Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0208—Specific bacteria not otherwise provided for
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
  • A61K31/429—Thiazoles condensed with heterocyclic ring systems
  • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
  • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
  • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/65—Tetracyclines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• Cystic Fibrosis is the most common inherited lethal disorder affecting
• cenocepacia is intrinsically resistant to polymyxins, aminoglycosides and most ⁇ -lactams, and can develop resistance to essentially all classes of antimicrobial drugs (Aaron et al. (2000) Am. J. Respir. Crit. Care Med. 161 : 1206-1212; Golini et al. (2006) Eur. J. Clin.
• B. cenocepacia gained notoriety as a pathogen in CF because it is difficult to identify and treat, and also due to its ability to readily spread between individuals with CF. Even outside CF, highly transmissible epidemic strains of B. cenocepacia have been shown to contaminate healthcare settings and spread nosocomially (Graindorge et al. (2010) Diagn. Microbiol. Infect. Dis. 66:29-40). It is also likely that multi-drug resistant B. cenocepacia can transfer mechanisms of resistance to other microbes present within the human airway, and particularly those co-colonizing the CF lung. This assumption is supported by the observation that 25-45% of adult CF patients are chronically infected with multiple multi-drug resistant bacteria (Lechtzin et al. (2006) Respiration 73:27-33).
• CF airway disease The pathogenesis of CF airway disease is multifactorial and includes defective antimicrobial activity in airway secretions, altered mucociliary clearance, abnormal submucosal gland function and overproduction of reactive oxygen species (ROS).
• ROS reactive oxygen species
• Chronic inflammation is most central to CF pathogenesis as a consequence of pulmonary infections and leads to lung damage resulting in 85% of deaths.
• Further contributing to enhanced lung pathology observed in CF patients is an overabundance of cytokine-secreting alveolar macrophages.
• Pro-inflammatory mediators such as IL- ⁇ , IL-8, TNF-a and anti-inflammatory IL-10 are detected in CF patients, even in young patients in the absence of culture-positive infection.
• CF macrophages are defective in this regard.
• This inherent weak autophagy (Luciani et al. (2010) Nat. Cell. Biol. 12:863-875; Luciani et al. (2011) Autophagy 7:104-106; Luciani et al. (2012) Autophagy 8) is further augmented by the ability of B. cenocepacia to downregulate essential autophagy molecules (Abdulrahman et al. (2011) Autophagy 7: 1359-1370).
• the inventors have identified a protein target for the development of novel approach for the treatment of CF infections.
• This protein target a member of the DNABII family of DNA binding proteins, is essential for biofilm formation and stability by multiple human pathogens (Goodman et al. (2011) Mucosal Immunol. 4:625-637; Justice et al. (2012) PLoS One 7:e48349; Gustave et al. (2012) J. Cyst. Fibros.) due to its contribution to the structural lattice of extracellular DNA (eDNA) within these bacterial communities.
• the DNABII family is a member of a class of proteins referred to as nucleoid associated proteins (NAPs), bacterial proteins that, in part, shape the intracellular bacterial nucleoid (Browning et al. (2010) Curr. Opin. Microbiol. 13:773-780). In addition, this family is ubiquitous, expressed by virtually all eubacteria. All characterized family members to date function as either a homodimer or heterodimer of subunits. The family is divided into two types, HU (histone- like protein) and IHF (integration host factor) with B.
• HU histone- like protein
• IHF integration host factor
• cenocepacia capable of expressing both (strain J2315 genes: BCAL3530, hupA; BCAL1585, hupB; BCAL1487, ihfA and BCAL2949, ihfb).
• strain J2315 genes BCAL3530, hupA; BCAL1585, hupB; BCAL1487, ihfA and BCAL2949, ihfb.
• the primary distinction between these family members is that HU binds DNA in a sequence independent manner, while IHF binds a consensus sequence
• cenocepacia induces peripheral damage by exacerbating IL- ⁇ production through the host receptor Pyrin by an as yet unknown mechanism (Grajin et al. (2012) Immunol. 188:3469-3477; Kotrange et al. (2011) J.
• DNABII proteins bind laminin and are thought to elicit direct contact to host cells (Winters et al. (1993) Infect. Immun. 61 :3259-3264).
• DNABII proteins are known to stabilize the structural integrity of eDNA within the extra-cellular polymeric matrix or substance (or EPS) of the biofilm of multiple pathogens (Goodman et al. (2011) Mucosal Immunol. 4:625-637).
• the inventors investigated whether there existed a role for the DNABII family of proteins in stabilization of these biofilms as well and thus, whether this family of proteins serve as a target for therapeutic intervention. Moreover, the presence of extracellular DNABII proteins, perhaps in association with bacterial cells, might influence the interaction of B. cenocepacia with host cells and particularly with its primary reservoir, the macrophage.
• the inventors herein disclose an immuno- therapeutic approach that targets the DNABII proteins, and particularly when coupled with existing conventional therapeutics, was found to be highly effective at both debulking biofilms formed by B. cenocepacia as well as rendering the bacteria now released from the biofilm EPS susceptible to the action of antibiotics.
• the methods can be used to diminish and eradicate reservoirs of B. cenocepacia from the lungs of CF patients.
• this method is shown to prevent recurrence of CF disease by inhibiting the ability of B. cenocepacia to multiply within macrophages isolated from CF mice, an important animal model of CF in humans.
• a method for inhibiting, competing or titrating a biofilm present or contributing to CF comprising, or alternatively consisting essentially of, or yet further consisting of contacting the biofilm with an interfering agent, thereby inhibiting, competing or titrating the biofilm.
• the contacting can be performed in vitro or in vivo.
• a method for inhibiting, preventing or titrating bacterial cells in a biofilm in a CF patient or a patient harboring an infection contributing to CF comprising, or alternatively consisting essentially of, or yet further consisting of, contacting the bacterial cells with an interfering agent, thereby inhibiting, preventing or titrating the bacterial cells and infection.
• the contacting can be performed in vitro or in vivo.
• the interfering agents can be combined with antimicrobials to further supplement the therapy, e.g., ceftazidime, ciprofloxacin, imipenem and minocycline.
• any of the above methods can further comprise, or consist essentially of, or yet further consist of administration or contacting with an effective amount of the antimicrobial, e.g., ceftazidime, ciprofloxacin, imipenem and minocycline.
• the administration or contacting of the interfering agent is performed in the absence of a DNase treatment.
• the DNAse treatment that is excluded from the therapy comprises an enzyme that catalyzes the cleavage of phosphodiester linkages in the DNA backbone.
• the DNase treatment that is excluded from the therapy comprises, or consists essentially of, or yet further consists of, Pulmozyme® (dornase alpha; Genentech, Inc.).
• any agent that occludes critical surfaces that are required for intercellular persistence, interferes or impedes the binding of the microbial DNA to the DNABII protein or polypeptide, is intended within the scope of this invention.
• interfering agents include:
• IHF integration host factor
• an antibody or antigen binding fragment that specifically recognizes or binds any one of (a) through (e), or an equivalent or fragment of each thereof; (i) an isolated or recombinant polynucleotide encoding the antibody or antigen binding fragment of (h); and j) a small molecule that competes with the binding of a DNA BII protein or polypeptide to a microbial DNA.
• an isolated or recombinant polypeptide consisting essentially of an amino acid sequence selected from SEQ ID NO. 1 to 5 or 12 to 27 or 30 to 33, or a DNA binding peptide identified in Figure 9.
• the methods are performed with an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, SEQ ID NO. 1 or 2, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11 , 28 or 29.
• the methods are performed with an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, SEQ ID NO. 3, 4 or 5, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28 or 29.
• the methods are performed with an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, SEQ ID NO. 12, 14, 16, 18, 20, 22, 24, 26, 30 or 32, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28 or 29.
• the methods are performed with an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of SEQ ID NO. 13, 15, 17, 19, 21, 23, 25, 27, 31 or 33, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28 or 29.
• the methods are performed with an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, SEQ ID NO. 12 and 13 or 14 and 15 or 16 and 17 or 18 and 19 or 20 and 21 or 22 and 23 or 24 and 25, or 26 and 27 or 30 and 31 or 32 and 33, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28 or 29.
• the methods are performed with an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, the c-terminal region or peptide of polypeptide of the group of a DNA BII polypeptide, an IHF polypeptide, SEQ ID NO. 6 through 11, 28, 29 or those identified in Table 1, or a fragment or an equivalent of each thereof.
• agents that can be used in the methods of this invention include:
• IHF integration host factor
• (k) a small molecule that competes with or occludes the binding of a DNABII protein or polypeptide to a microbial DNA.
• Also provided herein is a method for inducing an immune response in or conferring passive immunity in a CF subject in need thereof, comprising, or alternatively consisting essentially of or yet further consisting of, administering to the CF subject an effective amount of one or more agents of the group:
• IHF integration host factor
• Subjects in need of such immune response or support include those at risk of or suffering from an infection that produces a microbial biofilm.
• polypeptides and antibodies are also provided herein.
• antigen binding fragments are also provided herein.
• an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, an amino acid sequence selected from SEQ ID NO. 1 to 5 or 12 to 27, 30 to 35, 101-348 or a DNA binding peptide identified in FIG. 9.
• an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of SEQ ID NO. 1 or 2, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of SEQ ID NO. 3, 4 or 5, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, SEQ ID NO. 12, 14, 16, 18, 20, 22, 24, 26, 30 or 32, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of SEQ ID NO. 13, 15, 17, 19, 21, 23, 25, 27, 31 or 33, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of SEQ ID NO. 337, 338, 339, or 340, with the proviso that the polypeptide is none of SEQ ID NO, 6 to 11, 28, 29, or 42 through 100.
• an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, SEQ ID NO. 12 and 13 or 14 and 15 or 16 and 17 or 18 and 19 or 20 and 21 or 22 and 23 or 24 and 25, or 26 and 27 or 30 and 31 or 32 and 33, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, the C-terminal region containing at least 10, or alternatively at least 15, or alternatively at least 20, or alternatively at least 25, or alternatively at least 30, C-terminal amino acids of a polypeptide of the group of a DNABII polypeptide, an IHF polypeptide, an HU polypeptide, SEQ ID NO. 6 through 11, 28, 29 or those identified in Table 1, Table 2, the Arm fragment identified in Table 2, Table 4 or a fragment or an equivalent of each thereof, or a polypeptide of SEQ ID NOs.: 337 to 340, or an equivalent thereof.
• polypeptide comprising SEQ ID NO. 339 and 340; a polynucleotide or polypeptide comprising SEQ ID NOS. 341 to 348; with the proviso that the polypeptide is none of wild-type of any one of IHF alpha, IHF beta or SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• polypeptide consisting essential y of SEQ ID NO. 34 and 35 polypeptide consisting essential y of SEQ ID NO. 337 and 338; or polypeptide consisting essential y of SEQ ID NO. 339 and 340; a polynucleotide or polypeptide consisting essentially of any one of SEQ ID NOS . 341 to 348; with the proviso that the polypeptide is none of wild-type of any one of IHF alpha, IHF beta or SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• isolated or recombinant polypeptides comprising, or alternatively consisting essentially of or yet further consisting of, two or more, or three or more or four or more, or multiples of the above-identified isolated polypeptides, including fragments and equivalents thereof.
• isolated polypeptides comprising SEQ ID NO. 1 through 4 and/or 12 through 29, and/or 30 through 33, and/or 30 through 35 e.g., SEQ ID NO. 1 and 2, or alternatively 1 and 3 or alternatively 1 and 4, or alternatively 2 and 3, or alternatively SEQ ID NO.
• polypeptides can be in any orientation, e.g., SEQ ID NO. 1, 2, and 3 or SEQ ID NO. 3, 2 and 1 or alternatively SEQ ID NO. 2, 1 and 3, or alternatively, 3, 1 and 2, or alternatively 11 and 12, or alternatively 1 and 12, or alternatively 2 and 12, or alternatively, 1 and 12, or alternatively 2 and 13, or alternatively 12, 16 and 1, or alternatively 1, 16 and 12.
• this invention provides an isolated or recombinant polypeptide comprising SEQ ID NO. 1 or 2 and 3 or 4 or a polypeptide or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid corresponding to fragments of a DNABII protein such as the .beta.-3 and/or a-3 fragments of a Haemophilus influenzae IHF-a or IHF.beta. microorganism, non-limiting examples of which include SEQ ID NO. 12 through 27, or a fragment or an equivalent of each of the polypeptides, examples of which are shown in Tables 2 and 3.
• isolated wildtype polypeptides are specifically excluded, e.g.
• polypeptide is none of SEQ ID NO. 6 through 11 or a wildtype sequence identified in Table 1.
• SEQ ID NO. 1 or 2 or a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid corresponding to the .beta.-3 and/or a-3 fragments of an IHF. alpha, or IHF.beta. microorganism, non-limiting examples of which include SEQ ID NO. 12 through 27 and 30 through 33 or an equivalent of each thereof is located upstream or amino terminus from SEQ ID NO. 3 or 4 or a fragment or an equivalent thereof.
• the isolated polypeptide comprises SEQ ID NO.
• polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid corresponding to the .beta. -3 and/or .alpha.-3 fragments of an IHF. alpha, or IHF.beta.
• microorganism non-limiting examples of which include SEQ ID NO. 12 through 27, or an equivalent thereof located upstream or amino terminus to SEQ ID. NO. 1 or 2 or an equivalent thereof.
• a peptide linker can be added to the N-terminus or C-terminus of the polypeptide, fragment or equivalent thereof.
• the linker joins the polypeptides of this invention, e.g., SEQ ID NO. 1 to 4, 28, 29, 34, or 35 or 30 to 33, 34, or 35 or a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid corresponding to the .beta.-3 and/or .alpha.-3 fragments of a Haemophilus influenza IHF. alpha, or IHF.beta. microorganism, non- limiting examples of which include SEQ ID NO. 12 through 27 or an equivalent of each thereof.
• linker refers to a peptide sequence linked to either the N-terminus or the C-terminus of a polypeptide sequence.
• the linker is from about 1 to about 20 amino acid residues long or alternatively 2 to about 10, about 3 to about 5 amino acid residues long.
• An example of a peptide tinker is Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO: 37).
• polynucleotide that interferes with the binding of the microbial DNA with a polypeptide or fragment or equivalent thereof, e.g., SEQ ID 36, or a four- way junction polynucleotide resembling a Holliday junction, a 3 way junction polynucleotide resembling a replication fork, a polynucleotide that has inherent flexibility or bent polynucleotide; an isolated or recombinant polynucleotide encoding a polypeptide described above or an antibody or fragment thereof, which can be operatively linked to regulatory elements necessary for the expression and/or replication of the polynucleotide.
• the polynucleotide can be contained within a vector.
• an isolated host cell comprising, or alternatively consisting essentially of, or yet further consisting of an isolated or recombinant polypeptide described above, a four- way junction polynucleotide resembling a Holliday junction, a 3 way junction polynucleotide resembling a replication fork, a polynucleotide that has inherent flexibility or bent polynucleotide; an isolated or recombinant polynucleotide as described above, or a vector as described above.
• the methods are performed with an antibody or antigen binding fragment that specifically recognizes and binds the isolated or recombinant polypeptide as described above, including a fragment or an equivalent of the polypeptide.
• antibodies include a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, an antibody derivative, a veneered antibody, a diabody, a chimeric antibody, an antibody derivative, a recombinant human antibody, or an antibody fragment.
• the antibody is a monoclonal antibody.
• a hybridoma cell line that produces the monoclonal antibody.
• This invention also provides isolated or recombinant polynucleotides encoding one or more of the above -identified isolated or recombinant polypeptides or antibodies or a fragment thereof.
• Vectors comprising the isolated polynucleotides are further provided.
• the isolated polynucleotides can be contained within a polycistronic vector.
• Isolated host cells comprising one or more of an isolated or recombinant
• the isolated host cell is a eukaryotic cell such as antigen presenting cell, e.g. a dendritic cell.
• the antibodies, polynucleotides, polypeptides, vectors or host cells can further comprise a detectable label or a carrier, such as a pharmaceutically acceptable carrier.
• compositions comprising a carrier and one or more of an isolated or recombinant polypeptide of the invention, an isolated or recombinant polynucleotide of the invention, a vector of the invention, an isolated host cell of the invention, or an antibody of the invention are also provided.
• the carriers can be one or more of a solid support, a medical device like a stent or dental implant, or a liquid such as a pharmaceutically acceptable carrier.
• the compositions can further comprise an adjuvant, an antimicrobial or an antigenic peptide.
• compositions can further comprise additional biologically active agents.
• a antimicrobial agent such as other vaccine components (i.e., antigenic peptides) such as surface antigens, e.g. a Type IV Pilin protein (see Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875).
• This invention also provides a method for producing an antigenic peptide by growing or culturing a host cell comprising an isolated polynucleotide as described above under conditions that favor the expression of the polynucleotide.
• the polypeptide produced by this method can be isolating for further in vitro or in vivo use.
• a kit is also provided for diagnostic or therapeutic use comprising a composition as described above and instructions for use.
• a kit is also provided to perform screens for new drugs and/or combination therapies as provided herein.
• Al is V or I
• A2 is any one of K, Q, E, A, V or Y;
• A3 is any one of K, L, I, V or F;
• A4 is any one of S, I, R or V;
• A5 is any one of G or S;
• A6 is F;
• A7 is G;
• A8 is any one of N or S or T or K; and A9 is F.
• SEQ. ID NO. 2 is VK SGFGNF
• SEQ ID NO. 3 is B1-B2-B3-B4-B5-B5-B6-B7 wherein:
• Bl is absent or any one of G or K;
• B2 is absent or any one of R, I or K;
• B3 is N or V
• B4 is P or I
• SEQ. ID NO. 4 is NP(K/Q)TG SEQ. ID NO. 5 GRNP(K/Q)TG
• SEQ. ID NO. 6 Full Length Wild type (wt) 86-028NP Haemophilus influenzae IhfA;
• SEQ. ID NO. 8 Full Length wt R2846 Haemophilus influenzae IhfA, Genbank accession No.: AD096375, last accessed March 21, 2011 :
• SEQ ID NOS. 14 and 15 ⁇ -3 and a-3 portions of (IHFP)
• SEQ ID NOS. 16 and 17 ⁇ -3 and a-3 portions of SEQ ID NOS. 6, SEQ ID NOS. 16: TFKPGQ and SEQ ID NO. 17: KLRARVEKTK
• SEQ ID NOS. 18 and 19 ⁇ -3 and a-3 portions of 2019 Haemophilus influenzae IhfA, SEQ ID NO. 18: TFKPGQ and SEQ ID NO. 19: KLRARVENTK
• SEQ ID NOS. 20 and 21 ⁇ -3 and a-3 portions of SEQ ID NO. 8, SEQ ID NO. 20:
• SEQ ID NOS. 22 and 23 ⁇ -3 and a-3 portions of SEQ ID NO. 9, SEQ ID NO. 22:
• SEQ ID NOS. 24 and 25 ⁇ -3 and a-3 portions of SEQ ID NO. 10, SEQ ID NO. 24:
• TFRPGQ and SEQ ID NO. 25 KLKSRVENASPKDE
• SEQ ID NOS. 26 and 27 ⁇ -3 and a-3 portions of SEQ ID NO. 11, SEQ ID NO. 26:
• TFRPGQ and SEQ ID NO. 27 KLKARVEAYAGTKS
• SEQ ID NO. 28 E. coli hupA, Genbank accession No.: AP 003818, Last accessed March 21, 2011 : MNKTQLIDVIAEKAELSKTQAKAALESTLAAITESLKEGDAVQLVGFGTFK VNHRAERTGRNPQTGKEIKIAAANVPAFVSGKALKDAVK
• SEQ ID NO. 29 E. coli hupB, Genbank accession No.: AP 001090.1, Last accessed March 21, 2011 : MNKSQLIDKIAAGADISKAAAGRALDAIIASVTESLKEGDDVALVGFG TFAVKERAARTGRNPQTGKEITIAAAKVPSFRAGKALKDAVN
• SEQ ID NOS. 30 and 31 ⁇ -3 and a-3 portions of SEQ ID NO. 28, SEQ ID NO. 30:
• SEQ ID NOS. 32 and 33 ⁇ -3 and a-3 portions of SEQ ID NO. 29, SEQ ID NO. 32:
• SEQ. ID NO. 34 C-terminal 20 amino acids of IHF a: TFRPGQKLKSRVENASPKDE SEQ. ID NO. 35: C-terminal 20 amino acids of IHF ⁇ : KYVPHFKPGKELRDRANIYG SEQ. ID NO. 36: DNABII binding consensus sequence: WATCAANNNNTTR wherein W is A or T, N is any base and R is a purine
• E. coli IHFalpha GRNPKTGEDIPI
• SEQ. ID NO. 339 E. coli HUalpha: GRNPQTGKEIKI
• SEQ. ID NO. 340 E. coli HUbeta: GRNPQTGKEITI
• Table 1 is a non- limited summary of DNA binding proteins produced by gram(+) and gram (-) bacteria that can be used in the methods provided herein.
• Table 2 is a sequence alignment of relevant portions of the DNA binding proteins of various embodiments of this invention. Bold letters indicate an exact match to consensus, light gray lettering indicates a conservative amino acid change, and lightly or darkly shaded sequences are highly conserved across species. Gray shaded undefined sequences at the amino and/or carboxy-terminal are undefined amino acids that do not share consenus sequences.. Table 2 is based on information previously published in Obeto et al. (1994) Biochimie 76:901-908. The fragment "ARM" is denoted at the bottom of the table and polypeptide fragment comprising, or consisting essentially of, or yet further consisting of these fragments or their equivalents have biological activity as noted herein.
• Table 3 is a comparison of the 16 amino acid peptide motif to Liu et al. (2008) Cell Microbiol. 10(l):262-276.
• Table 4 is a listing of ⁇ , ⁇ , and C-terminal portions of DNABII proteins from the indicated organism.
• FIGS. 1A-1C show presence of both eDNA and IHF within a bio film formed by B. cenocepacia.
• FIG. 1A Unfixed twenty- four hr bio film formed by B. cenocepacia stained with FilmTracer FM 1-43.
• FIG. IB unfixed 24 hr bio film formed by B. cenocepacia and immunolabeled with monoclonal antiserum directed against dsDNA (white color in image).
• FIG. 1C unfixed 24 hr. bio film formed by B. cenocepacia labeled with both antiserum directed against dsDNA and rabbit anti-IHF serum to show the presence of IHF as well as dsDNA (white color) within the biofilm.
• FIG. 1 A Note robust biofilm formed by B. cenocepacia in FIG. 1 A.
• the presence of eDNA that is particularly dense at the bottom of the biofilm can be seen in FIG. IB.
• the 24-hr biofilm formed by B. cenocepacia is also rich in IHF as shown by the abundance in FIG. 1C. All images captured with a 63X objective.
• FIG. 2 shows labeling of IHF and eDNA within sputum collected from a CF patient culture-positive for B. cenocepacia.
• Immunolabeled light micrograph demonstrates heavy labeling of both eDNA (white strands) and the DNABII protein IHF (punctate labeling; see arrows) within a sputum sample recovered from a CF patient infected with B. cenocepacia.
• eDNA white strands
• DNABII protein IHF punctate labeling; see arrows
• FIGS. 3A-3H show disruption of pre-formed B. cenocepacia biofilms by incubation with antiserum directed against IHF.
• B. cenocepacia biofilms after 24 hr. growth in a chamber slide, then treated for 16 hrs with: FIG. 3 A- sterile medium.
• FIG. 3B - naive rabbit serum.
• FIG. 3C rabbit antiserum directed against isolated IHF.
• FIG. 3D -IgG enriched anti- IHF.
• FIG. 3E -serum effluent from enrichment column.
• FIG. 3F Western blot showing recognition of antibody directed against IHF as well as IgG-enriched anti-IHF to purified IHF and IHF within B.
• FIG. 3G plot of changes in average biofilm thickness ⁇ SEM following each of the indicated treatments.
• FIG. 3H plot of changes in biofilm biomass ⁇ SEM following each of the indicated treatments. Note that statistically significant disruption of pre-formed B.
• cenocepacia biofilms is mediated only by rabbit anti-IHF serum as well as a fraction of that serum that was enriched for IgG when compared to treatment with sterile medium, naive rabbit serum or serum effluent from IgG enrichment column.
• Asterisks indicate statistical significance (p ⁇ 0.05) compared to sterile medium, naive serum and enrichment column effluent.
• FIGS. 4A-4N show demonstration of the synergistic behavior of antibodies directed against IHF in combination with antimicrobials or antibiotics.
• FIGS. 4A-4C An untreated B. cenocepacia biofilm.
• FIGS. 4D-4F- a B. cenocepacia biofilm after treatment with a 1 :50 dilution of anti-IHF.
• FIG. 4M Average thickness of bio films after treatment with antibiotic or antibiotic plus anti-IHF ⁇ SEM.
• FIG. 4N Biofilm biomass following incubation with antibiotic or antibiotic plus anti-IHF ⁇ SEM. Asterisks indicate statistical significance between designated pairs (p ⁇ 0.05). Note significant reduction in both average biofilm thickness and biomass as mediated by a combination of anti-IHF serum plus ceftazidime, ciprofloxacin, imipenem and minocycline.
• FIGS. 5A-5E show induction of a more robust B. cenocepacia biofilm following exposure to Pulmozyme (DNase).
• FIG. 5A Treatment of a 24 hr B. cenocepacia biofilm with saline diluent alone.
• FIG. 5B Treatment of a 24 hr. B. cenocepacia biofilm with Pulmozyme (DNase) induced the formation of a markedly denser and thicker biofilm than that treated with saline diluent alone (compare panels A and B).
• FIG. 5C treatment of a 24 hr B. cenocepacia biofilm with both Pulmozyme and anti-IHF.
• Bio films were stained for viability and pseudocolored white (live cells) and dark (dead cells) and demonstrate minimal bacterial death upon any treatment.
• Mean relative biofilm thickness ⁇ SD and biomass ⁇ SEM are depicted graphically in FIGS. 5D & 5E.
• Asterisk indicates significantly thicker biofilm after exposure to Pulmozyme compared to treatment with saline diluent (p ⁇ 0.05).
• FIG. 6 shows pre-treatment of B. cenocepacia with anti-IHF induced significantly reduced survival in murine CF macrophages. Significantly fewer B. cenocepacia were detected 6 hrs after pre- treatment of the bacteria with anti-IHF compared to naive serum (* p ⁇ 0.05). Bacterial CFU/ml for each time point ⁇ SD are also shown.
• FIGS. 7A-7E show expression of a robust B. cenocepacia biofilm that incorporates IHF was dependent upon an active T6SS.
• FIG. 7A Biofilm formed by the parental isolate (B. cenocepacia strain K56-2).
• FIG. 7B biofilm formed by the type III secretion system mutant (strain JRL2) stained with propidium iodide.
• FIG. 7C biofilm formed by the type VI secretion system mutant (strain DFA2) stained with propidium iodide. All biofilms were labeled for the presence of a DNABII protein (IHF).
• IHF DNABII protein
• FIG. 7D IHF bound DNase footprint of the intergenic space (386 bp) between BCAL0339 and BCAL0340, part of the T6SS gene cluster for B. cenocepacia.
• the IHF footprint covers the region from 25 bp to 52 bp upstream of the BACL0340 start codon while a putative promoter was found 75 bp to 104 bp upstream of the start codon (FIG. 7E).
• HSS hyper-sensitive site indicative of DNA bending. This observation suggested that IHF might self-regulate its own release as well as perhaps that of eDNA that is incorporated into the biofilm matrix.
• FIGS. 8A-8F show demonstration of relative eDNA content of biofilms formed in chamber slides by either the parental isolate K56-2, its T3SS mutant or its T6SS mutant.
• Biofilms formed by either the parental B. cenocepacia strain or its T3SS and T6SS mutants followed by staining with FilmTracer FM 1-43 can be seen in FIGS. 8 A, 8C & 8E, respectively.
• Relative eDNA content of each unfixed biofilm can be ascertained via immunolabeling of each biofilm with a monoclonal antibody directed against dsDNA as in FIGS. 8B, 8D & 8F.
• FIG. 9A is a map indicating the amino acid residues of IHF that interact with or bind to another IHF in an IHF-IHF dimmer (indicated by triangles at the upper level) or interact with or bind DNA (indicated by triangles at the lower level).
• the peptide is divided by the short vertical bars into regions containing 3 amino acids.
• FIG. 9B graphically depicts the interaction of microbial DNA with an IHF.
• FIGS. 10A-10B show disruption of NTHI biofilms by anti-IHF £ coU .
• NTHI biofilms Representative images of NTHI biofilms and (B) calculated mean biomass.
• a medium; b, naive serum; c, anti-IHF £ cofi .
• Anti-IHF £ cofi significantly disrupted NTHI biofilms established for 16 hr to 2 weeks, compared to naive serum or medium.
• Data are expressed as the mean + SEM of three independent assays. */? ⁇ 0.05; **/? ⁇ 0.01 compared to respective naive serum treatment, one way ANOVA.
• FIGS. 11A-11C shows kinetics of biofilm disruption by anti-IHF £ co3 ⁇ 4 .
• FIGS. 12A-12F show that direct contact between anti-IHF ⁇ . co# and biofilm was not required to mediate disruption.
• A Representative images of bio films treated basolaterally with medium or serum.
• B-C Biofilms treated apically by placement of antibody coupled to agarose beads into a transwell,
• D NTHI biofilms after naked beads were layered under antibody-coupled beads apically,
• E biofilms after mixing of naked and antibody-coupled beads.
• F Biomass values after incubation with: a, medium; b, naive serum; c, anti-IHF ⁇ .
• co3 ⁇ 4 d, coupled IgG-enriched naive serum; e, coupled IgG-enriched anti-IHF £ co3 ⁇ 4 ; f, naked beads layered under coupled IgG-enriched naive serum; g, naked beads layered under coupled IgG- enriched anti-IHF £ co 3 ⁇ 4; h, mixed naked and coupled IgG-enriched naive serum; i, mixed naked and coupled IgG-enriched anti-IHF ⁇ . co #. Data are expressed as the mean + SEM of three independent assays. */? ⁇ 0.05 compared to respective naive serum or IgG-enriched naive serum conjugated to agarose beads treatment, one way ANOVA.
• FIGS. 13A-13B show adsorption of anti-IHF-specific antibody.
• A Representative images of biofilms incubated with IHF-adsorbed serum.
• B Changes in biofilm biomass and mean thickness, a, naive serum; b, anti-IHF ⁇ . co u .
• Adsorption of IHF- specific antibody counteracted biofilm disruption. Data are expressed as the mean + SEM of three independent assays. */? ⁇ 0.05 compared to naive serum, one way ANOVA.
• FIGS. 14A-14E show that anti-IHF ⁇ . co u acted synergistically with antibiotics.
• A- D Representative images biofilms treated with medium, antibiotic at the MIC 90 or antiserum.
• E Biomass and mean thickness of treated biofilms. a, medium; b, naive serum; c, anti-IHF ⁇ . cou- Incubation of NTHI biofilms with anti-IHF ⁇ . cof iplus antibiotic markedly altered biofilm architecture, significantly reduced biofilm biomass and mean thickness and negatively impacted viability (note yellow color of biofilms). Data are expressed as the mean + SEM of three independent assays. Bars indicate /? ⁇ 0.05, one way ANOVA. [0070] FIGS.
• 15A-15F shows that bacteria newly released from the bio film by treatment with anti-IHFg. co # showed enhanced sensitivity to antibiotics.
• Biofilms were incubated with ampicillin (A&D), amoxicillin-clavulanate (B&E), or cefdinir (C&F) in the absence or presence of a 1 :50 dilution of anti-IHF ⁇ co3 ⁇ 4 or naive serum.
• A-C CFU NTHI adherent within the biofilms.
• D-F Sum of the planktonic and adherent NTHI. a, medium alone; b, naive serum; c, anti-IHF ⁇ . co# . Data are expressed as the mean + SEM of three independent assays. Bars indicate p ⁇ 0.05, one way ANOVA.
• FIGS. 16A-16F show epitope mapping IHF NTHI and design of a minimal IHF NTH r targeted peptide.
• 3-D model depicting reactivity of (A) chinchilla anti-IHF ⁇ . co// and (B) chinchilla anti-IHF ⁇ . co #- complexed to DNA to synthetic peptides representing IHF NTHI - Regions with reactivity are indicated in grey, nonreactive regions in black.
• C 3-D model of DNA bound to IHF to show occlusion of tip-binding regions.
• D Localization of IhfA-3 N THi (yellow) and IhfA-5 NTHI (green) within IHF model.
• FIG. 17 shows IgG-enriched anti-IHFE. coli bound to agarose beads and placed into the apical chamber of a transwell did not diffuse into the basolateral chamber as determined by Western blot against purified IHFE. coli.
• Medium from the basolateral chamber was collected 24 hr after treatment with: a, naive serum added basolaterally; b, anti-IHFE. coli added basolaterally; c, IgG-enriched naive serum tethered to agarose beads added apically; d, IgG-enriched anti-IHFE. coli tethered to agarose beads added apically.
• FIGS. 18A-18B show adsorption of anti-IHF antibodies.
• A Band intensity was reduced by incubation of anti-IHF ⁇ . co u with purified IHF £. co u shown by slot blot and
• B quantitated by densitometry, a, naive serum; b, anti-IHF £ co3 ⁇ 4 ; c, anti-IHF £ co3 ⁇ 4 adsorbed with 0 ⁇ g IHF £ cou; d, anti-IHF £ cofi adsorbed with 2.2 ⁇ g IHF £ co3 ⁇ 4 e, anti-IHF £ cofi adsorbed with 4.4 ⁇ g IHF £. co3 ⁇ 4 f, anti-IHF ⁇ . co u adsorbed with 4.4 ⁇ g rsPilA.
• FIGS. 19A-19C show treatment with anti-IHF ⁇ .
• co u did not increase the sensitivity of planktonic cultures of NTHI to antibiotics.
• A-C Broth cultures of NTHI were incubated with ampicillin, amoxicillin-clavulanate, or cefdinir in the absence or presence of anti-IHF ⁇ .
• Data represent mean + SEM of three independent assays. Bars indicate p ⁇ 0.05, one way ANOVA.
• a polypeptide includes a plurality of polypeptides, including mixtures thereof.
• compositions and methods include the recited elements, but do not exclude others.
• Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the intended use.
• a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
• Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
• a "biofilm” intends a thin layer of microorganisms that adhere to the surface of a structure, that may be organic or inorganic, together with the polymers such as DNA that they secrete. They are very resistant to microbiotics and antimicrobial agents. They live on gingival tissues, teeth and restorations, causing caries and periodontal disease, also known as periodontal plaque disease. They also cause chronic middle ear infections. Biofilms can also form on the surface 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) estimate that over 65% of nosocomial (hospital-acquired) infections are caused by biofilms.
• Biofilms also frequently contaminate medical devices. They cause chronic vaginal infections and lead to life-threatening systemic infections in people with hobbled immune systems. Biofilms also are involved in numerous diseases. For instance, cystic fibrosis patients have Pseudomonas and B. cenocepacia infections that often result in antibiotic resistant biofilms.
• the term "inhibiting, competing or titrating" intends a reduction or prevention in the formation of the DNA/protein matrix that is a component of a microbial biofilm.
• a "DNA BII polypeptide or protein” intends a DNA binding protein or polypeptide that is composed of DNA-binding domains and thus have a specific or general affinity for microbial DNA. In one aspect, they bind DNA in the minor grove.
• a non-limiting example of a DNA BII protein is an integration host factor (IHF) protein.
• IHF integration host factor
• Other DNA binding proteins that may be associated with the biofilm 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).
• An "integration host factor” protein is a bacterial protein that is used by
• bacteriophages to incorporate their DNA into the host bacteria. They also bind extracellular microbial DNA.
• HMGB1 is an high mobility group box (HMGB) 1 protein that is reported to bind to and distort the minor groove of DNA and is an example of an interfering agent.
• HU refers to a class of heterodimeric proteins typically associate with E. coli. HU proteins are known to bind 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 to the HU protein are commercially available from Abeam.
• HU or "histone-like protein from E.coli strain U93” refers to a class of heterodimeric proteins typically associate with E. coli. HU proteins are known to bind 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 to the HU protein are commercially available from Abeam. The genes that encode the HU protein subunits in E. coli are hupA and hupB corresponding to SEQ ID Nos: 28 and 29, respectively.
• Microbial DNA intends single or double stranded DNA from a microorganism that produces a biofilm.
• An "interfering agent” intends an agent that any one or more of competes, inhibits, prevents, titrates or occludes a DNA BII polypeptide such as IHF to a microbial DNA or also breaks down a microbial biofilm. It can be any one or more of a chemical or biological molecule.
• IHF can specifically bind, bend or distorted DNA structures such as DNA containing four- way junctions, cis-platinum adducts, DNA loop or base bulges.
• agents include (1) small molecules that inhibit the DNA-binding activity of IHF, (2) small molecules such as polyamines and spermine that compete with IHF in DNA binding, (3) polypeptides such as peptide fragments of IHF that compete with IHF in DNA binding, (4) antibodies or fragments thereof directed to IHF, or (5) a four- way or bent polynucleotides or other types of polynucleotides containing bent or distorted DNA structures that compete in IHF-binding.
• a "small molecule that inhibits the binding of an IHF to a nucleic acid” refers to (1) or (2) above and include those that bind DNA in the minor grove, i.e., minor groove binding molecules.
• a "four-way polynucleotide” intends a polynucleotide that contains a four-way junction, also known as the Holliday junction, between four strands of DNA.
• a "bent polynucleotide” intends a double strand polynucleotide that contains a small loop on one strand which does not pair with the other strand.
• the loop is from 1 base to about 20 bases long, or alternatively from 2 bases to about 15 bases long, or alternatively from about 3 bases to about 12 bases long, or alternatively from about 4 bases to about 10 bases long, or alternatively has about 4, 5, or 6, or 7, or 8, or 9, or 10 bases.
• Polypeptides that compete with IHF in DNA binding intend proteins or peptides that occlude or compete with IHF in binding bent or distorted DNA structures but do not form a biofilm with the DNA. Examples, without limitation, include fragments of IHF that include one or more DNA binding domains of the IHF, or the biological equivalents thereof. DNA binding domains are shown in Figure 9.
• a "subject" of diagnosis or treatment is a cell or an animal such as a mammal, or a human.
• Non-human animals subject to diagnosis or treatment and are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals, and pets.
• protein protein
• peptide and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
• the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
• a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence.
• amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
• a "c-terminal polypeptide” intends the c-terminal half of a polypeptide. As an example, for polypeptides containing 90 amino acids, the c-terminal polypeptide would comprise amino acids 46 through 90. In another aspect, the term intends the c-terminal 20 amino acids from the carboxy terminus.
• polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
• polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
• a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
• 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.
• a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
• the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
• a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
• A adenine
• C cytosine
• G guanine
• T thymine
• U uracil
• polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
• isolated or recombinant refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides.
• isolated or recombinant nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
• isolated is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and
• the term "isolated or recombinant" means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
• an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.
• An isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome.
• a non- naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.
• an equivalent intends at least about 60%, or alternatively at least about 65%, or alternatively at least about 70%, or alternatively at least about 75%, or alternatively at least about 80 %, or alternatively about least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or sequence identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
• biologically equivalent polypeptides are provided in Table 2, and the Arm fragments identified in Table 2, which identify conservative amino acid substitutions to the preferred amino acid sequences.
• a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
• the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al, eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
• default parameters are used for alignment.
• a preferred alignment program is BLAST, using default parameters.
• Homology or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention. [0101] "Homology” or “identity” or “similarity” 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 that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
• the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
• the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
• 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.
• Examples of stringent hybridization conditions include: incubation temperatures of about 25° C. to about 37° C; hybridization buffer concentrations of about 6 X SSC to about lO.times.SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4xSSC to about 8 X SSC.
• Examples of moderate hybridization conditions include:
• incubation temperatures of about 40° C. to about 50° C; buffer concentrations of about 9xSSC to about 2> ⁇ SSC; formamide concentrations of about 30%> to about 50%>; and wash solutions of about 5> ⁇ SSC to about 2> ⁇ SSC.
• high stringency conditions include: incubation temperatures of about 55° C. to about 68° C; buffer concentrations of about l xSSC to about O.l xSSC; formamide concentrations of about 55%> to about 75%>; and wash solutions of about l xSSC, O. l xSSC, or deionized water.
• hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
• SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
• expression refers to the process by which polynucleotides are transcribed into mR A and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.
• encode refers to a polynucleotide which is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mR A for the polypeptide and/or a fragment thereof.
• the antisense strand is the
• the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect.
• the effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
• To prevent intends to prevent a disorder or effect in vitro or in vivo in a system or subject that is predisposed to the disorder or effect.
• An example of such is preventing the formation of a biofilm in a system that is infected with a microorganism known to produce one.
• composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
• a "pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
• “Pharmaceutically acceptable carriers” refers to any diluents, excipients, polymers, micelles, lipsomes, vectors, plasmids, or carriers that may be used in the compositions of the invention.
• Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable
• compositions suitable for injectable use can include sterile aqueous solutions (where the components are water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS).
• compositions for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• Oral compositions generally include an inert diluent or an edible carrier.
• the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
• Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
• compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate or Sterotes
• a glidant such as colloidal silicon dioxide
• a sweetening agent such as sucrose or saccharin
• the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• a "biologically active agent" or an active agent of this invention intends one or more of an isolated or recombinant polypeptide, an isolated or recombinant polynucleotide, a vector, an isolated host cell, or an antibody, as well as compositions comprising one or more of same.
• administering can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, nasal administration, injection, and topical application.
• An agent of the present invention can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
• the term "effective amount” refers to a quantity sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to
• the effective amount is the amount sufficient to result in a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is the amount sufficient to result in antibody generation against the antigen. In some embodiments, the effective amount is the amount required to confer passive immunity on a subject in need thereof. With respect to immunogenic compositions, in some embodiments
• the effective amount will depend on the intended use, the degree of
• the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations.
• the effective amount may comprise one or more administrations of a composition depending on the embodiment.
• conjugated moiety refers to a moiety that can be added to an isolated chimeric polypeptide by forming a covalent bond with a residue of chimeric polypeptide. The moiety may bond directly to a residue of the chimeric polypeptide or may form a covalent bond with a linker which in turn forms a covalent bond with a residue of the chimeric polypeptide.
• a "peptide conjugate” refers to the association by covalent or non-covalent bonding of one or more polypeptides and another chemical or biological compound.
• the "conjugation" of a polypeptide with a chemical compound results in improved stability or efficacy of the polypeptide for its intended purpose.
• a peptide is conjugated to a carrier, wherein the carrier is a liposome, a micelle, or a
• Liposomes are microscopic vesicles consisting of concentric lipid bilayers.
• liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter.
• Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer.
• PC phosphatidylcholine
• PE phosphatidylethanolamine
• PI phosphatidylinositol
• SM sphingomyelin
• DOPE dioleoylphosphatidylethanolamine
• lipids capable of producing a stable liposome are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol- amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan- olamine (DSPE),
• HSPC hydrogenated soy phosphatidylcholine
• lecithin phosphatidylethanolamine
• lysolecithin lysophosphatidylethanol- amine
• phosphatidylserine phosphatidylinositol
• sphingomyelin cephalin
• cardiolipin phosphatidic acid, cerebrosides
• DSPE distearoylphosphat
• DOPC dioleoylphosphatidylcholine
• DPPC dipalmitoylphosphatidylcholine
• palmitoyloleoylphosphatidylcholine POPC
• palmitoyloleoylphosphatidylethanolamine POPE
• dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-l-carb- oxylate (DOPE-mal).
• Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Choi).
• Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG),
• liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure.
• the three classifications as developed by the New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Medicine," December 1977, are multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large unilamellar vesicles (LUVs).
• MLVs multi-lamellar vesicles
• SUVs small uni-lamellar vesicles
• LUVs large unilamellar vesicles
• the biological active agents can be encapsulated in such for administration in accordance with the methods described herein.
• a "micelle” is an aggregate of surfactant molecules dispersed in a liquid colloid.
• a typical micelle in aqueous solution forms an aggregate with the hydrophilic "head” regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center.
• This type of micelle is known as a normal phase micelle (oil-in- water micelle).
• Inverse micelles have the head groups at the center with the tails extending out (water-in-oil micelle).
• Micelles can be used to attach a polynucleotide, polypeptide, antibody or composition described herein to facilitate efficient delivery to the target cell or tissue.
• pharmaceutically acceptable polymer refers to the group of compounds which can be conjugated to one or more polypeptides described here. It is contemplated that the conjugation of a polymer to the polypeptide is capable of extending the half-life of the polypeptide in vivo and in vitro. Non- limiting examples include polyethylene glycols, polyvinylpyrrolidones, polyvinylalcohols, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof.
• the biological active agents can be conjugated to a pharmaceutically acceptable polymer for administration in accordance with the methods described herein.
• a "gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
• Examples of gene delivery vehicles are liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
• a polynucleotide of this invention can be delivered to a cell or tissue using a gene delivery vehicle.
• Gene delivery “gene transfer,” “transducing,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction.
• Such methods include a variety of well-known techniques such as vector- mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun” delivery and various other techniques used for the introduction of polynucleotides).
• vector- mediated gene transfer by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes
• techniques facilitating the delivery of "naked" polynucleotides such as electroporation, "gene gun” delivery and various other techniques used for the introduction of polynucleotides.
• the introduced polynucleotide may be stably or transiently maintained in the host cell.
• Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
• a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
• a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
• a "plasmid" is an extra-chromosomal DNA molecule separate from the
• Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. 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 circumstances.
• Plasmids used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
• MCS multiple cloning site
• Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass- producing a gene or the protein it then codes for.
• a "yeast artificial chromosome” or “YAC” refers to a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. Built using an initial circular plasmid, they are linearized by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends.
• Yeast expression vectors such as YACs, Yips (yeast integrating plasmid), and YEps (yeast episomal plasmid), are extremely useful as one can get eukaryotic protein products with posttranslational modifications as yeasts are themselves eukaryotic cells, however YACs have been found to be more unstable than BACs, producing chimeric effects.
• a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
• viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
• Infectious tobacco mosaic virus (TMV)-based vectors can be used to manufacturer 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,
• a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
• retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
• the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
• retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
• Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
• the integrated DNA form is called a provirus.
• a vector construct refers to the adenovirus (Ad) or adeno-associated virus (AAV).
• Ads Adenoviruses
• Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Publication No. WO 95/27071. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Publication Nos. WO 95/00655 and WO 95/11984. Wild-type AAV has high infectivity and specificity integrating into the host cell's genome. See, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470 and
• Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5 ' of the start codon to enhance expression.
• Gene delivery vehicles also include DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
• direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins of this invention are other non-limiting techniques.
• eDNA refers to extracellular DNA found as a component to pathogenic, e.g., bacterial, biofilms.
• antibodies and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fab', F(ab) 2 , Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies and kappa bodies; multispecific antibody fragments formed from antibody fragments and one or more isolated CDRs or a functional paratope; 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 the immunoglobulin molecule contain a binding domain that interacts with an antigen.
• the constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulins
• antibody herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, human monoclonal antibody, recombinant human antibody, chimeric antibodies, antibody derivative, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, antigen binding fragment, so long as they exhibit the desired biological activity.
• monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies are highly specific, as each monoclonal antibody is directed against a single determinant on the antigen.
• the antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
• the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like.
• the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like.
• Monoclonal antibodies may be generated using hybridoma techniques or recombinant DNA methods known in the art.
• Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to antigens of interest, and screening of antibody display libraries in cells, phage, or similar systems.
• human antibody as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
• the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
• human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• human antibody refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C L , C H domains (e.g., C HI , C R2 , C R3 ), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
• antibodies designated primate monkey, baboon, chimpanzee, etc.
• rodent mouse, rat, rabbit, guinea pig, hamster, and the like
• other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies.
• chimeric antibodies include any combination of the above.
• a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies.
• an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
• linker peptides are considered to be of human origin.
• a human antibody is "derived from” a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, e.g., by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library.
• a human antibody that is "derived from” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of human germline immunoglobulins.
• a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences).
• 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.
• a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene.
• the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
• a "human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The term also intends recombinant human antibodies. Methods to making these antibodies are described herein.
• recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human
• Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. Methods to making these antibodies are described herein. [0144] As used herein, chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.
• humanized antibody or “humanized immunoglobulin” refers to a human/non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a variable region of the recipient are replaced by residues from a variable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and capacity.
• 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.
• the humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, a non-human antibody containing one or more amino acids in a framework region, a constant region or a CDR, that have been substituted with a
• Fc immunoglobulin constant region
• humanized antibodies are expected to produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody.
• the humanized antibodies may have conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions.
• Conservative substitutions groupings include: glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine- threonine and asparagine-glutamine.
• antibody derivative comprises a full-length antibody or a fragment of an antibody, wherein one or more of the amino acids are chemically modified by alkylation, pegylation, acylation, ester formation or amide formation or the like, e.g., for linking the antibody to a second molecule.
• the term "immunoconjugate” comprises an antibody or an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a fluorescent label, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
• a second agent such as a cytotoxic agent, a detectable agent, a fluorescent label, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
• the term "detectable label” intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histadine tags (N-His), magnetically active isotopes, e.g., 115 Sn, 117 Sn and 119 Sn, a non-radioactive isotopes such as 13 C and 15 N, polynucleotide or protein such as an antibody so as to generate a "labeled" composition.
• N-terminal histadine tags N-terminal histadine tags
• magnetically active isotopes e.g., 115 Sn, 117 Sn and 119 Sn
• a non-radioactive isotopes such as 13 C and 15 N
• polynucleotide or protein such as an antibody so as to generate a "labeled” composition.
• the term also includes sequences conjugated to the polynucleotide that will
• the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
• the labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to
• a response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property.
• a quantifiable e.g., numerically reportable
• the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
• luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence.
• Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal.
• Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).
• luminescent probes include, but are not limited to, aequorin and luciferases.
• fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, and Texas Red.
• suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).
• the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to,
• isothiocyanate groups amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
• the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
• fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue ® , and Texas Red ® .
• Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).
• the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
• Suitable functional groups including, but not are limited to,
• isothiocyanate groups amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
• the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
• Eukaryotic cells comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus.
• the term "host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human.
• Prokaryotic cells that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells' genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 ⁇ in diameter and 10 ⁇ long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coli bacterium, and Salmonella bacterium.
• a "native" or “natural” antigen is a polypeptide, protein or a fragment which contains an epitope, which has been isolated from a natural biological source, and which can specifically bind to an antigen receptor, in particular a T cell antigen receptor (TCR), in a subject.
• TCR T cell antigen receptor
• antigen and “antigenic” refer to molecules with the capacity to be recognized by an antibody or otherwise act as a member of an antibody-ligand pair.
• Specific binding refers to the interaction of an antigen with the variable regions of immunoglobulin heavy and light chains. Antibody-antigen binding may occur in vivo or in vitro.
• macromolecules including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to act as an antigen.
• nucleic acids encoding a protein with the potential to act as an antibody ligand necessarily encode an antigen.
• antigens are not limited to full-length molecules, but can also include partial molecules.
• antigenic is an adjectival reference to molecules having the properties of an antigen.
• the term encompasses substances which are immunogenic, i.e., immunogens, as well as substances which induce immunological unresponsiveness, or anergy, i.e., anergens.
• An "altered antigen” is one having a primary sequence that is different from that of the corresponding wild-type antigen.
• Altered antigens can be made by synthetic or recombinant methods and include, but are not limited to, antigenic peptides that are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand. (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).
• a synthetic or altered antigen of the invention is intended to bind to the same TCR as the natural epitope.
• a "self-antigen” also referred to herein as a native or wild-type antigen is an antigenic peptide that induces little or no immune response in the subject due to self-tolerance to the antigen.
• An example of a self-antigen is the melanoma specific antigen gplOO.
• MHC major histocompatibility complex
• HLA human leukocyte antigen
• the proteins encoded by the MHC are known as "MHC molecules" and are classified into class I and class II MHC molecules.
• Class I MHC includes membrane heterodimeric proteins made up of an a chain encoded in the MHC noncovalently linked with the ⁇ 2-microglobulin. Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8 + T cells. Class I molecules include HLA-A, B, and C in humans. Class II MHC molecules also include membrane heterodimeric proteins consisting of noncovalently associated a and ⁇ chains. Class II MHC molecules are known to function in CD4 + T cells and, in humans, include HLA-DP, -DQ, and DR. In a preferred embodiment, invention compositions and ligands can complex with MHC molecules of any HLA type.
• Immuno response broadly refers to the antigen-specific responses of lymphocytes to foreign substances.
• immunogen and “immunogenic” refer to molecules with the capacity to elicit an immune response.
• the response may involve antibody production or the activation of immune cells.
• the response may occur in vivo or in vitro.
• macromolecules including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to be immunogenic.
• nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen.
• immunogens are not limited to full-length molecules, but may include partial molecules.
• Passive immunity refers to the transfer of immunity from one subject to another through the transfer of antibodies. Passive immunity may occur naturally, as when maternal antibodies are transferred to a fetus. Passive immunity may also occur artificially as when antibody compositions are administered to non-immune subjects. Antibody donors and recipients may be human or non-human subjects. Antibodies 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 on an a subject in need thereof through the administration of antibodies or antigen binding fragments that specifically recognize or bind to a particular antigen. In some embodiments, passive immunity is conferred through the administration of an isolated or recombinant polynucleotide encoding an antibody or antigen binding fragment that specifically recognizes or binds to a particular antigen.
• a "ligand” is a polypeptide.
• the term "ligand” as used herein refers to any molecule that binds to a specific site on another molecule.
• the ligand confers the specificity of the protein in a reaction with an immune effector cell or an antibody to a protein or DNA to a protein.
• it is the ligand site within the protein that combines directly with the complementary binding site on the immune effector cell.
• solid phase support or “solid support”, used interchangeably, is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art.
• Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels.
• solid support also includes synthetic antigen-presenting matrices, cells, and liposomes. A suitable solid phase support may be selected on the basis of desired end use and suitability for various protocols.
• solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE.RTM. resin (obtained from Aminotech, Canada), polyamide resin
• An example of a solid phase support include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
• the nature of the carrier can be either soluble to some extent or insoluble.
• the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polynucleotide, polypeptide or antibody.
• the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
• the surface may be flat such as a sheet, test strip, etc. or alternatively polystyrene beads.
• suitable carriers for binding antibody or antigen or will be able to ascertain the same by use of routine experimentation.
• Cystic fibrosis is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia (B.
• cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option.
• the inventors have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins.
• Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm.
• IHF integration host factor
• cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing.
• This disclosure provides a method for inhibiting, competing or titrating a biofilm produced by Burkholderia, the method comprising, or alternatively consisting essentially of, or yet consisting of, contacting the biofilm with an effective amount of an anti- integration host factor (anti-IHF) antibody, thereby inhibiting, competing or titrating the biofilm.
• the method can be performed in vitro or in vivo.
• the contacting of the anti-IHF antibody is performed in the absence of a DNase treatment.
• the DNAse treatment that is excluded from therapy comprises an enzyme that catalyzes the cleavage of phosphodiester linkages in the DNA backbone.
• DNase enzymes that are excluded from therapy are known to target not only cruciform structures, but also a variety of secondary structure of DNA include DNAse I, T4 EndoVII and T7 Endo I.
• the DNase treatment that is excluded from therapy comprises, or consists essentially of, or yet further consists of, Pulmozyme® (dornase alpha; Genentech, Inc.).
• the method further comprises, or alternatively consisting essentially of, or yet further consists of, contacting the biofilm or Burkkholderia with an effective amount of an antimicrobial that inhibits the growth of the Burkkholderia causing the biofilm.
• an antimicrobial that inhibits the growth of the Burkkholderia causing the biofilm.
• Non-limiting examples of such include ampicillin, amoxicillin-clavulanate, ceftazidine, ciprofloxacin, imipenem, minocycline, and cefdinir.
• the contacting can be conducted in vitro in or in vivo.
• Also provided herein is a method of treating an infection or disease caused by a Burkholderia infection, in a subject, comprising, or alternatively consisting essentially of, or yet consisting of, administering to the subject an effective amount of an anti-IHF antibody, thereby treating the infection or disease caused by the Burkholderia infection.
• This disclosure also provides methods for treating or preventing the recurrence of an infection in a CF patient in need thereof, comprising, or alternatively consistin essentially of, or yet further consisting of, administering to the patient an effective amount of an anti-IHF antibody, thereby treating or preventing the recurrence of the infection in the CF patient.
• the administration of the anti-IHF antibody is in the absence of a DNase treatment.
• the DNAse treatment that is excluded from therapy comprises an enzyme that catalyzes the cleavage of phosphodiester linkages in the DNA backbone.
• DNase enzymes that are excluded from therapy and known to target not only cruciform structures, but also a variety of secondary structure of DNA include DNAse I, T4 EndoVII and T7 Endo I.
• the DNase treatment that is excluded from therapy comprises, or consists essentially of, or yet further consists of, Pulmozyme® (dornase alpha; Genentech, Inc.).
• the method further comprises, or yet further consists essentially of, or yet further consists of, administering to the subject an effective amount of an antimicrobial that inhibits the growth of the Burkholderia causing the biofilm.
• an antimicrobial that inhibits the growth of the Burkholderia causing the biofilm.
• Non-limiting examples of such include ampicillin, amoxicillin- clavulanate, ceftazidine, ciprofloxacin, imipenem, minocycline, and cefdinir.
• Burkholderia cenocepacia B. cenocepacia
• B. multivorans B. mallei, B. cepaci, or B. pseudomallei.
• Anti -IHF antibody for use in the above methods is one or more of an anti-IHFa or an anti-IHFp antibody.
• the anti-IHF antibody is an IgG antibody.
• the antibody can be any of the various antibodies described herein, non-limiting examples of such include a full-length monoclonal antibody, a polyclonal antibody, human antibodies, humanized antibodies, human monoclonal antibody, a recombinant human antibody, a chimeric antibody, a veneered antibody, a diabody, a humanized antibody, an antibody derivative, a recombinant humanized antibody, or a fragment or an antigen binding fragment thereof, so long it exhibits the desired biological activity.
• the fragment comprises, or alternatively consists essentially of, or yet further consists of the CDR of the antibody.
• the antibody is detectably labeled or further comprises a detectable label conjugated to it.
• Non-limiting examples of anti-IHF antibodies are described herein, and in one aspect, is one or more of an antibody that specifically recognizes and binds a polypeptide identified in Table 2, or the Arm fragment identified therein, or an equivalent of such polypeptide or a polynucleotide or polypeptide comprising one or more of the sequences: : TCTCAACGATTTA (SEQ ID NO. 341); WATCAANNNNTTR (where W is A or T, N is any nucleotide and R is a A or G; (SEQ ID NO. 342); MATITKLDIIEYLSDKYHLS (also referred to herein as hlFAl; (SEQ ID NO.
• YHLS QDTKNVVENFLEEI also referred to herein as hIFA2; (SEQ ID NO. 344); FLEEIRLSLESGODVKLSGF (also referred to herein as hIFA3; (SEQ ID NO. 345); KLSGFGNFELRD SSRPGRN (also referred to herein as hIFA4; (SEQ ID NO. 346); RPGRNPKTGDWPVSARRVV (also referred to herein as hIFA5; (SEQ ID NO. 347); ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQ ID NO.
• polypeptide sequences which is encoded by a polynucleotide or its complement that hybridizes under conditions of high stringency to a polynucleotide encoding such polypeptide sequences. Conditions of high stringency are described above and incoporrated herein by reference.
• equivalent polypeptides include, for example a polypeptide consisting of or comprising the above noted 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 on either the amine or carboxy termini (or on both).
• the infection is in vivo and in a mammalian host, such as a human patient, e.g., is an immature mammalian host or a pediatric patient.
• This disclosure also provides a method for inhibiting, competing or titrating a biofilm present or contributing to CF (e.g., a B. cenocepacia induced biofilm), the method comprising, or alternatively consisting essentially of, or yet further consisting of, contacting the biofilm with an interfering agent, thereby inhibiting, competing or titrating the biofilm.
• the contacting can be performed in vitro or in vivo.
• a method for inhibiting, preventing or breaking down a microbial biofilm in a CF patient or a patient harboring an infection contributing to CF comprising, or alternatively consisting essentially of, or yet further consisting of contacting the biofilm with an interfering agent, thereby inhibiting, preventing or breaking down the microbial biofilm.
• the patients can be an animal, mammal or a human patient.
• the methods are useful to screen for or confirm interfering agents having the same, similar or opposite ability as the polypeptides, polynucleotides, antibodies, host cells, small molecules and compositions of this invention. Alternatively, they can be used to identify which interfering agent is best suited to treat a microbial infection. For example, one can screen for new agents or combination therapies by having two samples containing for example, the DNA BII polypeptide and microbial DNA or biofilm and the agent to be tested. The second sample contains the DNA BII polypeptide and microbial DNA or biofilm and an agent known to active, e.g., an anti-IHF antibody or a small molecule to serve as a positive control.
• an agent known to active e.g., an anti-IHF antibody or a small molecule to serve as a positive control.
• a negative control containing the DNA BII polypeptide and the microbial DNA or biofilm can be provided.
• the DNA BII polypeptide and the microbial DNA or biofilm are detectably labeled, for example with luminescent molecules that will emit a signal when brought into close contact with each other.
• the samples are contained under similar conditions for an effective amount of time for the agent to inhibit, compete or titrate the interaction between the DNA BII polypeptide and microbial DNA or biofilm and then the sample is assayed for emission of signal from the luminescent molecules. If the sample emits a signal, then the agent is not effective to inhibit binding.
• the in vitro method is practiced in a miniaturized chamber slide system wherein the microbial (such as a bacterial) isolate causing a CF infection could be isolated from the human/animal then cultured to allow it to grow as a biofilm in vitro.
• the interfering agent such as anti-IHF antibody
• potential interfering agent biofilm is added alone or in combination with another agent to the culture with or without increasing dilutions of the potential interfering agent or interfering agent such as an anti-IHF (or other antibody, small molecule, agent etc.) to find the optimal dose that would likely be effective at treating that patient when delivered to the subject where the infection existed.
• a positive and negative control can be performed simultaneously.
• the method is practiced in a high throughput platform with the interfering agent (such as anti-IHF antibody) and/or potential interfering agent (alone or in combination with another agent) in a flow cell.
• the interfering agent such as anti-IHF antibody
• potential interfering agent biofilm is added alone or in combination with another agent to the culture with or without increasing dilutions of the potential interfering agent or interfering agent such as an anti-IHF (or other antibody, small molecule, agent etc.) to find the optimal dose that would likely be effective at treating that patient when delivered to the subject where the infection existed.
• Biofilm isolates are sonicated to separate biofilm bacteria from DNA BII polyeptide such as IHF bound to microbial DNA.
• the DNA BII polypeptide - DNA complexes are isolated by virtue of an anti-IHF antibody on the platform.
• the microbial DNA is then be released with e.g. a salt wash, and used to identify the biofilm bacteria added.
• the freed DNA is then identified, e.g., by PCR sequenced. If DNA is not freed, then the interfering agent(s) successfully performed or bound the microbial DNA. If DNA is found in the sample, then the agent did not interfere with DNA BII polypeptide- microbial DNA binding.
• a positive and/or negative control can be simultaneously performed.
• the methods can be used to identify and/or confirm interfering agents in an industrial setting.
• an antibiotic or antimicrobial known to inhibit growth of the underlying infection is added sequentially or concurrently, to determine if the infection can be inhibited. It is also possible to add the interfering agent to the microbial DNA or DNA BII polypeptide before adding the missing complex to assay for biofilm inhibition.
• the method When practiced in vivo in non-human animal, the method provides a pre-clinical screen to identify interfering agents that can be used alone or in combination with other agents to break down biofilms.
• a method for inhibiting, preventing or breaking down a microbial biofilm and/or clearing a microbial infection in a CF patient or a patient harboring an infection contributing to CF comprising, or alternatively consisting essentially of, or yet further consisting of contacting the biofilm with an interfering agent, thereby inhibiting, preventing or breaking down and/or clearing the microbial infection or the microbial biofilm.
• the patient can be an animal, mammal or human patient.
• the CF patient can be an animal, mammal or human patient.
• interfering agents can be combined with antimicrobials to further supplement the therapy.
• any of the above methods can further comprise, or consist essentially of, or yet further consist of administration or contacting with an effective amount of the antimicrobial.
• Non-limiting examples of such include
• the methods further comprise, or alternatively consist essentially of, or yet further consist of administering to the subject an effective amount of one or more of an antimicrobial, an antigenic peptide or an adjuvant.
• a non-limiting example of an antimicrobial agent is another other vaccine component such as a surface antigen, e.g. a Type IV Pilin protein (see Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875).
• a surface antigen e.g. a Type IV Pilin protein (see Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875).
• compositions of this invention can be concurrently or sequentially administered with each other or other antimicrobial agents and/or surface antigens.
• administration is locally to the site of the infection by direct injection or by inhalation for example.
• Other non-limiting examples of administration include by one or more method comprising transdermally, urethrally, sublingually, rectally, vaginally, ocularly, subcutaneous, intramuscularly, intraperitoneally, intranasally, by inhalation or orally.
• Microbial infections and disease that can be treated by the methods of this invention include infection that lead to or are associated with CF infection. These microbial infections may be present in the upper, mid and lower airway (otitis, sinusitis, bronchitis but also exacerbations of chronic obstructive pulmonary disease (COPD), chronic cough,
• COPD chronic obstructive pulmonary disease
• cystic fibrosis CF
• CAP community acquired pneumonia
• routes of administration applicable to the methods of the invention include intranasal, intramuscular, urethrally, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, inhalation, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. An active agent can be administered in a single dose or in multiple doses. Embodiments of these methods and routes suitable for delivery, include systemic or localized routes. In general, routes of administration suitable for the methods of the invention include, but are not limited to, direct injection, enteral, parenteral, or inhalational routes.
• Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital,
• intracapsular, intraspinal, intrasternal, and intravenous routes i.e., any route of administration other than through the alimentary canal.
• Parenteral administration can be conducted to effect systemic or local delivery of the inhibiting agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
• the interfering agents of the invention can also be delivered to the subject by enteral administration.
• Enteral routes of administration include, but are not limited to, oral, urethral and rectal (e.g., using a suppository) delivery.
• Methods of administration of the active through the skin or mucosa include, but are not limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration.
• a suitable pharmaceutical preparation for transdermal transmission, absorption promoters or iontophoresis are suitable methods.
• lontophoretic transmission may be accomplished using commercially available "patches" that deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
• the interfering agent will be administered by inhalation, injection or orally on a continuous, daily basis, at least once per day (QD), and in various embodiments two (BID), three (TID), or even four times a day.
• the therapeutically effective daily dose will be at least about 1 mg, or at least about 10 mg, or at least about 100 mg, or about 200 - about 500 mg, and sometimes, depending on the compound, up to as much as about 1 g to about 2.5 g.
• Dosing of can be accomplished in accordance with the methods of the invention using capsules, tablets, oral suspension, suspension for intra-muscular injection, suspension for intravenous infusion, gel or cream for topical application, or suspension for intra-articular injection.
• compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio
• compositions which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
• IC50 i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms
• levels in plasma may be measured, for example, by high performance liquid chromatography.
• an effective amount of a composition sufficient for achieving a therapeutic or prophylactic effect ranges from about 0.000001 mg per kilogram body weight per administration to about 10,000 mg per kilogram body weight per administration.
• the dosage ranges are from about 0.0001 mg per kilogram body weight per administration to about 100 mg per kilogram body weight per administration.
• Administration can be provided as an initial dose, followed by one or more "booster" doses.
• Booster doses can be provided a day, two days, three days, a week, two weeks, three weeks, one, two, three, six or twelve months after an initial dose.
• a booster dose is
• treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
• compositions and related methods of the present invention may be used in combination with the administration of other therapies. These include, but are not limited to, the administration of DNase enzymes, antibiotics, antimicrobials, or other antibodies.
• the methods and compositions include a deoxyribonuclease (DNase) enzyme that acts synergistically with the anti-DNABII antibody.
• DNase is any enzyme that catalyzes the cleavage of phosphodiester linkages in the DNA backbone.
• DNase enzymes Three non- limiting examples of DNase enzymes that are known to target not only cruciform structures, but also a variety of secondary structure of DNA include DNAse I, T4 EndoVII and T7 Endo I.
• the effective amount of anti-DNABII antibody needed to destabilize the biofilm is reduced when combined with a DNase.
• the DNase can be added directly to the assay or in a suitable buffer known to stabilize the enzyme.
• the effective unit dose of DNase and the assay conditions may vary, and can be optimized according to procedures known in the art.
• the methods and compositions can be combined with antibiotics and/or antimicrobials.
• Antimicrobials are substances that kill or inhibit the growth of microorganisms such as bacteria, fungi, or protozoans.
• bio films are generally resistant to the actions of antibiotics, compositions and methods described herein can be used to sensitize the infection involving a biofilm to traditional therapeutic methods for treating infections.
• the use of antibiotics or antimicrobials in combination with methods and compositions described herein allow for the reduction of the effective amount of the antimicrobial and/or biofilm reducing agent.
• antimicrobials and antibiotics useful in combination with methods of the current invention include amoxicillin, amoxicillin-clavulanate, cefdinir, azithromycin, and sulfamethoxazole- trimethoprim.
• the therapeutically effective dose of the antimicrobial and/or antibiotic in combination with the biofilm reducing agent can be readily determined by traditional methods.
• the dose of the antimicrobial agent in combination with the biofilm reducing agent is the average effective dose which has been shown to be effective in other bacterial infections, for example, bacterial infections wherein the etiology of the infection does not include a biofilm.
• the dose is 0.1, 0.15, 0.2, 0.25, 0,30, 0,35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.8, 0.85, 0.9, 0.95, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.5, 3.0 or 5 times the the average effective dose.
• the antibiotic or antimicrobial can be added prior to, concurrent with, or subsequent to the addition of the anti- DNABII antibody.
• the methods and compositions can be combined with antibodies that treat the bacterial infection.
• antibodies that treat the bacterial infection.
• One example of an antibody useful in the methods and compositions can be combined with antibodies that treat the bacterial infection.
• an antibody directed against an unrelated outer membrane protein i.e. OMP P5
• OMP P5 an unrelated outer membrane protein
• Treatment with this antibody alone does not debulk a biofilm in vitro.
• Combined therapy with this antibody and a biofilm reducing agent and/or a antimicrobial results in a greater effect than that which could be achieved by either reagent used alone at the same concentration.
• Other antibodies that may produce a synergistic effect when combined with a biofilm reducing agent or methods to reduce a biofilm include anti-rsPilA anti-OMP26, anti-OMP P2, and anti-whole OMP preparations.
• compositions and methods described herein can be used to sensitize the bacterial infection involving a biofilm to common therapeutic modalities effective in treating bacterial infections without a biofilm but are otherwise ineffective in treating bacterial infections involving a biofilm.
• the compositions and methods described herein can be used in combination with therapeutic modalities that are effective in treating bacterial infections involving a biofilm, but the combination of such additional therapy and biofilm reducing agent or method produces a synergistic effect such that the effective dose of either the biofilm reducing agent or the additional therapeutic agent can be reduced.
• the combination of such additional therapy and biofilm reducing agent or method produces a synergistic effect such that the treatment is enhanced.
• An enhancement of treatment can be evidenced by a shorter amount of time required to treat the infection.
• the additional therapeutic treatment can be added prior to, concurrent with, or subsequent to methods or compositions used to reduce the biofilm, and can be contained within the same formation or as a separate formulation.
• kits containing the agents and instructions necessary to perform the in vitro and in vivo methods as described herein are claimed. Accordingly, the invention provides kits for performing these methods which may include an interfering of this invention as well as instructions for carrying out the methods of this invention such as collecting tissue and/or performing the screen, and/or analyzing the results, and/or administration of an effective amount of an interfering agent as defined herein. These can be used alone or in combination with other suitable antimicrobial agents.
• kits can comprise, or alternatively consist essentially of, or yet further consist of any one or more agent of the group of an isolated or recombinant integration host factor (IHF) polypeptide, polynucleotide or a fragment or an equivalent of each thereof; an isolated or recombinant protein polypeptide identified in Table 1 , Table 2, the Arm fragments identified in Table 2, Table 3, Table 4 a DNA binding peptide identified in FIG. 9, or a fragment or an equivalent of each thereof; an isolated or recombinant polynucleotide or polypeptide of SEQ ID NO.
• IHF integration host factor
• a pharmaceutically acceptable polymer a liposome, a micelle, an implant, a stent, a paste, a gel, a dental implant, or a medical implant.
• interfering agent is of the group:
• IHF integration host factor
• the interfering agent is an isolated or recombinant DNABII polypeptide or a fragment or an equivalent of each thereof.
• Non-limiting examples of such are an IHF or HU alpha or beta polypeptide; an IHF .alpha, polypeptide; Moraxella catarrhalis HU; E. coli HupA, HupB, himA, himD; E. faecalis HU (such as V583), HMGB1 and those identified in Table 1.
• the interfering agent is an isolated or recombinant polypeptide consisting essentially of an amino acid sequence selected from SEQ ID NO. 1 to 5 or 12 to 27, 30 to 35, 101-340 or a DNA binding peptide identified in FIG. 9.
• the isolated or recombinant polypeptide comprises, or
• SEQ ID NO. 1 or 2 alternatively consists essentially of or yet further consists of SEQ ID NO. 1 or 2, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• the isolated or recombinant polypeptide comprises, or
• polypeptide alternatively consists essentially of, or yet further consists of SEQ ID NO. 3, 4 or 5, with the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• the isolated or recombinant polypeptide comprises, or
• polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• the isolated or recombinant polypeptide comprises, or
• polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• the isolated or recombinant polypeptide comprises, or
• polypeptide comprising SEQ ID NO. 12 and 13 alternatively consists essentially of, or yet further consists of an isolated or recombinant polypeptide of the group of: a polypeptide comprising SEQ ID NO. 12 and 13; a polypeptide comprising SEQ ID NO. 14 and 15; a polypeptide comprising SEQ ID NO. 16 and 17; a polypeptide comprising SEQ ID NO. 18 and 19; a polypeptide comprising SEQ ID NO. 20 and 21; a polypeptide comprising SEQ ID NO. 23 and 24; a polypeptide comprising SEQ ID NO. 25 and 26; a polypeptide comprising SEQ ID NO. 30 and 31 ; a polypeptide comprising SEQ ID NO. 32 and 33; a polypeptide comprising SEQ ID NO.
• polypeptide 34 and 35 a polypeptide comprising SEQ ID NO. 337 and 338; or a polypeptide comprising SEQ ID NO. 339 and 340; with the proviso that the polypeptide is none of wild-type of any one of IHF alpha, IHF beta or SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• the isolated or recombinant polypeptide is of the group: polypeptide consisting essential y of SEQ ID NO. 12 and 13; polypeptide consisting essential y of SEQ ID NO. 14 and 15; polypeptide consisting essential y of SEQ ID NO. 16 and 17; polypeptide consisting essential y of SEQ ID NO. 18 and 19; polypeptide consisting essential y of SEQ ID NO. 20 and 21; polypeptide consisting essential y of SEQ ID NO. 23 and 24; polypeptide consisting essential y of SEQ ID NO. 25 and 26; polypeptide consisting essential y of SEQ ID NO. 30 and 31; polypeptide consisting essential y of SEQ ID NO.
• polypeptide consisting essential y of SEQ ID NO. 34 and 35 polypeptide consisting essential y of SEQ ID NO. 337 and 338; or polypeptide consisting essential y of SEQ ID NO. 339 and 340; with the proviso that the polypeptide is none of wild-type of any one of IHF alpha, IHF beta or SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
• TCTCAACGATTTA SEQ ID NO. 341
• WATCAANNNNTTR where W is A or T, N is any nucleotide and R is a A or G;
• MATITKLDIIEYLSDKYHLS also referred to herein as hlFAl; (SEQ ID NO. 343);
• KYHLSKQDTKNVVENFLEEI also referred to herein as hIFA2; (SEQ ID NO. 344); FLEEIRLSLESGQDVKLSGF (also referred to herein as hIFA3; (SEQ ID NO. 345); KLSGFGNFELRD S SRPGRN (also referred to herein as hIFA4; (SEQ ID NO. 346); RPGRNPKTGDWPVSARRVV (also referred to herein as hIFA5; (SEQ ID NO. 347); ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQ ID NO.
• polypeptide sequences which is encoded by a polynucleotide or its complement that hybridizes under conditions of high stringency to a polynucleotide encoding such polypeptide sequences. Conditions of high stringency are described above and incoporrated herein by reference.
• equivalent polypeptides include, for example a polypeptide consisting of or comprising the above noted 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 on either the amine or carboxy termini (or on both).
• the isolated polypeptide is combined with one or more of a detectable label, a carrier such as a pharmaceutically acceptable carrier, or an adjuvant.
• fragments or an equivalent of the isolated or recombinant polypeptides described above are fragments or an equivalent of the isolated or recombinant polypeptides described above.
• An example of a fragment is a C-terminal polypeptide.
• the isolated or recombinant polypeptide comprises, or alternatively consists essentially of, or yet further consists of two or more of the isolated or recombinant polypeptides described above.
• the isolated or recombinant polypeptide comprises, or alternatively consists essentially of, or yet further consists of any one of SEQ ID. NO. 1 to 5, 12 to 27 or 30 to 33, or a fragment or an equivalent polypeptide, examples of which are identified in Table 1 or shown in Table 2 or the Arm fragment identified in Table 2, Table 3 or Table 4.
• isolated wild-type polypeptides are excluded, i.e., that the polypeptide is none of SEQ ID NO. 6 through 11, 28, 29, or a wildtype sequence identified in Table 1 or shown in Table 2.
• this invention provides an isolated or recombinant polypeptide consisting essentially of an amino acid sequence of the group SEQ ID. NO. 1 to 5, 12 to 27 or 30 to 35, 1 to 6 and 13 to 35, or a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid corresponding to the .beta.-3 and/or .alpha.-3 fragments of a Haemophilus influenzae IHFa or ⁇ , non-limiting examples of which include SEQ ID NO. 12 through 27, or a fragment or equivalent thereof of each thereof.
• the invention provides an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid sequence of the group SEQ ID NO. 1 to 4, or a fragment or an equivalent of each thereof, or a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid corresponding to the .beta.-3 and/or .alpha.-3 fragments of a Haemophilus influenzae IHF. alpha, or IHF.beta., non-limiting examples of which include SEQ ID NO.
• isolated wildtype DNA binding polypeptides are excluded, i.e., that the polypeptide is none of SEQ ID NO. 6 through 11, 28, 29, or 42 through 100 or an isolated wildtype polypeptide sequence listed in Table 1 or shown in Table 2.
• this invention provides an isolated or recombinant polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of, SEQ ID. NO 1 or 2 alone or in combination with a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid corresponding to the .beta.-3 and/or a-3 fragments of a Haemophilus influenzae IHF-a or IHF.beta., on-limiting examples of which include SEQ ID Nos. 12 through 27 or a fragment or a biological equivalent of each thereof.
• isolated wildtype DNA binding polypeptides are excluded, i.e., that the polypeptide is none of SEQ ID NO. 6 through 11, 28, 29, or 42 through 100 or an isolated polypeptide sequence listed in Table 1 or shown in Table 2.
• this invention provides an isolated or recombinant polypeptide comprising or alternatively consisting essentially of, or yet further consisting of, SEQ ID NO. 3 or 4 or a fragment or an equivalent of each thereof alone or in combination with a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an amino acid corresponding to the .beta. -3 and/or a-3 fragments of a Haemophilus influenzae IHF-a or IHF.beta., non- limiting examples of which include SEQ ID NO. 12 through 27, and 34-35 or a biological equivalent of each thereof.
• isolated wildtype DNA binding polypeptides are excluded, i.e., that the polypeptide is none of SEQ ID NO. 6 through 11, 28, 29, or 42 through 100 or an isolated wildtype polypeptide sequence listed in Table 1 or shown in Table 2.
• This invention also provides isolated or recombinant polypeptides comprising or alternatively consisting essentially of, or yet further consisting of, two or more, or three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more of all fourteen of the isolated polypeptides or a fragment or an equivalent of each thereof.
• isolated or recombinant polypeptides comprising SEQ ID NO. 1 through 4, e.g., SEQ ID NO. 1 and 2, or alternatively 1 and 3 or alternatively 1 and 4, or alternatively 2 and 3, or alternatively SEQ ID NO. 1, 2 and 3 or alternatively, 2, 3 and 4, or alternatively 1, 3 and 4.
• polypeptides can be in any orientation, e.g., SEQ ID NO. 1, 2, and 3 or SEQ ID NO. 3, 2 and 1 or alternatively 2, 1 and 3, or alternatively, 3, 1 and 2.
• Biological equivalents of these polypeptides are further included in this invention with the proviso that the sequences do not include isolated wildtype sequences such as those identified in Tables 1, 2 and 3.
• this invention provides an isolated or recombinant polypeptide comprising or alternatively consisting essentially of, or yet further consisting of, SEQ ID NO. 1 or 2 and 3 or 4, or a fragment or an equivalent of each thereof, with the proviso that the polypeptide is none of SEQ ID NO. 6 through 11, and they may further comprise any one or more of SEQ ID Nos. 11 through 26, e.g., 11 and 12, or alternatively 1 and 11, or
• SEQ ID NO. 1 or 2 is located upstream or amino terminus from SEQ ID NO. 3 or 4, with the proviso that the amino acid sequence is not an isolated wildtype polypeptide, e.g., none of SEQ ID NO. 6 through 11, 28 and 29.
• the isolated polypeptide comprises SEQ ID NO. 3 or 4 located upstream or amino terminus to SEQ ID NO. 1 or 2. Biological equivalents of these polypeptides are further included in this invention with the proviso that the sequence do not include isolated wildtype polypeptides.
• any polypeptide or protein having sequence identity to the wildtype polypeptides or those disclosed in Pedulla et al. (1996) PNAS 93: 15411-15416 is excluded from this invention.
• a peptide linker can be added to the N-terminus or C-terminus of the polypeptide.
• a “linker” or “peptide linker” refers to a peptide sequence linked to either the N-terminus or the C-terminus of a polypeptide sequence.
• the linker is from about 1 to about 20 amino acid residues long or alternatively 2 to about 10, about 3 to about 5 amino acid residues long.
• An example of a peptide linker is Gly-Pro-Ser- Leu-Lys-Leu (SEQ ID NO: 37).
• Gly-Gly-Gly Gly-Pro-Ser-Leu (SEQ ID NO: 38); Gly-Pro-Ser; Pro-Ser-Leu-Lys (SEQ ID NO: 39); Gly-Pro-Ser-Leu-Lys (SEQ ID NO: 40) and Ser-Leu-Lys-Leu (SEQ ID NO: 41).
• the isolated polypeptides of this invention are intended to include isolated wildtype and recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells, as well as muteins, analogs and fragments thereof, examples of such cells are described above. In some embodiments, the term also includes antibodies and anti-idiotypic antibodies as described herein. Such polypeptides can be isolated or produced using the methods known in the art and briefly described herein.
• polypeptides are conjugated or linked to a detectable label. Suitable labels are known in the art and described herein.
• polypeptides with or without a detectable label can be contained or expressed on the surface of a host prokaryotic or eukaryotic host cell, such as a dendritic cell.
• polypeptides are obtainable by a number of processes known to those of skill in the art, which include purification, chemical synthesis and recombinant methods.
• Polypeptides can be isolated from preparations such as host cell systems by methods such as immunoprecipitation with antibody, and standard techniques such as gel filtration, ion-exchange, reversed-phase, and affinity chromatography. For such methodology, see for example Deutscher et al. (1999) Guide To Protein Purification: Methods In
• this invention also provides the processes for obtaining these polypeptides as well as the products obtainable and obtained by these processes.
• polypeptides also can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin/Elmer/ Applied Biosystems, Inc., Model 430A or 431 A, Foster City, Calif, USA.
• a commercially available automated peptide synthesizer such as those manufactured by Perkin/Elmer/ Applied Biosystems, Inc., Model 430A or 431 A, Foster City, Calif, USA.
• polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC).
• HPLC high performance liquid chromatography
• this invention also provides a process for chemically synthesizing the proteins of this invention by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.
• proteins and polypeptides can be obtained by well-known recombinant methods as described, for example, in Sambrook et al. (1989) supra, using a host cell and vector systems described herein.
• polypeptides described herein conjugated to a detectable agent for use in the diagnostic methods are useful as detectably labeled polypeptides.
• detectably labeled polypeptides can be bound to a column and used for the detection and purification of antibodies. They also are useful as immunogens for the production of antibodies as described below.
• the polypeptides of this invention are useful in an in vitro assay system to screen for agents or drugs, which modulate cellular processes.
• amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
• a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
• Peptides of the invention can be modified to include unnatural amino acids.
• the peptides may comprise D-amino acids, a combination of and L-amino acids, and various "designer" amino acids (e.g., .beta.-methyl amino acids, C-a.-methyl amino acids, and N-a- methyl amino acids, etc.) to convey special properties to peptides.
• various "designer" amino acids e.g., .beta.-methyl amino acids, C-a.-methyl amino acids, and N-a- methyl amino acids, etc.
• peptides with a-helices .beta, turns, .beta, sheets, .gamma.-turns, and cyclic peptides can be generated.
• a-helical secondary structure or random secondary structure is preferred.
• polypeptides of this invention also can be combined with various solid phase carriers, such as an implant, a stent, a paste, a gel, a dental implant, or a medical implant or liquid phase carriers, such as beads, sterile or aqueous solutions, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, suspensions and emulsions.
• solid phase carriers such as an implant, a stent, a paste, a gel, a dental implant, or a medical implant or liquid phase carriers, such as beads, sterile or aqueous solutions, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, suspensions and emulsions.
• non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils.
• the carriers also can include an adjuvant that is useful to non-specifically augment a specific immune response. A skilled artisan can easily determine whether an adjuvant is required and select one.
• suitable adjuvants include, but are not limited to Freund's Complete and Incomplete, mineral salts and polynucleotides.
• suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of the heat labile enterotoxin of E. coli, mutant derivatives of cholera toxin, CPG oligonucleotides, and adjuvants derived from squalene.
• This invention also provides a pharmaceutical composition
• a pharmaceutical composition comprising or alternatively consisting essentially of, or yet further consisting of, any of a polypeptide, analog, mutein, or fragment of this invention, alone or in combination with each other or other agents, such an antibiotic and an acceptable carrier or solid support.
• compositions are useful for various diagnostic and therapeutic methods as described herein.
• This invention also provides isolated or recombinant polynucleotides encoding one or more of the above -identified isolated or recombinant polypeptides and their respective complementary strands.
• Vectors comprising the isolated or recombinant polynucleotides are further provided examples of which are known in the art and briefly described herein.
• the isolated or recombinant polynucleotides can be contained within a polycistronic vector.
• the polynucleotides can be DNA, RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA.
• this invention provides an interfering agent that is a four- way junction polynucleotide resembling a Holliday junction, a 3 way junction polynucleotide resembling a replication fork, a polynucleotide that has inherent flexibility or bent
• polynucleotide which can treat or inhibit DNA BII polynucleotide from binding to microbial DNA as well treat, prevent or inhibit biofilm formation and associated infections and disorders.
• One of skill in the art can make such polynucleotides using the information provided herein and knowledge of those of skill in the art. See Goodman and Kay (1999) J. Biological Chem. 274(52):37004-37011 and Kamashev and Rouviere-Yaniv (2000) EMBO J. 19(23):6527-6535.
• the invention further provides the isolated or recombinant polynucleotide operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA.
• a promoter of RNA transcription as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA.
• operatively linked means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule. Examples of such promoters are SP6, T4 and T7.
• cell-specific promoters are used for cell- specific expression of the inserted polynucleotide.
• Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are known in the art and commercially available. For general methodology and cloning strategies, see Gene
• polynucleotides derived from the polynucleotides of the invention encode polypeptides or proteins having diagnostic and therapeutic utilities as described herein as well as probes to identify transcripts of the protein that may or may not be present.
• These nucleic acid fragments can be prepared, for example, by restriction enzyme digestion of larger polynucleotides and then labeled with a detectable marker. Alternatively, random fragments can be generated using nick translation of the molecule. For methodology for the preparation and labeling of such fragments, see Sambrook, et al. (1989) supra.
• Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA.
• suitable expression vectors include plasmids, yeast vectors, viral vectors and liposomes.
• Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient transformation of cells both in vitro and in vivo.
• a nucleic acid When a nucleic acid is inserted into a suitable host cell, e.g., a prokaryotic or a eukaryotic cell and the host cell replicates, the protein can be recombinantly produced.
• suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using known methods. See Sambrook, et al. (1989) supra.
• the nucleic acid can be inserted into the host cell by methods known in the art such as
• this invention also provides a host cell, e.g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a prokaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody.
• a host cell e.g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a prokaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody.
• a pharmaceutically acceptable vector such as a replication-incompetent retroviral or adenoviral vector.
• Pharmaceutically acceptable vectors containing the nucleic acids of this invention can be further modified for transient or stable expression of the inserted polynucleotide.
• the term "pharmaceutically acceptable vector” includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells.
• An example of such a vector is a "replication-incompetent" vector defined by its inability to produce viral proteins, precluding spread of the vector in the infected host cell.
• LNL6 Miller et al. (1989) BioTechniques 7:980-990.
• the methodology of using replication-incompetent retroviruses for retroviral-mediated gene transfer of gene markers has been established.
• This invention also provides genetically modified cells that contain and/or express the polynucleotides of this invention.
• the genetically modified cells can be produced by insertion of upstream regulatory sequences such as promoters or gene activators (see, U.S. Patent No. 5,733,761).
• the polynucleotides can be conjugated to a detectable marker, e.g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell.
• a detectable marker e.g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell.
• detectable markers include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
• a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents.
• calorimetric indicator substrates can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
• this invention further provides a method for detecting a single-stranded polynucleotide or its complement, by contacting target single-stranded polynucleotide with a labeled, single-stranded polynucleotide (a probe) which is a portion of the polynucleotide of this invention under conditions permitting hybridization (preferably moderately stringent hybridization conditions) of complementary single-stranded polynucleotides, or more preferably, under highly stringent hybridization conditions.
• Hybridized polynucleotide pairs are separated from un-hybridized, single-stranded polynucleotides.
• the hybridized polynucleotide pairs are detected using methods known to those of skill in the art and set forth, for example, in Sambrook et al. (1989) supra.
• the polynucleotide embodied in this invention can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain a desired polynucleotide by employing a DNA synthesizer or ordering from a commercial service.
• the polynucleotides of this invention can be isolated or replicated using PCR.
• the PCR technology is the subject matter of U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: The Polymerase Chain Reaction (Mullis et al. eds., Birkhauser Press, Boston (1994)) or MacPherson et al. (1991) and (1995) supra, and references cited therein.
• one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA.
• this invention also provides a process for obtaining the polynucleotides of this invention by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides.
• these polynucleotides are further isolated.
• one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification.
• the DNA so amplified can be isolated from the cell by methods known to those of skill in the art.
• a process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained.
• RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell.
• the DNA can be delivered by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation.
• an appropriate gene delivery vehicle e.g., liposome, plasmid or vector
• electroporation e.g., liposome, plasmid or vector
• the RNA can then be isolated using methods known to those of skill in the art, for example, as set forth in Sambrook et al. (1989) supra.
• mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989) supra, or extracted by nucleic-acid-binding resins following the accompanying instructions provided by
• polynucleotide of this invention are useful as hybridization probes. Since the full coding sequence of the transcript is known, any portion of this sequence or homologous sequences, can be used in the methods of this invention.
• a probe useful for detecting the aforementioned mRNA is at least about 80% identical to the homologous region. More preferably, the probe is 85% identical to the corresponding gene sequence after alignment of the homologous region; even more preferably, it exhibits 90% identity.
• probes can be used in radioassays (e.g. Southern and Northern blot analysis) to detect, prognose, diagnose or monitor various cells or tissues containing these cells.
• the probes also can be attached to a solid support or an array such as a chip for use in high throughput screening assays for the detection of expression of the gene corresponding a polynucleotide of this invention.
• this invention also provides a probe comprising or corresponding to a polynucleotide of this invention, or its equivalent, or its complement, or a fragment thereof, attached to a solid support for use in high throughput screens.
• the total size of fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between at least 5 to 10 to about 100 nucleotides, or even full length according to the complementary sequences one wishes to detect.
• Nucleotide probes having complementary sequences over stretches greater than 5 to 10 nucleotides in length are generally preferred, so as to increase stability and selectivity of the hybrid, and thereby improving the specificity of particular hybrid molecules obtained. More preferably, one can design polynucleotides having gene-complementary stretches of 10 or more or more than 50 nucleotides in length, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology with two priming oligonucleotides as described in U.S. Patent No. 4,603,102 or by introducing selected sequences into recombinant vectors for recombinant production. In one aspect, a probe is about 50-75 or more alternatively, 50-100, nucleotides in length.
• the polynucleotides of the present invention can serve as primers for the detection of genes or gene transcripts that are expressed in cells described herein.
• amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase.
• a primer is the same length as that identified for probes.
• PCR PCR and kits for PCR amplification are commercially available. After amplification, the resulting DNA fragments can be detected by any appropriate method known in the art, e.g., by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
• Methods for administering an effective amount of a gene delivery vector or vehicle to a cell have been developed and are known to those skilled in the art and described herein.
• Methods for detecting gene expression in a cell include techniques such as in hybridization to DNA microarrays, in situ hybridization, PCR, RNase protection assays and Northern blot analysis. Such methods are useful to detect and quantify expression of the gene in a cell.
• expression of the encoded polypeptide can be detected by various methods. In particular it is useful to prepare polyclonal or monoclonal antibodies that are specifically reactive with the target polypeptide. Such antibodies are useful for visualizing cells that express the polypeptide using techniques such as immunohistology, ELISA, and Western blotting. These techniques can be used to determine expression level of the expressed polynucleotide.
• This invention also provides an antibody that binds and/or specifically recognizes and binds an isolated polypeptide for use in the methods of the invention.
• the antibody can be any of the various antibodies described herein, non-limiting examples of such include a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody derivative, a recombinant humanized antibody, or a derivative or antigen binding fragment thereof.
• the fragment comprises, or alternatively consists essentially of, or yet further consists of the CDR of the antibody.
• the antibody is detectably labeled or further comprises a detectable label conjugated to it.
• compositions comprising or alternatively consisting essentially of or yet further, consisting of one or more of the above embodiments are further provided herein. Further provided are polynucleotides that encode the amino acid sequence of the antibodies and fragments as well as methods to recombinantly produce the antibody polypeptides and fragments thereof.
• the antibody polypeptides can be produced in a eukaryotic or prokaryotic cell, or by other methods known in the art and described herein.
• Antibodies can be generated using conventional techniques known in the art and are well-described in the literature. Several methodologies exist for production of polyclonal antibodies. For example, polyclonal antibodies are typically produced by immunization of a suitable mammal such as, but not limited to, chickens, goats, guinea pigs, hamsters, horses, mice, rats, and rabbits. An antigen is injected into the mammal, which induces the
• Immunoglobulins may be purified from the mammal's serum.
• Antibodies specific to IHFa and IHFP can be generated by injection of polypeptides corresponding to different epitopes of IHFa and IHFp. For example, antibodies can be generated using the 20 amino acids of each subunit such as TFRPGQKLKSRVENASPKDE (SEQ ID NO. 34) for IHFa and
• KYVPHFKPGKELRDRANIYG (SEQ ID No. 35) for IHFp, or alternatively an antibody that specifically recognizes and binds a polynucleotide or peptide comprising one or more of the sequences: : TCTCAACGATTTA (SEQ ID NO. 341); WATCAANNNNTTR (where W is A or T, N is any nucleotide and R is a A or G; (SEQ ID NO. 342);
• MATITKLDIIEYLSDKYHLS also referred to herein as hlFAl; (SEQ ID NO. 343);
• KYHLSKQDTKNVVENFLEEI also referred to herein as hIFA2; (SEQ ID NO. 344);
• FLEEIRLSLESGODVKLSGF also referred to herein as hIFA3; (SEQ ID NO. 345);
• KLSGFGNFELRD S SRPGRN also referred to herein as hIFA4; (SEQ ID NO. 346); RPGRNPKTGDWPVSARRVV (also referred to herein as hIFA5; (SEQ ID NO. 347); ARRVVTFKPGQKLRARVEKTK (also referred to herein as hIFA6; (SEQ ID NO.
• polypeptide sequences which is encoded by a polynucleotide or its complement that hybridizes under conditions of high stringency to a polynucleotide encoding such polypeptide sequences. Conditions of high stringency are described above and incoporrated herein by reference.
• equivalent polypeptides include, for example a polypeptide consisting of or comprising the above noted 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 on either the amine or carboxy termini (or on both).
• Additional examples include antibodies that specifically reconginze and bind the polypeptide identified in Table 2 (and incorporated herein by reference). Variations of this methodology include modification of adjuvants, routes and site of administration, injection volumes per site and the number of sites per animal for optimal production and humane treatment of the animal.
• adjuvants typically are used to improve or enhance an immune response to antigens.
• Most adjuvants provide for an injection site antigen depot, which allows for a slow release of antigen into draining lymph nodes.
• Other adjuvants include surfactants which promote concentration of protein antigen molecules over a large surface area and
• Non- limiting examples of adjuvants for polyclonal antibody generation include Freund's adjuvants, Ribi adjuvant system, and Titermax.
• Polyclonal antibodies can be generated using methods known in the art some of which are described in U.S. Patent Nos. 7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746; 6,322,788;
• Monoclonal antibodies can be generated using conventional hybridoma techniques known in the art and well-described in the literature.
• a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SSI, Sp2 SA5, U397, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/0) or the like, or heteromyelomas, fusion products thereof, or any cell or fusion cell derived there from, or any other suitable cell line as known in the art (see, those at the
• Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing-heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present invention.
• the fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods.
• Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from various commercial vendors such as MorphoSys (Martinsreid/Planegg, Del), Biolnvent (Lund, Sweden), Affitech (Oslo, Norway) using methods known in the art. Art known methods are described in the patent literature some of which include U.S. Patent Nos. 4,704,692;
• Antibody derivatives of the present invention can also be prepared by delivering a polynucleotide encoding an antibody of this invention to a suitable host such as to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such antibodies in their milk.
• a suitable host such as to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such antibodies in their milk.
• antibody derivative includes post-translational modification to linear polypeptide sequence of the antibody or fragment.
• U.S. Patent Application includes post-translational modification to linear polypeptide sequence of the antibody or fragment.
• No. 6,602,684 Bl describes a method for the generation of modified glycol-forms of antibodies, including whole antibody molecules, antibody fragments, or fusion proteins that include a region equivalent to the Fc region of an immunoglobulin, having enhanced
• the antibodies of the invention include derivatives that are modified by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
• Antibody derivatives include, but are not limited to, antibodies that have been modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known
• the derivatives may contain one or more non-classical amino acids.
• Antibody derivatives also can be prepared by delivering a polynucleotide of this invention to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco, maize, and duckweed) that produce such antibodies, specified portions or variants in the plant parts or in cells cultured therefrom.
• transgenic plants and cultured plant cells e.g., but not limited to tobacco, maize, and duckweed
• transgenic plants and cultured plant cells e.g., but not limited to tobacco, maize, and duckweed
• Antibody derivatives have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain antibodies (scFv's), including tobacco seeds and potato tubers. See, e.g., Conrad et al.(1998) Plant Mol. Biol. 38: 101-109 and references cited therein. Thus, antibodies can also be produced using transgenic plants, according to know methods.
• Antibody derivatives also can be produced, for example, by adding exogenous sequences to modify immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half- life, or any other suitable characteristic. Generally part or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions are replaced with human or other amino acids.
• the CDR residues are directly and most substantially involved in influencing antigen binding.
• Humanization or engineering of antibodies can be performed using any known method such as, but not limited to, those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and
• Chimeric, humanized or primatized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared using standard molecular biology techniques.
• DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.
• the murine variable regions can be linked to human constant regions using methods known in the art (U.S. Patent No. 4,816,567).
• the murine CDR regions can be inserted into a human framework using methods known in the art (U.S. Patent No. 5,225,539 and U.S. Patents Nos. 5,530,101;
• the murine CDR regions can be inserted into a primate framework using methods known in the art (WO 93/02108 and WO 99/55369).
• the antibodies of this invention also can be modified to create chimeric antibodies.
• Chimeric antibodies are those in which the various domains of the antibodies' heavy and light chains are coded for by DNA from more than one species. See, e.g., U.S. Patent No.
• the antibodies of this invention can also be modified to create veneered antibodies.
• Veneered antibodies are those in which the exterior amino acid residues of the antibody of one species are judiciously replaced or "veneered" with those of a second species so that the antibodies of the first species will not be immunogenic in the second species thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein is primarily dependent on the nature of its surface, the immunogenicity of an antibody could be reduced by replacing the exposed residues which differ from those usually found in another mammalian species antibodies. This judicious replacement of exterior residues should have little, or no, effect on the interior domains, or on the interdomain contacts.
• ligand binding properties should be unaffected as a consequence of alterations which are limited to the variable region framework residues.
• the process is referred to as "veneering" since only the outer surface or skin of the antibody is altered, the supporting residues remain undisturbed.
• Non-limiting examples of the methods used to generate veneered antibodies include EP 519596; U.S. Patent No. 6,797,492; and described in Padlan et al. (1991) Mol. Immunol. 28(4-5):489-498.
• antibody derivative also includes “diabodies” which are small antibody fragments with two antigen-binding sites, wherein fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain.
• VH heavy chain variable domain
• VL light chain variable domain
• antibody derivative further includes engineered antibody molecules, fragments and single domains such as scFv, dAbs, nanobodies, minibodies, Unibodies, and Affibodies (Holliger & Hudson (2005) Nature Biotech 23(9): 1126-36; U.S. Patent
• antibody derivative further includes "linear antibodies".
• linear antibodies The procedure for making linear antibodies is known in the art and described in Zapata et al. (1995) Protein Eng. 8(10): 1057- 1062. Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-VH -C R I) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
• the antibodies of this invention can be recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction
• HPLC high performance liquid chromatography
• Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells, or alternatively from a prokaryotic host as described above.
• a eukaryotic host including, for example, yeast, higher plant, insect and mammalian cells, or alternatively from a prokaryotic host as described above.
• a number of antibody production systems are described in Birch & Radner (2006) Adv. Drug Delivery Rev. 58 : 671 -685.
• antibody also is intended to include antibodies of all immunoglobulin isotypes and subclasses. Particular isotypes of a monoclonal antibody can be prepared either directly by selecting from an initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class switch variants using the procedure described in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984; J. Immunol. Methods 74:307. Alternatively, recombinant DNA techniques may be used.
• antibodies can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like.
• a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like.
• Such labeled antibodies can be used for diagnostic techniques, either in vivo, or in an isolated test sample.
• the coupling of antibodies to low molecular weight haptens can increase the sensitivity of the antibody in an assay.
• the haptens can then be specifically detected by means of a second reaction.
• haptens such as biotin, which reacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See, Harlow and Lane (1988) supra.
• variable region of the antibodies of the present invention can be modified by mutating amino acid residues within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody.
• Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered.
• the mutations may be amino acid substitutions, additions or deletions.
• immunogenicity for example, by "backmutating" one or more framework residues to the corresponding germline sequence.
• antibodies of the invention may be engineered to include
• modifications within the Fc region to alter one or more functional properties of the antibody such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
• modifications include, but are not limited to, alterations of the number of cysteine residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody (U.S. Patent No. 5,677,425); and amino acid mutations in the Fc hinge region to decrease the biological half life of the antibody (U.S. Patent No. 6,165,745).
• the antibodies of the invention may be chemically modified.
• Glycosylation of an antibody can be altered, for example, by modifying one or more sites of glycosylation within the antibody sequence to increase the affinity of the antibody for antigen (U.S. Patents Nos. 5,714,350 and 6,350,861).
• a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures can be obtained by expressing the antibody in a host cell with altered glycosylation mechanism (Shields et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17: 176-180).
• the antibodies of the invention can be pegylated to increase biological half-life by reacting the antibody or fragment thereof with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
• Antibody pegylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
• the term "polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI -CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
• the antibody to be pegylated can be an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention (EP 0 154 316 and EP 0 401 384).
• antibodies may be chemically modified by conjugating or fusing the antigen-binding region of the antibody to serum protein, such as human serum albumin, to increase half-life of the resulting molecule.
• serum protein such as human serum albumin
• the antibodies or fragments thereof of the present invention may be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the development or progression of a disease and determine the efficacy of a given treatment regimen.
• diagnostic agents include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
• the detectable substance may be coupled or conjugated either directly to the antibody or fragment thereof, or indirectly, through a linker using techniques known in the art.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
• suitable prosthetic group complexes include
• fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
• An example of a luminescent material includes luminol.
• bioluminescent materials include luciferase, luciferin, and aequorin.
• radioactive material examples include 125 I, 13 X I, Indium- 111, Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-I l l, Gallium- 67, Praseodymium- 142, Samarium-153, Terbium-161, Dysprosium- 166, Holmium-166, Rhenium-186, Rhenium-188, Rhenium-189, Lead-212, Radium-223, Actinium-225, Iron-59, Selenium-75, Arsenic-77, Strontium-89, Molybdenum-99, Rhodium- 105, Palladium- 109, Praseodymium- 143, Promethium-149, Erbium-169, Iridium-194, Gold-
• Monoclonal antibodies may be indirectly conjugated with radiometal ions through the use of bifunctional chelating agents that are covalently linked to the antibodies.
• Chelating agents may be attached through amines (Meares et al. (1984) Anal. Biochem. 142:68-78); sulfhydral groups (Koyama (1994) Chem. Abstr. 120:217262t) of amino acid residues and carbohydrate groups (Rodwell et al. (1986) PNAS USA 83:2632-2636; Quadri et al. (1993) Nucl. Med. Biol. 20:559-570).
• the antibodies or fragments thereof of the present invention may be conjugated to a therapeutic agent for example a antimicrobial that will treat or prevent the recurrence of a Burklederia infection.
• a therapeutic agent for example a antimicrobial that will treat or prevent the recurrence of a Burklederia infection.
• Suitable therapeutic agents include ceftazidime, ciprofloxacin, imipenem and minocycline.
• Additional suitable conjugated molecules include ribonuclease (RNase), DNase I, an antisense nucleic acid, an inhibitory RNA molecule such as a siRNA molecule, an immunostimulatory nucleic acid, aptamers, ribozymes, triplex forming molecules, and external guide sequences.
• Aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G- quartets, and can bind small molecules, such as ATP (U.S. Patent No. 5,631,146) and theophiline (U.S. Patent No. 5,580,737), as well as large molecules, such as reverse transcriptase (U.S. Patent No. 5,786,462) and thrombin (U.S. Patent No. 5,543,293).
• Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. Triplex forming function nucleic acid molecules can interact with double-stranded or single- stranded nucleic acid by forming a triplex, in which three strands of DNA form a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules can bind target regions with high affinity and specificity.
• the functional nucleic acid molecules may act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules may possess a de novo activity independent of any other molecules.
• the therapeutic agents can be linked to the antibody directly or indirectly, using any of a large number of available methods.
• an agent can be attached at the hinge region of the reduced antibody component via disulfide bond formation, using cross-linkers such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP), or via a carbohydrate moiety in the Fc region of the antibody (Yu et al. (1994) Int. J. Cancer 56: 244; Upeslacis et al.
• the antibodies of the invention or antigen-binding regions thereof can be linked to another functional molecule such as another antibody or ligand for a receptor to generate a bi- specific or multi-specific molecule that binds to at least two or more different binding sites or target molecules.
• Linking of the antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, can be done, for example, by chemical coupling, genetic fusion, or noncovalent association.
• Multi-specific molecules can further include a third binding specificity, in addition to the first and second target epitope.
• Bi-specific and multi-specific molecules can be prepared using methods known in the art. For example, each binding unit of the bi-specific molecule can be generated separately and then conjugated to one another. When the binding molecules are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation.
• cross-linking agents examples include protein A, carbodiimide, N-succinimidyl-S-acetyl- thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohaxane-I-carboxylate (sulfo-SMCC) (Karpovsky et al., 1984 J. Exp. Med. 160: 1686; Liu et al, 1985 Proc. Natl. Acad. Sci. USA 82:8648).
• binding molecules are antibodies, they can be conjugated by sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains.
• the antibodies or fragments thereof of the present invention may be linked to a moiety that is toxic to a cell to which the antibody is bound to form "depleting" antibodies. These antibodies are particularly useful in applications where it is desired to deplete an NK cell.
• the antibodies of the invention may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
• solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• the antibodies also can be bound to many different carriers, for example pharmaceutically acceptable carriers.
• this invention also provides compositions containing the antibodies and another substance, active or inert.
• carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite.
• the nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for antibodies, or will be able to ascertain such, using routine experimentation.
• the following examples are intended to illustrate, but not limit the invention.
• B. cenocepacia strains (including strains K56-2, DFA2 and JRL2) were inoculated from a frozen stock into LB broth (BBL) and grown overnight at 37°C with shaking at 200 rpm. Cultures were then diluted 1 : 100 in fresh broth, the optical density read at 600 nm and adjusted to a final concentration of 106 CFU B. cenocepacia/ ml medium. The bacteria were further diluted 1 :2500 in LB broth and 200 ⁇ of the bacterial suspension was added to each well of a LabTek II 8-well chambered coverglass (LabTek).
• the DNA to bacterium ratio was determined using Zeiss image acquisition software wherein the relative fluorescent intensity of anti-dsDNA labeling to FilmTracerTM was compared.
• Biofilms formed by nontypeable Haemophilus influenzae were also established (Goodman et al. (2011) Mucosal Immunol. 4:625-637; Jurcisek et al. (2011) J. Vis. Exp.) and similarly stained.
• Sections were incubated with polyclonal anti-IHF or naive rabbit serum, and revealed with goat anti-rabbit IgG-Alexafluor 594 (Invitrogen). DNA was counterstained with Prolong Gold anti-fade reagent with DAPI (Molecular Probes). Images were viewed as described prior.
• cenocepacia were collected 16 hrs after treatment, stained the unfixed cells with LIVE/DEAD® i?acLightTM Bacterial Viability and Counting kit for flow cytometry (Molecular Probes) and distinguished between live and dead bacteria using a C6 calibrated flow cytometer (Accuri). Assays were performed a minimum of three times. [0310] Enrichment of IgG from whole serum. To confirm that biofilm resolution was mediated by antibody and not other serum components, IgG was purified from rabbit anti- IHF serum with HiTrap Protein G HP columns (GE Healthcare) according to manufacturer's instructions and collected both the eluent that passed through the column after application of serum and the IgG-enriched fraction.
• Membranes were blocked overnight at 4°C in 3% skim milk in Tris-buffered saline with 0.5% Tween20 (Fisher Scientific) prior to incubation with rabbit anti-IHF or IgG-enriched anti-IHF followed by goat anti-rabbit IgG- HRP (Invitrogen). Blots were developed with CN/DAB substrate kit (Pierce) and images captured with BioRad GS800 densitometer.
• cenocepacia were inoculated into LB broth containing two-fold serial dilutions of the following antibiotics: ceftazidime, ciprofloxacin, imipenem, meropenem, minocycline, sulfamethoxazole-trimethoprim or tobramycin (Sigma). Cultures were incubated static for 24 hrs, and turbidity of the culture assessed. The MIC for planktonic B. cenocepacia was identified as the concentration of antibiotic at which no bacterial growth was observed. With this information, established B.
• cenocepacia biofilms were treated with a 1 :50 dilution of anti-IHF, antibiotic at the determined MIC, anti-IHF plus antibiotic or medium alone for 16 hrs prior to viability stain with LIVE/DEAD® 5acLightTM Bacterial Viability kit and visualization by confocal microscopy as previously described.
• antibiotics ceftazidime- 16 ⁇ g/ml, ciprofloxacin- 2 ⁇ g/ml, imipenem- 32 ⁇ g/ml, meropenem- 16 ⁇ g/ml, minocycline- 4 ⁇ g/ml, sulfamethoxazole- trimethoprim- 16 ⁇ g/ml and tobramycin- 512 ⁇ g/ml.
• Quantitation of average thickness and mean biomass was determined via COMSTAT2 analysis. Assays were performed a minimum of three times.
• cenocepacia strain K56-2 biofilms with Pulmozyme® (dornase alpha; Genentech, Inc.), established biofilms were treated with a 1 :50 dilution of anti-IHF, 0.25 mg of Pulmozyme® , anti-IHF plus Pulmozyme® or medium alone for 16 hrs prior to viability stain and visualization by confocal microscopy. Quantitation of average thickness and mean biomass was determined via COMSTAT2 analysis. Assays were performed a minimum of three times.
• B. cenocepacia strain MHK1 (Hamad et al. (2010) Appl. Environ. Microbiol. 76:3170-3176) which has a mutation in an antibiotic efflux pump that confers gentamicin sensitivity but does not alter trafficking of the mutant in macrophages was used for this assay.
• Strain MHK1 was cultured as described above then treated with naive serum or anti-IHF serum at a
• B. cenocepacia strain K56-2 statically was grown in a chamber slide for 24 hrs before labeling with FilmTracer FM 1-43.
• FIG. 1A B. cenocepacia formed a robust biofilm of approx. 26 ⁇ height with characteristic towers.
• the unfixed biofilm was labeled with a monoclonal antibody to detect the presence of dsDNA (white).
• the biofilm formed contained an abundant amount of eDNA that was particularly dense at the base of the biofilm (FIG.
• cenocepacia To determine whether biofilms formed by B. cenocepacia strain K56-2 contained a member of the DNABII family of proteins, a biofilm formed in vitro by B.
• cenocepacia strain K56-2 was incubated with rabbit anti-IHF antibody [antiserum against E. coli IHF that cross-reacts with multiple DNABII family members (Goodman et al. (2011) Mucosal Immunol. 4:625-637)] and observed labeling throughout the biofilm (FIG. 1C).
• naive rabbit serum diluted 1 :50
• rabbit anti-IHF serum at an arbitrarily selected dilution of 1 :50
• the IgG enriched antiserum preparation retained the activity of the whole rabbit anti-IHF; however there was no ability of the effluent from the enrichment column to similarly disrupt the biofilm.
• Western blots were performed to demonstrate that antibodies contained within both whole anti-IHF serum as well as the fraction enriched for IgG recognized purified IHF protein as well as the mono-, di- and tri-meric forms of this protein within whole cell lysates of B. cenocepacia strain K56-2 (FIG. 3F); the DNABII family member, PG0121 ( ⁇ from Porphyromonas gingivalis), also shows the capacity to maintain multimers during SDS PAGE (S. Goodman, personal communication).
• cenocepacia at 2 hours post infection was not significantly different between those pre-treated with naive serum versus those pre-treated with anti-IHF serum (at a 1 : 1000 dilution) which suggested equivalent uptake by macrophages at this time point, by 6 hrs post infection B. cenocepacia pre-treated with anti-IHF serum were significantly impeded for growth within CF macrophages (p ⁇ 0.05) (FIG. 6).
• pre-treatment of B. cenocepacia with anti-IHF promoted their clearance by CF macrophages; however the mechanism which underlies this observation remains to be determined and is the subject of ongoing investigation.
• cenocepacia strain DFA2 (ABcsK; type VI secretion system mutant - T6SS) with anti-IHF antibody (Aubert et al. (2010) J. Biol. Chem. 285:35988-35998).
• positive labeling was distributed through the biofilm (FIG. 7A) as described earlier.
• TCTCAACGATTTA was identified, a near perfect match to the IHF binding consensus sequence WATCAANNNNTTR (where W is A or T, N is any nucleotide and R is a A or G).
• WATCAANNNNTTR where W is A or T, N is any nucleotide and R is a A or G.
• W is A or T
• N is any nucleotide
• R is a A or G
• Cystic Fibrosis is a hallmark example of a chronic and persistent disease that defies current treatment modalities.
• Biofilms resident within the lungs of CF patients contribute significantly to both pathogenesis and chronicity.
• a biofilm is a highly-organized, multicellular community encased in an extra-cellular polymeric matrix or substance (or EPS) that is affixed to an inert or biological surface and is the preferred lifestyle of all bacteria in nature.
• Bacterial populations within a biofilm as opposed to their planktonic or free-living counterparts, have a reduced growth rate (due to nutrient limitation), a distinct transcriptome (Post et al. (2007) Curr. Opin. Otolaryngol. Head Neck Surg.
• biofilms play a major role in pathogenesis and chronicity, such as CF, thus require novel methods for treatment and prevention.
• composition of the EPS of biofilms is highly diverse among genera, as well as influenced by the environment in which it is formed, a very common and critical component is the incorporation of extracellular DNA (eDNA).
• eDNA extracellular DNA
• Burkholderia cenocepacia incorporates an abundance of eDNA into the biofilms it forms.
• the presence of this wealth of eDNA is likely to provide exceptional protection to resident B. cenocepacia as a physical barrier and, as we have recently shown, eDNA within a biofilm can also bind effectors of innate immunity (Jones et al. (2012) J. Innate Immun), thus limiting or preventing their access to bacterial cells within.
• Peeters et al. Peeters et al. (2008) J. Hosp. Infect. 70:361-368
• cenocepacia were highly resistant to chlorhexidine, hydrogen peroxide and 5% bleach, even after treatment for 5 mins. Moreover, analysis of FDA product recall data for non-sterile pharmaceutical products from 1998-2006 showed that 48% of recalls were due to contamination by either B. cepacia, Pseudomonas species or Ralstonia picketti (Jimenez (2007) PDA J. Pharm. Sci. Technol. 61 :383-399). For non-sterile and sterile products, B. cenocepacia was the most frequently isolated species. Collectively, these data indicate that B. cenocepacia contamination of surfaces, equipment and
• B. cenocepacia incorporates an abundance of eDNA into their bio films.
• This eDNA is associated with a DNABII protein that interacts with antiserum directed against isolated native IHF produced by E. coli.
• B. cenocepacia biofilms are examined to determine the spatial distribution of this DNABII protein, it was found that as observed for NTHI (Goodman et al. (2011) Mucosal Immunol. 4:625-637), positive labeling was associated with each vertice of crossed strands of eDNA present within the biofilm.
• Biofilms produced by B. cenocepacia were susceptible to disruption by anti-IHF but not naive serum in an in vitro assay system.
• cenocepacia biofilm shows promise in providing a potential new approach for treatment of CF patients, and particularly those colonized with B. cenocepacia.
• the approach developed here is likely to have utility for other respiratory tract pathogens which often precede and facilitate B. cenocepacia infection of the CF lung (George et al. (2009) FEMS Microbiol. Lett. 300: 153-164).
• NTHI 86-028NP is a minimally passaged clinical isolate cultured from the nasopharynx of a child undergoing tympanostomy tube insertion for chronic otitis media. Formation of NTHI biofilms in 8-well chambered coverglass slides has been described (Jurcisek et al. (2011) J. Vis. Esp). For all biofilm assays, duplicate wells were viewed on a Zeiss 510 Meta-laser scanning confocal microscope, images compiled with Zeiss Zen software and biomass and/or mean biofilm thickness values calculated with COMSTAT2 software (Heydorn et al. (2000) Microbiology 146(Ptl0):2395-2407). All biofilm assays were repeated a minimum of three times, on separate days. Data represent mean + SEM.
• IHF-specific IgG was purified from polyclonal serum using HiTrap Protein G HP columns (GE Healthcare). Quantitation of IHF-specific IgG in both polyclonal and IgG- enriched anti-IHF ⁇ . co 3 ⁇ 4- was determined by slot blot versus purified IHF £ co 3 ⁇ 4. A standard curve was generated using rabbit reference serum versus purified rabbit IgG (Bethyl Laboratories, Inc.) and band intensity analyzed using Alpha View software (ProteinSimple).
• Biofilms were established for 24-, 48-, and 96 hr, or 1- and 2 weeks. To maintain bacterial viability, medium (brain heart infusion broth supplemented with 2 ⁇ g ml "1 each of ⁇ - NAD and heme) was changed twice daily. Biofilms were incubated with medium, anti-IHFg . con (a 1 :50 dilution, equivalent to 4.4 ⁇ g IHF-specific IgG well "1 for biofilms of ⁇ 48 hr or a 1 : 10 dilution, equivalent to 22.0 ⁇ g IHF-specific IgG well "1 for biofilms of >96 hr) or an equivalent volume of naive serum. After 16 hr, biofilms were stained with BacLightTM
• Bacterial Viability Kit (Molecular Probes), fixed in a solution of 1.6% paraformaldehyde, 2.5% glutaraldehyde and 4.0% acetic acid in 0.1 M phosphate buffer and viewed as described.
• Biofilms established for 24 hr were incubated with medium or either anti-IHF ⁇ . co3 ⁇ 4 or naive serum diluted 1 :50 for 0, 6, 12, 16 or 24 hr prior to viability staining and fixation as described. For the 0 hr time point, treatment was applied, and then immediately removed. To maintain bacterial viability of biofilms treated for 24 hr, treatments were replaced after 16 hr and incubated an additional 8 hr. To attempt to completely eradicate a 24 hr biofilm, a 1 :5 dilution of anti-IHF ⁇ . co u or an equivalent volume of naive serum was applied.
• IgG-enriched anti-IHF ⁇ was covalently coupled to agarose beads (>45 ⁇ diameter) via AminoLink Plus kit (Thermo Scientific). To determine whether direct contact of anti-IHF ⁇ . co u antibodies with the biofilm was required to induce resolution, 24 hr biofilms were established in optical bottom 96-well plates, then incubated with: medium, 1 :50 dilution of anti-IHF £ co u or an equivalent volume of naive serum, applied directly to the biofilm (80 ⁇ total volume) or after insertion of a 5 ⁇ pore size HTS Transwell (Corning) into the 96-well plate, medium, 0.5-, 5.0- or 50.0 ⁇ g IgG-enriched anti-IHF ⁇ .
• IHF-specific antibodies were adsorbed from serum by incubation with purified IHF £ cofi .
• Aliquots of anti-IHF £ cofi (4.4 ⁇ g IHF-specific IgG) were incubated with 2.2- or 4.4 ⁇ g purified IHF £. co 3 ⁇ 4 saline diluent, or a recombinant protein of equivalent molecular mass called 'rsPilA' (Novotny et al. (2009) PLoS One 8:e67629), for 1 hr.
• Western blot was performed to confirm adsorption of anti- IHF £ cou.
• the adsorbed sera were then applied to 24 hr NTHI biofilms per standard treatment and processing protocol.
• NTHI were prepared as described (Jurcisek et al. (2011) J. Vis. Exp.) and 10 6 CFU NTHI inoculated into wells of a 96-well plate prior to incubation with antibiotic at the MIC 90 or a 4- or 8-fold dilution thereof, with or without 1 :50 dilution of anti-IHF £ co3 ⁇ 4 or an equivalent volume of naive serum. After 16 hr, cultures were serially diluted and plated on to chocolate agar to semi-quantitate CFU NTHI/ well. Data represent mean + SEM of three independent assays.
• IhfA-3NTHi a non-reactive region
• IhfA-5 N THi reactive by antibodies against IHF £ cofi but nonreactive by anti-IHF ⁇ . co # prebound to DNA.
• NTHI biofilms established for 24 hr were treated with 1 :50 dilution of the following chinchilla sera: anti-IHF ⁇ . co3 ⁇ 4 anti-IHF ⁇ .
• co# pre -bound to DNA, naive serum, anti-IhfA-3NTHi or anti-IhfA-5NTHi for 16 hr prior to staining and assessment.
• Animal work was performed following the NIH Guide for the Care and Use of Laboratory Animals and under a protocol approved by the Nationalwide Children's Hospital Institutional Animal Care and Use Committee.
• co u were significantly reduced 94%, 86%, and 74%, respectively, compared to naive serum (/? ⁇ 0.05, FIG. 10B).
• anti-IHF ⁇ was diluted 1 : 10. Consequently, the biomasses for 96-hr or 1- or 2-week biofilms were significantly reduced by 74%, 43%, and 57%, respectively, compared to naive serum ( ⁇ 0.01 or 0.05).
• the relative quantity of eDNA increases (Jones et al. (2013) J. Innate Immun.
• ampicillin 32 ⁇ g ml "1 ), amoxicillin-clavulanate (1 ⁇ g ml “1 and 0.5 ⁇ g ml “1 , respectively) and cefdinir (0.25 ⁇ g ml "1 ).
• Twenty four hr NTHI biofilms were then exposed to anti-IHF £ cofi , diluted 1 :50, antibiotic or a combination of anti-IHF ⁇ . cof i plus antibiotic.
• anti- IHF ⁇ . co/ j mediated significant biofilm disruption (FIG. 14A), however incubation with any of the three antibiotics at the MIC 90 for planktonic NTHI did not result in bacterial death as determined by viability stain (FIGS.
• NTHI broth cultures were treated with a 1 :50 dilution of either anti-IHF ⁇ . co3 ⁇ 4 or naive serum, delivered alone or in the presence of ampicillin, amoxicillin/clavulanate, or cefdinir.
• Antibiotics were used at both the MIC 90 for planktonic NTHI and a 4-fold or 8-fold dilution thereof. No enhancement of antibiotic-mediated killing was observed upon exposure of planktonically grown NTHI to anti-IHF ⁇ . co u alone (FIGS. 20A-20C), suggesting that NTHI newly released from the biofilm by the action of anti-IHF ⁇ . co u were phenotypically unique from either their biofilm or planktonic counterparts.
• IHF from NTHI (referred to as 'IHF NTH i') was epitope mapped using a series of 20-mer overlapping peptides designed to mimic the deduced N- to C-terminus of the a-subunit of this DNABII protein.
• These peptides were screened with antisera recovered from chinchillas that had been immunized with either native IHF or IHF that had been complexed to an excess of DNA (Goodman et al. (201 1) Mucosal Immunol. 4:625-637).
• Antiserum against native ⁇ HF E . C oii was reactive with peptides predicted to represent the DNA-binding tip regions (FIG.
• NTHI biofilms incubated with anti-IHF£ CO /i complexed to DNA or anti-IhfA-3 NTHI were not altered in biofilm morphology or biomass (FIG. 16E & 16F).
• anti-IHF£. CO A incubation with anti-IhfA-5NTHi was equally efficacious to induce a significant reduction in biofilm biomass (p ⁇ 0.01) compared to naive serum.
• Bacterial biofilms are a significant contributor to most recurrent and chronic bacterial diseases, including those of the respiratory tract, urogenital tract and oral cavity. Biofilms are recalcitrant to action by the host immune system and antimicrobial agents, necessitating the development of novel treatment modalities for diseases with a biofilm component.
• eDNA is a prevalent component of the biofilm EPS of many microbes, and we previously demonstrated that members of the DNABII family of proteins play a crucial role in stabilizing the biofilm structure as exposure of biofilms to antibodies directed against IHF mediates significant disruption (Goodman et al. (201 1) Mucosal Immunol. 4:625-637).
• Table 3 SEQ ID NOS 129-139, respectively, in order of appearance
• Bacteria strain protein name ⁇ 3 sequence a3 sequence C-terminal 20 aa
• N. gonorrhoeae FA1090 (Oklahoma) IHFa TFHASQ KLKGMVEHYYDKQR TFHASQKLKGMVEHYYDKQR

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