BACKGROUND OF INVENTION Diseases caused by bacteria and fungi have been a plague on civilization for thousands of years, affecting not only man but animals and plants as well. The discovery of penicillin in the early 1940s and the quick succession by hundreds of other anti- microbial agents created an arsenal of drugs that offered unprecedented control over bacterial and fungal infections. However, within just a few years of the introduction and use of antibiotics, a troubling pattern emerged. The resistance of bacteria and fungi to antimicrobial agents has become a major medical and public health problem (Abraham 1997; Fishbane 1999). The resistance means that the bacteria outsmart the drugs that are supposed to kill them and consequently render these antibiotics useless.

The frequent treatment of infection and disease caused by bacteria and fungi with the same anti-microbial agent over a period of time in many instances results in the pathogen developing resistance to the drug. With a number of pathogens the acquired resistance is usually to an entire family of drugs and in some cases even to drugs that are structurally unrelated, giving rise to multiple drug resistance (Sensakovic and Smith 2001). The potent antibiotic vancomycin has offered a reliable last defense against the most virulent bacteria. However, in recent years, there have been an increasing number of reports of bacteria resistant to this drug (Gordon 2001). It is of common opinion that bacterial and fungal resistance to antibiotics is caused by injudicious use of antimicrobial agents. Furthermore, the acquired resistance is present only in certain strains of species or of a genus. The latter results from mutation in a gene located in the host chromosome or from acquisition of new genetic information by a bacterium mainly by conjugation or transformation with the introduction of more than one antibiotic into the environment, the multiple and fluctuating pressure produces the selection of bacterial variants that use multiple or multi-purpose mechanisms of resistance to survive under the variable environmental conditions.

Antibiotics have long been the primary therapeutic tool for the control and eradication of gram-positive and gram-negative infections. However, the continued incidence and severity of infections, the continual emergence of antibiotic resistant bacterial strains, and the inherent toxicity of some antibiotics, point to the limitations of antibiotic therapy. These observations have prompted the search for alternative prophylactic and therapeutic approaches.

In 1975, Kohler and Milstein reported that certain mouse cell lines could be fused with mouse spleen cells to create hybridomas that would secrete pure monoclonal cells with the advent of this technology, the potential existed to produce murine antibodies to any particular determinant or determinants on antigens. The ability to specifically bind a biologically important determinant demonstrated the potential for monoclonal antibodies use as therapeutic agents. The advantages of monoclonal antibody therapeutics over conventional pharmaceuticals include their exquisite selectivity, multiple effector functions, and ease of molecular manipulation such as radioisotope labeling and other types of conjugation.

One therapeutic application of monoclonal antibodies is passive immunotherapy in which the exogenously produced immunoglobulins are administered directly to the animal being treated by injection or by ingestion. To be successful, 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. 2000; Felts et al. 1999; Harrison et al., 1997; Kelly 2000; Korhonen et al 2000; Lin et al. 1998; Matsumoto et al. 1999; Otereo and Linares 1998; Saywer 2000;). Recent emergence of antibiotic resistant bacteria makes treatment of bacterial infections with passive immunotherapy desirable. Antibiotic treatment targeted to a single pathogen often involves eradication of a large population of normal microbes, and this can have undesired side effects. An alternative approach has been to utilize the inherent specificity of immunoglobulins to inhibit a specific pathogenic function in specific microbial populations. In this strategy, purified immunoglobulins of the appropriate specificity would be administered in order to provide a passive barrier to pathogen invasion. For example, monoclonal antibodies recovered from mice immunized with polysaccharides from Neisseria meningitidis Group B were observed to bind and opsonize several Kl-positive Escherichia coli strains regardless of their lipopolysaccharide (LPS) serotypes (Toropainen et al. 2001).

Moreover, the monoclonal antibodies were protective in mice against lethal challenges with E. coli Kl and Group B meningococcal organisms.

Although 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).

As an alternative to monoclonal antibodies obtained from non-human sources, the pooling of human immunoglobulins has been considered. However, commercial products are not yet readily available due to certain inherent limitations that have prevented their widespread use in the treatment of life-threatening bacterial disease.

One such limitation associated with immunoglobulin compositions is that they 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.

Another limitation is that the pre-selection process itself requires very expensive, continuous screening of the donor population to assure product consistency. Despite considerable effort, product lots can still vary between batches and geographic regions.

Yet another such limitation inherent in immunoglobulin compositions is that their use results in coincident administration of large quantities of extraneous proteinaceous substances (e. g., viruses) having the potential to cause adverse biologic effects. 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.

To overcome the potential limitations of pooling human immunoglobulins 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. Furthermore, 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 drug and has granted GRAS (generally accepted as safe) status. Finally, 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).

Recently, it was discovered that 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.

These 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 identification of broad protective antigens is no trivial task. The antigenic variation of a sub-population of bacteria within a given species demands the need for specific immunoglobulins for each serotype. The production of serotype specific human monoclonal antibodies to each of the many important bacterial pathogens would be impractical. Thus, there still exists a significant need for human monoclonal antibodies that are broadly (intergenus) protective against gram-positive and gram- negative bacterial diseases, as well as for methods for practical production and use of such antibodies. In a preferred embodiment antibodies should be capable of neutralizing disease-causing bacteria in a complement independent fashion, in consideration of immunocompromised patients.

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 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. Other bacteria, such as Bordetella pertussis secrete adhesion molecules that attach to both the bacterium and ciliated cells of the upper respiratory tract (Kerr JR 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).

Pathogenic bacteria must be able to proliferate and invade host tissue. 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. To circumvent these restrictive conditions, 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). These factors use of 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). However, in many cases, these antigens are weak immnunogens, 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).

More than half of the estimated 400,000 reported cases of bacterial sepsis are caused by gram-negative bacteria. At least 25% of these patients 100,000 ultimately die of septic shock. 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. Although the term 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). The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). 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). For example Canipylobacter 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).

In another case Helicobacter 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 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).

In spite of the advances attained in the diagnosis and early intervention with antibiotics, morbidity and mortality associated with sepsis caused by gram-negative bacteria are high. The mediators responsible for the pathogenesis of the sepsis are components derived from the own bacteria (endotoxins) and from the cells of the immune response of the host (tumor necrosis factor and some interleukins). The treatment traditionally used in sepsis is mainly directed against microorganisms by the use of increasingly potent antibiotics. However, it is clear that antibiotics are not a definitive solution, since even when they cause bacterial death they have no effects on endotoxin and may increase their liberation when cellular lysis occurs. New and successful treatments for sepsis have been tried since the 1980's, including the use of polyclonal and monoclonal antibodies of murine and human origin, directed against the lipid A of the endotoxin, as well as monoclonal antibodies against the tumor necrosis factor. Although these molecules are not completely efficient for the interruption of the chain of undesirable events provoked by the endotoxin, it is valid to accept the appearance of immunotherapies as an adjuvant treatment for a condition that threatens life.

SUMMARY OF DISCLOSURE 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). These claim are supported by 1) the recovery of immunoglobulins capable of inhibiting the proliferation of taxonomically distinct pathogenic bacteria in complement-dependent and complement independent fashions; and 2) in vitro diagnostics methods for detecting of pathogenic bacteria.

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.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Illustrates the predicted structure of btA gene of Pseudomonas aeruginosa using Swiss model.

Figure 2. Illustrates the localization of a TonBboxC in the fptA gene of Pseudomonas aeruginosa.

Figure 3. Illustrates the conserved regions and predicted ligand-binding region offptA 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.

Figures 5A-D. Show the localization of ligand-binding region of TonB dependent outer membrane receptors in gram-negative bacteria between Scott-Thomas domain and d2 domain 3 Figure 6. Illustrates the strategy used to produce D2-DLS01 antigen cocktail.

Figure 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.

DETAILED DESCRIPTION OF THE INVENTION It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis, and treatment of disease and infections caused pathogenic microorganisms. A further non- limiting discussion of such uses is further presented below. These examples are not meant to limit the scope of the invention that has been set forth in the foregoing description. Variation within the concepts of the invention is apparent to those skilled in the art. The disclosures of the cited references throughout the application are incorporated by reference herein.

1. Recovery of outer membrane associated polypeptides The identification of amino acid sequences that are useful, as immunogens for the protection of a host from pathogenic microorganisms requires information on the structural organization of outer membrane associated polypeptides. More'importantly, information regarding the role and genetic regulation of identified polypeptides is required. For the sake of clarification, hence forth in this document 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.

A number of studies have confirmed the close relationship between the availability of iron and pathogen virulence. The ability of a microbial invader to acquire iron from its vertebrate host has been recognized as an important virulence mechanism in some pathogenic bacteria. A number of reports have detailed the identification of outer membrane polypeptides that are up-regulated in environments that are low in iron. It becomes obvious that the propagation of pathogenic microorganisms in conditions of low iron availability can provide information about the composition and structural integrity of the outer membrane during virulence. There are a number of methods common to the art for mimicking the low available iron in vertebrate host. 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, deferoxamine, and other like agents. In a preferred embodiment, 2'2'- dipyridyl is added to M9 medium media at a concentration of about 50 uM-500 LM preferably about 100 pM.

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.

For example, 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).

Studies aimed at developing strategies for the purification of siderophores secreted by Stenotrophomonas maltophilia revealed that iron-binding ligands co-purified with membrane proteins (Scott 1995). In another study (Folschweiller 2000), FpvA expressed in P. aeruginosa grown in an iron-deficient medium was co-purified with a ligand X that was subsequently identified to be the fluorescent sideophore, pyoverdin (PaA). Fluorescence resonance energy transfer between iron-free PaA and the FpvA receptor furthered revealed the existence of an FpvA-pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin-loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferric-pyoverdin, it was shown that iron-free PaA binds to the outer membrane but it was not taken up into the cell, and that in vitro and, presumably, in vivo ferric-pyoverdin displaces the bound iron-free pyoverdin on FpvA-PaA to form FpvA-PaA-Fe complexes. In vivo, the kinetics of formation of this FpvA-PaA-Fe complex is more than two orders of magnitude faster than in vitro and depends on the presence of TonB. Thus to ensure that the purification scheme described enriches for membrane associated receptors we choose an assay that would identify the presence or absence of iron-binding ligands, i. e. siderophores. To determine at which step in the purification process is iron-binding ligands, i. e. siderophore, separated from their specified receptors we utilized the Chrome Azurol S (CAS) universal chemical assay of Schwyn and Nielands (1987). The CAS assay has been used previously to detect iron-reactive material in the culture supernatants of bacteria and fungi. At each step in the isolation procedure the OMAP solution is analyzed for iron-reactivity using the CAS assay.

Upon reaching the desired cell density cell cultures are concentrated, for example, by centrifugation, membrane concentration, and the like. For example, 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 (HEPES), 3-N (N-morpholino) propanesulfonic acid (MOPS), and the like. The washed pellet is resuspended and washed in a compatible buffer solution, i. e., TBS, HEPES, 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. In a preferred embodiment, 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 ira 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. To overcome these disadvantages, polyethyleneimine (PEI) or diethylaminoethyl (DEAE) groups have been used to absorb endotoxins form protein solutions. The adsorption process is primarily based on electrostatic interactions, which could be demonstrated by a significantly higher adsorption rate and binding capacity for endotoxins. In a preferred embodiment solubilized membranes are mixed with 10% Polyethyleneimine (PEI) by stirring until the concentration of PEI is 1% of the solubilized membrane solution. 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. To remove the PEI from the protein solution 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 strategies disclosed most commonly in the art, provide methods for the recovery of that maintain their native structure. For example, US Patents 5,830,479 (Emery) and 6,027,736 (Emery) reference thereto, a method for isolating high quantities of immunogenically effective siderophore receptor proteins from outer membranes of a single strain or species of gram-negative bacteria such as E. coli, Salmonella and/or Pasteurella. The method includes culturing the organism under conditions of low iron availability, that is, in a culture medium that lacks iron or includes an iron-chelating agent. 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.

Although, the aforementioned strategies may yield antibodies that bind to the cell surface of the target organism, these methods do not considered the strong affinity iron binding ligands share with their receptors. For example Scott (1995) demonstrated that a solubilization buffer containing 1% Triton X-100 and 1 M NaCl were ineffective in separating outer membrane polypeptides from iron-reactive material as determined using the CAS assay of Scwyn and Neiland (1987). In another example it required greater than 2% SDS to disrupt the complex comprised of ferric-exochelin, the major extracellular siderophore of Mycobacterium smegmatis, and a 29 kDa protein (Dover and Ratledge 1996).

Another limitation to these strategies is that they do not considered the conformation changes that siderophore receptor may undergo during the process of translocation of Fe-bound-siderophores to the cytoplasm of bacteria. As mentioned previously in this application there have been reports that demonstrate that suggest that siderophore receptors are loaded with its respective ligand even in the absence of iron.

Thus, 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 disrupt the siderophore receptor-ligand complex. In a preferred embodiment 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. Alternatively, 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.

2. Production of Anti-OMAP Polyclonal Antibodies To identify antigenic determinants for protection against a specified pathogen, from a target organism are used to immunize vertebrate host such as chickens, goats and rabbits that are known in the art for quantitative production of antibodies. They are administered in combination with a pharmaceutical carrier compatible with the sample and the animal. 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 THI or TH 2 cell-specific immune responses ; and (8) ability to selectively increase appropriate antibody isotype levels (for example, IgA) against antigens. 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 formulations 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.

In a preferential embodiment, 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.

3. Purification of Anti-OMAP Immunoglobulins A. Affinity Purification of Anti-OMAP Immunoglobulins To separate anti-OMAP immunoglobulins from other components of serum is accomplished by using protein A/G affinity purification. In a preferred embodiment, 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 1ll 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.

B. Production of Anti-OMAP Fab fragments Antibodies can completely suppress or enhance the antibody response to their specific antigen by several hundredfold. Immunoglobulin M (IgM) 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 (FcyRs). IgE enhances antibody responses via the low-affinity receptor for IgE (FceRII/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. In addition, 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. Hence, 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. On the contrary, 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.

Activation of complement: 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. Thus to ensure that anti-outer membrane associated polypeptides inhibit the proliferation of pathogens in a complement independent manner the Fc portion of these immunoglobulins are removed.

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 Pc fragments using an affinity-purification using an immobilized ProteinA column (Product# N. 20356, Pierce Chemical Co.).

4. Evaluation of the Affinity Anti-OMAP Immunoglobulins for Purified and Whole Cells The affinity of anti-OMAP antisera for the surface of a specified pathogen will be evaluated using a whole-cell enzyme linked immunoabsorbent assay (ELISA) method.

Briefly, pathogens will be diluted in an appropriate buffer commonly used to attached polypeptides or cells to the bottom of a well in a microtiter plate. To reduce the non- specific binding the microtiter a blocking step with a responsible blocking agent will be performed. Next, the polypeptides or cells will be probed with serial dilutions of anti-OMAP antisera. After thoroughly washing 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 the colorimetric substrate that liberates a color change when contacted by the appropriate reporter enzyme. The color change can be measured spectrophotometrically.

5. Characterization of A. Western Blot Analysis 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. Next the nitrocellulose filter is probed with appropriately diluted (as determined in the antibody binding assays) anti-OMAP antisera. After thoroughly washing 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.

6. I7z vitro growth Inhibition Studies In this invention 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. Thus, increasing dosage of purified immunoglobulins (monoclonal or polyclonal pools) are included in a defined medium supplemented with either 100 1M 2'2'-dipyridyl (iron-deplete) or 50 pM FeCl3 (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.

For example a B-D Falcon 48 well tissue culture plate (Fisher Scientific, Suwanee, GA) 1 ml of M9-minimal medium supplemented with maltose (lOg/ml) ; casamino acids (5 mg/ml) ; 1 M MgS04 (0.1%) and 120 uM 2'2'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 fzeoformafas, Staphylococcus aureus, and Stenotrophomonas maltophilia were serially diluted in increments of 100-fold. One-microliter aliquots of the serial diluted bacteria were inoculated into the M9 medium and incubated at 37 C for 18 h-24 h.

Simultaneously, 100 1ll 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 the D2 DLS05 polyclonal antibodies was evaluated by monitoring the absorbance spectrophotometrically at 600 nm. 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 it on the solid M9 medium.

7. Localization of Antigenic Determinants in We describe a strategy for predicting amino sequences that are potentially useful for the protection of a host from infection by pathogenic microorganisms, namely bacteria, fungi, and protozoans. 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 anti- microbial immunoglobulins is to identify polypeptides in the target pathogen that are essential to the proliferation and establishment of infection. The in vivo selection system (IVET) 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 drug 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 sequestration of the bio-available iron by the respiratory mucus creates an iron- deplete environment (Wang et al. 1996). In many instances low iron availability is a triggering signal for coordinated expression of virulence factors. Genetic regulators associated with the acquisition of iron have been directly linked to the up-regulation of pathogenic factors that include adhesions, lipases, proteases, siderophores, and toxins. IVET technology was used to identify genes in P. aeruginosa that are specifically inducible by respiratory mucus derived from cystic fibrosis (CF) patients (Wang et al.

1996). The interaction of bacteria with mucus from patients with CF resulted in marked induction of expression of several genes, including one that encodes a lipopolysaccharide biosynthetic enzyme, and a gene (fptA) for a protein responsible for uptake of the ferric pyochelin siderophore.

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. To evaluate the potential of FptA as target polypeptide on the surface P. aeruginosa the aforementioned criteria was assessed with anti-DLS01 rabbit antisera (see example 1).

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-DLS01bantisera were successful at detecting FptA (data not shown).

These results support the necessity of iron acquisition for the establishment of infection in a prescribed host. As a result of this study we choose the FptA as a model for the development of our scheme for localizing target amino acid sequences in essential proteins. 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. Furthermore, consideration should be given to the fact that in many instances, strains of the target microorganism will occur naturally. Therefore, various strains may be antigenically variable, i. e. differ from one another in the amino acid sequences of one or more of their antigenic determinants.

Thus, 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. Thus, 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.

Information about the location of the binding site for a group of sequences can clearly improve ligand-prediction methods. More specifically, the identification of amino acid sequences (including in the form of synthetic peptides) in the binding site of the siderophore receptors could lead to the production of anti-microbial immunoglobulins that could inhibit the proliferation of a target bacterium in a complement independent fashion. The mechanism of iron acquisition and transport in bacteria has been studied extensively in gram-negative bacteria, namely E. coli and P. aeruginosa. In these bacteria, the ferric-siderophore complex must cross both the outer membrane and the cytoplasmic membrane before delivering iron within the cytoplasm (Van der Helm 1998). The 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).

The x-ray crystallographic solution of general (OmpF) and specific (LamB) porin folding explained the foundations of outer membrane polypeptide structure (Sansom 1999), but the TonB-dependent gated porins, i. e. siderophores, present a formidable next step in the understanding of OM protein architecture. Recently, the crystal structures of ferric-enterobactin and ferrichrome outer membrane receptors from E. coli (FepA and FhuA, respectively) were solved (Clarke et al. 2000; Ferguson et al. 2000; Buchanan et al. 2000; Sansome 2000). These structures reveal two distinct functional domains: a 22-stranded antiparallel-barrel, and a N-terminal globular domain, which folds inside the-barrel, plugging the barrel pore (Armstron and McIntosh 1995; Cao et al 2000; Koebnik and Braun 1993; Moeck et al 1997; Murphy et al 1990; Newton et al 1999; Rutz et al. 1992; Braun et al 1998 ; Braun etal. 1999; Clarke et al. 2000; Ferguson et al. 2000; Groeger and Koster 1998; Buchanan et al. 2000; Sansome 2000 ; 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. Indeed, a conformational change of the secondary structure of the N-terminal sequence of the protein, which switches from a helix to an unwound structure, has been observed upon binding of ferric-siderophore to FhuA and has been proposed to signal the receptor-loading status to the protein TonB (Moeck et al. 1997). Whether TonB induces a second conformational change (such as opening of a channel in the N-terminal globular domain) is not known, but the strict requirement of TonB in ferric-siderophore transport is undeniable. Despite this recent structural elucidation of these two receptors, the molecular basis of the mechanism of ferric- siderophore transport remains hypothetical and obscure. 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.

A concern of protein analysis by mutagenesis is the differentiation of general structural perturbations from local, ligand-specific defects. Global conformational changes in OM proteins usually result in improper localization, degradation, and invariability. The site-directed substitutions in fepA did not exhibit these effects. All the mutants, including ABCD, were active in transport and/or nutrition (albeit in some cases at much reduced levels), indicating that FepA assembled normally and functioned in the OM. Furthermore, structural deformities in the mutants were not detectable by cytofluorimetric analysis of antibody binding to FepA surface epitopes (Newton et al 1999).

The first step in our scheme was to analyze the FptA gene for the presence of conserved and/or functional domains. First we wanted to determine if the FptA gene product possessed the common features of ferric-siderophore receptors, a 22-stranded antiparallel-barrel and a N-terminal globular domain, which folds inside the-barrel, plugging the barrel pore. We accomplished this task using 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). More specifically, the beta- structure content of fptA was typical of porins and in agreement with the 3D structures of the siderophore receptors such as FhuA and FepA. Next, we employed at least 2 different search programs for the identification of conserved amino acid sequences in the fptA gene. The domain search program Pfam, a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains, identified a Tond C motif at the C terminus of FptA (see Figure 2).

A second search program, B10CKS, also identified the Tond 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.

Thus 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. To further evaluate the structural organization of fptA we evaluated the sequences of the 2 regions of low sequence complexity and 3 the domains individually using a Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997). 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. Position-Specific Iterated BLAST (PSI-BLAST) provides an automated, easy-to-use version of a "profile"search, which is a sensitive way to look for sequence homologues. 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 FptA 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. Detailed analysis of these results from the blast analysis of the individual domains revealed that the amino acid sequences internal to domain 1 and domain 3 are variable (see Figure 4). These results were corroborated in separated study whereby monoclonal antibodies specific for binding epitopes to the E. coli siderophore receptor FepA inhibited the binding of ferric-enterobactin with purified FepA. However, these same antibodies failed to inhibit the binding of ferric enterobactin with purified FecA. These results demonstrated that the binding epitopes in FecA and FepA were different (Zhou et al. 1995). The d2 domains 1 and 3 contain sequence patterns that are independent of the iron-binding ligand (Figure 4). However, the fact 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. Alternative the difference in the number of returns may be a consequence of incomplete sequences being deposited in the databases used in these analyses.

As mentioned previous in this document binding of a ferric-siderophore to its recognition site is proposed to lead 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. Indeed, a conformational change of the secondary structure of the N-terminal sequence of the protein, which switches from a helix to an unwound structure, has been observed upon binding of ferric-siderophore to FhuA and has been proposed to signal the receptor-loading status to the protein TonB (Moeck et al 1997). For example it was demonstrated that the N- terminus of the P. aeruginosa FpvA receptor was not required for binding to its ligand (pyoverdin). In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N- terminal periplasmic extensions of FecA from E. coli and PupB from P. putida, which were previously shown to be involved in signal transduction (Folschweiller et al 2000).

The deleted 87 amino acids were analyzed using the BLAST tools. The results of the query (6 hits) showed that the sequence was not associated with a conserved function.

However, a direct comparison of the FpvA amino acid sequence with the d2 domain 1 using a pairwise BLAST tool showed that this sequence was present. Subsequent analysis of the consensus sequence of domain 1 revealed a conserved loop structure (hence forth called the Scott-Thomas domain).

We predict that the Scott-Thomas domain loop structure may play a role in signaling TonB that a ligand is loaded in the binding site of the siderophore receptor. This claim is supported by data that showed E. coli cells that possessed a mutant FhuA that was devoid of the N-terminus (residues 1-160) of the siderophore receptor FhuA resulted in a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB.

Higher concentrations of ferrichrome supported growth of a Ton B mutant that synthesized FhuADelta5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuADelta5-160.

However, growth of an FhuADelta5-160 TonB wild-type strain occurred at low ferrichrome concentrations, and ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of ferrichrome binding sites provided by the cork.

To test our claim that ligand-binding sites are internal to the Scott-Thomas domain and d2 domain 3 in FptA we scanned the polypeptide for continouous amino acid sequences using hydrophilicity/hydrophobicity plots. Four (4) candidate peptides were chosen using the Hopp and Woods algorithm and comparing the results to the method of Kyte and Doolittle. The peptides were synthesized commercially at Research Genetics (Huntsville, Al). Mice were immunized with individual peptides (50, ug) emulsified with Freund's complete adjuvant and injected intramuscularly. After two booster doses with the same amount of peptide 10 in incomplete Freund's adjuvant at +14 and +28 days, the anti-peptide antisera were collected on day +42 and tested by for the inhibited the proliferation of P. aeruginosa.

In a B-D Falcon 48 well tissue culture plate (Fisher Scientific, Suwanee, GA) 1 ml of M9-minimal medium supplemented with maltose (lOg/ml) ; methionine (40 pg/ml) ; and 1M MgS04 (0.1%). was added to individual wells. The minimal medium also included 100 ul of peptides 1-5 antisera. An overnight culture of P. aeruginosa was serially diluted in increments of 100-fold. One-microliter aliquots of a 10-8 dilution factor of bacteria were inoculated into the M9 medium and incubated at 37 C for 18 h - 24 h.

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. aerzlginosa (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.

As mentioned previously, monoclonal antibodies specific for binding epitopes to the E. coli siderophore receptor FepA inhibited the binding of ferric-enterobactin with purified FepA. However, these same antibodies failed to inhibit the binding of ferric enterobactin with purified FecA. These results demonstrated that the binding epitopes in FecA and FepA were different (Zhou et al. 1995). BLAST analysis of the third domain returned 272 hits from available databases. Subsequent BLAST analysis of the amino acid sequences that separate the Scott-Thomas domain and d2 domain 3 in the 272 hits could be categorized according to the structural class of the iron-binding ligand it recieves (SEQ ID NOS 3-8).

As mentioned previously, 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.

For example, a 100 ml culture of Pseudo7nonas 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-tract solution 2. The resuspended cells were then mixed with an equal volume of DNA X-traetTm 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 and then centrifuged at 10,000 x g for 10 min. The aqueous phase (top phase) was transferred to a new tube and the chloroform extraction step and centrifugation was repeated. The aqueous phase was again transferred to a new tube and mixed with 10.0 ml of DNA X-tract precipitation solution and incubated on ice for 30 min or longer. The P. aeruginosa genomic DNA was precipitated by centrifuging at 10,000 x g for 15 min in a microcentrifuge. 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 pl, 45. pL of 10x S1 nuclease buffer (2M NaCl, 50 mM NaOAc, pH 4.5,10 mM ZnS04, 5% glycerol), and 1.7 Ill of S1 nuclease at 100 U/pl 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 p1 of 100 mM MgCl2, 8 pi of dNTP mix (2.5 mM each of DATP, dCTP, dGTP, and dTTP), and 4. pi of 5 U/. pl Klenow. The mixture was incubated at 12 C. for 30 min. 450 p1 of STE (0. lM NaCl, 10 mM Tris-HCl, 1 mM EDTA ; pH 8.0) was added, and the mixture extracted once with phenol/chloroform, and once with chloroform, before adding 1 ml of ethanol to precipitate the DNA. The sample was incubated on ice for 10 min or at-20 C. overnight. The DNA was harvested by centrifugation in a microcentrifuge for 30 min., washed with 70% t ethanol and dried.

The DNA was resuspended in 7 pi of TE and to the solution was added 14 pi of phosphorylated Eco RI linkers (200 ng/pl), 3 pi of 10X ligation buffer, 3 pi of 10 mM ATP, and 3 pi ofT4 DNA ligase (4 U/pl). 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 p1 of H2 O, 45. pi of lOx Universal buffer, and 7 pi of Eco RI at 30 U/lll. After incubation at 37. C. for 1.5 h, 1.5 pi of 0. 5M EDTA 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 pi of TE buffer. 20 ng of insert DNA was ligated for 2-3 days at 4 C with Ipg of ZAP II vector in a final volume of 5 pl. 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 1 of SM buffer and 20 pu 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 MgS04 at a OD600 of 1. 0.

In a Falcon 2059 polypropylene tube 200 ul of the diluted XL1-Blue MRF'cells were mixed with 250 p1 of phage stock (> 1 x 105 phage particles), and 1 ul ofExAssist helper phage (> 1 x 106 pfu/lll) to release the phage particles at 37 C for 15 min. Next 3 ml LB broth was added to the tube, then incubated for 2.5-3.0 h at 37 C with shaking. At the completion of the incubation step 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 1 of freshly grown SOLR cells (OD600 1.0) to two 1.5 ml microcentrifue tubes. 100 ttl of the phage supernatant wasa added to 1 tube abd 10 al 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 jj. l of the cell mixture was plated on LB-amplicillin agar plates (50 llg/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-tract 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. The aqueous phase (top phase) was transferred to a new tube and mixed with 10.0 ml of DNA X-tract precipitation solution and incubated on ice for 30 min or longer. 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.

Siderophore binding proteins play a key role in the uptake of iron in many gram- positive and gram-negative bacteria. However the strategy listed above is limited to siderophore receptors of gram-negative bacteria. To identify target sequences that transcends across taxonomic lines that define gram-negative and gram-positive bacteria we evaluated members of the periplasmic binding protein family. Periplasmic binding protein sequences are conserved among gram-negative and gram-positive bacteria. Transport of certain ligands across the cytoplasmic membrane depends on a periplasmic binding-protein-dependent (PBT) system. The hydrophobic components from PBT systems involved in the uptake of siderophores, haem and vitamin B12 define a subclass of polytopic integral membrane proteins. The topology of these 'siderophore family'proteins differs from that of the equivalent components of other PBT systems in that each polypeptide (and each half of FhuB) consists of 10 membrane-spanning regions, with the N-and C-termini located in the cytoplasm. The conserved region at a distance of about 90 amino acids from the C-terminus, typical of all hydrophobic PBT proteins, is also oriented to the cytoplasm. However, in the 'siderophore family'proteins this putative ATPase interaction loop is followed by four instead of two transmembrane spans. (Groeger and Koster 1999; Kerppola and Aines 1992).

The siderophore receptor FhuD was used as a model to localize target sequences that are useful as immunogens. FhuD 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 structure 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.

8. Strategies for the identification of target sequences The discovery of 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.

Furthermore, diagnostic assays and kits using such conserved amino acid sequences are useful to detect many if not all bacteria that produce siderophore receptor.

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 structures.

9. Evaluation of kinetic constants of Anti-microbial Immunoglobulins In order to determine the kinetic parameters of antibodies that bind to the cell surface of pathogenic bacteria, specifically the on and off rates and their dissociation constants (KD), will be analyzed on a BIAcore instrument (Pharmacia).

10. hnmuotherapeutics Recently, it was discovered that 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.

These 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 mechanisms that regulate immunoglobulin G (IgG) catabolism are little understood. In a recent study the half-lives have radiolabelled mouse IgGl injected intravenously into beta 2-microglobulin-/-mice and wild-type or heterozygous siblings was compared. The clearance of lzsI-labelled IgGl was strikingly more rapid in the mice-lacking beta 2-microglobulin. beta 2-microglobulin-/-mice lack functional molecules of the MHC class I-related Fc receptor, FcRn. To determine whether the slower degradation of immunoglobulin in mice with beta 2-microglobulin correlated with the ability of the antibody to bind FcRn, the clearance of chicken IgY was measured which does not bind this receptor. The l25I-labelled IgY was catabolized equally as fast as beta 2-microglobulin-deficient and wild-type mice. These data suggest that FcRn can protect IgG from degradation, and is therefore important in maintaining IgG levels in the circulation.

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. Alternatively, IgY variable regions (heavy and light chains) 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.

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. (1994). Ultimately, clones from the library are identified which produce binding affinities of a desired magnitude for the antigen, and the DNA encoding the product responsible for such binding is recovered and manipulated for standard recombinant expression. 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, 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.

For therapeutic applications, 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.

For parenteral administration, the antibodies may be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. If the antibody is suitable for oral administration, 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. For prevention or treatment of disease, 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.

11. Animal Model A. Active Immunization OMAP antigen cocktails or peptides will be injected intramuscularly in vertebrate host, followed by three boosts of antigen at weekly intervals. One week after the last immunization the vertebrate host will be challenged with a predefined dose of desired pathogen. In addition, 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. One day later, 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.

B. Passive Immunization 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. One day after immunization, the animal is inoculated with a specified pathogen at a predetermined dosage. One day later, 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.

C. Calculating effective dosages of anti-microbial immunoglobulins As determine in example 5 the effectiveness of the antimicrobial immunoglobulins are dose dependent. More specifically it required 1 ug/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.

In a normal patient the volume (Vd) 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 ag./ml., patient weight Vd 0.25. For a 70 kg person the loading dose would be 175 mg., corresponding to the standard loading dose of 2.5 mg./kg commonly used by clinicians.

Several factors increase Vd up to 0.7 I./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. For example changing Vd decreases serum aminoglycoside concentrations by 25% to 40% if the dose is not increased appropriately. Vd 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. One can decrease the dosing interval to accommodate renal insufficiency, which is most easily done by calculating creatinine clearance and decreasing the interval appropriately.

Thus, to determine the number effective dosage of antimicrobial immuoglobulins in a patient the following equation is recommended: D = c (2h) bv Whereby, D = Dose of Antimicrobial Immunoglobulins c = number of cells as determined by Whole Cell ELISA h = numbers of hours expired after extraction of serum from patient b = amount of antibody required to neutralize one bacteria cell v = distribution volume Whereby, v*= patient weight in kg x 0.25 * The serum volume of patient can be affected several factors Ultimately, these simplistic models must be modified to incorporate mass action, distribution kinetics, antigen production rates, on and off rates for antigen-antibody interaction, affinities for soluble vs. cell-bound antigen, and antibody and radioisotope half-lives. Mass action specifies minimum free (unbound) antibody concentrations that must accompany a given degree of antigen saturation. Kinetics of tissue distribution means that substantial concentration gradients must be applied to achieve tumor penetration in reasonable time frames, which further diminishes the dose concentration in tumor by the antibody excesses and reduced specific activities implied. On and off rates determine the rapidity of antigen-antibody exchange, and the relative affinities determine the equilibrium binding ratios between soluble and cellular antigen. Finally, the tel, 2 of antibody survival and radioisotope decay both provide dimensions against which all other kinetic processes are measured.

It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis, and treatment of disease and infections caused pathogenic microorganisms. A further non- limiting discussion of such uses is further presented below. These examples are not meant to limit the scope of the invention that has been set forth in the foregoing description. Variation within the concepts of the invention is apparent to those skilled in the art. The disclosures of the cited references throughout the application are incorporated by reference herein.

Example 1 In this example used the method claimed in this application for the preparation of D2- DLS01 antigen cocktail An overnight culture of Stenotrophomonas maltophilia strain, designated D2-DLS01, was used to inoculate 500 ml of freshly prepared M9-minimal medium supplemented with maltose (lOg/ml) ; methionine (40 pg/ml) ; 1 M MgS04 (0.1%); and the iron- chelator, 2'2'dipyridyl (100 uM). 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.

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.

The solubilized membranes were mixed with 10% Polyethyleneimine (PEI) by stirring until the concentration of PEI was 1% of the solubilized membrane solution. 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.

Usually, 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.

Example 2 Preparation of anti-D2-DLS01 antisera D2-DLS01 antigen solution was diluted to 5 llg/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. Bleeding was performed by inserting the 19G needle of a winged infusion set into the central artery of the ear. 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 jug 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.

Four weeks later the rabbits were injected with 250 u, 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).

Example 3 In this example the immunogenicity of D2-DLS01 antigen cocktail was detemined using an enzyme linked immunoabsorbent assay (ELISA) method.

100 ng of 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. Next the membrane-associated polypeptides were probed with 100 1 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 u. l of 1 N NaOH. The color development was measured spectrophotometrically at 405 nm. The results are shown in figure 6. In a separate experiment the D2-DLS01 antisera reacted positively to the individual surface exposed immunogenic polypeptides or an immunogenic fragments (SEQ ID NOS 3-17) (Data not shown).

Next, increasing concentrations of immunogenic polypeptides from S. maltophilia D2- DLS01 were evaluated by polyacrylamide gel electrophoresis, followed by transfer step to a 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-HCl pH 7.4; 150 mM NaCl ; 0.5% Tween 20) the filter was probed with goat anti-rabbit alkaline phosphatase conjugate (Sigma-Aldrich, St. Louis, MO) for 1 h. 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).

Example 4 Purification of Immunoglobulins To separate the anit-D2 DLS01 immunoglobulins form other components of serum a protein A/G affinity column was used. Briefly, an Immobilized Protein A/G column (3 ml) (Pierce, Rockford, Ill.) 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 J. 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).

Example 5 In this example, anti-D2-DLS01 polyclonal antibodies were evaluatedfor their ability to inhibit the proliferation of S. maltophilia in iron-deplete medium In a B-D Falcon 48 well tissue culture plate (Fisher Scientific, Suwanee, GA) 1 ml of M9-minimal medium supplemented with maltose (lOg/ml) ; methionine (40 llg/ml) ; 1 M MgS04 (0.1%) and 120 gM 2'2'dipyridyl was added to individual wells. The minimal medium also included purified immunoglobulins at a concentration of 100 Lg/ml. An overnight culture of S. maltophilia was serially diluted in increments of 100-fold. One-microliter aliquots of the serial diluted bacteria were inoculated into the M9 medium and incubated at 37 C for 18 h-24 h. Simultaneously, 100 ul portion of the serial diluted bacteria were streaked on M9-maltose agar plates and incubated at 37 Cforl8h-24h.

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 pg of purified immunoglobulins are effective in neutralizing 100 or less bacteria.

Example 6 In this example, anti-D2-DLS01 polyclonal antibodies were evaluatedfor their ability to inhibit the proliferation of S. maltophilia in iron-replete medium In a B-D Falcon 48 well tissue culture plate (Fisher Scientific, Suwanee, GA) 1 ml of M9-minimal medium supplemented with maltose (lOg/ml) ; methionine (40 j-Lg/ml) ; 1 M MgS04 (0.1%); and the iron-chelator, 2'2'dipyridyl (100 uM) was added to a set of wells (3). In another set of wells 1 ml of M9-minimal medium supplemented with maltose (lOg/ml) ; methionine (40 J. g/ml) ; 1 M MgS04 (0.1%); and the FeC13 (50 uM) was added to individual wells. As a control 1 ml of M9-minimal medium supplemented with maltose (lOg/ml) ; methionine (40 pg/ml) ; and 1 M MgS04 (0.1%); was added to each. The iron-deplete, iron-replete and the control mediums were supplemented with purified immunoglobulins at a concentration of 100 J. g/ml.

An overnight culture of S. inaltophilia was serially diluted in increments of 100-fold.

One microliter aliquots of a the 10-8 dilution factor of bacteria were inoculated into the M9 medium and incubated at 37 C for 18 h-24 h.

The growth rate of S. maltophilia in the presence of the immunoglobulins in iron- deplete and iron-replete medium were evaluated by monitoring the absorbance spectrophotometrically at 600 nm. As seen in figure 10 the concentrations of immunoglobulins used in this experiment successfully inhibited the growth rate of S. maltophilia in iron-deplete and iron-replete conditions. After 48 h-72 h growth was notice in the iron-replete medium. This observation was overcome by increasing the concentration of immunoglobulin to 500 llg/ml.

Example 7 In this example, anti-D2-DLS01 polyclonal fab fragments were evaluated for their ability to inhibit the proliferatiora of S. fnaltophilia 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.

In a B-D Falcon 48 well tissue culture plate (Fisher Scientific, Suwanee, GA) 1 ml of M9-minimal medium supplemented with maltose (lOg/ml) ; methionine (40 pg/ml) ; 1 M MgS04 (0.1%); and the iron-chelator, 2'2'dipyridyl (100 uM) was added to individual wells. The minimal medium also included purified fab fragments at a concentration of 100 Fg/ml. An overnight culture of S. maltophilia was serially diluted in increments of 100-fold. One microliter portions of a the 10-8 dilution factor of bacteria were inoculated into the M9 medium and incubated at 37 C for 18 h-24 h.

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.

Example 8 In this example the ability of anti-D2-DLS01 polyclonal antibodies to recognize other pathogenic bacteria and fungi using an enzyme linked immunoabsorbent assay (ELISA) method.

100 ng of 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. Next the were probed with a 1 1000 dilution of anti-D2-DLS01 polyclonal antibodies. After thoroughly washing antibody binding was determine using an anti-rabbit alkaline phosphatase conjugate (diluted 1: 1000). 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 (J. 1 of 1 N NaOH. D2-DLS01 antisera only recognized the gram-negative bacteria (data not shown).

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