Composition for the treatment and / or prevention of mastitis

Abstract

To provide an improved method for reducing the onset and period of mastitis in a farm animal and, simultaneously, increasing production of milk and / or reducing the use of antibiotics.SOLUTION: A vaccine composition for reducing the severity and / or period of mastitis in a milk-producing animal comprises a chimeric polypeptide, which comprises a somatostatin-14 polypeptide with an amino acid sequence of SEQ ID NO: 1 and a chloramphenicol acetyltransferase polypeptide with a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, AND SEQ ID NO: 29.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Compositions are provided for reducing the severity and duration of mastitis in livestock while simultaneously increasing milk production. [Background technology]

[0002] Bovine somatotropin (BST) was approved by the United States Department of Agriculture (USDA) in 1993 under the brand name POSILAC™ (sometribove zinc suspension) for over-the-counter (OTC) use in dairy cows, beginning approximately two months after calving and continuing through the end of lactation. During this period, cows may receive subcutaneous injections every 14 days. POSILAC™ label ELANCO 2017. A typical lactation period begins immediately after calving and lasts approximately 10 months. Thus, cows may be treated for approximately eight months of the year. U.S. Food and Drug Administration BST Safety Information 2019; https: / / www.fda.gov / animal-veterinary / product-safety-information / bovine-somatotropin-bst. Healthy cows supplemented with POSILAC™ produce, on average, 10 pounds more milk per day. Recombinant bovine growth hormone (recombinant bovine somatotropin; rBST) is a synthetically derived hormone that may be identical to natural bovine growth hormone or may be slightly modified by adding additional amino acids.

[0003] Unfortunately, rBST has been reported to increase the incidence of clinical mastitis by 25% during the treatment period (Dohoo et al., 2003, Canadian J Vet. Res. 2003; 67:252-264). Cows treated with rBST also have an estimated 55% increased risk of clinical lameness and may also develop injection site reactions.

[0004] One alternative to BST or rBST for improving milk production is somatostatin immunization. Somatostatin (SEQ ID NO: 1) is known to have a relatively short half-life in the blood. To improve the immunological effect of somatostatin, vaccines have previously been developed to improve the half-life of the protein by conjugating somatostatin to a carrier protein. These conjugated somatostatin proteins are designed to have an increased half-life in the blood and improved antigenicity, thus providing improved benefits, particularly in terms of the cost of somatostatin preparation.

[0005] U.S. Patent No. 6,316,004 discloses various conjugated somatostatin-containing proteins that have been shown to have increased antigenicity and function with respect to livestock productivity compared to other conventional immunization or anabolic hormone-based methods.

[0006] U.S. Patent Nos. 7,722,881, 7,943,143, and 8,367,073 disclose chimeric polypeptides comprising the amino acid sequence of somatostatin-14 linked by a spacer sequence to a substantially inactive, truncated chloramphenicol acetyltransferase polypeptide, compositions thereof, and methods for increasing milk and meat productivity.

[0007] U.S. Patent No. 8,425,914 discloses a method for the treatment of obesity comprising administering a composition comprising a vaccine comprising an inactive chloramphenicol acetyltransferase (CAT) enzyme and a chimeric polypeptide of somatostatin-14 bound to an adjuvant.

[0008] U.S. Patent No. 10,441,652 discloses a method for improving the immunological response to a target antigen in an animal, comprising providing a polypeptide conjugate comprising the target antigen attached to a carrier polypeptide by a linker polypeptide, wherein the linker has a high percentage of predicted linear B-cell epitopes and the carrier polypeptide does not stimulate a large T-cell response.

[0009] Improved methods for reducing the incidence and duration of mastitis in livestock while simultaneously increasing milk production and / or reducing the use of antibiotics are desirable. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 6,316,004 [Patent Document 2] U.S. Patent No. 7,722,881 [Patent Document 3] U.S. Patent No. 7,943,143 [Patent Document 4] U.S. Patent No. 8,367,073 [Patent Document 5] U.S. Patent No. 8,425,914 [Patent Document 6] U.S. Patent No. 10,441,652 [Non-patent literature]

[0011] [Non-Patent Document 1] Dohoo et al., 2003, Canadian J Vet. Res. 2003; 67:252-264 Summary of the Invention [Means for solving the problem]

[0012] A vaccine composition is provided for reducing the severity and duration of mastitis in livestock while simultaneously increasing milk production.

[0013] In some embodiments, a vaccine composition for reducing the severity and / or duration of mastitis is provided, the composition comprising a chimeric polypeptide comprising a somatostatin-14 polypeptide having the amino acid sequence of SEQ ID NO:1 and a chloramphenicol acetyltransferase polypeptide having a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, wherein the composition reduces the severity and / or duration of mastitis in a milk-producing animal.

[0014] In some embodiments, the chimeric polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. Preferably, the composition wherein the chimeric polypeptide has the amino acid sequence of SEQ ID NO: 13.

[0015] In some embodiments, the milk-producing animal may be selected from the group consisting of dairy cows, beef cattle, buffalo, goats, sheep, camels, yaks, horses, reindeer, donkeys, and sows.

[0016] In some embodiments, the composition is at least 80% endotoxin-free.

[0017] In some embodiments, the composition is substantially endotoxin-free.

[0018] In some embodiments, the composition is formulated for use as a vaccine.

[0019] In some embodiments, the chloramphenicol acetyltransferase polypeptide lacks enzymatic activity.

[0020] In some embodiments, the somatostatin-14 polypeptide and the inactive chloramphenicol acetyltransferase polypeptide are linked by a spacer, hi some embodiments, the spacer comprises a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.

[0021] In some embodiments, the composition further comprises an adjuvant.

[0022] In some embodiments, the composition further comprises one or more antigens selected from the group consisting of natural polypeptides, recombinant polypeptides, synthetic polypeptides, polysaccharides, and glycoproteins.

[0023] In some embodiments, a composition for reducing the severity and / or duration of mastitis in a milk-producing animal is provided, the composition comprising a chimeric polypeptide comprising a somatostatin-14 polypeptide having the amino acid sequence of SEQ ID NO:1 and a chloramphenicol acetyltransferase polypeptide, wherein the chloramphenicol acetyltransferase polypeptide is modified at residue 192, residue 193, or both residues 192 and 193 relative to a wild-type chloramphenicol acetyltransferase polypeptide represented by residues 1 to 210 of SEQ ID NO:14, and the modification comprises, independently, substitution of one or both histidines with glycine or alanine.

[0024] In some embodiments, the chloramphenicol acetyltransferase polypeptide comprises a modification comprising a substitution of glycine for histidine at residue 193. In some embodiments, the chloramphenicol acetyltransferase polypeptide comprises a substitution of alanine for histidine at residue 193.

[0025] In some embodiments, the chloramphenicol acetyltransferase polypeptide modification comprises a histidine to glycine substitution at residue 192 and a histidine to glycine substitution at residue 193.

[0026] In some embodiments, the chloramphenicol acetyltransferase polypeptide is truncated by 10 amino acids at the C-terminus relative to the wild-type polypeptide.

[0027] In some embodiments, the animals exhibit reduced severity and / or duration of mastitis, a sharp reduction in SCC, reduced days of lost milk, reduced antibiotic use, improved antibiotic effectiveness, and / or reduced duration of antibiotic treatment compared to untreated control animals. In some embodiments, the antibiotic is selected from the group consisting of a beta-lactam (optionally, the beta-lactam is amoxicillin, ceftiofur, cephapirin, cloxacillin, hetacillin, or penicillin) and a lincosamide (optionally, the lincosamide is pirlimycin).

[0028] In some embodiments, cattle treated with the compositions provided herein exhibit a significant increase in milk production and do not exhibit side effects associated with rBST treatment selected from the group consisting of loss of muscle mass, increased bone growth, injection site reactions, and hoof disorders.

[0029] In some embodiments, milk producing animals exhibit a significant increase in milk production within four days of the first vaccination and do not exhibit loss of muscle mass, increased bone growth, injection site reactions, or hoof disorders. [Brief explanation of the drawings]

[0030] [Figure 1]1 is an exemplary schematic diagram of the pET30b CatSom plasmid, which may be used to prepare a polypeptide conjugate. The plasmid contains a kanamycin resistance marker, a Lac operator, a T7 promoter, a CAT coding sequence (all in accordance with an embodiment of the present invention), a linker region (in accordance with the present invention), and also contains a somatostatin coding region in accordance with the present invention. [Figure 2] 2 is an exemplary stained SDS-PAGE showing a 28 KD band corresponding to the predicted size of the codon-optimized CAT-deleted somatostatin polypeptide of the present invention. Lane 1 is LB + IPTG, reduced; lane 2 is LB, reduced; lane 3 is LB + IPTG; and lane 4 is LB. [Figure 3] 3A and 3B are tables showing the amount of milk produced in liters per day for vaccinated dairy cows (using a vaccine composition according to SEQ ID NO: 13, as described in the Examples) and control dairy cows (treated with rBST). Each cow had a specific identification number, as indicated in each table. [Figure 4] Figure 4A is a graph showing the increase in milk for vaccinated cows with each vaccination compared to control cows, according to an embodiment of the present invention. A sharp increase in average milk production is shown following administration of a vaccine comprising a polypeptide conjugate having the amino acid sequence of SEQ ID NO: 13. Figure 4B is a graph depicting the average actual difference in milk production between vaccinated and control cows. [Figure 5] 5 is a bar graph of days of milk lost after treatment in cows diagnosed with mastitis. In the 1× and 4× vaccine composition groups, cows diagnosed with mastitis lost significantly fewer days of milk than control cows diagnosed with mastitis that received saline injections. DETAILED DESCRIPTION OF THE INVENTION

[0031] (Sequence Identification and Sequence ID Number) Amino acid sequence of somatostatin 14: SEQ ID NO: 1 AGCKNFFWKTFTSC

[0032] A synthetic oligonucleotide encoding an inactive chloramphenicol acetyltransferase (CAT) polynucleotide having (His192->Gly, His193->Gly): SEQ ID NO: 2 atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagc acaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctc tggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggttttgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatt taaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag

[0033] Synthetic carrier polypeptide: inactive CAT enzyme (His192->Gly, His193->Gly): SEQ ID NO: 3: mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfssl wseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvggavcdgfhvgrmlnelqq (210 amino acids)

[0034] A synthetic oligonucleotide encoding an inactive chloramphenicol acetyltransferase (CAT) polynucleotide encoding (His193->Gly): SEQ ID NO: 4 atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagc acaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctc tggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggttttgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatt taaacgtggccaatatggacaacttcttcgccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag

[0035] Synthetic oligonucleotide encoding an inactive chloramphenicol acetyltransferase (CAT) polynucleotide encoding (1 His193->Ala): SEQ ID NO: 5 atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagc acaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctc tggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggttttgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatt taaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatgctgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag

[0036] Synthetic oligonucleotide encoding chloramphenicol acetyltransferase (CAT) polynucleotide (1 His+CAT wt): SEQ ID NO: 6 atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggcagaatgcttaatgaactgcagcag

[0037] Synthetic carrier polypeptide: Inactive CAT enzyme (one H->G): SEQ ID NO: 7 mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhgavcdgfhvgrmlnelqq (210 amino acids)

[0038] Inactive CAT enzyme with synthetic carrier polypeptide: (H->A) SEQ ID NO:8: mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfssl wseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhaavcdgfhvgrmlnelqq (210 amino acids)

[0039] Synthetic oligonucleotide encoding a model linker: SEQ ID NO: 9 tgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatg

[0040] Synthetic linker polypeptide: SEQ ID NO: 10 welhrsgprprprprpefm (19 amino acids)

[0041] Synthetic linker polypeptide: SEQ ID NO: 11 welhrsgprprpefm (15 amino acids)

[0042] Synthetic oligonucleotide encoding a CAT-deleted somatostatin fusion protein: SEQ ID NO: 12 atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattca cattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagttttctacacatatattcgc aagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgag tttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaag gtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcagtgggaac tgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatggccggctgcaagaacttcttttggaaaacctttacgagctgc

[0043] Synthetic CAT-deleted somatostatin fusion protein polypeptide conjugate: Carrier-Linker-SST: SEQ ID NO: 13 mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvf heqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqv gg avcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc (243 amino acids)

[0044] Synthetic unmodified CAT somatostatin fusion protein: SEQ ID NO: 14 mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvf heqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqv hh avcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc (243 amino acids)

[0045] Synthetic oligonucleotides; nucleic acid sequences encoding somatostatin-14 SEQ ID NO: 15 gctggctgcaagaatttcttctggaagactttcacatcctgt

[0046] Synthetic linker polypeptide: SEQ ID NO: 16 welhrsgprprprpefm (17 amino acids)

[0047] Synthetic linker polypeptide: SEQ ID NO: 17 welhrsgprprprprprpefm (21 amino acids)

[0048] Synthetic linker polypeptide: SEQ ID NO: 18 welhrsgprprprprprprpefm (23 amino acids)

[0049] Synthetic linker polypeptide: SEQ ID NO: 19 welhrsgprpefm (13 amino acids)

[0050] Synthetic linker polypeptide: SEQ ID NO: 20 welhrsgpkpkpkpkpefm (19 amino acids)

[0051] Synthetic linker polypeptide: SEQ ID NO: 21 welhrsgpkpkpkpefm (17 amino acids)

[0052] Synthetic linker polypeptide: SEQ ID NO: 22 welhrsgpkpkpefm (15 amino acids)

[0053] Synthetic linker polypeptide: SEQ ID NO: 23 welhrsgpkpefm (13 amino acids)

[0054] Synthetic linker polypeptide: SEQ ID NO: 24 welhrsgpkpkpkpkpkpefm (21 amino acids)

[0055] Synthetic linker polypeptide: SEQ ID NO: 25 welhrsgpkpkpkpkpkpkpefm (23 amino acids)

[0056] Inactive CAT enzyme corresponding to synthetic carrier polypeptide: (His192->Ala, His193->Ala): SEQ ID NO: 26: mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfssl wseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvaaavcdgfhvgrmlnelqq (210 amino acids)

[0057] Inactive CAT enzyme corresponding to synthetic carrier polypeptide: (His192->Ala, His193->Gly): SEQ ID NO: 27: mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfssl wseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvagavcdgfhvgrmlnelqq (210 amino acids)

[0058] Inactive CAT enzyme corresponding to synthetic carrier polypeptide: (His192->Gly, His193->Ala): SEQ ID NO: 28: mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfssl wseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvgaavcdgfhvgrmlnelqq (210 amino acids)

[0059] Synthetic carrier polypeptide: CAT enzyme with (His192, His193): SEQ ID NO: 29 mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfssl wseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhhavcdgfhvgrmlnelqq (210 amino acids)

[0060] Synthetic peptide-linker-SST fusion (linker-SST) SEQ ID NO: 30 welhrsgprprprprpefmAGCKNFFWKTFTSC

[0061] (definition) The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0062] The term "amino acid" refers to any of the 20 naturally occurring amino acids and any modified amino acid sequence. Modifications may involve natural processes (e.g., post-translational processing) or chemical modifications known in the art. Modifications include, but are not limited to, phosphorylation, ubiquitination, acetylation, amidation, glycosylation, covalent attachment of flavin, ADP-ribosylation, cross-linking, iodination, methylation, etc.

[0063] The term "polypeptide conjugate" or "chimeric polypeptide" or fusion protein refers to a first polypeptide linked to a second, heterologous polypeptide such that the first and second polypeptides are expressed in frame. Often, the two polypeptides can be joined by a linker or spacer moiety to optimize expression and function of the chimeric polypeptides of the invention.

[0064] The term "endotoxin" refers to a toxin associated with the cell wall of Gram-negative bacteria. In some cases, the toxin is a lipopolysaccharide component of the bacterial cell membrane, which is a component of the outer membrane of the cell wall of Gram-negative bacteria.

[0065] The term "host cell" or "host cells" refers to cells established in ex vivo culture. The host cells discussed herein are characterized by their ability to express the chimeric proteins of the present invention. Examples of suitable host cells useful in embodiments of the present invention include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Specific examples of such cells include SF9 insect cells (Summers and Smith, 1987, Texas Agriculture Experiment Station Bulletin, p. 1555), E. coli cells (BL21(DE3), Novagen), yeast (Pichia Pastoris, Invitrogen), and human hepatocytes (Hep G2 (ATCC HB8065)).

[0066] The term "nucleic acid sequence" refers to the order of the sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. The deoxyribonucleotide sequence encodes the amino acid sequence.

[0067] The terms "protein," "peptide," and "polypeptide" are used interchangeably and refer to an amino acid polymer or two or more interacting or linked amino acid polymers.

[0068] The term "substantially" refers to "to a significant extent," e.g., "substantially removed" means that at least 75%, more typically at least 80%, 85%, 90%, 95%, and most typically 96%, 97%, 98%, 99% of the target is removed; "substantially inactive" means that at least 75%, more typically 80%, 85%, 90%, 95%, and most typically 96%, 97%, 98%, 99% of the enzyme is inactivated. Such substantially inactive CAT enzymes are CAT enzymes in which 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of its activity has been removed. CAT activity can be determined using known functional assays (e.g., binding of n-butyryl coenzyme A to radiolabeled chloramphenicol followed by measurement by liquid scintillation counting (LCS)); 14 Determination of the amount of radiolabel transferred from [C]acetyl-CoA to chloramphenicol by thin-layer chromatography (Molecular Cloning: A Laboratory Model, 3 rd ed., J Sambrook and DW Russell, 2001. Cold Spring Harbor Press), or other known or similar assays).

[0069] The term "vector" is used in reference to a nucleic acid molecule that transfers DNA fragments from one cell to another. The term "vehicle" is sometimes used interchangeably with vector. Two common types of vectors are plasmids and viral vectors.

[0070] (mastitis) Mastitis is a significant problem in milk-producing animals. For example, the annual mortality rate of dairy cows due to mastitis is approximately 20%. Mastitis can be caused by many different pathogens. The main mastitis-causing pathogens include Escherichia coli (E. coli), Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, coagulase-negative staphylococci, and Arcanobacterium pyogenes. The most common mastitis pathogens can be found in udder tissue (spread from cow to cow (contagious pathogens)) or in the herd's immediate environment (environmental pathogens). Certain types of mastitis may be specific to a dairy herd. Contagious pathogens that cause mastitis can live on the skin of cows' udders and teats and can be transferred from cow to cow during milking. The pathogens attach to the skin, colonize the teats, and grow in the teat canal where infection occurs. Farms with high levels of contagious mastitis often have high somatic cell counts (SCCs). Identifying the causative pathogen in clinical mastitis without clinical testing of the milk may not always be easy. Mastitis can be clinical or subclinical. Cows with subclinical mastitis may not appear diseased but may experience yield loss due to high SCCs and may be a source of potential infection for other cows. In clinical mastitis, cows exhibit clear symptoms of the disease, such as udder swelling, heat, hardness, redness, or soreness. The appearance of the milk may be affected, such as a watery appearance, flakes, clots, or pus. Other symptoms may include reduced yield, elevated body temperature, loss of appetite, sunken eyes, signs of diarrhea, and decreased activity due to dehydration, painful distended udders, or poor physical condition.

[0071] Somatic cell counts (SCCs) are a key indicator of milk quality. The primary somatic cells are leukocytes (white blood cells), which increase in number in milk as an immune response to, for example, mastitis-causing pathogens, and numerous epithelial cells, which are milk-producing cells shed from the udder during infection. SCC is quantified as the number of cells per milliliter of milk. Generally, an SCC of less than 100,000 in a single cow indicates a healthy, "uninfected" cow, with no significant reduction in production due to subclinical mastitis. A threshold SCC of 200,000 may be used to indicate whether a cow is infected with mastitis. Cows with an SCC greater than 200,000 have at least a one-in-fourth chance of being infected. Cows infected with significant pathogens have an SCC greater than 300,000. Milk with an SCC greater than 400,000 is deemed unsuitable for human consumption by the European Union. SCC increases after calving, when colostrum is produced before the cow is accustomed to lactation, and at the end of lactation, possibly due to the lower amount of milk produced. In addition to SCC, Bactoscan, Milk Amyloid A test, or California Mastitis test may be performed. Automated measurement of electrical conductivity in milk may also be used.

[0072] Most symptoms of mastitis (e.g., swelling, redness, fever, and abnormal milk quality) are the result of the cow's immune system. In mild cases, the cow's immune response may be sufficient to self-heal. Other cases of mastitis can result in more serious illness, which can cause loss of a quarter or more of the udder, loss of internal tissue due to gangrene, or even death of the cow. Changes in milk composition in subclinical mastitis may be due to the composition of proteins in the milk. In some cases, the total protein content may not be affected, but changes in protein type may be due to the exudation of (low-quality) serum proteins into the milk. Casein, an important protein in healthy milk, may be significantly reduced in cows with subclinical mastitis. Casein is closely related to calcium levels in milk production. Milk pH (normally around 6.6) may rise to 6.8 or 6.9 in cows with mastitis. The presence of certain blood enzymes can affect the taste of milk and its ability to be made into other dairy products (e.g., cheese or yogurt).

[0073] Mastitis can be treated by the use of specific antibiotics, for example, by intramammary or systemic administration. The target site of antimicrobial therapy in clinical mastitis may vary depending on the pathogen. (Pyorala, Irish Vet. J. vol. 62, Suppl 40-44, 2009) For the treatment of Streptococci, penicillin G can be used by the IMM route, although the prognosis is poor. For Staphylococci, penicillin G, cloxacillin, macrolides, or lincosamides can be used by IMM and / or systemic therapy. The prognosis for S. aureus is poor. Cloxacillin is preferred against methicillin-resistant bacteria. For Escherichia coli (e.g., E. coli) or Klebsiella spp., fluoroquinolones or cephalosporins can be used. The most common route of treatment is intramammary administration (IMM). For example, intramammary antibiotics may be selected from, for example, beta-lactams (amoxicillin, ceftiofur, cephapirin, cloxacillin, hetacillin, and penicillin) and lincosamides (pirlimycin). Some intramammary antibiotics have been withdrawn from the U.S. market, but no new intramammary antibiotics have been introduced since 2006.

[0074] There is a trend towards reduced antibiotic use to reduce the likelihood of developing antibiotic resistance in pathogens. There is a need for alternative mastitis treatments to reduce the severity and duration of infection, reduce SCC acutely, reduce days of milk lost, reduce antibiotic use, improve antibiotic effectiveness, and / or reduce the duration of antibiotic treatment.

[0075] Surprisingly, it has been found that administration of a composition comprising an effective amount of a chimeric polypeptide comprising somatostatin-14, a spacer polypeptide, and a substantially inactive truncated chloramphenicol acetyltransferase carrier polypeptide to a milk-producing animal increases milk production while simultaneously reducing the severity and duration of mastitis, dramatically reducing SCC, reducing days of milk lost, reducing antibiotic use, improving antibiotic effectiveness, and / or reducing the duration of antibiotic treatment.

[0076] (somatostatin) Numerous studies have shown that animals immunized with somatostatin have a 10 to 20% increase in average daily gain, a 9% reduction in appetite, and an 11% increase in the efficiency of food utilization. Somatostatin-immunized animals and their offspring are of correct size, and the distribution of animal weight between muscle, bone, and fat is the same as that of control animals (see Reichlin, 1987). Therefore, somatostatin immunization provides a useful and safe method for improving the productivity of target animals. This is especially true when compared with the use of recombinant growth hormones, the use of which carries increased concerns about hormones in the milk or meat derived from treated animals, the safety of the animals themselves, or the buildup of hormones in ecosystems, particularly groundwater supplies. Another disadvantage of recombinant growth hormones is the increased incidence of mastitis in milk-producing animals.

[0077] Somatostatin (also known as growth hormone-inhibiting hormone, or GHIH) is a peptide hormone produced in the hypothalamus and certain parts of the gastrointestinal tract. Somatostatin is generally involved in the regulation of the endocrine system through its interaction with G protein-coupled somatostatin receptors. This somatostatin-based signaling cascade triggers numerous effects throughout the body.

[0078] Somatostatin is known to inhibit the release of growth hormone and thyroid-stimulating hormone from the anterior pituitary gland. (Patel YC and Srikant CB, Somatostatin and its receptors, Adv Mol Cell Endocrinol, 1999. 3: 43-73) Other hormones inhibited by somatostatin include insulin, glucagon, secretin, gastrin, pepsin, and maletin. (Patel YC and Srikant CB, Somatostatin and its receptors, Adv Mol Cell Endocrinol, 1999. 3: 43-73) Somatostatin's ability to regulate so many factors / hormones necessary for growth and food utilization makes it a central target for controlling animal growth in the livestock industry. In other words, inhibiting somatostatin leads to increased levels of growth hormone present in the target animal, thereby resulting in animals with improved performance, such as milk production and increased meat production.

[0079] Immunization of animals against somatostatin has been recognized as a means of neutralizing somatostatin in target animals, thereby eliminating the normal inhibitory effects of somatostatin on various aspects of animal productivity (e.g., milk production in dairy cows). (Reichlin S., ed., 1987, Somatostatin, Basic and Clinical Status, Plenum Press, New York (pp. 3-50, 121-136, 146-156, 169-182, 221-228, 267-274) Spencer GS, 1985, Hormonal systems regulating growth, review, Livestock Production Science, 12, 31-46) Importantly, these somatostatin-based immunization methods avoid the direct use of anabolic hormones (e.g., growth hormone) in animals, allowing for small changes in the concentration of endogenous anabolic factors, thereby providing an ecologically pure food.

[0080] (composition) In some embodiments, a composition is provided for treating, preventing, reducing the severity of, and / or reducing the duration of mastitis in an animal, comprising a polypeptide conjugate and a pharmaceutically acceptable diluent or adjuvant, wherein the polypeptide conjugate comprises somatostatin-14 covalently linked to an inactive chloramphenicol acetyltransferase (CAT) enzyme by a linker polypeptide.

[0081] In some embodiments, the animal is a dairy cow, beef cow, buffalo, goat, sheep, camel, yak, horse, reindeer, donkey, or sow.

[0082] In some embodiments, following administration of the composition to the animal, the animal exhibits a reduction in the incidence, duration, and / or severity of mastitis compared to a control animal that does not receive the composition.

[0083] Following administration of the composition to the animals, the animals exhibit a reduction in the incidence, duration, and / or severity of mastitis and also exhibit a significant increase in milk production compared to control animals not receiving the composition. In some embodiments, milk production increases within 4 days after administration of the composition. In some embodiments, milk production peaks within 8 to 14 days after administration of the composition. In some embodiments, the increase in milk production persists for at least 21 days.

[0084] In some embodiments, methods are provided for treating, preventing, reducing the severity of, and / or reducing the duration of mastitis in an animal, comprising administering to the animal an effective amount of a composition, wherein the composition comprises a polypeptide conjugate comprising somatostatin-14 covalently linked to an inactive chloramphenicol acetyltransferase (CAT) enzyme by a linker polypeptide.

[0085] In some embodiments, methods for treating mastitis in milk-producing animals are provided. Treating a milk-producing animal with a composition of the present disclosure results in a reduction in the incidence, severity, and / or duration of mastitis, and a concomitant rapid increase in milk production. The treatment method may include administering to the animal an effective amount of a composition comprising a polypeptide conjugate comprising somatostatin-14 covalently linked to an inactive chloramphenicol acetyltransferase (CAT) enzyme by a linker polypeptide, whereby milk production is significantly increased compared to a control animal not administered the vaccine. In some embodiments, the animal is a dairy cow, beef cow, goat, sheep, or sow. In some embodiments, milk production increases within 4 days after administration of the composition. In some embodiments, milk production peaks within 8 to 14 days after administration of the composition. In some embodiments, the increase in milk production persists for at least 21 days.

[0086] The polypeptide conjugates provided herein may exhibit improved antigenicity of somatostatin. The polypeptide conjugates of the present invention comprise somatostatin-14 fused to a substantially inactive chloramphenicol acetyltransferase protein via a functionally optimized linker. The polypeptide conjugates provided herein provide a highly effective, low-cost agent for use in the livestock industry for the treatment and prevention of mastitis, as described in more detail below. Embodiments of the present invention include the amino acid and nucleic acid sequences set forth in SEQ ID NOs: 1 to 30.

[0087] The present invention also provides production and purification methods for producing polypeptide conjugates of the present invention that are endotoxin-free and highly functional. Endotoxin-free polypeptides offer significant advantages for use in certain target animals, and small amounts of endotoxin, typically considered advantageous in terms of eliciting an immune response, can actually cause significant functional disadvantages. This is particularly true when the polypeptides of the present invention are used to immunize dairy cattle bred and raised in the United States. In addition, lower effective doses of the polypeptide conjugates may be required, and / or fewer doses may be used to treat / immunize the target animals. This reduction in required amounts also provides a consequent reduction in endotoxin in the compositions of the present invention. The combination of endotoxin-free isolation and lower effective doses of the polypeptide conjugates of the present invention allows for substantially endotoxin-free vaccines to be used herein.

[0088] Somatostatin-14 is a biologically active tetradecapeptide produced in the hypothalamus and gastrointestinal tract. The amino acid sequence of the tetradecapeptide is AGCKNFFWKTFTSC (SEQ ID NO: 1). The sequence of somatostatin-14 is highly conserved among mammals (Lin XW et al. Evolution of neuroendocrine peptide systems: gonadotropin-releasing hormone and somatostatin. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1998 119(3):375-88). The tetradecapeptide is encoded by the nucleic acid sequence GCTGGCTGCAAGAATTTCTTCTGGAAGACTTTCACATCCTGT (SEQ ID NO: 15). (Note that other nucleic acid sequences can be used to encode SEQ ID NO: 1; however, SEQ ID NO: 15 is provided for illustrative purposes.)

[0089] Somatostatin-14 is known to have a strong inhibitory effect on many hormones involved in growth and food utilization in animals. As previously described in U.S. Patents 6,316,004, 7,722,881, 7,943,143, 8,367,073, and 10,441,652 (incorporated herein by reference), chimeric forms of somatostatin-14 and somatostatin can be used in immunizing animals to increase daily body weight and, if desired, milk production. It should be noted that treatment of target animals with anti-somatostatin antibodies has proven expensive and functionally ineffective, thereby dismissing direct antibody treatment as impractical. Muromtsev GS, et al., 1990, Basics of Agricultural Biotechnology, Agropromizdat, Moscow, pp. 102-106. One aspect of the present invention is based on the concept that anti-somatostatin antibodies formed by the compositions and methods described herein will attenuate, but not completely eliminate, most of the inhibitory effects of somatostatin in target animals, a process that results in a natural and proportional increase in growth and productivity in the immunized target animals.

[0090] Surprisingly, as disclosed herein, it has been discovered that compositions comprising a polypeptide conjugate comprising somatostatin and an inactivated CAT carrier polypeptide reduce the severity and / or duration of mastitis, thereby reducing non-productive days in milk-producing animals (e.g., dairy cows).

[0091] Thus, aspects of the present invention facilitate somatostatin-based immunization by providing highly immunogenic materials for use in immunizing target animals. These somatostatin-based immunizing compounds have been optimized for expression and antigenicity. In some embodiments, somatostatin-14 is expressed as a codon-optimized, CAT-deleted somatostatin chimeric polypeptide. These materials offer an unexpected improvement over other methods of treating bovine mastitis, including the use of antibiotics, many of which are pathogen-specific, may promote the development of antibiotic-resistant pathogenic microorganisms, and require their removal from the product.

[0092] Embodiments of the present invention also provide novel adjuvant compositions for improved induction of humoral immunity in target subjects. These adjuvant compositions offer significant improvements over conventional materials for inducing humoral responses and are safe. The adjuvant compositions herein are used with recombinant protein-based antigens to produce vaccines of the present invention. The vaccines of the present invention are therefore useful in the treatment of numerous diseases or conditions.

[0093] In embodiments herein, all components of the adjuvant composition are non-animal-derived, thereby eliminating cross-contamination from potentially contaminated adjuvant components. For example, embodiments herein may utilize non-animal-derived Tween 80. Surprisingly, non-animal-derived Tween 80 has shown significantly better results in the use of the vaccines herein compared to animal-derived Tween 80, eliminating the possibility of animal contamination of the vaccine (e.g., bovine spongiform encephalopathy (BSE)). In addition, non-animal-derived Tween 80 exhibits better emulsification ability compared to animal-derived Tween 80, providing a further unexpected benefit for use in accordance with embodiments herein.

[0094] Adjuvant embodiments herein are also free of benzene and other carcinogenic compounds. These embodiments provide safety benefits not available in most conventional adjuvant compositions. For example, embodiments herein utilize Carbopol 974P, a benzene-free polycyclic acid.

[0095] In one embodiment, the immunological adjuvant comprises a Carbopol base, a squalene base, and an arabinogalactan solution. More specifically, the Carbopol base is prepared using Carbopol 974P in water or saline. The squalene base is prepared from a combination of squalene, non-animal-derived Tween 80, and Span 85. In some embodiments, the squalene base is MF59 (Chiron Corp., Emeryville, CA). The arabinogalactan is dissolved in PBS or saline. The adjuvant composition is combined with the chimeric polypeptide of the present invention to produce a vaccine of the present invention.

[0096] In yet another embodiment, the immunological adjuvant comprises a carbopol base, a squalene base, and a tragacanthin solution. More specifically, the carbopol base is prepared using Carbopol 974P in water or saline. The squalene base is prepared from a combination of squalene, non-animal derived Tween 80, and Span 85. Purified tragacanthin is dissolved in PBS or saline. The adjuvant composition is combined with the chimeric polypeptide of the present invention to produce a vaccine of the present invention.

[0097] Specific adjuvant combinations and concentrations are shown in the Examples below. Adjuvants according to the present invention are safe and effective for human use, free of animal products, free of petroleum-based hydrocarbons, and free of carcinogenic compounds.

[0098] (Vector and host cell) The present invention also relates to vectors containing the polynucleotide molecules of the present invention and host cells transformed with such vectors. Any polynucleotide molecule of the present invention may be placed into a vector for propagation in a target host, which generally includes a selectable marker and an origin of replication. Host cells are genetically engineered to contain these vectors and thereby express the polypeptides of the present invention. Generally, the vectors herein contain a polynucleotide molecule of the present invention operably linked to appropriate transcriptional or translational control sequences (e.g., sequences of bacterial or viral host cells). Examples of control sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences for controlling transcription and translation. A nucleotide sequence is operably linked when the control sequences herein functionally relate to the chimeric polypeptide encoded by the polynucleotide of the present invention.

[0099] Exemplary vehicles include plasmids, yeast shuttle vectors, baculovirus, inactivated adenovirus, etc. In one embodiment, the vehicle is a modified pET30b CatSom plasmid. Target host cells for use herein include bacterial hosts (e.g., E. coli), yeast, SF-9 insect cells, mammalian cells, plant cells, etc.

[0100] In one embodiment, the regulatory sequences include the T7lac, CAT, Trp, or T5 promoter for expression of the chimeric polypeptides of the invention in E. coli or other bacteria. These regulatory sequences are known in the art and may be used as appropriate and under known conditions.

[0101] When genetically modified green plant cells are used for expression, systems developed by Planet Biotechnology and others can be used.

[0102] Various plasmids of the present invention have been constructed for expression of the chimeric polypeptides of the present invention through the use of targeting control sequences. An exemplary plasmid can include a T7lac promoter.

[0103] Host cells for expression of the target chimeric polypeptide include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotic hosts include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as Pseudomonas and Streptomyces. In an exemplary embodiment, the host cell is of the genus Escherichia and may be Escherichia coli (E. coli).

[0104] As shown in the following examples, constructs of the present invention are provided for optimal expression of CAT-deleted recombinant proteins under a variety of circumstances. These constructs are particularly useful for expression in prokaryotic hosts, particularly bacteria of the genus Escherichia. Note that various plant expression systems, typically Agrobacterium tramefii, can also be used in the present invention.

[0105] (Purification of endotoxin-free fusion proteins) An embodiment of the present invention includes the use of an endotoxin-free, codon-optimized, CAT-deleted recombinant protein for use in vaccinating animals, particularly for vaccinating livestock, possibly dairy cows bred and raised in the United States. Endotoxin-free materials are particularly important for livestock bred and raised in the United States (see, e.g., Drackley, JK 2004. Physiological adaptations in transition dairy cows. Pp 74-87 in Proc. Minnesota Dairy Herd Health Conf., St. Paul, MN. University of Minnesota, St. Paul). Additionally, because the methods contemplated herein involve repeated vaccinations in animals, endotoxin-free compositions are increasingly important.

[0106] In one embodiment, the chimeric immunological recombinant protein-containing protein of the present invention is prepared by transforming a target cell with an appropriate recombinant protein-containing vehicle. As noted above, vehicles for use herein include known plasmid and vector systems suitable for expression in a selected target cell.

[0107] In some embodiments of the present invention, the chimeric immunological recombinant protein-containing protein is expressed in a target host cell. The expression of the chimeric protein is carried out using a target control sequence. In some embodiments, the chimeric polypeptide is optimized for expression in E. coli (particularly with respect to the linker sequence disclosed herein).

[0108] The chimeric protein can then be purified according to known protein purification techniques, including, for example, lysozyme lysis, differential centrifugation of inclusion bodies, sieve chromatography, etc. Refolding procedures can be carried out in guanidine chloride and urea at alkaline pH, after dialysis and lyophilization.

[0109] In one embodiment, E. coli is transformed with a plasmid containing the codon-optimized CAT-deleted recombinant protein with appropriate E. coli-based regulatory sequences for expression. In some cases, fermentation of approximately 10 liters of these cells provides a total biomass of at least 500 grams, and in some cases 600 grams, yielding approximately 4 to 6 grams of total protein. It is estimated from silver and Coomassie blue staining that up to half of the total protein may be the chimeric protein (data not shown).

[0110] In some embodiments herein, the chimeric proteins of the present invention are purified from transformed host cells in a substantially endotoxin-free state. The inventors' unexpected and surprising finding was the realization that in some animals, particularly dairy cows, multiple exposures to endotoxins, particularly endotoxins, can result in substantially impaired animals (e.g., mastitis and endotoxic shock in dairy cows bred and raised in the United States). This realization has resulted in attempts to eliminate or reduce the endotoxin dose or number of exposures in the vaccination of dairy cows. It should be noted that this endotoxin-based effect has not been observed to a large extent in cattle bred and raised in Russia and other countries because dairy cows are descendants of different bovine breeds (Holstein Association, 1 Holstein Place, Brattleboro, Vermont 05302-0808). This finding in U.S. dairy cows is contrary to the general expectation that vaccines should contain some small amount of endotoxin to help maximize the animal's immune response, as is the case with dairy cows vaccinated with somatostatin in some other European markets (see U.S. Patent 6,316,004).

[0111] As such, some embodiments herein are directed to the production of substantially endotoxin-free chimeric proteins for use in vaccines, and particularly for use in vaccines used in the livestock industry and in the livestock industry in the U.S. In certain embodiments, endotoxin levels are 1 EU / ml or less, and in other embodiments, endotoxin levels are substantially eliminated, i.e., the chimeric polypeptides of the present invention are substantially endotoxin-free.

[0112] In one embodiment, the IP recovered from the lysed host cells is washed multiple times with a wash solution that is completely endotoxin-free (i.e., endotoxin-free water or solution). The recovered IP precipitate may optionally be washed until the endotoxin level is below approximately 1 EU / ml (endotoxin testing can be performed using one or more known assays, including commercially available test kits from MP Biochemicals, Charles River, etc.). In some embodiments, the wash solution is endotoxin-free and contains one or more proteolytic protein inhibitors (e.g., phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), etc.). In some embodiments, the wash solution is phosphate-buffered saline (PBS) with an inhibitory-effective amount of PMSF, AEBSF, or a combination of both PMSF and AEBSF.

[0113] In some embodiments, the substantially endotoxin-free precipitate can be treated with a protein unfolding solution containing urea at pH 12.5 and refolded in a protein refolding solution containing arginine, glycerol, and / or sucrose and reduced molar concentrations of urea. The purified chimeric protein concentration can be varied to between 0.5 and 4, or 1 and 3 mg / ml, typically about 1.4 to 1.8 mg / ml. Optionally, the substantially endotoxin-free chimeric protein is provided in a vaccine formulation at a dose of about 0.5 to 5 mg / 2 ml, more typically 1 to 4 mg / 2 ml.

[0114] Other methods of endotoxin removal are contemplated within the scope of the present invention and can include, for example, commercially available ion exchange endotoxin removal columns, hydrophobic columns, etc. (see, e.g., Mustang E or G Columns (Millipore)).

[0115] (Improved immune response adjuvant) Embodiments of the present invention provide / use adjuvants, e.g., as disclosed in U.S. Patent No. 8,367,073, for improved induction of humoral immunity, i.e., aspects of immunity mediated by macromolecules found in extracellular fluids (e.g., secreted antibodies and antimicrobial peptides) as opposed to cell-mediated immunity, i.e., aspects of immunity associated with phagocytes, antigen-specific cytotoxic T cells, and cytokine responses. These adjuvants offer significant improvements over prior materials with respect to induction of humoral responses. While the adjuvants herein can be used with numerous vaccines, their use with polypeptides of the invention for vaccination in production animals (e.g., dairy cows) is demonstrated in the examples.

[0116] In some embodiments, all components of the adjuvant may be non-animal derived, thereby eliminating potential cross-contamination of vaccinated animals from potentially contaminated adjuvant compositions. For example, embodiments herein may utilize non-animal derived Tween 80. This is particularly important when the target animal is a dairy cow, given concerns about bovine spongiform encephalopathy (BSE) or other bovine diseases. Note that these concerns are equally relevant for human treatment, where non-animal derived adjuvants offer significant safety benefits. Additionally, adjuvant embodiments herein are free of benzene and other carcinogenic compounds. These embodiments offer safety benefits not available with most conventional adjuvant compounds. For example, embodiments herein may utilize Carbopol® 974P, or benzene-free polybasic acids.

[0117] In one embodiment, the immunological adjuvant may comprise an oil-in-water emulsion in combination with a selected antigen mixed into an emulsion premix.

[0118] An exemplary oil-in-water emulsion for use herein comprises a combination of mineral oil, Tween 80, Span 85, and a target polymer (benzene-free polyacrylic acid). In some cases, the target polymer is selected from the group consisting of Carbomer Homopolymer Type B. A typical oil-in-water emulsion comprises about 8 to 10% mineral oil (v / v), 0.003 to 0.004% Tween 80 (v / v), 0.007 to 0.008% Span 85 (v / v), and 0.04 to 0.06% polymer (w / v).

[0119] An exemplary premix emulsion of the present invention is approximately 50% oil-water based and is composed of a high molecular weight polymer, a surfactant, and an emulsifier. A high molecular weight polymer for use herein comprises acrylic acid crosslinked with an allyl ether of pentaerythritol. In some cases, the high molecular weight polymer has a Brookfield RVT viscosity between about 29,000 and 40,000, such as Carbopol® 974P (Noveon, Inc.).

[0120] (Method for obtaining an optimized immune response in dairy cows) In accordance with the compositions and methods of the present invention, the immunological composition described herein (endotoxin-free, codon-optimized, CAT-deleted somatostatin construct) is combined with the novel adjuvants described above to provide the vaccines of the present invention. In one embodiment, a total dose of about 1 to about 4 mg / 2 ml (of which 5% to 25% (v / v) is adjuvant, more typically 10% to 20% is adjuvant, and most typically about 20% is adjuvant) of the endotoxin-free, codon-optimized, CAT-deleted somatostatin construct is administered. Other conventional adjuvants may also be used with the codon-optimized, CAT-deleted somatostatin constructs of the present invention.

[0121] One purpose of the somatostatin chimeric polypeptide with an adjuvant is to reduce the severity or duration of mastitis while simultaneously increasing productivity in the target milk-producing animal (eg, dairy cow).

[0122] The preparation may be injected intramuscularly or subcutaneously preferably fewer than 12 times, more preferably fewer than 6 times, and in some cases as few as 1, 2, 3, 4, 5, or 6 times. When multiple injections are required in a target animal, an interval of 14 to 28 days before the next injection is typical. As noted above, embodiments herein avoid the use of recombinant hormone treatments in target animals, which is of great benefit in the livestock industry (where recombinant growth hormones are associated with various environmental concerns, such as the early onset of puberty in girls and the adverse effects of manure from treated cattle on both the surface and groundwater environments).

[0123] The sterile compositions of the present invention can be administered by subcutaneous or intramuscular routes. Typically, the site of administration is the neck or tail of the target animal, although other sites may be utilized. It should be noted that the site should be used such that side effects do not interfere with the animal's ability to move, eat, drink, etc.

[0124] As noted above, the vaccines herein, using the adjuvants described herein and typically in an endotoxin-free state, provide significant improvements in milk production in dairy cows, however, these treatments are not accompanied by an increase in feed consumption. [Example]

[0125] Example 1: Construction of a CAT-deleted somatostatin fusion protein The production of the CAT-deleted somatostatin fusion protein was carried out according to the method provided in U.S. Patent No. 7,722,881 (incorporated herein by reference). Site-directed mutagenesis was performed on the pET30b-Cat-Som plasmid to replace His192 and His193 with glycine residues (modified: Gly192 and Gly193). Inactivation of the His193 (and His192) residues eliminates the ability of the CAT enzyme to accept protons, thereby resulting in complete inactivation of CAT.

[0126] The spacer in the same pET30b-Cat-Som (with a His substitution) was codon-optimized for expression in E. coli in the absence of a co-expressed tRNA molecule.

[0127] The modified CAT-deleted somatostatin nucleic acid construct is shown as SEQ ID NO: 12. The CAT-deleted somatostatin fusion protein sequence is disclosed as SEQ ID NO: 13 (compared to the unmodified CAT-somatostatin fusion protein (SEQ ID NO: 14)).

[0128] Example 2: CAT-deleted somatostatin fusion proteins can be expressed at high levels The codon-optimized CAT-deleted somatostatin construct, as described in Example 1, was used to express the fusion protein in BL21(DE3) cells, as described in U.S. Patent 7,722,881. Transformed cells were grown in LB and induced with 0.4 mM IPTG for approximately 3 hours. One milliliter of cells from a culture at a density of OD 0.7 was pelleted and heated at 70°C for 10 minutes in 100 μl of SDS sample buffer. 40 μl of sample cell extract was loaded per lane for SDS PAGE.

[0129] As shown in Figure 2, a 28 KD band, corresponding to the expected size of the codon-optimized CAT-deleted somatostatin fusion protein, was observed in lanes 1 (LB + IPTG, reduced) and 3 (LB + IPTG) after induction with IPTG. No expression was observed in the controls, lanes 2 (LB, reduced) and 4 (LB). As expected, there was no difference in the size of the fusion protein when cultured under standard or reduced conditions.

[0130] Example 3: Compositions containing CAT-deleted somatostatin for use as vaccines Example 3A: Exemplary vaccines (e.g., as disclosed in U.S. Patent No. 10,441,652) comprising compositions containing fusion proteins of the preceding examples (e.g., using the endotoxin-free fusion protein of Example 2 (SEQ ID NO: 13)) in accordance with the present invention. Reagent solutions: 1. Carbopol Base a. Dissolve 0.5 grams of Carbopol 974P in water or saline. b. Mix and boil to dissolve, then autoclave. c. Store at 4°C. 2. Squalene-based a. Mix 58.1 ml of squalene, 4.6 ml of animal-free Tween 80, and 5.2 ml of Span 85. b. Filter the mixture through a 0.2μ filter. c. Store at 4°C. 3. Tragacanthin solution a. Tragacanth gum is extracted with methanol. b. Collect the methanol-insoluble fraction. c. Dry at room temperature. d. Store dry at room temperature. e. Add 1 gram of dried tragacanthin to water or saline. f. Mix and boil to dissolve, then autoclave. g. Store at 4°C.

[0131] Vaccine preparation 1. Prepare vaccine antigens in saline or PBS at 5 mg / ml or less. 2. Add 6.79ml of squalene base to the mixing bottle. 3. Add 10ml of Carbopol Base to Squalene Base (CS). 4. Mix well. 5. Add 10 ml of tragacanthin solution to the CS solution. 6. Mix well. 7. Add vaccine antigen, either undiluted or diluted for use in saline or PBS, to a final volume of 82 ml. 8. Add 1 ml of 1% thimerosal solution and mix well. 9. Store the vaccine at 4°C until use.

[0132] (Example 3B) A second exemplary vaccine according to the invention can be prepared using the fusion protein of Example 2 (SEQ ID NO: 13) (e.g., as disclosed in U.S. Patent No. 10,441,652). Reagent solutions: 1. Carbopol Base a. Dissolve 0.5 grams of Carbopol 974P in water or saline. b. Mix and boil to dissolve, then autoclave. c. Store at 4°C. 2. Squalene-based a. Mix 58.1 ml of squalene, 4.6 ml of animal-free Tween 80, and 5.2 ml of Span 85 and filter through a 0.2μ filter. b. Store at 4°C. 3. Arabinogalactan solution a. Add 1 to 10 grams of arabinogalactan to PBS or saline. b. Mix and boil to dissolve, then autoclave. c. Store at 4°C.

[0133] Vaccine preparation 1. Prepare vaccine antigens in saline or PBS at 5 mg / ml or less; 2. Add 6.79ml of squalene base to the mixing bottle; 3. Add 10ml of Carbopol Base to the Squalene Base; 4. Mix thoroughly and add 10 ml of arabinogalactan solution; 5. Add vaccine antigen, either undiluted or diluted for use in saline or PBS, to a final volume of 82 ml; 6. Add 1 ml of 1% thimerosal solution and mix; and 7. Store the vaccine at 4°C until use.

[0134] (Example 3C) A third exemplary vaccine composition was prepared using the endotoxin-free fusion protein of Example 2 (SEQ ID NO: 13) (eg, as disclosed in US Pat. No. 7,722,881). a. JH 14 (adjuvant) 24ml Mineral oil - 50% (v / v) Tween 80 - 0.1694% (v / v) Span 85 - 0.1915% (v / v) Carbopol 974NP - 0.125% (w / v) b. 95.6 ml of refolded protein of the present invention (2.96 mg / ml) - see Example 2 c. 35.6 ml of phosphate buffered saline d. Antibacterial / Antifungal Agent 0.36 mo 120ml

[0135] Example 4: Endotoxin-free chimeric peptide / adjuvant results in increased milk production As disclosed in U.S. Patent No. 7,722,881, a random pool of dairy cows (Holstein Crosses bred and raised in the United States) was identified, each 31 to 65 days postpartum (third through fifth lactation). Each cow was examined and determined to be in optimal health by a veterinarian.

[0136] The average cow weight in this study was approximately 1000 to 1200 lbs. Six lactating dairy cows were treated with a dose of 1.96 mg / chimeric protein / 2 ml in JH14. Alternatively, nine cows received the conventional rBST treatment. Treatment and milk production studies were conducted on a large scale, focusing on milk-producing dairy farms.

[0137] Vaccination was performed on day 0. Anti-SST serum antibodies and IGF-1 serum levels were tested at 4 weeks, and milk production and overall animal health were monitored periodically.

[0138] The six cows vaccinated with the inventive compositions described herein had a normal appearance and were free of endotoxin reactions and food withdrawal. All six cows had a positive serological response to SST with an average titer of 1:14. Milk production in the six cows was achieved with only one vaccination (see Figure 3A), demonstrating an average yield increase of 23.7%. The nine cows treated with conventional rBST injections on days 0 and 14 had an overall average increase in milk production of 2% (see Figure 3B).

[0139] The data in this example demonstrate a dramatic improvement in efficacy for the use of endotoxin-free compositions in combination with the adjuvants of the invention in dairy cows. These results showed a dramatic improvement in animal health and fecundity compared to cows injected twice with rBST.

[0140] Example 5: Multiple vaccinations with chimeric peptide / adjuvant increase milk production As described in U.S. Patent No. 10,441,652, 92 clinically healthy dairy cows (single and multiple parous, Girilander and Girilander / Holstein crossbreds) were identified, each 90 to 120 days post-partum. The cows were divided into four treatment groups: a group receiving a single vaccine dose, a group receiving a double dose, a group receiving a quadruple dose, and a saline control group. Treatment and milk production studies were conducted on a large scale, focusing on milk-producing dairy farms.

[0141] Cows were vaccinated intramuscularly in the neck region using alternating right-left-right injection sites on days 0, 21, and 42 of the study. Cows were milked three times daily according to farm practice, and milk production was recorded for each cow. Milk was analyzed for milk fat, lactose, protein, urea, and somatic cells (SCC). Cows were observed daily for injection site reactions, overall health, mastitis, and food problems, and body scores were obtained at 21-day intervals. The study ended 21 days after the third vaccination or 63 days from study initiation.

[0142] Treated cows demonstrated increased milk production compared to pre-vaccination production, beginning by day 4 post-vaccination and sustained for 21 days, with peak production occurring between days 8 and 14 post-vaccination. This trend was repeated with each of the three vaccinations, as shown in Figures 4A and 4B. Compared to control cows, vaccinated cows demonstrated a 5 to 20% increase in milk production during the post-vaccination period, with a mean increase in milk production of 13.6%. Using an LSmeans model, there were highly statistically significant differences in milk production between treatment weeks.

[0143] There were no significant differences between treatment groups with regard to site reactions, body scores, overall health, mastitis, or food problems. Vaccination (even at the quadruple dose) was well tolerated by the cows and resulted in no side effects compared to controls.

[0144] The data in this example demonstrate the surprising discovery that multiple repeated injections of the compounds of the present invention are highly effective in rapidly increasing milk production, and that the increased production can be maintained using multiple injections over an extended period of time. Multiple or repeated injections at regular intervals over a significant or indefinite period of time are expected to continue to result in improved milk production. This demonstrates the disclosure of a vaccine that is highly useful in the long-term enhancement of milk production.

[0145] Example 6: Clinical Study of Mastitis A clinical mastitis study was conducted in a Brazilian dairy herd over a 63-day period. In this study, mastitis was endemic to the herd. All cows had previously been injected with rBST (somatotropin) during the previous year. rBST is a bovine growth hormone that stimulates the production of IGF-1 (insulin-like growth factor 1), which increases milk production. The added milk and IGF-1 levels make the udder tissue more susceptible to mastitis, where inflammation increases somatic cells (SCC) in the milk. Other side effects of rBST include loss of muscle mass, increased bone growth, and hoof problems, including uneven hoof growth. In this herd, mastitis was chronic and caused by Escherichia coli.

[0146] In this study, a vaccine test composition was prepared containing a polypeptide conjugate fusion protein of somatostatin 14-linker peptide-inactive CAT carrier polypeptide having the amino acid sequence of SEQ ID NO: 13 in an adjuvant according to Example 3C, except that less chimeric protein was used. In this study, the single dose was 1 mg (1x dose) or 4 mg (4x dose) of SOMATOVAC™ (Braasch Biotech LLC) chimeric protein (SEQ ID NO: 13) in 2 mL of adjuvant containing JH14. Control cows received saline injections. The dosing schedule is shown in Table 1. Cows were first vaccinated 70 days after calving. Cows were injected intramuscularly (IM) on days 0, 21, and 42. No injection site reactions were observed.

[0147] [Table 1]

[0148] Cows were monitored for signs of mastitis and removed from milk production if they had clinical observations of SCC >400,000, udder expansion and / or udder discharge. Cows found to have mastitis received antibiotic infusions and were returned to production based on clinical signs, SCC less than 350,000, visual health, and ease of movement.

[0149] Individual cows excluded due to mastitis during the study in the control, 1x vaccine, and 4x vaccine groups are shown in Table 2.

[0150] [Table 2]

[0151] As shown in Figure 5 and Table 3, there was a significant reduction in days of milk lost in the 1x and 4x groups compared to the control group. Surprisingly, in mastitis cows treated with the 1x or 4x vaccine test composition, mastitis symptoms were less severe and the mastitis resolved very quickly. Even more surprising, the mastitis symptoms in two of the three cows receiving the 4x dose of vaccine test composition responded very rapidly to antibiotic treatment and were returned to the herd after only four days. The days of milk lost are calculated in Table 3. Cows receiving the 1x or 4x vaccine test composition showed a significant reduction in days of milk lost, a significant reduction in the % of days of milk lost (% lost milk days), and a significant reduction in the weight of milk lost (Kg) (Kg milk lost) compared to the control cows.

[0152] [Table 3]

[0153] Additionally, cows treated with the 1× or 4× vaccine test composition showed increased milk production and did not exhibit the side effects associated with rBST (e.g., loss of muscle mass, increased bone growth, or hoof disorders), as previously demonstrated. The composition of the milk in cows receiving the 1× or 4× vaccine test composition was the same as that of control cows, without an increase in SCC (a sign of mastitis).

Claims

1. A vaccine composition for reducing the severity and / or duration of mastitis, comprising a chimeric polypeptide comprising a somatostatin-14 polypeptide having the amino acid sequence of SEQ ID NO:1 and a chloramphenicol acetyltransferase polypeptide having an sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, wherein the composition reduces the severity and / or duration of mastitis in a milk-producing animal.

2. 2. The composition of claim 1, wherein the milk-producing animal is selected from the group consisting of dairy cows, beef cattle, buffalo, goats, sheep, camels, yaks, horses, reindeer, donkeys, and sows.

3. The composition of claim 1 , wherein the composition is endotoxin-free.

4. The composition of claim 1, wherein the chimeric polypeptide has an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

13.

5. The composition of claim 1 , wherein the chimeric polypeptide has the amino acid sequence of SEQ ID NO:

13.

6. 10. The composition of claim 1, formulated for use as a vaccine.

7. 10. The composition of claim 1, wherein the chloramphenicol acetyltransferase polypeptide lacks enzymatic activity.

8. The composition of claim 1, wherein the somatostatin-14 polypeptide and the inactive chloramphenicol acetyltransferase polypeptide are linked by a spacer.

9. 9. The composition of claim 8, wherein the spacer comprises a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:

25.

10. The composition of claim 1 further comprising an adjuvant.

11. 11. The composition of claim 10, further comprising one or more antigens selected from the group consisting of natural polypeptides, recombinant polypeptides, synthetic polypeptides, polysaccharides, and glycoproteins.

12. A composition for reducing the severity and / or duration of mastitis in a milk-producing animal, comprising a chimeric polypeptide comprising a somatostatin-14 polypeptide having the amino acid sequence of SEQ ID NO: 1 and a chloramphenicol acetyltransferase polypeptide, wherein the chloramphenicol acetyltransferase polypeptide is modified at residue 192, residue 193, or both residues 192 and 193 relative to a wild-type chloramphenicol acetyltransferase polypeptide represented by residues 1 to 210 of SEQ ID NO: 14, and the modification includes, independently, substitution of one or both histidines with glycine or alanine.

13. The composition of claim 12, wherein the modification comprises a substitution of histidine at residue 193 with glycine.

14. The composition of claim 12, wherein the modification comprises a substitution of histidine at residue 193 with alanine.

15. 13. The composition of claim 12, wherein the modification comprises a substitution of histidine at residue 192 with glycine and a substitution of histidine at residue 193 with glycine.

16. 13. The composition of claim 12, wherein the chloramphenicol acetyltransferase polypeptide is truncated by 10 amino acids at the C-terminus relative to the wild-type polypeptide.

17. 13. The composition of claim 12, wherein the composition is substantially endotoxin-free.

18. The composition of claim 12, wherein the composition is endotoxin-free.

19. 13. The composition of claim 12, wherein the animals exhibit reduced severity and / or duration of mastitis, a sharp reduction in SCC, reduced days of milk lost, reduced antibiotic use, improved antibiotic effectiveness, and / or reduced duration of antibiotic treatment compared to untreated control animals.

20. 20. The composition of claim 19, wherein the antibiotic is selected from the group consisting of a beta-lactam and a lincosamide, optionally the beta-lactam is amoxicillin, ceftiofur, cephapirin, cloxacillin, hetacillin, or penicillin, and optionally the lincosamide is pirlimycin.

21. 13. The composition of claim 12, wherein the milk-producing animals exhibit a significant increase in milk production and no loss of muscle mass, increased bone growth, injection site reactions, or hoof disorders within four days of the first vaccination.

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