Title:  Invaplex from gram negative bacteria, method of purification and methods of use

United States Patent:  6,680,374

Issued:  January 20, 2004

Inventors:  Oaks; Edwin V. (Gambrills, MD); Turbyfill; Kevin Ross (Waldorf, MD)

Assignee:  The United States of America as represented by the Secretary of the Army (Washington, DC)

Appl. No.:  772878

Filed:  January 31, 2001

Abstract

Isolated antibodies to Invaplex; novel compositions comprising immunoglobulins directed to invasin proteins and LPS from gram negative bacteria that selectively bind to Invaplex, and do not bind to the individual components of Invaplex.

SUMMARY OF THE INVENTION

The present invention fulfills the needs described above. In this application is described a novel composition comprising at least one invasin protein, a protein essential for bacterial invasion of a host cell, such as IpaA, IpaB, IpaC, and IpaD for Shigella. The invasin protein or proteins of the present invention are complexed with lipopolysaccharide (LPS). The complex of invasion protein or proteins and LPS is in a native conformation and has been termed Invaplex. The Invaplex described below is not only effective as a vaccine against gram-negative bacterial infection but it can also serve as a mucosal adjuvant and a diagnostic tool for detecting antibody response which correlates with protection against future infection.

Our initial experiments were aimed at isolating and purifying IpaC from a water extract of Shigella, a gram-negative bacteria. Usually, IpaC is extracted from growth culture medium. We chose to use the water extract, i.e. the solution resulting from incubating the bacteria with shaking in sterile water, because we hypothesized that the quantity of IpaC would be greater in such an extract. To our knowledge, no protein involved in the invasiveness of gram negative bacteria has been previously isolated from a water extract of gram negative bacteria. To our suprise, when water extract was subjected to various separation techniques such as gel filtration and ion-exchange chromatography, we found that whenever we could detect IpaC from the water extract, we also detected IpaB, IpaD and LPS in the same fractions. We proceeded to design a method to isolate this complex and characterize it. We have developed a method for purifying the Invaplex from intact invasive Shigellae or enteroinvasive E. coli (see FIG. 1 for general overview). Briefly, the Invaplex preparations are isolated from virulent, invasive shigellae. A crude mixture is extracted from the shigellae with water. The water extract consists of many proteins and lipopolysaccharide (LPS). The water extract material is then applied to a FPLC ion-exchange column which resolves two key protein peaks, called Invaplex (invasin complex) 24 and Invaplex 50. Fractions containing Invaplex 24 and Invaplex 50 are collected. We found that the complex was composed of many proteins, including IpaB, IpaC, IpaD in addition to LPS. The Invaplex 24 and Invaplex 50 preparations containing Ipa proteins and the LPS form a structure in a completely native configuration and environment. If such a structure is used to immunize animals, it will lead to an immune response directed against a native structure presented by gram-negative bacteria during infection. Mice and guinea pigs immunized with the Invaplex preparations showed a marked serum IgA and IgG response to several different antigens (including the water extract antigen, IpaC and LPS) present in the Invaplex 24 and Invaplex 50 preparations. The two Invaplex preparations were similar in that they both primed the mucosal immune system, but differed inthe specificity of the immune response generated. The animals were protected from challenge with gram negative bacteria and immunization with either Invaplex caused no visible distress to the animals. In addition, the highly immunogenic Invaplex can also serve as a very effective mucosal adjuvant very likely due to its ability to interact with the surfaces of host cells and present antigen to the immune system.

Our unpublished data where serum antibodies from recent vaccinees were found to be reactive with the Shigella water extract but poorly reactive with purified Ipa proteins indicates that the Invaplex may be recognized differently (conformationally) than the individual proteins. This suggested that a population of antibodies which specifically react with the complex and not the individual components are produced during an infection and may not be measured in an ELISA using individual purified proteins.

The Invaplex diagnostic assay of the present invention will measure a population of antibodies reactive with a virulence structure. Purified components of the invasin complex such as pure Ipa proteins or pure LPS, are unable to detect this population of antibodies.

Therefore, the present invention relates to a purified composition comprising Invaplex of gram-negative bacteria and methods of using the purified Invaplexes as adjuvants or diagnostic tools. The inventors have purified the Invaplex from two genera of gram-negative bacteria and determined that the complex comprises major antigenic proteins, the invasin proteins (e.g. for Shigella, IpaB, IpaC, and IpaD) as well as LPS. A novel method for purifying the Invaplex from intact bacteria is described.

The Invaplex containing invasin proteins and the LPS forms a structure in a completely native configuration and environment. This results in the ability to react with antibodies that are directed primarily toward epitopes involving the intact structure in addition to antibodies reactive with epitopes that are maintained on purified individual invasin proteins.

Experiments have shown that the Invaplex is also an effective adjuvant which results in very little reactogenicity or toxicity in addition to stimulating a potent mucosal and serum immune response when administered along with the desired antigen.

Therefore, it is one object of the present invention to provide a novel method for the isolation and purification of the Invaplex from gram negative bacteria.

It is another object of the present invention to provide a composition comprising isolated purified Invaplex as a diagnostic tool for detecting gram negative bacteria used as the source of the purified Invaplex.

It is yet another object of the present invention to provide a diagnostic assay for detecting gram-negative bacterial infection having the steps of contacting a sample from a subject suspected of having a gram negative bacterial infection with a purified Invaplex and detecting the presence or absence of a complex formed between the Invaplex and antibodies specific therefor, wherein the presence of a complex indicates presence of gram negative bacterial infection.

Further objects and advantages of the present invention will be clear from the description and claims that follow.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in part to a method for isolating purified Invaplex from gram-negative bacteria.

Invaplex can be prepared from any gram-negative bacteria including but not limited to those classified under the following genera, Shigella, Escherichia, Salmonella, Yersinia, Rickettsia, Brucella, Erhlichiae, Edwardsiella, Campylobacter, Legionella and Neisseria. These are all invasive bacteria that have a gram negative architecture (i.e. they have an inner or cytoplasmic membrane and an outer membrane surrounding the inner membrane).

In addition to wild type virulent gram-negative bacteria, mutants of these organisms may be useful, such as those which hyper-express quantities of invasin proteins and which might lead to the production of more Invaplex. The gene virF, for example is involved in the regulation of Ipa proteins in shigellae (Sakai et al. 1988, Mol. Microbiol. 2, 589-597). All documents cited herein supra and infra are hereby incorporated in their entirety by reference thereto. Furthermore, it may be beneficial to perpare Invaplex from bacteria mutated in toxin genes so that the organism does not produce toxin, for example Shiga toxin. Invaplex prepared from tox-strains would be potentially safer because potential contamination by the toxin would be eliminated

Increased levels of Invaplex may be achieved by extracting the complex in the presence of chemicals that stimulate secretion of the invasin proteins. Such chemicals include Congo Red, Evans Blue and direct orange (Bahrani, F. K, Infect Immun 65:4005-4010, 1997). These chemicals could be added during the water extraction or during the growth of the bacteria.

In order to isolate the invasin proteins (Ipa proteins or similar proteins in other invasive bacteria), the invasin proteins must be expressed by the bacteria. If the invasin proteins are not expressed on the surface or not expressed at all, the Invaplex will not be present. For example, in S. sonnei one must use form I cultures because they are virulent. Form II cultures do not express the Ipa proteins due to a large spontaneous deletion in the virulence plasmid.

Ipa protein presentation on the surface of shigellae may be decreased by mutating genes in the spa or mxi gene loci. The spa/mxi gene mutants make the Ipa proteins in normal quantities but the Ipa proteins are not presented or secreted to the exterior of the organism. Previously it has been shown that reduced amounts of IpaB and IpaC are in the water extract in spa mutants (Venkatesan et al., 1992, J. Bacteriol. 174, 1990-2001)

To overcome the possibility of using avirulent cultures, it is important that cultures used as a source of Invaplex be tested and proven to be virulent. This is usually done by the Sereny test (keratoconjunctivitis in guinea pigs as described in the Materials and Methods and Examples below). Another means in which to assure virulence is growing cultures of shigellae on Congo Red media in which colonies that are red (or bind the dye congo red) are almost always virulent. Assessment of virulence is extremely important with shigellae cultures as spontaneous mutations leading to avirulent cultures is commonplace. It is understood that even though some cultures may be partially virulent, it is possible to isolate Invaplex though the yield will be compromised.

In one embodiment, this invention relates to a method for isolating and purifying the Invaplex from gram negative bacteria. The present method uses an improvement of the water extraction technique described by Oaks et al., 1986 (Infect Immun 53, 57-63). These improvements were designed to increase yield of functional product by minimizing the time to prepare the water extract preparation. The improvements include using an ion-exchange column capable of concentrating the Invaplex thereby eliminating the need to concentrate the water extract by time-consuming ultrafiltration step prior to loading onto the ion-exchange column. Ultrafiltration often took overnight to perform during which time proteolytic degradation might occur. The Ipa proteins are extremely labile and degrade rather quickly. Another improvement is the amount of water used to extract the proteins. The present method uses a volume of water which is 1/20 the volume of the medium used to grow the culture instead of a ratio of 1/10 used previously (Oaks et al., 1986, supra). For example if the shigellae are grown in 20 liters of medium than one would use 1 liter of water for the extraction. Other modifications include filtration of the water extract with a 0.45 or 0.22 um membrane both before and/or after ultracentifugation. This step will remove bacteria from the water extract material and make it less likely to contain viable bacteria. This step will be essential for any product to be used in human trials. Also the original procedure described in Oaks, 1986 (supra) used PMSF (phenylmethylsulfonyl fluroide) a very potent and dangerous protease inhibitor. This protease inhibitor is no longer used because it is toxic. To minimize degradation of the proteins in the complex the water extract is maintained on ice to minimize proteolytic degradation.

The present method includes the steps of collecting bacterial cells, extracting the cells in sterile water, separating and discarding membrane fragments from the water extract resulting in a solution containing the Invaplex, and isolating the Invaplex from the solution. Growing gram negative bacteria in culture is known to a person with ordinary skill in the art. For a general reference, please see Manual of Methods for General Bacteriolog, 1981, Washington, D.C., P. Gephardt et al., eds. Media and conditions for growth are discussed for Shigella in detail in Materials and Methods below. After growth, the bacterial cells are extracted in sterile water, 20 mM Tris-HCl, normal saline (0.15M NaCl), or other buffers as long as the conditions allow binding of the Invaplex 24 and Invaplex 50 fractions to the desired column.

It is preferable to delete detergents in the extraction solution since detergents tend to form mixed micelles (micelles containing a variety of proteins) which may not behave in a consistent manner on the ion-exchange column. Detergents will also solubilize integral membrane proteins which may interfere with the Invaplex product. Finally, detergents may disrupt or denature the Invaplex and solubilize all of the indivdual components of the complex. Sterile water can be prepared by methods known in the art, for example, filtering through a 0.10, 0.22 or 0.45 micron filters. The concentration of bacterial cells to water is preferably 1x10⁷ to about 2x10¹² cells per ml of water or buffer, more preferably between 1x10⁸ to about 2x10¹¹.

Extraction time is preferably regulated since if it is too long, degradation of product may result, and too short will result in poor yield of product.

A protease inhibitor can be added, during the extraction step to aid in reducing the protein degradation of the final product. Examples of proteases which could be added include phenylmethylsulfonyl fluoride (PMSF). Other protease inhibitors are available such as serine protease inhibitors but they are usually somewhat toxic. If the Invaplex is to be administered to living cells, it would be preferable to delete the protease inhibitors or remove it from solution prior to administration due to the toxicity of the protease inhibitors. However, if the Invaplex is to be used for an ELISA reagent, the protease inhibitor could be left in the solution, in fact it would be preferable to have it in the solution.

Next, the cells and membrane fragments are removed from the solution by methods known in the art such as centrifugation, filtration, microfiltration, ultrafiltration, however, ultracentrifugation is preferable for removing the small membrane fragments before the solution is subjected ion-exchange chromatography. Following extraction, the complex may be separated from the cellular debris by any technique suitable for separation of particles in complex mixtures. The complex may then be purified by anion or cation exchange chromatography or other isolation techniques which may include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, isoelectric focusing, immunoadsorption, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC), high performance LC (HPLC), fast performance LC (FPLC), hydroxylapatite chromatography and lectin chromatography. Anion exchangers include diethylaminoethyl (DEAE) {--OCH2CH2N+H(CH2CH3)2}; quaternary aminoethyl (QAE) {--OCH2CH2N+(C2H5)-CH2CHOH--CH3}; and quaternary ammonium (Q) {--OCH2CHOH--CH3CHOH--CH2N+(CH3)C3}. Such functional groups are bound to various supports, each support varying in particle size, but also vary with respect to the support material. Examples of support material include: Monobeads, 10 um bead of hydrophilic polystyrene/divinylbenzene {i.e., Mono Q (Pharmacia, Uppsala, Sweden)}, Minibeads, 3 um bead of a hydrophilic polymer {i.e., Mini Q (Pharmacia)} SOURCE, 15 & 30 um monodispersed hydrophilized rigid, polystyrene/divinylbenzene beads {i.e., SOURCE Q (Pharmacia)} Sepharose, 34-50 um highly crosslinked agarose beads {i.e., HiTrap Q (Pharmacia) and Econo-Pac High Q (Bio-Rad)} Sepharose Fast Flow, 90 um agarose beads {i.e., QSepharose Fast Flow (Pharmacia)}, Sepharose Big Beads, 100-300 um agarose beads {i.e., QSepharose Big Beads (Pharmacia)}.

The chloride ion (Cl--) is the counterion of choice for anion exchange chromatography, with the choice of buffer dependent on the required pH interval. While Tris has a an effective buffering range of 7.6 to 8.0. Other buffers which may be used include: N-methyl-diethanolamine (pH 8.0-8.5), diethanolamine (pH 8.4-8.8), 1,3-diamino-propane (pH 8.5-9.0), ethanolamine (pH 9.0-9.5), and potentially piperazine (pH 9.5-9.8). These buffers are used at a low concentration, usually 20 mM, but could be as high as 50 mM.

Other columns or methods may be used as long as they maintain native structure of the Invaplex so that immunogenicity and function is intact, allow large volumes of a dilute protein solution to be loaded and concentrated, the buffers are biologically compatible, the method is rapid in order to minimize degradation of product and few processing steps are required.

It is preferable that each column be dedicated to a specific serotype and strain of gram-negative bacteria. The optimal protein concentration in the final product would be approximately 10 doses per ml. But the range could be as low as 0.1 dose per ml (protein conc. of 2.5 ug/ml) up to much higher levels of 5000 doses per ml (protein conc. of 125 mg/ml) as long as solubility is maintained, i.e. concentration not too high to cause precipitation and not too low to make filtration too costly and time consuming.

Ideally we are achieving 0.25 mg/ml to 5 mg/ml in peak fractions of Invaplex 24 and Invaplex 50. If protein concentration is less than 0.25 mg/ml than it must be concentrated by centrifugal size-exclusion filtration (mw cutoff of 10000 to 100,000 more preferably 30,000 mw cutoff).

Using the method described in the Materials and Methods below, the fractions containing the greatest amount of IpaB and IpaC were found in fractions eluted at 24% buffer and 50% buffer from the ion-exchange column, resulting in Invaplex 24, and Invaplex 50.

This method needs to be modified minimally for use with other gram-negative bacteria. For example, other ion-exchange columns can be used, and different antibodies must be used to probe for the target antigens. For example, antibodies for SipB and SipC would have to be used to identify peak fractions containing the complex from Salmonella. Yersinia would need anti YOP protein antibodies (Corneliz and Wolf-Watz, 1997, Mol. Microbiol. 23, 861-867).

Other methods for producing Invaplex include methods whereby individual subunit invasin proteins are combined with LPS in order to form a complex with a native configuration. In addition, the invasin/LPS complex can be further purified from other components in the Invaplex 24 and Invaplex 50 fractions by purification techniques as desribed above and below.

In another embodiment, the present invention relates to monoclonal or polyclonal antibodies specific for the above-described invasin complex. For instance, an antibody can be raised against a complex described above, or against a portion thereof of at least 10 amino acids, preferably, 11-15 amino acids. The peptides can be chosen to contain structural or conformational epitopes. Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to an invasin complex or polypeptide chosen of the present invention, or a unique portion thereof. Material and methods for producing antibodies are well known in the art (see for example Goding, in, Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986).

In a further embodiment, the present invention provides a method for detecting a gram-negative infection in a biological sample. By "biological sample" is intended any biological sample obtained from a subject. A subject is an insect, arthropod, animal, bird, fish, cell line, tissue culture, mammal including humans or other source, as well as other environmental samples such as water, plant, food, which may contain gram negative bacteria. Biological samples include body fluids (such as saliva, blood, plasma, urine, mucus, synovial fluid, stool etc.) tissues (such as muscle, skin, and cartilage) and any other biological source suspected of containing gram negative bacteria or the invasin complex of a gram negative bacteria. Methods for obtaining biological samples are known in the art.

Assaying for gram-negative bacteria infections can occur using any art-known method, such as antibody-based techniques. For example, the presence of Invaplex can be studies with classical immunohistological methods. In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunohistological staining of tissue section for pathological examination is obtained. Tissues can also be extracted, e.g. with urea and neutral detergent, for the liberation of invasin complex for Western-blot or dot/slot assay.

Other antibody-based methods useful for detecting gram negative infection include immunoassays, such as the ELISA and the radioimmunoassay (RIA). Using standard methodology well known in the art, a diagnostic assay can be constructed by coating on a surface (i.e. a solid support) for example, a microtitration plate, a membrane (e.g. nitrocellulose membrane), polymeric beads, or dip sticks, antibodies specific for invasin complex or invasin complex itself, or fragments thereof, and contacting it with a sample from a person suspected of having a gram negative bacterial infection. The presence of a resulting complex formed between invasin complex and antibodies specific therefor in the sample can be detected by any of the known detection methods common in the art such as fluorescent antibody spectroscopy or colorimetry. A good description of a radioimmune assay may be found in Laboratory Techniques and Biochemistry in Molecular Biology. by Work, T. S., et al. North Holland Publishing Company, N.Y. (1978), incorporated by reference herein. Sandwich assays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970. ELISA assays and other immunological methods included in the present invention can also be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed., 1988.

Invaplex or anti-invaplex antibodies, or fragments of ligands or antibodies capable of detecting Invaplex may be labeled using any of a variety of labels and methods of labeling for use in diagnosis and prognosis of gram negative bacterial infection. Examples of types of labels which can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non-radioactive isotopic labels, and chemiluminescent labels.

Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc.

Examples of suitable radioisotopic labels include ³ H, ¹¹¹ In, ¹²⁵ T, ³² P, ³⁵ S, ¹⁴ C, ⁵⁷ To, ⁵⁸ Co, ⁵⁹ Fe, ⁷⁵ Se, ¹⁵² Eu, ⁹⁰ Y, ⁶⁷ Cu, ²¹ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, ¹¹ C, ¹⁹ F, ¹²³ I, etc.

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Tr, ⁴⁶ Fe, etc.

Examples of suitable fluorescent labels include a ¹⁵² Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycodyanin label, an allophycocyanin label, a fluorescamine label, etc.

Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, etc.

Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to invasin complex, ligands or to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H., et al.,1976 (Clin. Chim. Acta 70:1-31), and Schurs, A. H. W. M., et al. 1977 (Clin. Chim Acta 81:1-40). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, and others, all of which are incorporated by reference herein.

The detection of the antibodies (or fragments of antibodies) of the present invention can be improved through the use of carriers. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to Invaplex or Invaplex antibodies. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Those skilled in the art will note many other suitable carriers for binding monoclonal antibody, or will be able to ascertain the same by use of routine experimentation.

The Invaplex and antibodies of the present invention, including fragments thereof may be used to detect gram-negative bacteria or gram-negative bacterial infection using bio chip and biosensor technology. Bio chip and biosensors of the present invention may comprise the polypeptides of the present invention to detect antibodies, which specifically recognize Invaplex. Bio chips or biosensors comprising polypeptides or antibodies of the present invention may be used to detect gram negative bacteria in biological and environmental samples and to diagnose an animal, including humans, with a gram negative bacterial infection. Thus, the present invention includes both bio chips and biosensors comprising polypeptides or antibodies of the present invention and methods of their use.

The Invaplex can be used to identify inhibitors of Invaplex. Natural and synthetic agents and drugs can be discovered which result in a reduction or elimination of the invasin complex. Knowledge of the mechanism of action of the inhibitor is not necessary as long as a dissociation of the complex is detected. Inhibitors may include agents or drugs which either bind or sequester the Invaplex. Agents or drugs related to this invention may result in partial or complete inhibition of virulence of gram negative bacteria, and possible inhibitors of Invaplex may be used in the treatment or amelioration of gram negative bacterial infections.

In providing a patient with agents which modulate the function of invasin complex to a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient) although a lower or higher dosage may be administered.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.

The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences [16th ed., Osol, A. ed., Mack Easton Pa. (1980)]. In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the compounds. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic-acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacrylate)-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

The present invention also provides kits for use in the diagnostic or therapeutic methods described above. Kits according to this aspect of the invention may comprise one or more containers, such as vials, tubes, ampules, bottles and the like, which may comprise one or more of the compositions of the invention.

The kits of the invention may comprise one or more of the following components, one or more compounds or compositions of the invention, and one or more excipient, diluent, or adjuvant.

One particularly important and significant advantage of the Invaplex 24 and Invaplex 50 is that, when used in an embodiment to modulate (i.e. regulate) the immune response, administration to an animal or an antigen-carrying Invaplex, or administration of Invaplex along with an antigen of interest, induces the animal to produce a cell-mediated as well as a humoral immune response against that antigen, without causing harmful side effects. Such a property has been much sought after without a great deal of success using killed microorganisms (e.g., bacteria or viruses), subunit vaccines, vaccines carried in viral or bacterial vehicles, and vaccines that include an adjuvant. An antigen refers to any compound heterologous to the Invaplex itself. The antigen is combined with the Invaplex by simply mixing the two substances together. However we fully anticipate making the Invaplex in the presence of the substance to be delivered but this would be more likely to be used for small molecules or pharmaceuticals not antigens.

The compound may be an organic or inorganic compound not found naturally in the gram-negative bacteria used in the production of the Invaplex, and/or a compound encoded by such a nucleic acid molecule (e.g., an RNA molecule or a protein). Organisms that can be protected from a disease are organisms that are susceptible to such a disease and preferably include animals and plants. Compounds can be carried while complexed to the Invaplex, inside, outside, or combinations thereof such that when the Invaplex is administered to an animal, it stimulates a desired immune response against the complexed compound. Any gram-negative invasive bacteria can be used to produce an Invaplex of the present invention.

Invaplex can be used as a vehicle, for the delivery of DNA, RNA, protein, or drug into a specific cell type that is interactive with the Invaplex. The Invaplex can be made in the presence of the substance to be delivered such as small molecules or pharmaceuticals. The stage that the Invaplex would be loaded with the compounds would be during the water extraction.

Claim 1 of 4 Claims

What is claimed is:

1. Isolated polyclonal or monoclonal antibodies that selectively bind to an Invaplex selected from Invaplex 24 or Invaplex 50, which antibodies selectively bind to the Invaplex and not the individual components thereof.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.