Compositions and methods for preventing, inhibiting, disrupting, or treating polymicrobial biofilms

Abstract

To provide compositions and methods for preventing, inhibiting, disrupting, or treating polymicrobial biofilms.SOLUTION: For example, methods are provided for one or more of the following: preventing, inhibiting, disrupting, dispersing, or treating invitro and / or invivo a polymicrobial biofilm comprising Haemophilus bacteria in a subject in need thereof. The method comprises or consists essentially of or yet further consists of contacting a multi-microbial biofilm with (i) an anti-DNA binding and bending protein (DNABII) antibody or a biologically active fragment thereof and (ii) an anti-type IV pilin (T4P) majority subunit (PII A) antibody or a biologically active fragment thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 108,421, filed November 1, 2020, and the benefit of 35 U.S.C. §365(c) of U.S. Application No. 17 / 364,578, filed June 30, 2021, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] background The Centers for Disease Control and Prevention and the National Institutes of Health estimate that biofilms contribute to the pathogenesis of approximately 80% of all bacterial infections (Dongari-Bagtzoglou, 2008). Among many others, biofilm-associated diseases, such as otitis media (OM), cystic fibrosis, chronic obstructive pulmonary disease, chronic rhinosinusitis, chronic wound infections, periodontitis, cystitis, and infections of medical implants and indwelling catheters, are typically chronic and / or recurrent due to the presence of bacteria within the biofilm, which are highly resistant to killing by host immune effectors and antibiotics (Costerton et al., 1999; Flemming et al., 1999). and Wingender, 2010). Therefore, there is a need to find effective therapies to treat these diseases. There is a need in the art for providing such a method. The present disclosure fulfills this need and provides related advantages as well. Summary of the Invention [Means for solving the problem]

[0003] Disclosure Overview Many diseases of the upper and lower respiratory tract are caused by nontypeable Haemophilus influenzae (NTHI), where biofilms contribute significantly to the respective disease course (Autio et al., 2015; Cardines et al., 2012; Zhang et al., 2012). One such example of a disease in which NTHI is the primary pathogen is the most common bacterial disease in children (Hassan, 2013; Mittal et al., 2018). The primary cause of otitis media (OM) is otitis media (Barkai et al., 2009; Cleary et al., 2018; Grevers et al., 2012; Vergison, 2008; Wiertsema et al., 2011). The role of biofilms in the progression and recurrence of OM is widely accepted. Nevertheless, like most NTHI-induced diseases, OM is still commonly treated with broad-spectrum oral antibiotics, which do not reach sufficient levels in the middle ear (or other sites) to eradicate biofilms or even bacteria growing planktonically within this anatomical niche (Belfield et al., 2015). While their use may be indicated or necessary, broad-spectrum antibiotics can also cause collateral damage in the form of skin rashes, diarrhea, and lifelong disruption of the gut microbiome, with associated immunological and / or developmental consequences (Gilbert et al., 2018; Kuehn et al., 2015; Lamont et al., 2020). Additionally, the common, indiscriminate and often ineffective use of antibiotics contributes significantly to the rapidly growing global problem of the emergence of multi-antibiotic resistant bacteria (Beekmann et al., 2005; Leibovitz et al., 2010;Song et al., 2012).

[0004] The delivery of vaccines is important as they target prevention (Andre et al., 2008) and infection. Biofilm-associated chronic or recurrent infections present in these children and adults, however, are greatly needed for effective therapeutic approaches.

[0005] By applying the discoveries disclosed herein, there is provided herein a method for one or more of preventing, inhibiting, disrupting, dispersing, or treating polymicrobial biofilms, including biofilms caused by, or that will be caused by, or that are generated by, Haemophilus bacteria in a subject in need thereof. The method comprises, consists essentially of, or even consists of administering to the subject (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof. In one embodiment, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or a biologically active fragment thereof. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In certain embodiments, the CDRs of the anti-chip chimer antibody are set forth in SEQ ID NOS: 1-3, 7-9, 13, and 14, shown below. The CDRs of the anti-tail chimer are set forth in SEQ ID NOS: 4-6 and 10-12. In one aspect, the antibody is a polyclonal antibody. In one aspect, the antibody is a monoclonal antibody. In another aspect, the antibody is a monoclonal antibody or a biologically active fragment thereof. In a further aspect, the anti-DNABII chip chimer antibody is a monoclonal, chimeric, or humanized antibody, e.g., as disclosed herein. In another aspect, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include the PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1).In some aspects, the polymicrobial biofilm is caused by Haemophilus, e.g., NTHI, and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis. In some embodiments, the method further comprises administering an antibiotic to the subject. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic and / or a sulfonamide antibiotic.

[0006] Also provided is a method for preventing, inhibiting, disrupting, dispersing, or treating one or more of diseases associated with polymicrobial biofilm infections caused by, or that will be caused by, or that involve Haemophilus bacteria in a subject in need thereof. The method comprises, consists essentially of, or even consists of administering to the subject (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof. In one embodiment, the antibody is a polyclonal antibody or a monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or a biologically active fragment thereof. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In certain embodiments, the CDRs of the anti-chip chimer antibody are set forth in SEQ ID NOS: 1-3, 7-9, 13, and 14, shown below. In further aspects, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, e.g., as disclosed herein. In another aspect, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include the PilA surface antigen or recombinant soluble PilA (Genbank Accession No. EFU96734.1) and type IV pilin (Genbank Accession No. Yp_003864351.1). In some aspects, the polymicrobial biofilm is composed of Haemophilus, e.g., NTHI, and Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella. In some embodiments, the disease is caused by one or more of: Cystic fibrosis (CF), pulmonary CF, chronic obstructive pulmonary disease (COPD), chronic rhinosinusitis (CRS), periodontitis, peri-implantitis, middle ear infection, otitis media (OM), acute otitis media (AOM), otitis media with effusion (OME), post-tympanostomy tube ear discharge (PTTO), ventilator-associated pneumonia (VAP), community-acquired pneumonia (CAP), chest tube / catheter / indwelling device biofilm infection, tonsillitis, pharyng ... The antibiotic is selected from laryngitis, epiglottitis, sinusitis, pneumonia, bronchitis, or other respiratory tract infections (RTIs). In some embodiments, the method further comprises administering an antibiotic to the subject. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic and / or a sulfonamide antibiotic.

[0007] In yet another aspect, methods are provided for one or more of preventing, inhibiting, disrupting, dispersing, or treating a polymicrobial biofilm caused by, or to be caused in the future by, or comprising Haemophilus bacteria. The methods comprise, consist essentially of, or even consist of contacting the biofilm with (i) an anti-DNA binding and bending protein (DNABII) antibody or a biologically active fragment thereof, and (ii) an anti-type IV pilus (T4P) majority subunit (PilA) antibody or a biologically active fragment thereof. In some aspects, the polymicrobial biofilm is caused by Haemophilus, e.g., NTHI, and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis. In one aspect, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or a biologically active fragment thereof. In some aspects, the anti-DNABII antibody is an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In certain embodiments, the CDRs of the anti-chip chimer antibody are set forth in SEQ ID NOS: 1-3, 7-9, 13, and 14 shown below. In some embodiments, the method further comprises contacting the polymicrobial biofilm with an antibiotic. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic and / or a sulfonamide antibiotic. The contacting step can be in vitro or in vivo.

[0008] In a further aspect, a method is provided for sensitizing a polymicrobial biofilm caused by, or to be caused in the future by, or containing Haemophilus bacteria due to antibiotic therapy, or for inducing a polymicrobial biofilm-forming bacterium into a newly released (NRel) state. The method comprises, consists essentially of, or even consists of contacting the biofilm with (i) an anti-DNA binding and bending protein (DNABII) antibody or a biologically active fragment thereof, and (ii) an anti-type IV pilus (T4P) majority subunit (PilA) antibody or a biologically active fragment thereof. The antibodies can be polyclonal or monoclonal antibodies, or biologically active fragments thereof. In one aspect, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or their respective biologically active fragments. In certain embodiments, the CDRs of the anti-chip chimer antibody are set forth in SEQ ID NOS: 1-3, 7-9, 13, and 14, shown below. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further aspect, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, e.g., as disclosed herein. In another aspect, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include the PilA surface antigen or recombinant soluble PilA (Genbank Accession No. EFU96734.1) and type IV pilin (Genbank Accession No. Yp_003864351.1). In some embodiments, the method further comprises contacting the polymicrobial biofilm with an antibiotic. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic and / or a sulfonamide antibiotic.In some aspects, the polymicrobial biofilm is caused by Haemophilus, e.g., NTHI, and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis. The contacting step can be in vitro or in vivo.

[0009] In yet a further aspect, a method is provided for sensitizing a polyclonal microbial biofilm caused by, or to be caused in the future by, or containing Haemophilus bacteria in a subject in need thereof for antibiotic therapy, or for inducing bacteria in a polymicrobial biofilm in a subject in need thereof to a newly released (NRel) state. The method comprises, consists essentially of, or even consists of administering to the subject (i) an anti-DNABII antibody or a biologically active fragment thereof, and (ii) a PilA polypeptide or a biologically active fragment thereof. The antibody can be a polyclonal antibody or a monoclonal antibody. In one aspect, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or a biologically active fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In certain embodiments, the CDRs of the anti-chip chimer antibody are set forth in SEQ ID NOS: 1-3, 7-9, 13, and 14, shown below. In a further aspect, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, e.g., as disclosed herein. In another aspect, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include the PilA surface antigen or recombinant soluble PilA (Genbank Accession No. EFU96734.1) and type IV pilin (Genbank Accession No. Yp_003864351.1). In some aspects, the polymicrobial biofilm is caused by one or more of Haemophilus, e.g., NTHI, and Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis. In some embodiments, the method further comprises administering an antibiotic to the subject.In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic and / or a sulfonamide antibiotic.

[0010] Kits and compositions are also provided, for example, for use in the methods disclosed herein. In one aspect, a kit is provided that comprises, consists essentially of, or even consists of at least two of: (i) an anti-DNABII antibody or a biologically active fragment thereof; (ii) an anti-PilA antibody or a biologically active fragment thereof; or a PilA polypeptide or a biologically active fragment thereof; or (iii) an antibiotic, and, optionally, instructions for use. The antibody can be a polyclonal or monoclonal antibody. In one aspect, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or a biologically active fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In certain embodiments, the CDRs of the anti-chip chimer antibody are set forth in SEQ ID NOs: 1-3, 7-9, 13, and 14, as shown below. In a further aspect, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, e.g., as disclosed herein. In another aspect, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include the PilA surface antigen or recombinant soluble PilA (Genbank Accession No. EFU96734.1) and Type IV pilin (Genbank Accession No. Yp_003864351.1). In some embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic and / or a sulfonamide antibiotic.

[0011] In another aspect, a composition is provided that comprises, consists essentially of, or even consists of at least two of the following: (i) an anti-DNABII antibody or a biologically active fragment thereof, (ii) an anti-PilA antibody or a biologically active fragment thereof, or one or both of a PilA polypeptide or a biologically active fragment thereof, or (iii) an antibiotic, and a carrier, optionally a pharmaceutically acceptable carrier. The antibody can be a polyclonal or monoclonal antibody. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In certain embodiments, the CDRs of the anti-DNABII chip chimer antibody are set forth in SEQ ID NOS: 1-3, 7-9, 13, and 14 shown below. In a further aspect, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another aspect, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank Accession No. EFU96734.1) and Type IV pilin (Genbank Accession No. Yp_003864351.1). The antibody may be detectably labeled. In some embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic and / or a sulfonamide antibiotic.

[0012] In some embodiments, antibodies, polypeptides, their respective biologically active fragments, or any combination thereof may be administered to a subject in a composition disclosed herein. In other embodiments, antibodies, polypeptides, their respective biologically active fragments, or any combination thereof may be administered to a subject as a polynucleotide encoding such antibody, polypeptide, or biologically active fragment, or a polynucleotide complementary thereto. In some embodiments, such a polynucleotide may be suitable for expressing the antibody, polypeptide, or biologically active fragment in a subject, for example, by further comprising a regulatory sequence directing expression. In further embodiments, a vector, e.g., a gene delivery vehicle, comprising a polynucleotide disclosed herein may also be used for administration. The vector may be a viral or non-viral vector disclosed herein. In still further embodiments, host cells comprising one or more of the polynucleotides and / or vectors disclosed herein are provided. Accordingly, kits and compositions comprising one or more of such polynucleotides and / or vectors and / or host cells are provided in addition to, or instead of, the corresponding expressed antibodies, peptides, or biologically active fragments thereof. Further provided are methods and compositions suitable for producing such polypeptides, antibodies, biologically active fragments, polynucleotides, vectors and host cells.

[0013] Additionally, methods are provided for selecting polymicrobial biofilms caused by, that will be caused by, or that include Haemophilus bacteria suitable for one or more of the following: prevention, inhibition, disruption, dispersion, treatment, or sensitization by the methods disclosed herein. In some embodiments, the polymicrobial biofilm is caused by Haemophilus, e.g., NTHI, and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis. The screening method includes the steps of (a) contacting a polymicrobial biofilm, or a polymicrobial biofilm isolated and grown therefrom, with any one of an anti-DNABII antibody or a biologically active fragment thereof, an anti-PilA antibody or a biologically active fragment thereof, or a PilA polypeptide or a biologically active fragment thereof, and (b) measuring the expression of one or more of the following genes: deaD, artM, fis, folA, folP, emrA, emrB, or acrR, and the proteins: AsnC, CyaA, GlpC, NrfA, TehB, TrpB, TrpC, TrpD, TruA, TrxA; AbgA, AroE, AroK, ArtP, AtpH, BioB, BioF, CcmA, CitD, CitT, CmK, CoaD, Dc D, DjlA, DksA, DnaE, ​​DnaQ, DsbE, FabG, FdhX, FtnB, FtsE, FtsI, FumC, Fur, GlpA, GlpB, GmK, GpsA, GreA, Hf q, HisI, HugZ, InfA, InfB, IspD, IspF, Lic2A, LicC, LicD, LolA, LpsA, menC, MenC, NapA, NrfB, NrfC, NTHI0 053, NTHI0249, NTHI0291, NTHI0319, NTHI0349, NTHI0436, NTHI0487, NTHI0490, NTHI0555, NTHI0732, NT HI0779, NTHI0820, NTHI1025, NTHI1085, NTHI1199, NTHI1437, NTHI1439, NTHI1503, NTHI1590, NTHI1748,OppF, PanF, PdxH, PepT, PhoB, PlsC, PolA, PpC, PpiB, PrfB, PurU, RadA, RelB, RibA, RplW, RpoE, RpoZ, RseA, SecF, Sxy, TesB, ThrB, TolA, UnG, UreG, Uua P, UvrB; ClpB, CydD, DeaD, DlD, DmsB, ExbD, GroES, HfeA, HfeB, HgpB, HisJ, HisJ, HitA, HktE, HxuC, LctP, LldD, LpxC, MurB, NdhA, NifS2, NTHI0043, NTHI 0052, NTHI0175, NTHI0364, NTHI1208, NTHI1214, NTHI1369, NTHI1703, OmpP2, OrfG, Pal, PdgX, RbfA, RecN, RplO, RplU, RpmE, RpsE, RpsJ, RpsL, RpsQ, RpsT, SodA, Tbp1, TolB, TonB, TrpA, TrxA, or ZnuA. In some embodiments, altered gene expression of one or more of the following genes: deaD, artM, fis, folA, folP, emrA, emrB, or acrR; altered levels of one or more of the following proteins: AsnC, CyaA, GlpC, NrfA, TehB, TrpB, TrpC, TrpD, TruA, or TrxA; altered levels of one or more of the following proteins: AbgA, AroE, AroK, ArtP, AtpH, BioB, BioF, CcmA, CitD, CitT, CmK, CoaD, DcD, DjlA, DksA, DnaE, ​​DnaQ, DsbE, FabG, FdhX, FtnB, FtsE, FtsI, FumC, Fur, GlpA, GlpB, GmK, GpsA, GreA, Hfq, HisI, HugZ, InfA, InfB, IspD, IspF, Lic2A, LicC, LicD, L olA, LpsA, menC, MenC, NapA, NrfB, NrfC, NTHI0053, NTHI0249, NTHI0291, NTHI0319, NTHI0349, NTHI0436, NTHI0487, NTHI0490, NTHI0555, NTHI0732,Low levels of one or more of the following proteins: NTHI0779, NTHI0820, NTHI1025, NTHI1085, NTHI1199, NTHI1437, NTHI1439, NTHI1503, NTHI1590, NTHI1748, OppF, PanF, PdxH, PepT, PhoB, PlsC, PolA, PpC, PpiB, PrfB, PurU, RadA, RelB, RibA, RplW, RpoE, RpoZ, RseA, SecF, Sxy, TesB, ThrB, TolA, UnG, UreG, UuaP, or UvrB; or the following proteins: ClpB, CydD, DeaD, DlD, DmsB, ExbD, GroES, HfeA, HfeB, H High levels of one or more of gpB, HisJ, HisJ, HitA, HktE, HxuC, LctP, LldD, LpxC, MurB, NdhA, NifS2, NTHI0043, NTHI0052, NTHI0175, NTHI0364, NTHI1208, NTHI1214, NTHI1369, NTHI1703, OmpP2, OrfG, Pal, PdgX, RbfA, RecN, RplO, RplU, RpmE, RpsE, RpsJ, RpsL, RpsQ, RpsT, SodA, Tbp1, TolB, TonB, TrpA, TrxA, or ZnuA indicate that the biofilm is suitable for the methods disclosed herein. The antibody can be a polyclonal or monoclonal antibody. In one aspect, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or a biologically active fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further aspect, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In certain embodiments, the CDRs of the anti-chip chimer antibody are set forth in SEQ ID NOS: 1-3, 7-9, 13, and 14 shown below. In another aspect, the anti-PilA antibody specifically recognizes and specifically binds to a PilA peptide, examples of which include:Examples of antibodies include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody may be detectably labeled. Optionally, high, low, or altered expression or levels are compared to the same but planktonically grown bacteria. Kits for use in the methods are further provided, comprising, consisting essentially of, or even consisting of instructions and probes suitable for the assay. In some embodiments, one or more of the probes are labeled. [Brief explanation of the drawings]

[0014] [Figure 1] Figures 1A-1B show quantification of NTHI released from biofilms by either anti-rsPilA or anti-IHF. NTHI biofilms established for 16 h were additionally incubated with rabbit anti-rsPilA IgG for 6 h (Figure 1A), or rabbit anti-IHF IgG for 15 min (Figure 1B), or with each of three negative controls (sBHI, IgG isolated from naive serum, or IgG isolated from anti-OMP P5 serum), followed by quantification of NTHI recovered from the supernatant above the biofilm. Anti-rsPilA and anti-IHF induced significant release of NTHI from biofilms to the newly released (NRel) state. Individual data points are shown, and bars represent the mean ± SEM. ****, P<0.0001, one-way ANOVA with Holm-Sidak correction.

[0015] [Figure 2-1]Figures 2A–2E show that anti-rsPilA NRel was released from the biofilm as individual cells, while anti-IHF NRel was aggregated. Figure 2A provides contour plots showing side and forward scatter profiles for two control samples: NTHI was briefly sonicated to generate individual cell suspensions, or NTHI colonies were harvested from agar plates to form bacterial aggregates, which were then evaluated by flow cytometry. (Figure 2B) Representative contour plots of anti-rsPilA NRel and (Figure 2C) anti-IHF NRel demonstrated unique scattering profiles between the two NRel populations. (Figure 2D) Forward scatter and (Figure 2E) side scatter distributions of anti-IHF NRel (right histogram) versus anti-rsPilA NRel (left histogram) were also clearly distinct. The percent increase in the cumulative distribution of anti-IHF NRel compared to anti-rsPilA NRel shown in (Figure 2D) and (Figure 2E) was determined by the Kolmogorov-Smirnov test (99% CI). These data revealed additional discriminative features of NRel populations generated via exposure to either anti-rsPilA or anti-DNABII antibody-mediated release of NTHI from biofilms. [Figure 2-2] Same as above.

[0016] [Figure 3]Figures 3A-3D show the release of NTHI from biofilms by incubation with anti-rsPilA or anti-IHF antibody-generated NRel populations, which importantly had distinct proteomic expression profiles compared to planktonically grown NTHI and each other. Figure 3A provides principal component analysis (PCA) plots generated from normalized spectral counts of each protein in anti-rsPilA NRel (center circle with dots), anti-IHF NRel (left circle with dots), and planktonically grown NTHI (right circle with dots). Triplicate samples of each population are surrounded by 95% confidence ellipses. The proteomic expression profiles of anti-rsPilA NRel and anti-IHF NRel were distinct from both planktonically grown NTHI and each other. Figure 3B provides a Venn diagram of the number of proteins with a significant (P < 0.05) 1.5-fold increase (above the dashed line) or 2 / 3 decrease (below the dashed line) specific to anti-rsPilA (left panel), anti-IHF (right panel), or common (center panel) compared to planktonic NTHI. Figure 3C shows that anti-rsPilA NRel (upper bar in each group) and anti-IHF NRel (lower bar in each group) demonstrated distinct protein expression patterns with a significant (P < 0.05) 1.5-fold increase or 2 / 3 decrease when compared to planktonic NTHI, represented by different COG categories. Figure 3D provides a direct comparison of the differences in protein expression profiles of anti-IHF NRel and anti-rsPilA NRel populations with a significant (P < 0.05) 1.5-fold increase or 2 / 3 decrease compared to each other, as shown by the volcano plots of anti-IHF NRel vs. anti-rsPilA NRel. Negative significant fold decreases represent proteins of higher abundance in anti-rsPilA NRel (dots in the upper left), while positive fold increases represent proteins of higher abundance in anti-IHF NRel (dots in the upper right) compared to each other.

[0017] [Figure 4]Figures 4A-4C show the relative gene expression differences supporting the observed distinct anti-IHF NRel and anti-rsPilA NRel phenotypes, including antibiotic susceptibility. Results of qRT-PCR assays to examine gene expression by NRel compared to planktonic NTHI. Figure 4A provides the relative expression of deaD, artM, and fis, genes associated with the lag phase of growth, which was significantly greater in anti-IHF NRel versus anti-rsPilA NRel. Figure 4B shows that enzymes targeted by TMP and SMX are encoded by folA and folP, respectively, and thus increased expression confers resistance. The relative expression of folA and folP by anti-rsPilA NRel was significantly less than that by anti-IHF NRel. Figure 4C shows that the relative expression of emrA and emrB, encoding subunits of the efflux pump that transports TMP-SMX, was significantly less in anti-rsPilA NRel versus anti-IHF NRel. Expression of acrR, which represents an efflux pump that transports AMC, was significantly elevated in anti-IHF NRel versus anti-rsPilA NRel. These patterns of relative gene expression support the enhanced sensitivity of anti-rsPilA NRel or anti-IHF NRel to TMP-SMX or AMC, respectively. *P<0.05, ***P<0.001, ****P<0.0001, Student's t-test.

[0018] [Figure 5]Figures 5A-5E show that NRel NTHI populations were more susceptible to killing than their planktonic counterparts, and this susceptibility differed significantly from one another. Figure 5A provides a diagram of the four populations of NTHI tested herein. NRel was generated by incubation of NTHI biofilms with rabbit polyclonal IgG isolated from anti-rsPilA serum (6 hours, bottom left) or anti-IHF serum (15 minutes, bottom right). Figures 5B and 5C show that anti-rsPilA NRel was significantly more susceptible to killing by trimethoprim / sulfamethoxazole than planktonic NTHI (0.94 μg and 4.7 μg TMP-SMX per ml, respectively; Figure 5B), but was only equally susceptible to killing by amoxicillin / clavulanate (2.5 μg and 1.25 μg AMC per ml, respectively; Figure 5C). Biofilm-resident NTHI exhibited minimal sensitivity to either TMP-SMX or AMC, as expected. In contrast, as shown in Figures 5D and 5E, anti-IHF NRel was only equally sensitive to killing by TMP-SMX as planktonic NTHI (0.09 and 0.45 μg / ml, respectively; Figure 5D), but significantly more sensitive to killing by AMC (0.30 and 0.15 μg / ml, respectively; Figure 5E). The inherently enhanced sensitivity of NTHI NRel to killing by either TMP-SMX or AMC depended on the mechanism by which they were released from biofilm localization. Individual data points are shown, and bars represent the mean ± SEM. ***P < 0.001, ****P < 0.0001, one-way ANOVA with Holm-Sidak correction.

[0019] [Figure 6]Figures 6A–6D show that the enhanced susceptibility of anti-rsPilA NRel NTHI or anti-IHF NRel NTHI to TMP-SMX or AMC, respectively, was independent of the timing of NTHI release from biofilms. Applicants exposed biofilms to rabbit polyclonal IgG isolated from either anti-rsPilA or anti-IHF sera for 2 hours, then harvested and assayed NRel NTHI for relative antibiotic susceptibility. (Figures 6A and 6B) show that anti-rsPilA NRel was significantly more susceptible to killing by TMP / SMX than planktonic NTHI (0.09 μg and 0.45 μg TMP-SMX per ml, respectively) (Figure 6A), but was only equally susceptible to killing by AMC (0.3 μg and 0.15 μg AMC per ml, respectively). In contrast, as shown in Figures 6C and 6D, anti-IHF NRel was only equally susceptible to killing by planktonic NTHI and TMP-SMX (0.94 and 4.7 μg / ml, respectively, Figure 6C), but was significantly more susceptible to killing by AMC (2.5 and 1.25 μg / ml, respectively, Figure 6D). These data indicated that time-matched anti-rsPilA NRel or anti-IHF NRel maintained the same distinct antibiotic susceptibility phenotype, as shown when comparing 15 min of anti-IHF NRel with 6 h of anti-rsPilA NRel (see Figure 5). Individual data points are shown, and bars represent the mean ± SEM. ****, P < 0.0001, one-way ANOVA with Holm-Sidak correction.

[0020] [Figure 7]Figure 7 shows the synergistic effect of anti-rsPilA and anti-IHF (e.g., anti-DNABII) antibodies. Wells were incubated with a 1:1:1 ratio of NTHI + Streptococcus pneumoniae + Staphylococcus aureus and allowed to form biofilms for 16 hours at 37°C and 5% CO2, followed by 2 hours of treatment to determine relative destruction compared to treatment with medium alone (see treatment marked in the figure). Bacteria were labeled with FM1-43 FX, and relative fluorescence was calculated per well. A clear synergistic outcome was achieved through combined treatment with anti-rsPilA and anti-chip chimer monoclonal antibodies, demonstrated here for the first time.

[0021] [Figure 8-1] Figures 8A-8C show the antibiotic susceptibility of NTHIs newly released from biofilms by anti-IHF NTHI. Biofilms were grown at 37°C for 16 hours and then treated with anti-IHF NTHI (1:50 dilution) or sBHI alone for 6 hours. Planktonic populations were collected and assayed for antibiotic susceptibility. The AC concentrations used were 1 / 0.5 μg / ml, and the TS concentrations used were 7.5 / 37.5 μg / ml. (Figure 8A) plots data from anti-IHF diluted 1 / 10 or no anti-IHF treatment. (Figure 8B) plots data from anti-IHF diluted 1 / 100 or no anti-IHF treatment. (Figure 8C) provides the percentage of kill. N=3. [Figure 8-2] Same as above.

[0022] [Figure 9-1]Figures 9A-9C show synergistic activity against multi-species biofilms. (Figure 9A) shows NTHI + Burkholderia cenocepacia. NTHI-only biofilms continued to function, as expected with clearly demonstrated cocktail synergy. For two-species biofilms, only both antisera were effective, but the cocktail induced statistically significant synergistic release of both species from the two-species biofilm. (Figure 9B) shows activity against NTHI + Staphylococcus aureus biofilms. (Figure 9C) shows NTHI + Streptococcus pneumonia. [Figure 9-2] Same as above. [Figure 9-3] Same as above.

[0023] [Figure 10-1]Figures 10A-10E show the synergistic activity of anti-rsPilA + anti-Tip chimers on release by Moraxella catarrhalis (Mcat) from dual-species biofilms. (Figure 10A) NRel released from either a two-species biofilm or an NTHI biofilm formed by NTHI + Mcat after 2 hours of exposure to 2.5 μg of either anti-rsPilA antibody, anti-Tip antibody, or a combination of both antibodies, compared with unrelated bacteria from untreated biofilms. (Figure 10B) NRel released from either a two-species biofilm or an NTHI biofilm formed by NTHI + Mcat after 6 hours of exposure to 2.5 μg of either anti-rsPilA antibody, anti-Tip antibody, or a combination of both antibodies, compared with unrelated bacteria from untreated biofilms. (Fig. 10C) NRel released from either two biofilms formed by NTHI+Mcat or an NTHI biofilm after 16 h of exposure to 2.5 μg of either anti-rsPilA antibody, anti-Tip antibody, or a combination of both antibodies, compared with unrelated bacteria from an untreated biofilm. (Fig. 10D) NRel released from either two biofilms formed by NTHI+Mcat or an NTHI biofilm after 6 h of exposure to 10 μg of either anti-rsPilA antibody, anti-Tip antibody, or a combination of both antibodies, compared with unrelated bacteria from an untreated biofilm. (Fig. 10E) NRel released from either two biofilms formed by NTHI+Mcat or an NTHI biofilm after 16 h of exposure to 10 μg of either anti-rsPilA antibody, anti-Tip antibody, or a combination of both antibodies, compared with unrelated bacteria from an untreated biofilm. [Figure 10-2] Same as above. [Figure 10-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description definition All technical publications and patent disclosures cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure by prior disclosure. Throughout this disclosure, various technical publications are referenced by their first author and publication year or other identifying citation. Full citations for publications identified by first author and publication year can be found immediately before the claims.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods, devices, and materials are now described.

[0026] 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., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition;the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology;the series Methods in Enzymology (Academic Press, Inc., NY);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press);MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5 th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3 rdSee, for example, the following publication: (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

[0027] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations, which are varied (+) or (-) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also 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.

[0028] The term "about," when used herein when referring to a measurement such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the stated amount.

[0029] 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 "an antibody" includes a plurality of antibodies, including mixtures thereof.

[0030] As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding any other elements that are of essential importance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, etc. "Consisting of" is intended to mean excluding trace elements of other components and more than substantial method steps for administering a composition of the present disclosure or process steps for producing the composition or achieving an intended result. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0031] "As needed" or "as needed" means that the subsequently described situation may or may not occur, and as a result, the description includes instances where the situation occurs and instances where it does not occur.

[0032] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0033] "Substantially" or "essentially" means nearly entirely or completely, e.g., 95% or more, of some given amount. In some embodiments, "substantially" or "essentially" means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0034] As used herein, the comparative terms used herein, such as high, low, increase, decrease, reduce, or any grammatical variations thereof, can refer to a certain variation from the reference.In some embodiments, such variation can refer to an increase of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1-fold, or about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or about 20-fold, or about 30-fold, or about 40-fold, or about 50-fold, or about 60-fold, or about 70-fold, or about 80-fold, or about 90-fold, or about 100-fold, or more. In some embodiments, such a variation may refer to a decrease of about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, etc., of the reference.

[0035] As used herein, the term "polymicrobial biofilm" refers to a biofilm caused by two or more species of biofilm-causing bacteria. In some embodiments, the polymicrobial biofilm is caused by one or more of Haemophilus, e.g., NTHI, and another bacterium, e.g., Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis.

[0036] In some embodiments, the term "destroy" or its grammatical variants refers to the disruption of the DNA / protein matrix that is a component of microbial biofilms, for example, targeting antigens in the structural basis of the biofilm on the outside of a microorganism such as bacteria, thus destroying the biofilm and reducing and / or eliminating the biofilm. In some embodiments, such destruction process is not provided by a microorganism such as bacteria. In some embodiments shown herein, anti-DNABII antibodies destroy biofilms. In further embodiments, such destruction is not microbial species-specific.

[0037] In certain embodiments, disrupting a biofilm refers to dispersing the biofilm, releasing the microorganisms from the DNA / protein matrix of the biofilm, and, if necessary, allowing the microorganisms to be killed by the host's immune effectors and / or antibiotics.

[0038] The term "disperse" or grammatical variations thereof refers to disrupting molecules on or within the surface of microorganisms in a biofilm, e.g., targeting antigens on the surface of the microorganisms, thus disrupting the biofilm and resulting in the reduction and / or elimination of the biofilm. In some embodiments provided herein, the anti-PilA antibody disperses the biofilm. In further embodiments, the biofilm is composed of non-typeable Haemophilus containing and / or produced by influenzae.

[0039] "Inhibiting, preventing, or disrupting" a biofilm contemplates a prophylactic or therapeutic reduction in biofilm structure.

[0040] "Microbial DNA" refers to single- or double-stranded DNA derived from microorganisms, such as bacteria, that are incorporated into biofilms. As used herein, the term "eDNA" refers to extracellular DNA found as a component of pathogenic biofilms.

[0041] The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. This term also refers to both double-stranded and single-stranded molecules.Unless otherwise specified or required, any embodiment of the present disclosure that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs.Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.

[0042] 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" refers to an alphabetic representation of a polynucleotide molecule. This alphabetic representation can be input into a database on a computer having a central processing unit and used for bioinformatics applications, such as functional genomics and homology searching. In some embodiments, the polynucleotides disclosed herein are RNA. In some embodiments, the polynucleotides disclosed herein are DNA. In some embodiments, the polynucleotides disclosed herein are hybrids of DNA and RNA.

[0043] The term "isolated," as used herein with respect to nucleic acids, e.g., DNA or RNA, refers to a molecule separated from other DNAs or RNAs, respectively, present in the natural source of the polymer. The term "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides, proteins, and / or host cells that are isolated from other cellular proteins and is intended to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated" refers to being separated from cellular constituents and other cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof, with which it is otherwise normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a dissimilar phenotype or genotype. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0044] In some embodiments, the term "engineered" or "recombinant" refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain, or parent host strain of the referenced species. In some embodiments, the term "engineered" or "recombinant" refers to being synthesized by human intervention.

[0045] When the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be assumed, without explicit description and unless otherwise intended, that equivalents or biological equivalents thereof are intended to be within the scope of the present disclosure. As used herein, the term "biological equivalent thereof" when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid is intended to be synonymous with "equivalent thereof," and is intended to be synonymous with "equivalent thereof," meaning that it has minimal homology but still maintains the desired structure or functionality. Unless specifically recited herein, any polynucleotide, polypeptide, or protein referred to herein is intended to include its equivalent. In some embodiments, an equivalent to a reference nucleic acid, polynucleotide, or oligonucleotide encodes the same sequence encoded by the reference. In some embodiments, an equivalent to a reference nucleic acid, polynucleotide, or oligonucleotide hybridizes to the reference, complement reference, reverse reference, and / or reverse complement reference, optionally under high stringency conditions.

[0046] Additionally or alternatively, an equivalent nucleic acid, polynucleotide, or oligonucleotide has at least 70%, or at least 75%, or at least 80% sequence identity, or alternatively, at least 85% sequence identity, or alternatively, at least 90% sequence identity, or alternatively, at least 92% sequence identity, or alternatively, at least 95% sequence identity, or alternatively, at least 97% sequence identity, or alternatively, at least 98% sequence identity to a reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively, an equivalent nucleic acid hybridizes to a reference polynucleotide or its complement under high stringency conditions. In one aspect, an equivalent should encode a functional protein that can be identified, as appropriate, by one or more assays described herein. In another embodiment, an equivalent has at least 70%, or at least 75%, or at least 80% sequence identity, or alternatively, at least 85% sequence identity, or alternatively, at least 90% sequence identity, or alternatively, at least 92% sequence identity, or alternatively, at least 95% sequence identity, or alternatively, at least 97% sequence identity, or alternatively, at least 98% sequence identity to a reference nucleic acid, polynucleotide, or oligonucleotide; or alternatively, an equivalent nucleic acid hybridizes to a reference polynucleotide or its complement under high stringency conditions, with the proviso that one or more mutated polynucleotides identified herein having one or more non-naturally occurring glycosylation sites are not mutated from the corresponding mutated polynucleotide in the disclosed sequences.

[0047] In one embodiment, an equivalent polynucleotide is one that hybridizes under stringent conditions to the polynucleotide or complement of a polynucleotide described herein for use in the described method. In another embodiment, an equivalent antibody or antigen-binding polypeptide is intended to bind to an antigen with at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% affinity, or higher, compared to a reference antibody or antigen-binding fragment. In another embodiment, the equivalent competes with the binding of the reference antibody or antigen-binding fragment to its antigen in a competitive ELISA assay. In another embodiment, an equivalent is intended to have at least about 80% homology or identity, and alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% percent homology or identity, and exhibit substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. Examples of biologically equivalent polypeptides are provided in Table 9 of WO2011 / 123396, which identifies conservative amino acid substitutions for the disclosed amino acid sequences.

[0048] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, the percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: Genetic coding = standard; Filter = none; Strand = both; Cutoff = 60; Expected = 10; Matrix = BLOSUM62; List = 50 sequences; Sort = high score; Database = non-redundant GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: www.ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0049] "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 in each sequence, which can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively, less than 25% identity, with one of the sequences of the present disclosure.

[0050] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions. "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can include two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can also constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0051] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonding between the bases of nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can include two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme. When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called "annealing," and these polynucleotides are described as "complementary." When hybridization can occur between one strand of a first polynucleotide and one strand of a second polynucleotide, a double-stranded polynucleotide can be "complementary" or "homologous" to another polynucleotide. "Complementarity" or "homology" (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion of bases in opposite strands that would be predicted to form hydrogen bonds with each other according to generally accepted rules of base pairing.

[0052] Hybridization reactions can be performed under conditions of different "stringency." Generally, low-stringency hybridization reactions are performed in 10xSSC at about 40°C, or in solutions of equivalent ionic strength / temperature. Medium-stringency hybridizations are typically performed in 6xSSC at about 50°C, and high-stringency hybridization reactions are generally performed in 1xSSC at about 60°C. Other 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, and wash incubation times of about 1, 2, or 15 minutes. SSC is a 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used. Hybridization reactions can also be performed under "physiological conditions," as known to those skilled in the art. Non-limiting examples of physiological conditions include temperature, ionic strength, pH, and Mg concentrations normally found in cells. 2+ is the concentration.

[0053] The term "amplification of polynucleotides" includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR) and amplification methods. These methods are well known and widely practiced in the art. See, for example, U.S. Patent Nos. 4,683,195 and 4,683,202, and Innis et al., 1990 (for PCR), and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR procedure describes a method of gene amplification consisting of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, extension, and denaturation using DNA polymerase, and (iii) screening of the PCR product for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide for the initiation of polymerization; i.e., each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

[0054] Reagents and hardware for performing PCR are commercially available. Primers useful for amplifying sequences from specific gene regions are preferably complementary to and specifically hybridize with sequences in the target region or its adjacent regions. The nucleic acid sequences generated by amplification can be directly sequenced. Alternatively, the amplified sequences can be cloned before sequence analysis. Methods for direct cloning and sequence analysis of enzymatically amplified genomic segments are known in the art.

[0055] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0056] The term "expression" refers to the production of a gene product. As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or 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.

[0057] The term "encoding," as applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when, in its natural state, or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0058] "Gene product" or alternatively "gene expression product" refers to the mRNA and / or amino acids (e.g., peptides or polypeptides) made when a gene is transcribed and translated.

[0059] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on it being operably linked to elements that contribute to or promote the initiation of transcription. By "operably linked" is intended that the polynucleotides are positioned in a manner that allows them to function in the cell.

[0060] The term "regulatory sequence," "expression control element," or "promoter," as used herein, refers to a polynucleotide that is operably linked to a target polynucleotide to be transcribed and / or replicated and facilitates the expression and / or replication of the target polynucleotide. A promoter is an example of an expression control element or regulatory sequence. A promoter can be located 5', or upstream of a gene or other polynucleotide, providing a control point for transcription of the regulated gene. Polymerase II and III are examples of promoters.

[0061] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is juxtaposed to a gene by means of genetic engineering (i.e., molecular biological techniques) so that transcription of that gene is directed by the linked enhancer / promoter.

[0062] "Probe", when used in the context of polynucleotide manipulation, refers to an oligonucleotide that serves as a reagent for detecting a target potentially present in a sample of interest by hybridizing with the target. Typically, a probe includes a detectable label or marker, or a means by which the label or marker can be attached either before or after the hybridization reaction. Alternatively, a "probe" can be a biological compound, such as a polypeptide, antibody, or fragment thereof, that can bind to a target potentially present in a sample of interest.

[0063] A "primer" is a short polynucleotide, generally a short polynucleotide with a free 3'-OH group, that binds to a target or "template" potentially present in a sample of interest by hybridizing with the target, and then promotes the polymerization of a polynucleotide complementary to the target. A "polymerase chain reaction" ("PCR") is a reaction in which a replicate copy of a target polynucleotide is made using a "primer pair" or "primer set" consisting of an "upstream" and a "downstream" primer, and a polymerization catalyst, such as DNA polymerase, and typically a thermostable polymerase enzyme. Methods for PCR are well known in the art and are taught, for example, in MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press). All processes that generate duplicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as "replication." Primers can also be used as probes in hybridization reactions, such as Southern or Northern blot analysis. Sambrook and Russell (2001), infra.

[0064] 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 esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein's or peptide's sequence. As used herein, the term "amino acid" refers to either natural and / or unnatural amino acids, or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0065] By "C-terminal polypeptide" is intended at least 10, or alternatively at least 15, or alternatively at least 20, or 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 to 90. In one embodiment, the term contemplates the carboxyl-terminal to C-terminal 20 amino acids.

[0066] As used herein, the amino acid (aa) or nucleotide (nt) residue position in a target sequence that "corresponds" to a specified position in a reference sequence refers to the residue position aligned with the specified position in the sequence alignment between the target sequence and the reference sequence. Various programs, such as Clustal Omega and BLAST, are available for performing such sequence alignments.

[0067] The term "conservative amino acid substitution" refers to the substitution of a natural amino acid residue with a standard residue, including naturally occurring and non-naturally occurring amino acids, such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue. Furthermore, any natural residue in a polypeptide may also be substituted with alanine according to the method of "alanine scanning mutagenesis." Naturally occurring amino acids are characterized based on their side chains as follows: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; uncharged polar: glutamine, asparagine, serine, threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline, methionine, leucine, norleucine, isoleucine. The general rules for amino acid substitution are set forth below. [Table 6]

[0068] The terms equivalent and biologically equivalent are used interchangeably, for example, when referring to a reference antibody, antibody fragment, protein, or polypeptide. In some embodiments, an equivalent antibody, antibody fragment, protein, or polypeptide is one that has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the reference protein or polypeptide. In some embodiments, an equivalent antibody, antibody fragment, protein, or polypeptide has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a polypeptide or protein disclosed herein. In some embodiments, an equivalent antibody, antibody fragment, protein, or polypeptide has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an antibody, antibody fragment, polypeptide, or protein encoded by an equivalent polynucleotide described herein. Additionally, or alternatively, polynucleotide equivalents encode antibodies, antibody fragments, proteins or polypeptides of the same or similar function as the reference or parent polynucleotide.

[0069] In some embodiments, equivalents are functional proteins that can be identified as needed by one or more assays described herein. In another aspect, equivalents have at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a reference protein or polypeptide.

[0070] In some embodiments, an equivalent or biologically equivalent antibody, antibody fragment, protein, or polypeptide performs a similar function as the wild-type and / or performs a similar level of function compared to the wild-type.

[0071] As used herein, "EC 50 " refers to the concentration of an antibody or fragment thereof that induces a response (e.g., binding between an antibody or fragment thereof and its target) that is intermediate between baseline and maximum after a specified exposure time.

[0072] Several parameters are used herein to describe the binding and non-binding reactions of receptor (R, e.g., an antibody or fragment thereof) and ligand (L, e.g., the target of the antibody or fragment thereof) molecules, which are formalized as R + L ⇔ RL. This reaction is expressed as M -1 seconds -1 and seconds -1 The on-rate constant k has units of on and the off-rate constant k off In equilibrium, the forward bonding transition R+L → RL must be balanced by the backward non-bonding transition RL → R+L. That is, k on [R][L]=k off [RL], where [R], [L], and [RL] represent the concentration of unbound free receptor, the concentration of unbound free ligand, and the concentration of the receptor-ligand complex. Furthermore, the equilibrium dissociation constant, K D" is [R] × [L] / [RL] k off / k on The equilibrium binding constant "K" can be calculated as A " is [RL] / ([R] × [L]) k on / k off It can be calculated as:

[0073] As used herein, the term "tip" or "conformational tip domain" of a polypeptide refers to a polypeptide that comprises a primary amino acid sequence whose structure has an antiparallel beta ribbon with a sharp turn typically mediated by a proline residue.

[0074] A "chimer" of a polypeptide refers to a polypeptide that contains two chips of one or more polypeptides. A chimer may contain two chips of the same polypeptide, or chips from two different polypeptides. A chimer of a DNABII polypeptide refers to a DNABII polypeptide that forms a two-arm protein, using IHF alpha and IHF beta as an example (see Figure 1 of U.S. Patent No. 11,104,723, incorporated by reference). Non-limiting examples include the IhfA chip fragment, also referred to herein as A chip fragment: NFELRDKSSRPGRNPKTGDVV, SEQ ID NO: , and the IhfB modified B4: SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: ) or FSLHHRQPRLGRNPKTGDSV, SEQ ID NO: , or two IhfA chip fragments, or two IhfB modified B4 chip fragments, also referred to herein as mB4 chip fragments. Additional examples are disclosed in PCT Publication No. WO2018 / 129078, which is incorporated herein by reference. In certain embodiments, the chip chimeric peptide comprises: [ka] (wherein "X" is an optional amino acid linker sequence comprising, consisting essentially of, or alternatively consisting of, 1 to 20 amino acids, and "X1" is any amino acid, or alternatively, "X1" is selected from the amino acids Q, R, K, S, or T), as used herein, comprising, consisting essentially of, or alternatively consisting of, a polypeptide sequence of IhfA5-mIhfB4. NTHI In a further aspect, "X1" is K or Q. In a further embodiment, the chip chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (wherein "X" is an optional amino acid linker sequence comprising, consisting essentially of, or alternatively consisting of 1-20 amino acids). In yet a further embodiment, the chip chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] In some embodiments, this may also be referred to herein as a chimer, in which GPSL serves as an amino acid linker sequence.

[0075] By "tail fragment" of a DNABII polypeptide is intended the region of either protein that is exposed to the bulk medium and is not obstructed by DNA or other polypeptides.

[0076] By immunodominant antigen is intended a region of a protein that is recognized by an antibody and that binds to the antibody with high affinity.

[0077] As used herein, "antibody" includes whole antibodies and any antigen-binding fragments or single chains thereof, variants, or derivatives thereof. Thus, the term "antibody" includes any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule. Examples 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, or at least a portion of a binding protein, any of which may be incorporated into the antibodies of the present disclosure. The term "antibody" is further intended to encompass fragments, defined portions thereof, derivatives, and variants, including antibody mimetics or portions of antibodies that mimic the structure and / or function of antibodies or defined fragments or portions thereof, including single-chain antibodies and fragments thereof. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include V, ... L , V H , C L and CH domains; F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; V H and C H Fd fragment consisting of domain; V of a single arm of an antibody L and V H Fv fragment consisting of domains, V H dAb fragments consisting of two domains, V and VD (Ward et al. (1989) Nature 341:544-546); and isolated complementarity-determining regions (CDRs). Furthermore, Fv fragments are fragments consisting of two domains, V and VD. L and V H Although the V are encoded by separate genes, they can be connected by a synthetic linker that allows them to be produced as a single protein chain using recombinant methods, where V L and V HThe domains pair to form monovalent molecules known as single-chain Fvs (scFvs). Bird et al. (1988) Science 242:423-426 and and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85: 5879-5883. Single-chain antibodies are also intended to be encompassed within the term "fragment of an antibody" or "biologically active fragment" of an antibody. Any of the above-mentioned antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as are intact antibodies.

[0078] The terms "antibody," "antibodies," and "immunoglobulin" include immunoglobulins of any isotype, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains, and fragments of antibodies that retain specific binding to an antigen; minibodies, diabodies, triabodies, tetrabodies, and kappabodies; multispecific antibody fragments formed from antibody fragments, and one or more isolated fragments. Examples of such fragments include, but are not limited to, heavy or light chain complementarity-determining regions (CDRs) or ligand-binding portions thereof, heavy or light chain variable regions (also referred to herein as variable domains), heavy or light chain constant regions (also referred to herein as constant domains), framework (FR) regions, or any portion thereof, at least a portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of an immunoglobulin molecule contain a binding domain that interacts with an antigen, and the constant regions of an antibody (Ab) may mediate the binding of the immunoglobulin to host tissue.

[0079] The terms "anti" or "antibody" when used before the name of a protein, e.g., anti-DNABII, anti-IHF, anti-HU, anti-chimer, anti-PilA, anti-OMP P5, refer to a monoclonal or polyclonal antibody, or a biologically active fragment thereof, that binds to and / or has affinity for a particular protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody may have affinity for or bind to proteins other than the protein against which it was raised. For example, anti-IHF was raised specifically against the IHF protein, but may also bind to other proteins related by either sequence or structural homology.

[0080] Complementarity-determining regions (CDRs) are portions of the variable regions of antibodies or T-cell receptors produced by B cells and T cells, respectively, where these molecules bind to their specific antigen (also called epitope). In certain embodiments, the terms "variable region" and "variable domain" are used interchangeably and refer to the polypeptides of the antibody light or heavy chains whose sequence of amino acid residues varies widely from antibody to antibody and determines the conformation of the binding site that confers the antibody's specificity for a particular antigen. In further embodiments, the variable region is about 90 to about 200 amino acids in length, for example, but not limited to, about 100 amino acids in length, or alternatively, about 110 amino acids in length, or alternatively, about 120 amino acids in length, or alternatively, about 130 amino acids in length, or alternatively, about 140 amino acids in length, or alternatively, about 150 amino acids in length, or alternatively, about 160 amino acids in length, or alternatively, about 170 amino acids in length, or alternatively, about 180 amino acids in length, or alternatively, about 190 amino acids in length. In certain embodiments, a variable region of an amino acid sequence, as used herein, refers to the first about 100 amino acids, or alternatively, about 110 amino acids, or alternatively, about 120 amino acids, or alternatively, about 130 amino acids, or alternatively, about 140 amino acids, or alternatively, about 150 amino acids of an amino acid sequence (including or excluding the signal peptide, if applicable) being the variable region.

[0081] A set of CDRs constitutes a paratope, also called an antigen-binding site, which is the portion of an antibody that recognizes and binds to an antigen. Three CDRs (CDR1, CDR2, and CDR3) are present in the amino acid sequence of the variable region of an antigen receptor, such as a heavy or light chain, optionally arranged non-contiguously from the amino terminus to the carboxyl terminus. As used herein, CDRn refers to a CDRn in an immunoglobulin chain or a CDRn derived from an immunoglobulin chain, where the number n is selected from 1 to 3. In one embodiment, CDRLn refers to a CDRn in a light chain or a CDRn derived from a light chain, where the number n is selected from 1 to 3, while CDRHn refers to a CDRn in a heavy chain or a CDRn derived from a heavy chain, where the number n is selected from 1 to 3. In certain embodiments, framework region (FR) refers to the portion of the variable region that is not a CDR. In certain embodiments, FRn refers to a FR in a heavy or light chain or a FR derived from a heavy or light chain, where the number n is selected from 1 to 4. In certain embodiments, the variable region comprises, consists essentially of, or alternatively consists of the following (optionally in the order provided, and optionally from amino terminus to carboxyl terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0082] The variable regions and / or CDRs of an antibody, or fragments thereof, can be determined by one of skill in the art using, for example, publicly available or commercially available tools. Non-limiting examples of such tools include IgBlast (accessible at www.ncbi.nlm.nih.gov / igblast / ), Scaligner (available from drugdesigntech at www.scaligner.com / ), IMGT rules and / or tools (see, e.g., www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html, also accessible at www.imgt.org / ), Chothia Canonical Assignment (accessible at www.bioinf.org.uk / abs / chothia.html), Antigen Receptor Numbering And Receptor Classification (ANARCI, accessible at opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / ), Kabat numbering method / scheme (e.g., Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) or the Paratome web server (accessible at www.ofranlab.org / paratome / ; see Vered Kunik, et al, Nucleic Acids Research, Volume 40, Issue W1, 1 July 2012, Pages W521-W524).

[0083] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific) and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, for example, mouse, rat, sheep and dog. As used herein, and unless specifically stated, the term "antibody" includes biologically active fragments thereof, polyclonal, monoclonal, human, humanized, variants, derivatized, chimeric, or other known modifications thereof.

[0084] The terms "polyclonal antibody" or "polyclonal antibody composition," as used herein, refer to a preparation of antibodies derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a particular antigen, each recognizing a different epitope.

[0085] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies are highly specific because each monoclonal antibody is directed against a single determinant on an antigen. The antibody may be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, or the like. The antibody may be further conjugated to another moiety, for example, a member of a specific binding pair, such as biotin (a member of the biotin-avidin specific binding pair). The antibody may also be bound to a solid support, including, but not limited to, a polystyrene plate or bead.

[0086] Monoclonal antibodies may be produced using hybridoma techniques or recombinant DNA methods known in the art. Hybridomas are cells produced in laboratories from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells, usually myelomas or lymphomas. Hybridomas grow and produce continuous samples of specific monoclonal antibodies. Alternative techniques for producing or selecting antibodies include in vitro exposure of lymphocytes to the antigen of interest and screening antibody display libraries in cell, phage, or similar systems.

[0087] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced randomly or by site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to an antibody that includes substantially all portions of the protein (e.g., CDRs, framework, C L , C H Domain (e.g., C H1 , C H2 , C H3 ), hinge, (V L , V H) refers to antibodies that are substantially non-immunogenic in humans and involve only minor sequence changes or modifications. Similarly, antibodies designated as primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals designate antibodies specific to that species, subgenus, genus, subfamily, or family. Furthermore, chimeric antibodies include any combination of the above. Such changes or modifications optionally retain or reduce immunogenicity in humans or other species compared to unmodified antibodies. Therefore, human antibodies are distinct from chimeric or humanized antibodies. It is noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Furthermore, when a human antibody is a single-chain antibody, it can include a linker peptide not found in native human antibodies. For example, an Fv can include a linker peptide, e.g., two to about eight glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region, and such a linker peptide is considered to be of human origin.

[0088] As used herein, a human antibody is "derived" from a particular germline sequence if the antibody is obtained from a system that uses human immunoglobulin sequences, for example, by immunizing a transgenic mouse with human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody "derived" from a human germline immunoglobulin sequence can itself be identified by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin. The selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a mouse germline sequence). In certain cases, a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence displays no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0089] 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 antibodies) in which residues from a variable region or fragment thereof (e.g., one, two, three, four, five, or all six CDRs) of the recipient are replaced by residues from a variable region or fragment thereof (e.g., one, two, three, four, five, or all six CDRs) of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. Humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. Humanized antibodies may also optionally comprise non-human antibodies containing one or more amino acids substituted with correspondingly positioned amino acids from a human antibody in at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, framework region, constant region, or CDR. Without wishing to be bound by theory, humanized antibodies generate a reduced immune response in a human host compared to a non-humanized version of the same antibody. Humanized antibodies may have conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions. Conservative substitution groupings include glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine. Specifically, the humanized antibodies disclosed herein may have one or more of the following substitutions within a certain range: EC 50 , K. on , K. off , K. A and / or K D In a further embodiment, a humanized antibody that specifically binds to the DNABII polypeptide or a fragment thereof (such as a tip chimeric peptide or a tail chimeric peptide) inhibits or releases a specific cytokine when treating a subject. In a further embodiment, a humanized antibody that specifically binds to the tip region (such as a tip chimeric peptide) of a DNABII polypeptide is Disrupts biofilms both in vivo and in vitro. Additionally, the humanization process, although a rational design process, can result in unexpected changes (positive or negative) in binding affinity, antigen specificity, or physical properties such as solubility or aggregation; therefore, the properties of humanized antibodies are not inherently predictable from those of the starting non-human antibody.

[0090] In one embodiment, an antibody, as used herein, may be a recombinant antibody. The term "recombinant antibody," as used herein, includes all antibodies prepared, expressed, generated, or isolated by recombinant means, e.g., antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for 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 antibody libraries, and antibodies prepared, expressed, generated, or isolated by any other means involving splicing of immunoglobulin (Ig) gene sequences into other DNA sequences. However, in certain embodiments, such recombinant antibodies can be subjected to in vitro mutagenesis (or, if animals transgenic for Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V of the recombinant antibody. H and V L The amino acid sequences of the regions are sequences that may not naturally occur within the antibody germline repertoire in vivo. Methods for making these antibodies are described herein.

[0091] In one embodiment, an antibody, as used herein, may be a chimeric antibody. As used herein, a chimeric antibody is an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.

[0092] As used herein, an antibody may be an "antibody derivative." In some embodiments, an antibody derivative includes a full-length antibody or a fragment of an antibody in which one or more of the amino acids have been chemically modified, for example, by alkylation, pegylation, acylation, ester formation, or amide formation, to link the antibody to a second molecule. This includes, but is not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof. In some embodiments, the term "antibody derivative" is intended to encompass molecules that bind to the epitope defined above and are modifications or derivatives of the native monoclonal antibodies of the present disclosure. Derivatives include, but are not limited to, bispecific, multispecific, heterospecific, trispecific, tetraspecific, multispecific antibodies, diabodies, chimeric, recombinant, and humanized antibodies.

[0093] An antibody, as used herein, may be an "antibody variant," which is intended to include antibodies produced in species other than mouse. It also includes antibodies containing post-translational modifications to the linear polypeptide sequence of the antibody or fragment. It further encompasses fully human antibodies.

[0094] The terms "antigen" and "antigenic" refer to a molecule capable of being recognized by an antibody or otherwise acting as a member of an antibody-ligand pair. "Specific binding" or "binding" refers to the interaction of an antigen with the variable regions of immunoglobulin heavy and light chains. Antibody-antigen binding can occur in vivo or in vitro. Those skilled in the art will understand that macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides, have the potential to act as antigens. Those skilled in the art will further understand that nucleic acids encoding proteins with the potential to act as antibody ligands necessarily encode antigens. Those skilled in the art will further understand that antigens are not limited to full-length molecules, but can also include partial molecules. The term "antigenic" is an adjective referring to a molecule having the properties of an antigen. This term encompasses substances that are immunogenic, i.e., immunogens, and substances that induce immunological unresponsiveness or anergy, i.e., anergens.

[0095] As used herein, the term "specific binding" refers to the interaction between a binding pair (e.g., an antibody and an antigen, or a receptor and a ligand). In various instances, specific binding occurs at a rate of about 10 -6 moles / liter, approximately 10 -7 moles per liter, or about 10 -8 This may be embodied in an affinity constant of moles / liter or less.

[0096] An immunodominant antigen is intended to be a region of a protein that recognizes and binds with high affinity to an antibody. In some embodiments, an immunoprotective antigen is intended to be a region of a protein that recognizes and binds with high affinity to an antibody that interferes with the function of the protein, and antibodies raised against the immunoprotective antigen are characterized by an enhanced or optimal effect on the target indicator as a result of interference with protein function, in some cases an improved ability to clear biofilms.

[0097] An "altered antigen" is one that has 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 antigenic peptides that have been differentially modified during or after translation, for example, but not limited to, by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, or linkage to antibody molecules, membrane molecules, or other ligands. See, e.g., Ferguson et al. (1988) Ann. See Rev. Biochem. 57:285-320. The synthetic or altered antigens disclosed herein are intended to bind to the same set of CDRs and / or TCR as the native epitope.

[0098] "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, however, not all antigens are immunogenic. The immune responses disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response can occur in vivo or in vitro. Those skilled in the art will understand that a variety of macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides, have the potential to be immunogenic. Those skilled in the art will further understand that 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 may include a partial molecule.

[0099] The term "passive immunity" refers to the transfer of immunity from one subject to another through the transfer of antibodies. Passive immunity can occur naturally when maternal antibodies are transferred to a fetus. Passive immunity can also occur artificially when an antibody composition is administered to a non-immune subject. The donor and recipient of the antibody can be human or non-human. The antibody may be polyclonal or monoclonal, may be generated in vitro or in vivo, and may be purified, partially purified, or unpurified, depending on the embodiment. In some embodiments described herein, passive immunity is conferred to a subject in need thereof by administration of an antibody or antigen-binding fragment that specifically recognizes or specifically binds to a particular antigen. In some embodiments, passive immunity is conferred by administration of an isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or specifically binds to a particular antigen.

[0100] The term "propagating" means growing a cell or population of cells. The term "growing" also refers to the proliferation of cells in the presence of a support medium, nutrients, growth factors, feeder cells, or any chemical or biological compounds necessary to obtain a desired number of cells or cell type.

[0101] The term "culturing" refers to the in vitro propagation of cells or organisms on or in various types of media. It is understood that the progeny of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0102] A "cell" or "host cell" refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell but are still included within the scope of the term as used herein. In some embodiments, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

[0103] A population of cells contemplates a collection of two or more cells that may or may not be phenotypically and / or genotypically identical (clonal). Populations may be purified, highly purified, substantially homogeneous, or heterogeneous, as described herein.

[0104] "Gene delivery vehicle" is defined as any molecule that can carry the inserted polynucleotide into host cells. Examples of gene delivery vehicles include liposomes, micelles, biocompatible polymers, including natural and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, and bacteria or viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art that have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy and simple protein expression.

[0105] The polynucleotides disclosed herein can be delivered to cells, tissues, or subjects using gene delivery vehicles. As used herein, "gene delivery," "gene transfer," "transduction," and the like refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for 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) and techniques that facilitate the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery, and various other techniques used for introducing polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically 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, several vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0106] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of it. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically provide a selective advantage under given environmental conditions. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the proteins produced may act as toxins under similar conditions.

[0107] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that makes the cell resistant to a particular antibiotic and a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites and allowing for the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid carrying the gene of interest. Just as bacteria produce proteins that confer antibiotic resistance, they can also be induced to produce large amounts of proteins from inserted genes. This is an inexpensive and easy way to produce large amounts of a gene, or the protein it encodes.

[0108] "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. Initially constructed using circular plasmids, they are linearized using restriction enzymes, and then DNA ligase can be used to add sequences or genes of interest into the linear molecule using cohesive ends. Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), are extremely useful because yeast itself is a eukaryotic cell, allowing for the production of eukaryotic protein products with post-translational modifications. However, YACs have been found to be more unstable than BACs and to produce chimeric effects.

[0109] A "viral vector" is defined as a recombinantly produced virus or viral particle containing a polynucleotide to be delivered to a host cell, either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, and the like. Vectors based on the infectious tobacco mosaic virus (TMV) can be used to produce proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009)). Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. Gene transfer has been demonstrated using retrovirus-based vectors. In vector-mediated aspects, vector construct refers to a polynucleotide comprising a retroviral genome or portion thereof and a therapeutic gene.

[0110] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process by which a gene or nucleic acid sequence is stably transferred into a host cell by a virus that enters the cell and integrates the viral genome into the host cell genome. The virus can enter the host cell through its normal infection mechanism, or the virus can be modified to bind to a different host cell surface receptor or ligand and enter the cell. As used herein, retroviral vector refers to a viral particle that can introduce exogenous nucleic acid into a cell via a viral or viral-like entry mechanism.

[0111] Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse transcribed into a DNA form that integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0112] In embodiments where gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide comprising a 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 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 possibility of recombination and the generation of wild-type viruses. See, for example, PCT International Application Publication Nos. WO95 / 00655 and WO95 / 11984, wild-type AAV has high infectivity and specificity for integration into the genome of host cells. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0113] Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. RNA can be transcribed in vitro or in vivo and is 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 extra, potentially inappropriate, alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to enhance expression.

[0114] Gene delivery vehicles also include DNA / liposome complexes, micelles, and target virus protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to the delivery of polynucleotides to cells or cell populations, the proteins described herein can be directly introduced into cells or cell populations by non-limiting techniques of protein transfection, or other non-limiting techniques include culturing conditions that can enhance the expression and / or activity of the proteins disclosed herein.

[0115] In embodiments where gene transfer is mediated by a lentiviral vector, the vector construct refers to a polynucleotide comprising a lentiviral genome or a portion thereof and a therapeutic gene. As used herein, "lentiviral-mediated gene transfer" or "lentiviral transduction" have the same meaning and refer to the process by which a gene or nucleic acid sequence is stably transferred into a host cell by a virus that invades the cell and integrates the viral genome into the host cell genome. Viruses can invade host cells through their normal infection mechanism, or they can be modified to bind to different host cell surface receptors or ligands and enter the cell. Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse-transcribed into DNA, which is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, lentiviral vector refers to a viral particle that can introduce exogenous nucleic acid into cells through a viral or viral-like entry mechanism. "Lentiviral vectors" are a type of retroviral vector known in the art that have certain advantages over other retroviral vectors in transducing non-dividing cells. Trono D. (2002) Lentiviral vectors, New See Spring-Verlag Berlin Heidelberg.

[0116] The lentiviral vector of the present disclosure is based on or derived from oncoretrovirus (a subgroup of retroviruses that includes MLV) and lentivirus (a subgroup of retroviruses that includes HIV).Examples include ASLV, SNV and RSV, all of which are separated into packaging and vector components for lentiviral vector particle production systems.The lentiviral vector particle of the present disclosure can be based on a specific retroviral version that has been genetically or otherwise modified (for example, by specific selection of packaging cell line).

[0117] That a vector particle according to the present disclosure is "based on" a particular retrovirus means that the vector is derived from that particular retrovirus. The genome of the vector particle includes components derived from that retrovirus as a backbone. The vector particle contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Typically, these include the gag and pol proteins derived from the particular retrovirus. Thus, while the majority of the structural components of a vector particle are typically derived from a retrovirus, they may be genetically or otherwise modified to provide desired, useful properties. However, certain structural components, particularly the env protein, may originate from different viruses. The host range of the vector and the cell types infected or transduced can be altered by using different env genes in the vector particle production system to confer different specificities to the vector particle.

[0118] The term "adeno-associated virus" or "AAV," as used herein, refers to a member of a class of viruses related to this name and belonging to the genus Dependoparvovirus in the family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes are capable of infecting cells from a variety of tissue types. At least 11 consecutively numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant or synthetic serotypes, e.g., AAV-DJ and AAV-PHP.B. AAV particles comprise, alternatively consist essentially of, or even further consist of the three major viral proteins: VP1, VP2, and VP3. In one embodiment, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. These vectors are commercially available or described in the patent or technical literature.

[0119] "Detectable label," "label," "detectable marker," or "marker" are used interchangeably and include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to polynucleotides, polypeptides, antibodies, or compositions described herein.

[0120] As used herein, the term "label" or detectable label refers to a directly or indirectly detectable compound or composition, e.g., an N-terminal histidine tag (N-His), a magnetically active isotope, e.g., a nucleotide sequence that is directly or indirectly conjugated to the composition to be detected to produce a "labeled" composition. 115 Sn, 117 Sn and 119 Sn, non-radioactive isotopes, e.g.13 C and 15 The term "label" contemplates a polynucleotide, polynucleotide, or protein, such as an antibody. The term also includes sequences conjugated to a polynucleotide that provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP). The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical change in a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. A label may be simply detected, or it may be quantified. A response that is simply detected generally includes a response whose presence is simply confirmed, while a response that is quantified generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response may be generated directly using a luminophore or fluorophore associated with the assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that generate a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally involves a change in a luminescent signal or the generation of a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0121] As used herein, the term "immunoconjugate" includes an antibody or antibody derivative associated with or linked to a second agent, e.g., a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semi-synthetic antibody, or a multispecific antibody.

[0122] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0123] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present in or on the surface of cells or tissues, such as cell surface markers.Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule.The choice of functional group of the fluorescent label depends on the site of attachment to either a linker, drug, marker, or second labeling agent.

[0124] As used herein, a purification tag or purification marker refers to a tag that can be used to purify a molecule or component to which the label is conjugated, such as an epitope tag (including but not limited to, Myc tag, human influenza hemagglutinin (HA) tag, FLAG tag), affinity tag (including but not limited to, glutathione-S-transferase (GST), polyhistidine (His) tag, calmodulin-binding protein (CBP), or maltose-binding protein (MBP)), or fluorescent tag.

[0125] The terms "acceptable," "effective," or "sufficient," when used to describe the selection of any component, range, dosage form, etc. disclosed herein, are intended to mean that said component, range, dosage form, etc. is suitable for the purpose disclosed.

[0126] In the context of the present disclosure, a "ligand" is a polypeptide. In one aspect, the term "ligand" as used herein refers to any molecule that binds to a specific site on another molecule. In other words, the ligand confers specificity to the protein in the reaction with immune effector cells, or antibodies against the protein, or DNA against the protein. In one aspect, this is a ligand site within the protein that directly combines with a complementary binding site on immune effector cells.

[0127] As used herein, the terms "treating," "treatment," and the like are used herein to mean obtaining a desired result, including beneficial or desired pharmacological and / or physiological effects and / or clinical outcomes. In some embodiments, the effect and / or result may be preventative in terms of one or more of completely or partially preventing a disorder or its signs or symptoms, and / or therapeutic in terms of a partial or complete cure of the disorder and / or adverse effects resulting from the disorder, whether detectable or undetectable, and / or reduction or amelioration of one or more symptoms, a decrease in the severity of a condition (including a disease), a stabilized (i.e., not worsening) state of a condition (including a disease), a delay or slowing of the progression of a condition (including a disease), an improvement or alleviation of the symptoms of a condition (including a disease), and remission (whether partial or total). Examples of "treatment" include, but are not limited to, preventing a disorder from occurring in a subject who may be susceptible to the disorder but has not yet been diagnosed as having it, inhibiting a disorder, i.e., preventing its development, and / or alleviating or ameliorating the symptoms of a disorder. In one aspect, treatment is the prevention of the onset of symptoms of disease or disorder. In some embodiments, it refers to (1) preventing symptoms or disease from occurring in subjects who are susceptible to disease or who have not yet shown symptoms of disease; (2) inhibiting disease or preventing its occurrence; or (3) improving disease or symptoms of disease or causing its regression. In one aspect, treatment excludes prevention or prevention.

[0128] In some embodiments, another beneficial or desired pharmacological and / or physiological effect achieved by treatment may include, consist essentially of, or further consist of one or more of the following: a reduced dose of antibiotics compared to treatment using antibiotics alone, a shorter duration of antibiotic use compared to treatment using antibiotics alone, a lower frequency of antibiotic use compared to treatment using antibiotics alone, or fewer side effects resulting from antibiotic use compared to treatment using antibiotics alone (milder side effects, no side effects, or less frequent or shorter duration of side effects). Such side effects may include, consist essentially of, or further consist of one or more of skin rash, diarrhea, and lifelong disruption of the gut microbiome.

[0129] By prevent is intended the prevention of a disease or disorder or effect in vitro or in vivo in a system or subject susceptible to the disorder or effect. Such an example would be preventing the formation of a biofilm in a system infected with a microorganism known to produce it.

[0130] In some embodiments, "treating a biofilm" or grammatical variations thereof refers to disrupting the biofilm (such as through dispersal and / or destruction). Additionally or alternatively, "treating a biofilm" can also refer to reducing and / or removing the microbial load (e.g., expressed as number and / or concentration) of the microorganisms that form the biofilm. In some embodiments, the microbial load is calculated based on microorganisms in any one, two, or all three of the following forms: planktonic, biofilm, or newly released. In some embodiments, the microbial load is calculated based only on microorganisms in the biofilm.

[0131] In one embodiment, the term "disease" or "disorder," as used herein, refers to diseases such as those caused by and / or related to polymicrobial biofilms, conditions diagnosed with, suspected of having, or high-risk for such diseases. In some embodiments, the diseases include pneumonia, sinusitis, sepsis, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chronic salpingitis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infections, mastoiditis, aortic graft infections, conjunctivitis, Brazilian purpura, occult bacteremia, and underlying lung diseases such as chronic bronchitis, bronchiectasis, and saccular ulcers. Additionally or alternatively, the disease is selected from cystic fibrosis (CF), pulmonary CF, chronic obstructive pulmonary disease (COPD), chronic rhinosinusitis (CRS), periodontitis, peri-implantitis, middle ear infection, otitis media (OM), acute otitis media (AOM), otitis media with effusion (OME), post-tympanostomy tube ear discharge (PTTO), ventilator-associated pneumonia (VAP), community-acquired pneumonia (CAP), chest tube / catheter / indwelling device biofilm infection, tonsillitis, pharyngitis, laryngitis, epiglottitis, sinusitis In some embodiments, the disease is selected from inflammatory bowel disease, pneumonia, bronchitis, or other respiratory tract infections (RTIs). In some embodiments, the disease is any condition involving H. influenzae (typeable and non-typeable strains), such as OM, pneumonia, sinusitis, sepsis, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chronic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infections, mastoiditis, aortic graft infections, conjunctivitis, Brazilian purpura, occult bacteremia, chronic obstructive pulmonary disease, and exacerbations of underlying lung diseases, such as chronic bronchitis, bronchiectasis, and cystic fibrosis. In some embodiments, the disease is any disorder of a subject caused directly or indirectly by a bacterial biofilm disclosed herein, such as NTHI. In some embodiments, the biofilm is a polymicrobial biofilm. In some embodiments, the polymicrobial biofilm is caused by Haemophilus, eg, NTHI, and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis.

[0132] "Administration" or "delivery" of antibodies, vaccines, peptides, cells, or vectors, or other agents and compositions containing the same, can be achieved in a single dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of the treatment, the target infection or biofilm being treated, and the subject being treated. Single or multiple administrations can be carried out, with the dose level and pattern selected by the treating physician or, in the case of animals, the treating veterinarian. Suitable dosage formulations and methods for administering agents are known in the art. The route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of administration routes include oral administration, intraperitoneal administration, infusion, intranasal administration, inhalation, injection, and topical application.

[0133] The term administration is intended to include, but is not limited to, oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection, or implant), nasal inhalation spray, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), or topical administration routes (e.g., gels, ointments, creams, aerosols, etc.), which, alone or together, can be formulated in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each administration route. The present disclosure is not limited by administration route, formulation, or dosing schedule.

[0134] "Administration" can be given in one dose, continuously or intermittently throughout the course of treatment.Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated.Single or multiple administrations can be carried out with the dose level and pattern selected by the treating physician.Suitable dosage formulations and methods for administering drugs are known in the art.

[0135] The agents of the present disclosure can be administered for therapy by any suitable route of administration, it will also be recognized that the most suitable route will vary with the condition and age of the recipient, and the disease being treated.

[0136] As used herein, the term "contacting" refers to the direct or indirect binding or interaction between two or more molecules. A specific example of direct interaction is binding. A specific example of indirect interaction is when one entity acts on an intermediate molecule, which then acts on a second reference entity. As used herein, contacting includes in solution, in solid phase, in vitro, ex vivo, in cells, and in vivo. In vivo contacting can be referred to as administering or administration.

[0137] A "composition" is intended to mean a combination of an active agent and another compound or composition, inert (e.g., a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipid-soluble solvent, preservative, adjuvant, etc., and includes a pharmaceutically acceptable carrier. Carriers also include pharmaceutical excipients and additives, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetra-oligosaccharides, and oligosaccharides; derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, including 1 to 99.99% by weight or volume of the combination. Exemplary protein excipients include serum albumins, e.g., human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / antibody components that may also function in a buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also contemplated within the scope of this technology, examples of which include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol.

[0138] A "pharmaceutical composition" is intended to include a combination of an active polypeptide, polynucleotide, or antibody with an inert or active carrier, e.g., a solid support, that makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

[0139] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water, and emulsions, such as oil-in-water or water-in-oil emulsions, as well as various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton. In some embodiments, a "pharmaceutically acceptable carrier" is , 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, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They should be used in accordance with the intended administration method. The form may be selected as to, ie, oral tablet, capsule, elixir, syrup, etc., and consistent with conventional pharmaceutical practice.

[0140] The term "pharmaceutically acceptable carrier (or vehicle)," which may be used interchangeably with the term "biologically compatible carrier or vehicle," refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with the cells and other agents being administered therapeutically, but also, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other complicating factors commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers suitable for use in the present disclosure include liquids, semi-solid (e.g., gels), and solid materials (e.g., cell scaffolds and matrices, tubesheets, and other such materials known in the art and described in more detail herein). These semi-solid and solid materials may be designed to resist degradation within the body (non-biodegradable), or they may be designed to degrade within the body (biodegradable, bioerodible). Biodegradable materials may also be bioabsorbable or bioresorbable, i.e., they may be dissolved and absorbed by bodily fluids (water soluble implants being one example), or they may be broken down and eventually eliminated from the body by either conversion to other materials or breakdown and excretion via natural pathways.

[0141] As used herein, "solid phase support" or "solid support," which are used interchangeably, are not limited to a particular type of support. Rather, numerous supports are available and known to those skilled in the art. Solid phase supports include silica gel, resins, derivatized plastic films, glass beads, cotton, plastic beads, and alumina gel. As used herein, "solid support" also includes synthetic antigen-presenting matrices, cells, and liposomes. A suitable solid phase support can be selected based on the desired end use and suitability for various protocols. For example, for peptide synthesis, a solid support may refer to a resin such as polystyrene (e.g., PAM resin available from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE® resin (available from Aminotech, Canada), polyamide resin (available from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tübingen, Germany) or polydimethylacrylamide resin (available from Milligen / Biosearch, Calif.).

[0142] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the support can be either soluble to some extent or insoluble. The support material can have virtually any possible structural configuration, so long as the coupled molecule can bind to a polynucleotide, polypeptide, or antibody. Thus, the support configuration can be spherical, as in beads, or cylindrical, as in the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, or alternatively, polystyrene beads. Those skilled in the art will know many other suitable supports for antibody or antigen binding, or will be able to ascertain such by use of routine experimentation.

[0143] Compositions used according to the present disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired response in conjunction with its administration, i.e., an appropriate route and regimen. The amount administered, both according to the number of treatments and the unit dose, depends on the desired outcome and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is unique to each individual. Factors affecting dosage include the subject's physical and clinical condition, the route of administration, the intended goal of treatment (relief of symptoms versus cure), and the efficacy, stability, and toxicity of the particular composition. Upon formulation, a solution is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, for example, the types of injectable solutions described herein.

[0144] A "subject" (used interchangeably herein with "individual" or "patient") of diagnosis or treatment is a cell or an animal, e.g., a mammal or a human. Non-human animals subjected to diagnosis or treatment are those subjected to infection or animal models, e.g., monkeys, murines, e.g., rats, mice, chinchillas, canines, e.g., dogs, lagomorphs, e.g., rabbits, livestock, game animals, and pets. The terms "subject," "host," "individual," and "patient," when used interchangeably herein, refer to an animal, typically a mammal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), livestock animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, a pediatric patient, an adolescent, a teenager, a pre-adolescent subject, a child, an infant, a fetus, or an in utero mammal). The mammal can be male or female. In some embodiments, the subject is a human. In one aspect, the subject is a pre-symptomatic subject. In another aspect, the subject has minimal clinical symptoms of disease. The subject can be a male or female adult, infant, or pediatric subject. In additional aspects, the subject is an adult. In some cases, the adult is an adult human, for example, an adult human over the age of 18.

[0145] As used herein, a biological sample or sample can be obtained from a subject, a cell line, or a cultured cell or tissue. Exemplary samples include, but are not limited to, cell samples, tissue samples, liquid samples, such as blood and other liquid samples of biological origin (including, but not limited to, ocular fluid (aqueous and vitreous), peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's or pre-ejaculatory fluid, female vaginal fluid, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions / washings, synovial fluid, mucosal secretions, fecal juice, pancreatic juice, lavage fluid from sinus cavities, bronchopulmonary aspirate, blastocyl cavity fluid, or umbilical cord blood). In one embodiment, the biological sample is suspected of having a biofilm. In another embodiment, the biological sample comprises a biofilm. In some embodiments, the sample is from the respiratory tract of a subject, e.g., mucus.

[0146] An "effective amount" is an amount sufficient to produce a beneficial or desired effect. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on several variables, including the duration for which individual dosage units are used, the bioavailability of the therapeutic agent, the route of administration, and the like. However, the specific dosage level of the therapeutic agent of the present disclosure for any particular subject will depend on various factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, the number of administrations, excretion rate, drug combination, and the severity and mode of administration of the particular disorder being treated. Treatment dosages can generally be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and / or in vivo studies can provide useful guidance initially on the appropriate dosage for patient administration. It is generally desirable to administer an amount of compound effective to achieve serum levels commensurate with concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks. In the context of immunogenic compositions, in some embodiments, an effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, an effective amount of an immunogenic composition is an amount sufficient to produce 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. One of skill in the art will be able to determine the appropriate amount depending on these and other factors.

[0147] An "immunogenic dose" of a composition is one that generates a detectable humoral (antibody) and / or cellular (T cell) immune response after administration compared to a detectable immune response before administration, or compared to a standard immune response before administration. In some embodiments, the immune response results in a manner that can be protective and / or therapeutic. In some embodiments, the antibody and / or T cell immune response protects an individual from a biofilm infection disclosed herein, particularly an infection of the middle ear and / or nasopharynx or lower respiratory tract. For this use, the exact dose will depend on the patient's health and weight, the mode of administration, the nature of the formulation, etc., but will generally range from about 1.0 μg to about 5000 μg per 70 kilogram patient, more typically from about 10 to about 500 μg per 70 kilogram body weight.

[0148] Humoral immune responses can be measured by many well-known methods, such as single radial immunodiffusion assay (SRID), enzyme immunoassay (EIA), and hemagglutination inhibition assay (HAI). In particular, SRID utilizes a layer of gel, such as agar, containing the immunogen to be tested. Wells are cut in the gel, and the serum to be tested is placed into the wells. Diffusion of the antibody into the gel results in the formation of a ring of precipitate, the area of ​​which is proportional to the concentration of antibody in the serum being tested. EIA, also known as ELISA (enzyme-linked immunoassay), is used to determine total antibody in a sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate, followed by an enzyme linked to an immunoglobulin, such as IgG. The enzyme activity attached to the plate is quantified by any conventional means, such as spectrophotometry, and is proportional to the concentration of antibody against the immunogen present in the test sample. HAI utilizes the ability of an immunogen, such as a viral protein, to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e., antibodies capable of inhibiting hemagglutination. Dilutions of test serum are incubated with a standard concentration of immunogen, followed by the addition of red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of red blood cells by the immunogen. Tests for measuring cellular immune responses include determining delayed-type hypersensitivity or measuring the proliferative response of lymphocytes targeting the immunogen.

[0149] In the case of in vitro application, in some embodiments, the effective amount depends on the size and characteristics of the application in question. This also depends on the characteristics and sensitivity of the in vitro target and the method of use. Those skilled in the art will be able to determine the effective amount based on these and other considerations. The effective amount may include one or more administrations of the composition depending on the embodiment. Methods for carrying out the present disclosure

[0150] In one aspect, a method is provided for preventing, inhibiting, disrupting, dispersing, or treating a polymicrobial biofilm caused by, or that will be caused by, or that contains Haemophilus bacteria in a subject in need thereof. The method comprises, consists essentially of, or even consists of administering to the subject (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof. The antibody may be a polyclonal or monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody includes one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or a biologically active fragment thereof. The antibody may be a polyclonal or monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip antibody or an anti-chip chimer antibody, or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and specifically binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled. In some embodiments, the polymicrobial biofilm is caused by one or more of Haemophilus, such as NTHI, and Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis.In some aspects, the polymicrobial biofilm is also caused by Gram-positive or Gram-negative bacteria, such as ESKAPE bacteria. In some embodiments, the method further comprises administering an antibiotic to the subject. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic or a sulfonamide antibiotic. In one aspect, the subject is a mammal or a human patient.

[0151] In another aspect, a method is provided for preventing, inhibiting, disrupting, dispersing, or treating one or more of diseases associated with polymicrobial biofilm infections caused by, or that will be caused by, or that involve Haemophilus bacteria in a subject in need thereof. The method comprises, consists essentially of, or even consists of administering to the subject (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or their respective biologically active fragments. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip antibody or an anti-DNABII chip chimer antibody, or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, e.g., as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and specifically binds to a PilA peptide, examples of which include the PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled. In some embodiments, the polymicrobial biofilm is composed of Haemophilus, e.g., NTHI, and Burkholderia. The polymicrobial biofilm is caused by one or more of the following: Staphylococcus aureus, Staphylococcus pneumoniae, Staphylococcus aureus, Staphylococcus pneumoniae, or Moraxella catarrhalis. In some aspects, the polymicrobial biofilm is also caused by gram-positive or gram-negative bacteria, such as ESKAPE bacteria. In some embodiments, the method further comprises administering an antibiotic to the subject. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic or a sulfonamide antibiotic. In some embodiments, the disease is as disclosed herein, e.g., cystic fibrosis (CF), pulmonary CF, chronic obstructive pulmonary disease (COPD), chronic rhinosinusitis (CRS), periodontitis, peri-implantitis, middle ear infection, otitis media (OM), acute otitis media (AOM), otitis media with effusion (OME), post-tympanostomy tube ear discharge (PTTO), ventilator-associated pneumonia (VAP), community-acquired pneumonia (CAP), chest tube / catheter / indwelling device biofilm infection, tonsillitis, pharyngitis, laryngitis, laryngopharyngitis, ... Ophthalmitis, sinusitis, pneumonia, bronchitis, or other respiratory tract infections In one embodiment, the subject is a mammal or a human patient.

[0152] In another aspect, a method is provided for sensitizing a polyclonal microbial biofilm caused by, or to be caused by, or containing Haemophilus bacteria in a subject in need thereof for antibiotic therapy, or for inducing bacteria in a polymicrobial biofilm in a subject in need thereof to a newly released (NRel) state. The method comprises, consists essentially of, or even consists of administering to the subject (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises one or more of an anti-DNABII antibody, an anti-DNABII chip antibody, an anti-DNABII chip chimer antibody, or their respective biologically active fragments. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody can be a polyclonal antibody or a monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled.In some aspects, the polymicrobial biofilm is caused by Haemophilus, e.g., NTHI, and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis. In some aspects, the polymicrobial biofilm is also caused by Gram-positive or Gram-negative bacteria, e.g., ESKAPE bacteria. In some embodiments, the method further comprises administering an antibiotic to the subject. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic or a sulfonamide antibiotic. In one aspect, the subject is a mammal or human patient.

[0153] In one aspect, a method is provided for preventing, inhibiting, disrupting, dispersing, or treating polymicrobial biofilms caused by, or that will be caused by, or that contain, Haemophilus bacteria. The method comprises, consists essentially of, or even consists of administering to a subject (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof. The antibody may be a polyclonal or monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody may be a polyclonal or monoclonal antibody. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and specifically binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled. In some embodiments, the polymicrobial biofilm is caused by one or more of Haemophilus, such as NTHI, and Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis.In some aspects, the polymicrobial biofilm is caused by Gram-positive or Gram-negative bacteria, such as ESKAPE bacteria. In some embodiments, the method further comprises contacting the biofilm with an antibiotic. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic or a sulfonamide antibiotic. In some embodiments, the contacting is in vitro or in vivo.

[0154] In another aspect, a method is provided for sensitizing a polymicrobial biofilm to antibiotic therapy or for inducing bacteria that form a polymicrobial biofilm caused by, or that will be caused by, or that includes Haemophilus bacteria, to a newly released (NRel) state. The method comprises, consists essentially of, or even consists of administering to a subject (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof. The antibody may be a polyclonal or monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody may be a polyclonal or monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and specifically binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled. In some embodiments, the polymicrobial biofilm is caused by one or more of Haemophilus, such as NTHI, and Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis.In some aspects, the polymicrobial biofilm is caused by Gram-positive or Gram-negative bacteria, such as ESKAPE bacteria. In some embodiments, the method further comprises contacting the biofilm with an antibiotic. In further embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic or a sulfonamide antibiotic. In some embodiments, the contacting is in vitro or in vivo.

[0155] In some embodiments of any aspect disclosed herein, an antibiotic is contacted with the biofilm or administered to a subject simultaneously with or after (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further aspect, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, e.g., as disclosed herein. In another aspect, the anti-PilA antibody specifically recognizes and specifically binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (GenBank Accession No. EFU96734.1) and Type IV pilin (GenBank Accession No. Yp_003864351.1). In some embodiments of any aspect disclosed herein, the method further comprises identifying bacteria in the subject or polymicrobial biofilm sample prior to the contacting or administering step. In some aspects, the polymicrobial biofilm is caused by Gram-positive or Gram-negative bacteria, e.g., ESKAPE bacteria. In still further embodiments, the bacteria have been identified as comprising, consisting essentially of, or even consisting of two or more Haemophilus bacteria. In some embodiments, the bacteria have been identified as comprising, consisting essentially of, or even consisting of Haemophilus bacteria and another non-Haemophilus bacterium.In some embodiments, the bacteria is a Haemophilus, eg, NTHI, and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis.

[0156] In some embodiments of any aspect disclosed herein, by applying the methods disclosed herein, polymicrobial biofilms caused by, or that will be caused by, or that include Haemophilus bacteria are one or more of: (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof; or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof; or an antibiotic alone are contacted or administered, and are prevented, inhibited, disrupted, dispersed, or treated in a synergistic manner. In some aspects, the polymicrobial biofilm is caused by Haemophilus, such as NTHI, and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis. In some aspects, the polymicrobial biofilm is also caused by Gram-positive or Gram-negative bacteria, such as ESKAPE bacteria.

[0157] In some embodiments of any aspect disclosed herein, the newly released (NRel) state of bacteria released from a biofilm is different from the same but planktonically grown bacteria. In some embodiments, the NRel bacteria comprise one or more of the following: altered gene expression of one or more of the following genes: deaD, artM, fis, folA, folP, emrA, emrB, or acrR; altered levels of one or more of the following proteins: AsnC, CyaA, GlpC, NrfA, TehB, TrpB, TrpC, TrpD, TruA, or TrxA; Proteins: AbgA, AroE, AroK, ArtP, AtpH, BioB, BioF, CcmA, CitD, CitT, CmK, CoaD, DcD, DjlA, DksA, DnaE, ​​DnaQ, DsbE, FabG, FdhX, FtnB, FtsE, FtsI, FumC, Fur, GlpA, GlpB, GmK, GpsA, GreA, Hfq, HisI, HugZ, InfA, InfB, IspD, IspF, Lic 2A, LicC, LicD, LolA, LpsA, menC, MenC, NapA, NrfB, NrfC, NTHI0053, NTHI0249, NTHI0291, NTHI0319, NTHI0349, NT HI0436, NTHI0487, NTHI0490, NTHI0555, NTHI0732, NTHI0779, NTHI0820, NTHI1025, NTHI1085, NTHI1199, NTHI143 7, NTHI1439, NTHI1503, NTHI1590, NTHI1748, low levels of one or more of OppF, PanF, PdxH, PepT, PhoB, PlsC, PolA, PpC, PpiB, PrfB, PurU, RadA, RelB, RibA, RplW, RpoE, RpoZ, RseA, SecF, Sxy, TesB, ThrB, TolA, UnG, UreG, UuaP, or UvrB;High levels of one or more of the following proteins: ClpB, CydD, DeaD, DlD, DmsB, ExbD, GroES, HfeA, HfeB, HgpB, HisJ, HisJ, HitA, HktE, HxuC, LctP, LldD, LpxC, MurB, NdhA, NifS2, NTHI0043, NTHI0052, NTHI0175, NTHI0364, NTHI1208, NTHI1214, NTHI1369, NTHI1703, OmpP2, OrfG, Pal, PdgX, RbfA, RecN, RplO, RplU, RpmE, RpsE, RpsJ, RpsL, RpsQ, RpsT, SodA, Tbp1, TolB, TonB, TrpA, TrxA, or ZnuA. In further embodiments, the higher (also referred to herein as increased), lower (also referred to herein as decreased), altered expression or levels are compared to the same bacteria but grown planktonically;

[0158] In one aspect, a method is provided for selecting a polymicrobial biofilm suitable for one or more of the following: prevention, inhibition, disruption, dispersal, treatment, or sensitization by the methods disclosed herein. The method comprises, or alternatively consists essentially of, or also consists of: (a) contacting the polymicrobial biofilm, or the polymicrobial biofilm isolated and grown therefrom, with one or both of (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof, as disclosed herein; and (b) assaying the bacteria released from the biofilm of (a) for expression of one or more of the genes and / or proteins disclosed herein. In some embodiments, the genes are selected from one or more of the following: deaD, artM, fis, folA, folP, emrA, emrB, or acrR.Additionally or alternatively, the protein may be one of the following: AsnC, CyaA, GlpC, NrfA, TehB, TrpB, TrpC, TrpD, TruA, TrxA; AbgA, AroE, AroK, ArtP, AtpH, BioB, BioF, CcmA, CitD, CitT, CmK, CoaD, DcD, DjlA, DksA, DnaE, ​​DnaQ, DsbE, FabG, FdhX, FtnB, FtsE, FtsI, FumC, Fur, GlpA, GlpB, GmK, GpsA, GreA, Hfq, HisI, Hu gZ, InfA, InfB, IspD, IspF, Lic2A, LicC, LicD, LolA, LpsA, menC, MenC, NapA, NrfB, NrfC, NTHI0053, NTHI0249, NTHI0291, NTHI0319, NTHI0 349, NTHI0436, NTHI0487, NTHI0490, NTHI0555, NTHI0732, NTHI0779, NTHI0820, NTHI1025, NTHI1085, NTHI1199, NTHI1437, NTHI1439, NTH I1503, NTHI1590, NTHI1748, OppF, PanF, PdxH, PepT, PhoB, PlsC, PolA, PpC, PpiB, PrfB, PurU, RadA, RelB, RibA, RplW, RpoE, RpoZ, RseA, Se cF, Sxy, TesB, ThrB, TolA, UnG, UreG, UuaP, UvrB; ClpB, CydD, DeaD, DlD, DmsB, ExbD, GroES, HfeA, HfeB, HgpB, HisJ, HisJ, HitA, HktE, HxuC , LctP, LldD, LpxC, MurB, NdhA, NifS2, NTHI0043, NTHI0052, NTHI0175, NTHI0364, NTHI1208, NTHI1214, NTHI1369, NTHI1703, OmpP2, OrfG, Pal, PdgX, RbfA, RecN, RplO, RplU, RpmE, RpsE, RpsJ, RpsL, RpsQ, RpsT, SodA, Tbp1, TolB, TonB, TrpA, TrxA, or ZnuA. The antibody can be a polyclonal or monoclonal antibody, or a derivative or variant fragment thereof.In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chimer antibody or a biologically active fragment thereof. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chimer antibody or a biologically active fragment thereof. The antibody can be a polyclonal or monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chimer antibody or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled.

[0159] In some embodiments, the method further comprises contacting the selected biofilm with (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof, and (iii) optionally an antibiotic, including a β-lactam antibiotic or a sulfonamide antibiotic. In some embodiments, the selected polymicrobial biofilm is in a subject. In further embodiments, the method further comprises administering to the subject (i) an anti-DNABII antibody or a biologically active fragment thereof, (ii) a PilA polypeptide or a biologically active fragment thereof, and (iii) optionally an antibiotic, including a β-lactam antibiotic or a sulfonamide antibiotic. The antibody may be a polyclonal antibody or a monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody can be a polyclonal antibody or a monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled.

[0160] In some embodiments of any aspect disclosed herein, one or more of the following indicates that the biofilm is suitable for, and selected as, a method disclosed herein: Altered gene expression of one or more of the following genes: deaD, artM, fis, folA, folP, emrA, emrB, or acrR; Altered levels of one or more of the following proteins: AsnC, CyaA, GlpC, NrfA, TehB, TrpB, TrpC, TrpD, TruA, or TrxA; The following proteins: AbgA, AroE, AroK, ArtP, AtpH, BioB, BioF, CcmA, CitD, CitT, CmK, CoaD, DcD, DjlA, DksA, DnaE, ​​DnaQ, DsbE, FabG, FdhX, FtnB, FtsE, FtsI, FumC, Fur, GlpA, GlpB, GmK , GpsA, GreA, Hfq, HisI, HugZ, InfA, InfB, IspD, IspF, Lic2A, LicC, LicD, LolA, LpsA, menC, MenC, NapA, NrfB, NrfC, NTHI0053, NTHI0249, NTHI0291, NTHI0319, NTHI0349, NTH low levels of one or more of I0436, NTHI0487, NTHI0490, NTHI0555, NTHI0732, NTHI0779, NTHI0820, NTHI1025, NTHI1085, NTHI1199, NTHI1437, NTHI1439, NTHI1503, NTHI1590, NTHI1748, OppF, PanF, PdxH, PepT, PhoB, PlsC, PolA, PpC, PpiB, PrfB, PurU, RadA, RelB, RibA, RplW, RpoE, RpoZ, RseA, SecF, Sxy, TesB, ThrB, TolA, UnG, UreG, UuaP, or UvrB; or High levels of one or more of the following proteins: ClpB, CydD, DeaD, DlD, DmsB, ExbD, GroES, HfeA, HfeB, HgpB, HisJ, HisJ, HitA, HktE, HxuC, LctP, LldD, LpxC, MurB, NdhA, NifS2, NTHI0043, NTHI0052, NTHI0175, NTHI0364, NTHI1208, NTHI1214, NTHI1369, NTHI1703, OmpP2, OrfG, Pal, PdgX, RbfA, RecN, RplO, RplU, RpmE, RpsE, RpsJ, RpsL, RpsQ, RpsT, SodA, Tbp1, TolB, TonB, TrpA, TrxA, or ZnuA.

[0161] In some embodiments of any aspect disclosed herein, altered expression refers to both increased and decreased expression.

[0162] In some embodiments of any aspect disclosed herein, high expression refers to expression that is increased by about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1-fold, or about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or about 20-fold, or about 30-fold, or about 40-fold, or about 50-fold, or about 60-fold, or about 70-fold, or about 80-fold, or about 90-fold, or about 100-fold or more, over a reference.

[0163] In some embodiments of any aspect disclosed herein, low expression refers to expression that is reduced by about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of reference.

[0164] In some embodiments of any aspect disclosed herein, the reference is the corresponding expression of the same but planktonically grown bacteria.

[0165] In one aspect, a kit is provided, for example, a kit for use in the methods disclosed herein. In some embodiments, the kit comprises, alternatively consists essentially of, or further consists of one or more of the following: (i) an anti-DNABII antibody or a biologically active fragment thereof and an anti-PilA antibody or a biologically active fragment thereof, or (ii) an anti-DNABII antibody or a biologically active fragment thereof and a PilA polypeptide or a biologically active fragment thereof, a polynucleotide encoding each of these elements, a vector comprising the polynucleotide, or a host cell comprising the polynucleotide and / or vector and optionally expressing the antibody or fragment thereof, or (iii) an antibiotic, optionally including a β-lactam antibiotic or a sulfonamide antibiotic, and optionally instructions for use. The antibody may be a polyclonal antibody or a monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody can be a polyclonal antibody or a monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled.

[0166] In another aspect, compositions are provided that comprise, alternatively consist essentially of, or even consist of one or more of: (i) an anti-DNABII antibody, a biologically active fragment thereof, a polynucleotide encoding it, a vector comprising the polynucleotide, or a host cell comprising the polynucleotide and / or vector and optionally expressing the antibody or fragment thereof; (ii) an anti-PilA antibody or a biologically active fragment thereof, a PilA polypeptide or a biologically active fragment thereof, a polynucleotide encoding it, a vector comprising the polynucleotide, or a host cell comprising the polynucleotide and / or vector and optionally expressing the antibody or fragment thereof; or (iii) an antibiotic, optionally including a β-lactam antibiotic or a sulfonamide antibiotic, and a carrier, optionally a pharmaceutically acceptable carrier. The antibody may be a polyclonal or monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody may be a polyclonal or monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chimer antibody or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, e.g., as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include the PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1).

[0167] Kits and compositions are also provided, for example, for use in the methods disclosed herein. In one aspect, a kit is provided that comprises, consists essentially of, or further consists of at least two of: (i) an anti-DNABII antibody or a biologically active fragment thereof; (ii) an anti-PilA antibody or a biologically active fragment thereof; or a PilA polypeptide or a biologically active fragment thereof; or (iii) an antibiotic, and, optionally, instructions for use. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-chip chimer antibody. In some embodiments, the antibiotic comprises, consists essentially of, or further consists of a β-lactam antibiotic and / or a sulfonamide antibiotic. The antibody may be a polyclonal or monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody may be a polyclonal or monoclonal antibody. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In one embodiment, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further embodiment, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, for example, as disclosed herein. In another embodiment, the anti-PilA antibody specifically recognizes and binds to a PilA peptide, examples of which include the PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody may be detectably labeled.

[0168] In another aspect, a composition is provided that comprises, consists essentially of, or even consists of at least two of the following: (i) an anti-DNABII antibody or a biologically active fragment thereof, (ii) an anti-PilA antibody or a biologically active fragment thereof, or one or both of a PilA polypeptide or a biologically active fragment thereof, or (iii) an antibiotic, and a carrier, optionally a pharmaceutically acceptable carrier. The antibody may be a polyclonal antibody or a monoclonal antibody, or a derivative or variant fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody. In one aspect, the anti-DNABII antibody comprises an anti-DNABII chip chimer antibody or a biologically active fragment thereof. In a further aspect, the anti-DNABII chip chimer antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody, e.g., as disclosed herein. In another aspect, the anti-PilA antibody specifically recognizes and specifically binds to a PilA peptide, examples of which include PilA surface antigen or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1). The antibody can be detectably labeled. In some embodiments, the antibiotic comprises, consists essentially of, or even consists of a β-lactam antibiotic and / or a sulfonamide antibiotic.

[0169] In some embodiments, antibodies, polypeptides, their respective biologically active fragments, or any combination thereof may be administered to a subject in a composition disclosed herein. In other embodiments, antibodies, polypeptides, their respective biologically active fragments, or any combination thereof may be administered to a subject as a polynucleotide encoding such antibody, polypeptide, or biologically active fragment, or a polynucleotide complementary thereto. In some embodiments, such a polynucleotide may be suitable for expressing the antibody, polypeptide, or biologically active fragment in a subject, for example, by further comprising a regulatory sequence directing expression. In further embodiments, a vector, e.g., a gene delivery vehicle, comprising a polynucleotide disclosed herein may also be used for administration. The vector may be a viral or non-viral vector disclosed herein. In still further embodiments, host cells comprising one or more of the polynucleotides and / or vectors disclosed herein are provided. Accordingly, kits and compositions comprising one or more of such polynucleotides and / or vectors and / or host cells are provided in addition to, or instead of, the corresponding expressed antibodies, peptides, or biologically active fragments thereof. Further provided are methods and compositions suitable for producing such polypeptides, antibodies, biologically active fragments, polynucleotides, vectors and host cells. Biofilm

[0170] "Biofilm" refers to an organized community of microorganisms that may adhere to the surface of an organic or inorganic structure, along with polymers such as DNA that become available in the extracellular environment due to secretion, release, and / or bacterial lysis. Biofilms are highly resistant to macrobiotics and antimicrobial agents. They survive on gum tissue, teeth, and restorations, causing tooth decay and periodontal disease, also known as periodontal plaque disease. They also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter lines, and contact lenses. They grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The Centers for Disease Control estimates that more than 65% of hospital-acquired infections are caused by biofilms. They cause chronic vaginal infections and lead to life-threatening systemic infections in people with compromised immune systems. Biofilms are also involved in many diseases. In one embodiment, a biofilm contains a DNABII polypeptide or protein. In a further embodiment, the biofilm comprises IHF and / or HU. In yet a further embodiment, the biofilm comprises IHFA and / or IHFB.

[0171] Bacteria may also exist planktonic, i.e., floating as single cells, and / or attached to a surface without forming a biofilm. In some embodiments, planktonic bacteria are artificially generated in a laboratory, for example, via sonication. In some embodiments, as used herein, planktonic bacteria does not include bacteria released from a biofilm due to treatment with the compositions disclosed herein, as applicants have discovered that such newly released (NRel) bacteria function differently compared to planktonic bacteria generated by sonication. More characteristics of NRel bacteria and their differences compared to planktonic ones are disclosed herein, for example, in Tables 2 and 3.

[0172] Biofilms include, but are not limited to, 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 (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 pyogenes (e.g., MGAS10270), Streptococcussobrinus (e.g., 6715), Salmonella enterica (e.g., typhi, CT18), Treponema denticola (e.g., ATCC® 35405), Treponema palladum (e.g., Nichols), Vibrio Biofilms are also implicated in numerous diseases, including those caused by Bacillus cholera (e.g., El Tor, N16961). Additional organisms known to be associated with and / or form biofilms include, but are not limited to, Campylobacter spp., Candida spp., Legionella pneumophila, and Listeria monocytogenes. For example, cystic fibrosis patients often have Pseudomonas infections, which result in antibiotic-resistant biofilms. Other diseases associated with biofilms include, but are not limited to, lung infections in cystic fibrosis patients, otitis media, ottorhea after tympanostomy tube insertion, chronic suppurative otitis media, native valve infective endocarditis, osteomyelitis, rhinosinusitis, prostatitis, urinary tract infections, wounds, dental caries, and periodontitis. Foodborne pathogens, including but not limited to some of the organisms listed above (e.g., Listeria monocytogenes, Escherichia coli, Salmonella enterica), can also form biofilms on the foods they contaminate. Diseases that cause biofilms in animals (e.g., Escherichia coli, Salmonella, and Shigella species) can also cause downstream food contamination and / or disease in the human host. Furthermore, biofilms need not be of a single homogenous microbial population, but may incorporate other pathogens and even host cells.

[0173] In addition to being associated with illnesses, both hospital-based and otherwise, and food contamination, biofilms are often a source of industrial contamination, most notably with respect to treating water and the surfaces they come into contact with. Complications involving organisms that form biofilms as industrial contaminants include, but are not limited to, biocorrosion, biofouling, and equipment damage as a result of biofilm formation. Non-limiting exemplary organisms associated with biofilms in industrial settings include those disclosed in Ferrera et al. (2015) Biofouling 31(2):173-180 and Desulfovibrio species. Additional details regarding biofilms can be found, for example, in Donlan (2002) Emerging Infectious Diseases 8(9):881-890.

[0174] Haemophilus is a genus of Gram-negative, pleomorphic coccobacilli belonging to the family Pasteurellaceae. These organisms infect the mucous membranes of the upper respiratory tract, mouth, vagina, and intestinal tract. This genus includes commensals as well as some important pathogenic species, such as Haemophilus influenzae and Haemophilus ducreyi, the causative agents of chancroid. All species are either aerobic or facultatively anaerobic.

[0175] The term "Haemophilus influenzae" refers to a pathogenic bacterium that can cause many different infections, such as ear infections, eye infections, OM, and sinusitis. Clinical isolates of H. influenzae are classified as either serotypes "a" through "f" or non-typeable (NTHI), depending on the presence or absence of a type-specific polysaccharide capsule on the bacterium. Many different strains of Haemophilus influenzae have been isolated and possess IhfA, ihfB, and hupA genes or proteins. Some non-limiting examples of different strains of Haemophilus influenzae include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019. The prototype NTHi isolate is the low-passage isolate 86-028NP, recovered from a child with chronic OM. 86-028NP was donated to the American Type Culture Collection, 10801, on October 16, 2001. It was deposited at University Blvd., Manassas, Va. 20110 and assigned deposit number PTA-4764.

[0176] In some embodiments of any aspect disclosed herein, the biofilms discussed herein are polymicrobial biofilms, i.e., polymicrobial biofilms that comprise, alternatively consist essentially of, or even consist of, two or more bacteria. In some embodiments, the biofilm comprises Haemophilus bacteria. In further embodiments, the polymicrobial biofilm comprises, alternatively consist essentially of, or even consist of, two or more Haemophilus bacteria. Additionally or alternatively, the polymicrobial biofilm comprises, alternatively consist essentially of, or even consist of one or more Haemophilus bacteria and non-Haemophilus bacteria (i.e., non-Haemophilus bacteria). In some embodiments, the Haemophilus bacteria comprise, alternatively consist essentially of, or even consist of Haemophilus influenzae (e.g., one or more of nontypeable Haemophilus influenzae (NTHI), Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019). In some aspects, the polymicrobial biofilm is caused by Haemophilus and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis.

[0177] In some embodiments, the Haemophilus bacteria comprise, alternatively consist essentially of, or even consist of non-typeable Haemophilus influenzae (NTHI). In one embodiment, the Haemophilus bacteria comprise, alternatively consist essentially of, or even consist of one strain of non-typeable Haemophilus influenzae (NTHI), e.g., 86-028NP, and optionally one or more bacteria other than NTHI (i.e., non-NTHI bacteria). Other NTHI strains, such as HI1716 or NTHI 2019, can be identified by those skilled in the art. See, e.g., Greiner et al. 2004. In another embodiment, the Haemophilus bacteria comprise, alternatively consist essentially of, or even consist of two or more strains of non-typeable Haemophilus influenzae (NTHI), and optionally one or more bacteria other than NTHI. In some embodiments, the polymicrobial biofilm is caused by NTHI and one or more of Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumonia, or Moraxella catarrhalis.

[0178] In some embodiments of any aspect disclosed herein, the non-Haemophilus bacteria and / or non-NTHI bacteria are selected from one or more of the following: Moraxella catarrhalis, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Burkholderia cenocepacia, ESKAPEE pathogens (selected from Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), 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), Helicobacter pylori (e.g., 26695), Klebsiella pneumoniae, Moraxella catarrhalis (e.g., RH4), Mycobacterium smegmatis (e.g., MC2), Mycobacterium tuberculosis (e.g., CDC1551), Neisseria 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 (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 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), Vibrio cholera (e.g., El Tor, N16961), Campylobacter spp., Candida spp., Legionella pneumophila, Vibrio vulnificus, Vibrio cholera, E.coli, Legionella pneumophila Salmonella, Shigella, Listeria, Aggregatibacter, Neisseria, Burkholderia cenocepacia (B. cenocepacia), B. multivorans, B. mallei, B. cepaci, B. pseudomallei, or Listeria monocytogenes.

[0179] In some embodiments of any aspect disclosed herein, the polymicrobial biofilm comprises, alternatively consists essentially of, or even consists of NTHI, Streptococcus pneumonia, Staphylococcus aureus, or Moraxella catarrhalis.

[0180] In some embodiments of any aspect disclosed herein, the polymicrobial biofilm is recurrent or resistant to antibiotic monotherapy. As used herein, a biofilm that is resistant to antibiotic monotherapy, i.e., referring to the use of only antibiotics in treating a biofilm, does not produce the desired effect and / or does not prevent the recurrence of the biofilm.

[0181] Biofilms contribute significantly to the chronicity and recurrence of bacterial disease due to the fact that biofilm-resident bacteria are highly resistant to killing by host immune effectors and antibiotics. Therefore, antibody-mediated release of bacteria from biofilms into the surrounding environment represents a powerful strategy for resolving otherwise difficult-to-treat biofilm-associated diseases. In previous studies, Applicant demonstrated that antibodies against two unique determinants of nontypeable Haemophilus influenzae (NTHI)—e.g., the NTHI type IV fimbria (T4P) or the bacterial DNABII DNA-binding protein, a species-independent target that provides structural integrity to bacterial biofilms—release biofilm-resident bacteria through distinct mechanisms. Here, Applicant demonstrated that the resulting phenotype of newly released (or NRel) NTHI depends on the specific mechanism of release. Using flow cytometry, proteomic profiling, and targeted transcriptomics, we demonstrated that the two NRel populations not only differed significantly from planktonic NTHI but, importantly, differed significantly from each other despite their genetic identity. Furthermore, each NRel population exhibited distinct and significantly increased susceptibility to killing by either sulfonamide or β-lactam antibiotics compared with planktonic NTHI, an observation consistent with their individual proteomes and further supported by relative differences in target gene expression. The distinct phenotypes of NTHI released from biofilms by antibodies against specific epitopes of T4P or DNABII-binding proteins offer new opportunities for developing targeted therapeutic strategies for biofilm eradication and disease resolution.It is also known that anti-rsPilA specifically targets an NTHI antigen that, unexpectedly, is not expressed by Moraxella catarrhalis, and that while anti-rsPilA has no effect on pure M. catarrhalis biofilms, it enables M. catarrhalis to assemble biofilms with NTHI's unique structural morphology, such that when M. catarrhalis assembles these "barrel"-like structures in the center of the NTHI biofilm lawn, NTHI then uses its type IV pili to twitch on the M. catarrhalis "barrels" and completely cover them. Treatment of NTHI + Mcat polymicrobial biofilms with anti-rsPilA showed that NTHI disperses itself.

[0182] In some embodiments, the biofilm is derived from Gram-negative or Gram-positive biofilm-producing bacteria. Non-limiting examples of conditions are selected from the group of chronic non-healing wounds, including venous ulcers and diabetic foot ulcers, ear infections, venous sinus infections, urinary tract infections, gastrointestinal diseases, lung infections, respiratory tract infections, cystic fibrosis, chronic obstructive pulmonary disease, catheter-related infections, indwelling device-related infections, infections associated with implanted prostheses, osteomyelitis, cellulitis, abscesses, and periodontal disease. PilA and Vaccines

[0183] In Applicants' long-standing efforts to develop a vaccine against respiratory tract disease caused by NTHI, Applicants focused on two unique biofilm-associated determinants. The first target is NTHI T4P, a key adhesin with multiple roles in adherence, colonization, biofilm formation, twitching motility, and transformation competence (Bakaletz et al., 2005; Carruthers et al., 2012; Das et al., 2017; Jurcisek et al., 2007; Mokrzan et al., 2019; Mokrzan et al., 2016; Toone et al., 2020). Antibodies against the majority subunit of NTHI T4P (PilA) and against a specific recombinant and soluble form of PilA ("rsPilA") induce dispersal of pre-existing NTHI and polymicrobial biofilms in vitro, as well as biofilms present within the middle ear in clinical models of NTHI-induced OM, where biofilm dispersal leads to rapid disease resolution (Mokrzan et al., 2018; Novotny et al., 2009; Novotny et al., 2013b; Novotny et al., 2016; Novotny et al., 2015; Ysebaert et al., 2019). The mechanism for this outcome requires the expression of both T4P and LuxS, the latter of which mediates quorum sensing in NTHI (Armbruster et al., 2009; Daines et al., 2005; Surette et al., 1999). Armbruster et al. We showed that luxS-induced production of autoinducer 2 (AI-2) resulted in increased biofilm formation in vitro and persistence in vivo in a chinchilla model of OM (Armbruster et al., 2009), and further demonstrated that NTHI absorbs AI-2 from its environment via RbsB (Armbruster et al., 2011). The role of luxS-mediated AI-2 signaling in preventing biofilm maturation and dispersal was further demonstrated by Pang et al., who used an NTHI construct inducible for luxS expression (Pang et al., 2018).

[0184] The study demonstrated that NTHI during biofilm dispersal induced by anti-rsPilA antibodies revealed an additional role for specific luxS quorum signaling that requires both T4P expression and luxS-induced production of AI-2 ( Novotny et al., 2015 ). NTHI is released in a "top-down" process, with maximum dispersion into the supernatant within 6 hours of incubation (Mokrzan et al., 2018; Novotny et al., 2015). Armbruster et al. also showed that Moraxella catarrhalis, which does not express AI-2, results in increased M. catarrhalis biofilm formation despite "interception" of the AI-2 signal generated by NTHI within polymicrobial biofilms formed by these two species (Armbruster et al., 2010). Interestingly, when applicants incubated preformed dual-species NTHI and M. catarrhalis biofilms with an antibody against rsPilA (targeting an antigen exclusively expressed by NTHI), both NTHI and M. catarrhalis dispersed from the biofilm (Mokrzan et al., 2018). The mechanism for M. catarrhalis dispersion is thought to be due to the interaction of M. catarrhalis with M. catarrhalis. revealed another example where arrhalis is intercepted in AI-2 produced by NTHI in response to exposure to anti-rsPilA ( Mokrzan et al., 2018 ).

[0185] PilA is a short for the major subunit protein of type IV pili. Type IV pili are typically 5-7 nm in diameter, several micrometers in length, and consist of a single protein subunit (Bardy et al., Microbiology, 149, 295-304, 2003; Wall and Kaiser, Mol. Microbiol., 32, 1-10, 1999). Type IV pili The subunits are typically 145-160 amino acids in length and may be glycosylated or phosphorylated. There are two classes of pilin subunits, type IVa and type IVb, which are distinguished from each other by the leader peptide and the average length of the mature subunit, in which this N-methylated amino acid occupies the N-terminal position and average length of the D region (due to the disulfide region) of the mature protein. Most respiratory pathogens express class IVa pilins, while enteropathogenic ones more typically express class IVb pilins. Type IVa pili contain a highly conserved hydrophobic N-terminally methylated phenylalatom. It is distinguished by the presence of 1C.

[0186] In some embodiments, a biologically active fragment of PilA has at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100%, or at least 1.5-fold, or at least 2-fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 7-fold, or at least 8-fold, or at least 9-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold or more greater biological activity than wild-type PilA. In some embodiments, a biologically active fragment of PilA comprises, consists essentially of, or even consists of, a fragment of PilA. In some embodiments, the biological activity comprises, consists essentially of, or further consists of inducing an antibody or other immune response in a subject against PilA. Such biological activity can be measured by one skilled in the art, for example, by quantifying antibody concentration and / or PilA affinity in a sample from a subject immunized with the biologically active fragment. Additionally or alternatively, the biological activity herein comprises, consists essentially of, or further consists of one or more of preventing, inhibiting, disrupting, dispersing, or treating a biofilm in a subject.

[0187] In some embodiments of any aspect disclosed herein, the PilA is a chimeric protein disclosed herein. In some embodiments of any aspect disclosed herein, the PilA, or biologically active fragment thereof, comprises, alternatively consists essentially of, or even consists of, a chimeric protein comprising a fragment of the nontypeable H. influenzae (NTHI) type IV pilus major subunit protein (PilA) and a fragment of the NTHI OMP P5 protein (also called P5-fimbrin, fimbrin, or OMP P5-homologous adhesin). In further embodiments, the PilA, or biologically active fragment thereof, comprises, alternatively consists essentially of, or even consists of, a chimeric protein comprising PilA modified to present a B-cell epitope of the LB1 peptide. Also provided herein are vaccine compositions comprising one or more chimeric proteins disclosed herein as PilA, or a biologically active fragment thereof, and methods of eliciting an immune response using the chimeric proteins disclosed herein. Such chimeric proteins are disclosed in US Pat. No. 7,811,591, which is incorporated herein by reference in its entirety.

[0188] In some embodiments of any aspect disclosed herein, PilA, or a biologically active fragment thereof, comprises, consists essentially of, or even consists of a chimeric protein described herein.

[0189] The LB1 peptide is a 40 amino acid synthetic chimeric P5-fimbrin-derived peptide that induces an immunogenic response against NTHI and is advantageous because it does not require tedious purification techniques. [ka] The LB1 peptide contains an N-terminal 19-amino acid peptide that is a B cell epitope (Arg Ser Asp Tyr Lys Phe Tyr Glu Asp Ala Asn Gly Thr Arg Asp His Lys Lys Gly, SEQ ID NO: ). The B cell epitope was derived from the predicted surface-exposed loop 3 of the outer membrane protein (fimbrin) of NTHi, designated OMP P5 (also known as P5-fimbrin or OMP P5-homologous adhesin). The LB1 peptide further contains a short 5-mer linker peptide and a 16-residue T cell promiscuous epitope. The T cell epitope was derived from the fusion protein of measles virus. The promiscuous T cell epitope induces a very strong T cell response in individuals exposed to this epitope.

[0190] In some embodiments, PilA or a biologically active fragment thereof comprises, consists essentially of, or even consists of a fragment of the LB1 peptide inserted into a safe and selective carrier protein that does not have reduced efficacy in inducing a B cell response. Preferably, the fragment of the LB1 peptide is inserted into a carrier that itself also confers protection against NTHi-induced disease. One such carrier that can induce protection against NTHi-induced disease is the NTHi type IV pilus (twitching pilus) protein, also known as the PilA protein. [ka] The PilA protein is a protein comprising the sequence of the pilA gene ( [ka] (which may comprise, alternatively consist essentially of, or alternatively consist of, the sequence of

[0191] In some embodiments, PilA or a biologically active fragment thereof comprises, consists essentially of, or further consists of a fragment of the LB1 peptide to present a peptide that induces an immunogenic response. Such a fragment of the LB1 peptide may be 12-35 amino acids in length, or 15-30 amino acids in length, or 18-19 amino acids in length, and is a subunit of the fimbrin protein. In some embodiments, the fragment of the LB1 peptide comprises, consists essentially of, or further consists of the N-terminal amino acid sequence RSDYKFYEDANGTRDHKKG (SEQ ID NO: ).

[0192] In another embodiment, PilA or a biologically active fragment thereof comprises, consists essentially of, or even consists of a PilA protein modified to present a 24-amino acid peptide. The 24-amino acid peptide may comprise a B-cell epitope of the LB1 peptide modified as set forth in the amino acid sequence LVRSDYKFYEDANGTRDHKKGRHT (SEQ ID NO: ), where leucine and valine are added to the N-terminus of the LB1 B-cell epitope and arginine, histidine, and threonine are at the C-terminus of the LB1 B-cell epitope. These modifications to the B-cell epitope are intended to aid protein folding and / or antigen presentation. Any modification to the LB1 B-cell epitope that will aid protein folding and / or antigen presentation may be used.

[0193] The amino acid sequence of the surface-exposed loop 3 of NTHi OMP P5 can vary between NTHi strains. PilA or a biologically active fragment thereof comprises, consists essentially of, or even consists of a fragment of a PilA protein modified to present a B-cell epitope of any variant amino acid sequence of loop 3 of NTHi OMP P5. In particular, PilA or a biologically active fragment thereof is selected from the group consisting of the following variant NTHi: The variant peptides may comprise, consist essentially of, or even consist of a PilA protein modified to present one of the OMP P5 amino acid sequences: RSDYKLYNKNSSSNSTLKNLGE (SEQ ID NO: ), RSDYKLYNKNSSTLKDLGE (SEQ ID NO: ), and RSDYKFYDNKRID (SEQ ID NO: ). Variant peptides may also be presented with leucine and valine added to the N-terminus, and arginine, histidine, and threonine added to the C-terminus, or any other modification that assists in protein folding and / or antigen presentation.

[0194] The chimeric proteins of the present invention comprise modified PilA amino acids in which native PilA amino acids are replaced with portions of the LB1 peptide. In addition, the chimeric proteins of the present invention comprise modified PilA amino acid sequences in which portions of the LB1 peptide are inserted within and in addition to native PilA amino acids. The chimeric proteins of the present invention have the ability to induce the formation of antibodies against the two proteins and are therefore more effective and specific vaccine candidates.

[0195] In one embodiment, the chimeric protein comprises the mature amino acid sequence (residues 13-149) of the NTHi PilA protein (SEQ ID NO: 51), wherein a portion of the LB1 peptide is inserted between the cysteine ​​residues at positions 62 and 72 of SEQ ID NO: 51; [ka] [ka] The chimeric protein may substitute naturally occurring amino acids, such as a chimeric protein having the amino acid sequence of SEQ ID NO: 51, which comprises residues 40-149 of SEQ ID NO: 51, with the B cell epitope of LB1 (Leu Val Arg Ser Asp Tyr Lys Phe Tyr Glu Asp Ala Asn Gly Thr Arg Asp His Lys Lys Gly Arg His Thr, SEQ ID NO: 52) inserted between residues 62 and 72 of SEQ ID NO: 51. In another embodiment, a portion of the LB1 peptide is inserted between the cysteine ​​residues at positions 131 and 144 of SEQ ID NO: 51, [ka] The chimeric protein may substitute naturally occurring amino acids, such as a protein having the amino acid sequence of SEQ ID NO: 51. This chimeric protein comprises residues 40-149 of SEQ ID NO: 51, with the B cell epitope of LB1 (SEQ ID NO: 52) inserted between residues 131 and 144 of SEQ ID NO: 51.

[0196] In another embodiment, the chimeric protein comprises the mature amino acid sequence (residues 13-149) of the NTHi PilA protein (SEQ ID NO: 51), wherein a portion of the LB1 peptide is inserted at the C-terminus of the PilA protein. [ka] This chimeric protein comprises residues 40 to 149 of SEQ ID NO:51, with the B cell epitope of LB1 (SEQ ID NO:52) inserted after residue 149 of SEQ ID NO:51.

[0197] In another embodiment, the chimeric protein comprises the mature amino acid sequence (residues 13-149) of the NTHi PilA protein (SEQ ID NO: 51), wherein a portion of the LB1 peptide is inserted at the N-terminus of the PilA protein. [ka] This chimeric protein comprises residues 40 to 149 of SEQ ID NO:2, with the B cell epitope of LB1 (SEQ ID NO:52) inserted before residue 40 of SEQ ID NO:51.

[0198] In further embodiments, the chimeric protein comprises a portion of an NTHi PilA protein and one or more of the LB1 peptides described herein. Chimeric proteins of the invention include those in which the same LB1 peptide is present two or more times within the portion of the NTHi PilA protein, and those in which two or more different LB1 peptides are present within the portion of the NTHi PilA protein.

[0199] The present disclosure further provides chimeric proteins comprising a portion of an NTHi PilA protein and any antigenic protein that elicits an immune response.

[0200] The chimeric protein may comprise the full length or a portion of the major subunit of the NTHi type IV pilus, encoded by the gene pilA. The PilA protein of NTHi isolate 86-028NP (e.g., SEQ ID NO: 51) is encoded by the nucleic acid sequence described in U.S. Patent No. 7,501,131, the entire contents of which are incorporated herein by reference. Polynucleotides encoding PilA polypeptides from NTHi clinical isolates 1728MEE, 1729MEE, 3224A, 10548MEE, 1060MEE, 1885MEE, 1714MEE, 1236MEE, 1128MEE, and 214NP are also provided. The amino acid sequences of these PilA polypeptides are, respectively: [ka] [ka] [ka] [ka] The possibilities of alternative codon usage are specifically contemplated in polynucleotides encoding polypeptides. In one embodiment, the polypeptides are encoded by the nucleotide sequences set forth in SEQ ID NOs: 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51, respectively, as disclosed in U.S. Patent No. 7,811,591 (each of which sequences is incorporated herein by reference in its entirety).

[0201] In some embodiments, the chimeric protein comprises a portion of an NTHi PilA protein. In one embodiment, the polypeptide comprises the NTHi 86-028NP amino acid sequence set forth in SEQ ID NO: 51, respectively. Polypeptides of the invention also include PilA polypeptides set forth in SEQ ID NOs: 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63. In additional embodiments, PilA polypeptides of the invention are from other non-typeable H. influenzae strains, as well as H. influenzae strains a, b, c, e, and f.

[0202] PilA polypeptides specifically include peptide fragments (i.e., peptides) or fragments of PilA polypeptides that retain one or more biological or immunogenic properties of the full-length polypeptides of the invention. In one embodiment, the PilA peptide fragments provided by the invention are designated TfpQ2, TfpQ3, TfpQ4, and OLP3, and comprise amino acids 35 to 68 of SEQ ID NO:51, amino acids 69 to 102 of SEQ ID NO:51, amino acids 103 to 137 of SEQ ID NO:51, and amino acids 21 to 35 of SEQ ID NO:51, respectively. Another PilA peptide fragment provided by the present disclosure comprises amino acids 40 to 149 of SEQ ID NO:51.

[0203] In some embodiments, a chimeric protein comprises a portion of a PilA polypeptide having one or more conservative amino acid substitutions that do not affect the biological and / or immunogenic activity of the PilA polypeptide. Alternatively, it is contemplated that the PilA polypeptide has conservative amino acid substitutions that may or may not alter biological activity.

[0204] In some embodiments, the chimeric protein comprises a variant portion of an NTHi PilA polypeptide of the invention (e.g., a polypeptide exhibiting at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity to the polypeptides of SEQ ID NOs: 51, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 that retain biological and / or immunogenic activity). Additionally, modifications in the sequence are readily made by substituting, adding, or deleting appropriate residues. For example, a cysteine ​​residue may be added to the carboxy terminus to provide a sulfhydryl group for convenient linkage to a carrier protein, or a spacer element such as an additional glycine residue may be incorporated into the sequence between the amino acid linked at the C-terminus of the peptide and the remainder of the peptide.

[0205] In some embodiments of any aspect disclosed herein, the PilA is a recombinant, soluble PilA (rsPilA). Recombinant PilA protein (rPilA) may be produced to serve as a more readily reproducible product. To do this, it is important that the rPilA folds properly to have the functional properties of the native pilin subunit as well, as described by Keiser et al., who also studied a pilin with four Cys residues. The published protocol of Keiser et al. (J. Biol. Chem., 276:24186-14193, 2001) is utilized. Briefly, a truncated pilin is engineered in which the first 28 residues are removed from the N-terminus to prevent aggregation, and this truncated pilin is further engineered for transport to the periplasm by incorporation of an OmpA leader sequence in the construct. Using this strategy, Keiser et al. generated a recombinant, soluble, monomeric P. aeruginosa pilin protein that was capable of binding to its receptor (asialo GM1) in in vitro assays and reduced morbidity and mortality in mice when the peptide was delivered 15 minutes before heterologous challenge. This soluble, monomeric, truncated NTHi PilA is useful in the studies described herein.

[0206] In some embodiments, chimeric proteins may be synthesized, purified, and sequenced using standard techniques. For example, chimeric proteins may be assembled semi-manually by stepwise Fmoc-tert-butyl solid-phase synthesis and purified by HPLC. The composition and amino acid sequence of recombinant, synthesized chimeric proteins may be confirmed by amino acid analysis and / or mass spectrometry. Anti-PilA antibody

[0207] In some embodiments, the anti-PilA antibody is a polyclonal antibody or a monoclonal antibody. In one aspect, the polyclonal antibody is in serum isolated from a subject immunized with a PilA polypeptide or a biologically active fragment thereof. In some embodiments, such serum can be purified and processed, e.g., digested, to produce Fab fragments of the antibody.

[0208] In one embodiment, the antibody specifically recognizes and binds to a PilA peptide, examples of which include surface antigen or recombinant soluble PilA (Genbank Accession No. EFU96734.1) and type IV pilin (Genbank Accession No. Yp_003864351.1). In one embodiment, the antibody is a polyclonal antibody. In another embodiment, the antibody is a monoclonal antibody or a biologically active fragment thereof. Methods for generating monoclonal antibodies, derivatives, fragments and variants thereof are known in the art.

[0209] In some embodiments, a biologically active fragment of an anti-PilA antibody has at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100%, or at least 1.5-fold, or at least 2-fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 7-fold, or at least 8-fold, or at least 9-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold or more greater biological activity than a reference anti-PilA antibody. In some embodiments, the biological activity comprises, consists essentially of, or even further consists of specifically recognizing and specifically binding to PilA. Such biological activity can be measured by one skilled in the art, for example, by quantifying the antibody's PilA affinity and / or specificity and / or competitive binding of PilA compared to a reference antibody. Additionally or alternatively, the biological activity herein includes, consists essentially of, or even consists of one or more of preventing, inhibiting, disrupting, dispersing, or treating biofilms in a subject. In some embodiments, the biological activity includes, consists essentially of, or even consists of the ability to kill H. influenzae bacteria. Other suitable activities may be selected from the following: reducing virulence, inhibiting adhesion, inhibiting twitching motility, inhibiting cell division, and / or inhibiting epithelial invasion and / or enhanced phagocytosis of H. influenzae bacteria. In some embodiments, the anti-PilA antibody or biologically active fragment thereof specifically binds to and specifically recognizes a PilA polypeptide or biologically active fragment thereof.A completely in vitro mediated bacterial assay system (Musher et al., Infect. Immun. 39:297-304, 1983; Anderson et al., J. Clin. Invest. 51:31-38, 1972) may be used to measure the biological activity of anti-chimeric protein antibodies. DNABII

[0210] Integration host factors (IHFs) are key structural elements of the bacterial biofilm matrix. IHFs and HUs (histone-like proteins) comprise two ubiquitous members of the DNABII family of bacterial DNA-binding proteins. Genes encoding IHFs and / or HUs are present in the genomes of every member of the eubacteria (Goodman et al., 2011). Therefore, this target is not unique to NTHIs, but instead is highly It is species-independent due to its presence in all pathogen-forming biofilms tested to date, including each of the priority ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) ( Devaraj et al., 2018 ; Devaraj et al., 2015 ; Novotny et al., 2013a ). Extracellular DNA (eDNA) and associated DNABII proteins are essential for the basic architecture and structural integrity of these biofilms ( Flemming and Wingender, 2010 ). ;Goodman et al., 2011;Jurcisek and Bakaletz, 2007;Whitchurch et al., Within the biofilm matrix, the cross-stranded eDNA is stabilized by IHF and HU (Swinger and Rice, 2004). The result is a biofilm structure. The eDNA matrix is ​​a lattice-like scaffold that supports and maintains the structure. Exposure of bacterial biofilms to antibodies against the DNABII protein destabilizes the eDNA matrix, causing the biofilm structure to collapse (Goodman et al., 2011). The mechanism for this outcome is DNABII molecules in the environment surrounding the biofilm are sequestered due to the formation of antibody complexes, thus inducing an equilibrium shift in which DNABII proteins within the eDNA matrix are released ( Brockson et al., 2014 ; Novotny et al., 2016 ). The result is the sudden and complete disintegration of the eDNA scaffold and the release of biofilm-resident bacteria, beginning within 3 minutes of exposure to anti-DNABII antibodies in vitro (Brockson et al. al., 2014; Novotny et al., 2019). Vaccines against DNABII proteins Induced antibodies disrupt pre-existing biofilms in chinchilla models of NTHI-induced OM, allowing clearance by host immune effectors (Goodman et al., 2011; Novotny et al., 2019; Novotny et al., 2016). Therapeutic treatment with acetaminophen resolves osteolytic peri-implantitis in a rat model of pre-existing Aggregatibacter actinomycetemcomitans biofilms (Freire et al., 2017) and also eradicates aggregated biofilms of P. aeruginosa from mouse lungs (Novotny et al., 2016).

[0211] The term "nucleoid-associated protein" or "NAP," as used herein, refers to a class of proteins that affect the dynamic spatial organization of nucleic acids in the nucleoid of prokaryotic cells. These proteins organize the genome by inducing DNA bending, binding, and aggregation. Certain NAPs are DNA-binding proteins and may be associated with biofilms, including the 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). Among NAPs, DNABII proteins are distinct and generally share strong sequence identity with an alpha-helical dimerization domain and may contain an antiparallel beta ribbon, which often contains a tip that binds to and inserts into the minor groove of DNA. [ka] (wherein X can be any amino acid sequence or can be selected from the amino acids Q, R, K, S, or T) and entangle it. A functional protomer is a dimer of identical or homologous subunits.

[0212] The DNABII family is a member of a class of bacterial proteins called nucleoid-associated proteins (NAPs), which in part shape the intracellular bacterial nucleoid (Browning et al. (2010) Curr. Opin. Microbiol. 13:773-780). In addition, this family The family members are ubiquitous and expressed by virtually all eubacteria. All characterized family members to date function as either homodimers or heterodimers of subunits. The family is divided into two types: HU (histone-like protein) and IHF (integration host factor), and B. cenocepacia can express both (strain J2315 genes: BCAL3530, hupA; BCAL1585, hupB; BCAL1487, ihfA, and BCAL2949, ihfb). The main difference between these family members is that HU binds to DNA in an independent manner, while IHF binds to a genus-conserved consensus sequence (WATCAANNNNTTR (where W is A or T and R is a purine), SEQ ID NO: ) (Swinger et al. (2004) Curr. Opin. Struct. Biol. 14:28-35). All DNABII proteins bind to DNA and bend it significantly; for example, E. coli IHF can effectively bend DNA into a U-turn (Rice et al. (1996) Cell 87:1295-1306). In addition, all family members exhibit bending They have a preference for bent or curved DNA structures, such as Holliday junctions, which are central cross-like structures in DNA recombination. Indeed, DNA BII proteins function as cofactors that promote all intracellular DNA functions, including gene expression, recombination, repair, and replication (Swinger et al. (2004) Curr. Opin. Struct. Biol. 14:28-35).

[0213] "DNABII polypeptide or protein" refers to a DNA-binding protein or polypeptide that is composed of a DNA-binding domain and thus has specific or general affinity for microbial DNA. In one embodiment, they bind to DNA in the minor groove. Non-limiting examples of DNABII proteins are the integration host factor (IHF) protein and the histone-like protein (HU) from E. coli strain U93. Other DNA-binding proteins that may be associated with biofilms include DPS (Genbank Accession No.: CAA49169), H-NS (Genbank Accession No.: CAA47740), Hfq (Genbank Accession No.: ACE63256), CbpA (Genbank Accession No.: BAA03950), and CbpB (Genbank Accession No.: NP_418813).

[0214] "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 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. In certain embodiments, the term "IHF" refers to one or both of the two IHF subunits: integration host factor subunit alpha (IHFA or IhfA) and integration host factor subunit beta (IHFB or IhfB).

[0215] "HU" or "histone-like protein from E. coli strain U93" typically refers to a class of heterodimeric proteins associated with E. coli. HU proteins are known to bind to DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU was reported by Laine et al. (1980) Eur. J. Biochem 103(3)447-481. Antibodies against the HU protein are commercially available from Abeam. The genes encoding the HU protein subunits in E. coli are hupA and hupB, which correspond to SEQ ID NOs: 29 and 30, respectively. Homologs for these genes are found in other organisms, and peptides corresponding to these genes from other organisms can be found in Table 10 of WO2011 / 123396.

[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 outer membrane proteins are, for example, OMP P5 (Genbank accession number: YP_004139079.1), OMP P2 (Genbank accession number: ZZX87199.1), and OMP P26 (Genbank accession number: YP_665091.1), while examples of surface antigens are rsPilA or recombinant soluble PilA (Genbank accession number: EFU96734.1) and type IV pilin (Genbank accession number: Yp_003864351.1).

[0217] In some embodiments of any aspect disclosed herein, the DNABII is selected from an integration host factor (IHF) or a histone-like protein (HU). In some embodiments of any aspect disclosed herein, the DNABII comprises, essentially consists of, or even consists of wild-type DNABII or a biologically active fragment thereof. In some embodiments, the biologically active fragment of DNABII comprises, essentially consists of, or even consists of a chimer disclosed herein. In some embodiments, the DNABII comprises, essentially consists of, or even consists of SEQ ID NOs: 1 to 348 of WO2014 / 201305 (each of the sequences is incorporated herein by reference in its entirety), or their respective fragments or equivalents. Other suitable DNABII or biologically active fragments thereof produced by Gram (+) and Gram (-) bacteria can be found in Table 8 of U.S. Patent No. 8,999,291, which is incorporated herein by reference, as well as in WO2014 / 201305, WO2017 / 023863, WO2017 / 066719, WO2018 / 129092, WO2018 / 129078, PCT Patent Application Nos. PCT / US2020 / 041082 and PCT / US2021 / 040576. Antibodies that bind to these proteins and their fragments are referred to as anti-DNABII antibodies.

[0218] A sequence alignment of relevant portions of the DNA-binding proteins of various embodiments is published in Table 9 of U.S. Pat. No. 8,999,291, which is incorporated herein by reference. Bold text indicates perfect agreement with the consensus, light gray text indicates conservative amino acid changes, and lightly or darkly shaded sequences are highly conserved across species. Gray-shaded undefined sequences at the amino and / or carboxy termini are undefined amino acids not shared with the consensus sequence. This table is based on information previously published in Obeto et al. (1994) Biochimie 76:901-908. The fragment "ARM" is shown at the bottom of the table. and polypeptide fragments comprising, consisting essentially of, or even consisting of these fragments or their equivalents have the biological activity described herein.

[0219] Non-limiting examples of such polypeptides include isolated or recombinant polypeptides comprising the amino acid sequences listed in the table below, or fragments or equivalents thereof, and their respective equivalents. Further non-limiting examples include isolated or recombinant polypeptides comprising, alternatively consisting essentially of, or even further of the amino acids AARKGINPKTKKSISIPARKVVRF (SEQ ID NO: ). In still further aspects, the present disclosure provides mature or recombinant HU antigenic polypeptides having amino acids corresponding to the P. gingivalis amino acids at positions 61 and / or 64 modified to I (or alternatively, another hydrophobic amino acid, e.g., V or F) and / or K. Biological equivalents of these polypeptides are further included in the present disclosure, with the proviso that the sequences do not include the entire isolated wild-type sequence. A non-limiting list of exemplary HU protein sequences from various species. [Table 7-1] [Table 7-2]

[0220] Non-limiting examples of suitable polypeptide fragments include, but are not limited to: (a) [ka] (b) [ka] (c) [ka] (d) [ka] (e) [ka] (f) [ka] (g) [ka] (h) [ka] (i) a polypeptide comprising the amino acid sequence NPXT, or (j) The equivalent of (a) to (i). wherein equivalents include amino acid sequences having at least about 80% homology or amino acid identity thereto, or amino acids encoded by a polynucleotide that hybridizes to a polynucleotide encoding the amino acid sequence or its complement under high stringency conditions, wherein 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.

[0221] As used herein, the term "chimer" or "chimeric peptide" refers to a recombinant polypeptide comprising, alternatively consisting essentially of, or further consisting of two or more fragments or domains of a DNABII polypeptide that are directly or indirectly (such as via a linker) conjugated to each other. In one embodiment, the domain is a conformational tip domain and / or a conformational tail domain. Additionally or alternatively, the two or more fragments or domains are derived from the same or different DNABII polypeptides. In one embodiment, the chimeric peptide comprises, alternatively consisting essentially of, or further consisting of the tip domain of IhfA and the tip domain of IhfB that are directly or indirectly (such as via a linker) conjugated to each other. See, for example, U.S. Patent No. 11,104,723, which is incorporated herein by reference. In another embodiment, the chimeric peptide comprises, alternatively consists essentially of, or even consists of the tail domain of IhfA and the tail domain of IhfB conjugated directly or indirectly (such as via a linker) to each other. See, for example, U.S. Patent No. 11,104,723, incorporated herein by reference. A "conformational tip domain" of a polypeptide refers to a polypeptide whose primary amino acid sequence has an antiparallel beta ribbon structure with a sharp turn typically mediated by a proline residue. The "tip" of an Ihf polypeptide is shown in Figure 1 of WO2018 / 129078.

[0222] A "conformation tip domain" of a polypeptide refers to a polypeptide that comprises a primary amino acid sequence whose structure has an antiparallel beta ribbon with a sharp turn typically mediated by a proline residue.

[0223] The table below shows examples of conformation tip domain polypeptides. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7]

[0224] The "chip fragment" of a DNABII polypeptide refers to a DNABII polypeptide that forms two arm proteins, using IHF alpha and IHF beta as examples. Non-limiting examples of such fragments include the chip fragment of IhfA (also referred to herein as A chip fragment): NFELRDKSSRPGRNPKTGDVV, SEQ ID NO: , and the chip fragment of IhfB (also referred to herein as B chip fragment): SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: ) or FSLHHRQPRLGRNPKTGDSV (SEQ ID NO: ).

[0225] By "tail fragment" of a DNABII polypeptide is intended the region of either protein that is exposed to the bulk medium and is not obstructed by DNA or other polypeptides.

[0226] In certain embodiments, the chip chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (wherein "X" is an optional amino acid linker sequence comprising, consisting essentially of, or alternatively consisting of, 1-20 amino acids, and "X1" is any amino acid, or alternatively, "X1" is selected from the amino acids Q, R, K, S, or T). In a further aspect, "X1" is K or Q. In a further embodiment, the chip chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (wherein "X" is an optional amino acid linker sequence comprising, consisting essentially of, or alternatively consisting of 1-20 amino acids). In yet a further embodiment, the chip chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (SEQ ID NO: , which in some embodiments is also referred to herein as a chimer).

[0227] In certain embodiments, the tail chimeric peptide IhfA3-IhfB2NTHI comprises, consists essentially of, or alternatively consists of the polypeptide sequence of FLEEIRLSLESGQDVKLSGF-X-TLSAKEIENMVKDILEFISQ (SEQ ID NO: ), where "X" is an optional amino acid linker sequence optionally comprising, consisting essentially of, or alternatively consisting of 1-20 amino acids. In certain embodiments, the linker is selected from any one or more of those disclosed herein. In one embodiment, the tail chimeric peptide IhfA3-IhfB2NTHI comprises, consists essentially of, or alternatively consists of the polypeptide sequence of FLEEIRLSLESGQDVKLSGF-X-TLSAKEIENMVKDILEFISQ (SEQ ID NO: ), where "X" is an optional amino acid linker sequence optionally comprising, consisting essentially of, or alternatively consisting of 1-20 amino acids. In certain embodiments, the linker is selected from any one or more of those disclosed herein. NTHIcomprises, consists essentially of, or alternatively consists of FLEEIRLSLESGQDVKLSGFGPSLTLSAKEIENMVKDILEFISQ (SEQ ID NO: ).

[0228] Further provided are polypeptides disclosed below as A1-A4 and A6, and B1-B6, which do not contain the conformation tip domain, and equivalents of these polypeptides from different organisms identified herein that produce DNABII polypeptides. MATITKLDIIEYLSDKYHLS (also referred to herein as A1; (SEQ ID NO: )); KYHLSKQDTKNVVENFLEEI (also referred to herein as A2; (SEQ ID NO: )); FLEEIRLSLESGQDVKLSGF (also referred to herein as A3; (SEQ ID NO: )); KLSGFGNFELRDKSSRPGRN (also referred to herein as A4; (SEQ ID NO: )); ARRVVTFKPGQKLRARVEKTK (also referred to herein as A6; (SEQ ID NO: )); MTKSELMEKLSAKQPTLSAK (also referred to herein as B1 (SEQ ID NO: )); TLSAKEIENMVKDILEFISQ (also referred to herein as B2 (SEQ ID NO: )); EFISQSLENGDRVEVRGFGS (also referred to herein as B3 (SEQ ID NO: )); RGFGSFSLHHRQPRLGRNPK (also referred to herein as B4 (SEQ ID NO: )); GRNPKTGDSVNLSAKSVPYF (also referred to herein as B5; (SEQ ID NO: )); and SVPYFKAGKELKARVDVQA (also referred to herein as B6; (SEQ ID NO: )) Includes:

[0229] Non-limiting examples of DNABII polypeptides include IHF or HU alpha or beta polypeptides; IHF alpha polypeptides; Moraxella catarrhalis HU; E. coli HupA, HupB, himA, himD; E. faecalis HU (such as V583).

[0230] Also provided are recombinant polypeptides comprising, alternatively consisting essentially of, or even consisting of, three to five conformation tip domains, which may be produced by the same or different bacterial species, and whose amino acid sequences may be the same (e.g., all A5 amino acid sequences), or have at least two, or at least three, or at least four, or all five different amino acid sequences (e.g., A5 and mB4, and their respective equivalents, and [ka] (various combinations of fragments containing X1), where X1 is any amino acid, or in one embodiment, an amino acid selected from the amino acids Q, R, K, S, or T. The conformational tip domains in the recombinant polypeptide can be in a linear or branched conformation. They can further comprise a detectable label and / or a purification label linked thereto. The structural orientation of the tip domains can be "head" to tail; tail to head (where the polypeptide contains any combination of three or more tip domains, head to tail, e.g., head-head-head); tail-head-head; or tail-head-tail, where the amine terminus of the wild-type sequence is the "head" and the carboxy terminus of the wild-type sequence is the "tail" of the polypeptide. In one embodiment, the polypeptide can be, in total, 41 to 120 amino acids in length.

[0231] Non-limiting examples of equivalent polypeptides include polypeptides having at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity thereto or in polypeptide sequence, or polypeptides encoded by polynucleotides or their complements that hybridize to polynucleotides encoding such polypeptide sequences under high stringency conditions. High stringency conditions are described herein and are incorporated by reference. Applicant has determined that the bold and underlined amino acids are heavily conserved and therefore, in one aspect, will not be modified or changed in the design of equivalent polypeptides. Additional examples of equivalent polypeptides include, for example, [ka] (wherein "X1" is any amino acid, or alternatively, "X1" is selected from the amino acids Q, R, K, S, or T).

[0232] Equivalent polypeptides, a type of biologically active fragment, also include polypeptides that consist of or comprise the above-described polypeptides with the addition of up to 25, or alternatively 20, or alternatively 15, or alternatively up to 10, or alternatively up to 5 random amino acids to either the amine or carboxy terminus (or both). In another embodiment, equivalent polypeptides also include polypeptides that consist of or comprise the above-described polypeptides with the addition of up to 25, or alternatively 20, or alternatively 15, or alternatively up to 10, or alternatively up to 5 amino acids to either the amine or carboxy terminus (or both) selected from the flanking amino acids of the corresponding wild-type sequence and equivalents of the wild-type flanking amino acids. Anti-DNABII antibody

[0233] In some embodiments, the anti-DNABII antibody or biologically active fragment thereof recognizes and binds to DNABII or a biologically active fragment thereof, optionally selected from one or more of the DNABII A5 peptide, the DNABII mB4 peptide, or a recombinant polypeptide comprising the A5 peptide and the mB4 peptide (i.e., a chimer).

[0234] In some embodiments, a biologically active fragment of DNABII has at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100%, or at least 1.5 times, or at least 2 times, or at least 3 times, or at least 4 times, or at least 5 times, or at least 6 times, or at least 7 times, or at least 8 times, or at least 9 times, or at least 10 times, or at least 20 times, or at least 50 times, or at least 100 times, or more, greater biological activity than a reference anti-DNABII antibody. In some embodiments, the biological activity comprises, consists essentially of, or even consists of, specifically recognizing and specifically binding to DNABII or a biologically active fragment thereof. Such biological activity can be measured by those skilled in the art, for example, by quantifying the antibody's DNABII affinity and / or specificity and / or competitive binding of DNABII or a biologically active fragment compared to a reference antibody. Additionally or alternatively, the biological activity herein comprises, or essentially consists of, or even consists of, one or more of preventing, inhibiting, destroying, dispersing, or treating biofilm in a subject.

[0235] Suitable anti-DNABII antibodies or biologically active fragments thereof can be found in U.S. Pat. No. 11,104,723, WO2014 / 201305, WO2017 / 023863, WO2017 / 066719, WO2018 / 129092, WO2018 / 129078, and PCT Publication No. WO2021 / 007260, each of which is incorporated by reference in its entirety.

[0236] Non-limiting examples of anti-IHF antibodies are described herein, and in one aspect are antibodies that specifically recognize and specifically bind to the polypeptides identified herein, or the Arm fragments identified therein (identified above), or such polypeptides or polynucleotides, or the sequences: TCTCAACGATTTA (SEQ ID NO:); WATCAANNNNTTR (where W is A or T, N is any nucleotide, and R is A or G; (SEQ ID NO: )); [ka] (also referred to herein as hIFA1; (SEQ ID NO: )); [ka] (also referred to herein as hIFA2; (SEQ ID NO: )); [ka] (also referred to herein as hIFA3; (SEQ ID NO: )); [ka] (also referred to herein as hIFA4; (SEQ ID NO:)); [ka] (also referred to herein as hIFA5; (SEQ ID NO: )); ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQ ID NO: )), or equivalents thereof, or one or more of polynucleotides or peptides having at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity thereto, or to a polypeptide sequence encoded by a polynucleotide or its complement that hybridizes to a polynucleotide encoding such a polypeptide sequence under high stringency conditions. High stringency conditions are described above and are incorporated herein by reference. Applicant has determined that the bolded and underlined amino acids are heavily conserved and therefore, in one aspect, will not be altered or changed in the design of an equivalent polypeptide. Additional examples of equivalent polypeptides include, for example, polypeptides that consist of or comprise the above-described polypeptides with the addition of up to 25, or alternatively 20, or alternatively 15, or alternatively up to 10, or alternatively up to 5 random amino acids to either the amine or carboxy terminus (or both).

[0237] In certain aspects, the present disclosure provides a polypeptide comprising the amino acid sequence Phe Leu Glu Glu Ile Arg Leu Ser Leu Glu Ser Gly Gln Asp Val Lys Leu Ser Gly Phe (SEQ ID NO: 64), [ka] The present invention 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 Lys Lys Gln Ala Lys Ala Ala Leu Glu Ala Thr Leu Asp Ala Ile Thr Ala Ser Leu Lys Glu Gly (SEQ ID NO: 67), a polypeptide comprising the amino acid sequence NPXT, or their respective equivalents. In a further aspect, 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 a further aspect, 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.

[0238] In addition, antibodies can be generated against the "tip" region of the DNABII protein, which in one aspect contains, or has been modified to contain, an antiparallel beta ribbon 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 may be generated using fragments of the DNABII protein containing the NPXT sequence, optionally flanked by about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids at one or both ends of said sequence. Non-limiting examples of such antibodies include the consensus amino acid sequence in underlined and bold text. [ka] In another aspect, antibodies may be generated against the chip region of the DNABII protein, which provides 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). One skilled in the art can identify the residue at position X (in the example below) in any one of these sequences. [ka] It will be appreciated that the amino acids (marked with a ) may be substituted with any amino acid, e.g., Q, R, K, S, or T. Examples include: SEQ ID NO: 68: Haemophilus influenzae IhfA, A5 fragment: [ka] SEQ ID NO: 69: Haemophilus influenzae HU, A5 fragment: [ka] SEQ ID NO: 65: Haemophilus influenzae IhfB, modified B4 (mB4) fragment: [ka] SEQ ID NO: 70: Haemophilus influenzae IhfA, A chip fragment: [ka] SEQ ID NO: 71: Haemophilus influenzae IhfB, B chip fragment: [ka] SEQ ID NO: 66 Haemophilus influenzae HU, fragment: VNER [ka]

[0239] In some embodiments, [ka] The polypeptide comprising the NPXT motif is at least about 20 amino acids long, and NPXT is located at the center of the sequence. Non-limiting examples of such sequences include SEQ ID NOs: 68, 69, and 66. Alternatively, polypeptides having an NPXT motif (e.g., as described above) can be modified to remove the NPXT motif by either substituting or deleting one or more amino acids, and monoclonal antibodies can be raised against these polypeptides. Applicant has determined that antibodies raised against DNABII polypeptides lacking an NPXT motif are useful in imaging and diagnostic methods for monitoring biofilm formation and / or destruction. In one embodiment, a kit is provided that includes an antibody raised against or binding to a DNABII having an NPXT motif, and an antibody raised against or binding to a DNABII lacking an NPXT motif (e.g., a modified polypeptide or a naturally occurring polypeptide). For example, the kit can include an antibody that recognizes and binds to polypeptides A5, B4, or mB4 (therapeutic), which can be combined in the kit with an antibody that recognizes and binds to polypeptides A3 or B2 (diagnostic). The kits are useful for the diagnosis, treatment, and monitoring of biofilm treatment.

[0240] 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 comprising or consisting essentially of an amino acid sequence selected from SEQ ID NOs: 64-65, 66, 67, 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 the table below. Hybridoma cell lines producing monoclonal antibodies that specifically recognize and bind to IhfA fragment A5 (SEQ ID NO: 68), IhfB fragment B4 (Arg Gly Phe Gly Ser Phe Ser Leu His His Arg Gln Pro Arg Leu Gly Arg Asn Pro Lys, SEQ ID NO: 72), and IhfB fragment mB4 (SEQ ID NO: 65) of Haemophilus influenzae were deposited with the American Type Culture Collection (ATCC) in accordance with the provisions of the Budapest Treaty on July 30, 2015 under the deposit numbers listed in the table below, and each hybridoma cell line is listed in the table below. Further non-limiting exemplary antibodies include those 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) pursuant to the provisions of the Budapest Treaty on August 1, 2016, under the accession numbers listed in the table below). Examples of antibodies that specifically recognize and bind to IhfA fragment A3 (SEQ ID NO: 64) or IhfB fragment B2 (Thr Leu Ser Ala Lys Glu Ile Glu Asn Met Val Lys Asp Ile Leu Glu Phe Ile Ser Gln SEQ ID NO: 73) of H. influenzae, as well as antibodies that specifically recognize or bind to a polypeptide comprising SEQ ID NO: 66, SEQ ID NO: 67, a polypeptide comprising the amino acid sequence NPXT, or their respective equivalents. [Table 9]

[0241] 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0242] In one aspect, the present 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 mIhfB4 NTHI 12E6.F8.D12.D5. In one aspect, the present 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0243] 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 LC 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.

[0244] In some embodiments of the antibodies provided herein, the HC variable domain sequence comprises an HC variable domain sequence that is at least 85% identical to the HC variable domain sequence of (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 mIhfB4 NTHI 12E6.F8.D12.D5, and / or the LC variable domain sequence comprises an HC variable domain sequence that is at least 85% identical to the HC variable domain sequence of (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 mIhfB4 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.

[0245] 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 and light chain immunoglobulin variable domain sequences form an antigen binding site that binds to an epitope of a DNABII protein.

[0246] In some embodiments, the heavy chain variable region comprises a CDRH1 sequence that comprises, alternatively consists essentially of, or even consists 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0247] In some embodiments, the heavy chain variable region comprises a CDRH2 sequence that comprises, alternatively consists essentially of, or even consists 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0248] In some embodiments, the heavy chain variable region comprises a CDRH3 sequence that comprises, alternatively consists essentially of, or even 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0249] In some embodiments, the heavy chain variable region comprises, alternatively consists essentially of, or even 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0250] In some embodiments, the light chain variable region comprises a CDRL1 sequence that comprises, alternatively consists essentially of, or even 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0251] In some embodiments, the light chain variable region comprises a CDRL2 sequence that comprises, alternatively consists essentially of, or even consists 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0252] In some embodiments, the light chain variable region comprises a CDRL3 sequence that comprises, alternatively consists essentially of, or even 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0253] In some embodiments, the light chain variable region comprises, alternatively consists essentially of, or even 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0254] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH1 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence comprising FSLTSYS (SEQ ID NO:), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of FSLTSYSV (SEQ ID NO:), FSLTSYSVH (SEQ ID NO:), GFSLTSYS (SEQ ID NO:), or their respective biological equivalents.

[0255] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH1 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence comprising FNIKDYY (SEQ ID NO: 110), for example, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of FNIKDYYM (SEQ ID NO: ), FNIKDYYMH (SEQ ID NO: ), GFNIKDYY (SEQ ID NO: ), or their respective biological equivalents.

[0256] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH2 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence comprising IWAGGST (SEQ ID NO:), for example, but not limited to, VIWAGGST (SEQ ID NO:), GVIWAGGST (SEQ ID NO:), LGVIWAGGST (SEQ ID NO:), WLGVIWAGGST (SEQ ID NO:), IWAGGSTN (SEQ ID NO:), VIWAGGSTN (SEQ ID NO:), GVIWAGGSTN (SEQ ID NO:), LGVIWAGGSTN (SEQ ID NO:), WLGVIWAGGSTN (SEQ ID NO:), IWAGGSTNY (SEQ ID NO:), VIWAGGSTNY (SEQ ID NO:), GVIWAGGSTNY (SEQ ID NO:), LGVIWAGGSTNY (SEQ ID NO:), WLGVIWAGGSTNY (SEQ ID NO:), or their respective biological equivalents.

[0257] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH2 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence that includes IDPENDDT (SEQ ID NO:), for example, but not limited to, WIDPENDDT (SEQ ID NO:), GWIDPENDDT (SEQ ID NO:), IGWIDPENDDT (SEQ ID NO:), WIGWIDPENDDT (SEQ ID NO:), IDPENDDTE (SEQ ID NO:), WIDPENDDTE (SEQ ID NO:), GWIDPENDDTE (SEQ ID NO:), IGWIDPENDDTE (SEQ ID NO:), WIGWIDPENDDTE (SEQ ID NO:), IDPENDDTEY (SEQ ID NO:), WIDPENDDTEY (SEQ ID NO:), GWIDPENDDTEY (SEQ ID NO:), IGWIDPENDDTEY (SEQ ID NO:), WIGWIDPENDDTEY (SEQ ID NO:), or their respective biological equivalents.

[0258] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH3 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence comprising AREDS (SEQ ID NO:), for example, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of AREDS (SEQ ID NO:), or a biological equivalent thereof.

[0259] In some embodiments, the heavy chain variable region of the antibody or fragment thereof is selected from the group consisting of TELGAY (SEQ ID NO: ), or a biological equivalent thereof.

[0260] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises the polynucleotide sequence set forth below: [ka] [ka] or a biological equivalent thereof.

[0261] 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.

[0262] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises the polynucleotide sequence set forth below: [ka] or a biological equivalent thereof.

[0263] 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.

[0264] In some embodiments, the light chain variable region of the antibody or fragment thereof is selected from the group consisting of QNVGTN (SEQ ID NO: ), for example, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of QNVGTNV (SEQ ID NO: ), QNVGTNVA (SEQ ID NO: ), or their respective biological equivalents.

[0265] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL1 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence comprising QSLLDSNGKTY (SEQ ID NO: ), for example, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of QSLLDSNGKTYL (SEQ ID NO: ), QSLLDSNGKTYLN (SEQ ID NO: ), or their respective biological equivalents.

[0266] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises an amino acid sequence comprising SAS (SEQ ID NO:), such as, but not limited to, YSAS (SEQ ID NO:), IYSAS (SEQ ID NO:), LIYSAS (SEQ ID NO:), ALIYSAS (SEQ ID NO:), SASY (SEQ ID NO:), YSASY (SEQ ID NO:), IYSASY (SEQ ID NO:), LIYSASY (SEQ ID NO:), ALIYSASY (SEQ ID NO:), SASYR (SEQ ID NO:). ), YSASYR (SEQ ID NO:), IYSASYR (SEQ ID NO:), LIYSASYR (SEQ ID NO:), ALIYSASYR (SEQ ID NO:), SASYRY (SEQ ID NO:), YSASYRY (SEQ ID NO:), IYSASYRY (SEQ ID NO:), LIYSASYRY (SEQ ID NO:), ALIYSASYRY (SEQ ID NO:), SASYRYS (SEQ ID NO:), YSASYRYS (SEQ ID NO:), IYSASYRYS (SEQ ID NO:), LIYSASYRYS (SEQ ID NO:), ALIYSASYRYS (SEQ ID NO:), or their respective biological equivalents.

[0267] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises an amino acid sequence comprising LVS (SEQ ID NO:), for example, but not limited to, YLVS (SEQ ID NO:), IYLVS (SEQ ID NO:), LIYLVS (SEQ ID NO:), RLIYLVS (SEQ ID NO:), LVSK (SEQ ID NO:), YLVSK (SEQ ID NO:), IYLVSK (SEQ ID NO:), LIYLVSK (SEQ ID NO:), RLIYLVSK (SEQ ID NO:), LVSKL (SEQ ID NO:). ), YLVSKL (SEQ ID NO:), IYLVSKL (SEQ ID NO:), LIYLVSKL (SEQ ID NO:), RLIYLVSKL (SEQ ID NO:), LVSKLD (SEQ ID NO:), YLVSKLD (SEQ ID NO:), IYLVSKLD (SEQ ID NO:), LIYLVSKLD (SEQ ID NO:), RLIYLVSKLD (SEQ ID NO:), LVSKLDS (SEQ ID NO:), YLVSKLDS (SEQ ID NO:), IYLVSKLDS (SEQ ID NO:), LIYLVSKLDS (SEQ ID NO:), RLIYLVSKLDS (SEQ ID NO:), or their respective biological equivalents.

[0268] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL3 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence that includes QQYNSYP (SEQ ID NO: ), for example, but not limited to, an amino acid sequence that begins with, ends with, or consists essentially of QQYNSYPT (SEQ ID NO: ), or a biological equivalent thereof.

[0269] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL3 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence comprising WQSTHFPH (SEQ ID NO: ), for example, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of WQSTHFPHT (SEQ ID NO: ), or a biological equivalent thereof.

[0270] In some embodiments, the light chain variable region of the antibody or fragment thereof has the polynucleotide sequence: [ka] or a biological equivalent thereof.

[0271] 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.

[0272] In some embodiments, the light chain variable region comprises the polynucleotide sequence: [ka] [ka] or a biological equivalent thereof.

[0273] In some embodiments, the light chain variable region has the amino acid sequence: [ka] or a biological equivalent thereof.

[0274] Exemplary antibodies comprising the disclosed CDR sequences and heavy and light chain variable sequences are respectively disclosed in the following tables. Alternative CDR predictions can be made based on the heavy and / or light chain sequences, for example, based on the Kabat, Clothia, AbM, or contact definitions of CDR specificity, and details of these CDR prediction methods are known in the art (see, for example, bioinf.org.uk / abs / #cdrid) and / or commercially available. The tables are the results of utilizing the CDR prediction algorithms provided by Ofran Lab (Paratome available at ofranservices.biu.ac.il / site / services / paratome / index.html) and Green Mountain Antibodies' CDR prediction program. [Table 10-1] [Table 10-2] [Table 11-1] [Table 11-2]

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] Further provided herein are isolated polynucleotides encoding the above-described polypeptides, vectors and host cells containing same, and methods for the recombinant production of the polypeptides using recombinant cell systems known in the art and described herein.

[0281] In another embodiment of the present technology, the isolated antibody comprises one or more of the following characteristics: (a) the light chain immunoglobulin variable domain sequence comprises one or more CDRs that are 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 that are 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 that binds to any of the disclosed sequences.

[0282] 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0283] 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 mIhfB4 NTHI 12E6.F8.D12.D5.

[0284] Non-limiting exemplary antibodies include those disclosed herein, including but not limited to: (a) [ka] (b) [ka] (c) [ka] (d) [ka] (e) [ka] (f) [ka] (g) [ka] (h) [ka] (i) a polypeptide comprising the amino acid sequence NPXT, or (j) The equivalent of (a) to (i). wherein equivalents include those generated for the disclosed polypeptide fragments comprising 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 to a polynucleotide encoding the amino acid sequence or its complement under high stringency conditions, wherein high stringency conditions include an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1x SSC to about 0.1x SSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1x SSC, 0.1x SSC, or deionized water.

[0285] The present disclosure provides antibody fragments (e.g., Fab fragments or antigen-binding fragments). The present disclosure also provides isolated polypeptides comprising, alternatively consisting essentially of, or even consisting of the amino acid sequence of a Fab fragment or antigen-binding fragment (e.g., Fab fragment), wherein the fragment or polypeptide binds to and / or specifically recognizes a DNABII polypeptide and / or biofilm component, including a DNABII protein or polypeptide.

[0286] The present disclosure provides an isolated antibody comprising a heavy chain (HC) variable domain sequence and a light chain (LC) variable domain sequence, wherein the heavy and light chain immunoglobulin variable domain sequences form an antigen-binding site that binds to an epitope of a DNABII protein. In certain embodiments, the antibody or fragment thereof binds to a DNABII peptide (including, but not limited to, the tip region of IHF or HU, the tip region of IHFA or IHFB, and / or the tip chimeric peptide IhfA5-mIhfB4). NTHI and / or the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail chimeric peptide IhfA3-IhfB2 NTHI In one embodiment, the antibody or fragment thereof binds to the chimeric peptide IhfA5-mIhfB4 NTHI In another embodiment, the antibody or fragment thereof binds to the tail chimeric peptide IhfA3-IhfB2 NTHI Combine with.

[0287] In one aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of amino acids (aa) 25 to aa 144 of SEQ ID NO: 1-6, 13, 24 or 26, or their respective equivalents; and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa 21 to aa 132 of SEQ ID NO: 7-9, 14 or 25, aa 21 to aa 126 of SEQ ID NO: 10-12 or 27, or their respective equivalents.

[0288] In a further aspect, antibodies and antigen-binding fragments thereof are provided that comprise, consist essentially of, or alternatively consist of a heavy chain (HC) comprising, consisting essentially of, or consisting of a sequence selected from the group of aa25 to aa473 of SEQ ID NO: 1 to 6, 13, 24 or 26, or their respective equivalents; and / or a light chain (LC) comprising, consisting essentially of, or consisting of a sequence selected from the group of aa21 to aa239 of SEQ ID NO: 7 to 9, 14 or 25, aa21 to aa233 of SEQ ID NO: 10 to 12 or 27, or their respective equivalents.

[0289] In a still further aspect, there is provided antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of a heavy chain (HC) comprising, consisting essentially of, or consisting of a sequence selected from the group of SEQ ID NOs: 1 to 6, 13, 24 or 26, or their respective equivalents; and / or a light chain (LC) comprising, consisting essentially of, or consisting of a sequence selected from the group of SEQ ID NOs: 7 to 12, 14, 25 or 27, or their respective equivalents.

[0290] In another aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of amino acids (aa) 25 to 144 of SEQ ID NO: 1 to 6, 13, 24 or 26, or an equivalent thereof; and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa 21 to aa 132 of SEQ ID NO: 6 to 9, 14 or 25, aa 21 to aa 126 of SEQ ID NO: 10 to 12 or 27, or their respective equivalents.

[0291] In yet another aspect, there is provided an antibody or fragment thereof comprising, consisting essentially of, or alternatively consisting of, any one or any two or all three CDRs of a sequence selected from the group of SEQ ID NOs: 1 to 6, 13, 24 or 26, or their respective equivalents; and / or any one or any two or all three CDRs of a sequence selected from the group of SEQ ID NOs: 7 to 12, 14, 25 or 27, or their respective equivalents.

[0292] In another aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa25 to aa144 of SEQ ID NO: 13, 24 or 26, or an equivalent thereof; and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa21 to aa132 of SEQ ID NO: 14 or 25, aa21 to aa126 of SEQ ID NO: 27, or an equivalent thereof. In a further aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa25 to aa144 of SEQ ID NO: 1-6, 13, 24 or 26, or their respective equivalents; and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa21 to aa132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10-12 or 27, or their respective equivalents.

[0293] Also provided are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7 to 9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10 to 12 or 27, or their respective equivalents. In another aspect, provided herein is an antibody or fragment thereof comprising, alternatively consisting essentially of, or consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10-12 or 27, or their respective equivalents. In a further aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10-12 or 27, or their respective equivalents.

[0294] In a still further aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10-12 or 27, or their respective equivalents. Also provided are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7 to 9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10 to 12 or 27, or their respective equivalents. Still further provided are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7 to 9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10 to 12 or 27, or their respective equivalents.

[0295] In another aspect, provided herein is an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or their respective equivalents, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 7, or their equivalents. Still further provided is an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or alternatively consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1 to 6, 13, 24 or 26, or their respective equivalents, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 8, or their equivalents. In another aspect, there is also provided an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or even further consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1 to 6, 13, 24 or 26, or their respective equivalents, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof.

[0296] In a further aspect, there is also provided an antibody or antigen-binding fragment thereof comprising, alternatively consisting essentially of, or alternatively consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or alternatively consisting of, the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1 to 6, 13, 24 or 26, or their respective equivalents, and a light chain (LC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. Also provided are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or alternatively consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1 to 6, 13, 24 or 26, or their respective equivalents, and a light chain (LC) immunoglobulin variable domain comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 11, or its equivalent. Also provided are antibodies and antigen-binding fragments thereof comprising, consisting essentially of, or even consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of, the amino acid sequence of aa25 to aa144 of any one of SEQ ID NOs: 1 to 6, 13, 24 or 26, or their respective equivalents, and a light chain (LC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa21 to aa126 of SEQ ID NO: 12, or its equivalent.

[0297] In one aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, alternatively consisting essentially of, or further consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or further consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or further consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In one embodiment, provided are antibodies and antigen-binding fragments thereof comprising, alternatively consisting essentially of, or further consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or further consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or further consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In another embodiment, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment is an anti-chip chimer (i.e., it specifically binds to and specifically recognizes a chip chimer).

[0298] In another aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, alternatively essentially consisting of, or consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In another aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, alternatively consisting essentially of, or consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, alternatively consisting essentially of, or even further consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In yet another aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO:2, or an equivalent, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO:9, or an equivalent. In some embodiments, the antibody or antigen-binding fragment is an anti-chip chimer (i.e., it specifically binds to and specifically recognizes a chip chimer).

[0299] In another aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even further consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In a further aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even further consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In one embodiment, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, or consisting essentially of, or consisting of an amino acid sequence from aa25 to aa144 of SEQ ID NO: 3, or an equivalent, and a light chain (LC) immunoglobulin variable domain sequence comprising an amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent. In some embodiments, the antibody or antigen-binding fragment is an anti-chip chimer (i.e., it specifically binds to and specifically recognizes a chip chimer).

[0300] Also provided are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In another aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In a further aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 12, or an equivalent. In some embodiments, the antibody or antigen-binding fragment is anti-tail chimer (i.e., specifically binds to and specifically recognizes a tail chimer).

[0301] In one embodiment, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In a further aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, the amino acid sequence of aa21 to aa126 of SEQ ID NO: 12, or an equivalent. In some embodiments, the antibody or antigen-binding fragment is anti-tail chimer (i.e., specifically binds to and specifically recognizes a tail chimer).

[0302] In another embodiment, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In one aspect, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In one embodiment, provided herein are antibodies and antigen-binding fragments thereof comprising, or alternatively consisting essentially of, or consisting of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 12, or an equivalent. In some embodiments, the antibody or antigen-binding fragment is anti-tail chimer (i.e., specifically binds to and specifically recognizes tail chimer).

[0303] In one aspect, an antibody or fragment thereof is provided that comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 24, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 25, or an equivalent thereof. In a further embodiment, the antibody or fragment thereof is directed against the tip region of a DNABII peptide (including but not limited to, the tip region of IHF or HU, the tip region of IHFA or IHFB, and / or the tip chimeric peptide IhfA5-mIhfB4 NTHI In one embodiment, the antibody or fragment thereof binds to the chimeric peptide IhfA5-mIhfB4 NTHIIn still further embodiments, the fragment is an antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is an anti-chimer (i.e., specifically binds to and specifically recognizes a chimer). In one embodiment, the antibody or fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa 25 to aa 144 of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa 21 to aa 132 of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In yet another embodiment, the antibody or fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof.In one embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In yet another embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, the antibody or fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof.In another embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In yet another embodiment, the antibody or fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof.

[0304] In another aspect, an antibody or fragment thereof is provided that comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 26, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 27, or an equivalent thereof. In a further embodiment, the antibody or fragment thereof is an antibody or fragment thereof that comprises the tail region of a DNABII peptide (including but not limited to, the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail chimeric peptide IhfA3-IhfB2 NTHI In one embodiment, the antibody or fragment thereof binds to the tail chimeric peptide IhfA3-IhfB2 NTHIIn still further embodiments, the fragment is an antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is anti-tail chimer (i.e., specifically binds to and specifically recognizes tail chimer). In one embodiment, the antibody or fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa 25 to aa 144 of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa 21 to aa 126 of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In yet another embodiment, the antibody or fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 12, or an equivalent thereof.In one embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In yet another embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 12, or an equivalent thereof. In one embodiment, the antibody or fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof.In another embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In yet another embodiment, the antibody or fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essen...

Claims

[Claim 1] The invention described in the specification.

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