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WO2002083843A2 - Polypeptides associes a la membrane externe - Google Patents

Polypeptides associes a la membrane externe Download PDF

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Publication number
WO2002083843A2
WO2002083843A2 PCT/US2002/011110 US0211110W WO02083843A2 WO 2002083843 A2 WO2002083843 A2 WO 2002083843A2 US 0211110 W US0211110 W US 0211110W WO 02083843 A2 WO02083843 A2 WO 02083843A2
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WIPO (PCT)
Prior art keywords
omaps
gram
immunogenic
bacteria
iron
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PCT/US2002/011110
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English (en)
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WO2002083843A3 (fr
Inventor
David L. Scott Jr.
Carl B. Thomas
Freeman Smalls
Myron Williams
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D-Squared Biotechnologies, Inc.
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Priority to AU2002258746A priority Critical patent/AU2002258746A1/en
Publication of WO2002083843A2 publication Critical patent/WO2002083843A2/fr
Publication of WO2002083843A3 publication Critical patent/WO2002083843A3/fr

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    • 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
    • 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
    • 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
    • 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

Definitions

  • the invention relates to the field of immunology, and in particular to the production of antibodies. More specifically, it concerns producing such antibodies by a process which includes the step of immunizing a mammal, poultry, or a transgenic animal with an antigen to which antibodies are desired.
  • passive immunotherapy In which the exogenously produced immunoglobulins are administered directly to the animal being treated by injection or by ingestion.
  • passive immunotherapy must deliver an appropriate amount of an immunoglobulin to the animal, because passive immunotherapy does not rely on an immune response in the animal being treated.
  • the immunoglobulins administered must be specific for the pathogen or the molecule desired to effect treatment.
  • One advantage of passive immunotherapy is the speed at which the antibody can be contacted with the target compared to a normal immune response.
  • Passive immunotherapy can also be used as a prophylaxis to prevent the onset of diseases or infections.
  • a major potential use of passive immunotherapy is in combating bacterial infections (Atici et al. 1996; Carlander et al.
  • mouse monoclonal antibodies are useful in treating infections in mice, their effectiveness in treating disease in other mammals (including humans) is limited.
  • the human immune system is capable of recognizing any mouse monoclonal antibody as a foreign protein. This can result in accelerated clearance of the antibody and thus abrogation of its pharmacological effect. More seriously, this could conceivably lead to shock and even death from allergic reactions analogous to "serum sickness".
  • Clinical experience has shown that anti-mouse immunoglobulin responses have limited the utility of these antibodies in approximately one-half of the patients receiving mouse monoclonal antibodies for treatment of various conditions (Tjandra et al., 1990).
  • immunoglobulin compositions are assembled from large pools of plasma samples that have been pre-selected for the presence of a limited number of particular antibodies. Typically, these pools consist of samples from a thousand donors who may have low titers to some pathogenic bacteria. Thus, at best, there is only a modest increase in the resultant titer of desired antibodies.
  • immunoglobulin compositions are used in coincident 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 titers of desired antibodies and high content of extraneous substances often limits, to sub-optimal levels, the amount of specific and thus beneficial immune globulin(s) administrable to the patient.
  • chicken antibodies offer an attractive alternative (Schade et al 1992).
  • Chicken antibodies offer many advantages over mammalian antibodies. Laying hens are highly cost-effective as producers of antibodies compared with the mammals traditionally used for production.
  • yolk antibodies show great acid and heat resistance. Extraction of yolk antibodies can be performed even on a large scale without costly investment, plus concentrating them from egg yolk is a relatively straightforward process. The antibodies are not harmed by pasteurization. More importantly, the FDA regards egg antibodies as a food rather than a drag and has granted GRAS (generally accepted as safe) status.
  • chicken antibodies have biochemical advantages over mammalian antibodies due to the phylogenetical differences between avian and mammalian species, resulting in increased sensitivity as well as a decreased background in immunological assays. In contrast to mammalian antibodies, chicken antibodies do not activate the human complement system nor will they react with rheumatoid factors, human anti-mouse IgG antibodies, or bacterial and human Fc (fragment crystallizable)-receptors (Carlander et al 2000).
  • IgY systemic effects of IgY relates to the absorption or translocation of fragments of orally administered antibody from the intestine into circulation.
  • the IgY molecule is disassembled by naturally occurring enzymes in the intestine into binding fragments, which comprise peptides of the highly variable portion of the terminal domain of the antibody.
  • the peptides of the highly variable portion of the antibody, the Fab chain are taken into circulation.
  • the constant, or Fc, portion of IgY is left in the intestine. Once in the circulation, these fragments randomly search out a pathogen with the matching lectan and neutralize it by binding to that site.
  • Fab moieties unlike the Fc portion, do not elicit an allergic reaction, presumably because they are either too small or are unrecognized as foreign for some other reason. These Fab moieties can be added to the terminal end of the host's circulating globulin, wherein they are hidden from destruction but available for neutralization.
  • the cell surface of invading bacteria contains structures that prevent the entry of noxious compounds into the cell and that help the cell evade recognition by host elements such as antibodies and complement while allowing the bacteria to obtain nutrients from the environment.
  • Surface components include appendages (such as capsules and f ⁇ mbriae), 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.
  • Other bacteria such as Bordetella pertussis secrete adhesion molecules that attach to both the bacterium and ciliated cells of the upper respiratory tract (Kerr J 1999).
  • 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 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. In microbes, iron is bound to low molecular weight carriers, designated siderophores.
  • pathogenic bacteria and fungi have evolved high affinity iron transport systems produced under low iron conditions, which consist of specific ferric iron chelators, siderophores, and iron-regulated outer membrane proteins and/or siderophore receptor proteins which 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). A number of factors that are associated with pathogenesis are up-regulated in iron- limited environments (Vasil and Oschner 1999).
  • outer membrane polypeptides such as adhesions, heamein binding proteins, siderophore receptors, and transferrin receptors have been described previously in the art to induce immunity to an infection or a disease (Flock 1999; Bracken et al 1999; Calderon et al. 1982; Genco et al; Harrison et al. 1997; Kelly 2000; Korhonen et al 2000; Lin et al. 1998; Matsumoto et al. 1999; Otero 1998; Perez-Perez and Israel 2000).
  • 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 endotoxin- induced excessive production and release of inflammatory cytokines by cells of the immune system, particularly macrophages (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.
  • 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.
  • endotoxin is occasionally used to refer to any "cell- associated" bacterial toxin, it should be reserved for the lipopolysaccharide complex associated with the outer envelope of gram-negative bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, and other leading pathogens (Issekutz 1983; McNeff et al. 1999; Montbriand and Malone 1996; Scott et al. 2000; Zhang et al. 1999).
  • 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. Thus, 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 infection 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. Different strains of Campylobacter as well as host factors likely play an important role in determining who develops GBS as well as the nerve targets for the host immune attack of peripheral nerves. (Nachamkin et al. 1998).
  • H. pylori lipopolysaccharide expresses Lewis x and/or y blood group antigens in mimicry with human gastric epithelial cells.
  • Mimicry may have two diverging roles in pathogenesis. Firstly, infection may break tolerance and anti-Lewis antibodies may be induced that bind to gastric mucosa and cause damage. Secondly, mimicry may cause "invisibility" of the pathogen to the host, thus aiding persistence of infection. For example pigs orally infected with H. pylori were specific for Lewis epitopes present on parietal cell ⁇ +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).
  • An object of the present invention is a purification strategy for the purification of that are endotoxin-free, ligand-free, and preserves their antigenic potential.
  • the strategy recovers membrane-associated polypeptides from the cell walls of pathogenic microorganisms in a sufficient quantity and immunogenic quality for formulating a vaccine against disease and infection.
  • Another object of the present invention is a strategy for the localization of amino acid sequences in that are required for the reception of iron-binding ligands. This approach uncovers protective epitopes other than lipopolysaccharide (LPS).
  • a further object of the present invention is to provide a method for the purification of membrane-associated polypeptide that is endotoxin-free, ligand-free, and preserves their antigenic potential. This method can be used to recover from bacteria, fungi, or protozoans.
  • a further object of the present invention is to provide purified and isolated DNA molecules containing amino acid sequences that are useful targets in membrane associated receptor polypeptides for the active and passive immunization of animal and humans against bacteria, fungi, and protozoa pathogens. The genes, DNA sequences, recombinant proteins and peptides are useful for diagnosis, immunization and the generation of diagnostic reagents and immunotherapeutic agents.
  • Figure 1 Illustrates the predicted structure of fpth gene of Pseudomonas aeruginosa using Swiss model.
  • Figure 2 Illustrates the localization of a TonB_boxC in the fptk gene of Pseudomonas aeruginosa.
  • Figure 3 Illustrates the conserved regions and predicted ligand-binding region of fpth gene of Pseudomonas aeruginosa using BLOCKS.
  • Figure 4 Shows the consensus amino acid sequences of the Scott-Thomas domain; d2 domain 3; and d2 domain 4.
  • FIGS 5A-D Show the localization of ligand-binding region of TonB dependent outer membrane receptors in gram-negative bacteria between Scott-Thomas domain ft ⁇ and d2 domain 3 ⁇
  • Figure 6 Illustrates the strategy used to produce D2-DLS01 antigen cocktail.
  • FIG. 7 Illustrates the immunogenicity anti-D2-DLS01 antisera.
  • Figure 8 Illustrates the size range OMAPS isolated from S. maltophilia.
  • Figure 9 Illustrates the antimicrobial activity of anti-D2-DLS01 polyclonal antibodies against S. maltophilia.
  • Figure 10 Illustrates the antimicrobial activity of anti-D2-DLS01 polyclonal antibodies against S. maltophilia in iron-replete conditions.
  • Figure 11 Illustrates the antimicrobial activity of anti-D2-DLS01 polyclonal antibodies against other bacteria and fungi.
  • Figure 12 Illustrates the antimicrobial activity of anti-D2-DLS01 polyclonal Fab fragments against S. maltophilia.
  • outer membrane associated polypeptides includes any polypeptide found on the surface of a bacteria, fungi, or protozoan. These polypeptides include but are not limited to adhesins, heamein binding proteins, siderophore receptors, and transferrin receptors.
  • Suitable culture ' media for providing low iron availability and promoting production of the membrane associated receptor polypeptides in bacteria include media such as M9 minimal media which has been combined with an iron-chelating agent, for example, 2' 2' -dipyridyl, deferoxatnine, and other like agents.
  • an iron-chelating agent for example, 2' 2' -dipyridyl, deferoxatnine, and other like agents.
  • 2' 2' - dipyridyl is added to M9 medium media at a concentration of about 50 ⁇ M -500 ⁇ M preferably about 100 ⁇ M.
  • the recovery of peptides from a specific pathogen is accomplished by first culturing the pathogen in media preferred for that organism using methodologies and apparatus known and used in the art, such as a fermenter, gyrator shaker, or other like apparatus.
  • a culture may be grown in a gyrator shaker in which the media is stirred continuously with aeration at about 300-600 rev/minute, for about 15-20 hours, at a temperature and pH appropriate for growth for that organism, i.e., about 35 C-45 C and about pH 7-7.6, preferably pH 6.5-7.5.
  • the bacterial culture is then processed to separate and purify the outer membrane associated polypeptides from other cell wall including lipolysaccharide (endotoxins).
  • cell cultures are concentrated, for example, by centrifugation, membrane concentration, and the like.
  • the cell culture may be centrifuged at about 2,450-20,000 x g, preferably at about 5,000-16,000 x g, for about 5-15 minutes at about 3 C-6 C.
  • the supernatant was removed by decanting, suctioning, pipetting and the like, and the concentrated cell pellet is collected and washed in a compatible buffer solution maintained at about pH 7-7.6, such as tris- buffered saline (TBS), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (H ⁇ P ⁇ S), 3-N (N-morpholino) propanesulfonic acid (MOPS), and the like.
  • TBS tris- buffered saline
  • H ⁇ P ⁇ S N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
  • MOPS 3-N (N-morpholino) propanesulfonic acid
  • the washed pellet is resuspended and washed in a compatible buffer solution, i.e., TBS, H ⁇ P ⁇ S, MOPS and the like.
  • a compatible buffer solution i.e., TBS, H ⁇ P ⁇ S, MOPS and the like.
  • Phenylmethyl sulfonyl fluoride and Traysylol (Sigma Chemical Co., St. Louis, Mo.), protease inhibitors, were added to the lysed cells at concentrations of 1 mM and 3%, respectively.
  • the supernatant from the cell culture was analyzed for iron- reactivity using the CAS assay.
  • the bacterial cells are then disrupted by sonication, freeze-fracture, french pressure, grinding with abrasives, glass bead vortexing, and other like methods known and used in the art, preferably at a temperature of about 3 C-6 C.
  • the cell homogenate is then centrifuged at about 10,000-20,000 x g for about 10-45 min to separate the cytoplasmic (supernatant) fraction from the membrane fraction (pellet).
  • the pellet is resuspended by suctioning and pipetting, or other like method in solubilization agents known to the art such as EDTA and Triton X- 100.
  • a solubilization buffer (10 mM Hepes [ph 7.4]; 1 mM EDTA; 1.2 M NaCl; 2% Triton X-100) that contains a detergent for solubilizing the outer membrane and sodium chloride to dissociate interactions between cellular components and membrane associated receptor polypeptides, preferably at a temperature of about 3 C-6 C for 1 h.
  • Endotoxins liberated by gram-negative bacteria are frequent contaminants of aqueous and physiological solutions (Issekutz 1983; McNeff et al. 1999; Montbriand and Malone 1996; Scott et al. 2000; Zhang et al. 1999). Because of their potent biological effects in vivo and in vitro, it is often necessary to eliminate even minute quantities of endotoxin from such solutions. Thus, the high binding affinity of polymyxin B for the lipid A moiety of most endotoxins is described in the art for the removal of endotoxins from solutions. However, thus far adsorbents have failed to bind endotoxins efficiently or have shown adverse biocompatibility characteristics.
  • PEI polyethyleneimine
  • DEAE diethylaminoethyl
  • solubilized membranes are mixed with 10% Polyethyleneimine (PEI) by stirring until the concentration of PEI is 1% of the solubilized membrane solution.
  • PEI Polyethyleneimine
  • the membrane-PEI solution is stirred for 1 h in a cold room at 4 C and then centrifuged for 15 min at 10,000 x g. Then PEI supernatant and PEI pellet are analyzed for iron- reactivity using the CAS assay.
  • ammonium sulfate (18.05 g/100 ml) is added by slow continuous stirring at 4 C for 1 h.
  • the ammonium sulfate precipitate is collected by centrifugation for 15 min at 10,000 x g.
  • the precipitate (resuspended in HE buffer) and supernatant are analyzed for iron-reactivity using the CAS assay and presence of endotoxins using anti-LPS or the Limulus amoebocyte lysate assay (Cohen J 2000).
  • the PEI extraction step is repeated until endotoxins are not detected in the supernatant.
  • the siderophore receptor proteins are then separated from the bacterial outer membrane and purified by use of the anionic detergent, sodium dodecyl sulfate, preferably under non-reducing conditions. It was found that the anionic detergent sodium dodecyl sulfate (0.2%), when used as a solubilizing detergent alone without a reducing agent such as 2-mercaptoethanol, is particularly effective for extracting a high quantity of the siderophore receptor proteins without denaturing or altering their immunogenicity such that the proteins will function in vivo as effective immunogens to elicit an antibody response against gram-negative bacteria. Still another claim, US Patent 6, 190,668 (Yang), and US Patent 6. 083, 743 (Chong) use's 0.5% Triton X-100 and 20 mM EDTA to solubilized .
  • the ammonium sulfate precipitate resuspended in HE buffer (10 mM Hepes ph 7.4; 1 mM EDTA) is mixed with a reducing agent and /or denaturants such as 2- mercaptoethanol, guanidine, urea, etc... to disrapt the siderophore receptor-ligand complex.
  • a reducing agent and /or denaturants such as 2- mercaptoethanol, guanidine, urea, etc...
  • solid urea is added to the suspension at a final concentration of 6M, and size fractionated by tangential-flow ultracentrifugation against a membrane with an apparent cut-off of 30 KDa.
  • dialysis tubing with an apparent molecular weight cut-off of 1300 will be used to separate the ligand, i.e.
  • siderophores from their respective receptors.
  • Siderophores are usually low molecular weight molecules ranging in molecular mass from about 500 to about 1000.
  • the filtrate and the retainate are analyzed for iron-reactivity using the CAS assay and presence of endotoxins using anti-LPS or the Limulus amoebocyte lysate assay.
  • the concentrated membrane associated receptor polypeptides (retainate) are reconstituted in a compatible buffer, i.e., TBS, HEPES, MOPS.
  • the purified proteins may be used immediately to prepare a vaccine, or may be stored for future use through lyophilization, cryopreservation, or other like technique known and used in the art.
  • Suitable pharmacological carriers include, for example, physiological saline (0.85%), phosphate-buffered saline (PBS), Tris (hydroxymethyl aminomethane (TRIS), Tris-buffered saline, and the like.
  • Adjuvants may be included in the vaccine to enhance the immune response in the animal.
  • Desirable characteristics of ideal adjuvants include: (1) lack of toxicity; (2) ability to stimulate a long-lasting immune response; (3) simplicity of manufacture and stability in long-term storage; (4) ability to elicit both CMI and HIR to antigens administered by various routes, if required; (5) synergy with other adjuvants; (6) capability of selectively interacting with populations of antigen presenting cells (APC); (7) ability to specifically elicit appropriate T H j or T H 2 cell-specific immune responses; and (8) ability to selectively increase appropriate antibody isotype levels (for example, IgA) against antigens.
  • APC antigen presenting cells
  • Such adjuvants include, for example, aluminum hydroxide, aluminum phosphate, Freund's Incomplete Adjuvant (FCA), liposomes, ISCOM, and the like.
  • the vaccine may also include additives such as buffers and preservatives to maintain isotonicity, physiological pH and stability.
  • Parenteral and intravenous foraiulations of the vaccine may include a carrier which is a biocompatible and can incorporate the protein and provide for its controlled release or delivery, for example, a sustained release polymer such as a hydrogel, acrylate, polylactide, polycaprolactone, polyglycolide, or copolymer thereof.
  • rabbits are used to generate antisera in New Zealand White rabbits using standard techniques. Briefly, rabbits are 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.
  • an Immobilized Protein A/G column (Pierce Chemical) (3 ml) equilibrated with 5 column volumes of Immunopure IgG Binding Buffer, then 6 ml of serum from immunized host diluted 1 : 1 with binding buffer was added to the column. The diluted serum was allowed to completely flow through the column. The column was washed with 10-15 column volumes of binding buffer. IgG was eluted from the column with 3-5 columns volumes of elution buffer.
  • the IgG was collected in 1.0 ml fractions and immediately neutralized by inclusion of 100 ⁇ l of 100 mM Tris-HCl, pH 7.5 in the collection vessel. Fractions containing IgG were identified by using absorbance at 280 nm, and pooled together. Pooled IgG fractions were desalted and concentrated by buffer exchange with a 5 ml desalting column. Briefly, 1.25 ml of pooled IgG was applied to and allowed to completely flow through column. Next, 10 x 1 ml aliquots of equilibration buffer was applied to the column and collected in 1 ml fractions. The protein concentration was monitored by absorbance at 280 nm.
  • Antibodies can completely suppress or enhance the antibody response to their specific antigen by several hundredfold.
  • Immunoglobulin M enhances antibody responses via the complement system, and complement activation by IgM probably starts the chain of events leading to antibody responses to suboptimal antigen doses.
  • IgG can enhance primary antibody responses in the absence of the complement system and seems to be dependent on Fc receptors for IgG (Fc ⁇ Rs).
  • IgE enhances antibody responses via the low-affinity receptor for IgE (Fc ⁇ RII/CD23). The precise effector mechanisms that cause enhancement are not known, but direct B-cell signaling, antigen presentation, and increased follicular localization are all possibilities.
  • IgG, IgE, and IgM may also suppress antibody responses when used in certain immunization regimes, and it seems reasonable that an important mechanism behind suppression is the masking of antigenic epitopes by antibodies.
  • FcgammaRIIB which contains a cytoplasmic inhibitory motif, acts as a negative regulator of antibody responses. This receptor, however, may prevent the antibody responses from exceeding a certain level rather than causing complete suppression.
  • the immunoglobulin G (IgG) molecule consists of three globular domains of two Fab segments and one Fc segment, which are mutually connected by two flexible polypeptide chains called hinge held together by a disulfide bond (Silverton et al., 1977), as shown in Fig. 1 A. Papain hydrolyzes IgG molecules on the hinge, and cysteine or mercaptoethanol reduces the disulfide bond of IgG molecules (Utsumi, 1969). IgG molecules are separated into the two Fab and one Fc fragments by this treatment.
  • the Fab region is the antigen-binding fragment of the antibody molecule.
  • a specific region of the antigen (called the antigenic determinant) will react stereochemically with the antigen-binding region at the amino terminus of each Fab.
  • the IgG molecule which has two antigen binding fragments [(Fab)2] is said to be divalent: it can bind to two Ag molecules.
  • the polypeptide composition of the Fc region of all IgGl antibody molecules is relatively constant regardless of antibody specificity; however, the Fab regions always differ in their exact amino acid sequences depending upon their antigenic specificity. Even though the antigen does not react with the Fc region of the IgGl molecule, this should not be taken to mean that the Fc region has no importance or biological activity.
  • specific amino acid regions of the Fc portion of the molecule are recognized by receptors on phagocytes and certain other cells, and the Fc domain contains a peptide region that will bind to and activate complement.
  • complement cascade antibodies combined with the surface of microorganisms or surfaces of Ag activate the complement cascade, which has four principal effects, related to host defense, induction of the inflammatory response chemotactic attraction of phagocytes to the site of immunological encounter opsonization of cells showing foreign Ag complement-mediated lysis of certain bacteria or viruses.
  • the removal of the Fc portion of anti-outer membrane associated polypeptides is achieved by incubating purified IgG (20 mg/ml) with immobilized papain (Pierce Chemical Company, Rockford, IL). Briefly, 0.5 ml of 50% Immobilized Papain slurry (0.25 ml of settled gel) was added to a glass test tube containing 4.0 ml of freshly prepared digestion buffer (20 mM phosphate; 20 mM cysteine-HCL; 10 mM EDTA- Na4, pH7.0). The gel was separated from the buffer by centrifugation. The buffer was discarded and the wash step was repeated once more.
  • the immobilized papain was resuspended in 0.5 ml of digestion buffer and mixed with 10 mg of purified IgG dissolved in 1 ml of digestion buffer and incubated for 5 hr (or overnight) at 37 C with at high speed in a gyrator shaker. Human IgG was incubated for 4 hr under the same conditions. 1.5 ml of 10 mM Tris-HCL, pH 7.5 was added to the digest and the generated Fab fragments were separated from the immobilized Papain by centrifugation. The Fab fragments were separated from undigested IgG and Fc fragments using an affinity-purification using an immobilized ProteinA column (Product# N. 20356, Pierce Chemical Co.).
  • ELISA enzyme linked immunoabsorbent assay
  • Preliminary characterization of immunogenic is accomplished by polyacrylamide gel electrophoresis, followed by transfer step to a nitrocellulose filter.
  • the nitrocellulose filter is blocked with a responsible blocking agent solution common to the art i.e., BSA, casein, milk proteins etc.
  • the nitrocellulose filter is probed with appropriately diluted (as determined in the antibody binding assays) anti-OMAP antisera.
  • antibody binding is determined using the appropriate secondary antibody conjugated to a reporter enzyme, i.e. alkaline phosphatases; peroxidases, etc.
  • Antibody binding is quantified by the addition of a substrate that liberates a signal when contacted by the appropriate reporter enzyme.
  • the substrate can be either colorimetric or chemiluminescence.
  • To estimate the size of bound membrane polypeptides a broad range protein marker is included.
  • target OMAP's are those that stimulate the production of antibodies that are capable of inhibiting the proliferation of pathogenic microorganisms in a complement independent manner.
  • increasing dosage of purified immunoglobulins are included in a defined medium supplemented with either 100 ⁇ M 2' 2' -dipyridyl (iron-deplete) or 50 ⁇ M FeC13 (iron-replete) and the growth of a specific pathogen is monitored by counting colony forming units (CFU) for solid medium or monitoring absorbance spectrophotometrically at 600 nm for liquid cultures.
  • CFU colony forming units
  • a B-D Falcon 48 well tissue culture plate (Fisher Scientific, Suwanee, GA) 1 ml of M9-minimal medium supplemented with maltose (10g/ ml); casamino acids (5 mg/ml); 1 M MgSO4 (0.1%) and 120 ⁇ M dipyridyl was added to individual wells.
  • 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.
  • the strategy utilizes bioinformatic tools, molecular biology techniques, and recombinant DNA methods to identify effective antigenic determinants in proteins that are essential to the propagation of the target bacteria.
  • the first step in the localization of target amino acids for the development of antimicrobial immunoglobulins is to identify polypeptides in the target pathogen that are essential to the proliferation and establishment of infection.
  • the in vivo selection system identifies bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes encode virulence factors, products specifically required for the infection process (Slauch and Camilli 2000).
  • the paradigm IVET system is based on complementation of an attenuating auxotrophic mutation by gene fusion and is designed to be of use in a wide variety of pathogenic organisms.
  • the IVET system has several applications in the area of vaccine and anti-microbial drag development. This technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components.
  • the strategy described in this application for the recovery of immunogenic polypeptides provides invaluable information in reference to what epitope's, on the surface of a pathogenic microorganism, are accessible by the immune system of the said host.
  • a desired target sequence must be 1) surfaced exposed; 2) immunogenic; 3) the target sequence should be essential to the ligand contacting its membrane associated receptor; and at least conserved at the species level.
  • Recombinant FptA was expressed and purified as described previously by Heinrichs and Poole 1996. The purified FptA was covalently attached to microtiter plate and probe with anti-D2-DLS01 antisera. The anti-D2-DLS01 " antisera were successful at detecting FptA (data not shown).
  • Candidate amino acid sequences in target proteins should be continuous, i.e., the determinant is an uninterrupted fragment of the primary structure of the protein on which the determinant occurs.
  • a vaccine based on a single strain may not provide immunity in a vaccinated individual against other strains of the same microorganism, as antibodies induced by the single strain may not be reactive with antigenic determinants on other strains.
  • This problem of antigenic variability has in fact been encountered with currently available anti-rabies vaccines (Luo et al. 1998).
  • Candidate target amino acid sequences must be conserved among all strains of the target microorganism.
  • a vaccine with a synthetic peptide with such a sequence will not be limited by antigenic variability and will be effective to provide protection against all strains of the target microorganism against which the vaccine is intended to provide protection.
  • a key advantage to these approaches is that the target of the immune system is known. Therefore the sequence can be monitored by methods common to the art such as PCR and ELISA, to ensure that the peptide is still a useful immunogen. Thus, if mutations should arise in the target sequence a new peptide can be synthesized.
  • ferric complexes are too large for passive diffusion or nonspecific transport across these membranes. Furthermore, ferric-siderophore uptake is both receptor and energy dependent. Therefore, the translocation of iron through the bacterial outer membrane as the ferric-siderophore requires the formation of an energy-transducing complex with the proteins TonB, ExbB, and ExbD, which couples the electrochemical gradient across the cytoplasmic membrane to a highly specific receptor and so promote transport of the iron complex across the outer membrane. Once in the periplasmic space, the ferric-siderophore binds to its cognate periplasmic binding protein and is then actively transported across the cytoplasmic membrane by an ATP-transporter system (Van der Helm 1998).
  • This structure is believed to be a common feature of any ferric- siderophore receptor and has been proposed to function like an air lock, involving two hatches.
  • the first hatch consists of the extracellular loops, which connect the strands of the barrel and fold toward the center of the pore, whereas the N-terminal globular domain forms the second hatch.
  • Binding of the ferric-siderophore to its recognition site leads to closure of the external loops (first hatch) and opening of a channel in the periplasmic side of the membrane (second hatch), allowing the transit of the ferric- siderophore complex.
  • These structural modifications are believed to be both ferric- siderophore and TonB induced.
  • TonB The instability, low abundance, energy dependence, and requirements for other proteins (i.e., TonB) increase the difficulty of relevant physical studies on siderophore receptors (van der Helm 1998).
  • the chemical lability of ferric enterobactin further complicates a crystallographic localization of its binding site in FepA.
  • Investigators have successfully used alanine-scanning mutagenesis of charged residues to analyze protein structures (Newton et al 1999) and double mutagenesis as a streamlined site-directed genetic method for identification of crucial residues in a multivalent binding event.
  • the first step in our scheme was to analyze the FptA gene for the presence of conserved and/or functional domains.
  • SWISS MODEL an automated protein modeling software package.
  • the results of the query of the amino acid sequence of FptA in SWISS MODEL revealed that the protein structure was similar to other ferric-siderophore receptors (See figure 1).
  • the beta- structure content of fptA was typical of porins and in agreement with the 3D stractures of the siderophore receptors such as FhuA and FepA.
  • the domain search program Pfam a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains, identified a 2 nB_C motif at the C terminus of FptA (see Figure 2).
  • a second search program, BIOCKS also identified the To ⁇ B_C box as well as 2 other conserved regions in the FptA gene (see Figure 3).
  • Blocks are multiply aligned ungapped segments corresponding to the most highly conserved regions of proteins.
  • the FptA gene product was defined to contain 3 domains and 2 regions of low complexicity. Preceding from the N terminus the structural orientation of FptA was as follows: the first and second domains followed the first region of low complexicity; a second region of low sequence complexicity separated the second and third domains. The third domain contained a TonB _C terminal box.
  • BLAST Basic Local Alignment Search Tool
  • BLAST is a set of search programs designed to explore all of the available sequence databases using a heuristic algorithm which seeks local as opposed to global alignments to detect relationships among sequences which share only isolated regions of similarity (Altschul et al., 1990).
  • the BLAST programs have been designed for speed, with a minimal sacrifice of sensitivity to distant sequence relationships.
  • the scores assigned in a BLAST search have a well-defined statistical interpretation, making real matches easier to distinguish from random background hits.
  • the Gapped BLAST algorithm allows gaps (deletions and insertions) to be introduced into the alignments that are returned. Allowing gaps means that similar regions are not broken into several segments. The scoring of these gapped alignments tends to reflect biological relationships more closely.
  • PSI-BLAST Position-Specific Iterated BLAST
  • the program first performs a gapped BLAST database search.
  • the PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching.
  • PSI-BLAST may be iterated until no new significant alignments are found.
  • the purpose of using the BLAST programs was to compare the blocked amino acid sequences of the EptA to polypeptide sequences in available databases to identify sequence patterns that may be conserved among receptors of iron binding ligands.
  • BLAST analysis of the first region of low complexity returned 24 hits from available databases.
  • BLAST analysis of the first domain returned 302 hits from available databases.
  • the results were receptors of iron binding ligands.
  • BLAST analysis of the second domain returned 69 hits from available databases.
  • BLAST analysis of the third domain returned 272 hits from available databases.
  • BLAST analysis of the second region of low complexity returned 1 hit from available databases. The lack of homologous sequences to this region makes it a likely candidate for a diagnostic target if the sequences are immunogenic.
  • domain 1 return more than domain 3 suggests that the conserved nature of these sequences may recognize sequences that are TonB independent since the BLAST results of domain 3 sequences contained a TonB_C site.
  • the difference in the number of returns may be a consequence of incomplete sequences being deposited in the databases used in these analyses.
  • the growth rate of P. aeruginosa in the presence of the immunoglobulins was evaluated by monitoring the absorbance spectrophotometrically at 600 nm. Only peptide 1 was effective in inhibiting the proliferation of P. aeruginosa (data not shown).
  • a pair wise BLAST tool was used to localize the position of peptide 1 in the amino sequence of FptA. These. results support our claim that amino acids sequences internal to D2 consensus domain 1 and 3 in Ton B dependent iron-ligand receptors.
  • the peptidel sequence was localized to the C-terminus of the Scott-Thomas domain as determined by Pair- Wise BLAST analysis. BLAST analysis of the other 3 peptides localized them to d2 domain 3. These observations suggest that surface exposed residues in Scott-Thomas domain and d2 domain 3 are not required for contacting siderophores.
  • target OMAP's are those that stimulate the production of antibodies that are capable of inhibiting the proliferation of pathogenic microorganisms in a complement independent manner. If the target pathogen genome has not been seqeunce, to identify OMAP's that meet these criteria expression libraries are created using genomic or cDNA depending upon the gene structure in the desired pathogen. For example, gene expression libraries from most bacteria can be constructed using size-fractionated genomic since prokaryotic gene are usually uninterrupted. However, with fungi (lower eukaryote) is necessary to produced cDNA for use in constructing expression libraries. The resulting expression library is probed with antisera obtained from a host inoculated with an antigen(s) (SEQ ID NOS 3-17) as detailed in section 1.
  • SEQ ID NOS 3-17 an antigen(s)
  • a 100 ml culture of Pseudomonas aeruginosa 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.
  • the concentrated staphylococci cells were 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.
  • DNA pellet was washed with 1 ml 70 % ethanol, air dried and resuspended in TE.
  • One hundred (100) micrograms P. aeruginosa 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 lOx SI nuclease buffer (2M NaCl, 50 mM NaOAc, pH 4.5, 10 mM ZnSO 4 , 5% glycerol), and 1.7 ⁇ l of SI 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 and 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.
  • the EcoRI sites in the DNA sequence were methylated using standard procedures. To this methylated DNA was added 5 ⁇ l of 100 mM MgCl 23 8 ⁇ l of dNTP mix (2.5 mM each of DATP, dCTP, dGTP, and dTTP), and 4. ⁇ l of 5 U/. ⁇ l Klenow. The mixture was incubated at 12 C. for 30 min.
  • the DNA was resuspended in 7 ⁇ l of T ⁇ and to the solution was added 14 ⁇ l of phosphorylated ⁇ co RI 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).
  • the sample was incubated at 4 C overnight, then incubated at 68 C for 10 min. to inactivate the ligase.
  • To the mixture was added 218 ⁇ l of H 2 O, 45. ⁇ l of lOx Universal buffer, and 7 ⁇ l of ⁇ co RI at 30 U/ ⁇ l. After incubation at 37.C. for 1.5 h, 1.5 ⁇ l of 0.5M ⁇ DTA was added, and the mixture placed on ice.
  • the DNA was size fractionated on a sucrose gradient, pooling fractions containing DNA of 6-10 kb.
  • the pooled DNA was ethanol precipitated and resuspended in 5 ⁇ l of T ⁇ buffer.
  • 20 ng of insert DNA was ligated for 2-3 days at 4 C with l ⁇ g of ZAP II vector in a final volume of 5 ⁇ l.
  • the ligation mixture was packaged using GIGAPACK II GOLD (Trademark of Stratagene) and plated on E. coli SURE (Trademark) 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.
  • coli SURE cells and 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-HCl, 150 mM NaCl, pH 7.5, prior to being probed with the appropriate polyclonal antibodies for the identification of a target recombinant OMAP'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 XLl-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 XLl-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, then 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 pagemenids were plated by adding 200 ⁇ l of freshly grown SOLR cells (OD600 1.0) to two 1.5 ml microcentrifiie tubes. 100 ⁇ l of the phage supernatant wasa added to 1 tube abd 10 ⁇ l was added to the other then the tubes were incubated at 37 c for 15 min. At the completion of the incubation step the 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 is 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 is precipitated by centrifuging at 10,000 x g for 15 min in a microcentrifuge.
  • DNA pellet is washed with 1 ml 70 % ethanol, air dried and resuspended in TE or water.
  • the purified phagemid DNA is sequenced and amino acid sequences deduced.
  • each polypeptide and each half of FhuB consists of 10 membrane-spanning regions, with the N- and C-termini located in the cytoplasm.
  • this putative ATPase interaction loop is followed by four instead of two transmembrane spans.
  • the siderophore receptor Fh ⁇ D was used as a model to localize target sequences that are useful as immunogens.
  • Fh ⁇ D is a soluble periplasmic binding protein that transports ferrichrome and other hydroxamate siderophores.
  • the crystal structure of FhuD from Escherichia coli in complex with the ferrichrome homolog gallichrome has been determined at 1.9 A resolution, the first stracture of a periplasmic binding protein involved in the uptake of siderophores.
  • Gallichrome is held in a shallow pocket lined with aromatic groups; Arg and Tyr side chains interact directly with the hydroxamate moieties of the siderophore.
  • FhuD possesses a novel fold, suggesting that its mechanisms of ligand binding and release are different from other structurally characterized periplasmic ligand binding proteins (Clarke et al 2000).
  • a periplasmic binding protein conserved domain was identified in the amino acid sequence of FhuD using Reverse Position Specific BLAST.
  • a consensus sequence internal to this domain was defined by multiple sequence alignment of periplasmic binding proteins using cobbler software programs.
  • the unique consensus sequence identified by COBBLER was lysine rich (SEQ ID NO 3). Using this sequence to query available databases using BLAST it was realized that the sequence was localized to gram-negative bacteria, gram-positive bacteria and mycobacteria.
  • conserved sequences within the siderophore receptor of a range of bacterial pathogens allows the selection of a minimal number of antigens having particular amino acid sequences (including in the form of synthetic peptides) to immunize against the disease caused by pathogens that have such receptors.
  • Such conserved amino acid sequences among many bacterial pathogens permits the generation of siderophore receptor specific antibodies, including monoclonal antibodies that recognize most if not all siderophore receptors.
  • Such antisera are useful for the detection and neutralization of most if not all bacteria that produce siderophore receptors and are also useful for both active and passive immunization against the diseases caused by such pathogens.
  • Hydrophilicity/hydrophobicity plots of this region was particularly useful in identifying a 19 bp epitopes (SEQ ID NOS 3-16) immunogens that protect a specified host from diseases caused by gram negative bacteria and gram positive bacteria and mycobacteria in a complement dependent fashions.
  • the preferred method for determining target sequences is to compare the results of Kyte and Doolittle (1982) and the Hopp and Woods algorithm (1981).
  • the production of antibodies to these region may physical impair the iron bound ligand from contacting its receptor.
  • diagnostic assays and kits using such conserved amino acid sequences are useful to detect many if not all bacteria that produce siderophore receptor.
  • SEQ ID NOS 3-16 As an alternative to the strategy provided above for the localization of the immunogenic amino acid sequences in SEQ ID NOS 3-16. Briefly, individual (epitopes (SEQ ID NOS 3-16) are used to generate specific antisera in a vertebrate host. The specific antisera are attached to a microtiter plate as described in example 3. Next, the antisera are probed with peptide libraries as described in the art. The recovered peptides are absorbed with pre-immune antisera obtained from the vertebrate host prior to immunization. The pre-immune antisera are separated from the peptides by immunoprecipitation or affinity column purification using protein A as described in the art. The resulting clones are sequenced and used as immunogens to generate eptiope specific antisera. A key advantage of this strategy is that the recovered peptide sequences represent surface available sequences or mimic unique surface stractures.
  • IgY systemic effects of IgY relates to the absorption or translocation of fragments of orally administered antibody from the intestine into circulation.
  • the IgY molecule is disassembled by naturally occurring enzymes in the intestine into binding fragments, which comprise peptides of the highly variable portion of the terminal domain of the antibody.
  • the peptides of the highly variable portion of the antibody, the Fab chain are taken into circulation.
  • the constant, or Fc, portion of IgY is left in the intestine. Once in the circulation, these fragments randomly search out a pathogen with the matching lectan and neutralize it by binding to that site.
  • Fab moieties unlike the Fc portion, do not elicit an allergic reaction, presumably because they are either too small or are unrecognized as foreign for some other reason. These Fab moieties can be added to the terminal end of the host's circulating globulin, wherein they are hidden from destruction but available for neutralization. A potential limitation of using the Fab moieties of chicken IgY is the rapid clearance.
  • the enhancement of the half-life of IgY when providing therapy in mammals, including man can be achieved by genetically removing the stop codons at the end of a gene encoding anti-microbial chicken immuoglobulins and inserting a linker and a second gene encoding a human IgG.
  • IgY variable regions can replace the variable regions of human IgG using recombinant DNA techniques known in the art. These methods will lead to a longer half-life of therapeutic chicken IgY.
  • phage display techniques For therapeutic applications that would require the use of human immunoglobulins, advantage can be taken of phage display techniques to provide libraries containing a repertoire of antibodies with high affinities for the desired antigen. For production of such repertoires, it is necessary to immortalize the B cells from an immunized mouse expressing human immuoglobulins; the resulting B cells are then used as a source of DNA. The mixture of cDNAs obtained from B cells, e.g., derived from spleens, are used to prepare an expression library, for example, a phage display library transfected into E. coli. The resulting cells are tested for immunoreactivity to the desired antigen. Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths et al.
  • Phage display libraries may also be constructed using previously manipulated nucleotide sequences and screened in similar fashion. In general, the cDNAs encoding heavy and light chain are independently supplied or are linked to form F v analogs for production in the phage library. The phage library is then screened for the antibodies with highest affinity for the antigen and the genetic material recovered from the appropriate clone. Further rounds of screening can increase the affinity of the original antibody isolated. The manipulations described above for recombinant production of the antibody or modification to form a desired analog can then be employed.
  • the antibodies may be administered in a pharmaceutically acceptable dosage form. They may be administered by any means that enables the active agent to reach the desired site of action, for example, intravenously as by bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes.
  • the antibodies may be administered as a single dose or a series of treatments.
  • parenteral administration the antibodies may be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
  • the formulation may contain suitable additives such as, for example, starch, cellulose, silica, various sugars, magnesium carbonate, or calcium phosphate.
  • suitable vehicles are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
  • the appropriate dosage of antibody will depend upon known factors such as the pharmacodynamic characteristics of the particular antibody, its mode and route of administration, the age, weight, and health of the recipient, the type of condition to be treated and the severity and course of the condition, frequency of treatment, concurrent treatment and the physiological effect desired.
  • OMAP antigen cocktails or peptides will be injected intramuscularly in vertebrate host, followed by three boosts of antigen at weekly intervals.
  • the vertebrate host will be challenged with a predefined dose of desired pathogen.
  • each group receiving a different antigen preparation will be housed separately in order to avoid contamination of the vaccinated, coccidia free layers.
  • Two-day fecal samples will be collected five days after infection and oocyst output will be determined.
  • blood samples are collected via cardiac puncture under methoxyflurane anesthesia and plated on the appropriate selection agar plates.
  • the number of colony forming units (cfu) per/ml, of blood is quantified after 24 hr.
  • the statistical significance of differences observed in the levels of disease causing organism relative to controls is analyzed by the Student's t-test.
  • Vertebrate hosts such as chicken, mice, rats etc. will be immunized with immunoglobulins (1 mg/ml) obtained from hyperimmune serum. Pre-immune sera are used as negative controls.
  • immunoglobulins (1 mg/ml) obtained from hyperimmune serum.
  • Pre-immune sera are used as negative controls.
  • One day after immunization the animal is inoculated with a specified pathogen at a predetermined dosage.
  • blood samples are collected via cardiac puncture under methoxyflurane anesthesia and plated on the appropriate selection agar plates.
  • the number of colony forming units (cfu) per ml, of blood is quantified after 24 hr.
  • the statistical significance of differences observed in the levels of disease causing organism relative to controls is analyzed by the Student's t-test.
  • the effectiveness of the antimicrobial immunoglobulins are dose dependent. More specifically it required 1 ⁇ g/cell to inhibit the proliferation of gram-negative bacteria when using antimicrobial immunoglobulins recovered from immunized rabbits. To determine the effect dose of D2-DLS01 or treating infection intravenously. Since the antimicrobial immunoglobulins inhibit the proliferation of the tested bacteria in a dose dependent manner it is critical to employ a method for calculating effective dosages, that considers the multiplicity rate (doubling time) of the pathogen, time expired between diagnosis and first treatment, and most importantly the volume of distribution.
  • Vd volume (kg.) is estimated by multiplying patient weight (kg.) by 0.25.
  • a usual loading dose can be calculated by multiplying a target therapeutic peak value, such as 10 ⁇ g./ml., patient weight Vd 0.25.
  • a target therapeutic peak value such as 10 ⁇ g./ml.
  • the loading dose would be 175 mg., corresponding to the standard loading dose of 2.5 mg./kg commonly used by clinicians.
  • V d up to 0.7 l./kg., which requires changes in the loading dose, including ascites (0.32 65 to 0.44), surgery (0.35), trauma (0.35), sepsis (0.4), granulocytopenia (0.4), burns (0.5 to 0.7), cystic fibrosis, critical illness requiring an intensive care unit stay, hematological malignancy, cancer and AIDS.
  • changing V d decreases serum aminoglycoside concentrations by 25% to 40% if the dose is not increased appropriately.
  • V d can also be decreased to the 0.05 to 0.15 l./kg range in cases of severe dehydration but dosage is rapidly normalized after treatment with intravenous fluids.
  • D2-DLS01 Stenotrophomonas maltophilia strain
  • M9-minimal medium supplemented with maltose (10g/ ml); methionine (40 ⁇ g/ml); 1 M MgSO4 (0.1%); and the iron- chelator, 2' 2' dipyridyl (100 ⁇ M).
  • the culture was incubated in gyration water bath 37 C until the growth density of the culture read an optical density (OD) 0.6 at A600.
  • OD optical density
  • the S. maltophilia cells 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.
  • the supernatant was stored at -20 C and the bacteria cells were gently resuspended in 250 ml of ice cold HE (10 mM Hepes [ph 7.4] and 1 mM EDTA) buffer and the centrifugation step was repeated.
  • the cell culture supernatant was analyzed for the presence of siderophores (henceforth iron reactive material) using the Chrome Azural S (CAS) the presence lipopolysaccharides was determined Limulus amoebocyte lysate assay. As seen in table 1, the D2-DLS01 supernatant was positive for iron-reactivity and LPS.
  • the concentrated bacteria were resuspended in 17 ml of HE buffer in a 50 ml sterile tube, then frozen in liquid nitrogen and thawed at room temperature. This step was repeated until the solution became viscous.
  • Ten milliliters (10 ml) of the viscous lysate was layered on a 0.5 ml 60% sucrose shelf and centrifuge for 1 h at 38,000-x g in a model L8-70M ultracentrifuge (Beckman Instruments, Inc.) in a SW 41 swinging bucket rotor (Beckman Instruments, Inc.) to differentiate the cytoplasmic and membrane fractions.
  • the cytoplasmic and membrane fractions were analyzed for iron- reactive material and the presence of LPS as described previously. The iron reactivity and the LPS contamination were localized to the membrane fraction (see Figure 6 1).
  • the membrane fraction was resuspended in 50 ml of solubilization buffer (10 mM Hepes [ph 7.4]; 1 mM EDTA; 1.2 M Nail; 2% Triton X-100) and incubated 1 h at 4 C.
  • solubilized membranes were mixed with 10% Polyethyleneimine (PEI) by stirring until the concentration of PEI was 1% of the solubilized membrane solution.
  • PEI Polyethyleneimine
  • the membrane-PEI solution was stirred for 1 h in a cold room at 4 C and then centrifuged for 15 min at 10,000-x g in a model J2-21 centrifuge (Beckman Instruments, Inc.).
  • 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 supernatant (50 ml) was mixed by slow stirring with 18.05 g of ammonium sulfate and incubated with continuous stirring at 4 C for 1 h.
  • the ammonium sulfate precipitate was collected by centrifugation for 15 min at 10,000-x g in a model J2-21 centrifuge (Beckman Instruments, Inc.).
  • the ammonium sulfate supernatant and pellet (resuspended in HEU buffer) were analyzed for iron-reactivity and LPS contamination.
  • the iron-reactive fraction was recovered in the Ammonium sulfate pellet no LPS was detectable
  • the ammonium sulfate precipitate 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 presence of LPS.
  • 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 contrast the iron- reactivity was found in the retainate, no LPS were detected in either fraction.
  • the iron-reactivity was transferred to the filtrate by the addition of solid urea to a final concentration of 6 M.
  • the retainate, hence for D2-DLS01 antigen cocktail, was modified with .02% sodium azide and stored at - 20 C.
  • D2-DLS01 antigen solution was diluted to 5 ⁇ g/ml with HEU (10 mM Hepes [ph 7.4]; 1 mM EDTA; 6 M urea), and then incubated for 10 min in boiling water. Next the denatured D2-DLS01 antigen solution was dialyzed against 6 changes of HE buffer (250 ml). The HE buffer was changed every 12 h. D2-DLS01 antigen solution was used to generate antisera in four to five month old female New Zealand White rabbits (4) using standard techniques. Briefly, the rabbits were bled to obtain 10 ml of non- immune serum before immunization.
  • HEU HEU
  • Methyl Salicylate (2- Hydroxybenzoic methyl ester; oil of wintergreen) was used to encourage the artery to dilate.
  • the blood was collected by gravitational flow through the tubing connected to the needle into a collection vessel.
  • the rabbits were initially inoculated with 500 ⁇ g of outer membrane associated polypeptides in saline plus Freunds's complete adjuvant. The mixture was administrated in 0.1 ml aliquots in several sites of each rabbit's back subcutaneously.
  • the rabbits were injected with 250 ⁇ g of outer membrane associated polypeptides in saline plus Freund's incomplete adjuvant subcutaneously as previously indicated.
  • Rabbits were boosted at 4-week intervals as in detailed previously and bled as described earlier, 10 days after each boost.
  • the central ear artery chosen for bleeding was alternated with each bleeding.
  • a total of 50 ml of blood was taken at each bleed.
  • Antibody binding assays were performed after each bled (see Example 3).
  • the immunogenicity of D2-DLS01 antigen cocktail was determined using an enzyme linked immunoabsorbent assay (ELISA) method.
  • ELISA enzyme linked immunoabsorbent assay
  • membrane associated polypeptides in carbonate buffer were covalently attached to a microtiter plate and blocked with a 5 % milk fat proteins in TBS to eliminate non-specific binding of the probe antibody.
  • the membrane-associated polypeptides were probed with 100 ⁇ l of 100-fold serial dilutions of serum from the immunized rabbits. After thoroughly washing antibody binding was determine using an anti-rabbit alkaline phosphatase conjugate (diluted 1:1000).
  • Antibody binding was determined by adding the colorimetric substrate para-nitrophenyl phosphate (PNP) and allowing the reaction to proceed for 1 h. At the completion of the lh incubation the reaction was terminated by the addition of 100 ⁇ l of 1 N NaOH.
  • PNP para-nitrophenyl phosphate
  • the filter was washed with TTBS. After the wash step the colorimetric substrate BCIP/NBT(Pierce Chemical) was added and the colorimetric assay was allowed to proceed for 30 min at room temperature. The reaction was terminated by the rinsing of the filter with sterile water. As seen in figure 7. the anti-D2-DLS01 antisera recognized S. maltophilia ranging in size from 70 kDa -100 kDa as determine using a broad range protein marker (BioRad).
  • an Immobilized Protein A/G column (3 ml) (Pierce, Rockford, 111.) equilibrated with 5 column volumes of Immunopure IgG Binding Buffer, then 6 ml of serum from immunized host diluted 1 : 1 with binding buffer was added to the column. The diluted serum was allowed to completely flow through the column. The column was washed with 10-15 column volumes of binding buffer. IgG was eluted from the column with 3-5 columns of elution buffer.
  • the IgG was collected in 1.0 ml fractions and immediately neutralized by inclusion of 100 ⁇ l of 100 mM Tris-HCl, pH 7.5 in the collection vessel. Fractions containing IgG were identified by using absorbance at 280 nm, and pooled together. Pooled IgG fractions desalted and concentrated by buffer exchange with a 5 ml desalting column. Briefly, 1.25 ml of pooled IgG was applied to and allowed to completely flow through column. 10 x 1 ml aliquots of equilibration buffer was applied to the column and collected in 1 ml fractions. The protein concentration in each fraction was monitored spectrophometrically at 280 nm. The fractions that contained high reading were pooled and the exact concentration of IgG was determined by using a Pierce BCA protein assay (Pierce).
  • anti-D2-DLS01 polyclonal antibodies were evaluated for their ability to inhibit the proliferation ofS. maltophilia in iron-deplete medium
  • the growth rate of S. maltophilia in the presence of the immunoglobulins was evaluated by monitoring the absorbance spectrophotometrically at 600 nm. As seen in figure 9 the concentrations of immunoglobulins did reduce the growth rate of S. maltophilia until we reached the 10 ⁇ 8 dilution factor. Evaluation of the colony forming units present at this dilution factor it on the solid M9-maltose medium demonstrated that 100 ⁇ g of purified immunoglobulins are effective in neutralizing 100 or less bacteria.
  • anti-D2-DLS01 polyclonal antibodies were evaluated for their ability to inhibit the proliferation ofS. maltophilia in iron-replete medium
  • anti-D2-DLS01 polyclonal fab fragments were evaluated for their ability to inhibit the proliferation ofS. maltophilia in iron-deplete medium
  • Anti-D2-DLS01 Fab fragments were generated by incubating purified anti-D2-DLS01 IgG (20 mg/ml) with immobilized papain (Pierce Chemical Company, Rockford, IL). Briefly, 0.5 ml of 50%) Immobilized Papain slurry (0.25 ml of settled gel) was added to a glass test tube containing 4.0 ml of freshly prepared digestion buffer (20 mM phosphate; 20 mM cytokine-HCL; 10 mM EDTA-Na4, pH7.0). The gel was separated from the buffer by centrifugation. The buffer was discarded and the wash step was repeated once more.
  • the immobilized papain was resuspended in 0.5 ml of digestion buffer and mixed with 10 mg of purified IgG dissolved in 1 ml of digestion buffer and incubated for 5 hr (or overnight) at 37 C with at high speed in a gyrator shaker. Human IgG was incubated for 4 hr under the same conditions. 1.5 ml of 10 mM Tris- HCL, pH 7.5 was added to the digest and the generated Fab fragments were separated from the immobilized Papain by centrifugation. The Fab fragments were separated from undigested IgG and Fc fragments using an Immobilized Protein A column (Product# N. 20356, Pierce Chemical Co.) as described in example 4.
  • the growth rate of S. maltophilia in the presence of the fab fragments was evaluated by monitoring the absorbance spectrophotometrically at 600 nm. As seen in figure 11 the concentrations of anti-D2DLS01 Fab fragments used in this experiment successfully inhibited the growth rate of S. maltophilia. These results demonstrate that the anti-D2-DLS01 inhibit S. maltophilia in a complement independent manner.
  • OMAP preparations diluted in carbonate buffer from B. cepcia, C. neoformans, E. coli, P. aeruginosa, S. aureus, S. epidermis, and S. maltophilia were covalently attached to a microtiter plate and blocked with a 5 % milk fat proteins in TBS to eliminate non-specific binding of the probe antibody.
  • Binding was quantitated by adding the colorimetric substrate para-Nitrophenyl phosphate (pNPP) colorimetric substrate and monitoring the color development spectrophotometrically at 405 nm. The assay was stopped after 1 h by the addition of 100 ⁇ l of 1 N NaOH. D2-DLS01 antisera only recognized the gram-negative bacteria (data not shown). LIST OF REFERENCES
  • PchR a regulator of ferripyochelin receptor gene (fptA) expression in Pseudomonas aeruginosa, functions both as an activator and as a repressor. J Bacteriol. 178:2586-92.
  • Nissim A Hoogenboom HR, Tomlinson IM, Flynn G, Midgley C, Lane D, Winter G (1994) Antibody fragments from a 'single pot' phage display library as immunochemical reagents. EMBO J. 13:692-8.
  • Patruta SI Horl WH (1999) Iron and infection. Kidney Int Suppl. 69:S 125-30. Review.

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Abstract

La présente invention concerne des procédés de localisation et de récupération de polypeptides associés à la membrane externe convenant particulièrement pour l'immunisation tant active que passive des mammifères, de la volaille et des humains aux fins de protection contre des affections et des infections d'origine bactérienne, fongique et/ou protozoaire. Ces polypeptides sont séparés des endotoxines contaminantes et de ligands cibles. Des peptides purifiés et/ou synthétiques peuvent être utilisés dans des stratégies d'immunisation pour une protection large ou spécifique d'une espèce contre des bactéries, des champignons u des protozoaires. L'invention concerne également des techniques de diagnostic de bactéries, de champignons ou de protozoaires.
PCT/US2002/011110 2001-04-10 2002-04-10 Polypeptides associes a la membrane externe WO2002083843A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006672A3 (fr) * 2001-07-10 2007-11-22 Squared Biotechnologies Inc D Anticorps d'acide nucleique antimicrobien, materiaux et procedes de fabrication et d'utilisation correspondants
WO2009005040A1 (fr) * 2007-06-29 2009-01-08 Meiji Seika Kaisha Ltd. Protéine pa0427 de membrane externe de pseudomonas aeruginosa
WO2019217665A1 (fr) * 2018-05-09 2019-11-14 West Virginia University Administration de peptide récepteur d'acquisition de fer pour la vaccination contre pseudomonas aeruginosa
EP4116316A1 (fr) * 2015-07-04 2023-01-11 Evaxion Biotech A/S Protéines et acides nucléiques utiles dans des vaccins ciblant pseudomonas aeruginosa

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDRADE ET AL.: 'The iron uptake mechanisms of enteroinvasive escherichia coli' BRAZILIAN JOURNAL OF MICROBIOLOGY vol. 31, 2000, pages 200 - 205, XP002961604 *
CARNIEL ET AL.: 'Purification, location and immunological characterization of the iron-regulated high-molecular-weight proteins of the highly pathogenic yersiniae' INFECTION AND IMMUNITY vol. 57, no. 2, February 1989, pages 540 - 545, XP002961603 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006672A3 (fr) * 2001-07-10 2007-11-22 Squared Biotechnologies Inc D Anticorps d'acide nucleique antimicrobien, materiaux et procedes de fabrication et d'utilisation correspondants
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
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
WO2019217665A1 (fr) * 2018-05-09 2019-11-14 West Virginia University Administration de peptide récepteur d'acquisition de fer pour la vaccination contre pseudomonas aeruginosa

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