HK1066031A - Composition and method for destruction of infectious prion proteins - Google Patents

Description

Compositions and methods for destroying infectious prion proteins

Technical Field

The present invention relates to compositions and methods for destroying infectious prion protein associated with Transmissible Spongiform Encephalopathies (TSEs), such as Bovine Spongiform Encephalopathy (BSE) and scrapie in sheep. More particularly, the invention relates to the use of proteases for destroying infectious prion proteins in animal tissues, and/or for the disinfection and sterilization of medical devices and similar articles contaminated with such infectious prion proteins.

Background

Prion proteins are conformationally irregular proteins that are associated with infectious neurodegenerative diseases in human as well as non-human mammalian species.

Prion diseases in non-human mammalian species include scrapie (sheep), transmissible mink encephalopathy (mink), chronic wasting disease (elk, deer), Bovine Spongiform Encephalopathy (BSE) (bovine), feline spongiform encephalopathy (feline), and simian spongiform encephalopathy (monkey).

Various neurodegenerative diseases of humans that are etiologically related to prion proteins include: Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, severe insomnia, Kuru and variant Creutzfeldt-Jakob disease. The pathogenesis of human prion diseases is associated with carnivores (BSE-infected cattle, causing new creutzfeldt-jakob disease variants), human growth hormone administration (causing iatrogenic creutzfeldt-jakob disease), and forced malignant cell phagocytosis (causing kuru).

Over 180,000 BSE cases and 100 human creutzfeldt-jakob disease cases have been reported in europe since 1992, and it is expected that human creutzfeldt-jakob disease cases will increase significantly. Since there is currently no treatment for this disease and pathogenic prion proteins are recalcitrant and non-immunogenetic, it is difficult to control the spread of the disease. Isoforms of pathogenic and infectious prion proteins are very stable, have an abundant beta-sheet structure, and are thermostable and resistant to common proteolytic enzymes (Prusiner, S.B., Proc. Natl.Acad, Sci.U.S.A., 95, 11363 (1998); Cohen, F.E., and Prusiner, S.B., Ann.Rev.biochem., 67, 793 (1998); and Pan, K-M, Baldwin, M.N., Nguyen, J., Gasset, M.Serban, A.G., Groth, D.Mehlhorn, I.Huang, Z.Fletterick, R.J., Cohen, F.E., and Prusiner, S.B., Proc.Natl.Acad.Sci.90, S.10962 (1993)).

Research is currently focused primarily on BSE, bovine-derived human food contaminated with prion protein, and the reproduction and proliferation of prion protein diseases in bovine species. Infection in cattle herds is associated with cattle fed with feed containing bone meal and organs and tissues extracted from infected cattle, sheep and other ruminants.

Currently, in many countries, animal products that are not human food supplies, and animal by-products that are not live sources of raw materials and nutritional supplements for animal feed, are incinerated and their ash residues buried underground to prevent the transmission of prion protein from animals in the presence of infectious prion protein.

In europe, meat and bone meal from animal by-products has been banned for use in feed. In the united states, although there has been no report of an outbreak of BSE, the animal husbandry and its refining and processing industries have enacted strict measures to prevent the onset and spread of the disease (Franco, b.a., feed stuffs, 2 months and 12 days 2001). In addition, the united states has banned the import of meat and meat by-products.

Various assays have been developed for the presence of infectious prion protein in animal tissues, including western blot assays, sandwich immunoassays, enzyme-linked immunosorbent assays (ELISA), fluorescence immunoassays, capillary immunoelectrophoresis assays, and plasminogen-limiting assays (Genetic Engineering News, vol.21, No.6, 3/15 2001), but to date, the corresponding ability to remove infectious prion protein from infected animal tissues has not been developed.

Infectious prion protein is difficult to destroy by conventional methods such as autoclaving (even at temperatures as high as 200 ℃ which are not effective in inactivating infectious prion protein), boiling, freezing, and exposure to reagents such as formaldehyde, carbolic acid, and chloroform, which denature, rather than degrade, conformationally regular proteins. Typically, incineration or bleaching treatments are used to destroy the pathogenic isoforms of prion proteins.

Accordingly, it would be a significant advance in the art to provide a composition and method for destroying infectious prion protein that is suitable for treating biological materials, such as animal tissue that contains or is contaminated with infectious prion protein.

In addition, cross-contamination caused by the repeated use of medical devices previously exposed to prion-infected tissue becomes increasingly dangerous and a potential factor in the spread of infection.

Sterilization of health care devices and tools using antimicrobial, disinfectant agents is critical to prevent cross-contamination by medical instruments during health care. Medical devices or instruments may be sterilized and disinfected by a variety of conventional methods using a variety of physical and chemical methods that destroy infected biological materials, such as bacteria or viruses. For example, medical devices may be disinfected and sterilized using chemical disinfectants such as peracetic acid, hydrogen peroxide, sodium hydroxide, formic acid, bleach, alcohol, ethylene oxide, formaldehyde, formalin, and glutaraldehyde. Incineration, high pressure steam treatment, freezing, dry heat, boiling, ultraviolet radiation, and microwave radiation may also be used to destroy common infectious agents such as bacteria and viruses.

However, as discussed above, it is well known that infectious prion eggs are difficult to destroy by conventional methods, and thus it is not effective to disinfect or sterilize prion-contaminated medical devices or similar items by conventional methods.

It is therefore another object of the present invention to provide a composition and method for effectively disinfecting or sterilizing prion-infected medical devices such as surgical instruments or similar items such as kitchen utensils and laboratory instruments.

Summary of The Invention

The present invention provides methods and compositions for destroying infectious prion protein.

In one aspect, the invention relates to a method of treating a site contaminated with infectious prion protein to reduce the infectious prion protein, the method comprising the steps of:

(a) heating the site to a temperature and for a time sufficient to increase the proteolytic susceptibility of infectious prion protein at the site; and

(b) exposing the heated site to a proteolytic enzyme effective to at least partially reduce the infective prion at the site.

The site may be a tissue containing or contaminated with infectious prion protein, or may be an item susceptible to contamination by infectious prion protein.

The temperature of step (a) is no more than about 150 ℃, preferably in the range of about 100 ℃ to about 150 ℃, more preferably in the range of about 125 ℃ to about 140 ℃.

The temperature of step (b) is lower than the temperature of step (a), for example: (1) in the range of about 35 ℃ to about 100 ℃, (2) in the range of about 40 ℃ to about 75 ℃, (3) in the range of about 50 ℃ to about 60 ℃. Step (b) is preferably carried out at a temperature above about 40 ℃, more preferably above about 50 ℃.

Proteolytic enzymes useful in step (b) include, but are not limited to, keratinase, proteinase K, trypsin, chymotrypsin, pepsin, chymosin, cathepsin, subtilisin, elastase, collagenase, endopeptidase, peptidase, oligopeptidase, thermolysin, bactolysin, myclysins, carboxypeptidases, leuminamidopeptidase, aminopeptidase, hyperthermophilic protease, carbonyl hydrolase, papain, pancreatin, streptokinase, ficin, carboxypeptidase, chymopapain, and bromelain. Keratinases and/or active fragments thereof are particularly preferred for the practice of the present invention. Keratinases are generally a group of known proteolytic enzymes capable of decomposing keratin, and they are the main components of feathers, horns, hooves, and hair. The inventors of the present application have surprisingly and surprisingly found that: keratinases are also effective in destroying infectious prion proteins, particularly if the infectious prion proteins have been processed to make them more susceptible to proteolysis. More preferably, such proteolytic enzymes are Bacillus Licheniformis (Bacillus Licheniformis) PWD-1 keratinase and/or active fragments thereof. Alternatively, the proteolytic enzyme is a variant of wild-type Bacillus amyloliquefaciens subtilisin (Bacillus amyloliquefaciens subtilisin) comprising one or more amino acid substitutions, additions, or deletions.

In addition, the method described above may further comprise the step of (c) testing the site to confirm there has been a reduction in infectious prion protein, the step comprising: the site is tested using a method selected from the group consisting of western blot assay, sandwich immunoassay assay, ELISA, fluoroimmunoassay test, capillary immunoelectrophoresis assay, and plasminogen-limiting assay. The present invention is preferably tested by western blotting.

As described above, a particular aspect of the invention relates to a treatment for reducing infectious prion protein at a site contaminated or suspected of being contaminated with infectious prion protein, wherein the site comprises tissue containing or contaminated with infectious prion protein. These tissues include animal tissues, preferably mammalian tissues such as bovine tissues, ovine tissues, and the like. It can also be from any part of the body of these animals, such as brain, pituitary, intestine, lung, heart, kidney, and spleen tissue. The animal tissue preferably comprises nervous system tissue, more preferably BSE-infected or scrapie-infected tissue. It can be obtained from animals carrying infectious prion protein.

As described above, another particular aspect of the invention relates to a treatment for reducing infectious prion protein at a site contaminated or suspected of being contaminated with infectious prion protein, wherein the site comprises an article susceptible to contamination by infectious prion protein. These items may include surgical instruments such as forceps (clamps), forceps (forceps), scissors, knives, cables, reamers, forceps (tweeters), cannulas, calipers, sculptors, curettes, scrapers, dilators, clip applicators, constriction devices, constrictors, scoopers, needle holders, pipettes, coagulation electrodes, depth electrodes for electroencephalographs, rib and sternum dilators, bipolar probes, and costal scissors. In addition, these items may include knives and kitchen utensils such as knives, forks, scissors, peelers, paring knives, slicers, spatulas and meat cutters, or laboratory instruments such as containers, filtration devices, centrifuges, spectrophotometers and fluorometers, or veterinary devices such as forceps, tweezers, knives, saws, probes and electronic stunning equipment.

When the treated site contains items, the proteolytic enzyme used in step (b) is preferably provided in the form of a solution in order to decontaminate and disinfect the items. When keratinase is used as the proteolytic enzyme, the enzyme solution is characterized by a low effective concentration, preferably in the range of about 0.2g/L to about 1.0 g/L.

Another aspect of the invention relates to a method for increasing the degradability of infectious prion protein by proteolytic enzymatic degradation treatment, comprising (a) heating the prion protein to below the pyrolytic destruction temperature of the prion protein, followed by (b) performing an enzymatic degradation treatment of the prion protein.

A still further aspect of the invention relates to a method for removing infectious prion protein from bovine tissue comprising or contaminated with infectious prion protein, the method comprising (a) cooking bovine tissue at a temperature in the range of about 100 ℃ to about 150 ℃, followed by (b) exposing the bovine tissue to a heat-stable proteolytic enzyme at a temperature in the range of about 35 ℃ to about 100 ℃, wherein the proteolytic enzyme is effective to proteolytically hydrolyze the infectious prion protein, at least partially destroying the infectious prion protein in the bovine tissue. Cooking is preferably carried out for about 5 minutes to about 5 hours, and the proteolytic enzyme used in step (b) preferably comprises Bacillus licheniformis PWD-1 keratinase.

Thermostable proteolytic enzymes are particularly useful in the practice of the present invention. Accordingly, a particular aspect of the present invention relates to a method of at least partially degrading infectious prion protein in a tissue comprising or contaminated with infectious prion protein by the steps comprising: heating the tissue while exposing it to a heat-stable proteolytic enzyme at a temperature and for a time sufficient to at least partially degrade the infectious prion protein; another particular aspect of the invention relates to a tissue composition comprising tissue containing or contaminated with infectious prion protein and a proteolytic enzyme that is thermally stable at a temperature in the range of about 35 ℃ to about 100 ℃. Such heat stable proteolytic enzymes are preferably thermostable proteases useful for treating meat animal products or by-products thereof at a sufficiently high temperature and for a sufficiently long time to destroy infectious prion protein therein that causes BSE.

Another aspect of the invention relates to a method of treating tissue to reduce infectious prion protein. The method comprises the following steps:

(a) heating the tissue at a temperature and for a time sufficient to increase the proteolytic degradability of infectious prion protein in the tissue; and

(b) exposing said heated tissue to a proteolytic enzyme effective to at least partially reduce infectious prion protein in said tissue.

Another aspect of the invention relates to a method of sterilizing an article susceptible to contamination by infectious prion protein, the method comprising the steps of:

(a) heating said article at a sufficiently high temperature and for a sufficiently long time to increase the proteolytic susceptibility of prion protein on said article; and

(b) exposing said heated article to a proteolytic enzyme effective to at least partially reduce infective prion protein on said article.

In another aspect, the present invention relates to a method for removing infectious prion protein from a surgical instrument contaminated with infectious prion protein, the method comprising (a) heating the surgical instrument at a temperature in the range of about 100 ℃ to about 150 ℃, for example, for about 5 minutes to about 5 hours, followed by (b) exposing the heated surgical instrument to a heat-stable proteolytic enzyme at a temperature in the range of about 35 ℃ to about 100 ℃, to at least partially effectively destroy the infectious prion protein contaminating the surgical instrument.

A still further aspect of the present invention relates to a cleaning composition for disinfecting an article susceptible to contamination by infectious prion protein, said composition comprising:

(i) one or more proteolytic enzymes selected from the group consisting of keratinase, proteinase K, trypsin, chymotrypsin, pepsin, chymosin, cathepsin, subtilisin, elastase, collagenase, endopeptidase, peptidase, oligopeptidase, thermolysin, bacillus lysin, myclysins, carboxypeptidase, leucinemidopeptidase, aminopeptidase, hyperthermophilic protease, carbonyl hydrolase, papain, pancreatin, streptokinase, ficin, carboxypeptidase, chymopapain, and bromelain; and

(ii) a solvent.

The cleaning compositions of the present invention preferably include keratinase at a concentration in the range of about 0.2g/L to about 1.0 g/L. The present invention may employ various solvents such as distilled water, buffer solutions, washing solutions, alcohols, or any other inorganic or organic solvent for enzymatic detergents, which can be readily determined by one of ordinary skill in the art without undue experimentation. More preferably, the cleaning composition further comprises one or more chemical additives to enhance disinfecting/sterilizing effects, including but not limited to: surfactants, builders, co-accelerators, fillers and other adjuvants.

Other aspects, features and embodiments of the invention will become more fully apparent from the following disclosure and appended claims.

Brief description of the drawings

FIGS. 1 and 2 show the results of gel electrophoresis/Western blotting on SDS-PAGE gels demonstrating the efficacy of the method of the invention for the destruction of infectious prion protein.

Detailed description of the invention and preferred embodiments

The present invention and its features, aspects and embodiments, the published technical literature cited in the background of the invention, and the following patents and technical literature, each of which is hereby incorporated by reference in their entirety, will be described in more detail below: U.S. patent nos.: 4,908,220, respectively; 4,959,311, respectively; 5,063,161, respectively; 5,171,682, respectively; 5,186,961, respectively; and 5,712,147; deslys, J.P., "Screening strained title for BSE", Nature, Vol.409, pp.476-477, 1/25/2001; and Cohen, F.E. "Protein profiling and Prion diseases", J.mol.biol. (1999), Vol.293, pp.313-320.

The present invention uses proteolytic enzymes to degrade infectious prion protein in tissue or to disinfect or sterilize prion-contaminated articles such as surgical instruments, knives and kitchen utensils, veterinary equipment, and laboratory instruments.

The efficacy of the method of the present invention in degrading infectious prion proteins is unexpected because exposure of infectious prion proteins to high temperatures alone (e.g., at 200 ℃) does not change their pathogenic properties; in addition, conventional proteolytic enzymes such as proteinase K, which completely digest non-infectious PrPc, also do not destroy the corresponding infectious isoform. Thus, it has been very surprisingly found that enzymatic treatment can be carried out using temperatures below the incineration temperature (which temperature heretofore has been required to destroy infectious PrPSc) to completely remove infectious PrPSc from tissues containing or contaminated with infectious prion proteins.

The term elevated temperature as used herein means a temperature of at least 35 ℃. The term proteolytic sensitivity refers to the ability to degrade infectious prion proteases to non-infectious products.

The site, e.g., tissue or article, can be treated to reduce infectious prion protein therein using various techniques described below.

For example, in one embodiment of the invention, a tissue or article (which may comprise, or be contaminated with, or is suspected of comprising, or being contaminated with, infectious prion protein) is heated at a temperature and for a time sufficient to increase the susceptibility of proteolysis of infectious prion protein, if present, while exposing the tissue or article to a proteolytic enzyme, effective to at least partially destroy any infectious prion protein present therein.

This treatment may be carried out in a two-step sequence by first heating the tissue or article at an elevated temperature and then exposing the heated tissue or article to an enzymatic reagent at a lower elevated temperature to effect proteolytic degradation of the infectious prion protein.

In such a two-step treatment process, the tissue or article may first be heat treated at an elevated temperature and then cooled to a lower elevated temperature, for example by subjecting the tissue or article to exothermic, convective or other suitable means, to perform a second enzymatic treatment step, the tissue or article being at a suitable temperature when it is inoculated with or exposed to proteolytic enzymes.

In the second step of the two-step treatment process, the tissue or article is exposed to a proteolytic enzyme effective to at least partially destroy infectious prion protein in the tissue or article.

Thus, the method can be carried out in various embodiments wherein the proteolytic susceptibility of infectious prion protein in the tissue or article is increased by heating the tissue or article to an elevated temperature for subsequent proteolytic enzyme treatment. The elevated temperature in the heating step can be any suitable temperature, such as at least 35 ℃, at least 40 ℃, at least 60 ℃, at least 75 ℃ and/or no more than 150 ℃ (or other lower temperature, if desired), with one illustrative specific temperature range being from about 100 ℃ to about 150 ℃, more preferably from about 125 ℃ to about 140 ℃.

Alternatively, the present invention may be used to destroy prion protein in a one-step process, in which case the proteolytic enzyme is stable and effective at the temperatures involved in the process, thus eliminating the need for an initial heating step.

In the one-step process, animal tissue or articles and proteolytic enzymes are heated together to a suitable elevated temperature to effect enzymatic degradation of infectious prion protein.

For example, infectious prion protein in a tissue or article containing or contaminated with infectious prion protein can be at least partially degraded by heating the tissue or article while exposing the tissue or article to a heat-stable proteolytic enzyme.

Regardless of the particular sequence used in the method of the present invention, the tissue or article is exposed to a proteolytic enzyme (the second of the two-step processes, or the enzymatic degradation step of the one-step process) to at least partially effectively destroy infectious prion therein.

The enzymatic degradation step of the present invention may be carried out at any suitable temperature. For example at a temperature above about 35 ℃, above about 40 ℃ or above about 50 ℃ depending on the thermostable properties of the proteolytic enzyme used.

As an illustrative example, the enzymatic degradation step may be performed at a temperature in the range of about 35 ℃ to about 100 ℃, about 40 ℃ to about 100 ℃, about 50 ℃ to about 100 ℃, about 40 ℃ to about 75 ℃, about 50 ℃ to about 60 ℃, depending on the stability of the proteolysis and the enzymatic activity of the particular proteolytic enzyme used.

In the enzymatic degradation step, the proteolytic enzyme at least partially, preferably completely, destroys the infectious prion in the tissue or article to be treated.

It will be appreciated that any of a wide variety of proteases may be used in the present invention and that the choice of a particular proteolytic enzyme for proteolytic degradation will influence the choice of temperature and the choice of any high temperature treatment of the tissue or article prior to exposure to the proteolytic enzyme.

The particular temperature conditions for the enzymatic treatment, as well as the appropriate temperature conditions necessary or desired for any high temperature preliminary treatment step prior to enzymatic treatment, can be readily determined by one skilled in the art without undue experimentation.

Useful proteolytic enzymes for use in the present invention include those which are active and effective under the conditions of use. For high temperature enzymatic treatment, suitable proteolytic enzymes are thermostable under the conditions of use.

In this connection, widely varying proteolytic enzymes with thermostable properties are known. For example, the various proteolytic enzymes used in particular embodiments of the invention are also thermostable at 35 ℃, 40 ℃, 50 ℃, 60 ℃, or even at 100 ℃.

The proteolytic enzyme may be any suitable type of proteolytic enzyme, and may comprise a single enzyme species or a mixture of enzymes. The enzymes can be used in pure form and in concentrated form, or else in diluted form. Preferably, the enzyme is dissolved in a solvent to form an enzyme solution having a concentration of about 0.2g/L to about 1.0 g/L.

Illustrative proteolytic enzymes useful in the invention include, but are not limited to, keratinase, proteinase K, trypsin, chymotrypsin, pepsin, chymosin, cathepsin, subtilisin, elastase, collagenase, endopeptidase, peptidase, oligopeptidase, thermolysin, bacillus lysin, myclysins, carboxypeptidases, leuminamidopeptidase, aminopeptidase, hyperthermophilic protease, carbonyl hydrolase, papain, pancreatin, streptokinase, ficin, carboxypeptidase, chymopapain, and bromelain.

Preferred enzyme species include keratinases. A particularly preferred keratinase enzyme comprises Bacillus licheniformis PWD-1 keratinase. The proteolytic enzyme species used in the practice of the invention include active fragments of proteolytic enzymes, for example active fragments of keratinases such as Bacillus licheniformis PWD-1 keratinase. When the present invention uses keratinase, the effective concentration required for the enzyme solution is significantly lower than for conventional enzyme detergents or disinfectants. In addition, keratinase is characterized by an optimal temperature range of activity of keratinase from about 50 ℃ to about 65 ℃, well above those most common enzymatic detergents, in the pH range from about 6.0 to about 9.5. Thus, the decontamination temperature of the method of the present invention can be significantly increased, which more efficiently increases the proteolytic sensitivity of infectious prion protein on surgical instruments.

When treating tissue by the method of the invention, the tissue treated may be any suitable type of tissue, including mammalian tissue, non-mammalian tissue, and even plant tissue that actually or potentially contains infectious prion protein. Mammalian tissue may include human as well as non-human mammalian tissue.

In a particular aspect, tissues suitable for treatment by the methods of the invention include, but are not limited to, bovine tissue, ovine tissue, simian tissue, and human tissue, as well as including different tissue types, such as brain, pituitary, intestinal, lung, heart, kidney, and/or spleen tissue. In one aspect, the methods of the invention are used to treat nervous system tissue, which may be central nervous system tissue and/or peripheral nervous system tissue.

In accordance with the present invention, the method for removing infectious prion protein from tissue or sterilized/disinfected articles, such as surgical instruments and the like, with a proteolytic enzyme after the proteolytic enzymatic treatment is complete may further comprise one or more steps of examining the tissue or article to confirm destruction of the infectious prion protein therein. The detection of infectious prion protein in the tissue or article can be carried out in any suitable manner and using any suitable detection technique or method, such as subjecting the tissue or article to a western blot assay, a sandwich immunoassay assay, an ELISA, a fluorescence immunoassay assay, a capillary immunoelectrophoresis assay, a plasminogen threshold assay, or other suitable assay, each of which is effective to determine the presence or absence of infectious prion protein in the treated tissue.

The enzymatic treatment process of the invention may be carried out in any suitable manner, using any suitable sequence of treatment steps.

For example, in one embodiment, the tissue or article to be treated is subjected to an initial non-enzymatic heat treatment followed by an enzymatic treatment to destroy infectious or contaminating prion proteins, followed by rinsing and non-enzymatic treatment to examine the treated tissue or article, as needed or desired; if desired, if the post-treatment test shows that the infectious prion protein has not been completely removed from the tissue or article, a further heat/enzyme treatment (e.g., a non-enzymatic heat treatment and an enzymatic high temperature treatment in an alternating and repeating cycle) may be performed.

In another embodiment, the method comprises the steps of: the tissue or article is heated at a sufficiently high temperature and for a sufficiently long time while being exposed to a heat-stable proteolytic enzyme to at least partially degrade infectious prion protein in the tissue or article comprising or contaminated with infectious prion protein. The treated tissue or article is then examined to determine whether infectious prion protein has been removed.

Additionally, prior to any heat/enzyme treatment, the methods of the present invention may include first determining the presence or absence of infectious prion protein in the tissue or article to be treated, such that the treatment is directed only to tissue or articles that contain infectious prion protein.

Alternatively, although the presence of infectious prion protein has not been determined explicitly in advance, tissues or articles that may potentially contain or be contaminated with infectious prion protein may be subjected to a heat/enzyme treatment, followed by examination of the treated product to determine the presence of any infectious prion protein therein.

The process of the present invention is useful for destroying infectious prion protein in meat and meat by-products that are susceptible to contamination with or contain infectious prion protein that mediates TSEs, such as BSE.

Thus, the present invention provides a reliable method of treating cattle and other livestock products and by-products, rather than incinerating them to avoid the spread of BSE and other infectious prion protein disease, by subjecting the products to further treatment in food processing and/or processing operations.

Thus, in one embodiment, the invention provides a method of treatment of livestock products and animal by-products, wherein prion protein is subjected to non-enzymatic heat treatment, followed by enzymatic degradation of infectious prion protein, for example by heating to a temperature below the pyrolytic destruction temperature (< 200 ℃), at which infectious prion protein is typically incinerated.

In an illustrative embodiment of the invention, bovine tissue is cooked at a temperature in the range of about 100 ℃ to about 150 ℃, for example about 5 minutes to about 5 hours, and then exposed to a proteolytic enzyme in the temperature range of about 35 ℃ to about 100 ℃, wherein the proteolytic enzyme is thermally stable and effective to hydrolyze proteins to destroy infectious prion protein in the bovine tissue, thereby removing infectious prion protein from bovine tissue comprising infectious prion protein.

The cooking process may be carried out in a suitable tank or vessel in which elevated temperature conditions are suitably maintained and pressure is selectively controlled during the cooking operation to provide a desired atmospheric, subatmospheric, or superatmospheric pressure.

Following cooking, bovine tissue is treated with proteolytic enzyme using Bacillus licheniformis PWD-1 keratinase to destroy all infectious prion protein therein. The tissue may be treated in any suitable manner, according to the heat/enzyme treatment method.

For example, after testing or assaying to confirm complete destruction of infectious prion protein, for example, bovine tissue can be processed to obtain animal feed ingredients (e.g., meat and bone meal) or feed additives.

In a particularly preferred embodiment, the bovine tissue is first cooked at an elevated temperature in the range of about 125 ℃ to about 150 ℃ and then enzymatically treated with Bacillus licheniformis PWD-1 keratinase at an elevated temperature in the range of about 40 ℃ to about 60 ℃.

The use of the process of the present invention enables the processed beef by-products which would otherwise (suspected or confirmed to be infectious prion protein) need to be incinerated and landfilled to be utilized.

The method of the present invention therefore makes a significant advance in the art, allowing materials to be used as nutrients, which would otherwise constitute a biohazard if not disposed of. The process of the present invention avoids both the cost and infrastructure required to incinerate and land-fill infected or contaminated animal tissue.

The invention is embodied in a simple method for removing infectious prion protein, e.g., BSE-mediated prion protein, from a tissue by exposing the tissue to a thermostable proteolytic enzyme at a sufficiently high temperature and for a sufficiently long time to at least partially remove the infectious prion protein from the tissue.

The methods of the invention are broadly applicable to the destruction of prion proteins that can cause Transmissible Spongiform Encephalopathies (TSEs) and/or other prion protein-mediated diseases, including, but not limited to, Bovine Spongiform Encephalopathy (BSE) and scrapie in sheep.

The process of the invention is suitable for the treatment of animal meat in human food and animal by-products of animal feed or animal feed ingredients processing.

In a composition aspect, the invention includes a tissue composition comprising (i) a tissue, such as bovine tissue, comprising an infectious prion protein, such as a prion protein that mediates BSE, and (ii) a proteolytic enzyme, such as bacillus licheniformis PWD-1 keratinase, that is heat stable over a temperature range used for enzymatic treatment, such as from about 40 ℃ to about 60 ℃.

Such tissue compositions may be present at elevated temperatures. The composition is enzymatically active at a suitable elevated temperature to yield a product composition comprising a proteolytic enzyme and a treated tissue free of infectious prion protein.

Although in the foregoing the invention has illustratively been described in terms of the method of the invention being applied primarily to the treatment of infected or contaminated harvested animal tissue, such as parts of processed animals used in the production of animal feed ingredients, the invention also includes the use of proteolytic enzymes to treat prion diseases in vivo.

In one such embodiment, the present invention provides a therapeutic composition comprising a proteolytic enzyme as an active ingredient for treating prion disease in vivo, the proteolytic enzyme being effective against prion disease.

Another embodiment includes a therapeutic composition comprising a combination of keratinase and a non-infectious prion protein (e.g., PrPc, or a modified PrPSc such that it has the same non-infectious, or non-infectious PrPSc fragment) as a molecular recognition protein for an infectious prion protein in vivo.

It will be appreciated that other therapeutic compositions include keratinase or other vectorized configuration proteolytic enzymes or combinations.

Another illustrative therapeutic composition comprises a combination of a pyrogenic agent, such as a non-toxic modified endotoxin analog, and a proteolytic enzyme, which is effective against prion diseases that cause fever in vivo.

The invention also includes in vivo therapeutic compositions comprising a polynucleotide sequence as part of a recombinant polynucleotide expression vector, the polynucleotide sequence comprising a first region encoding for the production of a proteolytic enzyme or active fragment thereof and a second region encoding for the production of a pyrogenic peptide. Following metastatic infection, in vivo expression of thermostable proteolytic enzymes or active fragments thereof and pyrogenic peptides has an effect against infectious prion proteins.

As a further example, the therapeutic composition may be formulated with a blood-brain barrier crossing agent, such as an amphiphilic drug oligoconjugate capable of crossing the blood-brain barrier, for the treatment of infectious spongiform encephalopathy. Such compositions may include a therapeutic compound, such as a keratinase, other proteolytic enzyme, or active fragment thereof, conjugated to an oligomer, wherein the oligomer includes a lipophilic moiety coupled to a hydrophilic moiety. The oligomers used to formulate such therapeutic compositions are described in more detail in International publication WO00/09073 (published 24/2/2000).

Another important application of the method of the invention is the disinfection and/or sterilization of items such as surgical instruments, knives, kitchen utensils, laboratory instruments and veterinary devices. This method is widely applicable to the destruction of prion-contaminated articles:

(a) surgical instruments such as forceps, tweezers, scissors, knives, cables, drills, forceps, cannulas, calipers, sculptors, curettes, dilators, clip applicators, retractors, scoops, needle holders, pipettes, coagulation electrodes, electroencephalograph depth electrodes, rib and sternal dilators, bipolar probes, and costal scissors, and the like;

(b) knives and kitchen utensils, such as knives, forks, scissors, peelers, paring knives, slicers, spatulas, and meat cutters;

(c) laboratory instruments, such as filtration devices, centrifuges, spectrophotometers, fluorometers, and various containers; and

(d) veterinary devices such as forceps, tweezers, knives, saws, probes, and electronic stunning equipment.

The above list is merely illustrative of several applications of the invention and should not be considered as limiting the scope of the invention in any way.

The following table represents disinfection/sterilization cycles according to embodiments of the present invention:

TABLE 1

Step (ii) of	Temperature of	Time of day
			Prewashing (Cold water)	At room temperature	2-5 minutes
Heating of	35-100℃	20-40 minutes
			Cooling down	34-51℃	2-10 minutes
Enzymatic washing	34-51℃	20-120 minutes
			Ultrasonic treatment	34-51℃	5 minutes
Detergent washing	51-57℃	2-5 minutes
			Cleaning and drying	At room temperature	5 minutes
High pressure steam treatment	200-500℃	---

In another embodiment, a heat stable proteolytic enzyme is used in this sterilization/disinfection process so that the heating and enzymatic washing steps can be performed simultaneously, with the sterilization/disinfection treatment being performed at a sufficiently high temperature and for a sufficiently long time to completely destroy infectious prion protein and sterilize the treated items.

In a composition aspect, the invention includes a cleaning composition comprising (i) a proteolytic enzyme that is heat stable within a temperature range for use in enzyme treatment, e.g., a temperature range of from about 40 ℃ to about 60 ℃, e.g., bacillus licheniformis PWD-1 keratinase; and (ii) a solvent.

Any solvent suitable for use in enzymatic detergents may be used in the compositions of the present invention. Distilled water is a preferred solvent in view of biocompatibility and low cost. Other conventional inorganic and organic solvents such as alcohols, buffer solutions, detergent solutions may also be used in the present invention, and those skilled in the art can readily select and use solvents that are compatible with the particular enzyme. Conventional chemical additives may also be added to the cleaning compositions of the present invention, including, but not limited to, surfactants, builders, co-accelerators, fillers and other adjuvants.

The cleaning compositions of the present invention maintain their effectiveness at destroying infectious prion protein even at very low concentrations, e.g., less than 0.3 g/L. When a keratinase enzyme is used in the cleaning composition, the enzyme concentration of such compositions is preferably in the range of about 0.2g/L to about 1.0 g/L.

The cleaning composition of the present invention is enzymatically active at high temperatures, so that it can be used at high temperatures to completely destroy infectious prion protein on surgical instruments, knives, kitchen utensils, veterinary equipment, and laboratory instruments.

The features and advantages of the present invention will become more fully apparent by reference to the following illustrative examples.

Example 1

In this example, the feather degrading bacterium, Bacillus licheniformis strain PWD-1, isolated from a thermophilic anaerobic digester of poultry manure, was used as the source of keratinase. (see M.Williams and J.C.H.Shih, J.appl.Bacteriol., 67, 25 (1989); J.C.H.Shih, Poultry Sci.72, 1617(1993)) (see X.Lin, C.G.Lee, E.S.Casale, and J.C.H.Shih, appl.environ.Microbiol.58, 3271 (1992)).

The gene encoding this keratinase was isolated and sequenced (see x.lin, d.w.kelemen, e.s.miller and j.c.h.shih, appl.env.microbiol., 61, 1469(1995)) and fermentative production of this enzyme on a large scale (see j.j.wang and j.c.h.shih, j.ind.micro.biotech., 22, 608 (1999)). This enzyme is a serine protease.

Crude and purified keratinases were prepared as previously described (see X.Lin, C.G.Lee, E.S.Casale, and J.C.H.Shih, appl.environ.Microbiol.58, 3271(1992) and J.J.Wang and J.C.H.Shih, J Ind.Microb.Biotech.22, 608(1999)) and obtained from the Fermentation Facility at the Carolina State University, Raleigh, North Carolina (NCSU). The test of the action of keratinase on PrP was carried out in Lelystad (ID-Lelystad), the society of zootechnics and health in the Netherlands.

Purified keratinase was compared to other proteases including elastase, collagenase, proteinase K and trypsin (all from Sigma chemical company) in reactions with various substrates. Hydrolysis of keratin, elastin, and collagen with ninhydrin color reaction with free amino groups added (A)₄₅₀) Assays were performed (see x.lin, c.g.lee, e.s.casale, and j.c.h.shih, appl.environ.microbiol.58, 3271 (1992)). The free amino equivalent is calculated based on free leucine. A in supernatant for casein hydrolysis₂₈₀Increase in (see Price and Johnson, 1989). The results are shown in table 1 below. For each given substrate, the relative activity of all proteases was determined. Cumulative Relative Activity (CRA) indicates: keratinases have a wide range of substrates and high activity.

TABLE 1 relative specific Activity of proteases on different substrates^a

Substrate	Keratinase	Elastase	Collagenase	Proteinase K	Trypsin
						Keratin proteinb	1.00	0.29	0.00	0.36	0.09
Elastinb	2.52	1.00	0.43	0.57	0.61
						Collagen proteinb	2.58	1.15	1.00	0.70	0.38
Casein proteinc	1.28	0.80	0.02	1.00	0.40
						CRAd	7.38	3.24	1.45	2.63	1.48

^aAll enzyme activities were determined and compared under their optimal conditions, respectively.

^bProteolysis was determined by the ninhydrin reaction (Lin et al, 1992).

^cIncreased soluble A for proteolysis₂₈₀Assay (Price and Johnson 1989).

^dRelative activity was accumulated.

The test for the effect of keratinase on pathogenic PrP was performed in a separate device in the molecular recognition laboratory of ID-Lelystad. Pathogenic PrP was determined using the European Union-differentiation Procedure of Prionics Check (prion AG, Zurich). The prion testing procedure is based on Western blotting techniques and uses the 6H4 monoclonal antibody to visualize the specific form of PrP [ Prionin AG, Test for BSE-prion in Cattle, (Test for Detection of B SE-Prionin Cattle) Practical ProductInformation, zurich (2000) ].

To mimic the meat and bone meal processing, the original procedure was modified. First, proteinase K in the standard method was replaced by keratinase. Crude keratinase was used. Second, the effect of the pre-cooked BSE tissue was examined. The homogenized tissue was cooked for 40 minutes at 115 ℃ with a Vulcain pressure cooker. Third, antioxidant, sodium sulfite (Na) was tested₂SO₃) The function of (1). The procedure was the same as the Practical Product Information [ Prionin AG, examination of BSE-prions in Cattle, (Test for Detection of BSE-Prionin Cattle) Practical Product Information, Zurich (2000)]。

The following protocol was used: 1g of BSE positive brain tissue was mixed with 9ml of prion buffer and homogenized. Dividing into two parts, each 5ml, adding Na into one part₂SO₃Final concentration of 0.1%, the other part was not added with Na₂SO₃. Divided into 4X 2.0ml portions, which were each charged to a high pressure Forken (Fa)lcon) tube. An additional 1.0ml was used as a positive control, treated with standard prion methods, and an additional 1.0ml was used for a control without keratinase. Will have and not have Na₂SO₃Two tubes of (a) were retorted under pressure for 40 minutes, and the other two tubes were not retorted. Samples, 150. mu.l each, were treated with keratinase (150. mu.g, 1,000EU/mg) in wells of a PCR plate at 50 ℃ for zero-time or 4 hours. Keratinase was pre-dissolved in phosphate buffer, 0.05M, pH 7.5. The reaction was stopped by the addition of Prionics Pefabloc, a serine protease inhibitor. At the end of the enzyme incubation, 10. mu.l of each sample mixture was loaded onto SDS-PAGE gels, followed by electrophoresis, Western blotting, and immunochemical luminescence detection of prions.

The results of this experiment are shown in FIG. 1 (degradation of BSE prion protein by keratinase) where bands 1-17 are as follows:

belt 1: only the buffer solution.

The belt 2: BSE brain tissue was used for the experiments.

The belt 3: with Na₂SO₃Precooking, keratinase stop at zero-time.

The belt 4: as with band 3, except for 4 hours of keratinase digestion.

The belt 5: in the absence of Na₂SO₃Precooking with keratinase stopping at zero-time.

The belt 6: as with band 5, except that the keratinase digestion was 4 hours.

The belt 7: without pre-cooking, with Na₂SO₃Keratinase stops at zero-time.

The belt 8: as with band 7, except that the keratinase digestion was 4 hours.

The belt 9: no pre-cooking and no Na₂SO₃Keratinase stops at zero-time.

The belt 10: as with band 9, except that the keratinase digestion was 4 hours.

The belt 11: without pre-cooking, with Na₂SO₃Without keratinase.

The belt 12: as with band 11, except for 4 hours of incubation (note: keratinase was added ad libitum).

The belt 13: purified scrapie PrP, Na₂SO₃Keratinase stops at zero-time.

The belt 14: as with band 13, except that the keratinase digestion was 4 hours.

The belt 15: purified scrapie PrP, Na₂SO₃And (3) a keratinase.

The belt 16: as with belt 15, except for 4 hours of incubation.

The belt 17: a PrP standard.

As shown in FIG. 1, the digestion of infectious PrP by keratinase is evident, especially when the sample is precooked at 115 ℃ for 40 minutes (lanes 3-6). Without prior cooking (band 7-10), the potency of the keratinase is lower, but the keratinase still degrades more than half of the infectious PrP-positive material. Keratinase was also found to be active on purified scrapie PrP (lanes 13-16). Na (Na)₂SO₃The presence alone or together with keratinase does not seem to differ much (bands 15-16). The 12-band sample was arbitrarily keratinase-added and thus showed positive.

Example 2

The efficacy of the present invention is further demonstrated by tests conducted according to the following experimental procedures.

PWD-1 keratinase, a serine protease derived from Bacillus licheniformis strain PWD-1, used to degrade PrP in infected bovine and ovine brainstem tissue^Sc. Prion assay (prion AG, Zurich, Switzerland) for the determination of PrP in a sample^ScPrion testing was performed as follows: precookingTissue samples, which were digested with purified PWD-1 keratinase as a digestive enzyme.

In each case, 1g of BSE positive brainstem tissue was mixed and homogenized with 9ml of prion homogenization buffer, followed by cooking in a Vulcan pressure cooker at 115 ℃ for 40 minutes. In a 96-well plate, 150. mu.l of the sample was added to each well and treated with PWD-1 keratinase (0.05M, pH 7.5) pre-dissolved in phosphate buffer. The enzyme concentration used was 250. mu.g/ml. Enzymatic digestion was carried out at 50 ℃ for 60 minutes. The reaction was stopped by adding 15. mu.l of Pefabloc , a serine protease inhibitor. Time-zero samples were processed as follows: inhibitors are added first prior to the addition of PWD-1 keratinase. At the end of the enzyme incubation, 10. mu.l of each sample was loaded onto SDS-PAGE gels. Detection is carried out in a prion detection kit by electrophoresis, a western blotting method, an immunochemiluminescence method and the like.

Brain stem tissue samples, positive for 3 BSE (sample B1, B2, B3), negative for 1 (bovine sample N), positive for 2 scrapie (samples S1 and S2) and negative for 1 (ovine sample N) were tested with pure PWD-1 keratinase and corresponding controls.

The results are shown in FIG. 2, with the left panel ("keratinase treatment") being the result of keratinase digestion and the right panel ("control") being the result of a standard prion test. Keratinase is able to hydrolyze all PrP tested^ScBSE (lanes 3-5), and scrapie (lanes 8 and 9).

FIG. 2 shows that infectious prion protein was completely destroyed in bovine tissue samples containing infectious prion protein (samples B1, B2, B3) and ovine samples containing infectious prion protein (samples S1 and S2) when these samples were treated with heat and enzyme binding according to the invention.

The results demonstrate the versatility of keratinases in degrading various types of test proteins. The results were positive in examining the potency of keratinase on BSE PrP, especially when BSE brain tissue samples were pre-cooked. This is the first experimental demonstration that pathogenic PrP can be degraded by enzymes.

Pressure cooking at about 125 is a conventional step in converting livestock by-products to meat and bone meal. Post-digestion treatment with keratinase according to the present invention for destruction of infectious PrP provides an efficient method to control the spread of BSE. The keratinase treated meat and bone meal is easily tested for the absence of PrP, so the meat and bone meal can be recycled for feed.

Thus, the process of the present invention provides a simple and useful method for enzymatic treatment of livestock products and by-products.

While the present invention has been described with respect to various illustrative features, aspects and embodiments, it will be appreciated that the present invention is not thus limited, as those of ordinary skill in the art will readily appreciate that the invention extends to other variations, modifications, and other embodiments, as may be included within the scope of the present invention.

Accordingly, the invention is to be broadly construed and understood to include other variations, modifications, and other embodiments, within the spirit and scope of the invention.

Industrial applicability of the invention

The methods and compositions of the present invention are useful for destroying infectious prion protein and are suitable for treating biological materials, such as animal tissue containing or contaminated with infectious prion protein. The present invention enables the treatment of biological material (suspected or confirmed presence of infectious prion protein) that requires incineration and destruction to make it a useful and safe animal feed or other nutritional end product. The methods and compositions of the present invention are also useful for disinfecting and/or sterilizing articles, such as surgical instruments, knives, kitchen utensils, laboratory instruments, and veterinary equipment, and effectively prevent cross-contamination and proliferation of infectious prion protein due to repeated use of such articles.

Claims (61)

1. A method of treatment for reducing infective prion protein at a site contaminated or suspected of being contaminated with infective prion protein, the method comprising the steps of:

(a) heating the locus to a temperature and for a time sufficient to increase the proteolytic susceptibility of infectious prion protein at the locus;

(b) exposing the heated site to a proteolytic enzyme such that the site is at least partially effective in reducing the infectious prion.

2. The process of claim 1 wherein the temperature of step (a) does not exceed about 150 ℃.

3. The process of claim 1, wherein the temperature of step (a) is between about 100 ℃ to about 150 ℃.

4. The process of claim 1, wherein the temperature of step (a) is between about 125 ℃ and about 140 ℃.

5. The method of claim 1, wherein step (b) is performed at a temperature between about 35 ℃ and about 100 ℃.

6. The method of claim 1, wherein step (b) is performed at a temperature greater than about 40 ℃.

7. The method of claim 1, wherein step (b) is performed at a temperature greater than about 50 ℃.

8. The process of claim 1, wherein step (b) is carried out at a lower temperature than step (a).

9. The method of claim 1, wherein step (b) is performed at a temperature between about 40 ℃ and about 75 ℃.

10. The method of claim 1, wherein step (b) is performed at a temperature between about 50 ℃ and about 60 ℃.

11. The method of claim 1, wherein the proteolytic enzyme comprises an enzyme selected from the group consisting of: keratinase, proteinase K, trypsin, chymotrypsin, pepsin, chymosin, cathepsin, subtilisin, elastase, collagenase, endopeptidase, peptidase, oligopeptidase, thermolysin, bacillus lysin, myclysins, carboxypeptidase, leuminopeptidase, aminopeptidase, hyperthermophilic protease, carbonyl hydrolase, papain, pancreatin, streptokinase, ficin, carboxypeptidase, chymopapain, and bromelain.

12. The method of claim 1, wherein the proteolytic enzyme comprises a keratinase and/or an active fragment thereof.

13. The method of claim 1, wherein the proteolytic enzyme comprises a bacillus licheniformis PWD-1 enzyme and/or an active fragment thereof.

14. The method of claim 1, further comprising the step of (c) testing said site to confirm that infectious prion protein has been reduced.

15. The method of claim 14, wherein the testing step (c) comprises testing the locus by western blot assay, sandwich immunoassay assay, ELISA, fluorescence immunoassay assay, capillary immunoelectrophoresis assay, and plasminogen limit assay.

16. The method of claim 14, wherein the testing step (c) comprises testing the locus by western blot assay.

17. The method of claim 1, wherein said site comprises tissue containing or contaminated with infectious prion protein.

18. The method of claim 17, wherein said tissue comprises mammalian tissue.

19. The method of claim 17, wherein said tissue comprises nervous system tissue.

20. The method of claim 17, wherein said tissue comprises bovine tissue.

21. The method of claim 17, wherein the tissue comprises BSE-infected tissue.

22. The method of claim 17, wherein said tissue comprises ovine tissue.

23. The method of claim 17, wherein said tissue comprises scrapie-infected tissue.

24. The method of claim 17, wherein said tissue is selected from the group consisting of brain, pituitary, intestine, lung, heart, kidney, and spleen tissue.

25. The method of claim 17, wherein said tissue is from an animal carrying infectious prion protein.

26. The method of claim 1, wherein said locus comprises an article susceptible to contamination by infectious prion protein.

27. The method of claim 26, wherein the article comprises a surgical instrument.

28. The method of claim 27, wherein the surgical instrument is selected from the group consisting of forceps, tweezers, scissors, knives, cables, reamers, forceps, cannulas, calipers, sculptors, curettes, scrapers, dilators, clip applicators, constriction devices, constrictors, scoops, needle holders, pipettes, coagulation electrodes, depth electrodes for electroencephalographs, rib and sternum dilators, bipolar probes, and rib scissors.

29. The method of claim 26, wherein said articles comprise knives and kitchen utensils.

30. The method of claim 29, wherein said knives and kitchen utensils are selected from the group consisting of knives, forks, scissors, peelers, paring knives, slicers, spatulas, and meat cutters.

31. The method of claim 26, wherein said article comprises a laboratory instrument.

32. The method of claim 31, wherein said laboratory instrument is selected from the group consisting of a container, a filtration device, a centrifuge, a spectrophotometer, and a fluorometer.

33. The method of claim 26, wherein said item comprises a veterinary device.

34. The method of claim 33, wherein said veterinary device is selected from the group consisting of forceps, tweezers, knives, saws, probes, and electronic stunning equipment.

35. The method of claim 1, wherein the proteolytic enzyme comprises a protease.

36. The method of claim 35, wherein the protease comprises a carbonyl hydrolase.

37. The method of claim 36, wherein the carbonyl hydrolase comprises subtilisin.

38. The method of claim 37, wherein the subtilisin comprises a variant of wild-type Bacillus amyloliquefaciens subtilisin comprising one or more amino acid substitutions, additions, or deletions.

39. A method for increasing the degradability of infectious prion protein by proteolytic enzymatic degradation treatment, said method comprising (a) heating the prion protein to a temperature below the pyrolytic destruction temperature of the prion protein, followed by (b) performing an enzymatic degradation treatment of the prion protein.

40. A method for removing infectious prion protein from bovine tissue comprising or contaminated with infectious prion protein, said method comprising (a) cooking said bovine tissue at a temperature in the range of about 100 ℃ to about 150 ℃, followed by (b) exposing said bovine tissue to a heat-stable proteolytic enzyme at a temperature in the range of about 35 ℃ to about 100 ℃ effective to proteolytically at least partially destroy infectious prion protein in said bovine tissue.

41. The method of claim 40, wherein said cooking is carried out for about 5 minutes to about 5 hours.

42. The method of claim 40, wherein the proteolytic enzyme comprises Bacillus licheniformis PWD-1 keratinase.

43. A method for degrading infectious prion protein, the method comprising (a) heating the infectious prion protein in a first high temperature range, followed by (b) cooling the infectious prion protein to a lower high temperature in a second high temperature range, and (c) effectively exposing the infectious prion protein to a proteolytic enzyme at the lower high temperature to degrade the infectious prion protein to benign degradation products.

44. A method for at least partially degrading infectious prion protein in a tissue or article comprising or contaminated with infectious prion protein, said method comprising the steps of: heating the tissue or article while exposing the tissue or article to a heat-stable proteolytic enzyme at a temperature and for a time sufficient to at least partially degrade infectious prion protein in the tissue or article.

45. A method of processing a meat animal product or by-product thereof to destroy infectious prion protein that mediates BSE, the method comprising: treating the meat animal product or by-products thereof with a thermostable protease at a sufficiently high temperature and for a sufficiently long time to destroy BSE-mediating infective prion protein.

46. A tissue composition comprising a tissue containing or contaminated with infectious prion protein and a proteolytic enzyme that is thermally stable at a temperature in the range of about 35 ℃ to about 100 ℃.

47. A method of treating tissue to reduce infectious prion protein, the method comprising the steps of:

(a) heating the tissue to a temperature and for a time sufficient to increase the proteolytic susceptibility of the infectious prion protein in said tissue; and

(b) exposing the heated tissue to a proteolytic enzyme such that the tissue is at least partially effective in reducing infectious prion.

48. A method of sterilizing an article susceptible to contamination by infectious prion protein, the method comprising the steps of:

(a) heating said article at a temperature and for a time sufficient to increase the proteolytic susceptibility of prion protein on said article; and

(b) exposing the heated article to a proteolytic enzyme effective to at least partially reduce infective prion protein on the article.

49. A method for removing infectious prion protein from a surgical instrument contaminated with infectious prion protein, said method comprising (a) heating said surgical instrument at a temperature in the range of about 100 ℃ to about 150 ℃, and then (b) exposing said heated surgical instrument to a heat-stable proteolytic enzyme at a temperature in the range of about 35 ℃ to about 100 ℃ to at least partially effectively destroy infectious prion protein contaminating said surgical instrument.

50. A cleaning composition for disinfecting an article susceptible to contamination by infectious prion protein, said composition comprising:

(i) one or more proteolytic proteins selected from the group consisting of keratinase, proteinase K, trypsin, chymotrypsin, pepsin, chymosin, cathepsin, subtilisin, elastase, collagenase, endopeptidase, peptidase, oligopeptidase, thermolysin, bacillus lysin, myclysins, carboxypeptidase, leucinemidopeptidase, aminopeptidase, hyperthermophilic protease, carbonyl hydrolase, papain, pancreatin, streptokinase, ficin, carboxypeptidase, chymopapain, and bromelain;

and

(ii) a solvent.

51. The cleaning composition of claim 50 comprising a keratinase.

52. The cleaning composition of claim 51, wherein the concentration of the keratinase is in the range of about 0.2g/L to about 1.0 g/L.

53. The cleaning composition of claim 50, wherein the solvent is selected from the group consisting of distilled water, alcohols, buffer solutions, and washing solutions.

54. The cleaning composition of claim 50, further comprising one or more chemical additives selected from the group consisting of surfactants, builders, boosters and fillers.

55. A method of tissue treatment to reduce infectious prion protein therein, the method comprising the steps of:

(a) heating the tissue at a temperature of at least 40 ℃ for a time sufficient to increase the proteolytic susceptibility of infectious prion protein in the tissue; and

(b) exposing the heated tissue to a proteolytic enzyme such that the tissue is at least partially effective in reducing infectious prion.

56. A method for degrading infectious prion protein, the method comprising (a) heating the infectious prion protein in a first high temperature range, said first high temperature being greater than 60 ℃ but less than the pyrolytic destruction temperature of said prion protein, followed by (b) cooling the infectious prion protein to a lower high temperature in a second high temperature range, followed by (c) effectively exposing the infectious prion protein to a proteolytic enzyme at the lower high temperature to degrade the infectious prion protein to benign degradation products.

57. A method for at least partially degrading infectious prion protein in a tissue comprising or contaminated with infectious prion protein, the method comprising the steps of: the tissue is heated above 40 ℃ but below the pyrolytic destruction temperature of prion protein and for a sufficient period of time while being exposed to a heat stable proteolytic enzyme to at least partially degrade infectious prion protein.

58. A method of processing animal meat products or by-products thereof to remove infectious prion protein from parenchymal BSE therein, the method comprising: treating the meat animal product or by-product thereof with a thermostable protease at a temperature above 60 ℃ but below the pyrolytic destruction temperature of prion protein for a time sufficient to destroy infectious prion protein therein that mediates BSE.

59. Tissue compositions comprising tissue containing or contaminated with infectious prion protein and a proteolytic enzyme selected from the group consisting of keratinase, chymotrypsin, pepsin, chymosin, cathepsin, subtilisin, elastase, collagenase, endopeptidase, peptidase, oligopeptidase, thermolysin, bacillus lysin, myclysins, carboxypeptidase, leumidopeptidase, aminopeptidase, and hyperthermophilic protease.

60. A method for processing a meat animal product or by-product thereof, said method comprising treating said meat animal product or by-product thereof with a protease effective to destroy any infectious prion protein associated therewith at a temperature greater than 40 ℃ but less than the pyrolytic destruction temperature of the prion protein for a time sufficient to destroy any infectious prion protein associated therewith.

61. A method of treatment for reducing infective prion protein at a site contaminated or suspected of being contaminated with infective prion protein, comprising the steps of:

(a) heating the locus at a temperature of at least 40 ℃ for a time sufficient to increase the proteolytic susceptibility of infectious prion protein at the locus; and

(b) exposing the heated site to a proteolytic enzyme such that the site is at least partially effective in reducing infective prion.