# Patent

## Title
Degradation of prion protein and reduced prion infectivity by earthworm homogenates

## Abstract

Methods for the degradation of prion proteins are described. Items that may be contaminated with prions are treated with earthworm-derived extracts to degrade prion proteins and reduce their infectivity in animals. Methods of using an earthworm-derived protein extract for treating foodstuffs and surfaces to inhibit or disable prion infectivity are disclosed. Also disclosed are methods for treating medical instruments, tools used to butcher animals, and laboratory equipment.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 61/099,944, filed Sep. 25, 2008, the entire contents of which are hereby incorporated by reference in its entirety.


GOVERNMENT SUPPORT
This invention was made with United States government support awarded by the following agencies: ARMY/MRMC DAMD17-03-1-0369. The United States Government has certain rights in this invention.


TECHNICAL FIELD
The present technology relates generally to the field of biological decontamination. The technology finds particular application in preventing or inhibiting the spread of prion-related diseases.
BACKGROUND
The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.
Transmissible spongiform encephalopathies (TSE) are untreatable and fatal neurodegenerative diseases that affect a number of mammalian species. Variants of TSE affect humans and many wild and domesticated animals. Examples of TSE are bovine spongiform encephalopathy (BSE) in cattle (also known as “mad cow disease”), scrapie in sheep, chronic wasting disease (CWD) in moose, deer and elk, and Creutzfeldt-Jakob disease (CJD) in humans. (Prusiner, Brain Pathol, 8:499-513 (1998).) All these diseases attack the neurological system and are characterized by initially long incubation followed by a short period of neurological symptoms, including dementia, loss of coordination, and eventually death.
The infectious agents that cause and transmit TSE are called prions. Prions are abnormally folded variants (PrPSc) of naturally occurring and normally harmless cellular proteins (PrPC). Prions are not believed to be associated with cells or virus particles, and most evidence shows that prions have no associated nucleic acids or other genetically transmissible material. In humans, the prion protein is encoded by the PRNP gene, which resides on chromosome 20. The protein is most commonly found in neurons, and in lower amounts in other cells such as leukocytes, monocytes, and platelets. (Holada et al., Lancet 356:1772 (2000).) When the normal cellular form of the prion protein is misfolded, the prion is able to infect and propagate by converting other normal molecules of the protein into the abnormally folded form. Thus, diseases caused by prions are characterized by accumulation of the misfolded form of the normal cellular prion protein. (Prusiner, PNAS, 95:13363-13383 (1998).) The misfolded form is extremely stable and accumulates as plaques in infected tissue, causing cell death and tissue damage that manifests as holes and a spongy appearance. The misfolded disease form of the prion protein is predominantly found in the brain, but has also been detected in the tonsil, spleen, and lymph nodes of infected humans.
Horizontal transmission is implicated in spreading the infectious agent causing scrapie in sheep and chronic wasting disease (CWD) in free-ranging cervids. (Hoinville, Rev Sci Tech, 15:827-852 (1996); Miller & Williams, Nature, 425:35-36 (2003).) The recent expansion of endemic regions of CWD in North America emphasizes the need to identify transmission routes. Several studies have advanced soil as a contributor to horizontal transmission, suggesting it acts as an environmental reservoir for retaining and facilitating exposure of susceptible animals to the infectious agent. (Johnson et al., PLoS Pathog, 2(4) (2006); Johnson et al., PLoS Pathog, 3(7) (2007), Seidel et al., PLoS ONE, 2(5) (2007).) TSE infectious agents can enter the soil environment from the decomposing carcasses of infected animals as well as from the excreta and saliva shed from infected animals. In addition, prion diseases can be transmitted by certain high-risk tissues, including the brain, spinal cord, cerebral spinal fluids, and the eye. After a surgical procedure on a prion-infected patient, or the butchering of a prion-infected animal, prion-containing residue may remain on the instruments and tools. During the long incubation period of prion-associated diseases, it is extremely difficult to determine if a person or animal is a prion carrier.
TSE infectious agents can survive in the environment for years (Seidel et al., PLoS ONE, 2(5) (2007)), subsequently serving as a long-term source of infection. Deer can become infected with CWD when exposed to facilities that previously contained decomposed infected carcasses and/or soil contaminated with excreta from infected animals. (Miller et al., Emerg Infect Dis, 10:1003-1006 (2004).) In addition, clay-bound prions are significantly more infectious than unbound PrPSc. (Johnson et al., PLoS Pathog, 2(4) (2006); Johnson et al., PLoS Pathog 3(7) (2007).)
SUMMARY
The present technology generally relates to biological decontamination, and to aspects of contacting surfaces with a composition to minimize or eliminate contamination with infectious prions. In one aspect, this disclosure provides a method for degrading prions, the method comprising contacting a surface that carries or is suspected to carry prion-infected material with an effective amount of composition comprising an earthworm extract or an active fraction thereof. In one embodiment, the degradation of prions results in a reduction of prion infectivity.
In one embodiment, the composition comprises about 10-30% by weight earthworm extract. In one embodiment, the earthworm extract is an aqueous extract. In one embodiment, the earthworm extract is an earthworm-extracted supernatant. In one embodiment, the earthworm-extracted supernatant is prepared by wet-grinding earthworms and filtering the supernatant. In one embodiment, the earthworm extract is lumbrokinase enriched from a crude earthworm extract. In one embodiment, the earthworm extract has been lyophilized. In one embodiment, the lyophilized earthworm extract is prepared by wet-grinding earthworms, filtering the supernatant, followed by freeze-drying. In one embodiment, the earthworm extract is derived from Lumbricus rubellus, Eisenia fetida, or Lumbricus bimastus. 
In one embodiment, wherein the earthworm extract comprises one or more proteases having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to an earthworm protease selected from the group consisting of SEQ ID NOs: 1-29. In one embodiment, the earthworm extract comprises one or more proteases selected from the group consisting of SEQ ID NOs: 1-29.
In one embodiment, the active fraction of an earthworm extract is a protease-containing fraction from size exclusion chromatography of an earthworm extract. In another embodiment, the active fraction is a protease-containing fraction from ion exchange chromatography of an earthworm extract. In one embodiment, the active fraction is capable of reducing infectivity of prions by at least 75% compared to a control, as measured by an infectivity assay of a living mammalian subject. In one embodiment, the subject is a hamster or a mouse.
In one embodiment, the pH of the composition is from about 6 to 8. In one embodiment, the contacting step is performed at a temperature of from about 20 to about 50° C.
In one embodiment, the surface is the surface of a medical device. In one embodiment, the surface is the surface of butchering equipment. In one embodiment, the surface is the surface of laboratory equipment used in prion research.
In one embodiment, the step of contacting comprises soaking the surface in the composition. In one embodiment, the soaking has a duration of at least 30 minutes. In one embodiment, the step of contacting comprises spraying or wiping the surface with the composition. In one embodiment, following the treating of the surface, the prion-infected material is unable to infect a live mammal.
In another aspect, this disclosure provides a method for inhibiting infection by an infectious prion, the method comprising adding a composition that degrades prions to an animal foodstuff containing or suspected of containing a prion-infected material, wherein the composition comprises an effective amount of an earthworm extract or an active fraction thereof, and wherein prions in the foodstuff, if present, are degraded.
In another aspect, this disclosure provides a foodstuff that has been treated with a composition that degrades prions, wherein the composition comprises an effective amount of an earthworm extract or an active fraction thereof, and wherein prions in the foodstuff, if present, are degraded.
In another aspect, this disclosure provides a method for identifying an active fraction of an earthworm extract that reduces prion infectivity, comprising the steps of: (a) contacting one or more earthworm extracts or fractions thereof with a prion-containing material; (b) following step (a), contacting the prion-containing material with an animal host; and (c) measuring the level of prion infectivity in the animal host, wherein a reduced level of prion infectivity in the animal host compared to a control sample indicates that the one or more earthworm extracts or fractions thereof are effective in reducing prion infectivity. In one embodiment, the method further comprises the step of comparing levels of prion infectivity from different fractions of earthworm extracts. In one embodiment, the method further comprises the step of comparing levels of prion infectivity from different earthworm extracts.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows western blots demonstrating the degradation of prion proteins by earthworms. FIG. 1A shows whole worm protein extracts assayed for hamster prion protein from earthworms exposed to infectious brain material in soil. FIG. 1B shows protein extracts from prion-exposed earthworms bisected into anterior and posterior sections and total proteins extracted (a=anterior; p=posterior).
FIG. 2 shows western blots demonstrating in vitro degradation of prion proteins isolated from CWD infected deer by earthworm protein extracts and lumbrokinase. FIG. 2A shows digestion of PrPCWD with dilutions of clarified earthworm protein extracts. FIG. 2B shows digestion of PTA-enriched PrPCWD with dilutions of lumbrokinase.
FIG. 3 is a western blot demonstrating in vitro degradation of prion proteins isolated from a hamster model by earthworm protein extracts and lumbrokinase. FIG. 3A shows 1, 2, 4 and 8 day incubations of PrPHY and earthworm protein. FIG. 3B shows dilutions of PrPHY treated for 8 days with earthworm protein extracts.
FIG. 4 is a bar graph showing the reduced in vivo infectivity of prion protein that was previously treated in vitro by lumbrokinase or earthworm protein homogenate.



DETAILED DESCRIPTION
The technology relates to compositions of earthworm-derived extracts or homogenates that degrade prion proteins, methods of using the compositions to decontaminate surfaces, instruments, tools, and foodstuffs, and a foodstuff prepared by applying the methods and compositions. It will be appreciated that the compositions and methods disclosed are amenable for treating of a wide range of items that may be contaminated with prion infected material.
In practicing the present technology, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, and microbiology are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press (1986)); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. Enzymol. (Academic Press, Inc. (1984)); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY (1987)); and Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu, Eds., respectively. Units, prefixes, and symbols may be denoted in their accepted SI form.
In the description that follows, a number of terms are utilized extensively. Definitions are herein provided to facilitate understanding of the invention. The terms described below are more fully defined by reference to the specification as a whole.
As used herein, the terms “a” and “an” mean “one or more” unless the singular is expressly specified.
As used herein, when referring to a numerical value or other quantity, the term “about” means within plus or minus 10% of the enumerated value unless stated otherwise.
As used herein, the term “activity” describes quantitatively the conversion of a given substrate under defined reaction conditions.
As used herein, “to degrade” or “to digest” refers to the process of breaking down a protein or other macromolecule into simpler components, which causes a decrease in that macromolecule's abundance or activity.
As used herein, “effective amount” of a composition refers to a quantify sufficient to achieve a desired standard of prion removal, degradation, or destruction, or a desired standard of reduced infectivity.
As used herein, “horizontal transmission” refers to the transmission of an infectious agent by a means other than by genetic inheritance from parent to child.
As used herein, “infectivity” refers to the ability of a prion-contaminated substance, item, or surface to cause a prion-associated disease in an individual.
As used herein, the terms “preventing” or “prevention” refer to a reduction in the risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to the disease but does not yet experience or display symptoms of the disease). In the context of the present technology, prevention includes interfering with the mechanism of transmitting prion-based diseases.
As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms polypeptide, peptide, and protein are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, carboxylation, hydroxylation, ADP-ribosylation, and addition of other complex polysaccharides.
As used herein, the term “protease” means any protein molecule catalyzing the hydrolysis of peptide bonds. It includes naturally-occurring proteolytic enzymes, as well as protease variants. It also comprises any fragment of a proteolytic enzyme, or any molecular complex or fusion protein.
As used herein, the term “PrPC” refers to the native prion protein molecule, which is naturally and widely expressed within the bodies of mammals. Its structure is highly conserved and is not believed to be associated with a disease state.
As used herein, the term “PrPCWD” refers to a prion protein isolated from an animal diagnosed as having chronic wasting disease.
As used herein, the term “PrPHY” refers to a prion protein purified from a prion-infected hamster.
As used herein, the term “PrPSc” refers to the conformationally altered form of the PrPC molecule that is believed to be associated with TSE and prion diseases. PrPSc has the same amino acid sequence as normal, cellular PrPC, but has an altered folded conformation in which some of the α-helix is converted to β-pleated sheet.
As used herein, the term “PrP” refers to a prion protein in general.
As used herein, the term “PrPTSE” refers to a PrP that causes a transmissible spongiform encephalopathy.
As used herein, the term “sequence identity” means that two polypeptide sequences are identical (i.e., on a amino acid-by-amino acid basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
As used herein, “transmissible spongiform encephalopathy” (TSE) refers to a group of inevitably fatal progressive disease conditions that affect the brain and nervous system of animals, causing the deterioration of mental and physical abilities and causing tiny holes to appear in the cortex causing it to appear like a sponge. TSE diseases include, but are not limited to, classic Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, kuru, fatal familial insomnia, GSS, scrapie, BSE, CWD, and other TSEs, including rare TSEs of captive and experimental animals. These conditions form a spectrum of diseases with overlapping signs and symptoms.
Overview
Described herein are compositions and methods for degrading prions to minimize or eliminate their infectivity. The extreme stability of the protein causes prions to be resistant both to degradation by the infected host and to denaturation by many physical and chemical means outside the host. In addition, prions are resistant to routine methods of decontamination and sterilization. Infectious particles possessing nucleic acid (DNA or RNA) are dependent upon it to direct their continued replication. Prions, however, are infectious by their effect on normal versions of the protein. Therefore, sterilizing prions involves the denaturation or degradation of the protein to a state where the molecule is no longer able to induce the abnormal folding of normal proteins. Glutaraldehyde, formaldehyde, ethylene oxide, liquid hydrogen peroxide, most phenolics, alcohols, dry heat, boiling, freezing, ultraviolet radiation, ionizing radiation, and microwave radiation have generally been reported to be ineffective. More extreme conditions are required to inactivate prions—for example, a one-hour exposure to a NaOCl solution containing 20,000 ppm of Cl2 or autoclaving in 1 N NaOH are methods that are recommended for inactivating prions.
Common earthworms have the capacity to ingest soil particles and, in the process of digestion, degrade prion proteins and decrease their associated infectivity. The earthworm species Lumbricus rubellus and Eisenia fotida originated as exotic species introduced from Europe, but now have successfully competed with native species and range from the northeast and south-central United States to the Western Great Lakes and Pacific coast. (Reynolds et al., Earthworm Ecology and Biogeography in North America, Hendrix, ed. CRC Press, Inc., Florida, USA (1995) 1-28; Hendrix & Bohlen, Bioscience, 52:801-811 (2002).) Extracts and active fractions from these and other earthworm species are useful in the methods described below.
Preparation of Compositions
In one aspect, the compositions disclosed herein contain an earthworm homogenate, an earthworm extract, an active fraction of an earthworm extract, and/or one or more purified proteases found in earthworm extracts. The extracts, fractions, and purified proteases can be prepared by a variety of methods. For example, an earthworm homogenate can be produced by mechanical means such as grinding, wet-grinding, pulverizing, blending, smashing, or mixing whole earthworms into an homogenous mixture. Chemical processes might also be suitable. One method of producing an earthworm homogenate is to quick freeze worms in liquid nitrogen, pulverize the frozen worms with a mortar and pestle, and then resuspend the resulting powdered earthworm tissue in a suitable buffer, for example, phosphate-buffered saline. In one embodiment, a useful resuspension contains from about 1-75%, from about 5-60%, from about 5-50%, or from about 10-30% by weight of earthworm tissue. The resuspension may be filtered (e.g., using diatomaceous silica), centrifuged, or treated in any other suitable manner known to those of the art to remove insoluble or other particulate matter that may be present in the preparation. The preparation may be stored as a liquid resuspension or as a frozen solid.
Those of skill in the art are able to select and prepare a buffer suitable for resuspending the homogenate. Some components of buffers are salts, chelating agents to inhibit enzymatic degradation of active components, and preservatives to inhibit contamination by microbial growth. Another important parameter of a buffer is its pH. One buffer that is useful for preparing an earthworm homogenate contains about 130 mM NaCl, about 3 mM KCl, about 10 mM NaHPO4, and about 1.8 mM KH2PO4. Many biologically-derived products typically function at a pH of about 7. Thus, a suitable pH for such a buffer is from about 6 to about 8.
In one embodiment, the earthworm extract or homogenate may be lyophilized and stored as a powder. Lyophilization is a dehydration process typically used to preserve a perishable material or to make a material more convenient to transport or store. The process involves freezing the material under low pressure and using a small amount of heat to remove water from the material. Other names for the process are freeze drying and cryodesiccation. Cryoprotectants or lyoprotectants may be added to provide additional protection to the lyophilized preparation. Some lyoprotectants are certain sugars, such as the natural substances sucrose and trehalose, and polyalcohols. The preparation may be lyophilized before or after resuspension in a suitable buffer. Furthermore, the lyophilized preparation may then be used in the disclosed methods either directly as a powder or after reconstitution by adding water or another appropriate buffer or solvent. For example, the lyophilized powder may be added to an animal feed or other foodstuff to inhibit the infectivity of any prions that may be present. Alternatively, a liquid preparation may be more suitable for treatment of a surface.
In one embodiment, the protein extract is derived from earthworms categorized in the class or subclass Oligochaeta, which are members of the phylum Annelida. In accordance with the methods disclosed, one of skill in the art could determine the effectiveness of a preparation derived from any particular variety of earthworm. In particular, members of the genera Lumbricus (red worms) and Eisenia (tiger worms) are useful for preparing an effective composition. Moreover, in one embodiment of the technology, the earthworm protein homogenate is derived from the earthworm species Lumbricus rubellus or Eisenia fetida. 
In one embodiment, a composition useful in the present methods is lumbrokinase. Enzymes extracted from earthworms are collectively called lumbrokinase (LK). (Nakajima et al. Biosci Biotechnol Biochem, 63:2031-2033 (1993); Cho et al., J Biochem Mol Biol, 37:199-205 (2004).) Lumbrokinase is commercially available as a semi-purified earthworm extract from L. rubellus containing a complex of earthworm fibrinolytic enzymes. One method of preparing LK is disclosed in publication number JP 2005230013. Briefly, an earthworm homogenate slurry is filtered, centrifuged, and passed through an anion exchange or affinity chromatography column, followed by concentrating, desalting, and lyophilizing the eluant. Alternative methods of production using chromatography are described in JP 2007039404, and additional purification and preparation steps are described in publication number CN 1587398.
In some embodiments, lumbrokinase is used as an agent to degrade PrP or reduce the infectivity of PrP. LK degrades abnormally folded proteins, such as the infective prion protein PrPSc, with an effectiveness similar to earthworm homogenates generally. In another embodiment, the earthworm homogenate is an active fraction of lumbrokinase. Active fractions of LK (e.g., LrP II and LrP III), and similar preparations from E. fetida may also be used in the present methods.
Active fractions of an earthworm extract can include, but are not limited to, proteins, carbohydrates, or combinations thereof (e.g., glycoproteins) that, alone or in combination with other components, can reduce prion infectivity (as measured using the assays described herein). Numerous separation procedures can be employed to further purify desired components or remove unwanted or contaminating components, including decanting, filtration, sedimentation, centrifugation, heating, adsorption, precipitation, chromatography, or ion exchange. The resulting active fraction can be subsequently concentrated by evaporation, vaporization, lyophilization or vacuum drying.
One method includes obtaining an extract of earthworm material and size-fractionating the extract, e.g., through a size exclusion column. Another method includes obtaining an extract of earthworm material and fractionating the extract using ion exchange chromatography. Chromatography may be performed in column or batch mode. In column chromatography, a sample is loaded onto the column and eluted with successively higher concentrations of a solvent. Other purification methods which may be used in addition to reverse phase chromatography include low pressure column chromatography, batch chromatography, precipitation, specific adsorbent chromatography, gel filtration, HPLC and combinations of these methods. Using these or similar methods of fractionating a earthworm extract, one or more active fractions capable of degrading prions and/or reducing prion infectivity may be obtained.
In one embodiment, the composition comprises one or more earthworm proteases. In some embodiments, the one or more earthworm proteases are selected from the group consisting of SEQ ID NOs: 1-29, shown in Table 1.







TABLE 1







Exemplary Earthworm Proteases












GenBank





Accession
SEQ ID


Name
Species
No.
NO:













Fibrinolytic enzyme
Lumbricus rubellus
BAB40767
1


Fibrinolytic enzyme
Lumbricus rubellus
BAB40768
2


Fibrinolytic protease 0
Eisenia fetida
ABG68022
3


Fibrinolytic protease 1
Eisenia fetida
ABD76397
4


Fibrinolytic protease 2
Eisenia fetida
ABG68023
5


Fibrinolytic protease P-III-1
Eisenia fetida
ABB19359
6


Fibrinolytic enzyme
Lumbricus bimastus
AAD05563
7


Lumbrokinase
Lumbricus bimastus
AAL28118
8


Lumbrokinase
Lumbricus rubellus
AAN58692
9


Lumbrokinase
Lumbricus bimastus
AAN78282
10


Lumbrokinase
Lumbricus bimastus
AAP04532
11


Lumbrokinase
Eisenia fetida
AAT74899
12


Lumbrokinase
Eisenia fetida
AAT74900
13


Lumbrokinase
Eisenia fetida
AAW27919
14


Lumbrokinase
Eisenia fetida
ABQ23217
15


Lumbrokinase
Eisenia fetida
ABW04903
16


Lumbrokinase
Eisenia fetida
ABW04904
17


Lumbrokinase
Eisenia fetida
ABW04905
18


Lumbrokinase
Eisenia fetida
ABW04906
19


Lumbrokinase-1T4
Lumbricus rubellus
AAA96502
20


Lumbrokinase-3
Eisenia fetida
AAR13224
21


Lumbrokinase-3(1)
Lumbricus rubellus
AAA96503
22


Lumbrokianse-3T2
Lumbricus rubellus
AAQ13828
23


Lumbrokinase-4
Eisenia fetida
AAR13225
24


Lumbrokinase-5
Eisenia fetida
AAR13226
25


Lumbrokinase-7T1
Lumbricus rubellus
AAQ13829
26


Lumbrokinase-7T2
Lumbricus rubellus
AAQ13830
27


Lumbrokinase-Da2
Lumbricus rubellus
AAQ13831
28


Lumbrokinase-P2(2)
Lumbricus rubellus
AAQ15052
29









In some embodiments, the one or more earthworm proteases will possess a certain amount of sequence identity to the proteases of SEQ ID NOs: 1-29. For instance, earthworm proteases useful in the claimed methods includes derivatives of the amino acid sequences of SEQ ID NOs: 1-29 having one or more substitutions, additions and/or deletions, including one or more non-naturally occurring amino acids. Preferably, derivatives exhibit at least about 50% identity to any wild type or reference sequence, preferably at least about 70% identity, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any wild type or reference sequence described herein. Sequence (or percent) identity can be determined according to methods known in the art. Derivatives can also include post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like.
Peptide derivatives can also include any of these modifications so long as the polypeptide maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. Furthermore, modifications may be made that have increase affinity and/or specificity for prion proteins. Polypeptides described herein can be made recombinantly, synthetically, purified from natural sources, or in tissue culture.
Methods of Degrading Prions and Reducing Prion Infectivity
In one aspect of the present technology, the composition is effective in reducing prion infectivity. In general, infectivity refers to the ability of a pathogen to establish an infection in an individual. Thus, infectivity refers to the relative capability of a prion-contaminated object, substance, or surface to transmit one or more prions to, or cause a prion-associated disease (i.e., a TSE), in an animal; thus, reduced infectivity is marked by a decreased ability to transmit a prion or cause a prion disease. A reduction in infectivity can be expressed in relative terms as a ratio, fraction, or percentage describing the infectivity after a treatment relative to the infectivity prior to the treatment. Thus, reduced infectivity can be expressed by saying the infectivity is about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or 0.001% of the untreated control.
Several measures can be used to demonstrate reduced infectivity. For example, two similar prion-free, healthy populations or test subjects are exposed to a prion-contaminated object or substance that either has or has not been treated with an earthworm homogenate under certain conditions. A reduced infectivity is indicated by disease symptoms not manifesting or manifesting after a longer interval of time in the population or subject that was exposed to the object or substance that was treated. In some embodiments, the present methods can be used to degrade prions such that the incubation time for the disease is substantially lengthened and/or infectivity is reduced. In particular, treatment with earthworm homogenate or an active fraction thereof may reduce prion infectivity to less than about 0.01% (one-ten thousandth) of the value observed from an untreated control.
In addition, a reduced incidence (rate of occurrence of initial infections) of prion-associated disease in a controlled population indicates a reduced infectivity. When two similar prion-free, healthy populations are exposed to a prion-contaminated object or substance that either has or has not been treated by a method described herein, a reduced infectivity is indicated by observing a smaller infected subset of subjects among the population that was exposed to the object or substance that was treated.
In an illustrative embodiment, the activity of one or more earthworm extracts or active fractions thereof may be measured using a live animal assay. For example, purified hamster prions (PrPHY) are incubated in the presence of earthworm extract or an active fraction thereof. The samples are then intracerebrally inoculated into weanling Syrian hamsters. A dilution series of PrPHY may also be inoculated as a control. Hamsters are monitored on a regular basis for the onset of clinical symptoms. Brains from clinically positive hamsters and uninfected controls are analyzed for protease-resistant PrP by immunoblotting.
Methods of Decontaminating Surfaces
Aspects of the present technology relate to contacting an object, surface, or substance with a composition containing an earthworm homogenate, an active fraction thereof, or one or more purified earthworm proteases to degrade prions that may be present. In one embodiment of the methods disclosed, the contacting is performed by soaking the surface in the composition. Other methods of contacting the surface with the composition can also be used such as immersing or dunking the surface in the composition, or coating, spraying, wiping, layering, or wetting the surface with the composition. The length of time of contacting the surface with the composition can be varied. In some embodiments, the contact is maintained by soaking for up to 30 minutes. In other embodiments, the contact is maintained by soaking for at least 30 minutes. In yet other embodiments, the contact is maintained until an effective or desired reduction in infectivity has been achieved.
Many types of surfaces may become contaminated with prions during their use. Any tool or instrument that is used to cut, slice, chop, or otherwise touch in any way the tissues or fluids of an animal, including human tissue or fluid, could become contaminated. Examples of items that could be exposed to prions and would need decontamination are medical devices used in surgical procedures, such as scalpels, scissors, endoscopes, forceps, catheters, retractors, clamps, spatulas, and the like, equipment used for butchering an animal, such as a knife, and laboratory equipment that is used in prion research such as homogenizers, tubes, spatulas, and the like. Prions can also be present in animal excreta, saliva, mucus, blood, lymph, tears, and other fluids originating from a live infected animal, and these substances can attach to and contaminate a surface. Surfaces that can become contaminated are tables and other fixtures that are used to support a patient or animal for surgery, butchering, or other procedures, i.e., medical equipment, surgical implements, pharmaceutical equipment, mortuary equipment, meat processing equipment, food handling equipment, cookware, and the like. Contamination can occur on surfaces present in professional environments such as operating rooms, hospitals, veterinary clinics, ambulances, and the like, in commercial environments such as those involved with food processing, fur production, animal husbandry, and the like, and in home environments such as a garage, barn, or kitchen, for instance, where a person stores or butchers a deer or other game.
In addition, other substances and surfaces can become contaminated with prions. For instance, foodstuffs, feed, and animal chow can contain prions, and this food can subsequently infect animals and humans that ingest the prion-contaminated food. For example, the “mad cow disease” (BSE) scare and the incidence of a new variant of human CJD (vCJD) are considered to be a consequence of dietary exposure to scrapie sheep carcasses rendered for animal feed in the case of BSE (Brown et al., JAMA 278:1008-1011 (1997)), and to beef from cattle affected with BSE in the case of vCJD. (Bruce et al., Nature 389:498-501 (1997).) There is also evidence of cross-species infectivity of prions. In the case of BSE and vCJD, epidemiological data places the majority of vCJD cases in the UK where the overwhelming majority of BSE cases have been reported. This link is supported by evidence obtained from studies in macaques and mice demonstrating cross-species infectivity of prions, and additional studies showing transmission of mouse-adapted strains to hamsters, transmission of human strains to ferrets and goats after passage through primates and cats, and transmission of a bovine strain to hamsters after passage through mice. Some measures have been introduced to limit human exposure to prions—ruminant protein feed has been banned in the US and UK to reduce the number of BSE cases, and other measures have been taken to prevent potentially infected meat from entering the human food chain. Also, the FDA and CBER have declined to accept blood from donors who stayed in the UK for more than 6 months during 1980-1996.
Despite these precautions and because modes of prion transmission are not well understood, many potential means of food contamination are still possible. For instance, animal feed can become contaminated by contacting prion-containing soil. A human could then become infected by ingesting meat from an animal previously infected by prion-contaminated feed. Foodstuffs can become contaminated by coming into contact with equipment, implements, tools, or machines that had previously been used to prepare prion-infected matter. Thus, the present technology includes methods for adding an effective amount of an earthworm homogenate composition to an animal foodstuff to degrade any prions that may be present.
The activities of biologically-derived compositions can vary with physical conditions. Temperature and pH are two common parameters that may influence the activity of such a composition. Most biological systems maintain a neutral pH of 7 or a near-neutral pH, and thus compositions prepared from a biological system often are used at a neutral or near-neutral pH. In one embodiment of the methods described, the pH of the composition is from about 6 to 8. The temperature of 37° C. is human body temperature, and is often used with biological preparations. Most pieces of laboratory or medical equipment can maintain this temperature. In one embodiment of the method, the contacting step is performed at a temperature of 37° C. In other embodiments, the contacting step is performed at a temperature of from about 20 to about 50° C. Earthworms live and grow in the soil, where environmental conditions may vary over a wide range of temperatures. Thus, in yet other embodiments of the methods disclosed, the contacting step is performed at a temperature of from about 0-30° C., from about 10-40° C., or from about 30-60° C.
EXAMPLES
The present invention is further illustrated by the following examples, which should not be construed as limiting in any way.
Materials and Methods
Earthworms. The earthworms used in this study were commercially raised, common garden earthworms (Lumbricus rubellus and Eisenia fetida). Earthworm homogenates were prepared by quick-freezing worms in liquid nitrogen, then pulverizing the sample using a mortar and pestle. The powdered tissue was resuspended in buffered saline (PBS, 130 mM NaCl, 3 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4) to 30% (w/v). Earthworm homogenates were centrifuged for 5 min at 3000 rpm to remove particulates. Clarified extracts were transferred to clean tubes and stored at −20° C. For anterior and posterior extracts, earthworms were bisected and each half processed as above.
Lumbrokinase. Lumbrokinase (LK), in capsule form, was purchased from the Allergy Research Group (Alameda, Calif.). The enzyme powder was removed from the gelatin capsule and resuspended in ddH2O to make a 400 mg/ml (w/v) stock solution. The LK suspension was centrifuged at 3000 rpm for 5 min and the clarified supernatant was collected.
Purified TSE agents. Brains from deer affected with CWD were homogenized with PBS in a Dounce homogenizer to 20% (w/v). For other experiments, brains from Syrian hamsters clinically affected with the Hyper strain of hamster-adapted transmissible mink encephalopathy agent were homogenized in PBS to 10% (w/v). Enriched PrPTSE was prepared using a variation of the phosphotungstic acid (PTA) precipitation. Equal volumes of brain homogenate and sarkosine were mixed with CaCl2 and DNAse (final concentration: 2% sarkosine, 10 mM CaCl2, and 100 μg/ml DNAse) and incubated overnight at 37° C. on a platform rocker. After centrifuging 3000 rpm for 5 min, the supernatant was transferred to a clean tube. PTA (4% in 170 mM MgCl2) was added to the supernatant (final concentration 0.2%) and incubated on a platform rocker overnight at 37° C. The enriched preparations were then centrifuged at 14,000 rpm for 30 min, the resulting pellet resuspended in PBS ( 1/10 starting volume) by sonication and finally incubated with proteinase K (50 μg/ml) for 30 min at 37° C.
Earthworm/TSE environment. Earthworms (n=6 to 10) were placed in a small container with 50 g of commercial top-soil (soil, peat moss, humus, and sand). Hamster brain tissue ( ̃1 g) from a clinically affected hamster (Hyper strain) was added to the soil and the container was covered for 1-10 days at room temperature. At specified time intervals, earthworms were removed from this environment, rinsed in ddH2O and homogenized in PBS. A duplicate container of earthworms with no infectious agent served as a control.
Prion Degradation. Earthworm homogenates (30% w/v) and dilutions of the clarified homogenate (30%, 10%, 5%, 1% in PBS) were spiked with a constant volume of PTA-enriched PrPSc (PrPCWD or PrPHY), vortexed, and incubated at 37° C. Diluted LK solutions (200, 100, 50, 25, and 4 mg/ml in PBS) were incubated at 37° C. with matching amounts of PTA-enriched PrPSc. Equivalent amounts of earthworm homogenates and LK without added PrPSc, as well as PrPSc alone, were included as controls. Samples were incubated 0-8 days.
Samples were prepared for western blot analysis by boiling in SDS-PAGE sample buffer (100 mM Tris (pH 8.0), 10% SDS, 7.5 mM EDTA, 100 mM dithiothreitol, and 30% glycerol) for 10 minutes. The samples were separated on 4-20% precast gels (BioRad, Hercules, Calif.), proteins were transferred to polyvinyl difluoride membranes and probed with mAbs 3F4 (hamster) or 6H4 (deer). Detection was achieved using an HRP-conjugated goat anti-mouse immunoglobulin G (IgG) (BioRad) and visualized with SuperSignal Chemiluminescence (Pierce, Rockford, Ill.).
Animal bioassay. PTA-purified hamster agent (PrPHY) was incubated in the presence of earthworm proteins and LK. The samples were diluted in PBS (pH 7.4), and 30 μl were intracerebrally inoculated into male, weanling Syrian hamsters (Harlan, Indianapolis, Ind.). A dilution series of PTA-enriched PrPHY was also prepared and similarly inoculated. In addition, animals were inoculated with earthworm homogenate or LK (no infectious agent) as controls. Hamsters were monitored on a regular basis for the onset of clinical symptoms. Brains from clinically positive hamsters and uninfected controls were analyzed for protease-resistant PrP by immunoblotting.
Example 1
Earthworm Digestion of Infectious Agent in Soil
To examine the impact of earthworm digestion of infectious agent, earthworms were removed from the prion infected soil, rinsed in water, and homogenized. Protein extracts were prepared and examined for the presence of PrPSc by western blot analysis. The detected prion protein migrated faster ( ̃20 KDa) than the brain homogenate control in the SDS-PAGE gel (FIG. 1A). These results suggested that partial degradation of the prion protein occurred within the earthworm. To examine the movement of prion protein through the digestive tract of these earthworms, we bisected individual worms and assayed anterior and posterior sections for the presence of PrPSc. Detectable levels of prion protein in these samples were observed only in the anterior sections (FIG. 1B) with a slight size shift (<31 KDa), suggesting abnormal protein is undergoing degradation prior to entering the earthworm's hindgut. Prion protein was not identified in the posterior sections suggesting complete digestion of detectable prion in the digestive tract of the earthworm. Accordingly, earthworms can ingest infectious tissue, and prion proteins present within that tissue are subsequently digested in the earthworm digestive tract. Lanes in Panel A are as follows: Lane 1: 5% brain homogenate; Lane 2: earthworm in absence of infectious material; Lanes 3 and 4: earthworm homogenate. In Panel B, each pair of lanes shows the prion protein detected in anterior (a) and posterior (p) sections from 4 individual worms.
Example 2
Digestion of CWD Infectious Agent by Earthworm Protein Extracts and Lumbrokinase In Vitro
In vitro analyses using PTA-enriched CWD agent incubated with earthworm protein extracts were performed. In these experiments, PTA-PrPCWD was incubated with clarified earthworm protein extracts at 37° C. for set times (t=0, 2, 4, or 8 days) and then immunoblot-tested for prion protein levels. Prion protein immunoreactivity was observed only with the t=0 day sample. For subsequent studies, the digestion times were decreased and dilutions of the earthworm extracts were used. After incubation at 37° C. for 16 h, no prion signal was detected when 30% earthworm protein extract was used, and a weakened signal was seen with a 10% earthworm extract (FIG. 2A). The signal from the control CWD starting agent diluted 1:50 (lane 7) matches the intensity of the 5% earthworm protein dilution sample. The above experiment was repeated with anterior and posterior clarified earthworm protein extract. Anterior derived extract reduced PrPCWD signal similar to the whole worm extract, while protein extract prepared from posterior sections had little effect on the PrPCWD. The lanes in Panel A are as follows: Lane 1: PrPCWD; Lane 2: worm extract, Lanes 3-6: 30%, 10%, 5%, 1%, respectively, worm extract with CWD agent. Migration of molecular weight markers (KDa) is provided to the left.
Lumbrokinase (LK) is a commercially available, semi-purified earthworm extract containing a complex of earthworm enzymes from L. rubellus. The ability of LK to degrade purified prion protein from CWD-infected deer (PrPCWD) was assessed. The results are shown in FIG. 2B. The lanes in Panel B are as follows: Lane 1: PrPCWD; Lane 2: lumbrokinase, no PrPCWD; Lanes 3-7: 200, 100, 50, 25, 4 mg/ml lumbrokinase, respectively. Incubation of LK resulted in loss of PrPCWD signal with the highest enzyme concentrations tested (200 and 100 mg/ml; FIG. 2B). Both earthworm homogenate and LK can digest prions isolated from a deer infected with CWD. Accordingly, earthworm homogenate and LK are effective in methods to degrade or digest prion proteins.
Example 3
Digestion of Hamster Prion Infectious Agent by Earthworm Protein Extracts and Lumbrokinase In Vitro
The ability of earthworm homogenates and LK to degrade prion protein isolated from a hamster-adapted prion model was examined in vitro. Purified hamster prions (PrPHY) were incubated in the presence of earthworm protein extracts and (in separate experiments) LK. Similar to purified deer (CWD) prions, both the earthworm extract and the LK degraded PrPHY (FIG. 3). The lanes in Panel A are as follows: Lanes 1-4: 1, 2, 4 and 8 day incubations of PrPHY and earthworm protein, respectively. Lane 5: earthworm extract; Lane 6: PrPHY. The lanes in Panel B are as follows: Lanes 1-5: 1:10; 1:20; 1:40; 1:100 and 1:200 (respectively). Migration of molecular weight markers (KDa) is provided to the left. The results show that a considerable decline in prion protein signal was detected after eight days (lane 6). After 8 days of digestion, the amount of PrPHY detected was reduced to approximately one-one hundredth ( ̃0.01) of the untreated sample (comparing lanes 3 & 4, panel A with lanes 4&5, panel B). Thus, in a similar manner to that demonstrated by Example 2, earthworm homogenate and LK can digest prions isolated from a hamster model of prion disease. Accordingly, earthworm homogenate and LK are useful in methods to degrade or digest prion proteins.
Example 4
In Vivo Activity of Infectious Agent after In Vitro Degradation
Animal bioassays were performed to determine if in vitro degradation of prion protein affected infectivity. Increased incubation periods were observed with both the LK and earthworm homogenate-digested prion samples. Compared to control samples (infectious agent only), which had incubation period of 77 days, incubation of infectious agent with LK extended the incubation period to 93 days, while digestion of agent with total earthworm proteins extended incubation period to 110 days. Dilution of infectivity controls indicates that both the LK and earthworm homogenate digestions resulted in a greater than 3 log reduction in infectivity. Accordingly, earthworm homogenate and LK are effective in methods to decrease the infectivity of prion proteins.
Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.
As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 units refers to groups having 1, 2, or 3 units.
All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
Other embodiments are set forth within the following claims.

## Claims

1. A method for degrading infectious prion proteins or reducing prion infectivity, the method comprising contacting a surface that carries or is suspected to carry prion-infected material with an effective amount of a composition comprising a whole earthworm tissue homogenate or an active fraction thereof, wherein the composition comprises one or more earthworm tissue-derived prion-degrading enzymes selected from the group consisting of a fibrinolytic enzyme, a fibrinolytic protease, and a lumbrokinase, wherein the active fraction does not consist solely of earthworm digestive tract luminal contents, and wherein the active fraction does not consist solely of lumbrokinase.

2. The method of claim 1, wherein the degradation of prions results in a reduction of prion infectivity.

3. The method of claim 1, wherein the composition comprises about 10-30% by weight of the earthworm tissue homogenate or active fraction thereof.

4. The method of claim 1, wherein the active fraction is an earthworm tissue homogenate supernatant.

5. The method of claim 4, wherein the supernatant is prepared by wet-grinding earthworms and filtering the homogenate.

6. The method of claim 1, wherein the earthworm tissue homogenate or active fraction thereof has been lyophilized.

7. The method of claim 6, wherein the preparation of the lyophilized earthworm tissue homogenate or active fraction thereof comprises wet-grinding earthworms and freeze-drying the tissue homogenate or active fraction thereof.

8. The method of claim 1, wherein the earthworm tissue homogenate or active fraction thereof comprises lumbrokinase.

9. The method of claim 1, wherein the earthworm tissue homogenate or active fraction thereof is derived from an earthworm selected from the group consisting of: Lumbricus rubellus, Eisenia fetida, and Lumbricus bimastus.

10. The method of claim 1, wherein the one or more enzymes has at least 95% sequence identity to an earthworm protease selected from the group consisting of SEQ ID NOs: 1-29.

11. The method of claim 1, wherein the earthworm tissue homogenate or active fraction thereof has been subjected to size exclusion chromatography.

12. The method of claim 1, wherein the earthworm tissue homogenate or active fraction thereof has been subjected to ion exchange chromatography.

13. The method of claim 1, wherein the surface is selected from the group consisting of: a medical device, butchering equipment, and laboratory equipment.

14. The method of claim 1, wherein the step of contacting comprises soaking the surface in the composition.

15. The method of claim 1, wherein the step of contacting comprises spraying or wiping the surface with the composition.

16. The method of claim 1, wherein following the treating of the surface, the prion-infected material is unable to infect a live mammal.

17. A method for inhibiting infection by an infectious prion, the method comprising adding a composition that degrades prions to an animal foodstuff containing or suspected of containing a prion-infected material, wherein the composition comprises an effective amount of a whole earthworm tissue homogenate or an active fraction thereof, wherein the composition comprises one or more earthworm-derived prion-degrading enzymes selected from the group consisting of a fibrinolytic enzyme, a fibrinolytic protease, and a lumbrokinase, wherein prions in the foodstuff, if present, are degraded, wherein the active fraction does not consist solely of earthworm digestive tract luminal contents, and wherein the active fraction does not consist solely of lumbrokinase.