# Patent

## Title
Extracellular matrix materials as vaccine adjuvants for diseases associated with infectious pathogens or toxins

## Abstract

Disclosed are vaccines and vaccine adjuvants useful in the treatment and/or prevention of infection and diseases associated with infectious pathogens, such as tetanus, as well as diseases associated with biological toxins. Also provided are methods of preparing an adjuvant and the vaccine containing the adjuvant. Methods are also provided for vaccinating/immunizing an animal against infection and diseases associated with infectious pathogens, such as tetanus, and other diseases associated with biological toxins. Adjuvant materials are presented that are prepared from an extracellular matrix material. The adjuvants are demonstrated to enhance the immunogencity of an infectious pathogen antigen or biological toxin antigen of interest, as well as to enhance the survival of an immunized animal.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 11/583,771 entitled “Extracellular Matrix Cancer Vaccine Adjuvant”, filed Oct. 20, 2006, which claims priority from U.S. Provisional Patent Application No. 60/730,379 entitled “Use of Extracellular Matrix Materials as a Vaccine Carrier and Adjuvant”, filed Oct. 27, 2005. The entire disclosure and contents of the above applications are hereby incorporated by reference.


STATEMENT OF JOINT RESEARCH AGREEMENT
In compliance with 37 C.F.R. §1.71(g) (1), disclosure is herein made that the claimed invention was made pursuant to a Joint Research Agreement as defined in 35 U.S.C. 103 (c) (3), that was in effect on or before the date the claimed invention was made, and as a result of activities undertaken within the scope of the Joint Research Agreement, by or on the behalf of the University of Notre Dame and Cook Biotech, Inc. (West Lafayette, Ind.).
BACKGROUND
1. Field of the Invention
The present invention relates generally to vaccines that include an adjuvant, and to adjuvants alone. In particular, the invention relates to adjuvants derived or obtained at least in part from biological tissues, such as extracellular matrices, particularly small intestinal submucosa (SIS). The invention also relates to the field of methods for immunizing an animal against diseases associated with infectious pathogens, and infections by said pathogens, or toxins using a vaccine preparation that includes a tissue-derived adjuvant. The invention also relates to the field of methods for preparing adjuvants, as a method for preparing an adjuvant from small intestinal tissue for use as a part of a vaccine to immunize an animal against disease associated with an infectious agent, and in particular, against tetanus, as a vaccine for the treatment and/or prevention of tetanus, is provided.
2. Related Art
Aluminum hydroxide and aluminum phosphate (collectively referred to as alum) are routinely used as adjuvants in human and veterinary vaccines (1). The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxins is well established (2) and Hepatitis B virus antigen vaccine has been adjuvinated with alum (3). While the usefulness of alum is well established for some applications, it has limitations. For example, alum is a poor inducer of Th1 cellular immune responses and stimulates the production of antibodies, which is consistent with Th2 cellular immune response (4-6). Unfortunately, a Th2 based immune response is not likely to offer optimal protection against several important infectious diseases, including tuberculosis (TB), human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Alum is poorly effective for influenza vaccination and inconsistently elicits a cell mediated immune response. The antibodies elicited by alum-adjuvinated antigens are mainly of the IgG1 isotope in the mouse, which may be optimal for protection by some vaccinal agents.
Tetanus is an important human and animal disease characterized by painful, uncontrolled muscle spasms, and death due to paralysis of the respiratory muscles. This disease is associated with infection by Clostridium tetani and prophylactic vaccination is common. Tetanus vaccines typically use alum as an adjuvant.
A need continues to exist in the medical arts for materials that may be used to enhance and/or improve existing clinical alternatives to the treatment and prophylaxis of disease associated with infectious agents and toxins, for example, to improve existing forms of tetanus treatment vaccines and tetanus vaccine adjuvants with improved immunogenicity.
SUMMARY OF THE INVENTION
The present invention was developed in part by the inventors' recognition of the robust inflammatory response invoked by an extracellular matrix material (ECM) preparation, such as matrix isolated from the small intestinal submucosa (SIS). While not intending to be limited to any particular mechanism of action, the extracellular matrix material appears to provide the robust inflammatory response through, among other things, it's contribution of pro-inflammatory species that drive the immune response to the antigenic species that it is co-administered with. The present invention harnesses the inflammatory-provoking activity of ECM, such as SIS, and preparations from other forms of ECM, in the design of an immunopotent infectious agent vaccine preparation and infectious agent adjuvant.
The crafting of infectious agent vaccine preparations using ECM, and materials like it, may be used in combination with many different infectious pathogens or biological toxins. By way of example, and in some embodiments, the biological toxin is tetanus toxin. By further way of example, and in some embodiments, the biological toxin is ricin.
The present invention is unique in that, among other things, it involves the modification and use of a three-dimensional extracellular matrix material, and modified preparations thereof, to provide a vaccine. By way of example, and in some embodiments, the vaccine is a tetanus vaccine. The invention thus provides in some embodiments highly improved infectious agent preparations with an adjuvant material having an acceptable biocompatibility.
The adjuvant effect of the ECM, such as the SIS adjuvant preparation, extends to a vaccine administered to protect against diseases associated with an infectious pathogen or biological toxin. In some embodiments, the present invention provides an adjuvant comprising an SIS gel or particulate SIS. Administered together with tetanus toxoid, these preparations confer protective immunity in vivo to animals challenged with tetanus toxin.
Infectious Agent Adjuvant
In one aspect, the present invention provides an extracellular matrix (ECM) material, such as a modified preparation of SIS, as an infectious agent vaccine adjuvant. In some embodiments, these preparations may be described as essentially free of alum. In some embodiments, the ECM materials may be described as a modified preparation of SIS (diluted) about 2-fold to about 20-fold.
Infectious Agent Vaccine
In another aspect, the present invention provides an infectious agent vaccine comprising a preparation of an extracellular matrix material together with a preparation of an antigen of an infectious pathogen.
In one aspect of the invention, there is provided an adjuvant composition comprising an immunogenically enhancing preparation characteristic of an extracellular matrix material (ECM), particularly a preparation comprising an extracellular matrix derived from small intestinal submucosa (SIS) or renal capsule material (RCM). In particular embodiments, the adjuvant composition comprises an extracellular matrix material comprising a small intestinal submucosa tissue preparation. In some embodiments, the adjuvant composition comprises 1 part of an extracellular matrix material (ECM) and 9 parts of a pharmaceutically acceptable carrier solution. By way of example, such a carrier is sterile saline.
According to another aspect, there is provided a composition comprising an adjuvant and a antigen of interest. In some embodiments, the antigen is a toxoid antigen. In some embodiments, the vaccine may be described as a vaccine to protect against infectious pathogens, such as a tetanus vaccine, an influenza vaccine, a rabies vaccine, a viral hepatitis vaccine, a diphtheria vaccine, an anthrax vaccine, a Streptococcus pneumonia infection vaccine, a malaria vaccine, a leishmaniasis vaccine, or a Staphylococcal enterotoxin B toxicosis vaccine.
Biological Toxins
Examples of the biological toxins that may be used in the preparation of the vaccines of the present invention are provided below:

    Abrin
    Aflatoxins
    Botulinum toxins
    Clostridium perfringens episilon toxin
    Conotoxins
    Diacetoxyscirpenol
    Ricin
    Saxitoxin
    Shigatoxin
    Staphylococcal enterotoxins
    Tetrodotoxin
    T-2 Toxin
    Diptheria toxin
    Streptococcal toxins
    Cholera toxin
    Pertussis toxin
    Pneumolysin


In particular embodiments, the vaccine may be described as a vaccine to protect against diseases associated with biological toxins, such as ricin.
Prion-Associated Diseases:
In some embodiments, the invention provides an adjuvant preparation that is suitable for use in combination with a prion-associated disease. By way of example, such prion associated diseases include, all of which are classified as transmissible spongiform encephalopathies, bovine spongiform encephalopathy, scrapie, cervid chronic wasting disease and Creutzfeld-Jakob disease.
In other embodiments, the invention may be described as providing a vaccine to protect against disease associated with a viral infection. By way of example, the vaccines of the present invention may be formulated to provide a composition useful in the treatment and/or prevention of viral infections associated with influenza, rabies and viral hepatitis.
In other embodiments, the invention may be described as providing a vaccine to protect against diseases associated with a bacterial infection. By way of example, the vaccines of the present invention may be formulated to provide a composition useful in the treatment and/or prevention of bacterial infections associated with diseases such as diphtheria, anthrax, sepsis, pneumonia, otitis media and meningitis.
In other embodiments, the invention may be described as providing a vaccine to protect against diseases associated with a parasitic infection. By way of example, the vaccines of the present invention may be formulated to provide a composition useful in the treatment and/or prevention of a parasitic infection associated with the diseases of malaria and leishmaniasis.
In other embodiments, the invention may be described as providing a vaccine to protect against illnesses and/or diseases associated with exposure to a biological toxin. By way of example, the vaccines of the present invention may be formulated to provide a composition useful in the treatment and/or prevention of illness associated with exposure to biological toxins such as ricin (that causes respiratory distress) or exposure to Staphylococcal enterotoxin B (SEB) (that results in food poisoning).
Method of Preparing a Adjuvant and a Vaccine
In another aspect, the invention provides a method for preparing an infectious agent vaccine. In some embodiments, the method comprises preparing an adjuvant for vaccination against infectious agents and biological toxins as described herein, and combining the adjuvant with an immunizing antigen of interest. In some embodiments, the antigen of interest is a tetanus toxoid preparation.
Methods of Preventing, Treating, Inhibiting, and/or Immunizing an Animal against an Infectious Pathogen
According to yet another broad aspect of the invention, a method for treating an animal having an infectious disease or at risk of contracting a disease or illness associated with exposure to an infectious pathogen. By way of example, diseases associated with exposure to an infection pathogen include tetanus, malaria, diphtheria, anthrax, sepsis, pneumonia, otitis media and meningitis. In some embodiments, the invention provides a method for immunizing an animal against tetanus. In yet another embodiment, the invention provides a method for inhibiting the severity of tetanus and/or preventing the onset of tetanus altogether.
Clinical Infectious Pathogen Treatment Preparations
In yet another aspect, the invention provides a variety of unique infectious pathogen treatment preparations. These infectious agent treatment preparations may take the form of a gel, a sheet, or an injectable preparation suitable for parenteral administration, combined with an appropriate antigen of interest.
The following abbreviations are used throughout the description of the present invention:
ECM—Extracellular Matrix;
HCV—Hepatitis C Virus;
HV—Human Immunodeficiency Virus
RCM—Renal Capsule Material
SIS—Small Intestinal Submucosa;
TB—Tuberculosis



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1, according to some embodiments of the invention, presents a remnant of SIS extracellular matrix material in a rat 28 days after surgical implantation. The remaining biomaterial is surrounded by macrophages with occasional lymphocytes. Stained with H & E, 400×.
FIG. 2, according to some embodiments of the invention, presents the focus of mononuclear inflammation at the interface of an implant/tendon surface in a rat which underwent repair of Achilles tendon defect with RCM 7 days earlier. Stained with H & E, 200×.
FIG. 3, according to some embodiments of the invention, presents the survival data of mice, vaccinated with 0.03 micrograms of tetanus toxoid, following challenge with 1 ng/mouse of tetanus toxin intraperitoneally. Treatment groups are untreated (None); SIS gel (SG); SIS particulate (SP); tetanus toxoid (TT); TT with alum (TT/Alum); TT with SIS gel (TT/SG); and TT with SIS particulate (TT/SP). Each group consisted of 15 mice. All mice which were untreated or vaccinated with only SIS gel or SIS particulate died; six mice vaccinated with unadjuvanted tetanus toxoid survived, and all mice vaccinated with tetanus toxoid in alum, SIS gel, or SIS particulate survived. This represents a significant (P≦0.001) increase in number of mice surviving for the latter three groups compared to all other groups.
FIG. 4, according to some embodiments of the invention, presents survival data of mice, vaccinated with 0.05 micrograms of tetanus toxoid, following challenge with 1 ng/mouse of tetanus toxin intraperitoneally. Treatment groups are untreated (None); SIS gel (SG); SIS particulate (SP); tetanus toxoid (TT); TT with alum (TT/Alum); TT with SIS gel (TT/SG); and TT with SIS particulate (TT/SP). Each group consisted of 15 mice. All mice which were untreated or vaccinated with only SIS gel or SIS particulate died; eight mice vaccinated with unadjuvanted tetanus toxoid survived; and all mice vaccinated with tetanus toxoid in alum, SIS gel, or SIS particulate survived. This represents a significant (P≦0.001) increase in number of mice surviving for the latter three groups compared to all other groups.



DETAILED DESCRIPTION
It is advantageous to define several terms before describing the invention. It should be appreciated that the following definitions are used throughout this application.
Definitions
Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated.
For the purposes of the present invention, the term “adjuvant” is defined as a substance which enhances the immune response to an antigen.
For purposes of the present invention, the term, “adjuvancy” is defined as the ability of an agent to enhance and/or promote the immune response of animal to a particular antigen.
For the purposes of the present invention, the term “biosynthetic material” is defined as a material that is in part or whole made up from or derived from a biological tissue.
For purposes of the present invention, the term “biological tissue” is defined as an animal tissue, including human, or plant tissue that is or that once was (cadaver tissue, for example) part of a living tissue or organism.
For the purposes of the present invention, the term “extracellular matrix” is defined as a tissue derived or bio-synthetic material that is capable of supporting the growth of a cell or culture of cells.
For the purposes of the present invention, the term “infectious agent” is defined as any bacterial, viral, prion or parasitic agent capable of causing disease in humans or animals subsequent to infection or secretion of a substance, such as the production of a toxin or toxins. This term also includes the toxic products of such agents. By way of example, such an infectious agent includes clostridium botulinum, the causative agent of tetanus.
For the purposes of the present invention, the term “biological toxin” is a poisonous substance, especially a protein that is produced by living cells or organisms and is capable of causing disease when introduced into the body tissues, such as ricin or Staphylococcal enterotoxin B and tetanus toxin.
For the purposes of the present invention, the term, “immunogenic amount” is an amount of an infectious pathogen antigen preparation of interest or amount of a biological toxin that elicits a clinically detectable protective response in an animal. By way of example, a clinically detectable protective response in an animal may be the production of an elevated titer of antibodies in the animal specific for the infectious pathogen antigen or biological toxin.
Description
A method for providing a preparation having an enhanced activity for inhibiting and protecting against an infectious pathogen provided. In particular embodiments, the infectious pathogen is tetanus.
A method for the treatment and/or inhibition of an infection caused by an infectious pathogen is also provided. In some embodiments, the method employs a composition comprising a vaccine, the vaccine comprising an adjuvant having a menu of pro-inflammatory species characteristic of an extracellular matrix (ECM) material together with an antigen associated with an infectious pathogen or a biological toxin. These preparations are found to be more immunogenic than use of the infectious pathogen antigen or biological toxin antigen vaccine alone in the treatment and/or prophylaxis against an infectious pathogen or biological toxin, such as tetanus.
The immune response to the described tetanus vaccine is enhanced by use of SIS as an adjuvant.
The description of the present invention is enhanced by the various examples that follow.
EXAMPLE 1
Materials and Methods for ECM as an Adjuvant for a Vaccine against Diseases Associated with an Infectious Pathogen
The present example provides some examples of materials and methods that may be used in the practice of the present invention.
Small Intestinal Submucosa (SIS)
Small Intestinal Submucosa (SIS) was obtained from Cook Biotech, Inc. (West Lafayette, Ind.). Experimental grade material was provided for use in the present studies of an SIS preparation that was described as having been prepared by harvesting porcine jejunum and placing 10- to 20-cm lengths into saline solution (31-33). Following removal of all mesenteric tissues, the jejunal segment was everted and the tunica mucosa abraded using a longitudinal wiping motion with a scalpel handle and moistened gauze. The serosa and tunica muscularis were then gently removed using the same procedure. The remaining tissue was disinfected with peracetic acid, rinsed extensively in high purity water, and sterilized using ethylene oxide. SIS Particulate is supplied by Cook Biotech, Inc. (West Lafayette, Ind.) and is SIS material ground and sieved. The size particles are in the range from 45 micron to 335 micron. SIS gel is supplied by Cook Biotech, Inc. (West Lafayette, Ind.) and is produced from SIS material via an acid digestion and purification process.
Tetanus Toxin and Tetanus Toxoid
Tetanus toxin and tetanus toxoid were purchased from List Biological Laboratories (Campbell, Calif.).
Alum
Alum was purchased as Alhydrogel®, an aluminum hydroxide gel adjuvant (Brenntak Biosector, Frederikssund, Denmark).
Animals (Mice)—Statistical Analysis
Results of survival versus non-survival following challenge with tetanus toxin were compared between groups using the Chi-square test with two degrees of freedom. Differences were considered significant when p≦0.001.
EXAMPLE 2
Use of Adjuvant in the Inhibition of Tetanus
To determine if SIS can act as an adjuvant for vaccines against diseases associated with infectious pathogens or biological toxins, preparations were made with tetanus toxoid, an inactivated form of tetanus toxin. Both gel SIS and SIS particles produced from a sheet of single layer SIS were evaluated as adjuvants. Briefly, groups of 15 Balb/C female mice (Harlan, Inc., Indianapolis, Ind.) were vaccinated initially (0.1 ml volume/dose) and again, five weeks later with one of the following:
Particulate SIS
Gel SIS
Tetanus toxoid (TT; 0.03 ug/dose)
TT (0.05 ug/dose)
TT (0.03 ug/dose)+alum (alhydrogel)
TT (0.05 ug/dose)+alum (alhydrogel)
TT (0.03 ug/dose)+particulate SIS
TT (0.05 ug/dose)+particulate SIS
TT (0.03 ug/dose)+gel SIS
TT (0.05 ug/dose)+gel SIS
Untreated control group
Five weeks after the second vaccination, mice were challenged with a lethal dose of tetanus toxin (1 ng/mouse) given intraperitoneally in 0.2 ml of sterile saline. Mice were then observed over the next 96 hours and the number of surviving mice recorded for each group.
The results of this study are as follows:











Treatment Group
Number Surviving/Total at 96 h







Untreated
 0/15


Gel SIS
 0/15


Particulate SIS
 0/15


Tetanus toxoid (TT; 0.03 ug/dose)
 6/15


TT (0.05 ug/dose)
 8/15


TT (0.03 ug/dose) + alum (alhydrogel)
15/15


TT (0.05 ug/dose) + alum (alhydrogel)
15/15


TT (0.03 ug/dose) + particulate SIS
15/15


TT (0.05 ug/dose) + particulate SIS
15/15


TT (0.03 ug/dose) + gel SIS
15/15


TT (0.05 ug/dose) + gel SIS
15/15









A significantly greater number of mice survived challenge with tetanus toxin in groups vaccinated with either 0.03 or 0.05 μg/dose of tetanus toxoid administered in alhydrogel, SIS gel, or particulate SIS compared to all other vaccination groups.
These results demonstrate the ability of both particulate and gel SIS to act as an adjuvant for a vaccine against disease associated with an infectious pathogen, such as tetanus.
EXAMPLE 3
Exemplary Infectious Pathogens
The present example demonstrates the utility of the present invention with disease associated with a wide variety of infectious pathogens and biological toxins, including by way of example and not exclusion, tetanus, influenza, rabies, viral hepatitis, diphtheria, anthrax, Streptococcus pneumoniae infection, malaria, leishmaniasis, ricin toxicosis, and Staphylococcal enterotoxin B toxicosis.







TABLE 2







Classification of Common Vaccines for Humans










Disease or Pathogen
Type of Vaccine







Whole Organisms:




Bacterial cells:



Cholera
Inactivated



Plague
Inactivated



Tuberculosis
Attenuated BCG+



Salmonella typhi
Attenuated



Viral Particles:



Influenza
Inactivated



Measles
Attenuated



Mumps
Attenuated



Rubella
Attenuated



Polio (Sabin/OPV)
Attenuated



Polio (Salk/IPV)
Inactivated



V. zoster
Attenuated



Yellow fever
Attenuated



Type of Vaccine



(Purified) Macromolecules



Toxoids:



Diphtheria
Inactivated exotoxin



Tetanus
Inactivated exotoxin



acellular Pertussis
Inactivated exotoxins



Capsular polysaccharide:



Haemophilus influenzae b
polysaccharide + protein carrier



Neisseria meningidis
Polysaccaride



Streptococcus pneumoniae
23 distinct capsular polysaccharides



Surface antigen:



Hepatitis B
Recombinant surface antigen




(HbsAg)







+Bacillus Calmette-Guerin (BCG) is an antiviral strain of Mycobacterium bovis.






Vaccines for Disease Associated with Viral Infections
1. Influenza—Influenza is an acute febrile respiratory disease resulting from infection with the influenza virus. Current influenza vaccines use aluminum adjuvants. To enhance the efficacy of vaccines, several adjuvants have been examined. For example, the oil-in-water emulsion MF59 has been reported to improve vaccine immunity (Higgins (1996)1; Martin (1997)2), though it does not completely solve the low efficiency of the influenza vaccine in the elderly (Banzhoff (2003)3). A synthetic peptide, GK1, derived from Taenia crassiceps cysticerci was reported to enhance the immune response accompanying influenza vaccination in both young and aged mice (Segura-Velásquez (2006)4), but trials in humans have not been published.
As part of the present invention, an influenza vaccine may be provided that comprises the extracellular matrix material described herein as the vaccine adjuvant combined with an immunologically effective amount of an influenza antigen. By way of example, such an influenza antigen may comprise a current influenza virus combination of antigens of an H5N1 (hemagglutinin [HA] subtype 1; neuraminidase [NA] subtype 1), and H3N2 influenza A virus, and an influenza B virus. This preparation and other influenza antigen preparations are described in Palese (2006)33). This article and all of its teachings are incorporated herein by reference.
2. Rabies—Rabies is a devastating neurological disease that is caused by infection with the rabies virus. Vaccination against rabies typically utilizes inactivated virus and an aluminum adjuvant. A lipoid adjuvant of the oil-in-water type, based on squalene, significantly increased the immunologic response of mice to vaccination with an inactivated virus vaccine when compared to vaccination using an aluminum salt adjuvant (Suli, 2004). An adjuvant based on glycopeptidolipids extracted from Mycobacterium cheloniae enhanced the immune response of mice to vaccination with an inactivated rabies virus vaccine (de Souza Matos (2000)6).
As part of the present invention, a rabies vaccine may be provided that comprises the extracellular matrix material as the vaccine adjuvant combined with an immunologically effective amount of a rabies antigen. By way of example, a rabies antigen may comprise an inactivated rabies virus. One example of an inactivated rabies virus vaccine antigen that may be used in the present formulations is described in de Souza Matos (2000)6.
3. Viral Hepatitis—Viral hepatitis, particularly that caused by Hepatitis B virus, is a serious health problem with over 300 million people affected worldwide. Vaccination offers hope for effective prophylaxis. Peptide epitopes of the virus stimulated a significant immune response when fused with heat shock protein 70 from Mycobacterium tuberculosis as an adjuvant (Peng (2006)7). Unmethylated CpG dinucleotides were effective as an adjuvant with hepatitis B antigen in aged mice (Qin (2004)8); and a vaccine consisting of hepatitis B virus antigens and an immunostimulatory DNA sequence is in human clinical trials (Sung (2006)9). In development of an intranasal vaccine, it was shown that DL-lactide/glycolide copolymer microspheres with chitosan were an effective adjuvant for a vaccine based on recombinant Hepatitis B surface protein (Jaganathan (2006)10).
As part of the present invention, a viral hepatitis vaccine may be provided that comprises the extracellular matrix material as the vaccine adjuvant combined with an immunologically effective amount of a viral hepatitis antigen. By way of example, such a hepatitis antigen may comprise recombinant hepatitis B surface protein. By way of example, such a hepatitis B surface protein antigen is described in Jaganathan, (2006)10), which reference is specifically incorporated herein by reference.
Vaccines for Disease Associated with Bacterial Infections:
1. Diphtheria—A respiratory disease characterized by dysnepea, weakness, and pyrexia, diphtheria is the result of infection with Corynebacterium diphtheriae, bacteria which produces a toxin that is carried hematogenously through the body. Immunization against diphtheria is frequently combined with immunization against tetanus and pertussis; these vaccines typically contain aluminum salt adjuvants (Sugai (2005)11). Unmethylated CpG dinucleotides were effective as an adjuvant in a diphtheria-tetanus-pertussis vaccine and shifted the immune response toward cell-mediated immunity in mice immunized intraperitoneally (Sugai (2005)11). Trials to reduce adverse side-effects related to the aluminum salt adjuvant of a vaccine consisting of diphtheria toxoid, tetanus toxoid, and purified Bordetella pertussis antigens including pertussis toxoid showed that reduction of the aluminum salt content of the vaccine resulted in reduced geometric mean antibody concentrations to the relevant antigens, but did not result in reduction of local or general side effects (Theeten (2005)12). Monophosphoryl lipid A was shown in mice to effectively serve as an adjuvant for diphtheria toxin in mice (Caglar (2005)13).
As part of the present invention, a diphtheria vaccine may be provided that comprises the extracellular matrix material as the vaccine adjuvant combined with an immunologically effective amount of a diphtheria antigen. By way of example, a diphtheria antigen may comprise a diphtheria toxoid. One example of a diphtheria toxoid that may be used in the practice of the present invention is described in Theeten (2005)12.
2. Anthrax—Anthrax is a disease caused by the bacterium, Bacillus anthracis. Specifically, the bacterium produces a toxin which results in hemorrhagic necrosis of lymph nodes, hematogenous spread, shock, and death. A vaccine consisting of one subunit (protective antigen) of this toxin was shown to protect mice when combined with a microparticle adjuvant administered by either the intramuscular or intranasal routes (Flick-Smith (2002)14. Further, vaccination protected mice against infection with B. anthracis spores. While the aluminum salt-adjuvanted anthrax-vaccine-adsorbed is the only anthrax vaccine licensed in the United States, major drawbacks exist, including a very lengthy and complicated dosing schedule, followed by annual booster injections. Further, the aluminum adjuvant of anthrax vaccine has been linked to Gulf War Illness among veterans of the 1991 conflict (Petrik (2007))15.
As part of the present invention, an anthrax vaccine may be provided that comprises the extracellular matrix material as the vaccine adjuvant combined with an immunologically effective amount of an anthrax antigen. By way of example, such an anthrax antigen may comprise the one subunit (protective antigen) of the Bacillus anthracis bacterium. One such particular antigenic subunit is described in Flick-Smith (2002)14.
3. Streptococcus pneumoniae—A bacterial pathogen of particular importance to the elderly and young adults, Streptococcus pneumoniae causes disease including sepsis and pneumonia, otitis media and meningitis. Vaccines typically involve adsorption of S. pneumoniae antigens to aluminum salt adjuvants, and reduced aluminum salt content led to reduced immunogenicity of S. pneumoniae vaccines (Levesque (2006)16. In human trials, IL-12 failed to improve the immune response to a pneumococcal polysaccharide vaccine; and IL-12 was associated with a high incidence of local and systemic side effects in humans (Hedlund (2002)17. Intranasal immunization against S. pneumoniae has been shown to be an effective method for preventing infection and disease, with unmethylated CpG dinucleotides serving as an effective adjuvant for an intranasal polysaccharide-protein conjugate vaccine (Sen (2006)18). Likewise, IL-12 and the B-subunit of cholera toxin were both shown to enhance efficacy of intranasally-administered preparations of S. pneumoniae antigens (Sabirov (2006)19; Pimenta (2006)20).
As part of the present invention, a pneumonia vaccine may be provided that comprises the extracellular matrix material described herein as the vaccine adjuvant combined with an immunologically effective amount of a pneumococcal antigen. By way of example, such a pneumococcal antigen may comprise a pneumococcal polysaccharide antigen. One form of a pneumococcal polysaccharide antigen is described in Hedlund (2002)17. This pneumococcal antigen may used as part in combination with the herein described adjuvants in a vaccine preparation.
Vaccines for Diseases Associated with Parasitic Infections
1. Malaria—Malaria affects millions of people worldwide and each year, 1-2 million people die from the disease caused by Plasmodium falciparum. Thus, the need for prophylactic measures has led to great interest in anti-malaria vaccines. The apical membrane antigen, a malaria vaccine candidate, was reported to have an enhanced immunogenicity by the aluminum salt adjuvant Alhydrogel (HCl Biosector, Denmark); and this adjuvant effect was further enhanced, and shifted from a Th1 response to a mixed Th1/Th2 response, by inclusion of the adjuvant CpG oligodeoxynucleotide (Mullen (2006)21). Alhydrogel and Montanide ISA 720 (Seppic, France) were compared in rhesus monkeys as adjuvants for a vaccine based on protective epitopes from the circumsporozoite protein of P. falciparum. Though Montanide ISA 720 induced superior immune responses, the formation of sterile abscesses at injection sites were noted as a significant disadvantage (Langermans (2005)22). Other studies with a circumsporozoite protein vaccine conducted in rhesus monkeys showed that some novel oil-in-water adjuvants with components of immunostimulants 3-deacetylated monophosphoryl lipid A (3D-MPL) and the saponin Quillaja saponaria 21 (QS21) were safe and stimulated improved antibody responses (Stewart (2006)23). Some of these same oil-in-water adjuvants improved the immune response to a vaccine constructed of the P. falciparum antigen, Liver Stage Antigen-1 (Brando (2006)24).
As part of the present invention, a malarial vaccine may be provided that comprises the extracellular matrix material as the vaccine adjuvant combined with an immunologically effective amount of a malarial antigen. By way of example, such a malarial antigen may comprise a P. falciparum antigen Liver Stage Antigen-1. This antigen is described in detail in Brando (2006)24, this article being specifically incorporated herein by reference. This antigen may be combined with the extracellular matrix material described herein as an adjuvant to provide an anti-malarial vaccine as described herein.
2. Leishmaniasis—Leishmaniasis is a parasitic disease associated with infection by a species of parasites from the Leishmania genus. A large spectrum of clinical disease forms can result from infection, ranging from cutaneous lesions to fatal visceral forms. In the absence of effective, non-toxic treatments, great effort has been given to vaccine development. Vaccines based on DNA of the parasite have been shown to induce partial protection; aluminum phosphate adjuvant has no effect on the humoral response to this vaccine, but has been reported to slightly increase the cellular immune response and protection against infection in a mouse model (Rosado-Vallado (2005)25). In evaluations in rhesus monkeys using a soluble Leishmania antigen and alum with IL-12 as adjuvants, it was shown that the adjuvants improved protective immunity, though transient nodules developed at the site of subcutaneous injection (Kenney (1999)26). CpG oligodeoxynucleotides served as an effective adjuvant for a vaccine consisting of live, nonattenuated L. major organisms alone or in combination with lysates of heat-killed L. major promastigotes, either without or bound to alum (Mendez (2003)27). Partial protective immunity was stimulated, but mice receiving alum-containing vaccines developed large dermal lesions that required up to 10 weeks to heal.
As part of the present invention, an anti-parasitic infection associated disease vaccine may be provided that comprises the extracellular matrix material as the vaccine adjuvant combined with an immunologically effective amount of a Leishmaniasis antigen, or any of the other antigenic species described above. By way of example, a Leishmaniasis antigen may comprise the Leishmaniasis antigen described in detail in Kenny (1999)26, which article is specifically incorporated herein by reference.
Vaccines for Disease Associated with Biological Toxins
1. Ricin—Ricin is a toxin produced naturally by the seeds of the castor bean plant, Ricinus communis. When humans or animals are exposed to the toxin, severe respiratory distress and death may result. Because of its potency and ability to be administered via aerosol, ingestion, or injection, ricin is considered a powerful bioweapon. Though there is presently no approved commercial vaccine for ricin, pilot trials in humans have examined the use of recombinant, non-toxic forms of one of the subunits of ricin (Vitetta (2006)28). This preparation was administered without an adjuvant and elicited ricin-neutralizing antibodies in some of those tested, particularly at higher doses. However, all dose groups were found to result in significant side-effects, including myalgia and headache. Ricin toxoid adjuvantized by liposomal encapsulation was found to induce a stronger immune response when administered intra-tracheally than the vaccine adjuvantized with an aluminum salt adjuvant (Griffiths (1997)29). A vaccine consisting of a deglycosylated chain A ricin (DCAR) and the adjuvant LTR72, a mutant of the heat-labile enterotoxin of Escherichia coli, resulted in a stronger antibody response of vaccinated mice to ricin, but did not result in improved protection against lung injury when challenged with ricin (Kende (2006)30).
As part of the present invention, an anti-ricin vaccine may be provided that comprises the extracellular matrix material as the vaccine adjuvant as described herein combined with an immunologically effective amount of a ricin toxoid antigen. By way of example, such a ricin toxoid antigen is described in detail in Griffiths (1997)29, which article is specifically incorporated herein by reference.
2. Staphylococcal enterotoxin B (SEB)—SEB is produced by the bacteria, Staphylococcus aureus and is associated with food poisoning. Incorporation of SEB toxoid into biodegradable poly(DL-lactide-co-glycolide) microspheres enhanced the immune response of mice to a degree similar to SEB toxoid adsorbed to alum and combined with complete Freund adjuvant (Eldridge, 1991)31). Similarly, SEB toxoid was effectively adjuvantized by incorporation into polylactic polyglycolic acid copolymer nanospheres; the resulting immune response was comparable to that achieved by using alum as an adjuvant (Desai (2000)32).
As part of the present invention, an anti-toxin-associated disease vaccine may be provided that comprises the extracellular matrix material as the vaccine adjuvant combined with an immunologically effective amount of an antigen such as ricin toxoid or SEB toxoid as antigen. By way of example, such antigens are described in detail in Vitetta (2006)28 and Eldridge (1991)31, the teachings of which are specifically incorporated herein by reference.
Vaccines for Diseases Associated with Prions:
In some embodiments, the invention provides an adjuvant preparation that is suitable for use in combination with a prion-associated disease. By way of example, such prion associated diseases include, all of which are classified as transmissible spongiform encephalopathies, bovine spongiform encephalopathy, scrapie, cervid chronic wasting disease and Creutzfeld-Jakob disease.
Although prions use immune and lymphoreticular cells to gain access to the brain (Aguzzi, 2003)36, existing evidence suggests that humoral immune responses can suppress infection. In particular, antibodies to the cellular prion protein (PrPc) are known to inhibit prion propagation (Petetz, 200137; Enari, 200138). Still, host tolerance to endogenous PrPc remains a major obstacle to active vaccination. In mice, vaccination with recombinant PrPc antigens such as peptides and polypeptides stimulated only weak immune responses. Co-administration of prion antigens with adjuvants such as Freund's (Polymenidou, 200439; Koller, 2002 40; Sigurddson, 200241; Gilch, 200342; Hanan, 200143; Hanan, 200144; Souan, 200145; Arbel, 200346); Montanide IMS-1313 (Schwartz, 200347); TiterMax®, a combination of a proprietary block copolymer CRL-8941, squalene, a metabolizable oil, and a unique microparticulate stabilizer (Gilch, 200342); and CpG oligonucleotides (Rosset, 200448) all failed to induce strong immune responses.
It is anticipated that the presently described adjuvant preparations of an extracellular matrix material may be used with the prion protein (PrPc) to provide an improved vaccine against prion-associated infections.
All documents, patents, journal articles and other materials cited in the present application are hereby incorporated by reference.
Although the present invention has been fully described in conjunction with several embodiments thereof with reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims, unless they depart therefrom.
BIBLIOGRAPHY
The references listed below as well as all references cited in the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques and/or compositions employed herein.

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## Claims

1. A composition suitable for use against an infectious pathogen comprising:
an immunogenic amount of an infectious pathogen or biological toxin antigen preparation of interest; and
an adjuvant of heterologous extracellular matrix, wherein the immunogenic amount of the infectious pathogen or biological toxin antigen preparation of interest in the presence of the adjuvant is less than the immunogenic amount of the infectious pathogen or biological toxin antigen preparation of interest that elicits a detectable protective response in the absence of the adjuvant, wherein the infectious pathogen antigen preparation of interest comprises an inactivated preparation of the infectious pathogen antigen of interest or toxic products thereof.

2. The adjuvant of claim 1, wherein said infectious pathogen or biological toxin is tetanus, influenza, rabies, viral hepatitis, diphtheria, anthrax, Streptococcus pneumoniae infection, malaria, leishmaniasis, ricin toxicosis, prions, or Staphylococcal enterotoxin B toxicois.

3. The composition of claim 1, wherein the infectious pathogen or-biological toxin antigen preparation of interest is tetanus toxoid.

4. The composition of claim 1, wherein the extracellular matrix is a heterologous acellular collagenous tissue preparation.

5. The composition of claim 1, prepared by a method comprising:
obtaining an adjuvant of an extracellular matrix capable of enhancing the immunogenicity of an infectious pathogen or biological toxin antigen of interest; and
combining said adjuvant with an immunogenic amount of an infectious pathogen or biological toxin antigen of interest.

6. The composition of claim 1, wherein the infectious pathogen is tetanus.

7. The composition of claim 1, wherein the extracellular matrix comprises a heterologous acellular collagenous tissue preparation.

8. The composition of claim 1, wherein the biological toxin antigen preparation of interest comprises an inactivated preparation of the biological toxin antigen of interest.

9. The composition of claim 1, wherein the extracellular matrix consists essentially of a heterologous acellular collagenous tissue preparation.

10. The composition of claim 9, wherein the adjuvant comprises a 1:10 ratio of the extracellular matrix and a pharmaceutically acceptable carrier solution.

11. The composition of claim 1, wherein the immunogenic amount of the biological toxin antigen preparation of interest is 0.03 μg to 0.05 μg of a tetanus toxoid.

12. The composition of claim 1 comprising a vaccine.

13. A method for immunizing an animal against an infectious pathogen of interest comprising:
administering an immunogenic amount of the composition of claim 1 composition comprising an infectious pathogen antigen of interest and an infectious pathogen vaccine adjuvant sufficient to stimulate an immune response in said animal for said infectious pathogen of interest or a toxic product produced by said infectious pathogen of interest, wherein the immunogenic amount of the composition sufficient to stimulate an immune response in the animal is less than the immunogenic amount of the infectious pathogen antigen of interest in the absence of the infectious pathogen vaccine adjuvant sufficient to stimulate an immune response.

14. The method of claim 13 wherein the infectious pathogen antigen of interest comprises a tetanus toxoid preparation.

15. The method of claim 13 wherein the immunogenic amount of the composition comprises 0.03 μg to 0.05 μg of the tetanus toxoid.

16. The method of claim 13 wherein an immunized animal has an enhanced survival rate compared to a non-immunized animal.