Patent Application: US-69944807-A

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 .

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 . 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 . 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 . 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 ) 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 were purchased from list biological laboratories ( campbell , calif .). alum was purchased as alhydrogel ®, an aluminum hydroxide gel adjuvant ( brenntak biosector , frederikssund , denmark ). 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 . 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 : 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 , 2001 37 ; enari , 2001 38 ). 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 &# 39 ; s ( polymenidou , 2004 39 ; koller , 2002 40 ; sigurddson , 2002 41 ; gilch , 2003 42 ; hanan , 2001 43 ; hanan , 2001 44 ; souan , 2001 45 ; arbel , 2003 46 ); montanide ims - 1313 ( schwartz , 2003 47 ); titermax ®, a combination of a proprietary block copolymer crl - 8941 , squalene , a metabolizable oil , and a unique microparticulate stabilizer ( gilch , 2003 42 ); and cpg oligonucleotides ( rosset , 2004 48 ) 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 . 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 . 1 . higgins d a , et al . 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