable excipients, such as the excipients disclosed herein.

[0499] For purposes of the above-referenced formulations and combination formulations, the interfering agent is: (a) an isolated or recombinant integration host factor (IHF) polypeptide, or their respective fragments or equivalents; (b) an isolated or recombinant histone-like protein (HU) polypeptide or their respective fragments or equivalents; (c) an isolated or recombinant polypeptide of SEQ ID NO: 12 to 17, SEQ ID NO: 33, a polypeptide comprising the amino acid sequence NPXT, or a fragment or equivalent thereof; (d) a polypeptide that competes with integration host factors for binding to microbial DNA; (e) an isolated or recombinant polynucleotide encoding any one of (a) through (f), or a polynucleotide that hybridizes under stringent conditions to the above polynucleotides, their equivalents, or their complements; (f) an antibody or antigen-binding fragment, or an equivalent or fragment of each antibody or antigen-binding fragment thereof, that specifically recognizes or specifically binds to any one of (a) to (e); (g) an isolated or recombinant polynucleotide encoding the antibody or antigen-binding fragment of (h), or its complement; (i) a small molecule that competes with the binding of a DNABII protein or DNABII polypeptide to microbial DNA; (j) (i) an antibody produced by hybridoma cell line IhfA5 nTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line mIhfmB4 Antibodies from the group of antibodies produced by NTHI 12E6.F8.D12.D5; (k) an antibody or antigen-binding fragment comprising one or more of the CDR sequences disclosed herein, including the CDR sequences disclosed in SEQ ID NOs: 58-106, 108, and 109, the heavy chain sequence disclosed in SEQ ID NO: 51, and / or the light chain sequence disclosed in SEQ ID NO: 55, or their respective equivalents; or (l) An antibody or antigen-binding fragment comprising one or more of the CDR sequences disclosed herein, including the CDR sequences disclosed in SEQ ID NOs: 110-159, the heavy chain sequence disclosed in SEQ ID NO: 53, and / or the light chain sequence disclosed in SEQ ID NO: 57, or their respective equivalents. is an interference agent of the group

[0500] In some embodiments, the antibody or antigen-binding fragment of (f), (j), (k), and / or (l) is not a polyclonal antibody.

[0501] In a specific embodiment, the present disclosure provides a formulation or combined formulation comprising an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of an amino acid sequence selected from SEQ ID NOs: 12 to 17, SEQ ID NO: 33, or their respective equivalents. The antibodies disclosed herein can be selected to have a high level of epitope binding specificity and high binding affinity for biofilms. Generally, the greater the binding affinity of the antibody, the more stringent washing conditions can be performed in an immunoassay to remove non-specifically bound material without removing the target. Thus, antibodies of the present technology useful in the disclosed methods typically have a binding affinity of at least 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , or 10 -12M. In certain embodiments, the antibody has a kinetic on-rate sufficient to reach equilibrium under standard conditions in at least 12 hours, at least 5 hours, at least 1 hour, or at least 30 minutes. In other embodiments, the affinity of the antibody or antigen-binding fragment is less than or about 1000 picomolar (pM), 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, about 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, or 4 pM.

[0502] In some embodiments, the antibody is present in the formulation at a concentration of from about 0.1 mg / mL to about 200 mg / mL, or alternatively, from about 1 to about 150 mg / mL, or alternatively, from about 2 mg / mL to about 100 mg / mL, or alternatively, from about 3 mg / mL to about 80 mg / mL, or alternatively, from about 4 mg / mL to about 50 mg / mL, or alternatively, from about 5 mg / mL to about 20 mg / mL. In some embodiments, the antibody has a concentration of at least about 1 mg / mL, or alternatively, at least about 2 mg / mL, at least about 3 mg / mL, or alternatively, at least about 4 mg / mL, or alternatively, at least about 5 mg / mL, or alternatively, at least about 6 mg / mL, or alternatively, at least about 7 mg / mL, or alternatively, at least about 8 mg / mL, or alternatively, at least about 9 mg / mL, or alternatively, at least about 10 mg / mL, or alternatively, at least about 15 mg / mL, or alternatively, at least about 20 mg / mL, Or alternatively, it is present at a concentration of at least about 30 mg / mL, or alternatively at least about 40 mg / mL, or alternatively at least about 50 mg / mL, or alternatively at least about 60 mg / mL, or alternatively at least about 70 mg / mL, or alternatively at least about 80 mg / mL, or alternatively at least about 90 mg / mL, or alternatively at least about 100 mg / mL, or alternatively at least about 120 mg / mL, or alternatively at least about 150 mg / mL, or alternatively at least about 200 mg / mL.In some embodiments, at least one of the plurality of antibodies has a concentration of at least about 1 mg / mL, or alternatively, at least about 2 mg / mL, or alternatively, at least about 3 mg / mL, or alternatively, at least about 4 mg / mL, or alternatively, at least about 5 mg / mL, or alternatively, at least about 6 mg / mL, or alternatively, at least about 7 mg / mL, or alternatively, at least about 8 mg / mL, or alternatively, at least about 9 mg / mL, or alternatively, at least about 10 mg / mL, or alternatively, at least about 15 mg / mL, or alternatively, at least In some embodiments, the hydroxybenzoates are present at a concentration of at least about 20 mg / mL, or alternatively at least about 30 mg / mL, or alternatively at least about 40 mg / mL, or alternatively at least about 50 mg / mL, or alternatively at least about 60 mg / mL, or alternatively at least about 70 mg / mL, or alternatively at least about 80 mg / mL, or alternatively at least about 90 mg / mL, or alternatively at least about 100 mg / mL, or alternatively at least about 120 mg / mL, or alternatively at least about 150 mg / mL, or alternatively at least about 200 mg / mL.

[0503] In some embodiments in which multiple different antibodies are included in the antibody combination formulation, the different antibodies may be present at substantially equal concentrations. In another aspect of such embodiments, one or more of the different antibodies may be present at a substantially higher concentration than the other antibodies, for example, in a ratio of about 1.5:1, or alternatively about 1.5:1:1, or alternatively about 1.5:1:1:1, or alternatively about 2:1, or alternatively about 2:1:1, or alternatively about 2:1:1:1, or alternatively at least about 2.5:1, or alternatively at least about 2.5:1:1, or alternatively at least about 2.5:1:1.

[0504] In some embodiments, the combined formulation comprises, alternatively consists essentially of, and / or further comprises (i) an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of SEQ ID NO: 13 and / or SEQ ID NO: 14, or their respective equivalents, and (ii) an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of SEQ ID NO: 16 and / or SEQ ID NO: 17 and / or SEQ ID NO: 31 and / or SEQ ID NO: 33, or their respective equivalents. In some embodiments, one or more antibodies in the formulation are not polyclonal. In some embodiments, the formulation is used as a therapeutic agent.

[0505] In some embodiments, the combined formulation comprises, alternatively consists essentially of, and / or further comprises (i) an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of SEQ ID NO: 12 and / or SEQ ID NO: 31 and / or SEQ ID NO: 33, or an equivalent thereof, and (ii) an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of SEQ ID NO: 15 and / or SEQ ID NO: 31 and / or SEQ ID NO: 33, or an equivalent thereof. In some embodiments, one or more antibodies in the formulation are not polyclonal. In some embodiments, the formulation is used as a diagnostic agent.

[0506] Methods for stably formulating antibody formulations and combined formulations can be devised according to techniques disclosed in the art (see, e.g., U.S. Patent Application No. 12 / 875,083 (published as US2011 / 0059079)).

[0507] Diagnostic and Therapeutic Methods Also provided is a method for inhibiting, competing with or gradually decreasing the binding of DNABII polypeptide or DNABII protein to microbial DNA, by contacting DNABII polypeptide or DNABII protein or microbial DNA with an interfering agent, thereby inhibiting, competing with or gradually decreasing the binding of DNABII protein or DNABII polypeptide to microbial DNA.In another embodiment, DNABII polypeptide and microbial DNA are detectably labeled, for example, with a luminescent molecule that generates a signal when they are in close contact with each other.Contacting can be carried out in vitro or in vivo.

[0508] In another aspect, a method is provided for inhibiting, preventing or destroying microbial biofilms by contacting the biofilm with an interfering agent, thereby inhibiting, preventing or destroying the microbial biofilms.In a further aspect, the DNABII polypeptide and the microbial DNA are detectably labeled, for example, with a luminescent molecule that generates a signal when they are in close contact with each other.Contacting can be carried out in vitro or in vivo.

[0509] When carried out in vitro, the method is useful for screening or identifying interfering agents that have the same, similar, or opposite abilities as the polypeptides, polynucleotides, antibodies, host cells, small molecules, and compositions disclosed herein. Alternatively, the method can be used to identify which interfering agent is optimal for treating microbial infections. For example, by having two samples containing a DNABII polypeptide, microbial DNA, and a test agent, new agents or combination therapies can be screened, for example. The second sample contains a DNABII polypeptide, microbial DNA, and a known active agent, such as an anti-IHF antibody or a small molecule used as a positive control. In a further embodiment, several samples are provided, and the interfering agent is added to the system at increasing dilutions to determine the optimal dose that is likely to be effective for treating subjects in a clinical setting. As will be apparent to those skilled in the art, a negative control containing a DNABII polypeptide and microbial DNA can be provided. In a further embodiment, the DNABII polypeptide and microbial DNA are detectably labeled with a luminescent molecule that generates a signal when they are brought into close contact with each other. The sample is maintained under similar conditions for a time period during which the agent is effective to inhibit, compete with, or reduce the interaction between the DNABII polypeptide and the microbial DNA, and then the sample is assayed for the generation of a signal from the luminescent molecule. If the sample generates a signal, the agent is not effective in inhibiting binding.

[0510] In another embodiment, an in vitro method is performed in a miniaturized chamber slide system in which isolates of infectious disease-causing microorganisms (e.g., bacteria) can be isolated from humans / animals and then cultured and grown as biofilms in vitro; see, for example, Experiment 1 below. An interfering agent (such as an anti-IHF antibody) or potential interfering agent is added to the culture, with or without increasing dilutions of anti-IHF (or other antibodies, small molecules, agents, etc.), potential interfering agent, or interfering agent, alone or in combination with another agent, to find an optimal dose that, when delivered to a subject with an infection, is likely to be effective in treating the patient. As will be apparent to one of skill in the art, positive and negative controls can be run simultaneously.

[0511] In a further embodiment, the method is carried out in a high-throughput platform with an interfering agent (such as, for example, an anti-IHF antibody) and / or a potential interfering agent (alone or in combination with another agent) in a flow cell. The interfering agent (such as, for example, an anti-IHF antibody) or the potential interfering agent is added to the culture with or without increasing dilutions of, for example, anti-IHF (or other antibodies, small molecules, agents, etc.), the potential interfering agent, or the interfering agent, alone or in combination with another agent, to find the optimal dose that is likely to be effective in treating the patient when delivered to a subject with an infection. The biofilm isolate is sonicated to separate the biofilm bacteria from the DNABII polypeptide, such as IHF, that is bound to microbial DNA. The DNABII polypeptide-DNA complex is isolated by the anti-IHF antibody on the platform. The microbial DNA is then released, for example, by salt washing, and used to identify the added biofilm bacteria. The released DNA is then identified, for example, by PCR sequencing. If no DNA is released, the interfering agent has successfully functioned or bound to microbial DNA. If DNA is found in the sample, the agent did not interfere with the binding between the DNABII polypeptide and the microbial DNA. As will be apparent to those skilled in the art, positive and / or negative controls can be performed simultaneously.

[0512] In another aspect, one or more of the interfering agents or antibodies disclosed herein are used in a method for detecting biofilms in vivo.In a further embodiment, the interfering agent or antibody is detectably labeled, for example, with a luminescent or fluorescent molecule.A further application of the method disclosed herein includes the use of such interfering agents or antibodies, for example, to image biofilms using a detectably labeled primary interfering agent or antibody that produces a detectable signal when bound to biofilms, or a detectably labeled secondary antibody that binds to the primary interfering agent or primary antibody when it binds to biofilms.

[0513] The above method can also be used as a diagnostic test, as a given bacterial species may respond better to one agent than another for reversal of its biofilm, and this rapid, high-throughput assay system would allow one skilled in the art to assay a panel of potential anti-IHF-like agents to identify the most effective group.

[0514] An advantage of these methods is that most clinical microbiology laboratories in hospitals are already equipped to perform these types of assays (i.e., determining MIC and MBC values) using bacteria grown in liquid culture (or in planktonic state). As will be apparent to those skilled in the art, bacteria generally do not grow in planktonic state when causing disease. Instead, they grow as stable biofilms, and these biofilms are significantly more resistant to treatment with antibiotics, antibodies, or other therapeutic agents. This resistance is the reason why most MIC / MBC values ​​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 will become a more reliable predictor of clinical efficacy, even in personalized medicine applications.

[0515] In addition to clinical settings, this method can also be used to identify infectious disease-causing microorganisms and / or to identify effective interfering agents in industrial settings.

[0516] In a further embodiment of the above method, an antibiotic or antimicrobial agent known to inhibit the progression of the underlying infection is added sequentially or simultaneously to determine whether the infection can be inhibited.To assay biofilm inhibition, an interfering agent can be added to the microbial DNA or DNABII polypeptide before adding the complex.

[0517] When performed in vivo in non-human animals such as chinchillas, the method provides a preclinical screen for identifying interfering agents that can be used alone or in combination with other agents to degrade biofilms, examples of which are provided in Examples 4-9 below.

[0518] In another aspect, provided herein are methods for inhibiting, preventing, or disrupting a biofilm in a subject by administering to the subject an effective amount of an interfering agent, thereby inhibiting, preventing, or disrupting the microbial biofilm, examples of which are shown in Examples 4-7 below.

[0519] Alternatively or additionally, methods for inhibiting, preventing, or disrupting biofilms can be performed in vitro and / or ex vivo and involve providing a biofilm sample (obtained from a subject or generated in vitro) and administering an effective amount of an interfering agent, thereby inhibiting, preventing, or disrupting the microbial biofilm. Similarly, the compositions disclosed herein can also be used in method embodiments for inhibiting, preventing, or disrupting microbial biofilms on surfaces colonized by biofilms, such as, but not limited to, hospital instruments, industrial equipment, and other materials not contained in living tissue.

[0520] For purposes of the in vitro and in vivo methods referred to above, an interfering agent is (a) an isolated or recombinant integration host factor (IHF) polypeptide, or their respective fragments or equivalents; (b) an isolated or recombinant histone-like protein (HU) polypeptide or their respective fragments or equivalents; (c) an isolated or recombinant polypeptide of SEQ ID NO: 12 to 17, SEQ ID NO: 31, SEQ ID NO: 33, a polypeptide comprising the amino acid sequence NPXT, or a fragment or equivalent thereof; (d) a polypeptide that competes with integration host factors for binding to microbial DNA; (e) an isolated or recombinant polynucleotide encoding any one of (a) through (f), or a polynucleotide that hybridizes under stringent conditions to the above polynucleotide, its equivalent, or its complement; (f) an antibody or antigen-binding fragment, or an equivalent or fragment of each antibody or antigen-binding fragment thereof, that specifically recognizes or specifically binds to any one of (a) to (e); (g) an isolated or recombinant polynucleotide encoding the antibody or antigen-binding fragment of (h), or its complement; (i) a small molecule that competes with the binding of a DNABII protein or DNABII polypeptide to microbial DNA; (j) (i) an antibody produced by hybridoma cell line IhfA5 nTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line mIhfmB4 Antibodies from the group of antibodies produced by NTHI 12E6.F8.D12.D5; (k) an antibody or antigen-binding fragment comprising one or more of the CDR sequences disclosed herein, including the CDR sequences disclosed in SEQ ID NOs: 58-106, 108, and 109, the heavy chain sequence disclosed in SEQ ID NO: 51, and / or the light chain sequence disclosed in SEQ ID NO: 55, or their respective equivalents; or (l) An antibody or antigen-binding fragment comprising one or more of the CDR sequences disclosed herein, including the CDR sequences disclosed in SEQ ID NOs: 110-159, the heavy chain sequence disclosed in SEQ ID NO: 53, and / or the light chain sequence disclosed in SEQ ID NO: 57, or their respective equivalents. is an interference agent of the group

[0521] In some embodiments, the antibody or antigen-binding fragment of (f), (j), (k), and / or (l) is not a polyclonal antibody.

[0522] In some embodiments, the methods disclosed herein comprise, or alternatively consist essentially of, and / or further comprise, administering one or more antibodies, alone or in combination. In some embodiments, such methods comprise, or alternatively consist essentially of, and / or further comprise, administering (i) an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of SEQ ID NO: 13 and / or SEQ ID NO: 14, or their respective equivalents, and (ii) an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of SEQ ID NO: 16 and / or SEQ ID NO: 17, or their respective equivalents. In some embodiments, such methods comprise, or alternatively consist essentially of, and / or further comprise, administering (i) an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of SEQ ID NO: 12, or its equivalent, and (ii) an antibody that specifically recognizes or specifically binds to an isolated or recombinant polypeptide consisting essentially of SEQ ID NO: 15, or its equivalent. In further embodiments of the disclosed methods, the antibodies can be administered simultaneously. In alternative embodiments, the antibodies are administered sequentially.

[0523] Also provided herein is a method for inducing an immune response in a subject in need thereof or conferring passive immunity to a subject in need thereof, comprising administering to the subject an effective amount of: (a) an isolated or recombinant integration host factor (IHF) polypeptide, or their respective fragments or equivalents; (b) an isolated or recombinant histone-like protein (HU) polypeptide or their respective fragments or equivalents; (c) an isolated or recombinant polypeptide of SEQ ID NO: 12 to 17, SEQ ID NO: 31, SEQ ID NO: 33, a polypeptide comprising the amino acid sequence NPXT, or a fragment or equivalent thereof; (d) a polypeptide that competes with integration host factors for binding to microbial DNA; (e) an isolated or recombinant polynucleotide encoding any one of (a) through (f), or a polynucleotide that hybridizes under stringent conditions to the above polynucleotides, their equivalents, or their complements; (f) an antibody or antigen-binding fragment, or an equivalent or fragment of each antibody or antigen-binding fragment thereof, that specifically recognizes or specifically binds to any one of (a) to (e); (g) an antibody or antigen-binding fragment comprising one or more of the CDR sequences disclosed herein, including the CDR sequences disclosed in SEQ ID NOs: 58-106, 108, and 109, a heavy chain sequence disclosed herein including the heavy chain sequence disclosed in SEQ ID NO: 51, and / or a light chain sequence disclosed in SEQ ID NO: 55, or their respective equivalents; (h) an antibody or antigen-binding fragment comprising one or more of the CDR sequences disclosed herein, including the CDR sequences disclosed in SEQ ID NOs: 110-159, the heavy chain sequence disclosed herein including the heavy chain sequence disclosed in SEQ ID NO: 53, and / or the light chain sequence disclosed herein including the light chain sequence disclosed in SEQ ID NO: 57, or their respective equivalents; or (i) An isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment of (f) through (h), or a complement thereof, or an equivalent thereof. Also provided are methods comprising, alternatively consisting essentially of, and / or further consisting of administering one or more of the group of:

[0524] In some embodiments, the antibody or antigen-binding fragment of (f), (g), and / or (h) is not a polyclonal antibody.

[0525] In a further aspect, the method comprises, or alternatively consists essentially of, and / or further consists of, administering to the subject an effective amount of one or more of an antimicrobial agent, an antigenic peptide, or an adjuvant.

[0526] Non-limiting examples of antibacterial agents are antibodies directed against surface antigens, e.g., vaccine components such as OMP P5, rsPilA, OMP 26, OMP P2, or type IV Pilin protein (see Jurcisek and Bakaletz (2007), J. Bacteriology, 189(10):3868-3875; Murphy et al. (2009), The Pediatric Infectious Disease Journal, 28:S121-S126; Novotny et al. (2015), Mol Microbiol., 96(2):276-92).

[0527] The agents and compositions disclosed herein can be administered simultaneously or sequentially with other antibacterial agents and / or surface antigens. In a specific embodiment, administration is local administration to the site of infection, for example, by direct injection or inhalation. Other non-limiting examples of administration include administration by one or more methods, including transdermal, urethral, ​​sublingual, rectal, vaginal, ocular, subcutaneous, intramuscular, intraperitoneal, intranasal, by inhalation, or orally.

[0528] Microbial infections and diseases that can be treated by the methods disclosed herein include, but are not limited to, infections by a variety of organisms associated with biofilm formation, including, but not limited to, those disclosed in Examples 4-9. Non-limiting examples of responsible organisms (and their exemplary strains in parentheses) include Aggregatibacter actinomycetemcomitans, Borrelia burgdorferi (e.g., B31), Bordetella pertussis (e.g., Tohama I), Burkholderia pseudomallei (e.g., 668), Burkholderia cenocepacia (e.g., HI2424), Escherichia coli (e.g., K12 MG1655), Enterococcus faecalis (e.g., V583), Haemophilus influenzae (e.g., Rd KW20), Helicobacter pylori (e.g., 26695), Klebsiella pneumoniae, Moraxella catarrhalis (e.g., RH4), Mycobacterium smegmatis (e.g., MC2), Mycobacterium tuberculosis (e.g., CDC1551), Neisseria gonorrhoeae, and the like. gonorrhoeae (e.g., FA1090), Neisseria meningitidis (e.g., MC58), Pseudomonas aeruginosa, Porphyromonas gingivalis (e.g., W83), Prevotella intermedia (e.g., 17), Prevotella melaninogenica (e.g., ATCC® 25845), Staphylococcus aureus (e.g., MW2), Staphylococcus epidermidis (e.g., RP62A), Streptococcus agalactiae (e.g., 2603V / R), Streptococcus bovis, Streptococcus gallolyticus (e.g., UCN34), Streptococcus gordonii (e.g., NCTC 7868(Challis), Streptococcus mutans (e.g., UA159), Streptococcus pneumoniae (e.g., R6), Streptococcus pyogenes (e.g., MGAS10270), Streptococcus sobrinus (e.g., 6715), Salmonella enterica (e.g., typhi, CT18), Treponema denticola (e.g., ATCC® 35405), Treponema palladum (e.g., Nichols), and Vibrio cholera (e.g., El Tor, N16961). Additional organisms known to be associated with and / or form biofilms include, but are not limited to, Campylobacter species, Candida species, Legionella pneumophila, and Listeria monocytogenes. For example, cystic fibrosis patients have Pseudomonas infections, which often result in antibiotic-resistant biofilms. Exemplary diseases associated with biofilms include, but are not limited to, lung infections in cystic fibrosis patients, otitis media, native valve infective endocarditis, osteomyelitis, rhinosinusitis, prostatitis, recurrent urinary tract infections, wounds, dental caries, and periodontitis. Conditions such as infected prosthetic devices, joints, catheters, stents, or other surgical implants are also associated with biofilm formation.

[0529] These microbial infections can be present in the upper, middle, and lower respiratory tract (such as otitis, sinusitis, bronchitis, but also 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 disclosed herein, these diseases and complications resulting from these infections can also be prevented or treated.

[0530] Infections can also occur in the oral cavity (dental caries, periodontitis) and are caused by Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Infections can also be localized to the skin (abscesses, "staph" infections, impetigo, secondary infections of burns, Lyme disease) and are caused by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Borrelia burdorferi. Infections of the urinary tract (UTI) are typically caused by Escherichia coli and can also be treated. Infections of the gastrointestinal tract (GI) (diarrhea, cholera, gallstones, stomach ulcers) are typically caused by Salmonella enterica serovar, Vibrio cholerae, and Helicobacter pylori. Genital tract infections typically involve and are caused by Neisseria gonorrhoeae. The infection can be a bladder infection or an indwelling device infection caused by Enterococcus faecalis. Infections associated with implanted prosthetic devices, such as artificial hip or knee joints, or dental implants, or medical devices, such as pumps, catheters, stents, or monitoring systems, are typically caused by various bacteria and can be treated by the methods disclosed herein. These devices can also be coated or conjugated with the agents described herein. Thus, by practicing the in vivo methods disclosed herein, these diseases and complications resulting from these infections can also be prevented or treated.

[0531] Infections caused by Streptococcus agalactiae, which is a leading cause of bacterial sepsis in newborns, can also be treated with the methods disclosed herein. Infections caused by Neisseria meningitidis, which can also cause meningitis, can also be treated.

[0532] Thus, administration routes applicable to the methods disclosed herein 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 and tailored depending on the agent and / or desired effect. Active agents can be administered in a single dose or multiple doses. Suitable embodiments of these methods and routes of delivery include systemic or local routes. In general, administration routes suitable for the methods disclosed herein include, but are not limited to, direct injection, enteral, parenteral, or by inhalation.

[0533] 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 typically involves invasive or systemically absorbed topical or mucosal administration of the pharmaceutical preparation.

[0534] The interfering agents disclosed herein can also be delivered to a subject by enteral administration, including, but not limited to, oral and rectal delivery (e.g., using a suppository).

[0535] Methods of administering active substances through the skin or mucosa include, but are not limited to, topical application of appropriate pharmaceutical preparations, transcutaneous transmission, transdermal transmission, injection, and epidermal administration. For transdermal penetration, absorption enhancers or iontophoresis are suitable methods. Iontophoretic penetration can be achieved using commercially available "patches" that deliver their products continuously through intact skin via electrical pulses for periods of several days or longer.

[0536] In various embodiments of the methods disclosed herein, the interfering agent is administered by inhalation, injection, or orally continuously, daily, at least once daily (QD), and in various embodiments twice daily (BID), three times daily (TID), or even four times daily. A daily therapeutically effective dose will typically be 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 in some cases as much as about 1 g to about 2.5 g, depending on the compound.

[0537] The present disclosure provides methods and compositions for inhibiting or preventing infection of a host or host cells by bacteria that export DNABII protein, comprising, alternatively consisting essentially of, and / or further consisting of, administering to a tissue exposed to or infected with the bacteria an effective amount of a bacteria-associated antibody that specifically recognizes and specifically binds to the DNABII protein, thereby inhibiting or preventing infection of a host or host cells by the bacteria. The antibody may be a polyclonal antibody, a monoclonal antibody, or a derivative of an antibody that recognizes and binds to the bacteria-associated DNABII protein. Multiple antibodies may be administered concurrently or sequentially, along with the supportive therapy referred to herein. Non-limiting exemplary antibodies include the antibodies disclosed herein, e.g., (a) SEQ ID NO: 12 [ka] (b) SEQ ID NO: 13 [ka] (c) SEQ ID NO: 14 [ka] (d) SEQ ID NO: 15 [ka] (e) SEQ ID NO: 16 [ka] (f) SEQ ID NO: 17 [ka] (g) SEQ ID NO: 33 [ka] (h) SEQ ID NO: 31 [ka] (i) a polypeptide comprising the amino acid sequence NPXT; or (j) Equivalents of (a) through (i), wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complement, wherein the high stringency conditions comprise an incubation temperature of from about 55° C. to about 68° C.; a buffer concentration of from about 1×SSC to about 0.1×SSC; a formamide concentration of from about 55% to about 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. The disclosed polypeptide fragments include, but are not limited to, antibodies generated against the disclosed polypeptide fragments.

[0538] In some embodiments, the antibody is not a polyclonal antibody.

[0539] Due to the ubiquitous nature of these DNABII proteins, which associate with the surface of bacteria throughout the cellular life cycle, including in all states, e.g., individual aggregated biofilm bacteria or inhabited biofilm bacteria, antisera directed against DNABII proteins can interfere with interactions with host cell surfaces, including attachment and subsequent invasion. Bacteria that are able to invade and multiply within host cells are protected from the host's immune system and antimicrobial treatment. The inability of bacteria to bind and invade makes them susceptible to elimination by the immune system and administered antimicrobials. Sources of antibodies against DNABII proteins can be induced by active vaccination of the host with DNABII proteins or fragments thereof, or by passive transfer of antisera or antibodies against DNABII proteins. Non-limiting examples of suitable polypeptide fragments include: (a) SEQ ID NO: 12 [ka] (b) SEQ ID NO: 13 [ka] (c) SEQ ID NO: 14 [ka] (d) SEQ ID NO: 15 [ka] (e) SEQ ID NO: 16 [ka] (f) SEQ ID NO: 17 [ka] (g) SEQ ID NO: 33 [ka] (h) SEQ ID NO: 31 [ka] (i) a polypeptide comprising the amino acid sequence NPXT; or (j) Equivalents of (a) through (i), wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complement, wherein the high stringency conditions comprise an incubation temperature of from about 55° C. to about 68° C.; a buffer concentration of from about 1×SSC to about 0.1×SSC; a formamide concentration of from about 55% to about 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. Including but not limited to:

[0540] The present disclosure provides a method for inhibiting or preventing infection of cells by bacteria that export DNABII protein. The method comprises, alternatively, consists essentially of, and / or further consists of, administering to a tissue infected with the bacteria an effective amount of an antibody that specifically recognizes and specifically binds to the DNABII protein, thereby inhibiting or preventing infection by the bacteria. The source of antibodies against the DNABII protein can be induced by active vaccination of the host with the DNABII protein, or by passive transfer of antisera or antibodies against the DNABII protein. The antibody can be a polyclonal antibody, a monoclonal antibody, or a derivative of an antibody that recognizes and binds to the bacteria-associated DNABII protein. Multiple antibodies can be administered simultaneously or sequentially, along with the supportive therapy referred to herein. Non-limiting exemplary antibodies include the antibodies disclosed herein, e.g., (a) SEQ ID NO: 12 [ka] (b) SEQ ID NO: 13 [ka] (c) SEQ ID NO: 14 [ka] (d) SEQ ID NO: 15 [ka] (e) SEQ ID NO: 16 [ka] (f) SEQ ID NO: 17 [ka] (g) SEQ ID NO: 33 [ka] (h) SEQ ID NO: 31 [ka] (i) a polypeptide comprising the amino acid sequence NPXT; or (j) Equivalents of (a) through (i), wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complement, wherein the high stringency conditions comprise an incubation temperature of from about 55° C. to about 68° C.; a buffer concentration of from about 1×SSC to about 0.1×SSC; a formamide concentration of from about 55% to about 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. The disclosed polypeptide fragments include, but are not limited to, antibodies generated against the disclosed polypeptide fragments.

[0541] In some embodiments, the antibody is not a polyclonal antibody.

[0542] The administration can be in vitro, in culture, or in vivo, to a patient infected with bacteria. When performed in vivo, the method can be used to treat a subject infected with bacteria by administering an effective amount of an antibody to the infected subject. In addition, when the subject is a non-human animal, the method can be used to investigate possible treatments or combination therapies before administration to humans. When performed in vitro, the method is useful for screening other therapeutic agents and combination therapies, such as small molecule drugs that inhibit or prevent bacterial infections in tissues.

[0543] Also provided is a method of treating a bacterial infection in a subject in need thereof infected with a bacterium containing a DNABII protein, comprising, alternatively consisting essentially of, and / or further consisting of, administering to the subject an effective amount of an antibody that specifically recognizes and specifically binds to the DNABII protein, thereby inhibiting or preventing bacterial infection. The antibody may be a polyclonal antibody, a monoclonal antibody, or a derivative of an antibody that recognizes and binds to the bacteria-associated DNABII protein. The source of antibodies against the DNABII protein may be induced by active vaccination of the host with the DNABII protein, or by passive transfer of antisera or antibodies against the DNABII protein. Multiple antibodies may be administered concurrently or sequentially, along with the supportive therapy referred to herein. Non-limiting exemplary antibodies include the antibodies disclosed herein, e.g., (a) SEQ ID NO: 12 [ka] (b) SEQ ID NO: 13 [ka] (c) SEQ ID NO: 14 [ka] (d) SEQ ID NO: 15 [ka] (e) SEQ ID NO: 16 [ka] (f) SEQ ID NO: 17 [ka] (g) SEQ ID NO: 33 [ka] (h) SEQ ID NO: 31 [ka] (i) a polypeptide comprising the amino acid sequence NPXT; or (j) Equivalents of (a) through (i), wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complement, wherein the high stringency conditions comprise an incubation temperature of from about 55° C. to about 68° C.; a buffer concentration of from about 1×SSC to about 0.1×SSC; a formamide concentration of from about 55% to about 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. The disclosed polypeptide fragments include, but are not limited to, antibodies generated against the disclosed polypeptide fragments.

[0544] In some embodiments, the antibody is not a polyclonal antibody.

[0545] Still further, there is provided a method for treating a condition in a subject in need thereof, the condition being associated with bacterial infection, wherein the bacteria expresses a DNABII protein, comprising, alternatively consisting essentially of, and / or further consisting of, administering to the subject an effective amount of an antibody that specifically recognizes and specifically binds to the DNABII protein, thereby inhibiting or preventing bacterial infection. The source of antibodies against the DNABII protein can be induced by active vaccination of the host with the DNABII protein, or by passive transfer of antisera or antibodies against the DNABII protein. The antibody can be a polyclonal antibody, a monoclonal antibody, or a derivative of an antibody that recognizes and binds to the bacteria-associated DNABII protein. Multiple antibodies can be administered simultaneously or sequentially, along with the supportive therapy referred to herein. Non-limiting exemplary antibodies include the antibodies disclosed herein, such as (a) SEQ ID NO: 12 [ka] (b) SEQ ID NO: 13 [ka] (c) SEQ ID NO: 14 [ka] (d) SEQ ID NO: 15 [ka] (e) SEQ ID NO: 16 [ka] (f) SEQ ID NO: 17 [ka] (g) SEQ ID NO: 33 [ka] (h) SEQ ID NO: 31 [ka] (i) a polypeptide comprising the amino acid sequence NPXT; or (j) Equivalents of (a) through (i), wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complement, wherein the high stringency conditions comprise an incubation temperature of from about 55° C. to about 68° C.; a buffer concentration of from about 1×SSC to about 0.1×SSC; a formamide concentration of from about 55% to about 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. The disclosed polypeptide fragments include, but are not limited to, antibodies generated against the disclosed polypeptide fragments.

[0546] In some embodiments, the antibody is not a polyclonal antibody.

[0547] Any of the above methods may further comprise, alternatively consist essentially of, or even consist of administering to the subject or to the in vitro tissue or cell culture an effective amount of one or more of an antimicrobial agent, an antigenic peptide, or an adjuvant. In some aspects, the subject is a non-human animal or a human patient.

[0548] The antibody, polypeptide, or composition is administered locally or systemically by any suitable method, for example, topically to the site of infection or biofilm, rectally, vaginally, ophthalmically, subcutaneously, intramuscularly, intraperitoneally, urethrally, intranasally, by inhalation, or orally.

[0549] In some aspects, the subject is a pediatric patient and the antibody is administered in a pediatric formulation.

[0550] Also disclosed are screens for identifying potential therapeutic agents that inhibit or prevent infection of cells by bacteria that export DNABII proteins and / or disrupt or prevent biofilm formation. The screening method comprises, alternatively, essentially consists of, and / or further consists of contacting an agent with a bacterially infected tissue in vitro, or administering an agent to a bacterially infected tissue in vivo, and determining whether the agent binds to the DNABII protein. Methods for determining binding are known in the art, and some non-limiting examples are described herein. In one aspect, if the agent binds to the protein, the agent is a potential therapeutic agent; if the agent does not bind to the protein, the agent is not a potential therapeutic agent. In another aspect, if infection or biofilm is inhibited, disrupted, or prevented in vivo, the agent is a potential therapeutic agent; if infection is not inhibited or prevented, the agent is not a potential therapeutic agent. Methods for determining whether an infection is inhibited or prevented are known in the art, some non-limiting examples of which are described herein; methods for determining whether a biofilm is disrupted or prevented are also known in the art, and are further disclosed herein. Non-limiting examples of potential therapeutic agents are selected from the group of antibodies, antibody derivatives, polypeptides, or small molecules. Multiple antibodies can be administered contemporaneously or sequentially, along with the supportive therapies referred to herein. Non-limiting exemplary antibodies include the antibodies disclosed herein, e.g., (a) SEQ ID NO: 12 [ka] (b) SEQ ID NO: 13 [ka] (c) SEQ ID NO: 14 [ka] (d) SEQ ID NO: 15 [ka] (e) SEQ ID NO: 16 [ka] (f) SEQ ID NO: 17 [ka] (g) SEQ ID NO: 33 [ka] (h) SEQ ID NO: 31 [ka] (i) a polypeptide comprising the amino acid sequence NPXT; or (j) Equivalents of (a) through (i), wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complement, wherein the high stringency conditions comprise an incubation temperature of from about 55° C. to about 68° C.; a buffer concentration of from about 1×SSC to about 0.1×SSC; a formamide concentration of from about 55% to about 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. The disclosed polypeptide fragments include, but are not limited to, antibodies generated against the disclosed polypeptide fragments.

[0551] In some embodiments, the antibody is not a polyclonal antibody.

[0552] In a further embodiment, the agent binds to the protein and binding is compared to binding of an anti-DNABII antiserum, eg, an antiserum directed against the DNABII protein, to the protein.

[0553] It will be appreciated that any of the general properties envisioned for interference agents that inhibit, reduce, or compete with the binding of DNABII protein to microbial DNA also apply to the methods disclosed above for bacterial infections.

[0554] Dosing can be achieved using capsules, tablets, oral suspensions, intramuscular injection suspensions, intravenous injection suspensions, topical application gels or creams, or intra-articular injection suspensions according to the methods disclosed herein.

[0555] The dosage, toxicity, and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as determining the 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 effects and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. In certain embodiments, the compositions exhibit a high therapeutic index. Compounds that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the affected tissue site to minimize potential damage to uninfected cells and thereby reduce side effects.

[0556] 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 lies (in certain embodiments) within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form and route of administration utilized. For any compound used in the methods, a therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound at which half-maximal inhibition of symptoms is achieved) 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.

[0557] 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 administration to about 10,000 mg per kilogram of body weight per administration. The dosage is suitably from about 0.0001 mg per kilogram of body weight per administration to about 100 mg per kilogram of body weight per administration. Administration can be as an initial dose followed by one or more "booster" doses. The booster doses can be administered 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months after the initial dose. In some embodiments, the booster dose is administered after assessing the subject's response to the previous dose.

[0558] Those skilled in the art will recognize that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or can include a series of treatments.

[0559] Antibody-based functional analysis The antibodies disclosed herein can be used to purify the polypeptides disclosed herein and identify biologically equivalent polypeptides and / or polynucleotides. The antibodies disclosed herein can also be used to identify agents that modify the function of the polypeptides disclosed herein, such as aptamers, polynucleotides, and small molecules. These antibodies include polyclonal antisera, monoclonal antibodies, and various reagents derived from these preparations that are familiar to those skilled in the art and are described above.

[0560] Antibodies that neutralize the activity of the proteins encoded by the identified genes can also be used to confirm in vivo and in vitro function by adding such neutralizing antibodies to in vivo and in vitro test systems. Such neutralizing antibodies are also useful as pharmaceutical agents that modulate the activity of the polypeptides disclosed herein.

[0561] Various antibody preparations can also be used in analytical methods such as ELISA assays or Western blots to confirm the expression of the proteins encoded by the identified genes by the test cells in vitro or in vivo.Fragments of such proteins generated by metabolic protease degradation can also be identified by using appropriate polyclonal antisera with samples derived from experimental samples.

[0562] Furthermore, in some embodiments, the antibodies disclosed herein can be used to visualize and / or detect biofilms. In such embodiments, the antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, or the like, or can be conjugated to other moieties, such as a member of a specific binding pair, for example, biotin (a member of the biotin-avidin specific binding pair). The detectably labeled antibody can then be introduced into a sample suspected of being colonized with a biofilm, and visualized and / or detected by microscopy or other methods known to detect the associated label, such as spectroscopy, cytometry, or other common techniques. Conjugated or unlabeled antibodies targeting the conjugated moiety or antibody, respectively, can also be identified by known analytical methods. For example, in some embodiments, detectably labeled secondary antibodies specific to the antibody isotypes disclosed herein may also be used in visualizing and / or detecting biofilms; such embodiments may involve administering an antibody specific to the "tail" of the DNABII protein to a surface suspected of being colonized by a biofilm, followed by administration of a detectably labeled secondary antibody and subsequent detection of the associated label.

[0563] In some embodiments, the "tail" of the DNABII protein (e.g., SEQ ID NOs: 12, 15, and 33), the consensus amino acid sequence [ka] The use of antibodies specific for the amino acid sequence of the DNABII protein, or their respective equivalents, which do not contain or have been engineered to exclude the sequence, is envisaged for visualizing or detecting the presence of biofilms on surfaces according to the methods described hereinabove. In some embodiments, the antibody is not a polyclonal antibody. In further embodiments, one or more of the antibodies disclosed hereinabove can be used for treating biofilms detected according to the methods described hereinabove. Non-limiting examples of antibodies that can be used in this step include those specific for the "tip" of the DNABII protein (e.g., SEQ ID NOs: 13, 14, 16, and 17), amino acid sequences that correspond to a sharp turn as part of an antiparallel beta ribbon, amino acid sequences [ka] In some embodiments, the antibody is not a polyclonal antibody.

[0564] The antibodies disclosed herein can be used for vaccination alone, in combination with peptide or protein-based vaccines or dendritic cell-based vaccines, or to boost vaccinations.

[0565] Screening assays The present disclosure provides methods for screening for equivalent agents, such as monoclonal antibodies and fragments thereof, equivalent to the exemplary antibodies described herein, as well as various agents that modulate the activity of the active agents and pharmaceutical compositions disclosed herein or the function of the polypeptide or peptide products encoded by the polynucleotides disclosed herein. For purposes of this disclosure, "agent" is intended to include, but is not limited to, simple or complex organic or inorganic molecules, peptides, proteins (e.g., antibodies), polynucleotides (e.g., antisense polynucleotides), or ribozymes. A wide variety of compounds can be synthesized, including, for example, polymers such as polypeptides and polynucleotides, and synthetic organic compounds based on various core structures; these are also encompassed by the term "agent." In addition, various natural sources, such as plant or animal extracts, can also provide compounds for screening. Although not always explicitly stated, it is understood that an agent may be used alone or in combination with another agent that has the same or different biological activity as the agent identified by the screens of the present invention.

[0566] To carry out in vitro screening method, first provide suitable cell culture or tissue that is infected with the microorganism to be treated.Cell is cultured for an appropriate length of time under the conditions (temperature, growth medium or culture medium, and gas (CO2)) to achieve exponential growth without density-dependent constraints.In addition, it is also desirable to maintain another separate cell culture that is not infected as a control.

[0567] As will be apparent to one of skill in the art, suitable cells can be cultured in microtiter plates and multiple agents can be assayed simultaneously by looking for genotypic changes, phenotypic changes, or reduction in microbial titer.

[0568] If the agent is a composition other than DNA or RNA, such as a small molecule as described above, the agent can be added directly to the cell culture or to the culture medium for supplementation. As will be apparent to those skilled in the art, an "effective" amount must be added, which can be determined empirically.

[0569] If the agent is an antibody or antigen-binding fragment, the agent can be contacted with or incubated with the target antigen and polyclonal antibody described herein under conditions for performing a competitive ELISA. Such methods are known to those skilled in the art.

[0570] The assay can also be performed in a subject. When the subject is an animal such as a rat, chinchilla, mouse, or monkey, the method provides a convenient animal model system that can be used before clinical testing of an agent in human patients. In this system, if the symptoms of disease or microbial infection are alleviated or eliminated compared to untreated animals with the same infection, the candidate agent is a potential drug. It can also be useful to have a separate negative control group of healthy, untreated cells or animals to provide a basis for comparison.

[0571] Additional screening assays specific for the antibodies contemplated herein include methods suitable for identifying antibodies or agents that specifically bind to one or more of the polypeptides disclosed herein, which involve identifying antibodies that bind to the site on the antigen to which an antibody of the invention binds.

[0572] The binding site of an antibody can be determined by methods known to those skilled in the art. For example, if an antibody binds to or recognizes a partial conformation of an antigen, the binding site of the antibody can be determined by using X-ray structural analysis to identify amino acid residues on the antigen adjacent to the antibody. For example, an antibody or a fragment thereof and an antigen or a fragment thereof are bound to each other and crystallized, and then structural analysis is performed to identify each amino acid residue on the antigen within interaction distance with the antibody. The interaction distance is 8 angstroms or less, preferably 6 angstroms or less, and more preferably 4 angstroms or less. One or more such amino acid residues within interaction distance with the antibody may constitute the site on the antigen to which the antibody binds (epitope). Two or more such amino acid residues cannot be adjacent to each other in the primary sequence. Such structural analysis can reveal the three-dimensional structure of the protein and thus provide both the sequence and conformation of the identified epitope.

[0573] A test antibody or test agent can be contacted with one or more of the polypeptides disclosed herein.The distance between the substance and the amino acid residues that make up the epitope to which any one of the antibodies disclosed herein binds is then measured.The test antibody or test agent can be determined to specifically bind if it is within an appropriate interaction distance from each of the residues.In some embodiments, the test antibody is not a polyclonal antibody.

[0574] In some embodiments, a test antibody or test agent is analyzed based on its interaction with a conformational epitope identified by structural analysis of antibody-antigen interactions. A non-limiting example of such a conformational epitope is a beta-hairpin, which may be contained within one or more of the polypeptides disclosed herein. A further non-limiting exemplary epitope is an amino acid sequence that conforms to a sharp turn as part of an antiparallel beta ribbon, such as the consensus amino acid sequence NPXT, where "X" refers to any amino acid. In some embodiments, X is selected from the amino acids Q, R, K, S, or T. Non-limiting examples of such antibodies are disclosed herein by showing the consensus amino acid sequence NPXT underlined and in bold. One skilled in the art will recognize that in any one of these sequences, the residue at position X (marked with a double underlin...

Claims

[Claim 1] The invention described in this specification.

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