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WO2003006672A2 - Anticorps d'acide nucleique antimicrobien, materiaux et procedes de fabrication et d'utilisation correspondants - Google Patents

Anticorps d'acide nucleique antimicrobien, materiaux et procedes de fabrication et d'utilisation correspondants Download PDF

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Publication number
WO2003006672A2
WO2003006672A2 PCT/US2002/021663 US0221663W WO03006672A2 WO 2003006672 A2 WO2003006672 A2 WO 2003006672A2 US 0221663 W US0221663 W US 0221663W WO 03006672 A2 WO03006672 A2 WO 03006672A2
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Prior art keywords
nucleic acid
antibodies
seip
seips
membrane
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PCT/US2002/021663
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English (en)
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WO2003006672A3 (fr
Inventor
David L. Scott, Jr.
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D-Squared Biotechnologies, Inc.
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Priority to AU2002316619A priority Critical patent/AU2002316619A1/en
Publication of WO2003006672A2 publication Critical patent/WO2003006672A2/fr
Publication of WO2003006672A3 publication Critical patent/WO2003006672A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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

Definitions

  • the present invention provides methods for inhibiting the proliferation of disease- causing pathogens. More particularly, the present invention provides methods for the localization and recovery of surface exposed immunogenic polypeptides (SEIP) of microbes, such as bacteria, fungi, and protozoa, which are involved in the adhesion of such pathogens to host cell surfaces, and the adhesion of iron-binding molecules to pathogen cell surfaces. Further, the SEIPs of the present invention are used as substrates to recover high affinity nucleic acid antibodies. Such isolated high-affinity nucleic acid antibodies are then used to bind pathogenic SEIPs so as to inhibit proliferation of the pathogen. The nucleic acid antibodies of the present invention are hence useful for the prevention, diagnosis, and treatment of pathogenic diseases and infections.
  • SEIP surface exposed immunogenic polypeptides
  • the cell surface of invading bacteria contains structures that prevent the entry of noxious compounds into the bacterial cell, and help bacteria evade recognition by host elements, such as antibodies and complement, while simultaneously allowing such bacteria to obtain nutrients from the host environment.
  • Bacterial cell surface components include appendages (such as capsules and fimbriae), lipopolysaccharides, porins, and receptors possessing a variety of functions. Some bacteria have surface structures or molecules that enhance their ability to attach to host cells. A number of bacteria possess pili (long hairlike projections), which enable them to attach to the membrane of the intestinal or geritourinary tract.
  • Microbial pathogens frequently take advantage of host systems for their pathogenesis. For example shedding of cell surface molecules as soluble extracellular domains (ectodomains) by P. aeruginosa is one of the host responses activated during tissue injury (Vasil and Ochsner 1999).
  • Iron (Fe3+) is an essential nutrient for the proliferation of bacteria in vivo, but is virtually unavailable (the concentration of bio-available iron is approximately 10 "18 M) in avian, animal or mammalian tissues because the iron is either intracellular or extracellular, complexed with high affinity, iron-binding proteins (Brown 1998; Fishbane 1999; Perez and Israel 2000; Calderon et al 1982; Crosa 1997;hacker and Kaper 2000; Weinnberg 1999; Ratledge and Dover 2000). Due to its extreme insolubility, Fe3+ is not transported as a monatomic ion. Hence, in microbes, iron is bound to low molecular weight carriers called siderophores.
  • pathogenic bacteria and fungi have evolved high affinity iron transport systems produced under low iron conditions, which include siderophores, specific ferric iron chelators, and iron-regulated outer membrane proteins and/or siderophore receptor proteins that are receptors for siderophores on the outer membrane of the bacterial cell (Neilands). Siderophores are synthesized by and secreted from the cells of bacteria under conditions of low iron (Neilands 1983).
  • Siderophores are low molecular weight proteins ranging in molecular mass from about 500 to about 1000 MW, which chelate ferric iron and then bind to its appropriate receptor in the outer bacterial membrane, which in turn transport the iron into the bacterial cell (Calderon 1982; van der Helm 1998; Rutz et al 1991 ; Rutz et al. 1992; Dover and Ratledge 1996).
  • these antigens are weak immunogens, i.e., the immune response generated by a specific antigen, while directed against the desired target, is not of a sufficient magnitude to confer immunity (Toropainen et al 2001). More importantly, the strategies used to prepare these antigens from membrane fractions usually contain immuno-suppressive components that can stimulate the production of auto-antibodies (Issekutz 1983; McNeff et al. 1999; Montbriand and Malone 1996; Scott et al. 2000; Zhang et al. 1999).
  • Gram-negative sepsis and septic shock primarily result from excessive endotoxin-induced production and release of inflammatory cytokines by cells of the immune system, particulariymacrophages (Zhang et al. 1999).
  • Tumor necrosis factor alpha (TNF) is the primary mediator of the systemic toxicity of endotoxin (Zhang et al. 1999). Consequently, neutralization of endotoxins represents an important aspect of a logical, multifaceted approach to treating this complex clinical syndrome. This approach is potentially specific since it does not interfere with the host defense.
  • LPS lipopolysaccharide
  • Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components.
  • the cell wall antigens (O antigens) of gram- negative bacteria are components of LPS.
  • Lipopolysaccharides are capable of eliciting a variety of inflammatory responses in an animal.
  • LPS is often considered a part of the pathology of gram-negative bacterial infections (Issekutz 1983; McNeff et al. 1999; Montbriand and Malone 1996; Scott et al. 2000; Zhang et al. 1999).
  • Campylobacter infections are known as one of the most identifiable antecedent infections associated with the development of Guillain-Barre syndrome (GBS).
  • Campylobacter is thought to cause this autoimmune disease through a mechanism called molecular mimicry, whereby Campylobacter contains ganglioside-like epitopes in the lipopolysaccharide moiety that elicit autoantibodies reacting with peripheral nerve targets.
  • Campylobacter is associated with several pathologic forms of GBS, including the demyelinating (acute inflammatory demyelinating polyneuropathy) and axonal (acute motor axonal neuropathy) forms.
  • demyelinating acute inflammatory demyelinating polyneuropathy
  • axonal acute motor axonal neuropathy
  • H ⁇ licobacter pylori lipopolysaccharide expresses Lewis x and/or y blood group antigens that mimic human gastric epithelial cells.
  • Such mimicry may have two diverging roles in pathogenesis. First, infection may break tolerance and anti-Lewis antibodies may be induced that bind to gastric mucosa and cause damage. Second, mimicry may cause "invisibility" of the pathogen to the host, thus aiding persistence of the infection.
  • pigs orally infected with H. pylori were specific for Lewis epitopes present on parietal cell H+K(+)-ATPase. In contrast, in infected patients the autoantibodies were directed to protein epitopes of H+K(+)-ATPase not induced through mimicry (Vandenbroucke-Grauls and Appelmelk 1998).
  • Acquired bacterial resistance results from mutations in a gene located in the host chromosome, or from the acquisition of new genetic information by a bacterium mainly by conjugation or transformation.
  • multiple antibiotics are introduced into an environment, multiple fluctuating pressures are created that drive the selection of certain bacterial variants that utilize multiple or multi-purpose resistance mechanisms to survive in such fluctuating environmental conditions.
  • antibiotics have long been the primary therapeutic tool for the control and eradication of bacterial infections, the continued incidence and severity of infections, the continual emergence of antibiotic- resistant bacteria, and the inherent toxicity of some antibiotics has prompted the search for alternative prophylactic and therapeutic treatments.
  • monoclonal antibody therapy is superior to conventional pharmaceutical treatments in that monoclonal antibodies are highly selective, have multiple effector functions, and are easily manipulated (e.g. radioisotope labeling etc.).
  • one therapeutic application of monoclonal antibodies is passive immunotherapy. In passive immunotherapy, exogenously produced immunoglobulins are administered directly to the recipient by injection or by ingestion.
  • passive immunotherapy must deliver an appropriate amount of an immunoglobulin to the recipient, because passive immunotherapy does not rely on an immune response in the animal being treated. Moreover, the immunoglobulins administered must be specific for the pathogen to effect treatment. Passive immunotherapy is superior to treatments that rely upon the normal immune response because of the speed at which antibodies contact the target as compared to a normal immune response. Additionally, passive immunotherapy can be used prophylactically to prevent the onset of disease or infection.
  • Clostridium difficile is the bacterial pathogen identified as the cause of pseudomembranous colitis and is principally responsible for nosocomial antibiotic-associated diarrhea (AAD) and colitis.
  • AAD results from antibiotic-induced alteration of the normal flora of the intestine, allowing C. difficile to proliferate.
  • purified immunoglobulins of the appropriate specificity are administered to act as a passive barrier to pathogenic invasion.
  • monoclonal antibodies recovered from mice immunized with polysaccharides from Neisseria meningitidis Group B bind and opsonize several K1 -positive Escherichia coli strains regardless of their lipopolysaccharide (LPS) serotypes (Toropainen et al. 2001 ).
  • LPS lipopolysaccharide
  • monoclonal antibodies protect against lethal challenges with E. coli K1 and Group B meningococcal organisms in mice.
  • mouse monoclonal antibodies are useful in treating infections in mice, their effectiveness in treating disease in other animals (including humans) is limited.
  • the human immune system is capable of recognizing any mouse monoclonal antibody as a foreign protein resulting in an accelerated clearance of the antibody, and thus abrogation of its pharmacological effect.
  • a more serious consequence of using mouse monoclonal antibodies in humans (or other animals) could be shock or even death (analogous to "serum sickness").
  • anti-mouse immunoglobulin responses limit the utility of mouse monoclonal antibodies in approximately one-half of the patients receiving such antibodies for treatment of various conditions (Tjandra et al., 1990).
  • pooled human immunoglobulin compositions Another limitation associated with pooled human immunoglobulin compositions is that the pre-selection process itself requires very expensive, continuous screening of the donor population, to assure product consistency, and despite considerable effort, the product varies from batch to batch and according to trie geographic location of donors.
  • a further limitation inherent in pooled immunoglobulin compositions is that their use results in the coincidental administration of large quantities of extraneous proteinaceous substances (e.g., viruses) having the potential to cause adverse biologic effects.
  • extraneous proteinaceous substances e.g., viruses
  • the combination of low and inconsistent titers of desired antibodies, and high content of extraneous substances in the composition often limits the amount of specific immunoglobulins administrable to the patient to suboptimal levels.
  • nucleic acid antibodies offer an attractive alternative.
  • nucleic acid antibodies can be employed to target biological structures, such as cell surfaces or viruses, through specific interactions with a molecule that is an integral part of that biological structure.
  • nucleic acid antibodies are advantageous in that they are not limited by self-tolerance, as are conventional antibodies.
  • nucleic acid antibodies do not require animals or cell cultures for synthesis or production.
  • nucleic acids can bind to complementary nucleic acid sequences. This property of nucleic acids has been extensively utilized for the detection, quantification and isolation of nucleic acid molecules. Moreover, it is also well known in the art that a number of proteins function by binding to nucleic sequences, for example regulatory proteins that bind to nucleic acid operator sequences. The ability of certain nucleic acid binding proteins to bind to their targets has been used in the detection, quantification, isolation and purification of such proteins. In the present application, novel, non-naturally-occurring nucleic acid sequences that bind to nucleic acid binding proteins, are developed using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). See U.S. Patent 5,270,163.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • SELEX is a method for isolating oligonucleotide ligands of a chosen target molecule that involves admixing a target molecule with a pool of oligonucleotides (e.g., RNA) of diverse sequences, retaining complexes formed between the target and oligonucleotides, recovering the oligonucleotides bound to the target, reverse-transcribing the RNA into DNA, amplifying the DNA with polymerase chain reactions (PCR), transcribing the amplified DNA into RNA, and repeating the cycle with ever increasing binding stringency.
  • oligonucleotides e.g., RNA
  • PCR polymerase chain reactions
  • the present invention allows the general use of nucleic acid molecules for the detection, quantification, isolation, and purification of novel proteins, which have not previously been identified as nucleic acid binding proteins in the art.
  • nucleic acid antibodies isolated by SELEX are used to affect the function of specific target molecules or structures, for example by inhibiting, enhancing, or activating specific proteins.
  • SEIPs are used as substrate to select high affinity oligonucleotide-ligands. This strategy has led to the identification of a specific oligoncucleotides (nucleic antibodies) that inhibits the reception of iron-binding molecules as well as adhering of pathogenic microbe to target tissue. Oligonucleotides having specificity for receptors for iron-binding ligands and adhesin proteins on gram-positive and gram-negative bacteria have not been previously described in the art.
  • Objects of the present invention are accomplished by a novel method for the production of nucleic acid antibodies and anti-microbial agents against target surface exposed immunogenic polypeptides (SEIP) recovered from microbes comprising the following steps:
  • SEIP's with individual recombinant SEIP's, then adding the mixture to growth medium containing the target microbe, the successful growth of the microbe is indicative of a SEIP that can be used to generate anti-
  • target SEIPs are recovered from bacteria, fungi or protozoa.
  • the membrane-associated receptors preferably include siderophore receptors.
  • the host comprises a vertebrate host.
  • SEIP novel surface exposed immunogenic polypeptides
  • (j) identify and select recombinant SEIPs that reverse the effects of anti- SEIP on a target microbe; this is accomplish by incubating the anti- SEIP's with individual recombinant SEIP's, then adding the mixture to growth medium containing the target microbe, the successful growth of the microbe is indicative of a SEIP that can be used to generate anti-
  • it is an object of the present invention to provide a method for recovering high-affinity nucleic acid antibodies that bind one or more SEIP's comprising the following steps: a. forming an antibody-SEIP complex by contacting a candidate mixture containing random nucleic acid antibodies to at least one polypeptide fragment, or synthetic polypeptide fragment, of a SEIP; D. separating the antibody-SEIP complex from unbound nucleic acid antibodies; and, c. detecting and identifying the antibodies bound to SEIP.
  • antibody-SEIP complexes are separated from unbound nucleic acid antibodies using various techniques including affinity purification, magnetic separation, or size exclusion. Moreover, detection and identification of antibodies bound to SEIPs, in a preferred embodiment, is performed using techniques including polymerase chain reaction (PCR) or DNA sequencing.
  • PCR polymerase chain reaction
  • the candidate mixture contains single-stranded nucleic acid antibodies.
  • the candidate mixture contains oligonucleotide antibodies, wherein each oligonucleotide antibody comprises a conserved region and a randomized region, and is between 25 to 40 base pairs in length.
  • the SEIPs are receptors of iron-binding molecules. These receptors of iron-binding molecules are comprised of the sequences of SEQ ID NO:1 - SEQ ID NO:22.
  • recovered high-affinity nucleic acid antibodies are modified to make them substantially resistant to endogenous nuclease activity by a method comprising, a. modifying a nucleotide base; b. modifying at least one phosphodiester bond; and c. modifying the ⁇ '-terminus, the 3'-terminus, or both the 5' and 3'- terminus of said antibodies.
  • the high-affinity nucleic acid antibodies are protonated or acidified. It is an object of the present invention to provide a method for treating infections and diseases caused by microbes, including bacteria, fungi and protozoa, comprising administering a composition that comprises of a therapeutically effective dose of one or more purified and isolated, novel high-affinity nucleic acid antibodies and a pharmaceutically acceptable carrier or diluent to a patient in need of such treatment. It is also an object of the invention to provide a pharmaceutical composition comprising, a therapeutically effective dose of one or more purified and isolated, novel high-affinity nucleic acid antibodies, and a pharmaceutically acceptable carrier or diluent.
  • the patient comprises a mammal, including human.
  • the patient comprises a vertebrate organism other than a mammal, such as, but not limited to avians, reptiles, amphibians, and fish.
  • SEIPs also include micribial pili (long hairlike projections), which enable microbes to attach to the membrane of the intestinal or geritourinary tract, adhesion molecules that attach to both the bacterium and ciliated cells of the upper respiratory tract, or cell surface components that are shed as soluble extracellular domains (ectodomains) by microbes in response to host immune responses activated during tissue injury.
  • micribial pili long hairlike projections
  • Endotoxins are invariably associated with gram-negative bacteria as constituents of the outer membrane of the cell wall, and are commonly co-purified with outer membrane polypeptides.
  • the term "endotoxin” is also occasionally used to refer to any "cell-associated” bacterial toxin, and specifically encompasses lipopolysaccharide complexes associated with the outer envelope of gram-negative bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria,
  • Membrane-associated polypeptides are any polypeptides that are localized to the outermost protective layer of a target microbe and include surfaced exposed immunogenic polypeptides (SEIP's) such as membrane-associated receptors complexed with their iron-binding ligands.
  • SEIP's surfaced exposed immunogenic polypeptides
  • Ligands are defined as molecules that bind to other molecules; in normal usage a ligand is a soluble molecule, such as a hormone or neurotransmitter that binds to a receptor.
  • antibodies refers to a highly selective molecule that can bind to a specific target among millions of irrelevant sites.
  • Monoclonal antibodies are produced by a single clone of hybridoma cells, and therefore represent a single species of an antibody molecule.
  • polyclonal antibodies is used to represent antibodies raised in immunized animals, or created from multiple clones. Hence, the term is used to characterize multiple species of an antibody molecule.
  • High-affinity nucleic acid antibodies are single-stranded DNA oligonucleotides that bind to specific molecular targets.
  • a "therapeutically effective amount” is intended to mean that amount of an agent that, when administered to an animal in need of such treatment, is sufficient to effect treatment for a pathogenic disease mediated by SEIPs.
  • a therapeutically effective amount of a nucleic acid antibody is a quantity sufficient to modulate, regulate, or inhibit the activity of one or more SEIPs such that a disease condition which is mediated by that activity is reduced or alleviated.
  • Treating is intended to mean at least the mitigation of a disease condition in an animal, such as a human, that is affected, at least in part, by the activity of one or more SEIPs, and includes: preventing the disease condition from occurring in an animal, particularly when the animal is found to be predisposed to having the disease condition but has not yet been diagnosed as having it; modulating and/or inhibiting the disease condition; and/or alleviating the disease condition.
  • compositions of this invention comprise an effective modulating, regulating, or inhibiting amount of a nucleic acid antibody and an inert, pharmaceutically acceptable carrier or diluent.
  • the compositions are prepared in unit-dosage form appropriate for the mode of administration, e.g., parenteral or oral administration.
  • An inventive agent is administered in conventional dosage form prepared by combining a therapeutically effective amount of an agent (e.g., a nucleic acid antibody) as an active ingredient with appropriate pharmaceutical carriers or diluents according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
  • an agent e.g., a nucleic acid antibody
  • the pharmaceutical carrier employed may be either a solid or liquid.
  • solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • liquid carriers are syrup, peanut oil, olive oil, water and the like.
  • the carrier or diluent may include time-delay or time-release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
  • a variety of pharmaceutical forms can be employed.
  • a solid carrier used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
  • the amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g.
  • a liquid carrier is used, the preparation will be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension.
  • the agent may be dissolved in a suitable cosolvent or combinations of cosolvents.
  • suitable cosolvents include, but are not limited to, alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, gylcerin and the like in concentrations ranging from 0-60% of the total volume.
  • the composition may also be in the form of a solution of the active ingredient in an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution. It will be appreciated that the actual dosages of the agents used in the compositions of this invention will vary according to the particular complex being used, the particular composition formulated, the mode of administration and the particular site, host and disease being treated. Optimal dosages for a given set of conditions can be ascertained by those skilled in the art using conventional dosage- determination tests in view of the experimental data for an agent.
  • compositions of the invention may be manufactured in manners generally known for preparing pharmaceutical compositions, e.g., using conventional techniques such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing.
  • Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, which may be selected from excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • compositions for oral use can be obtained using a solid excipient in admixture with the active ingredient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.
  • Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the antibodies or antimicrobial agents according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the antimicrobial agents or nucleic acid antibodies may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit-dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g, containing conventional suppository bases such as cocoa butter or other glycerides.
  • the antibodies may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the antibodies may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for hydrophobic agents is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the cosolvent system may be a VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:5W) contains VPD diluted 1 :1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic agents well, and itself produces low toxicity upon systemic administration.
  • co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.
  • hydrophobic pharmaceutical agents may be employed.
  • Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
  • compositions also may comprise suitable solid- or gel- phase carriers or excipients.
  • suitable solid- or gel- phase carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • homologous refers to a nucleotide or protein sequence that is substantially similar to another such that it is complimentary to the other sequence and capable of hybridizing to it.
  • Equivalent is used when referring to two nucleotide sequences, wherein the two nucleotide sequences in question encode the same sequence of amino acids. When “equivalent” is used in referring to two peptides, it means that the two peptides will have substantially the same amino acid sequence (i.e. at least 70% homologous).
  • equivalent refers to a property, the property does not need to be present to the same extent (e.g., two peptides can exhibit different rates of the same type of enzymatic activity), but the properties are preferably substantially the same.
  • the term “substantially” varies with the context as understood by those skilled in the relevant art and generally means at least 70%, preferably means at least 80%, more preferably at least 90%, and most preferably at least 95%.
  • the phrase “substantially similar” includes complete identity as well as less than complete identity (e.g., of amino acid sequences or enzymatic activity).
  • isolated refers to, e.g., a peptide, DNA, or RNA separated from other peptides, DNAs, or RNAs, respectively, and being found in the presence of (if anything) only a solvent, buffer, ion or other component normally present in a biochemical solution of the same. "Isolated” does not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure substances or as solutions.
  • SEIP surface exposed immunogenic polypeptides
  • the cells from each culture were concentrated by centrifuging the culture in 250 ml tubes for 10 min at 5,000 x g in a model J2-21 Beckman centrifuge, and the membrane associated polypeptides from each pathogen were recovered separately as described below.
  • the supernatant was stored at -20 C, and the bacteria and fungi cells were gently resuspended in 250 ml of ice-cold HE (10 mM Hepes [ph 7.4] and 1 mM EDTA) buffer. The centrifugation step was then repeated.
  • the supernatant Prior to freezing the cell culture, the supernatant was analyzed for the presence of siderophores (henceforth iron- reactive material) using the Chrome Azural S (CAS). Each one of the culture supernatants tested positive for iron-reactivity.
  • the culture supernatants were also analyzed for the presence of endotoxins (LPS).
  • the concentrated cells were resuspended in 17 ml of HE buffer in a 50 ml sterile tube, and then frozen in liquid nitrogen and thawed at room temperature. This step was repeated until the solution became viscous. Next the lysates were processed to separate and purify the membrane-associated polypeptides from other cell wall components, including lipolysaccharides (endotoxins).
  • solubilization buffer 10 mM Hepes [ph 7.4]; 1 mM EDTA; 1.2 M NaCI; 2% Triton X-100
  • solubilized membranes were mixed with 10% Polyethyleneimine (PEI) by stirring until the concentration of PEl was 1% of the solubilized membrane solution.
  • PEI Polyethyleneimine
  • the PEI supernatant and pellet (resuspended in HE buffer) were analyzed for iron-reactive material and the presence of LPS as described previously.
  • the iron-reactivity was identified in the PEI supernatant while the LPS contamination molecules were localized to the PEI pellet.
  • the iron-reactive PEI supernatants 50 ml were mixed by slow stirring with
  • the ammonium sulfate precipitate obtained from each pathogen was resuspended in 25 ml of HE buffer (10 mM Hepes [ph 7.4]; 1 mM EDTA) and size- fractionated by tangential-flow ultra centrifugation against a membrane with an apparent cut-off of 30 Kda.
  • the filtrate and retainate were analyzed for iron-reactivity (using the CAS assay), and the presence of LPS molecules.
  • the iron- reactive material is low in molecular weight (500-1000 daltons). Thus the iron- reactivity was expected to be found in the filtrate. In stead, iron-reactivity was found in the retainate, and no LPS contamination molecules were detected in either fraction.
  • polypeptide preparations as described above or individual SEIPs are used to immunize a host, for example chicken, goat and rabbit, which is known in the art for quantitative production of antibodies.
  • D2 DLS-05 antigen cocktail was used to generate antisera in laying hens commercially by Aves Lab (Tigard, Oregon, www.aveslab.com). Briefly, laying hens were immunized 3 times subcutaneously at intervals of two weeks, using complete Freund's adjuvant for the first injection and incomplete Freund's adjuvant for subsequent injections. Aves Labs purifies Immunoglobulins (IgY) from egg yolks using a proprietary method. See also Lee, et al., Preparative Biochemistry and Biotechnology, 27(4):227-37 (1997).
  • IgY Immunoglobulins
  • the recovery of antisera from a host that has been previously immunized with purified membrane-associated polypeptides provides information useful for the identification of determinants of these polypeptides that are surface exposed. Moreover, the ability of the antisera to recognize these antigenic determinants in intact cells provides information as to which membrane-associated polypeptides should be targeted for diagnostic and therapeutic purposes.
  • the polyclonal antibodies were diluted 1 :10 in carbonate buffer and covalently attached to a microtiter plate by incubation at 37 C for 2 h.
  • a 5 % milk-fat protein-TBS solution was used as a blocking agent to eliminate non-specific binding of staphylococci cells.
  • 100 ⁇ l aliquots of serial dilutions (100-fold) S. aureus D2 DLS-03 cells were added to each well coated with D2 DLS-05 antisera and incubated for 1 h. After thoroughly washing away any unbound S.
  • nitrocellulose filter As described by Maniatis et al.
  • the nitrocellulose filter was blocked with 5% milk fat proteins for 1 h. After blocking, the filter was probed with a 1 :1000 dilution of D2-DLS01 antisera for 1 h. After thoroughly washing with TTBS (60 mM Tris-HCI pH 7.4; 150 mM NaCI; 0.5% Tween 20) the filter was probed with anti-lgY alkaline phosphatase conjugate (Sigma-Aldrich, St. Louis, MO) for 1 h.
  • TTBS 60 mM Tris-HCI pH 7.4; 150 mM NaCI; 0.5% Tween 20
  • the filter was washed with TTBS. After washing, the colorimetric substrate BCIP/NBT (Pierce Chemical) was added, and a colorimetric assay was allowed to proceed for 30 min at room temperature and then terminated by rinsing the filter with sterile water.
  • BCIP/NBT Pieris Chemical
  • the D2-DLS05 antisera recognized S. aureus SEIP's ranging in size from 30 kda -100 kda as determined using a broad range protein marker (BioRad).
  • target SEIP's are those that stimulate the production of antibodies that are capable of inhibiting the proliferation of pathogenic microbes in a complement-independent manner.
  • increasing dosages of purified immunoglobulins are included in a defined medium supplemented with either 100 ⁇ M 2' 2' -dipyridyl (iron-deplete) or 50 ⁇ M FeCI3 (iron- replete), and the growth of a specific pathogen is monitored by counting colony forming units (CFU) for solid medium or monitoring absorbance using spectrophotometry at 600 nm for liquid cultures.
  • CFU colony forming units
  • a B-D Falcon 48 well tissue culture plate (Fisher Scientific, Suwanee, GA) was used by placing 1 ml of M9-minimal medium supplemented with maltose (10g/ ml), casamino acids (5 mg/ml), 1 M MgSO4 (0.1%), and 120 ⁇ M 2'2' dipyridyl in each individual well.
  • the minimal medium also included D2 DLS-05 antisera at a concentration of 1 mg/ml.
  • Overnight cultures of Bacillus cereus, Cryptococcus neoformans, Staphylococcus aureus, and Stenotrophomonas maltophilia were serially diluted in increments of 100-fold.
  • serially diluted bacteria were inoculated into the M9 medium and incubated at 37 C for 18 h -24 h. Simultaneously, 100 ⁇ l portion of the serial diluted bacteria were streaked on M9- maltose agar plates and incubated at 37 C for 18 h -24 h.
  • the growth rate of each pathogen in the presence of D2 DLS05 polyclonal antibodies was evaluated by monitoring the absorbance spectrophotometrically at 600nm.
  • the proliferation of each pathogen was inhibited when less than 100 cells were inoculated in the growth medium as determined by evaluation of the colony forming units present at this dilution factor on the solid M9 medium.
  • SEIP's that stimulate the production of anti-microbial antibodies
  • target SEIP's are those that stimulate the production of antibodies that are capable of inhibiting the proliferation of pathogenic microbes in a complement-independent manner.
  • expression libraries are created using techniques known in the art using genomic DNA or cDNA depending upon the gene structure of a desired pathogen. For example, gene expression libraries from most bacteria can be constructed using size-fractionated genomic DNA since prokaryotic genes are usually uninterrupted. However, with fungi (lower eukaryote) it is necessary to use cDNA to construct expression libraries. Once an expression libraries is created, it is probed with antisera obtained from a host inoculated with an antigen cocktail as detailed in section 1.
  • a 100 ml culture of Staphylococcus aureus D2 DLS-03 grown in the appropriate selection medium was harvested at an optical density (OD) 0.6 at A600.
  • the cells were concentrated by centrifuging the culture in 50 ml tubes for 10 min at 5,000 x g in a model J2-21 Beckman centrifuge. Concentrated staphylococci cells were then resuspended in 5.0 ml DNA X-tractTM solution 2. The resuspended cells were then mixed with an equal volume of DNA X-tractTM solution 2 in a 50 ml polypropylene tube. Next 10 ml of molecular grade chloroform was added to the lysate and the mixture was made homogenous by inversion.
  • the aqueous phase (top phase) was transferred to a new tube and the chloroform extraction step and centrifugation was repeated. The aqueous phase was then transferred again to a new tube and mixed with 10.0 ml of DNA X-tractTM precipitation solution and incubated on ice for 30 min or longer.
  • the Staphylococcus genomic DNA was precipitated by centrifuging at 10,000 x g for 15 min in a microcentrifuge. The DNA pellet was washed with 1 ml 70 % ethanol, air dried and resuspended in TE.
  • Staphylococcus genomic DNA in TE was mechanically sheared in a 1 ml syringe with a 25-gauge needle.
  • the sheared DNA was made blunt-ended by adding water to a final volume of 405 ⁇ l, 45. ⁇ L of 10x S1 nuclease buffer (2M NaCI, 50 mM NaOAc, pH 4.5, 10 mM ZnSO 4 , 5% glycerol), and 1.7 ⁇ l of S1 nuclease at 100 U/ ⁇ l and incubating at 37 C. for 15 min.
  • the sample was extracted once with phenol/chloroform and once with chloroform. Then, 1 ml of ethanol was added to precipitate the DNA.
  • the sample was incubated on ice for 10 min or at -20 C overnight and the DNA was harvested by centrifugation in a microcentrifuge for 30 min.
  • the DNA was washed with 70% ethanol and dried. EcoRI sites in the DNA sequence were then methylated using standard procedures. 5 ⁇ l of 100 mM MgCI 2 , 8 ⁇ l of dNTP mix (2.5 mM each of DATP, dCTP, dGTP, and dTTP), and 4. ⁇ l of 5 U/. ⁇ l Klenow was then added to this methylated DNA.
  • the mixture was incubated at 12 C for 30 min.
  • the DNA was resuspended in 7 ⁇ l of TE, and 14 ⁇ l of phosphorylated Eco Rl linkers (200 ng/ ⁇ l), 3 ⁇ l of 10X ligation buffer, 3 ⁇ l of 10 mM ATP, and 3 ⁇ l of T4 DNA ligase (4 U/ ⁇ l) was added to the solution.
  • the sample was then incubated at 4 C overnight, then incubated at 68 C for 10 min. to inactivate the ligase. 218 ⁇ l of H 2 O, 45. ⁇ l of 10x Universal buffer, and 7 ⁇ l of Eco Rl at 30 U/ ⁇ l were then added to the mixture. After incubation at 37 C for 1.5 h, 1.5 ⁇ l of 0.5M EDTA was added, and the mixture was placed on ice.
  • the DNA was size fractionated on a sucrose gradient, pooling fractions containing DNA ranging in size from 6-10 kb.
  • the pooled DNA was ethanol- precipitated and resuspended in 5 ⁇ l of TE buffer.
  • 20 ng of insert DNA was ligated for 2-3 days at 4 C with 1 ⁇ g of ZAP II vector in a final volume of 5 ⁇ l.
  • the ligation mixture was packaged using GIGAPACK II GOLD (Stratagene) and plated on E. coli SURE cells on NZY plates.
  • the library was titrated, amplified, and stored at 4 C under 0.3% chloroform.
  • the target pathogen-lambda-ZAP library was plated on E.
  • plaques were transferred onto nitrocellulose membranes, which had been pre-soaked in 10 mM IPTG to induce expression from the pBluescript lacZ promoter. Filters were blocked using 0.5% skim milk in 50 mM Tris-HCI, 150 mM NaCI, pH 7.5, prior to being probed with the anti-D2DLS05 polyclonal antibodies for the identification of recombinant SEIP's.
  • the plaques of interest were picked using a sterile toothpick from the agar plate and transferred to a sterile microcentrifuge tube containing 500 ⁇ l of SM buffer and 20 ⁇ l of cloroform.
  • the solution was vortexed to release the phage particles into the SM buffer, then incubated for 1-2 hours at room temperature or overnight at 4 C. Simultaneously, overnight cultures of XL1-Blue MRF' and SOLR cells in LB broth, supplemented with 0.2% (w/v) maltose and 10 mM MgSo4, at 30°C, were grown. The next day, the cells were gently concentrated by centrifugation at 1000 x g and resuspended in 10 mM MgSO4 at a OD600 of 1.0.
  • a Falcon 2059 polypropylene tube 200 ⁇ l of the diluted XL1-Blue MRF' cells were mixed with 250 ⁇ l of phage stock (> 1 x 105 phage particles), and 1 ⁇ l of ExAssist helper phage (> 1 x 106 pfu/ ⁇ l) to release the phage particles at 37 C for 15 min.
  • 3 ml LB broth was added to the tube, and incubated for 2.5-3.0 h at 37 C with shaking.
  • the tube was placed at 65 C for 20 min, then centrifuged at 100 x g for 15 min. The supernatant was decanted into a fresh tube.
  • the excised phagemids were plated by adding 200 ⁇ l of freshly grown SOLR cells (OD600 1.0) to two 1.5 ml microcentrifue tubes. A 100 ⁇ l aliquot of the phage supernatant was added to one 1 ml tube, and 10 ⁇ l was added to another. The tubes were then incubated at 37 C for 15 min. At the completion of the incubation step, 200 ⁇ l of the cell mixture was plated on LB-amplicillin agar plates (50 ⁇ g/ml) and incubated overnight at 37 C. The next day individual colonies were picked and grown in 10 ml of LB-amplicillin medium.
  • the cells were concentrated by centrifuging the culture in 50 ml tubes for 10 min at 5,000 x g in a model J2-21 Beckman centrifuge.
  • the concentrated bacteria were resuspended in 2.5 T.E. buffer ph 8.0 then mixed with an equal volume of DNA X-tractTM solution 1 in a 50 ml polypropylene tube.
  • the mixture was made homogenous by inversion, and then centrifuged at 10,000 x g for 10 min.
  • the supernatant was transferred to a new tube containing an equal volume of molecular grade chloroform extraction.
  • the mixture was made homogenous by inversion and then centrifuged at 10,000 x g for 10 min.
  • aqueous phase top phase
  • DNA X-tractTM precipitation solution 10.0 ml
  • DNA X-tractTM precipitation solution 10.0 ml
  • DNA was precipitated by centrifuging at 10,000 x g for 15 min in a microcentrifuge.
  • the DNA pellet was washed with 1 ml 70 % ethanol, air dried and resuspended in TE or water.
  • the purified phagemid DNA was then sequenced and its amino acid sequence was deduced. A 100 ⁇ l aliquot of 10 X M9-minimal medium, supplemented with glucose
  • nucleic acid antibodies offer an attractive alternative.
  • nucleic acid antibodies can be employed to target biological structures, such as cell surfaces or viruses, through specific interaction with a molecule that is an integral part of that biological structure.
  • Nucleic acid antibodies can be selected against many kinds of targets, including proteins, small organic molecules, and carbohydrates (see Klug and Famulok, Molecular Biology Reports 1994, 20, 97-107). Thus, selection of nucleic acid antibodies from a candidate mixture of nucleotide sequences offers a simple and flexible mechanism for obtaining a probe nucleic acid sequence against virtually any target molecule.
  • oligonucleotides antibodies in a candidate mixture contain a variable region flanked by one or two constant regions.
  • the variable region contains randomized sequences while the constant region(s) contains primer binding sites for amplification and/or sequencing of the oligonucleotide antibody, and/or restriction sites for cloning.
  • the variable region is designed to contain molecular diversity from which specific ligands can be selected. For example, Ferric Technologies Aptamer Library 25 (n25) or Ferric
  • n40 Feric Technologies, Inc., Atlanta, Georgia
  • a SEIP for example a receptor of iron-binding ligands, or adhesin proteins
  • purified LPS from a target pathogen in PBS and incubated at room temperature for 15 min.
  • Unbound aptamers are separated using a spin column with a specified MW limitation. For example, a micron 30 filter will retain nucleic acid sequences greater than 100 bases, while allowing nucleic acid sequences less than 100 bp to be collected in the filtrate.
  • the retainate containing the aptamer-SEIP's complex(es) are used as substrate for PCR.
  • the PCR is assay is for a total of twenty-five to thirty cycles under the following conditions: denaturing at 94 C for 45 sec, annealing at 60 C for 45 sec, and chain extension at 72 C for 1 min.
  • the PCR products are verified on a 2% agarose TBE electrophoresis gel, and digested with restriction enzymes Kpn 1 and BamHL
  • the digested fragments are ligated and cloned into vector pBlueScript KS (Stratagene), and transformed into bacterial host DH5-alpha. Individual colonies were identified and sequenced.
  • the resulting anti-SEIP's aptamers are synthesized or recovered by restriction digestion from the pBlueScript KS (Stratagene) vector and used in in vitro growth inhibition assays.
  • the minimal medium also included purified 1-10 ⁇ g of individual anti-SEIP aptamers.
  • the final volume of the medium was 1 ml by adding an appropriate volume of sterile water.
  • An overnight culture of a target pathogen was serially diluted in increments of 100-fold.
  • One-microliter aliquots of the serial diluted pathogen were used to inoculate M9 medium, and it was incubated at 37 C for 18-24 h.
  • the growth rate of the pathogen in the presence of the nucleic antibodies was evaluated by monitoring absorbance spectrophotometrically at 600 nm.
  • Blocking the primary stages of infection may be the most effective strategy to prevent bacterial infections.
  • Bacterial attachment usually involves an interaction between a bacterial surface protein called an adhesin protein and the host cell receptor.
  • Nucleic acid antibodies can be evaluated for their ability to prevent a target pathogen from attaching to surface components of the target host cell or solid supports by methods described in the art.
  • a target pathogen is inoculated on the appropriate solid medium 24-48 hrs at the appropriate temperature.
  • Cells are scraped off the plates and resuspended in cold phosphate-buffered saline (pH 7.5) to an optimal density of 10 9 cfu/ml.
  • 100 ul of cells are added to an assay tube containing 1 ml of PBS, 0.1% Tween 80, and 100 ul of a collagen, fibronection, or laminin modified with a detectable label. The mixture is incubated at room temperature for 1 hr. Tubes are then centrifuged in Eppendorf centrifuge for five min. The supernatants are carefully aspirated, and the pellets are measured for activity as determined by the type of label used to modified the target polypeptide.
  • a nucleic acid is considered pure when it has been isolated so as to be substantially free of incomplete nucleic acid products produced during the synthesis of the desired nucleic acid.
  • a purified nucleic acid will also be substantially free of contaminants, which may hinder or otherwise mask the antibacterial activity of the oligonucleotide.
  • a nucleic acid is able to bind to, or gain entry into, a target cell to modulate a physiological activity of interest, it shall be deemed as substantially free of contaminants that would render the nucleic acid less useful.
  • a variety of standard methods were used to purify/produce the presently described antibacterial nucleic acids.
  • nucleic acids are synthesized using commercial phosphoramidites on commercially purchased DNA synthesizers from ⁇ 1 uM to >1 mM scales using standard phosphoramidite chemistry and methods that are well known in the art.
  • the nucleic acids are deprotected following phosphoramidite manufacturer protocols. Unpurified nucleic acids are either dried down under vacuum or precipitated and then dried.
  • Nucleic acid antibodies are then purified by chromatography on commercially available reverse phase or ion exchange media. Peak fractions are combined and the samples are concentrated and desalted via alcohol (ethanol, butanol, isopropanol, and isomers and mixtures thereof, etc.) precipitation, reverse phase chromatography, diafiltration, or gel filtration.
  • an appropriate dosage of nucleic acid antibodies, or mixture thereof may be determined by any of several well-established methodologies. For instance, animal studies are commonly used to determine the maximal tolerable dose, or MTD, of bioactive agent per kilogram weight. In general, at least one of the animal species tested is mammalian. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human. Additionally, therapeutic dosages may also be altered depending upon factors such as the severity of infection, and the size or species of the host.
  • the presently described antibacterial nucleic acids may be administered to a patient by virtually any means used to administer conventional antibiotics. A variety of delivery systems are well known in the art for delivering bioactive compounds against bacteria in an animal.
  • nucleic acid antibodies are preferably administered with a pharmaceutically acceptable carrier, via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, intracranial, subdermal, transdermal, intratracheal methods, or the like.
  • the carrier may take a wide variety of forms depending on the form of the preparation desired for administration, e.g., intravenous, oral, topical, aerosol (for topical or pulmonary delivery), suppository, parenteral, or spinal injection.
  • nucleic acid antibodies are formulated in a water, ethanol, and propylene glycol base for topical administration.
  • nucleic acid antibodies are formulated in a water, ethanol, and propylene glycol base for topical administration.
  • preparations of acid stable protonated/acidified nucleic acids may be provided by an oral dose.
  • pulmonary infections may be treated both parenterally and by direct application of suitably formulated forms of the antibacterial nucleic acids to the lung by inhalation therapy or intranasal administration.
  • nucleic acids antibodies can accumulate to relatively high levels in the kidneys, liver, spleen, lymph glands, adrenal gland, aorta, pancreas, bone marrow, heart, and olivary glands. Nucleic acids antibodies tend to accumulate to a lesser extent in skeletal muscle, bladder, stomach, esophagus, duodenum, fat, and trachea. Still lower concentrations are typically found in the cerebral cortex, brain stem, cerebellum, spinal cord, cartilage, skin, thyroid, and prostate (Crooke 1993). Interestingly, pathogenic microbes also tend to accumulate in many of the above organs.
  • nucleic acid antibodies can be used to target infections caused by pathogenic microbes in specific target organs and tissues.
  • the presently described nucleic acid antibodies can also be effective in combating bacterial contamination of laboratory cultures, consumables (food or beverage preparations), or industrial processes.
  • bacterial infection is a particularly problematic complication in immuno-compromised individuals, such as patients suffering from acquired immuno-deficiency disease syndrome (AIDS), HIV infected individuals, patients undergoing chemotherapy or radiation therapy, etc.
  • an additional embodiment of the presently described invention is the use of the presently described nucleic acid antibodies to treat immuno-compromised patients.
  • the nucleic acid antibodies of the invention may also find usility as disinfectants, and particularly as liquid disinfectant preparations having biostatic or preferably biocidal properties.
  • the disinfectant solution contains at least a sufficient amount of nucleic acid antibodies of the invention, and may also contain other active ingredients with biostatic and/or biocidal properties.
  • the nucleic acid antibodies of the invention may also be used as disinfection solutions for skin.
  • Such compositions contain the nucleic acid antibodies in a solution that is in a vehicle suitable for topical use.
  • the disinfectant may be of the quick- drying variety, in which case it is desirable for the nucleic acid to be in an ethanol base.
  • Such solutions preferably contain an emollient for the skin as well, since the alcohol tends to be extremely drying to skin.
  • suitable emollients include, but are not limited to: a polyhydric alcohol such as polyethylene glycol, glycerin, diglycerin, propylene glycol, butylene glycol, erythritol, dipropylene glycol, and sorbitol.
  • the amount of emollient may be in the range of 0.1-3.0 w/w %, and more preferably in the range 0.2-1.5 w/w %. In the case where the content of the emollient is less than 0.1 wt % it may not be very effective, and over 3.0% the solution may be overly sticky.
  • Disinfectant solutions for the skin are especially useful as hand disinfectants, following medical treatment or waste management. Such hand disinfectants may also be useful in surgical settings, both for the medical staff and to sterilize the area of surgery on the patient. SEQUENCE LISTING
  • MOLECULE TYPE protein
  • FEATURE share homology with periplamic-binding protein class of siderophore receptors

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Abstract

L'invention se rapporte à des procédés d'inhibition de la prolifération d'agents pathogènes entraînant des maladies. Plus particulièrement, l'invention se rapporte à des procédés de localisation et de récupération de polypeptides immunogéniques exposés en surface (SEIP) de microbes, tels que les bactéries, les champignons, et les protozoaires, qui sont impliqués dans l'adhésion de ces agents pathogènes à des surfaces de cellule hôte, et dans l'adhésion de molécules de fixation de fer à des surfaces de cellule pathogène. Par ailleurs, les SEP selon l'invention sont utilisés en tant que substrats afin d'obtenir des anticorps d'acide nucléique à affinité élevée. De tels anticorps d'acide nucléique à affinité élevée isolés sont ensuite utilisés pour lier les SEIP pathogènes afin d'inhiber la prolifération de l'agent pathogène. Ces anticorps d'acide nucléique selon l'invention sont par conséquent utiles dans la prévention, le diagnostic et le traitement des maladies et des infections pathogènes.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009005040A1 (fr) * 2007-06-29 2009-01-08 Meiji Seika Kaisha Ltd. Protéine pa0427 de membrane externe de pseudomonas aeruginosa
CN101138319B (zh) * 2007-09-28 2010-04-14 中国科学院华南植物园 星果藤的组织培养繁殖方法
WO2016036839A1 (fr) * 2014-09-02 2016-03-10 The Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions et méthodes de traitement de la gonorrhée
US10900043B2 (en) 2014-09-02 2021-01-26 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for treating bacterial disease
EP4116316A1 (fr) * 2015-07-04 2023-01-11 Evaxion Biotech A/S Protéines et acides nucléiques utiles dans des vaccins ciblant pseudomonas aeruginosa

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002258746A1 (en) * 2001-04-10 2002-10-28 D-Squared Biotechnologies, Inc. Outer membrane associated polypeptides
AU2002353764A1 (en) * 2001-06-17 2003-03-18 D-Squared Biotechnologies, Inc. Staphylococci surface-exposed immunogenic polypeptides

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009005040A1 (fr) * 2007-06-29 2009-01-08 Meiji Seika Kaisha Ltd. Protéine pa0427 de membrane externe de pseudomonas aeruginosa
AU2008272087B2 (en) * 2007-06-29 2013-06-13 Meiji Seika Kaisha, Ltd. Pseudomonas aeruginosa outer membrane protein PA4710
CN101138319B (zh) * 2007-09-28 2010-04-14 中国科学院华南植物园 星果藤的组织培养繁殖方法
WO2016036839A1 (fr) * 2014-09-02 2016-03-10 The Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions et méthodes de traitement de la gonorrhée
US10286016B2 (en) 2014-09-02 2019-05-14 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for treating gonorrhea
US10900043B2 (en) 2014-09-02 2021-01-26 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for treating bacterial disease
EP4116316A1 (fr) * 2015-07-04 2023-01-11 Evaxion Biotech A/S Protéines et acides nucléiques utiles dans des vaccins ciblant pseudomonas aeruginosa
US12006342B2 (en) 2015-07-04 2024-06-11 Evaxion Biotech A/S Proteins and nucleic acids useful in vaccines targeting Pseudomonas aeruginosa

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