WO2010078478A1

WO2010078478A1 - Tissue adjuvants and medical products including the same

Info

Publication number: WO2010078478A1
Application number: PCT/US2009/069894
Authority: WO
Inventors: Paul J. Hall; Michael C. Hiles
Original assignee: Cook Biotech Incorporated
Priority date: 2008-12-31
Filing date: 2009-12-31
Publication date: 2010-07-08

Abstract

Described are adjuvants and medical products including such adjuvants having increased immunogenicity towards a particular antigen. In certain embodiments, the adjuvant includes a CpG oligonucleotide and a collagenous extracellular matrix (ECM) material, preferably in a synergistically effective amount. Medical products including at least one antigen derived from a bacterial pathogen and a collagenous ECM material are also described herein. Such medical products can further include a CpG oligonucleotide. Medical products as described herein can find use as a vaccine preparation to treat or prevent a medical condition, and preferably a medical condition caused by a bacterial pathogen. Related methods of manufacture and use are also described.

Description

TISSUE ADJUVANTS AND MEDICAL PRODUCTS INCLUDING THE

SAME

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of United States Provisional

Patent Application Serial No. 61/141,945 filed December 31, 2008 entitled "Tissue Adjuvant and Medical Products Including the Same" which is hereby incorporated by reference in its entirety.

BACKGROUND

The present invention resides generally in the field of medical products and in particular aspects to adjuvants and vaccines for the prevention and/or treatment of a particular medical condition.

As further background, adjuvants have been used in the medical field to enhance the immune response to a particular antigen. In many cases, adjuvants are administered in conjunction with a vaccine to either prevent or treat a particular medical condition. Common adjuvants include alum (aluminum hydroxide and aluminum phosphate), Freund's adjuvant (both complete and incomplete), calcium phosphate, and oil emulsions. More recently, tissue vaccines have been established that include an extracellular matrix (ECM) material as an adjuvant. Such vaccines have been disclosed to prevent or treat disease caused by infectious pathogens, biological toxins, and even cancer. See International PCT Application Publication Nos. WO 2008/112344, WO 2008/094276, and WO 2008/051852.

Although these adjuvants and vaccines containing such adjuvants have found use in particular applications, a need remains for alternative and improved adjuvants and vaccines containing such adjuvants to treat or prevent a wide range of medical conditions The present invention provides such medical products, as well as methods of preparation and use related thereto. SUMMARY

In one aspect, the present invention provides a medical product. The medical product includes at least one antigen derived from a bacterial pathogen, a CpG oligonucleotide including at least one CpG site, and a collagenous extracellular matrix (ECM) material. The CpG oligonucleotide and collagenous ECM material are present in a synergistically effective amount to increase the immunogenicity of the antigen(s).

In another aspect, the present invention provides a medical product. The medical product includes at least one antigen derived from Staphylococcus aureus or Escherichia coli and a collagenous extracellular matrix (ECM) material.

In another aspect, the present invention provides a vaccine preparation. The vaccine preparation includes at least one antigen derived from a bacterial pathogen, a CpG oligonucleotide including at least one CpG site, a particulate of a collagenous extracellular matrix (ECM) material, and an aqueous carrier. The antigen, CpG oligonucleotide, and collagenous ECM material are dispersed within the carrier. The CpG oligonucleotide and collagenous ECM material are present in a synergistically effective amount to increase the immunogenicity of the antigen(s).

In another aspect, the present invention provides an adjuvant for increasing the immunogenicity of an antigen. The adjuvant includes a CpG oligonucleotide including at least one CpG site and a collagenous extracellular matrix (ECM) material. The CpG oligonucleotide and collagenous ECM material are present in a synergistically effective amount to increase the immunogenicity of the antigen(s).

In another aspect, the present invention provides a method for treating or preventing a medical condition in a patient. The method includes providing a medical product as described herein to the patient. The medical condition to be prevented or treated is caused by a bacterial pathogen, and the medical product includes an antigen derived from the bacterial pathogen.

In another aspect, the present invention provides a method for treating or preventing a medical condition in a patient. The method includes providing a vaccine preparation as described herein to the patient. The medical condition to be prevented to treated is caused by a bacterial pathogen, and the vaccine preparation includes an antigen derived from the bacterial pathogen.

In another aspect, the present invention provides a method for inducing an antigen specific immune response in a patient. The method includes providing a medical product as described herein to a patient. The immune response is specific for the pathogenic antigen contained in the medical product.

In another aspect, the present invention provides a method for preparing a medical product. The method includes providing at least one antigen derived from a bacterial pathogen, a CpG oligonucleotide including at least one CpG site, and a collagenous extracellular matrix (ECM) material. The at least one antigen, CpG oligonucleotide and ECM material are combined with one another such that the CpG oligonucleotide and said collagenous ECM material are present in a synergistically effective amount to increase the immunogenicity of the antigen(s). In the case of a vaccine preparation, the at least one antigen, CpG oligonucleotide, and ECM material are dispersed in an aqueous carrier.

In another aspect, the present invention provides a method for preparing a medical product. The method includes culturing a bacterial pathogen on a collagenous extracellular matrix (ECM) material such that the pathogen deposits at least one immunogenic component having antigenicity thereon. The bacterial pathogen is then rendered biologically inert. In certain embodiments, a CpG oligonucleotide including at least one CpG site can be provided to the material either as a separate component or is exposed by virtue of the bacterial pathogen being rendered biologically inert (e.g., by lysing).

Additional embodiments as well as features and advantages of the invention will be apparent from the further descriptions herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 provides a flow diagram showing a preferred method for preparing a medical product as described herein.

DETAILED DESCRIPTION

As disclosed above, in certain aspects, the present invention provides medical products useful in a wide variety of medical applications. In its broadest scope, such medical products include at least one antigen derived from a bacterial pathogen and a collagenous extracellular matrix (ECM) material. In preferred embodiments, a CpG oligonucleotide including at least one CpG site is included in the medical product. The CpG oligonucleotide and collagenous ECM material can be present in a synergistically effective amount to increase the immunogenicity of the at least one antigen.

It is advantageous to use a remodelable material in the medical products and methods of the present invention, and particular advantage can be provided by including a remodelable collagenous material. Such remodelable collagenous materials can be provided, for example, by collagenous materials isolated from a suitable tissue source from a warm-blooded vertebrate, and especially a mammal. Reconstituted or naturally-derived collagenous materials can be used in the present invention. Such materials that are at least bioresorbable will provide advantage in the present invention, with materials that are bioremodelable and promote cellular invasion and ingrowth providing particular advantage. Remodelable materials may be used in this context to promote cellular growth within the site in which a medical product of the invention is implanted. Moreover, the properties (e.g., thickness) of a medical product can be adjusted to control the extent of cellular ingrowth.

Suitable bioremodelable materials can be provided by collagenous extracellular matrix materials (ECMs) possessing biotropic properties, including in certain forms angiogenic collagenous extracellular matrix materials. For example, ECMs include materials such as submucosa, renal capsule membrane, dermal collagen, dura mater, pericardium, fascia lata, serosa, peritoneum or basement membrane layers, including liver basement membrane. Suitable submucosa- containing materials for these purposes include, for instance, materials that include intestinal submucosa, including small intestinal submucosa, stomach submucosa, urinary bladder submucosa, and uterine submucosa. These identified submucosa or other layers can occur in the ECM material alone, or in combination with other materials such as those derived from one or more adjacent layers in the source tissue.

The submucosa-containing ECM can be derived from any suitable organ or other biological structure, including for example submucosa derived from the alimentary, respiratory, intestinal, urinary or genital tracts of warm-blooded vertebrates. Submucosa-containing materials useful in the present invention can be obtained by harvesting such tissue sources and delaminating the submucosa (alone or combined with other materials) from smooth muscle layers, mucosal layers, and/or other layers occurring in the tissue source. For additional information as to submucosal materials useful in the present invention, and its isolation and treatment, reference can be made, for example, to U.S. Patent Nos. 4,902,508, 5,554,389, 5,993,844, 6,206,931, and 6,099,567, as well as International PCT Application Publication No. WO 08/067085.

As prepared, the submucosal material and any other ECM used may optionally retain growth factors or other bioactive components native to the source tissue. For example, the submucosal or other ECM may include one or more native growth factors such as basic fibroblast growth factor (FGF-2), transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), and/or platelet derived growth factor (PDGF). As well, submucosa or other ECM used in the invention may include other biological materials such as heparin, heparin sulfate, hyaluronic acid, fibronectin and the like. Thus, generally speaking, the submucosa or other ECM material may include a native bioactive component that induces, directly or indirectly, a cellular response such as a change in cell morphology, proliferation, growth, protein or gene expression.

Submucosal or other ECM materials of the present invention can be derived from any suitable organ or other tissue source, usually sources containing connective tissues. The ECM materials processed for use in the invention will typically include abundant collagen, most commonly being constituted at least about 80% by weight collagen on a dry weight basis. Such naturally-derived ECM materials will for the most part include collagen fibers that are non-randomly oriented, for instance occurring as generally uniaxial or multi- axial but regularly oriented fibers. When processed to retain native bioactive components, the ECM material can retain these components interspersed as solids between, upon and/or within the collagen fibers. Particularly desirable naturally-derived ECM materials for use in the invention will include significant amounts of such interspersed, non- collagenous solids that are readily ascertainable under light microscopic examination. Such non-collagenous solids can constitute a significant percentage of the dry weight of the ECM material in certain inventive embodiments, for example at least about 1%, at least about 3%, and at least about 5% by weight in various embodiments of the invention.

The submucosal or other ECM material used in the present invention may also exhibit an angiogenic character and thus be effective to induce angiogenesis in a host engrafted with a device including the material. In this regard, angiogenesis is the process through which the body makes new blood vessels to generate increased blood supply to tissues. Thus, angiogenic materials, when contacted with host tissues, promote or encourage the formation of new blood vessels. Methods for measuring in vivo angiogenesis in response to biomaterial implantation have recently been developed. For example, one such method uses a subcutaneous implant model to determine the angiogenic character of a material. See, C. Heeschen et al., Nature Medicine 7 (2001), No. 7, 833-839. When combined with a fluorescence microangiography technique, this model can provide both quantitative and qualitative measures of angiogenesis into biomaterials. C. Johnson et al., Circulation Research 94 (2004), No. 2, 262-268.

Further, in addition or as an alternative to the inclusion of native bioactive components, non-native bioactive components such as those synthetically produced by recombinant technology or other methods, may be incorporated into the submucosal or other ECM tissue. These non-native bioactive components may be naturally-derived or recombinantly produced proteins that correspond to those natively occurring in the ECM tissue, but perhaps of a different species (e.g. human proteins applied to collagenous ECMs from other animals, such as pigs). The non-native bioactive components may also be drug substances, such as a pharmaceutical agent. Illustrative drug substances that may be incorporated into and/or onto the ECM materials used in the invention include, for example, antibiotics, thrombus-promoting substances such as blood clotting factors, e.g. thrombin, fibrinogen, and the like. These substances may be applied to the ECM material as a premanufactured step, immediately prior to the procedure (e.g. by soaking the material in a solution containing a suitable antibiotic such as cefazolin), or during or after engraftment of the material in the patient.

A non-native bioactive component can be applied to a submucosal or other ECM tissue by any suitable means. Suitable means include, for example, spraying, impregnating, dipping, etc. The non-native bioactive component can be applied to the ECM tissue either before or after the adhesive is applied to the material, or both. Similarly, if other chemical or biological components are included in the ECM tissue, the non-native bioactive component can be applied either before, in conjunction with, or after these other components.

Submucosal or other ECM tissue used in the invention is preferably highly purified, for example, as described in U.S. Patent No. 6,206,931 to Cook et al. Thus, preferred ECM material will exhibit an endotoxin level of less than about 12 endotoxin units (EU) per gram, more preferably less than about 5 EU per gram, and most preferably less than about 1 EU per gram. As additional preferences, the submucosal or other ECM material may have a bioburden of less than about 1 colony forming units (CFU) per gram, more preferably less than about 0.5 CFU per gram. Fungus levels are desirably similarly low, for example less than about 1 CFU per gram, more preferably less than about 0.5 CFU per gram. Nucleic acid levels are preferably less than about 5 μg/mg, more preferably less than about 2 μg/mg, and virus levels are preferably less than about 50 plaque forming units (PFU) per gram, more preferably less than about 5 PFU per gram. These and additional properties of submucosa or other ECM tissue taught in U.S. Patent No. 6,206,931 may be characteristic of the submucosal tissue used in the present invention.

In additional embodiments, medical products of the invention can include

ECM' s or other collagenous materials that have been subjected to processes that expand the materials. In certain forms, such expanded materials can be formed by the controlled contact of an ECM material with one or more alkaline substances until the material expands, and the isolation of the expanded material. Illustratively, the contacting can be sufficient to expand the ECM material to at least 120% of (i.e. 1.2 times) its original bulk volume, or in some forms to at least about two times its original volume. Thereafter, the expanded material can optionally be isolated from the alkaline medium, e.g. by neutralization and/or rinsing. The collected, expanded material can be used in any suitable manner in the preparation of a medical device. Illustratively, the expanded material can be enriched with bioactive components, dried, and/or molded, etc., in the formation of a graft construct of a desired shape or configuration.

Expanded collagenous or ECM materials can be formed by the controlled contact of a collagenous or ECM material with an aqueous solution or other medium containing sodium hydroxide. Alkaline treatment of the material can cause changes in the physical structure of the material that in turn cause it to expand. Such changes may include denaturation of the collagen in the material. In certain embodiments, it is preferred to expand the material to at least about three, at least about four, at least about 5, or at least about 6 or even more times its original bulk volume. The magnitude of the expansion is related to several factors, including for instance the concentration or pH of the alkaline medium, exposure time, and temperature used in the treatment of the material to be expanded.

ECM materials that can be processed to make expanded materials can include any of those disclosed herein or other suitable ECM's. Typical such ECM materials will include a network of collagen fibrils having naturally-occurring intramolecular cross links and naturally-occurring intermolecular cross links. Upon expansion processing as described herein, the naturally-occurring intramolecular cross links and naturally-occurring intermolecular cross links can be retained in the processed collagenous matrix material sufficiently to maintain the collagenous matrix material as an intact collagenous sheet material; however, collagen fibrils in the collagenous sheet material can be denatured, and the collagenous sheet material can have an alkaline -processed thickness that is greater than the thickness of the starting material, for example at least 120% of the original thickness, or at least twice the original thickness.

Illustratively, the concentration of the alkaline substance for treatment of the remodelable material can be in the range of about 0.5 to about 2 M, with a concentration of about 1 M being more preferable. Additionally, the pH of the alkaline substance can in certain embodiments range from about 8 to about 14. In preferred aspects, the alkaline substance will have a pH of from about 10 to about 14, and most preferably of from about 12 to about 14. In addition to concentration and pH, other factors such as temperature and exposure time will contribute to the extent of expansion, as discussed above. In this respect, in certain variants, the exposure of the collagenous material to the alkaline substance is performed at a temperature of about 4 to about 45 ⁰C. In preferred embodiments, the exposure is performed at a temperature of about 25 to about 40 ⁰C, with 37 ⁰C being most preferred. Moreover, the exposure time can range from at least about one minute up to about 5 hours or more. In some embodiments, the exposure time is about 1 to about 2 hours. In a particularly preferred embodiment, the collagenous material is exposed to a 1 M solution of NaOH having a pH of 14 at a temperature of about 37 ⁰C for about 1.5 to 2 hours. Such treatment results in collagen denaturation and a substantial expansion of the remodelable material. Denaturation of the collagen matrix of the material can be observed as a change in the collagen packing characteristics of the material, for example a substantial disruption of a tightly bound collagenous network of the starting material. A non-expanded ECM or other collagenous material can have a tightly bound collagenous network presenting a substantially uniform, continuous surface when viewed by the naked eye or under moderate magnification, e.g. 10Ox magnification. Conversely, an expanded collagenous material can have a surface that is quite different, in that the surface is not continuous but rather presents collagen strands or bundles in many regions that are separated by substantial gaps in material between the strands or bundles when viewed under the same magnification, e.g. about 10Ox. Consequently, an expanded collagenous material typically appears more porous than a corresponding non-expanded collagenous material. Moreover, in many instances, the expanded collagenous material can be demonstrated as having increased porosity, e.g. by measuring for an increased permeability to water or other fluid passage as compared to the non-treated starting material. The more foamy and porous structure of an expanded ECM or other collagenous material can allow the material to be cast or otherwise prepared into a variety of sponge or foam shapes for use in the preparation of medical materials and devices. After such alkaline treatments, the material can be isolated from the alkaline medium and processed for further use. Illustratively, the collected material can be neutralized and/or rinsed with water to remove the alkalinity from the material, prior to further processing of the material to form a medical graft material of the invention.

A starting ECM material (i.e., prior to treatment with the alkaline substance) can optionally include a variety of bioactive or other non-collagenous components including, for example, growth factors, glycoproteins, glycosaminoglycans, proteoglycans, nucleic acids, and lipids. Treating the material with an alkaline substance may reduce the quantity of one, some or all of such non-collagenous components contained within the material. In certain embodiments, controlled treatment of the remodelable material with an alkaline substance will be sufficient to create a remodelable collagenous material which is substantially devoid of nucleic acids and lipids, and potentially also of growth factors, glycoproteins, glycosaminoglycans, and proteoglycans. This may be true for other processing techniques as discussed herein, such as the controlled treatment of the material with a detergent.

In certain embodiments, one or more bioactive components, exogenous or endogenous, for example, similar to those removed from an ECM material (i.e., both non-expanded and expanded materials) during processing, can be returned to the material. For example, an ECM material can include a collagenous material which has been depleted of nucleic acids and lipids, but which has been replenished with growth factors, glycoproteins, glycosaminoglycans, and/or proteoglycans. These bioactive components can be returned to the material by any suitable method. For instance, in certain forms a tissue extract, such as is discussed in U.S. Patent No. 6,375,989, containing these components can be prepared and applied to an ECM collagenous material. In one embodiment, the ECM can be incubated in a tissue extract for a sufficient time to allow bioactive components contained therein to associate with the ECM material. The tissue extract may, for example, be obtained from non-expanded collagenous tissue of the same type used to prepare the expanded material. Other means for returning or introducing bioactive components to an ECM material include spraying, impregnating, dipping, etc. as known in the art. By way of example, an ECM material may be modified by the addition of one or more growth factors such as basic fibroblast growth factor (FGF-2), transforming growth factor beta (TGF beta), epidermal growth factor (EGF), platelet derived growth factor (PDGF), and/or cartilage derived growth factor (CDGF), As well, other biological components may be added to an ECM material, such as heparin, heparin sulfate, hyaluronic acid, fibronectin and the like. Thus, generally speaking, an ECM material may include a bioactive component that induces, directly or indirectly, a cellular response such as a change in cell morphology, proliferation, growth, protein or gene expression similar to a non-expanded collagenous material.

With respect to the above, a collagenous ECM material can be provided in any suitable form prior to or after becoming a component of a medical product. Suitable forms include, for example, as one or more sheets or layers, as a foam, a sponge, a fluidized form (e.g., a vaccine preparation), as a particulate or combinations thereof. The form used will typically depend on a variety of factors including, but not limited to, the end use of the medical product and the type of medical product to be prepared (e.g., fluidized vs. sheet form).

In one embodiment, the processed ECM material is made into a fluidized composition, for instance using techniques as described in U.S. Patent Nos. 5,275,826 and 5,516,533. Such a form is typically desired in the formation of a vaccine preparation where at least one antigen and, optionally, a CpG oligonucleotide including at least one CpG site, is/are dispersed in a fluidized form of the ECM material. In this regard, solutions or suspensions of the ECM material can be prepared by comminuting and/or digesting the material with a protease (e.g. trypsin or pepsin), for a period of time sufficient to solubilize the material and form substantially homogeneous solution. The ECM material is desirably comminuted by tearing, cutting, grinding, shearing or the like. Grinding the material in a frozen or freeze-dried state is advantageous, although good results can be obtained as well by subjecting a suspension of pieces of the material to treatment in a high speed blender and dewatering, if necessary, by centrifuging and decanting excess waste. The comminuted material can be dried, for example freeze dried, to form a powder. Thereafter, if desired, the powder can be hydrated, that is, combined with water or buffered saline and optionally other pharmaceutically acceptable excipients, to form a fluid tissue graft composition, e.g. having a viscosity of about 2 to about 300,000 cps at 25 ⁰C. The higher viscosity graft compositions can have a gel or paste consistency.

In one illustrative preparation, an ECM material prepared as described herein is reduced to small pieces (e.g. by cutting) which are charged to a flat bottom stainless steel container. Liquid nitrogen is introduced into the container to freeze the specimens, which are then comminuted while in the frozen state to form a coarse powder. Such processing can be carried out, for example, with a manual arbor press with a cylindrical brass ingot placed on top of the frozen specimens. The ingot serves as an interface between the specimens and the arbor of the press. Liquid nitrogen can be added periodically to the specimens to keep them frozen.

Other methods for comminuting ECM material specimens can be utilized to produce a powder usable in accordance with the present invention. For example, ECM material specimens can be freeze-dried and then ground using a manual arbor press or other grinding means. Alternatively, ECM material can be processed in a high shear blender to produce, upon dewatering and drying, a powder. Further grinding of the ECM material powder using a prechilled mortar and pestle can be used to produce consistent, more finely divided product. Again, liquid nitrogen is used as needed to maintain solid frozen particles during final grinding. The powder can be easily hydrated using, for example, buffered saline to produce a fluidized tissue graft material of this invention at the desired viscosity.

To prepare another preferred fluidized material, an ECM material powder can be sifted through a wire mesh, collected, and subjected to proteolytic digestion to form a substantially homogeneous solution. For example, the powder can be digested with 1 mg/ml of pepsin (Sigma Chemical Co., St. Louis Mo.) and 0.1 M acetic acid, adjusted to pH 2.5 with HCl, over a 48 hour period at room temperature. After this treatment, the reaction medium can be neutralized with sodium hydroxide (NaOH) to inactivate the peptic activity. The solubilized submucosa can then be concentrated by salt precipitation of the solution and separated for further purification and/or freeze drying to form a protease- solubilized collagenous ECM material in powder form.

As an alternative to the above fluidized compositions, a particulate ECM material can be prepared as described above. For example, a bacterial pathogen can be cultured onto one or more sheets of a collagenous ECM material so as to deposit an immunogenic component having antigenicity thereon, The pathogen can be rendered biologically inert. The ECM material including the bacterial pathogen can be formed into a particulate using the techniques described above. Once formed, the particulate ECM material including the immunogenic components from the bacterial pathogen can then be combined with a pharmaceutically acceptable carrier, such as water, buffered saline, and the like, to form a vaccine preparation. In another embodiment, the ECM material can be formed into a particulate, and the bacterial pathogen and, if desired, CpG oligonucleotide, can be combined with the particulate in a pharmaceutically acceptable carrier each as separate components. In embodiments of the invention where a medical product is provided in sheet form, the medical product will have a thickness in the range of about 50 to about 1000 microns, more preferably about 100 to 600 microns, and most preferably about 100 to about 350 microns. If necessary or desired, a multilaminate medical product can be used. For example, a plurality of (i.e. two or more) layers of a biocompatible material, for example submucosa-containing or other ECM material, can be bonded together to form a multilaminate structure. Illustratively, two. three, four, five, six, seven, or eight or more layers of a biocompatible material can be bonded together to provide a multilaminate material. In certain embodiments, two to six collagenous, submucosa-containing layers isolated from intestinal tissue of a warm-blooded vertebrate, particularly small intestinal tissue, are bonded together to provide the material. Porcine-derived small intestinal tissue is preferred for this purpose. The layers of collagenous tissue can be bonded together in any suitable fashion, including dehydrothermal bonding under heated, non-heated or lyophilization conditions, using adhesives as described herein, glues or other bonding agents, crosslinking with chemical agents or radiation (including UV radiation), or any combination of these with each other or other suitable methods.

Generally, when configured for attachment to tissue, the medical product of the invention is cut or otherwise configured to a desired size for its end use. Once the medical product has been placed on, in, or around the tissue, the medical product can be more securely attached to the surrounding tissue using any of several known suitable attachment means. Suitable attachment means include, for example, stapling, suturing, and the like. A medical grade adhesive can also be used. In many embodiments, the medical material will be more securely attached to the surrounding tissue by sutures. There are a variety of synthetic materials currently available in the art for use as sutures. For example, sutures comprising Prolene™, Vicryl™, Mersilene™, Panacryl™, and Monocryl™, are contemplated for use in the invention. Other suture materials will be well known to those skilled in the art. The aforementioned materials therefore serve merely as examples and, consequently, are in no way limiting.

The medical product of the invention can be in a dehydrated or hydrated state. Dehydration of a medical product of the invention can be achieved by any means known in the art. Preferably, dehydration is accomplished by lyophilization. drying in a vacuum, air drying, heated (e.g. oven) drying, or any combination of these. Typically, the medical product will be dehydrated when it is to be stored for a period of time. Any suitable solution can then be used to rehydrate the medical material prior to use. Preferably, the rehydration solution comprises water or buffered saline. The above-described methods of dehydration and rehydration of the medical product allow for an effective shelf life and convenient packaging.

In certain embodiments, the collagenous ECM material can be crosslinked. A collagenous ECM material can be crosslinked once combined with either the CpG oligonucleotide and/or the at least one antigen, or can be crosslinked separately before any other component is provided to a medical product, or both. Increasing the amount (or number) of crosslinkages within the medical product or between two or more layers of the medical material can be used to enhance its strength. However, when a remodelable material is used, the introduction of crosslinkages within the material may also affect its resorbability or remodelability. Consequently, in certain embodiments, a remodelable ECM material used in a medical product will substantially retain its native level of crosslinking, or the amount of added crosslinkages within the medical material will be judiciously selected depending upon the desired treatment regime. In many cases, the medical material will exhibit remodelable properties such that the remodeling process occurs over the course of several days or several weeks. In certain preferred embodiments, the remodeling process occurs within a matter of about 5 days to about 12 weeks.

For use in the present invention, introduced crosslinking of the medical product may be achieved by photo-crosslinking techniques, or by the application of a crosslinking agent, such as by chemical crosslinkers, or by protein crosslinking induced by dehydration or other means. Chemical crosslinkers that may be used include for example aldehydes such as glutaraldehydes, diimides such as carbodiimides, e.g. , 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, diisocyanates, such as hexamethylene-diisocyanate, ribose or other sugars, acyl-azide, sulfo-N-hydroxysuccinamide, or polyepoxide compounds, including for example polyglycidyl ethers such as ethyleneglycol diglycidyl ether, available under the trade name DENACOL EX810 from Nagese Chemical Co., Osaka, Japan, and glycerol polyglycerol ether available under the trade name DENACOL EX 313 also from Nagese Chemical Co. Typically, when used, polyglycerol ethers or other polyepoxide compounds will have from 2 to about 10 epoxide groups per molecule.

When multiple layers of a medical material are used to form a laminate material, the layers of the laminate can be additionally crosslinked to bond multiple layers of medical material to one another, Thus, additional crosslinking may be added to individual layers prior to coupling to one another, during coupling to one another, and/or after coupling to one another.

Turning now to a discussion of the at least one antigen used in a medical product of the invention, any antigen can be used and preferably the antigen is derived from, or otherwise related to, the medical condition being treated or prevented. In this manner, a medical product can be formed that can elicit an antigen specific immune response in a patient. In one embodiment, a bacterial pathogen is grown on a collagenous ECM material such that the pathogen deposits one or more immunogenic components having antigenicity thereon. The pathogen is lysed or otherwise rendered biologically inert, which can expose residues having antigenicity. These immunogenic component(s) and residues can be the same or different. Alternatively or additionally, CpG oligonucleotides can also be exposed during this process. In a particularly preferred embodiment shown in Fig. 1. a bacterial pathogen can be cultured onto a collagenous extracellular matrix (ECM) material such that the pathogen deposits one or more immunogenic components having antigenicity. The pathogen can then be rendered biologically inert and/or removed from the material. The ECM material including the immunogenic components can then be combined with a CpG oligonucleotide as described herein. The ECM material containing the immunogenic component(s) and CpG oligonucleotide(s) can be then prepared in any suitable form. For example, the ECM material can be formed as one or more sheets or layers, as a foam, a sponge, a fluidized form, as a particulate or combinations thereof as discussed previously. In another embodiment, at least one antigen can be prepared separately from the CpG oligonucleotide and collagenous ECM material and can be combined with these other components prior to administration to a patient.

In certain embodiments, it may be desirable to include a CpG oligonucleotide as a component of a medical product described herein, Such oligonucleotides are generally known in the art as having adjuvanticity. These adjuvants are formed from CpG sites within DNA where a cytosine nucleotide is linked to a guanine nucleotide by a phosphate group. A CpG oligonucleotide as used herein can be any desired length. Preferably, the CpG oligonucleotide is in the range of about 2 to about 100 nucleotides in length. More preferably, the CpG oligonucloetide is in the range of about 5 to about 25 nucleotides in length. As well, a CpG oligonucleotide can include any number of unmethylated CpG sites. The CpG oligonucleotides can be single stranded or double stranded. Any number of CpG oligonucleotides can be included on or within a medical product. If more than one CpG oligonucleotide is included, such oligonucleotides can be the same or different. The CpG oligonucleotide(s) can be included on or within a medical product before, in conjunction with, or after any of the other components as described herein.

A CpG oligonucleotide as used herein can be synthetically produced or can occur naturally. For example, unmethylated CpG sites can be exposed after a bacterial pathogen has been cultured on a collagenous ECM material and subsequently rendered biologically inert (e.g., by lysing) as previously described. Alternatively, a CpG oligonucleotide can be synthetically produced separately from the antigen and collagenous ECM material and can be combined with these other components prior to administration to a mammal, For example, a collagenous ECM material can be soaked in a solution containing one or more CpG oligonucleotides including at least one CpG site.

The CpG oligonucleotide and collagenous ECM material can be administered alone as an adjuvant, or can be administered together with any suitable amount of an antigen. For example, a CpG oligonucleotide and/or collagenous ECM material can be administered with a priming dose of an antigen or a boost dose of an antigen. By "priming dose" is meant the initial dose of an antigen given to a patient. In one embodiment, a priming dose of the antigen can be administered with one or both of these components followed by a boost dose of the antigen. By "boost dose" is meant any subsequent dose of antigen administered after the priming dose. In another embodiment, a priming dose of the antigen can initially be administered, and one or both of these components can subsequently be administered in conjunction with a boost dose of the antigen.

Medical products of the invention are, in certain embodiments, designed to elicit an antigen specific immune response. Such an immune response can be cell- mediated or humoral. Alternatively or additionally, a medical product of the invention can elicit a non-specific immune response. In this regard, a medical 99

product of the invention can elicit components of the innate immune system and/or the acquired immune system of a mammal, and preferably a human.

In preferred embodiments, the CpG oligonucleotide and collagenous ECM material are present in a synergistically effective amount. By "synergistically effective amount" is meant an amount of CpG oligonucleotide and collagenous ECM material such that the two components act together to provide an antigen specific immune response. Such a response is greater than it would otherwise be when only the CpG oligonucleotide or the collagenous ECM material is administered alone. It will be understood that the amount of CpG oligonucleotide and collagenous ECM material can vary depending on a variety of factors including, but not limited to, the type of antigen(s) included in the medical product, the particular CpG oligonucleotide used, the form of collagenous ECM material used, and/or the type of medical condition to be treated or prevented.

Medical products of the invention can be used to treat or prevent a variety of medical conditions (including veterinary). Preferably, the medical condition will be caused, at least in part, by pathogenic bacteria. Any bacterial pathogen that can cause a medical condition is contemplated for use in the present medical products, although certain bacteria may provide a greater immune response with combined with a collagenous ECM material and/or CpG oligonucleotide. For example, the bacterial pathogen can be Mycobacterium leprae, Clostridium tetani, Bordetella pertussis, Salmonella typhi, Salmonella paratyphi, Vibrio cholorae, Yersinia pestis, Mycobacterium tuberculosis, Neisseria meningitides, Streptococcus pneumoniae, Bacillus anthracis, Clostridium botulinum, Shigella dysenteriae, Escherichia coli, Salmonella enteriditis, Clostridium perfringens, Treponema pallidum, Corynebacterium diphtheriae, Neisseria gonorrhoeae, Coxiella burnetti, Rochalimaea quintana, Rochalimaea vinsonii, Haemophilus influenzae, Reckettsia prowazekii, Borrelia burgdorferi, Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Streptococcus mitis, Streptococcus pyogenes, Streptococcus pyogenes, Clostridium perfringens, Bacillus cereus, Staphylococcus aureus, Orientia tsutsugamushi, Rickettsii riskettsii, Erhlichia chajfensis, Erhlichia equi, Legionella pneumophilia, Campylobacter spp,, Listeria monocytogenes, Helicobacter pylori, and Chlamydia trachomatis In preferred embodiments, the bacterial pathogen is Staphylococcus aureus or Escherichia coli.

The medical products as described herein can be administered to a mammal in either solid or sheet form, by surgical implantation alone or in combination with other art-recognized implant compositions, or in fluidized form, by injection. In this respect, preferred routes of administration include, for example, oral, intranasal, transdermal, and parenteral injection or infusion, including subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, and intrathecal, etc. In a preferred embodiment, a medical product is administered by intramuscular or intradermal injection. Medical products as described herein can also include a pharmaceutically acceptable carrier. Such carriers will generally be known to those skilled in the art and can include, for example, water, buffered saline, and the like. For instance, a vaccine preparation can be prepared by combining a particulate collagenous ECM material, at least one antigen, and a CpG oligonucleotide including at least one CpG site in an aqueous carrier.

The medical products of the invention can be provided in sterile packaging suitable for medical products. Sterilization may be achieved, for example, by irradiation, ethylene oxide gas, or any other suitable sterilization technique, and the materials and other properties of the medical packaging will be selected accordingly.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. In addition, all publications cited herein are indicative of the abilities of those of ordinary skill in the art and are hereby incorporated by reference in their entirety as if individually incorporated by reference and fully set forth.

Claims

WHAT IS CLAIMED IS:

1. A medical product, comprising: at least one antigen derived from a bacterial pathogen; a CpG oligonucleotide including at least one CpG site; and a collagenous extracellular matrix (ECM) material, wherein said CpG oligonucleotide and said collagenous ECM material are present in a synergistically effective amount to increase the immunogenicity of said at least one antigen.

2. The medical product of claim 1 , wherein said medical product further comprises a bioactive component and/or pharmaceutical agent.

3. The medical product of claim 1 , wherein said collagenous ECM material is in the form of a sheet, a gel, a particulate, or combinations thereof.

4. The medical product of claim 3, wherein said collagenous ECM material is in the form of a gel and the at least one antigen is dispersed within said gel.

5. The medical product of claim 3, wherein said antigen is formed by rendering a bacterial pathogen grown on a sheet of a collagenous ECM material biologically inert.

6. The medical product of claim 5, wherein said at least one bacterial pathogen is rendered biologically inert by being lysed.

7. The medical product of any of claims 1-6, wherein said ECM comprises submucosa.

8. The medical product of claim 7, wherein said submucosa is intestinal, urinary bladder or stomach submucosa.

9. The medical product of claim 8, wherein said submucosa is small intestinal submucosa (SIS).

10. A medical product, comprising: at least one antigen derived from Staphylococcus aureus or Escherichia coli; and a collagenous extracellular matrix (ECM) material.

11. The medical product of claim 10, wherein said medical product further comprises a bioactive component and/or pharmaceutical agent.

12. The medical product of claim 10, wherein said collagenous ECM material is in the form of a sheet, a gel, a particulate, or combinations thereof.

13. The medical product of claim 12, wherein said collagenous ECM material is in the form of a gel and the at least one antigen is dispersed within said gel.

14. The medical product of claim 12, wherein said antigen is formed by rendering Staphylococcus aureus or Escherichia coli grown on a sheet of a collagenous ECM material biologically inert.

15. The medical product of claim 14, wherein said Staphylococcus aureus or Escherichia coli is rendered biologically inert by being lysed.

16. The medical product of any of claims 10-15, wherein said ECM comprises submucosa.

17. The medical product of claim 16, wherein said submucosa is intestinal, urinary bladder or stomach submucosa.

18. The medical product of claim 17, wherein said submucosa is small intestinal submucosa (SIS).

19. A vaccine preparation comprising: at least one antigen derived from a bacterial pathogen; a CpG oligonucleotide including at least one CpG site; a particulate of a collagenous extracellular matrix (ECM) material; and an aqueous carrier, wherein said antigen, CpG oligonucleotide, and particulate are dispersed within the carrier, and wherein said CpG oligonucleotide and said ECM material are present in a synergistically effective amount to increase the immunogenicity of said at least one antigen.

20. The vaccine preparation of claim 19, wherein said preparation further comprises a bioactive component and/or pharmaceutical agent.

21, The vaccine preparation of claim 19, wherein said ECM comprises submucosa.

22. The vaccine preparation of claim 21, wherein said submucosa is intestinal, urinary bladder or stomach submucosa.

23. The vaccine preparation of claim 22, wherein said submucosa is small intestinal submucosa (SIS).

24. An adjuvant for increasing the immunogenicity of an antigen, comprising: a CpG oligonucleotide including at least one CpG site; and a collagenous extracellular matrix (ECM) material, wherein said CpG oligonucleotide and said collagenous ECM material are present in a synergistically effective amount to increase the immunogenicity of said antigen.

25. The adjuvant of claim 24, wherein said collagenous ECM material is in the form of a sheet, a gel, a particulate, or combinations thereof.

26. The adjuvant of claim 25, wherein said collagenous ECM material is in the form of a gel and the CpG oligonucleotide is dispersed within said gel,

27. The adjuvant of any of claims 24-26, wherein said ECM comprises submucosa.

28. The adjuvant of claim 27, wherein said submucosa is intestinal, urinary bladder or stomach submucosa.

29. The adjuvant of claim 28, wherein said submucosa is small intestinal submucosa (SIS).

30. A method for treating or preventing a medical condition in a patient, comprising: providing a medical product of any of claims 1-18 to a patient, wherein said medical condition is caused by a bacterial pathogen, and wherein said medical product includes an antigen derived from said bacterial pathogen.

31. A method for treating or preventing a medical condition in a patient, comprising: providing a vaccine preparation of any of claims 19-23 to a patient, wherein said medical condition is caused by a bacterial pathogen, and wherein said vaccine preparation includes an antigen derived from said bacterial pathogen.

32. A method for inducing an antigen specific immune response in a patient, comprising: providing a medical product of any of claims 1-18 to a patient, wherein said immune response is specific for the pathogenic antigen contained in the medical product.

33. A method for inducing an antigen specific immune response in a patient, comprising: providing a vaccine preparation of any of claims 19-23 to a patient, wherein said immune response is specific for the pathogenic antigen contained in the medical product.

34. A method for preparing a medical product, comprising: providing at least one antigen derived from a bacterial pathogen; providing a CpG oligonucleotide including at least one CpG site; providing a collagenous extracellular matrix (ECM) material; and combining said at least one antigen, CpG oligonucleotide, and ECM material such that the CpG oligonucleotide and said collagenous ECM material are present in a synergistic ally effective amount to increase the immunogenicity of said at least one antigen.

35. The method of claim 34, wherein said medical product further comprises a bioactive component and/or pharmaceutical agent.

36. The method of claim 34, wherein said collagenous ECM material is in the form of a sheet, a gel, a particulate, or combinations thereof.

37. The method of any of claims 34-36, wherein said ECM comprises submucosa.

38. The method of claim 37, wherein said submucosa is intestinal, urinary bladder or stomach submucosa.

39. The method of claim 38, wherein said submucosa is small intestinal submucosa (SIS).

40. A method for preparing a medical product, comprising: culturing a bacterial pathogen on a collagenous extracellular matrix (ECM) material such that the pathogen deposits at least one immunogenic component having antigenicity on said material; and rendering said pathogen biologically inert.

41. The method of claim 40, wherein said medical product further comprises a bioactive component and/or pharmaceutical agent.

42, The method of claim 40, wherein said collagenous ECM material is in the form of a sheet, a gel, a particulate, or combinations thereof.

43. The method of claim 40, wherein a CpG oligonucleotide is provided to the material by being exposed after the bacterial pathogen is rendered biologically inert.

44. The method of any of claims 40-43, wherein said ECM comprises submucosa.

45. The method of claim 44, wherein said submucosa is intestinal, urinary bladder or stomach submucosa.

46. The method of claim 45, wherein said submucosa is small intestinal submucosa (SIS).