Pure anthelmintic compositions and related methods

Abstract

Disclosed are compositions and methods for treating or reducing the severity of parasitic or helminthic infections in a subject. The methods involve administering to the subject a therapeutically effective amount of a composition containing isolated, natural, bioactive nematicidal crystals formed from a single species of nematicidal crystal protein. The isolated, natural, bioactive nematicidal crystals are substantially free of bacterial spores or host bacterial proteins other than the crystalline form of the nematicidal crystal protein. Also disclosed are methods for producing isolated, natural, bioactive nematicidal crystals. The crystal protein can be a full-length Cry protein, a truncated Cry protein, a Cry protein variant, or a Cry protein subvariant. Examples of crystal proteins include Cry5B, Cry21, Cry14A, Cry6A, and Cry13A.

Description

[Technical field]

[0001] [Federally sponsored research identified] This invention was made with government support under Grant No. NIAID 5R01AI056189-13 awarded by the National Institutes of Health (NIH) of the U.S. Department of Health and Human Services (HHS), and Grant No. NIFA 2016-67015-24861 awarded by the National Institute of Food and Agriculture (NIFA) of the U.S. Department of Agriculture (USDA). The government has certain rights in this invention.

[0002] [Related Applications] This application claims priority to U.S. Provisional Patent Application No. 62 / 510,081, filed May 23, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0003] Soil-transmitted helminths (STHs) that infest the human gastrointestinal tract infect 2.3 billion of the world's poorest people and over 400 million of the poorest children. Crystalline (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are potential drugs that provide safe and effective STH treatment. However, while the anthelmintic biological activity of Cry proteins has been established, significant challenges remain in effectively delivering intact, bioactive Cry proteins to the gastrointestinal (GI) tract of humans and animals for STH treatment. Application of Cry proteins as practical STH treatments in developing countries, where treatments must be available in large quantities at very low prices (less than $1 per dose) to treat large numbers of poor patients, is limited by the cost and scale-up issues associated with expressing and purifying Cry proteins.

[0004] A cheap, simple and scalable way to provide Cry proteins is to express them in B. thuringiensis. The bacterium is well suited to express Cry proteins at very high levels and has already been cultivated on a large scale and inexpensively for environmental applications. However, high-level production of Cry proteins in Bt requires sporulation. Thus, Cry protein compositions currently applied to the environment are in the form of "spore-crystal lysate" (SCL) that contains both crystal proteins and spores from the bacteria. Application of such SCL compositions to humans is problematic due to the presence of bacterial spores that may contain many enterotoxin genes that cause food poisoning in humans. Furthermore, since spores are difficult to handle and less effective, any formulation containing Bt spores will necessarily contain a significant amount of inactive ingredients (spores) along with active ingredients (crystals) per unit weight, making it more difficult to administer to both humans and animals. Separating spores and crystal proteins is difficult because they are roughly the same size. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need in the art for new approaches to deliver protein therapeutics, such as antiparasitic proteins, to the gastrointestinal tract. [Means for solving the problem]

[0006] The present disclosure is based on the discovery that bacteria that lack sporulation or have no sporulation ability can be used to produce highly pure nematicidal crystal preparations suitable for pharmaceutical use. The pure nematicidal crystal preparations of the present invention provide superior anti-helminthic activity to crystal proteins purified from spore crystal lysate (SCL). Moreover, such preparations are substantially free of impurity spores and host bacterial proteins that are unsuitable for administration to humans. In some exemplary embodiments, the preparations of the present invention are substantially free of soluble cellular components, lipids, and cell wall debris.

[0007] In one aspect, the disclosure provides a pharmaceutical composition comprising an isolated, naturally occurring, bioactive nematicidal crystal formed from a single species of nematicidal crystal protein, the nematicidal crystal protein being produced by a host bacterium in a non-sporulating form, and the pharmaceutical composition being substantially free of bacterial spores or host bacterial proteins other than the nematicidal crystal protein in a crystalline form.

[0008] In some embodiments, the pharmaceutical composition of the present invention comprises an excipient suitable for oral administration to a human subject. In some embodiments, the host bacterium is a Bacillus species. In some embodiments, the host bacterium is Bacillus thuringiensis (Bt). In some embodiments, the host bacterium is an Escherichia coli or Pseudomonas fluorescens species.

[0009] In some embodiments, the non-sporulating host bacterium is genetically modified to have a genetic mutation that results in a deficiency in sporulation, such that the naturally occurring bioactive nematicidal crystals are trapped in the cytosol of the bacterium. In some embodiments, the genetic mutation that results in a deficiency in sporulation is a deletion or inactivation of one or more genes selected from the group consisting of: kinA, kinB, spoOA, spoOB, spo0E, spo0F, spo0J, spoOM spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. In one embodiment, the genetic mutation resulting in a sporulation defect is a deletion or inactivation of the spoOA gene.

[0010] In some embodiments, the host bacterium is genetically modified to express a single nematicidal crystal protein under the control of a sporulation specific promoter. In some embodiments, the sporulation specific promoter is Cry3A, GerA, GNAT or TadA promoter. In some embodiments, the single nematicidal crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, Cry55B and variants and truncations thereof. In some embodiments, the nematicidal crystal protein is Cry5B or its variant or fragment. In some embodiments, the nematicidal crystal protein is Cry5B variant Ser407Cys.

[0011] In some embodiments of the pharmaceutical compositions of the present disclosure, the pharmaceutical composition further comprises a second crystal protein in the form of isolated natural bioactive nematicidal crystals, formed solely from the second crystal protein.

[0012] In some embodiments, the pharmaceutical composition comprises isolated natural bioactive nematicidal crystals in an amount of at least 95%.

[0013] In some embodiments, the isolated natural bioactive nematicidal crystals are in an orally available dosage form. In some embodiments, the pharmaceutical composition is in a dry powder form and is encapsulated in a pharmaceutical capsule.

[0014] In another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) growing a non-sporulating host bacterium engineered to express a single species of nematicidal crystal protein; wherein the non-sporulating host bacterium produces natural, bioactive nematicidal crystals formed from the single species of nematicidal crystal protein, the host bacterium being grown in a growth medium, and optionally the non-sporulating host bacterium releasing the natural, bioactive nematicidal crystals into the growth medium; and (b) isolating and concentrating the natural, bioactive, nematicidal crystals to form isolated, natural, bioactive, nematicidal crystals. The present invention provides a method for producing a pharmaceutical composition, comprising:

[0015] In some embodiments of the method, the host bacterium is a Bacillus species. In some embodiments, the host bacterium is Bacillus thuringiensis (Bt). In some embodiments, the host bacterium is an E. coli or P. fluorescens species.

[0016] In some embodiments of the method, the non-sporulating host bacterium is genetically modified to have a genetic mutation that results in a deficiency in sporulation, such that the naturally occurring bioactive nematicidal crystals are trapped in the cytosol of the bacterium. In some embodiments, the genetic mutation that results in a deficiency in sporulation is a deletion or inactivation of one or more genes selected from the group consisting of: kinA, kinB, spoOA, spoOB, spoOE, spoOF, spoOJ, spoOM spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. In some embodiments, the genetic mutation that results in a sporulation defect is a deletion or inactivation of the spoOA gene.

[0017] In some embodiments of the above method, the host bacterium is genetically modified to express a single nematicidal crystal protein under the control of a sporulation-specific promoter. In some embodiments, the sporulation-specific promoter is Cry3A, GerA, GNAT, or TadA promoter. In some embodiments, the single nematicidal crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, Cry55B, and variants and truncations thereof. In some embodiments, the nematicidal crystal protein is Cry5B or a variant or fragment thereof. In some embodiments, the nematicidal crystal protein is Cry5B variant Ser407Cys.

[0018] In some embodiments, the method further comprises exposing the non-spore-forming host bacterium to an antimicrobial compound to inactivate the host bacterium. In some embodiments, the antimicrobial compound is iodine. In some embodiments, the antimicrobial compound is an antibiotic medication. In some embodiments, the antimicrobial compound is a β-lactam antibiotic. In some embodiments, the antimicrobial compound is an organic solvent selected from the group consisting of a terpene, hexane, and formaldehyde. In some embodiments, the antimicrobial compound is a terpene. In some embodiments, the terpene is selected from the group consisting of thymol, eugenol, geraniol, carvacrol, and citral, or combinations thereof. In some embodiments, the terpene is carvacrol. In some embodiments, the antimicrobial compound is hexane.

[0019] In some embodiments, the method includes adding an edible oil to the inactivated host bacteria to extract the inactivating agent from the nematicidal crystal protein. In some embodiments, the edible oil is selected from the group consisting of corn oil, soybean oil, coconut oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, safflower oil and sunflower oil. In some embodiments, the edible oil is corn oil. In some embodiments, the edible oil is corn oil. In some embodiments, the method includes adding an organic solvent to the inactivated host bacteria to extract cellular components from the nematicidal crystal protein. In some embodiments, the organic solvent is hexane. In some embodiments, hexane is added to the inactivated host bacteria up to 50% v / v. In some embodiments, the method includes centrifuging a mixture of the organic solvent and the inactivated host bacteria to pellet the nematicidal crystal protein.

[0020] In some embodiments, the method further comprises homogenizing the inactivated host bacteria to form a bacterial lysate containing natural bioactive nematicidal crystals. In some embodiments, the method further comprises concentrating the bacterial lysate. In some embodiments, the step of concentrating the bacterial lysate is selected from the group consisting of centrifugation, ultrafiltration and dialysis.

[0021] In some embodiments, the method further comprises formulating the isolated, natural, bioactive nematicidal crystals into an orally available dosage form. In some embodiments, the formulating comprises freeze-drying or spray-drying the isolated, natural, bioactive nematicidal crystals. In some embodiments, the formulating comprises filling the isolated, natural, bioactive nematicidal crystals into a pharmaceutical capsule.

[0022] In a further aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) growing a nonsporulating host bacterium engineered to express a single species of nematicidal crystal protein; wherein the nonsporulating host bacterium produces natural, bioactive nematicidal crystals formed from the single species of nematicidal crystal protein; (b) inactivating the grown nonspore-forming host bacteria by exposure to an antimicrobial compound; (c) homogenizing the inactivated nonsporulating host bacteria to form a bacterial lysate; and (d) concentrating the natural, bioactive, nematicidal crystals in the bacterial lysate to form isolated, natural, bioactive, nematicidal crystals. The present invention provides a method for producing a pharmaceutical composition, comprising:

[0023] In some embodiments, the method further comprises concentrating the grown nonspore-forming host bacteria prior to inactivation. In some embodiments, the host bacteria is a Bacillus species. In some embodiments, the host bacteria is Bacillus thuringiensis (Bt). In some embodiments, the host bacteria is an E. coli or P. fluorescens species.

[0024] In some embodiments of this method, the non-sporulating host bacterium is engineered to have a genetic mutation that results in a deficiency in sporulation such that the naturally occurring bioactive nematicidal crystals are trapped in the cytosol of the bacterium. In some embodiments, the genetic mutation that results in a deficiency in sporulation is a deletion or inactivation of one or more genes selected from the group consisting of: kinA, kinB, spoOA, spoOB, spo0E, spo0F, spo0J, spoOM spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. In some embodiments, the genetic mutation that results in a sporulation defect is a deletion or inactivation of the spoOA gene.

[0025] In some embodiments of the above method, the host bacterium is genetically modified to express a single nematicidal crystal protein under the control of a sporulation-specific promoter. In some embodiments, the sporulation-specific promoter is Cry3A, GerA, GNAT, or TadA promoter. In some embodiments, the single nematicidal crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, Cry55B, and variants and truncations thereof. In some embodiments, the nematicidal crystal protein is Cry5B or a variant or fragment thereof. In some embodiments, the nematicidal crystal protein is Cry5B variant Ser407Cys.

[0026] In some embodiments of the method, the antimicrobial compound is iodine. In other embodiments of the method, the antimicrobial compound is an antibiotic medicament. In some embodiments of the method, the antimicrobial compound is a β-lactam antibiotic. In some embodiments, the antimicrobial compound is an organic solvent selected from the group consisting of a terpene, hexane, and formaldehyde. In some embodiments, the antimicrobial compound is a terpene. In some embodiments, the terpene is selected from the group consisting of thymol, eugenol, geraniol, carvacrol, and citral, or combinations thereof. In some embodiments, the terpene is carvacrol. In some embodiments, the antimicrobial compound is hexane.

[0027] In some embodiments, the method further comprises extracting antimicrobial compounds from the inactivated host bacteria. In some embodiments, the method comprises adding an edible oil to the inactivated host bacteria to extract the inactivating agent from the nematicidal crystal protein. In some embodiments, the edible oil is selected from the group consisting of corn oil, soybean oil, coconut oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, safflower oil and sunflower oil. In some embodiments, the edible oil is corn oil. In some embodiments, the method comprises adding an organic solvent to the inactivated host bacteria to extract cellular components from the nematicidal crystal protein. In some embodiments, the organic solvent is hexane. In some embodiments, hexane is added to the inactivated host bacteria up to 50% v / v. In some embodiments, the method comprises centrifuging a mixture of the organic solvent and the inactivated host bacteria to pellet the nematicidal crystal protein.

[0028] In some embodiments, the method further comprises formulating the isolated, natural, bioactive nematicidal crystals into an orally available dosage form. In some embodiments, the method comprises a formulation step comprising freeze-drying or spray-drying the isolated, natural, bioactive nematicidal crystals. In some embodiments, the formulation step comprises filling the isolated, purified, pure, natural, bioactive nematicidal crystals into a pharmaceutical capsule.

[0029] In a further aspect, the present disclosure provides a method of treating a parasitic infection in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising isolated, naturally occurring, bioactive nematicidal crystals formed from a single species of nematicidal crystal protein, and which is free of bacterial spores.

[0030] In some embodiments of the treatment method, the nematicidal crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, Cry55B, and variants and truncations thereof.In some embodiments, the nematicidal crystal protein is Cry5B.In some embodiments, the nematicidal crystal protein is Cry5B variant Ser407Cys.

[0031] In some embodiments of the treatment method, the composition further comprises a second crystal protein in the form of isolated, natural, bioactive nematicidal crystals formed only from the second crystal protein. In some embodiments, the pharmaceutical composition comprises at least about 95% isolated, natural, bioactive nematicidal crystals. In some embodiments, the composition is in dry powder form and filled into a pharmaceutical capsule. The embodiments of the present invention are further described below: [Section 1] A pharmaceutical composition comprising isolated, naturally occurring, biologically active nematicidal crystals formed from a single species of nematicidal crystal protein, the crystal protein being produced by a host bacterium in a non-sporulating form, the pharmaceutical composition being substantially free of bacterial spores or host bacterial proteins other than the nematicidal crystal protein in the form of crystals. [Section 2] Item 1. The pharmaceutical composition according to item 1, further comprising an excipient suitable for oral administration to a human subject. [Section 3] Item 2. The pharmaceutical composition according to item 1, wherein the host bacterium is a Bacillus species. [Section 4] Item 2. The pharmaceutical composition according to item 1, wherein the host bacterium is Bacillus thuringiensis (Bt). [Section 5] Item 2. The pharmaceutical composition according to item 1, wherein the host bacterium is an E. coli or P. fluorescens species. [Section 6] Item 2. The pharmaceutical composition according to item 1, wherein a non-sporulating host bacterium has been genetically modified to have a genetic mutation that results in a lack of sporulation, such that the natural bioactive nematicidal crystals are trapped in the cytosol of the bacterium. [Section 7] The pharmaceutical composition described in item 6 above, wherein the genetic mutation resulting in a defect in sporulation is a deletion or inactivation of one or more genes selected from the group consisting of kinA, kinB, spoOA, spoOB, spo0E, spo0F, spo0J, spoOM spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. [Section 8] 7. The pharmaceutical composition according to item 6, wherein the genetic mutation resulting in a defect in sporulation is a deletion or inactivation of the spoOA gene. [Section 9] Item 2. The pharmaceutical composition according to item 1, wherein the host bacterium is genetically modified to express the single species of nematicidal crystal protein under the control of a sporulation-specific promoter. [Section 10] Item 10. The pharmaceutical composition according to item 9, wherein the sporulation-specific promoter is a Cry3A, GerA, GNAT, or TadA promoter. [Section 11] Item 2. The pharmaceutical composition according to item 1, wherein the single species of nematicidal crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, Cry55B, and variants and truncations thereof. [Section 12] Item 12. The pharmaceutical composition according to item 11, wherein the nematicidal crystal protein is Cry5B or a variant or fragment thereof. [Section 13] Item 13. The pharmaceutical composition according to item 12, wherein the nematicidal crystal protein is Cry5B variant Ser407Cys. [Section 14] Item 1. The pharmaceutical composition according to item 1, further comprising a second crystal protein in the form of isolated natural bioactive nematicidal crystals, the crystals being formed solely from the second crystal protein. [Section 15] 15. The pharmaceutical composition according to any one of items 1 to 14, wherein the pharmaceutical composition comprises an isolated natural bioactive nematicidal crystal in a content of at least 95%. [Section 16] 16. The pharmaceutical composition according to any one of items 1 to 15, wherein the isolated natural bioactive nematicidal crystals are in a dosage form that can be administered orally. [Section 17] 17. The pharmaceutical composition according to any one of items 1 to 16, wherein the pharmaceutical composition is in the form of a dry powder and is encapsulated in a pharmaceutical capsule. [Section 18] (a) growing a nonsporulating host bacterium genetically engineered to express a single species of nematicidal crystal protein; wherein the nonsporulating host bacterium produces natural, bioactive nematicidal crystals formed from the single species of nematicidal crystal protein, the host bacterium being grown in a growth medium, and optionally the nonsporulating host bacterium releasing the natural, bioactive nematicidal crystals into the growth medium; and (b) isolating and concentrating the natural, bioactive, nematicidal crystals to form isolated, natural, bioactive, nematicidal crystals. A method for producing a pharmaceutical composition comprising: [Section 19] 19. The method according to claim 18, wherein the host bacterium is a Bacillus species. [Section 20] 19. The method according to item 18, wherein the host bacterium is Bacillus thuringiensis (Bt). [Section 21] 19. The method according to claim 18, wherein the host bacterium is of the species E. coli or P. fluorescens. [Section 22] 19. The method according to claim 18, wherein the non-sporulating host bacterium has been genetically modified to have a genetic mutation that results in a deficiency in sporulation, such that the natural bioactive nematicidal crystals are trapped in the cytosol of the bacterium. [Section 23] 23. The method according to claim 22, wherein the genetic mutation resulting in a defect in sporulation is a deletion or inactivation of one or more genes selected from the group consisting of kinA, kinB, spo0A, spo0B, spo0E, spo0F, spo0J, spo0M spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. [Section 24] 23. The method according to claim 22, wherein the genetic mutation resulting in a defect in sporulation is a deletion or inactivation of the spoOA gene. [Section 25] 19. The method according to claim 18, wherein the host bacterium is genetically modified to express a single species of nematicidal crystal protein under the control of a sporulation-specific promoter. [Section 26] 26. The method according to claim 25, wherein the sporulation-specific promoter is a Cry3A, GerA, GNAT, or TadA promoter. [Section 27] 20. The method according to claim 18, wherein the single nematicidal crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, Cry55B, and variants and truncations thereof. [Section 28] 28. The method according to claim 27, wherein the nematicidal crystal protein is Cry5B or a variant or fragment thereof. [Section 29] 29. The method according to claim 28, wherein the nematicidal crystal protein is Cry5B variant Ser407Cys. [Section 30] 20. The method according to claim 18, further comprising exposing the non-spore-forming host bacterium to an antibacterial compound to inactivate the host bacterium. [Section 31] 31. The method according to claim 30, wherein the antibacterial compound is iodine. [Section 32] 31. The method according to claim 30, wherein the antibacterial compound is an antibiotic drug. [Section 33] 31. The method of claim 30, wherein the antibacterial compound is a β-lactam antibiotic. [Section 34] 31. The method according to claim 30, wherein the antibacterial compound is an organic solvent selected from the group consisting of terpenes, hexane and formaldehyde. [Section 35] 35. The method of claim 34, wherein the antibacterial compound is a terpene. [Section 36] 36. The method according to claim 35, wherein the terpene is selected from the group consisting of thymol, eugenol, geraniol, carvacrol and citral or a combination thereof. [Section 37] 37. The method according to claim 36, wherein the terpene is carvacrol. [Section 38] 35. The method of claim 34, wherein the antibacterial compound is hexane. [Section 39] 31. The method of claim 30, further comprising the step of adding an edible oil to the inactivated host bacteria to extract the antimicrobial compounds, soluble cell components, lipids and cell wall debris from the nematicidal crystal protein. [Section 40] 40. The method according to claim 39, wherein the edible oil is corn oil. [Section 41] 31. The method of claim 30, further comprising the step of adding an organic solvent to the inactivated host bacteria to extract the antimicrobial compound from the nematicidal crystal protein. [Section 42] 42. The method according to claim 41, wherein the organic solvent is hexane. [Section 43] 43. The method of claim 42, wherein hexane is added to the inactivated host bacteria up to 50% v / v. [Section 44] 44. The method of claim 43, further comprising centrifuging the mixture of organic solvent and inactivated host bacteria to pellet the nematicidal crystal protein. [Section 45] 31. The method of claim 30, further comprising homogenizing the inactivated host bacteria to form a bacterial lysate containing natural bioactive nematicidal crystals. [Section 46] 46. ​​The method of claim 45, further comprising concentrating the bacterial lysate. [Section 47] 47. The method according to claim 46, wherein the step of concentrating the bacterial lysate is selected from the group consisting of centrifugation, ultrafiltration and dialysis. [Section 48] 20. The method according to claim 18, further comprising the step of formulating the isolated natural bioactive nematicidal crystals into an orally available dosage form. [Section 49] 49. The method according to claim 48, wherein the formulating step comprises freeze-drying or spray-drying the isolated natural bioactive nematicidal crystals. [Section 50] 49. The method according to claim 48, wherein the formulating step comprises filling the isolated natural bioactive nematicidal crystals into a pharmaceutical capsule. [Section 51] (a) growing a nonsporulating host bacterium engineered to express a single species of nematicidal crystal protein; wherein the nonsporulating host bacterium produces natural, bioactive nematicidal crystals formed from the single species of nematicidal crystal protein; (b) inactivating the grown nonspore-forming host bacteria by exposure to an antimicrobial compound; (c) homogenizing the inactivated nonsporulating host bacteria to form a bacterial lysate; and (d) concentrating the natural, bioactive, nematicidal crystals in the bacterial lysate to form isolated, natural, bioactive, nematicidal crystals. A method for producing a pharmaceutical composition comprising the steps of: [Section 52] 52. The method according to claim 51, further comprising a step of concentrating the grown non-sporulating host bacteria prior to inactivation. [Section 53] 52. The method according to claim 51, wherein the host bacterium is a Bacillus species. [Section 54] 52. The method according to item 51, wherein the host bacterium is Bacillus thuringiensis (Bt). [Section 55] 52. The method according to claim 51, wherein the host bacterium is of the species E. coli or P. fluorescens. [Section 56] 52. The method of claim 51, wherein a non-sporulating host bacterium has been genetically modified to have a genetic mutation that results in a lack of sporulation, such that the natural bioactive nematicidal crystals are trapped in the cytosol of the bacterium. [Section 57] 57. The method according to claim 56, wherein the genetic mutation resulting in a defect in sporulation is a deletion or inactivation of one or more genes selected from the group consisting of kinA, kinB, spo0A, spo0B, spo0E, spo0F, spo0J, spo0M spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. [Section 58] 57. The method according to claim 56, wherein the genetic mutation resulting in a defect in sporulation is a deletion or inactivation of the spoOA gene. [Section 59] 57. The method according to claim 56, wherein the host bacterium has been genetically modified to express the single species of nematicidal crystal protein under the control of a sporulation-specific promoter. [Section 60] 60. The method according to claim 59, wherein the sporulation-specific promoter is a Cry3A, GerA, GNAT, or TadA promoter. [Section 61] 52. The method according to claim 51, wherein the single cytoplasmic crystal protein is selected from the group consisting of Cry5B, Cry6A, Cry5C, Cry5D, Cry13A, Cry14A, Cry21A, Cry21B, Cry55B, and variants and truncations thereof. [Section 62] 62. The method according to claim 61, wherein the nematicidal crystal protein is Cry5B or a variant or fragment thereof. [Section 63] 63. The method according to claim 62, wherein the nematicidal crystal protein is Cry5B variant Ser407Cys. [Section 64] 52. The method according to claim 51, wherein the antibacterial compound is iodine. [Section 65] 52. The method according to claim 51, wherein the antibacterial compound is an antibiotic drug. [Section 66] 52. The method of claim 51, wherein the antibacterial compound is a β-lactam antibiotic. [Section 67] 52. The method of claim 51, wherein the antibacterial compound is an organic solvent selected from the group consisting of terpenes, hexane and formaldehyde. [Section 68] 68. The method of claim 67, wherein the antibacterial compound is a terpene. [Section 69] 69. The method according to claim 68, wherein the terpene is selected from the group consisting of thymol, eugenol, geraniol, carvacrol and citral or a combination thereof. [Section 70] 70. The method according to claim 69, wherein the terpene is carvacrol. [Section 71] 71. The method of claim 70, wherein the antibacterial compound is hexane. [Section 72] 52. The method of claim 51, further comprising the step of extracting the antibacterial compound and cell wall debris from the inactivated host bacteria. [Section 73] 52. The method of claim 51, further comprising the step of adding an edible oil to the inactivated host bacteria to extract the inactivating agent from the nematicidal crystal protein. [Section 74] 74. The method according to claim 73, wherein the edible oil is corn oil. [Section 75] 52. The method of claim 51, further comprising the step of adding an organic solvent to the inactivated host bacteria to extract the antimicrobial compound, soluble cell components, lipids and cell wall debris from the nematicidal crystal protein. [Section 76] 76. The method according to claim 75, wherein the organic solvent is hexane. [Section 77] 77. The method of claim 76, wherein hexane is added to the inactivated host bacteria up to 50% v / v. [Section 78] 76. The method of claim 75, further comprising centrifuging the mixture of organic solvent and inactivated host bacteria to pellet the nematicidal crystal protein. [Section 79] 79. The method according to any one of the above items 51 to 78, further comprising the step of formulating the isolated natural bioactive nematicidal crystals into an orally available dosage form. [Section 80] 80. The method of claim 79, wherein the formulating step comprises freeze-drying or spray-drying the isolated natural bioactive nematicidal crystals. [Section 81] 80. The method according to claim 79, wherein the formulating step comprises filling the isolated and purified natural bioactive nematicidal crystals into a pharmaceutical capsule. [Section 82] 1. A method for treating a parasitic or helminthic infection in a subject, comprising: A method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising isolated, naturally occurring, bioactive nematicidal crystals formed from a single species of nematicidal crystal protein, wherein the pharmaceutical composition is substantially free of bacterial spores or host bacterial proteins other than the nematicidal crystal protein in the form of a crystal. [Section 83] 83. The method of claim 82, wherein the nematicidal crystal protein is selected from the group consisting of Cry5B, Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, Cry55B, and variants and truncations thereof. [Section 84] 83. The method according to claim 82, wherein the nematicidal crystal protein is Cry5B. [Section 85] 83. The method according to claim 82, wherein the nematicidal crystal protein is Cry5B variant Ser407Cys. [Section 86] 83. The method of claim 82, wherein the pharmaceutical composition further comprises a second crystal protein in the form of isolated natural bioactive nematicidal crystals, said crystals being formed solely from said second crystal protein. [Section 87] 83. The method according to claim 82, wherein the pharmaceutical composition comprises an isolated natural bioactive nematicidal crystal content of at least about 95%. [Section 88] 83. The method according to claim 82, wherein the pharmaceutical composition is in dry powder form and is encapsulated in a pharmaceutical capsule. [Brief description of the drawings]

[0032] [Figure 1]Figures 1A-1B show the conserved amino acid blocks in wild-type crystal proteins. Figure 1A shows the location of the conserved blocks among several Cry proteins. de Maagd, RA, et al. "How Bacillus thuringiensis has evolved specific toxins to colonize the insect world." Trends in Genetics 17(4): 193-99, 195 (Figure 2a) (April 2001). Figure 1B shows the location of the conserved blocks among several Cry proteins. Schnepf, E., et al. "Bacillus thuringiensis and Its Pesticidal Crystal Proteins." Microbiology and Molecular Biology Reviews 62(3): 775-806, 781 (Figure 3) (Sept. 1998). [Diagram 2] FIG. 2 shows the amino acid sequence of Cry5Ba1 [SEQ ID NO:1]. [Diagram 3] FIG. 3 shows the amino acid sequence of Cry13Aa1 [SEQ ID NO:2]. [Figure 4] FIG. 4 shows the amino acid sequence of Cry14Aa1 [SEQ ID NO:3]. [Diagram 5] Figures 5A to 5C show the amino acid sequences of other crystal proteins. Figure 5A shows the amino acid sequence of Cry21Aa1 [SEQ ID NO: 4]. Figure 5B shows the amino acid sequence of Cry21Aa2 (98% identity with Cry21Aa1) [SEQ ID NO: 5]. Figure 5C shows the amino acid sequence of Cry6A [SEQ ID NO: 6]. [Figure 6] FIG. 6 is a flow chart showing the steps for purifying nematicidal crystal protein from inactivated BaCC (IBaCC). [Figure 7] FIG. 7 is an image of a protein gel with samples from several steps of the purification process of Cry5B PCC. [Figure 8]FIG. 8 is a phase contrast micrograph of Cry5B PCC at 1000× magnification. [Figure 9] Figure 9A is a graph showing the time course of Trichuris motility following exposure to Cry5B PCC in vitro, and Figure 9B is a histogram showing in vivo Trichuris burden in hamsters treated with Cry5B PCC and water control. [Figure 10] Figure 10A is a graph showing the time course of hookworm motility after exposure to Cry5B PCC in vitro. Figure 10B is a histogram showing hookworm burden in hamsters treated with Cry5B PCC and water control. Figure 10C is a histogram showing hookworm burden in hamsters before and after treatment with Cry5B PCC and water control. [Figure 11] Figure 11A shows a plot of hookworm burden in hamsters treated with either Cry5B spore-crystal lysate (SCL) or water control, and Figure 11B shows a plot of eggs per gram in hamsters treated with either SCL or water control. [Figure 12] FIG. 12 is a histogram showing the toxicity of various concentrations of Cry5B PCC against Ancyclostoma ceylanicum hookworm eggs; some Cry5B PCC were treated with the addition of lysozyme. [Figure 13] Figure 13A is a diagram showing the protocol for purifying PCC from BaCC. Figure 13B is a microscopic image of BaCC before and after treatment with 10% hexane (top), and a bar graph showing the cell proliferation rate (measured using OD600) of BaCC before and after treatment with 10% hexane (bottom). Figure 13C is an image of an SDS-PAGE gel showing the starting BaCC material and the final purified PCC product. [Figure 14] FIG. 14 is a line graph showing the biological activity of PCC obtained by treating BaCC with 10% hexane to kill the bacteria and with 50% hexane to remove other microbial contaminants (PCC-Hex / Hex). [Figure 15]FIG. 15 is a bar graph showing the biological activity of PCC obtained by treating BaCC with carvacrol to kill the bacteria (IBaCC) and with 50% hexane to remove other microbial contaminants (PCC-Carv&Hex), and PCC obtained by treating BaCC with 10% hexane to kill the bacteria and 50% hexane to remove other microbial contaminants (PCC-Hex&Hex). [Figure 16] FIG. 16 is a line graph showing the biological activity of PCC obtained by treating BaCC with carvacrol to kill the bacteria and with 50% hexane to remove other microbial contaminants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Disclosed are compositions of pure nematicidal crystal proteins, methods for producing such pure nematicidal crystal proteins, and methods for treating or preventing STH infections by administering a formulation of the pure nematicidal crystal proteins to a subject.

[0034] microorganisms In some embodiments, the bacterium of the present disclosure is a non-sporulating bacterium. As used herein, the term "non-sporulating bacterium" encompasses wild-type bacteria that are not capable of sporulation (e.g., certain gram-negative bacteria) and genetic variants of sporulating bacteria that are engineered to lack sporulation (e.g., certain gram-positive bacteria). As used herein, "mutations that result in the absence of sporulation" or "genetic mutations that result in the absence of sporulation" refer to any genetic mutation that results in the absence of a member of the sporulation pathway and / or any genetic mutation that prevents the formation of viable spores. For example, non-sporulating bacteria and their preparation are described in International Patent Application No. PCT / US2017 / 013436, which is incorporated herein by reference.

[0035] In some embodiments, sporulation-deficient bacteria are preferred. An example of a sporulation-deficient bacterium is spoOA-Bacillus thuringiensis. Any mutation or combination of mutations that results in sporulation deficiency but does not substantially affect viability or heterologous gene expression can be used. Such mutations include, but are not limited to, mutations in the following genes: kinA, kinB, spoOA, spoOB, spo0E, spo0F, spo0J, spoOM spoIIB, spoIID, spoIIE, spoIIF, spoIIG, spoIIL, spoIIM, spoIIIA, spoIIIB, spoIIIE, spoIVA, spoIVC, spoIVD, spoVG, spoVK, spoVL, spoVM, spoVN, spoVP, spoVQ, spoVID, σH, σF, σE, σG, and σK. (Silvaggi, J., et al. Unmasking novel sporulation genes in Bacillus subtillus. J Bacteriol. 186, 8089-8095, 2004; Sandman, K., et al. Genetic Analysis of Bacillus subtilis spo Mutations Generated by Tn917-Mediated Insertional Mutagenesis. Genetics. 117, 603-617, 1987; Malvar and Baum, Tn5401 Disruption of the spoOF Gene, Identified by Direct Chromosomal Sequencing, Results in CrylIlIA Overproduction in Bacillus thuringiensis. J Bacteriol. 176, 4750-4753, 1994)

[0036] In some embodiments, recombinant sporulation deletion may also result in the deletion of production of other crystal proteins and other virulence-related sporulation products in the host bacterium. For example, spo0A-Bt does not produce Cry5B or other endotoxins, such as Cry1, Cry4, or Cry8. In some embodiments, genes for other Cry proteins and / or accessory proteins may be deleted or inactivated to ensure that additional Cry proteins are not used to form protein crystals.

[0037] In such embodiments of sporulation-deficient bacteria, the sporulation-deficient bacteria may be engineered to express a single crystal protein gene, such as Cry5B, under the control of a promoter that is actively and / or highly expressed during the vegetative growth or stationary phase of the bacteria prior to the sporulation phase of the bacteria. Such recombinant bacteria may express only a single crystal protein, and the resulting nematicidal crystals will be homogeneously composed of only a single type of crystal protein. In some embodiments, the promoter is heterologous (i.e., a sporulation-deficient specific promoter). In one embodiment, the promoter is a Cry3A, GerA, GNAT or TadA promoter.

[0038] In some embodiments, strains of non-spore-forming bacteria that autolyse at the end of the growth cycle may be used, such that the homogenization step in the purification process may be avoided or reduced.

[0039] Bacteria are particularly applicable to the control of STHs for the following reasons: 1) recombinant bacteria can inexpensively express large amounts of Cry proteins before being administered to the digestive tract of a mammalian subject, and Cry proteins so expressed have been shown to act significantly against STHs, independent of Cry proteins that may be secreted by bacteria in the digestive tract. 2) Studies using purified Cry proteins to combat hookworms, whipworms, and H. bakeri, all conducted in infected rodents, have shown that STHs in the mammalian digestive tract can ingest and be killed / detoxified by the Cry proteins. 3) Recombinant bacteria that express therapeutic proteins without purifying the proteins are cheaper to produce, since no protein purification steps are required. 4) Recombinant bacteria that deliver STH-treating proteins (e.g., Cry5B) are more efficient at treating infections at the same bioactive protein dose (e.g., total Cry5B) than purified proteins (e.g., Cry5B).

[0040] The microorganisms of the disclosed compositions and methods include killed and inactivated forms of Bacillus species, such as Bacillus subtilis (e.g., B. subtilis natto, and B. subtilis PY79), B. cereus (e.g., B. cereus var. Toyoi (Toyocerin), B. cereus var. toyoii), B. toyonensis, B. clausii, B. pumilus, and B. thuringiensis. B. subtilis has been widely characterized as a food additive that is safe for human consumption. In some exemplary embodiments, killed and inactivated forms of B. thuringiensis are used.

[0041] Other useful bacteria include, but are not limited to, non-sporulating variants of the following bacteria: Lactococcus sp., Bifidobacterium sp., Streptococcus sp., Clostridium sp., Sporolactobacillus sp., Sporosarcina sp., Brevibacillus sp., Leuconostoc sp., Pedicoccus sp., Enterococcus sp., and Escherichia sp. Lactobacillus sp. includes, but is not limited to, L. lactis, L. casei, L. paracasei, L. acidophilus, L. bulgaricus, L. delbrueckii subsp. bulgaricus, L. helveticus, L. plantarum, L. salivarius, L. reuteri, L. gasseri, and L. animalis. Bifidobacterium sp. includes, but is not limited to, B. animalis, B. bifidum, B. breve, B. infantis, and B. longum. Streptococcus sp. includes, but is not limited to, S. thermophilus. Clostridium sp. includes, but is not limited to, Clostridium butyricum. Sporolactobacillus sp. includes, but is not limited to, Sporolactobacillus vineae. Sporosarcina sp. includes, but is not limited to, Sporosarcina pasteurii. Brevibacillus sp. includes, but is not limited to, Brevibacillus laterosporus.

[0042] Further bacteria useful in the context of the present disclosure include forms of gram-negative bacteria. In some exemplary embodiments, gram-negative bacteria include E. coli species (e.g., NISSLE 1917) and Pseudomonas species (e.g., Pseudomonas fluorescens). Examples of Cry-expressing gram-negative bacteria that can be killed or inactivated by the methods of the present disclosure include the Cry-expressing E. coli strain of Ge et al. ("Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product", Gene, 93: 49-54 (1990)) and the P. fluorescens strain of Peng et al. ("A Delta-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity", App. Microbiol Biotech., (2003), 63:300-306).

[0043] Nematicidal Protein As used herein, the term "nematicidal protein" refers to any protein that exhibits toxic activity against nematodes or helminths, unless the context makes clear otherwise. Examples of nematicidal proteins include crystal proteins such as bacterial antiparasitic Cry proteins (e.g., Crickmore et al., 1998 Microbiology and Molecular Biology Reviews 62(3): 807-813; Schnepf et al., 1998 Microbiology and Molecular Biology Reviews 62(3): 775-806; including but not limited to B. thuringiensis Cry proteins Cry5B (e.g., SEQ ID NO: 1) and sub-variants thereof, Cry13A (e.g., SEQ ID NO: 2) and sub-variants thereof, Cry14A (e.g., SEQ ID NO: 3) and sub-variants thereof, Cry21A (e.g., SEQ ID NOs: 4-5) and sub-variants thereof, and Cry6A and sub-variants thereof (e.g., SEQ ID NO: 6)) for delivery by oral administration (ingestion, drink, food, pill, capsule, powder, etc.) to the digestive tract of a vertebrate infected with a helminth (e.g., roundworm). Cry proteins are expressed in the cytosol of bacteria, forming crystals, and the anthelmintic protein becomes accessible upon lysis or release of the bacteria. Nematicidal crystals formed from nematicidal crystal proteins (e.g., Cry proteins) induce toxicity in worms by releasing the individual nematicidal crystal proteins upon solubilization or decrystallization, allowing the crystal proteins to act directly on the worms.

[0044] The nematicidal crystals of the present invention are more stable and resistant to proteolysis than the individual crystal proteins that form them. The crystal proteins expressed by the bacteria of the present invention form crystals of about 100 kDa to about 170 kDa, about 110 kDa to about 160 kDa, about 120 kDa to about 150 kDa, about 125 kDa to about 145 kDa, or about 130 kDa to about 140 kDa. The crystal proteins aggregate to form larger nematicidal crystals.

[0045] Each nematicidal crystal may have a size along its longest axis of about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, about 1400 nm, about 1500 nm, about 1600 nm, about 1700 nm, about 1800 nm, about 1900 nm, or about 2000 nm.

[0046] In some embodiments, the length of the longest axis of each nematicidal crystal of the present invention may be about 100 nm to about 2000 nm, about 200 nm to about 2000 nm, about 300 nm to about 2000 nm, about 400 nm to about 2000 nm, about 500 nm to about 2000 nm, about 600 nm to about 2000 nm, about 700 nm to about 2000 nm, about 800 nm to about 2000 nm, about 900 nm to about 2000 nm, or about 1000 nm to about 2000 nm.

[0047] In some embodiments, the length of the longest axis of each nematicidal crystal of the present invention may be about 100 nm to about 1000 nm, about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 900 nm to about 1000 nm, or about 1000 nm to about 1000 nm.

[0048] In some embodiments, the pure nematicidal crystal formed from a first type of nematicidal crystal protein (e.g., Cry5B) can be combined with the nematicidal crystal formed from a second type of nematicidal crystal protein (e.g., Cry5C, Cry5D, Cry6A, Cry13A, Cry14A, Cry21A, Cry21B, or Cry55B) in one pharmaceutical preparation. In such an embodiment, it is possible to simultaneously deliver multiple forms of pure nematicidal crystal to the digestive tract in one preparation. For example, the simultaneous administration of Cry5B and Cry21A to the digestive tract can prevent the development of parasite resistance to the combination therapy, since there is no cross-resistance between Cry5B-resistant roundworms and Cry21A-resistant roundworms.

[0049] In long-term practice, to contain recombinant bacteria environmentally, it may be desirable to eliminate antibiotic selection capability (e.g., genetic selection markers) from the plasmid used to introduce the heterologous Cry protein coding sequence and to use bacterial strains that are unable to replicate outside of a vertebrate host. For example, LAB (Lactic Acid Bacteria) have been engineered to be autotrophic in thymidine or thymine synthesis, such that they will not grow in an environment without thymidine or thymine, but can only grow in the intestine of a vertebrate where thymidine and thymine are present. See, e.g., Steidler L, et al. "Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10." Nat Biotechnol 21: 785-789 (2003).

[0050] Cry transformed bacteria, such as Bacillus or LAB, can be cultured and expression of intracellular, membrane-anchored, or secreted Cry proteins by such bacteria can be confirmed using antibodies directed against each Cry protein and standard Western blotting or ELISA techniques.

[0051] To assess the biological activity of all constructs, recombinant bacteria expressing Cry proteins (full length, truncated, or variant) can be fed to C. elegans. Cry protein toxicity to C. elegans can then be quantified by LC50, brood size, and growth inhibition assays on solid media and in liquid wells. C. elegans can access the Cry protein via secreted proteins on solid media / in liquid wells or by the ability of the worms to crush, liberate, and digest bacteria. Confirmation can be obtained that the recombinant bacteria are producing biologically active Cry proteins. Additionally, biological activity (e.g., LC50 in μg / mL) can be measured.50 ) can be quantified to determine the construct exhibiting the highest activity.

[0052] Truncation, variants, and subvariants Crystal proteins can be truncated to improve their effectiveness. The usefulness of Bt toxins (e.g., crystal proteins) for STH control can be limited by the size of the protein that the STH can ingest. Some parasitic roundworms have difficulty ingesting proteins larger than about 40 kDa. Thus, the effectiveness of any particular Bt toxin can be limited by the size exclusion of the protein that the STH ingests, and therefore should be small enough to be easily absorbed into the STH intestine while maintaining toxic activity. Truncated toxins can be easier to express in bacteria. Producing truncated toxins also relaxes the requirement that the target STH must have the correct protease for correct processing of the full-length protoxin (inactive) to the truncated active toxin form. Thus, the truncated toxin is immediately available for intoxication, regardless of whether the correct protease processing enzyme is present in the STH target. Also, truncated toxins may be expressed at higher levels in microorganisms because they are soluble in the cells expressing them and are less likely to form insoluble inclusion bodies (which may be toxic to the cells or result in misfolding of the toxin). Thus, it is desirable to produce truncated Bt toxin fragments (e.g., crystal protein fragments). "Truncated," when referring to Bt toxin proteins (crystal proteins), refers to Bt toxin proteins that are not full length but that retain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the toxin activity of the corresponding full-length Bt toxin protein.

[0053] A "variant" or "subvariant" of a Cry protein includes a polypeptide having one or more substitutions, such as 20 or fewer substitutions or 10 or fewer substitutions, or substitutions at 10% or fewer residues, relative to the corresponding wild-type polypeptide or a truncated version thereof. The variant, subvariant, or truncated polypeptide has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity, such as toxic activity, of the corresponding wild-type polypeptide or truncated version. Conservative substitutions include substitutions within the following groups: glycine, alanine, threonine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, cysteine; lysine, arginine; aspartic acid, glutamic acid; serine, threonine; asparagine, glutamine; phenylalanine, tyrosine. One example of a variant Cry protein is Cry5B (SEQ ID NO: 7), in which the serine at position 407 has been replaced with a cysteine ​​(Ser407Cys).

[0054] The crystal proteins may be full-length, truncated, variant, or subvariant. Truncated crystal proteins may include any truncation of the N- and C-terminus that maintains toxin activity. The truncated forms are not full-length but maintain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the toxic activity of the corresponding full-length Bt toxin protein. For example, the truncated portion may be truncated between the end of conserved block 5 and the C-terminus of the full-length protein. Figures 1A and 1B show a diagram comparing the numbered conserved amino acid blocks (1-5) in various Cry proteins.

[0055] In one embodiment, the truncated crystal protein may comprise the toxin domain of the crystal protein and optionally up to 5, 10 or 20 additional amino acids. The truncated crystal protein may be truncated after the conserved amino acid sequence of block 5 and may optionally comprise up to 5, 10 or 20 additional amino acids. The conserved amino acid sequence of block 5 may comprise the motif DRIEF (SEQ ID NO:23), DRLEF (SEQ ID NO:24) or some other related sequence followed by amino acid residues, for example 3 amino acids upstream and 2 amino acids downstream of the motif. Table 1 shows the block 5 sequences of various Cry proteins. See, e.g., Schnepf, E., et al., Bacillus thuringiensis and Its Pesticidal Crystal Proteins, Microbiology and Molecular Biology Reviews 62(3): 775-806, (e.g., p. 781, Figure 3) (Sept. 1998); and Crickmore et al., Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins, Microbiology and Molecular Biology Reviews 62(3): 807-813 (Sept. 1998). The truncated crystal protein may be truncated at the N-terminus. For example, the truncated crystal protein may not include about the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids at the N-terminus.

[0056] Cry protein variants may exhibit at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any of the known Cry protein sequences, e.g., those disclosed in Crickmore et al., 1998 Microbiology and Molecular Biology Reviews 62(3): 807-813, or Schnepf et al., 1998 Microbiology and Molecular Biology Reviews 62(3): 775-806, including full-length Cry proteins and truncated Cry proteins, Cry protein variants or sub-variants thereof. Also contemplated in some embodiments are polynucleotides encoding such Cry proteins and truncations and variants thereof.

[0057] [Table 1]

[0058] In Table 1, the consensus sequence indicates the position where at least 75% of the aligned proteins of the group have the same or a conservative amino acid sequence. A capital letter in the sequence indicates that at least 75% of the residues at that position are identical. A lower case letter indicates that at least 75% of the residues at that position are conservative. Conserved amino acids are those belonging to the following groups: a (A, G, S, T or P); d (D, E, N or Q); f (F, W or Y), I (I, L, M or V), and k (K or R).

[0059] The truncated crystal protein may be a truncated form of Cry5B, such as B. thuringiensis Cry5B (Figure 2). The truncated Cry5B may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 to at least about amino acid 693. The truncated form of Cry5B may further include up to 5, 10, 20, 30, 40 or 50 amino acids from the C-terminus after conserved block 5, for example up to about amino acid 698, 703, 713, 733 or 743.

[0060] The truncated crystal protein may be a truncated form of Cry13A, such as B. thuringiensis Cry13A (Figure 3). The truncated Cry13A may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 to at least about amino acid 688. The truncated form of Cry13A may further include 5, 10, 20, 30, 40 or 50 amino acids from the C-terminus after conserved block 5, for example to about amino acid 693, 698, 708, 718, 728 or 738.

[0061] The truncated crystal protein may be a truncated form of B. thuringiensis Cry14A (Figure 4). The truncated Cry14A may extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 to at least about amino acid 675. The truncated form of Cry14A may further include 5, 10, 20, 30, 40 or 50 amino acids from the C-terminus after conserved block 5, for example to about amino acid 680, 685, 695, 705, 715 or 725.

[0062] The truncated crystal protein can be a truncated form of Cry21A, such as B. thuringiensis Cry21Aa1 (FIG. 5A) or Cry21Aa2 (FIG. 5B). The truncated Cry21A can extend from about amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to at least about amino acid 685. The truncated form of Cry21A can further include 5, 10, 20, 30, 40, or 50 amino acids from the C-terminus after conserved block 5, for example, to about amino acid 690, 695, 705, 715, 725, or 735.

[0063] Nucleic acid molecules encoding amino acid sequence variants, truncated versions, or both of Cry proteins are produced by various methods known in the art. Such methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or production by, for example, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis in previously produced variant or non-variant versions of the protein. In addition, the technology includes synthetic nucleic acid molecules in which nucleotides are modified to include codons that are preferred in a particular organism, to eliminate codons that are less frequently used in a particular organism, or to eliminate sequences that may inhibit transcription or RNA processing, etc.

[0064] Cry protein truncations may include at least conserved blocks 1-5. As shown in Figures 1A and 1B, an alignment of known Cry toxins reveals five conserved sequence blocks (blocks 1-5) that are common to many of the proteins and are believed to be located in the active toxin domain. See de Maagd, RA, et al. "How Bacillus thuringiensis has evolved specific toxins to colonize the insect world." Trends in Genetics 17(4): 193-99 (April 2001). Comparison of the sequences at the carboxy-terminal end suggests that there are three additional blocks outside the active toxin core. See Schnepf, E., et al. "Bacillus thuringiensis and Its Pesticidal Crystal Proteins." Microbiology and Molecular Biology Reviews 62(3): 775-806 (Sept. 1998). Thus, the Cry protein truncation can be truncated after the conserved block 5 amino acid sequence (e.g., DRIEF (SEQ ID NO:23) or DRLEF (SEQ ID NO:24)). Alternatively, the Cry protein truncation can be truncated after the conserved block 5 amino acid sequence (e.g., DRIEF (SEQ ID NO:23) or DRLEF (SEQ ID NO:24)) plus approximately 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of the C-terminal domain.

[0065] The complete amino acid sequence of Cry5Ba1 is shown in Figure 2. In Cry5B, the conserved amino acid sequence DRIEF (SEQ ID NO:23) ends at amino acid number 693. Thus, truncated forms of Cry5B may include at least amino acids 50 to about 693. Truncated forms of Cry5B may extend from about amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 to at least about amino acid 693. Alternatively, or in addition, truncated forms of Cry5B may include about 5, 10, 15, 20, 25, 30, 35 or 40 additional amino acids of the C-terminal domain.

[0066] The complete amino acid sequence of Cry13Aa1 is shown in Figure 3. In Cry13A, the conserved amino acid sequence DRLEF (SEQ ID NO:24) ends at amino acid number 688. Thus, truncated forms of Cry13A may include at least amino acids 50 to about 688. Truncated forms of Cry5B may extend from about amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 to at least about amino acid 688. Alternatively, or in addition, truncated forms of Cry13A may include about 5, 10, 15, 20, 25, 30, 35 or 40 additional amino acids of the C-terminal domain.

[0067] The complete amino acid sequence of Cry14Aa1 is shown in Figure 4. In Cry14A, the conserved amino acid sequence DRIEF (SEQ ID NO:23) ends at amino acid number 675. Thus, truncated forms of Cry14A may include at least amino acid 50 to approximately 675. Truncated forms of Cry5B may extend from approximately amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 to at least approximately amino acid 675. Alternatively, or in addition, truncated forms of Cry14A may include approximately 5, 10, 15, 20, 25, 30, 35 or 40 additional amino acids of the C-terminal domain.

[0068] The complete amino acid sequences of Cry21Aa1 and Cry21Aa2 are shown in Figures 5A and 5B, respectively. The amino acid sequence of Cry21Aa2 is about 98% identical to the amino acid sequence of Cry21Aa1. In Cry21A, the conserved amino acid sequence DRIEF (SEQ ID NO:23) ends at amino acid number 685. Thus, a truncated form of Cry21A may include at least amino acids 50 to about 685. A truncated form of Cry5B may extend from about amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 to at least about amino acid 685. Alternatively or in addition, a truncated form of Cry21A may include about 5, 10, 15, 20, 25, 30, 35 or 40 additional amino acids of the C-terminal domain.

[0069] Anthelmintic experiments Once heterologous Cry protein expression and biological activity is confirmed in a desired bacterium, the recombinant bacteria may be used for therapeutic and prophylactic antiparasitic experiments.

[0070] Antibody production: Antibodies against recombinant Cry proteins (e.g., Cry5B, Cry21A, Cry14A, Cry13A and Cry6A, full-length and truncated proteins) may be produced and purified according to standard methods (e.g., Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009)).

[0071] Biological activity testing: To test the biological activity of all constructs, recombinant Bacillus or other bacteria expressing heterologous Cry proteins are fed to the free-living nematode C. elegans. C. elegans can access the Cry proteins via proteins secreted onto solid media / into liquid wells, by proteins naturally released when bacteria break open, or by the worm's ability to disrupt and digest bacteria to release bacterial cells.

[0072] Rodent parasite studies: Three intestinal nematodes are studied: H. bakeri (small intestinal nematode) in mice, Trichuris muris (whipworm) in mice, and A. ceylanicum (hookworm) in hamsters. The studies examine 1) whether and for how long heterologous Cry-expressing bacteria can colonize the gut; and 2) how the bacteria affect the acquisition and progression of intestinal nematode infection.

[0073] Parasite testing: The best heterologous Cry protein expressing recombinant bacterial strain based on expression and bioactivity is orally ingested into naive (uninfected) mice.

[0074] Course amelioration test: Mice are infected with H. bakeri. After 2 weeks, infected mice are treated with heterologous Cry protein expressing bacteria or control bacteria, respectively. The burden of worms in the intestine and the number of eggs in the feces are used to determine whether the recombinant bacteria have an eradicating effect in mice already infected with nematodes.

[0075] Examples of parasitesThe methods of the disclosure relate to the control of parasites, such as nematodes and flatworms, using crystal proteins from Bacillus and their derivatives. Parasites included within the scope of the present invention include, but are not limited to, those belonging to the Class Adenophorea, such as the Orders Mononchida, Family Plectidae, and Orders Stichosomida, Family Mermithidae, and Tetradonematidae; the Class Secernentea, such as the Orders Rhabditida, Family Carabonematidae, Cephalobidae, Chambersiellidae, Heterorhabditidae, Oxyuridae, Panagrolaimidae, Rhabditidae, Steinernematidae, Syrphonematidae, Syrphonematidae, or Thelastomatidae; the Orders Spirurida, Family Filariidae, Onchocercidae, Physalopteridae, Syngamidae, Spiruridae, Subuluridae, or Thelaziidae; the Orders Diplogasterida, Family Diplogasteridae; and the Order Tylenchida, Family Allantonematidae, Aphelenchidae, Aphelenchoididae, Entaphelenchidae, Fergusobiidae, Phaenopsitylenchidae, Sphaerulariidae, Anguinidae, Dolichodoridae, Belonolaimidae, Pratylenchidae, Hoplolamidae, Heteroderidae, Criconematidae, Tylenchulidae, or Tylenehidae.In one embodiment, the parasite belongs to the Class Secernentea, Order Ascaridida, Family Ascarididae; Class Adenophorea, Order Trichurida, Family Trichuridae; Class Secernentea, Order Strongylida, Family Ancylostomatidae (ancylostomidae) or Trichostrongylidae; or Class Secernentea, Order Spirurida, Family Dracunculidae, Filariidae, or Onchocercidae.

[0076] The parasite may be a helminth. Helminths within the scope of the present invention include, but are not limited to, those belonging to the following: Phylum Annelida, Class Polychaetae, Class Myzostomida, Class Clitellata, Subclass Hirudinea, Order Gnathobdellidae, Order Rhynchobdellidae; Phylum Platyhelminthes (Flatworms), Class Turbellaria, Class Monogenea, Order Monopisthocotylea, Order Polyopisthocotylea, Class Trematoda, Subclass Aspidogasrea, Subclass Digenea; Super Order Anepitheliocystida, Order Strigeatida, Family Schistosomatidae, Subfamily Schistosomatinae, Genus Schistosoma, Order Echinostomatida, Family Fasciolidae, Family Paramphistomatidae, Family Echinostomatidae; Super Order Epitheliocystida, Order Plagiorchiida, Family Dicrocoeliidae, Family Troglotrematidae, Order Opisthorchiida, Family Heterophyidae, Family Opisthorchiidae, Class Cestoda, Subclass Cestodaria, Subclass Eucestoda, Order Pseudophyllidea, Family Diphyllobothriidae, Order Cyclophyllidea, Family Taeniidae, Family Hymenolepididae, Family Dilepididae, Family Mesocestoididae, Order Tetraphyllidea, Order Proteocephalata, or Order Spatheobothridea.For example, Cry proteins within the scope of the present invention may be used to inhibit, suppress or combat roundworms, whipworms, hookworms, schistosomes or flukes.

[0077] The parasite may be a gastrointestinal parasitic roundworm / nematode.Gastrointestinal parasitic roundworm / nematode includes, but is not limited to, the following species: Haemonochus, Cooperia, Ostertagia, Trichostrongylus, Teladorsagia, Nematodirus, Ancylostoma, Cyathostominea / Cyathostomin / Cyathostome, Strongylus, Parascaris, Ascaris, Trichuris, Oesophagostomum / Oesophagustomum, Trichiuris, Bunostomum, Oxyuris, Chabertia, Habronema, Draschia, Triodontophorus, Toxocara, Toxascaris, and Uncinaria.Haemonochus species includes, but is not limited to, Haemonchus contortus and Haemonchus placei. Cooperia species include, but are not limited to, Cooperia oncophora, Cooperia pectinata, and Cooperia curticei. Ostertagia species include, but are not limited to, Ostertagia ostertagi, Ostertagia (Teladorsagia) circumcincta, and Ostertagia trifurcate. Trichostrongylus species include, but are not limited to, Trichostrongylus axei, Trichostrongylus colubriformis, and T. circumcincta. Teladorsagia species include, but are not limited to, Teladorsagia (Ostertagia) circumcincta. Nematodirus species include, but are not limited to, Nematodirus spathiger. Ancylostoma species include, but are not limited to, Ancylostoma caninum, Ancylostoma braziliense, and Ancylostoma tubaeforme.Cyathostominea / Cyathostomin / Cyathostome nematodes are also included. Strongylus species (small and large) include, but are not limited to, Strongylus vulgaris, Strongylus equinus, and Strongylus edentatus. Parascaris species include, but are not limited to, Parascaris equorum. Strongyloides species include, but are not limited to, Strongyloides westeri. Ascaris species include, but are not limited to, Ascaris suum. Trichuris species include, but are not limited to, Trichuris globulosa, Trichuris suis, Trichuris campanula, and Trichuris vulpis. Oesophagostomum / Oesophagustomum species include, but are not limited to, Oesophagustomum dentatum, Oesophagustomum quadrispinulatum, Oesophagostomum columbianum, and Oesophagostomum venulosum. Trichiuris species include, but are not limited to, Trichiuris ovis. Bunostomum species include, but are not limited to, Bunostomum trigonocephalum. Oxyuris species include, but are not limited to, Oxyuris equi (pinworm). Chabertia species include, but are not limited to, Chabertia ovina. Habronema species include, but are not limited to, Habronema microstoma, and Habronema muscae. Draschia species include, but are not limited to, Draschia megastoma. Triodontophorus species include, but are not limited to, Triodontophorus minor, and Triodontophorus serrates.Toxocara species include, but are not limited to, Toxocara canis and Toxocara cati. Toxascaris species include, but are not limited to, Toxascaris leonine. Uncinaria species include, but are not limited to, Uncinaria stenocephala. Roundworms that infest the human digestive tract include, but are not limited to, the hookworms Ancylostoma duodenale and Necator americanus, the whipworm Trichuris trichiura, the roundworm Ascaris lumbricoides, the nematode Strongyloides stercoralis, and the pinworm Enterobius vermiculari.

[0078] Further Treatment Agents In some embodiments, the pharmaceutical composition of the present invention is administered in combination with at least one additional therapeutic agent. The additional therapeutic agent can be, for example, a small molecule or a polypeptide (including antibodies and fragments thereof). In further embodiments, the additional therapeutic agent is a nicotinic acetylcholine receptor agonist. In some embodiments, the additional therapeutic agent is administered simultaneously with the pharmaceutical composition of the present invention. In some embodiments, the additional therapeutic agent is administered before or after (in any order) the pharmaceutical composition of the present invention. In some embodiments, the nicotinic acetylcholine receptor agonist is selected from the levamisole family of nicotinic acetylcholine receptor agonists. In some embodiments, the nicotinic acetylcholine receptor agonist is levamisole. In some embodiments, the levamisole is administered in an amount of about 0.1 mg / kg to about 5.0 mg / kg. In some embodiments, the nicotinic acetylcholine receptor agonist is pyrantel or tribendimidine. In some embodiments, the pyrantel is administered in an amount of about 1.0 mg / kg to about 15.0 mg / kg. In some embodiments, tribendimidine is administered in an amount of about 0.25 mg / kg to about 10 mg / kg.

[0079] Administration, dosage forms, pharmaceutical compositions The present invention contemplates the administration of pure crystalline protein to the digestive tract of a subject. Thus, the pharmaceutical composition of the present invention can be formulated for oral administration. Oral administration is preferably performed as an aqueous suspension, emulsion, powder or solid. The composition of the present invention can be incorporated into food or added to food by the user before consumption. Administration to the digestive tract can also be in the form of a rectal suppository (e.g., a gel or semi-solid formulation). Any of these preparations can be prepared using standard methods.

[0080] The treatment method of the present invention is typically carried out on any animal for which it is desired to suppress pathogens or parasites. In some embodiments, the animal is a human. However, the animal can be any livestock or zoological individual for which parasite / pathogen suppression provides economic and health benefits. The method of the present invention can be beneficial to any animal, including birds, reptiles, mammals, such as horses, cattle, sheep, goats, pigs and other livestock, or various animals of zoological interest. Other objectives are readily apparent to those familiar with the art of nutrient absorption, dietary utilization and bioavailability.

[0081] The present invention further contemplates a treatment system for treating, alleviating and / or preventing parasitic infections. The system is typically in the form of a package that contains the treatment composition of the present invention or in combination with a packaging material. The packaging material includes a label or instructions for use of the components of the package. The instructions indicate the intended use of the packaged components as described herein for the method or composition of the present invention. For example, the system may include, but is not limited to, one or more unit doses of the treatment composition of the present invention. Alternatively, the system may include a bulk amount of the treatment composition. The label includes instructions for using the treatment composition in appropriate unit dose or bulk form, and may also include information regarding storage of the composition, applicable diseases, dosage, route and method of administration, etc.

[0082] Additionally, the system of the present invention may include one or more of the following ingredients in integral or separate packaging depending on the particular intended use: ingredients such as bifidogenic oligosaccharides, flavorings, carriers, etc. One embodiment includes a unit dose package of pure nematicidal crystal protein for use in combination with a conventional liquid product, together with instructions for combining said pure nematicidal crystal protein with said product for use in a treatment method.

[0083] Different dosing regimens can be used in the method of the present invention. In some embodiments, daily dosing is performed once, twice, three or four times a day for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. In some embodiments, daily dosing is performed once or twice a day on alternate days.

[0084] Administration of a composition containing an active ingredient effective in inhibiting parasitic proliferation in the intestine and feces typically involves the administration of 1 to 10 unit doses of the composition, with each dose containing 10 mg to 10 g, for a human weighing about 100 kg, for one day to one month. The administration of a unit dose is typically performed every 12 to 4 hours. Preferably, administration of 2 to 4 doses of the composition per day (each containing about 0.1 g to 50 g per dose) for one to seven days is sufficient to achieve the desired results.

[0085] In various specific embodiments, an effective dose of the composition of the present invention may range from about 0.01 mg / kg to about 100 mg / kg of the composition for an adult patient, preferably from about 0.1 mg / kg to about 10 mg / kg of the composition. The effective dose may be administered to the subject at any suitable frequency, for example at least once a week, preferably once a day. Pediatric doses may range from 15% to 90% of the adult dose.

[0086] In some other embodiments, the compositions of the present invention may be administered at a constant continuous dosage, for example, about 2 g to about 4 g / day to about 6 g to about 10 g / day, depending on the severity of the physiological condition. Once the infection has been effectively alleviated, in many cases, the dosage administered to the subject may be reduced to about 2 g to about 4 g / day for maintenance purposes. The desired dose may be administered as multiple (e.g., 2, 3, 4, 5, 6 or more) divided doses at appropriate intervals throughout the day.

[0087] The pharmaceutical composition of the present invention can be administered by any acceptable mode or means of administration known in the art. However, oral administration is preferred because this route of administration delivers the pure nematicidal crystal protein to the digestive tract. The dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage form, such as tablets, pills, soft or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, etc., and can be in the form of a unit dose suitable for convenient administration at a precise dosage. An exemplary embodiment of the dosage form is a capsule containing the pure nematicidal crystal protein of the present invention in dry form in combination with a pharmaceutical carrier. The capsule for such dosage form can be of any suitable type, such as a conventional variety of gelatin capsules.

[0088] The physiologically compatible carrier medium used in combination with the pure nematicidal crystal protein can be of any simple type, such as a pharma- ceutically acceptable carrier, such as a fructooligosaccharide (FOS) medium, or other soluble fiber, sugar, nutrient or base for the composition that can be used to formulate the bacterial species (e.g., into an oral dosage form). Other carrier media include mannitol, inulin (a polysaccharide), polydextrose, arabinogalactan, the polyols lactulose, lactitol, etc. As will be apparent to those skilled in the art, a variety of materials can be used as carrier media in the practice of the present invention based on the description herein.

[0089] In various embodiments, the carrier medium, if present, can be combined with the bacterial species in any suitable amount, for example, 5% to 95% by weight of the carrier medium based on the total weight of the bacterial species and the carrier medium. In some other embodiments, the amount of the carrier medium can range from a lower limit of 5%, 10%, 12%, 15%, 20%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, or 75% to an upper limit of 20%, 22%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% that is greater than the lower limit. The amount of the carrier medium in a particular embodiment can be determined by considering the particular dosage form, the relative amount of pure nematicidal crystal protein, the total weight of the composition including the carrier medium and the bacterial species, and the physical and chemical properties of the carrier medium, as well as other factors, as will be understood by those skilled in the art of probiotics and pharmaceutical formulations.

[0090] In some embodiments, the pure nematicidal crystal protein is formulated into a composition that protects the Cry protein from the acidic environment of the stomach. Thus, the present invention encompasses a composition comprising a pure nematicidal crystal protein and a pharma- ceutically acceptable acid-resistant ("enteric") carrier. By acid-resistant, it is meant that the carrier or coating does not dissolve in an acidic environment. The acidic environment is characterized by a pH of less than 7. The acid-resistant carrier is resistant to acids below a pH of about 4.0. Preferably, the acid-resistant carrier does not dissolve at a pH of 2-3. Most preferably, the acid-resistant carrier does not dissolve at a pH of less than 2. For protection of the pure nematicidal crystal protein from stomach acid, the pure nematicidal crystal protein is coated with or encapsulated in an acid-resistant carrier.

[0091] In some embodiments, the coating is pH sensitive. For example, the coating can dissolve when the pH is increased above 4.0. For example, the coating dissolves in a neutral environment such as in the small intestine, but does not dissolve in an acidic environment such as in the stomach. Alternatively, the enteric coating dissolves when exposed to a specific metabolic event, such as contact with the digestive enzymes present in the small intestine. For example, the coating is digested by pancreatic enzymes, such as trypsin, chymotrypsin or pancreatic lipase. The formulation is hydrated in the small intestine. The digestion or dissolution of the coating can release pure nematicidal crystal protein, such as pure Cry5B, into the intestine.

[0092] In some other embodiments, the pure nematicidal crystal protein is stabilized in a gel or paste, such as an anhydrous carbohydrate paste. In other formulations, the pure nematicidal crystal protein is freeze-dried and / or suspended in a gel or paste. Enteric coating materials are known in the art, examples of which include malic acid-propane 1,2-diol. Cellulose derivatives, such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate (HPMCP), are also useful in enteric acid-resistant coatings. Other suitable enteric coatings include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and anionic polymers of methacrylic acid and methyl methacrylate. Yet another suitable enteric coating is ethyl acrylate-methylacrylic acid copolymer in aqueous emulsion form, or hydroxypropyl methylcellulose acetate succinate (HPMAS). (See, e.g., U.S. Pat. No. 5,591,433.) The enteric coating is designed to resist dissolution in the stomach and dissolve in neutral or alkaline intestinal fluids. In some embodiments, the pure nematicidal crystal protein is preferably prepared into a dry powder. Suitable drying methods include air drying, forced air drying, spray drying, freeze drying, and the like. Of these, spray drying, drum drying, or forced air drying are preferred. Protectants, such as skim milk, monosodium glutamate, and sugar, may be used during drying. Sugar, glucose, and trehalose may be used. In one example of freeze drying, the pure crystal protein may be frozen at -80°C and then placed into a FreeZone 1L benchtop freeze drying system (Labconco catalog number 7740020). The condenser is set to -60°C and the vacuum is set to 22 mTor. The sample is freeze-dried overnight. In one example of spray drying, a PCC sample at 10% w / v solids was spray dried using a Yamato Pulvis GB22 (or any other spray drying system) at 5 mL / min through a 100μ atomizer nozzle with 1 Kgf / m of atomizing air. 2 0.21 m of dry air 3Spray dry at 98°C / min with inlet / outlet temperatures set at 98°C / 59°C, respectively.

[0093] Examples of auxiliary and adjuvant substances include preservatives, wetting agents, suspending agents, sweeteners, flavoring agents, aromatic agents, emulsifying agents, and dispersing agents. If necessary, the prevention of the action of contaminating microorganisms can be achieved by using various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents, such as sugars, sodium chloride, etc., can also be included.

[0094] Solid dosage forms can be prepared with coatings and shells, such as enteric coatings well known in the art. It can contain a deterrent and can be a composition that releases active compound in a delayed manner in a certain part of the intestinal tract. Examples of embedding compositions that can be used are polymeric materials and waxes. Optionally, active compound can be in microencapsulated form with one or more of the above excipients.

[0095] Liquid dosage forms for oral administration include pharma- ceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the active ingredient (e.g., pure nematicidal crystal protein) and, optionally, pharmaceutical adjuvants in a carrier, such as water, saline, aqueous dextrose, glycerol, ethanol, etc.; solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide; oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters; or mixtures of such substances, thereby forming a solution or suspension.

[0096] Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art, see, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, Easton, Pa., 1990).

[0097] method The method of the present invention is for treating a parasitic helminth infection in a subject, and comprises administering to the subject a therapeutically effective amount of a composition comprising an isolated, natural, bioactive nematicidal crystal formed from a single species of nematicidal crystal protein. The pharmaceutical composition of the present invention is substantially free of bacterial spores or host bacterial proteins other than the nematicidal crystal protein in the form of crystals.

[0098] The method of the present invention is also for reducing the severity of a parasitic helminth infection and comprises administering to a subject a therapeutically effective amount of a composition comprising isolated, natural, bioactive nematicidal crystals formed from a single species of nematicidal crystal protein, the pharmaceutical composition being substantially free of bacterial spores or host bacterial proteins other than the nematicidal crystal protein in crystalline form.

[0099] definition As used herein, unless otherwise clear from the context, the term "treatment," and similar terms such as "treated," "treating," and the like, refer to an approach for obtaining beneficial or desired results, which include, and preferably are, clinically desirable results. Treatment can optionally involve amelioration of the symptoms or condition of a disease, or slowing the progression of a disease or condition.

[0100] As used herein, unless the context indicates otherwise, a "subject" refers to a vertebrate, such as a mammal. A mammal can be a cat, rodent, dog, cow, horse, pig, goat, sheep, or primate. In some embodiments, the subject is a human.

[0101] As used herein, unless otherwise clear from the context, "reducing the likelihood of onset," "prevention," and similar terms, such as "prevented," "preventing," and the like, encompass approaches to inhibiting or reducing the likelihood of onset or recurrence of a disease or condition with statistical significance, such as in comparison to the results obtained when the indicated method step is omitted. Similarly, inhibiting or reducing the likelihood of onset or recurrence of symptoms of a disease or condition, or, in some cases, delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition, are also encompassed. As used herein, "prevention" and similar terms also encompass reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to onset or recurrence of the disease or condition. The methods of such and related embodiments may be carried out using an effective or treatment-effective amount of an agent that substantially prevents onset, reduces the severity, or reduces the likelihood of soil-transmitted helminth (STH) infection. As used herein, an "effective amount" or "therapeutically effective amount" of a composition, agent or substance is an amount sufficient to effect a desired biological effect, eg, a beneficial outcome, eg, a clinical outcome.

[0102] In this document, unless the context makes it clear that the opposite is true, "edible oil" includes oils suitable for human or animal consumption. Examples of edible oils include, but are not limited to, oils extracted from plants, such as corn oil, soybean oil, coconut oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil or canola oil, safflower oil, sunflower oil. Edible oils include, but are not limited to, nut oils, such as almond oil, beech nut oil, Brazil nut oil, cashew oil, hazelnut oil, macadamia nut oil, mongongo nut oil, pecan oil, pine nut oil, pistachio oil, walnut oil, and pumpkin seed oil. Edible oils include oils derived from citrus fruits, fruits, melons and gourd seeds, or any edible plant.

[0103] In some preferred embodiments, the compositions of the present invention for treating or reducing the severity or likelihood of STH infection are prepared as pharmaceutical compositions. They can be preferably prepared for oral delivery. The pharmaceutical compositions are prepared so that the drugs contained therein are bioavailable after administration of the compositions to humans.

[0104] In carrying out the several aspects of the invention, it will be understood that, unless otherwise indicated, conventional methods in virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art may be employed, many of which are described below for purposes of illustration, and such techniques are explained at length in the literature. See, for example: Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Animal Cell Culture (R. Freshney, ed., eds., 1984); 1986); Perbal, A Practical Guide to Molecular Cloning (1984), and other similar publications.

[0105] Standard methods may be used for DNA recombination, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification methods may be performed according to manufacturer's specifications or as commonly practiced in the art or as described herein. These and related techniques and procedures may generally be performed according to conventional methods known in the art and described in various general and more specialized texts, which are cited and described herein. Unless otherwise defined, the technical terms used herein in connection with molecular biology, analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry, and the laboratory procedures and techniques of molecular biology, analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry herein, are those known and commonly used in the art. Standard methods may be used for recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery and patient treatment.

[0106] As used herein, the terms "pure" and "purity" in combination with a number (e.g., 95% pure) refer to the compounds and substances of the present disclosure present in weight / weight amounts relative to other compounds and substances. For example, a composition that is 95% pure nematicidal crystals means that 95% (weight / weight) of the composition is nematicidal crystal protein and 5% (w / w) of the composition is one or more other substances.

[0107] As used herein, the term "crystal content" in combination with a percentage (e.g., 95% crystal content) means that the nematicidal crystals of the present disclosure are present in the composition at the stated percentage, based on the total weight of the composition. In some embodiments, the compositions of the present invention have a crystal content of at least 80%, at least 90%, or at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%).

[0108] As used herein, the term "substantially free of" with respect to a composition or compound (e.g., a compound that is substantially free of spores) means that the composition or compound contains 5% w / w or less of other substances (e.g., a compound contains 5% w / w, 2% w / w, 1% w / w, 0.5% w / w, 0.1% w / w, 0.01% w / w, 0.001% w / w or 0.0001% w / w or less).

[0109] In this specification and claims, the singular includes the plural, unless the context clearly dictates otherwise. Throughout this document, unless the context requires otherwise, the term "comprises" or variations thereof, such as "comprising," are understood to be intended to include a recited element or integer or groups of elements or integers, but not to exclude other elements or integers or groups of elements or integers. Unless otherwise specified, each embodiment herein applies to every other embodiment, mutatis mutandis.

[0110] In this specification, the description of a quantity using the term "about" refers to a range of ±5% of the associated value (if the value cannot be further divided by the number of molecules, nucleotides, or amino acids, etc., it is rounded up to the nearest integer). For example, "about 20%" can include 15-20%, including the upper and lower limits, and "about 80%" can include 75-85%, including the upper and lower limits. Furthermore, when the term "about" is used in this specification with respect to a quantity, it is understood that in addition to a value of ±5%, the described numerical value itself is also intended and described. For example, the description "about 23%" clearly intends, describes, and includes exactly 23%. EXAMPLES

[0111] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0112] Preparation of IBaCC To produce Cry5B in sporulation-deficient cells, B. thuringiensis (Bt) strain 4D8, a strain lacking crystal protein expression (Identification of Bacillus thuringiensis subsp. kurstaki strain HD1-Like bacteria from environmental and human samples after aerial spraying of Victoria, British Columbia, Canada, with Foray 48B. Appl Environ Microbiol. 2001 Mar;67(3):1035-43), in which the master spo0A regulator of sporulation (spo0A-) was deleted by homologous recombination. This composition is further designated Cry5B-BaCC (Bacillus with Cytosolic Crystal).

[0113] To inactivate Cry5B-BaCC, the transformed B. thuringiensis 4D8 strain was aerobically cultivated in 3x concentrated Luria-Bertani medium (LB) supplemented with 10 μg / mL erythromycin and 200 μg / mL kanamycin in a baffled 2L flask with a volume of 200 mL for 48 hours with shaking at 30°C. The transformed B. thuringiensis cells were centrifuged at 4500 rpm for 1 hour at 4°C, resuspended in pre-cooled sterile double distilled water in a quarter of the original cell culture volume, and then treated with 1 mg / mL carvacrol (food grade antibacterial agent) for 15 minutes with shaking at 4°C. The carvacrol-treated cells were centrifuged and washed three times with pre-cooled sterile double distilled water. The final pellet was concentrated 40 times and stored at -80°C until use. This dead Bacillus with biologically active Cry5B crystals is referred to as IBaCC (Inactivated Bacillus with Cytosolic Crystal). The 4D8 strain is also capable of autolysis, naturally lysing and releasing the crystals at the end of the growth cycle. Nevertheless, killing with carvacrol and homogenization can be performed to kill any remaining cells and break open any remaining intact IBaCC. EXAMPLES

[0114] Purification of Cry5B from IBaCC by homogenization Figure 6 shows a flow chart of the method used to purify Cry5B crystal protein from IBaCC. To purify large amounts of Cry5B protein from IBaCC, a 10 L culture harvest was performed (Figure 6, step 610). 10 L of BaCC was grown to 250-350 OD / mL. The BaCC was then concentrated to 0.5 L-1 L by centrifugation (8000 x g, 30 min) and then inactivated by adding 1 mL / L carvacrol and stirring at room temperature for 15 min (step 612) to obtain IBaCC. Concentration of BaCC prior to the inactivation step may also be performed by other methods, such as ultrafiltration or dialysis.

[0115] The IBaCC was homogenized at 15,000 psi for 5-7 passes (step 614), and the resulting lysate was centrifuged to concentrate the bacterial lysate (step 616). The IBaCC may be concentrated by other methods, such as ultrafiltration or dialysis. The resulting pellet was resuspended in 0.5 L to 1 L of 1 M NaCl solution (step 618). The concentrated lysate was then mixed with 250 mL / L of oil at room temperature for 2 hours to extract carbachol (step 620). After this oil extraction, 9 L of water was added (step 622), and the mixture was again concentrated by centrifugation (step 624). The concentrated pellet was resuspended in 0.5 L to 1 L of NaCl solution (step 626). The mixture was then washed three times with 10 L of water and concentrated by centrifugation (8000×g, 30 min) (step 628). The resulting composition is pure Cry5B crystals (PCC). In some embodiments, the composition may be spray dried or freeze dried (step 630).

[0116] Figure 7 shows a protein gel with samples applied at various stages of the purification method. Lane 1 of the gel is 5 μL of homogenized IBaCC after growth to 200 nm OD. Lane 2 is a 15 μL sample of homogenized IBaCC concentrated supernatant. Lane 3 is a 15 μL sample of the aqueous phase of the oil extracted IBaCC lysate wash. Lane 4 is a 15 μL sample of the 1 M NaCl wash (step 626). Lane 5 is a 15 μL sample of the phosphate buffered saline wash of pelleted IBaCC. Lane 6 is a 15 μL sample of PCC after step 628 (from a separate batch of BaCC grown to an OD of 312 absorbance 600 units).

[0117] FIG. 8 shows a phase contrast micrograph of the PCC particles at 1000× magnification. EXAMPLES

[0118] Purification of Cry5B from autolysed IBaCC Some BaCC strains, such as 4D8, autolyse at the end of the growth cycle. This autolysis may eliminate or reduce the need for a homogenization step after inactivation with an antimicrobial compound. Thus, in the purification flow chart of FIG. 6, the homogenization step 614 may be omitted or may require fewer passes if an autolysing bacterial strain is used. In such an embodiment, a harvest of a 10 L culture of autolysed BaCC may be performed (FIG. 6, step 610). The 10 L of BaCC may be grown to 250-350 OD / mL. The BaCC may then be concentrated to 0.5 L-1 L by centrifugation and then inactivated by adding 1 mL / L carvacrol and stirring at room temperature for 15 minutes (step 612) to obtain IBaCC. Since IBaCC was autolysed before and / or after inactivation, there was no need for homogenization. Concentration of BaCC before the inactivation step may be performed by other methods, such as ultrafiltration or dialysis.

[0119] The lysate of autolyzed IBaCC may then be concentrated by centrifugation, ultrafiltration, or dialysis (step 616). The resulting pellet may then be resuspended in 0.5 L to 1 L of 1 M NaCl solution (step 618). The concentrated lysate may then be mixed with 250 mL / L of oil at room temperature for 2 hours to extract carbachol (step 620). After this oil extraction, 9 L of water may be added (step 622) and the mixture may again be concentrated by centrifugation, ultrafiltration, or dialysis (step 624). The concentrated mixture may then be resuspended in 0.5 L to 1 L of NaCl solution (step 626). The mixture may then be washed three times with 10 L of water and concentrated by centrifugation, ultrafiltration, or dialysis (step 628). The resulting composition may be pure Cry5B crystals (PCC). In some embodiments, the composition may be spray-dried or freeze-dried (step 630). EXAMPLES

[0120] PCC intoxicates Trichuris in vitro In vitro, whipworm motility was examined after treatment with PCC prepared as described in Example 2. Worms were placed in triplicate per well in RPMI medium containing antibiotics. Doses of PCC(Cry5B) were 20, 100 and 500 μg / mL with four wells per dose. Figure 9A shows a graph of the dose-response effect of PCC(Cry5B) on whipworm motility measured at the indicated time points. Data are expressed as the average motility index per well, where 3=highly motile, 2=slowly motile, and 1=immobile when touched. EXAMPLES

[0121] PCC reduces Trichuris burden in vivo The ability of PCC to reduce whipworm burden was investigated in hamsters. Mice were infected with the whipworm Trichuris muris. Mice were divided into two groups (four mice per group) and treated with water and PCC oil wash with approximately 500 mg / kg Cry5B, prepared as described in Example 2. Both groups were administered water and PCC oil wash by gavage as a single dose in a volume of 0.5 ml. On day 34 post-infection, fecal egg counts before treatment were measured and mice were divided into two groups with equal fecal egg counts. On day 35 post-infection, mice were treated by gavage. On day 41 post-infection (six days after treatment), post-treatment evaluation was performed by dissecting all mice to measure total whipworm counts (cecum). A 45% reduction in whipworm burden was observed (mean 5.5 whipworm counts for water; mean 3 whipworm counts for PCC) (p=0.02).

[0122] FIG. 9B shows a histogram from this study comparing whipworm burden in hamsters treated with water (control) and 500 mg / kg of PCC prepared as described in Example 2. EXAMPLES

[0123] PCC intoxicates hookworms in vitro In vitro hookworm (Necator americanus) motility was examined following treatment with PCC prepared as described in Example 2. Three hookworms were placed in each well, with four wells per dose, at a dose of 250 μg / mL. Figure 10A is a graph showing the time course of hookworm motility following in vitro exposure to Cry5B PCC. PCC showed a dose-dependent effect on hookworm intoxication. Data are expressed as the average motility index per well, where 3 = highly motile, 2 = slowly motile, 1 = immobile unless touched, and 0 = immobile even when touched. EXAMPLES

[0124] PCC reduces hookworm burden in vivo PCC was used at a dose of 10 mg / kg to treat hamsters infected with the hookworm Nector americanus. Six hamsters were infected with N. americanus and placed on an immunosuppressive regimen (dexamethasone in drinking water and injected twice weekly) for the duration of the experiment to prevent the parasite from being eradicated. After hookworm feeding (approximately 60 days later), three hamsters were treated with a single dose of oral PCC 10 mg / kg, and three hamsters were treated with water as a control. Fecal egg count measurements were taken before treatment to confirm worm infestation, and the hamsters were divided into two groups to ensure equal fecal egg counts before treatment. Five days after treatment, the animals were killed and the intestinal hookworm burden was examined.

[0125] Figure 10B is a histogram showing hookworm burden in hamsters treated with Cry5B PCC and water control. Figure 10C is a histogram showing hookworm egg counts in hamster feces before and after treatment with Cry5B PCC and water control. A single dose of 10 mg / kg PCC reduced hookworm burden by 92% (P=0.016) and fecal egg counts by 98% (FEC; P=0.0-19).

[0126] In contrast, spore-containing lysate (SCL) of Cry5 must be administered at four times the dose to achieve comparable hookworm burden reduction in hamsters. Hamsters were infected with the hookworm Nector americanus and treated with a single dose of 40 mg / kg Cry5B. Figure 11A shows a plot of hookworm burden in hamsters after treatment with oral Cry5B SCL or water control. Figure 11B shows a plot of eggs / g in hamsters after treatment with oral Cry5B SCL or water control. A single dose of 40 mg / kg Cry5B SCL resulted in an 88% reduction in hookworm burden and a 95% reduction in fecal egg counts. EXAMPLES

[0127] PCC is an effective toxin against hookworm eggs FIG. 12 is a histogram showing the toxicity of various concentrations of Cry5B PCC (three preparations) compared to Cry5B IBaCC on egg-to-larval development of Ancyclostoma ceylanicum hookworms. Some Cry5B PCCs were treated with the addition of lysozyme, but this did not appear to affect the toxicity of the Cry5B PCC. A. ceylanicum eggs (approximately 65 eggs / well, multiple wells / dose) were placed in buffer with E. coli food source and grown for 7 days at 25°C. On the seventh day, the number of eggs that hatched and developed to the third larval infective stage is determined. Even at low concentrations, PCC is able to inhibit the development of the hookworms with greater activity than IBaCC (IC 50 is approximately 15ng / mL). EXAMPLES

[0128] Improved purification method for pure PCC from BaCC B. thuringiensis expressing Cry5B was cultivated as described in Example 1, i.e., at a 10 L scale (BaCC) (step 631) (Figure 13A). After concentrating the cells 10-fold by centrifugation (4500 rpm, 4°C for 60 min) (step 632), the washed BaCC was suspended in 500 mL (final volume) of water, hexane was added to a final concentration of 10% v / v, and mixed for 1 h at room temperature in a closed vessel to prepare IBaCC (step 633). Figure 13B shows microscopic images illustrating the preparation of IBaCC from BaCC. The left microscopic image shows BaCC, and the right microscopic image shows the destruction of the rod-like structure, indicating the effect of hexane on BaCC. The bar graph in Figure 13B shows the values ​​of cell growth percentage for BaCC (100% cell growth) and BaCC treated with 10% hexane (0% cell growth, complete inactivation). Figure 13C shows an image of an SDS-PAGE gel of the BaCC starting material and the final purified PCC product after being subjected to the methodology shown in the flow chart of Figure 13 A. Other cell inactivation means can be used in place of hexane, such as detergents, antibacterial agents, biocides, etc.

[0129] Pure Cry5B crystals (PCC) were prepared from IBaCC as described in Example 2. Briefly, IBaCC was lysed by homogenization at 15,000 psi for 5-7 passes to release PCC from IBaCC cell wall ghosts (step 634).

[0130] To purify the PCC from soluble cellular components, lipids and cell wall debris by phase partitioning, hexane was added to 50% v / v and mixed vigorously in a closed container at room temperature for 1 hour. The emulsion was transferred to a closed bottle and centrifuged at 4500 rpm at 4°C for 1 hour (step 635). The top layer of hexane was taken and used as waste solvent. The middle layer containing the cell wall debris was taken and discarded. The aqueous layer containing the water-soluble cellular components was taken and discarded, leaving a pellet of PCC. The PCC pellet was washed with water and then frozen and stored. The resulting PCC had a protein content of 98% w / w. Other methods of PCC recovery are possible, such as continuous centrifugation where PCC is collected in the aqueous phase and hexane and interface are collected in the supernatant phase. The final PCC can be stored frozen or freeze-dried or spray-dried and stored at room temperature, 4°C or freezing temperatures. EXAMPLES

[0131] PCC purified by an improved method is an effective toxin against nematodes. In another example, the method of Example 9 was combined with the hexane phase partition method of Example 9. Figure 14 is a graph showing the biological activity of PCC prepared from BaCC using 10% hexane for bacterial inactivation and 50% hexane for removal of other bacterial contaminants (PCC-Hex / Hex). The graph shows unlyophilized PCC as diamonds and lyophilized PCC as squares. Values ​​shown are the average reproductive size after 3 days of single C. elegans starting at larval stage L4 (n=3 per condition). The efficacy of PCC is excellent, as it completely inhibits the emergence of reproductive size at 10 μg / mL. In contrast, literature data shows an average inhibition rate of 70% at this dose (Hu, Y, et al., Proc Natl Acad Sci USA 107: 5955-60 (2010)). These data indicate that the PCC Hex / Hex method is well compatible with lyophilization. EXAMPLES

[0132] PCC purified by an improved method is an effective toxin against hookworms. Figure 15 shows the biological activity of PCC prepared from BaCC using carvacrol for bacterial inactivation (IBaCC) and 50% hexane phase partitioning to remove other bacterial contaminants (PCC-Carv&Hex). Values ​​shown are the percentage of hookworm A. ceylanicum eggs that reached the mature infective larval stage at day 7. Lower bars indicate greater inhibition of hookworm growth. The control was hexane alone in the medium at the same final concentration as in the PCC samples. The data show that hexane alone has no effect on the parasite.

[0133] Figure 16 shows the bioactivity of PCC prepared from BaCC using carvacrol to kill bacteria and 50% hexane to remove other bacterial contaminants. Values ​​shown are motility of A. ceylanicum adult hookworms in vitro assessed at 24 hour intervals, where 3 = active and highly motile; 2 = slow motility; 1 = motionless unless touched (highly intoxicated); 0 = motionless even when touched (presumed dead). The data show a clear dose response with higher PCC doses decreasing motility, indicating that PCC treated with hexane is more bioactive.

[0134] All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated herein by reference. Although the foregoing invention has been described in some detail by way of example for purposes of clarity of understanding, it will be apparent to those skilled in the art in light of the teachings of the invention that changes and modifications may be made thereto without departing from the scope or spirit of the claims.

[0135] While specific steps, elements, aspects and applications of the invention have been shown and described herein for purposes of illustration, it is to be understood that the invention is not limited thereto, as modifications may be made by those skilled in the art, especially in light of the above teachings, without departing from the scope or spirit of the invention. Accordingly, the invention is not to be limited, except as by the appended claims.

[0136] The various embodiments described above can be combined to represent further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature cited herein and / or listed in the application data sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, as necessary, to use concepts from various patents, applications, and documents to represent further embodiments.

[0137] Such and other changes can be made to the embodiments in light of the above detailed description. In general, the terms used in the claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full range of equivalents allowed by the claims.

Claims

1. 1. A method for preparing a pharmaceutical composition comprising isolated, naturally occurring, bioactive nematicidal crystals, comprising: (a) growing a non-sporulating host bacterium genetically engineered to express a single nematicidal crystal protein; wherein said non-sporulating host bacterium produces natural, bioactive nematicidal crystals formed from the single nematicidal crystal protein, said host bacterium being grown in a growth medium; (b) concentrating the nonspore-forming host bacteria; (c) exposing the concentrated nonsporulating host bacteria to an antimicrobial compound comprising a terpene, hexane or formaldehyde to inactivate the concentrated nonsporulating host bacteria; and (d) adding an edible oil to the inactivated nonsporulating host bacteria to extract the antimicrobial compounds, soluble cell components, lipids and cell wall debris from the nematicidal crystals. Including, the host bacterium is Bacillus thuringiensis genetically engineered to have a genetic mutation that results in a sporulation defect such that the natural bioactive nematicidal crystals are trapped in the cytosol of the bacterium, wherein the genetic mutation that results in a sporulation defect is a deletion or inactivation of the spoOA gene; method.

2. The host bacterium has been genetically engineered to express the single nematicidal crystal protein under the control of a sporulation specific promoter, The single nematicidal crystal protein is Cry5B or a variant or truncation thereof having an amino acid sequence with at least 90%, at least 95% or at least 98% sequence identity with SEQ ID NO:1, or is Cry5B variant Ser407Cys or a variant or truncation thereof having an amino acid sequence with at least 90%, at least 95% or at least 98% sequence identity with SEQ ID NO:7; The sporulation specific promoter is a Cry3A, GerA, GNAT or TadA promoter; The method of claim 1.

3. 3. The method of claim 1 or 2, further comprising homogenizing the inactivated host bacteria to form a bacterial lysate containing the natural bioactive nematicidal crystals.

4. The method according to any of claims 1 to 3, further comprising the step of formulating said isolated natural bioactive nematicidal crystals into an orally available dosage form.

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