JP2021127334A - Composition for treating and/or preventing mastitis - Google Patents

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

Compositions are provided for reducing the severity and duration of mastitis in livestock while increasing milk production.

Bovine somatotropin (BST) is a POSILAC ™ (sometribove zinc suspension) for OTC (over-the-counter) use in dairy cows starting approximately 2 months after birth and ending at the end of the lactation period. Under the brand name, it was approved by the United States Department of Agriculture (USDA) in 1993. During this period, cows may be injected subcutaneously every 14 days. POSILAC ™ label ELANCO 2017. A typical lactation period begins shortly after the calf is born and is about 10 months long. Therefore, cattle can be treated for about 8 months of the year. US 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 an average of more than 10 pounds of milk per day. Recombinant bovine growth hormone (recombinant bovine somatotropin; rBST) may be identical to natural bovine growth hormone or may be slightly modified by the addition of additional amino acids, synthetically induced. It is a hormone.

Unfortunately, rBST has been reported to increase the frequency 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 manifestations of lameness and may exhibit injection site reactions.

One alternative to BST or rBST for improving milk production is somatostatin immunity. Somatostatin (SEQ ID NO: 1) is known to have a relatively short half-life in blood. To improve the immunological effects of somatostatin, vaccines were previously 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 and improved antigenicity in the blood and therefore provide improved benefits, especially in terms of the cost of preparing somatostatin. do.

US Pat. No. 6,316,004 provides a variety of 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. It is disclosed.

US Pat. Nos. 7,722,881, 7,943,143, and 8,367,073 are chimeric polypeptides comprising the amino acid sequence of somatostatin-14 linked by a spacer sequence to a substantially inert and truncated chloramphenicol acetyltransferase polypeptide. The composition, as well as methods for increasing the productivity of milk and meat, are disclosed.

US Pat. No. 8,425,914 describes the treatment of obesity, including administration of a composition comprising a vaccine comprising an inactive chloramphenicol acetyltransferase (CAT) enzyme and a chimeric polypeptide of somatostatin-14 conjugated to an adjuvant. Discloses the method for.

U.S. Pat. No. 10,441,652 is a method for improving an immunological response to a target antigen in an animal, including providing a polypeptide conjugate comprising a target antigen bound to a carrier polypeptide by a linker polypeptide. It discloses a method in which the linker has a high rate of predicted linear B cell epitopes and the carrier polypeptide does not stimulate large T cell reactions.

An improved method is desired that reduces the onset and duration of mastitis in livestock while at the same time increasing milk production and / or reducing the use of antibiotics.

U.S. Pat. No. 6,316,004 U.S. Pat. No. 7,722,881 U.S. Pat. No. 7,943,143 U.S. Pat. No. 8,367,073 U.S. Pat. No. 8,425,914 U.S. Pat. No. 10,441,652

Dohoo et al., 2003, Canadian J Vet. Res. 2003; 67: 252-264

Vaccine compositions for increasing milk production while reducing the severity and duration of mastitis in livestock are provided.

In some embodiments, a somatostatin-14 polypeptide having the amino acid sequence of SEQ ID NO: 1, a vaccine composition for reducing the severity and / or duration of mammitis, and SEQ ID NO: 3, SEQ ID NO: 7 , A composition comprising a chimeric polypeptide comprising a chloramphenicol acetyltransferase polypeptide having a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. Provided are compositions that reduce the severity and / or duration of mammitis in milk-producing animals.

In some embodiments, the chimeric polypeptide has an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 13. Preferably, the chimeric polypeptide has the amino acid sequence of SEQ ID NO: 13.

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

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

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

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

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

In some embodiments, the somatostatin-14 polypeptide and the inactive chloramphenicol acetyltransferase polypeptide are linked by spacers. In some embodiments, the spacers are 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, Includes a sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 25.

In some embodiments, the composition further comprises an adjuvant.

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

In some embodiments, a composition for reducing the severity and / or duration of mammitis in milk-producing animals, a somatostatin-14 polypeptide having the amino acid sequence of SEQ ID NO: 1, and chloramphenicol. A composition comprising a chimeric polypeptide containing an acetyltransferase polypeptide, wherein the chloramphenicol acetyltransferase polypeptide is a wild-type chloramphenicole acetyltransferase represented by residues 1 to 210 of SEQ ID NO: 14. The polypeptide has been modified at residues 192, 193, or both residues 192 and 193, and the modification is with one or both histidines, independent of glycine or alanine. A composition comprising the substitution of is provided.

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

In some embodiments, the chloramphenicol acetyltransferase polypeptide modification comprises the substitution of histidine at residue 192 with glycine and the substitution of histidine at residue 193 with glycine.

In some embodiments, the chloramphenicol acetyltransferase polypeptide has 10 amino acids truncated at the C-terminus to the wild-type polypeptide.

In some embodiments, animals have a reduced severity and / or duration of mastitis, a sharp decrease in SCC, a decrease in lost milk days, as compared to untreated control animals. , Reduced use of antibiotics, improved efficacy of antibiotics, and / or reduced duration of antibiotic treatment. In some embodiments, the antibiotics are beta-lactams (in some cases, beta-lactams are amoxicillin, cefthiofur, cepapirin, cloxacillin, hetacillin, or penicillin), and lincosamides (in some cases, lincosamides). Is selected from the group consisting of (is pyrrimycin).

In some embodiments, cows treated with the compositions provided herein show a significant increase in milk production, loss of muscle mass, increased bone growth, injection site reactions, and hoof disorders. No side effects associated with rBST treatment selected from the group consisting of.

In some embodiments, milk-producing animals show a significant increase in milk production within 4 days of initial vaccination, resulting in loss of muscle mass, increased bone growth, injection site reactions, or hoof disorders. Does not show.

FIG. 1 is an exemplary schematic of a pET30b CatSom plasmid that can 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 according to embodiments of the invention), a linker region (according to the present invention), and a somatostatin coding region according to the present invention. FIG. 2 is an exemplary stained SDS-PAGE showing a band of 28 KD corresponding to the expected size of the codon-optimized CAT-deficient somatostatin polypeptide of the invention. Lane 1 is LB + IPTG, reduction; Lane 2 is LB, reduction; Lane 3 is LB + IPTG; and Lane 4 is LB. 3A and 3B show liters of milk production per day for vaccinated dairy cows (using the vaccine composition according to SEQ ID NO: 13 as described in the Examples) and control dairy cows (treated with rBST). It is a table showing the amount in. Each cow had a specific identification number, as shown in each table. FIG. 4A is a graph showing the increase in milk of vaccinated cows with each vaccination as compared to control cows according to aspects of the invention. A sharp increase in average milk production is shown after administration of a vaccine containing a polypeptide conjugate having the amino acid sequence of SEQ ID NO: 13. FIG. 4B is a diagram showing the average difference in actual milk production between vaccinated cows and control cows. FIG. 5 is a bar graph of the number of days of milk lost after treatment in cows diagnosed with mastitis. In the 1x and 4x vaccine compositions, cows diagnosed with mastitis lost significantly less milk days than control cows diagnosed with mastitis who received saline injections. showed that.

(Sequence identification and sequence identification number)
Amino acid sequence of somatostatin 14: SEQ ID NO: 1
AGCKNFFWKTFTSC

Synthetic oligonucleotide encoding an inactive chloramphenicol acetyltransferase (CAT) polynucleotide with (His192-> Gly, His193-> Gly): SEQ ID NO: 2
atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag

Synthetic carrier polypeptide: Inactive CAT enzyme (His192-> Gly, His193-> Gly): SEQ ID NO: 3:
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnff

Synthetic oligonucleotide encoding the Inactive Chloramphenicol Acetyltransferase (CAT) polynucleotide encoding (His193-> Gly): SEQ ID NO: 4
atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag

Synthetic oligonucleotide encoding the Inactive Chloramphenicol Acetyltransferase (CAT) polynucleotide encoding (1 His193-> Ala): SEQ ID NO: 5
atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatgctgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag

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

Synthetic carrier polypeptide: Inactive CAT enzyme (one H-> G): SEQ ID NO: 7
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnff

Synthetic carrier polypeptide: Inactive CAT enzyme with (H-> A) SEQ ID NO: 8:
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnff

Synthetic oligonucleotide encoding the model linker: SEQ ID NO: 9
tgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatg

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

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

Synthetic oligonucleotide encoding a CAT-deficient somatostatin fusion protein: SEQ ID NO: 12
atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcagtgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatggccggctgcaagaacttcttttggaaaacctttacgagctgc

Synthetic CAT-deficient somatostatin fusion protein polypeptide conjugate:
Carrier-linker-SST: SEQ ID NO: 13
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqv gg avcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc (243 amino acids)

Synthetic unmodified CAT somatostatin fusion protein: SEQ ID NO: 14
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqv hh avcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc (243 amino acids)

Synthetic oligonucleotide; nucleic acid sequence encoding somatostatin-14 SEQ ID NO: 15
gctggctgcaagaatttcttctggaagactttcacatcctgt

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

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

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

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

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

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

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

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

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

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

Synthetic carrier polypeptide: Inactive CAT enzyme corresponding to (His192-> Ala, His193-> Ala): SEQ ID NO: 26:
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnff

Synthetic carrier polypeptide: Inactive CAT enzyme corresponding to (His192-> Ala, His193-> Gly): SEQ ID NO: 27:
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnff

Synthetic carrier polypeptide: Inactive CAT enzyme corresponding to (His192-> Gly, His193-> Ala): SEQ ID NO: 28:
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnff

Synthetic carrier polypeptide: CAT enzyme with (His192, His193): SEQ ID NO: 29
mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnff

Synthetic Peptide-Linker-SST Fusion (Linker-SST) SEQ ID NO: 30
welhrsgprprprprpefmAGCKNFFWKTFTSC

(Definition)
The following definitions are provided to facilitate the understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure.

The term "amino acid" refers to any 20 natural amino acids and any modified amino acid sequence. Modifications may include natural processes (eg, post-translational processing) or may include chemical modifications known in the art. Modifications include, but are not limited to, phosphorylation, ubiquitination, acetylation, amidation, glycosylation, flavin covalent binding, ADP-ribosylation, cross-linking, iodolation, methylation and the like.

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

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.

The term "host cell" or "host cells" refers to cells established in ex vivo culture. The ability to express the chimeric proteins of the invention is a feature of host cells discussed herein. Examples of suitable host cells useful in aspects of the invention include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Specific examples of such cells are SF9 insect cells (Summers and Smith, 1987, Texas Agriculture Experiment Station Bulletin, p1555), E.Coli cells (BL21 (DE3), Novagen), yeast (Pichia Pastoris, Invitrogen). ) And human hepatocytes (Hep G2 (ATCC HB8065)).

The term "nucleic acid sequence" refers to the sequence of sequences of deoxyribonucleotides along the strand of deoxyribonucleotide. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. The deoxyribonucleotide sequence encodes an amino acid sequence.

The terms "protein", "peptide", and "polypeptide" are used paraphrasically to mean an amino acid polymer, or two or more interacting or linked amino acid polymers.

The term "substantially" refers to "to a considerable extent", for example, "substantially removed" is at least 75%, more typically at least 80%, 85%, 90% of the target. , 95%, and most typically 96%, 97%, 98%, 99% are removed, and "substantially inert" means at least 75% of the enzyme, more typically. It means that 80%, 85%, 90%, 95%, and most typically 96%, 97%, 98%, 99% are inactivated. Such substantially inactive CAT enzymes have had 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of their activity removed. It is a CAT enzyme. CAT activity can be determined using known functional assays (eg, binding of n-butyrylcoenzyme A to radiolabeled chromatographic phenicol, and subsequent measurement by liquid scintillation measurement (LCS); ^[14 C] determined by determining the amount of radiolabel moved from 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).

The term "vector" is used with respect to nucleic acid molecules that transfer DNA fragments from one cell to another. The term "vehicle" is sometimes used paraphrased as a vector. Two common types of vectors are plasmids and viral vectors.

(Mastitis)
Mastitis is a serious problem in milk-producing animals. For example, the annual mortality rate of dairy cows due to mastitis is about 20%. Mastitis can be caused by many different pathogens. The main causative agents of mammitis are Escherichia coli (E. coli), Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Coagulase-negative staphylococcus. -Negative staphylococci), and includes Arcanobacterium pyogenes. The most common mastitis pathogens can be found in udder tissue (bovine to bovine (contact infectious pathogen)) or in the surrounding environment of the herd (environmental pathogen). Certain types of mastitis may be specific to the dairy cow herd. Contact-borne pathogens that cause mastitis can inhabit the skin of bovine udders and teats and can be transferred from bovine to bovine during milking. The pathogen attaches to the skin, colonizes the papillae, and grows in the papillary duct where the infection occurs. Farms with high levels of infectious mastitis often have high somatic cell counts (SCCs). Identifying the causative agent in clinical mastitis without clinical testing of milk may not always be easy. Mastitis may be clinical mastitis or latent mastitis. Cattle with latent mastitis appear to be disease-free, but can result in reduced yields due to high SCC and can be the source of possible infections for other cattle. .. In clinical mastitis, cows exhibit definite symptoms of the disease, such as udder swelling, fever, hardness, redness, or pain. The appearance of milk can be affected, for example, watery appearance, flakes, clots, pus. Other symptoms may include decreased yield, increased body temperature, loss of appetite, depressed eyes, signs of diarrhea, and decreased exercise due to dehydration, swollen breast pain or poor physical condition.

Somatic cell counts (SCCs) are a major indicator of milk quality. The major somatic cells are, for example, white blood cells (leukocytes, white blood cells) that increase and become present in milk as an immune response to the causative agent of mammitis, and detached from the breast when an infection occurs. A large number of epithelial cells that are milk-producing cells. SCC is quantified as the number of cells per milliliter of milk. In general, an SCC of 100,000 or less in an individual cow refers to a healthy "non-infected" cow, and there is no significant reduction in production due to latent mastitis. The threshold SCC of 200,000 may be used to indicate whether a cow is infected with mastitis. Cows with more than 200,000 SCCs are at least one-quarter likely to be infected. Cattle infected with significant pathogens have more than 300,000 SCCs. Milk with an SCC of more than 400,000 has been determined by the European Union to be unsuitable for human consumption. SCC increases after calving, when first milk is produced before the cow becomes accustomed to lactation, and at the end of lactation, probably due to a smaller amount of milk produced. In addition to SCC, Bactoscan, Milk Amyloid A Test, or California Mastitis Test may be performed. Automatic measurement of electrical conductivity in milk may be used.

Most symptoms of mastitis (eg, swelling, redness, fever, and milk abnormalities) are the result of the immune system in cows. In mild cases, the bovine immune response may be sufficient to cause self-healing. Other cases of mastitis can be a more serious illness that can lead to the loss of more than a quarter of the udder, the loss of body tissue due to gangrene, or the death of cattle. The changes in the milk composition in latent mastitis may be the composition of the protein in the milk. In some cases, total protein content may not be affected, but changes in protein type may be affected by exudation of (poor quality) serum protein into milk. Casein, an important protein in healthy milk, can be significantly reduced in cows with latent mastitis. Casein is closely associated with calcium levels in milk production. The pH of milk (usually around 6.6) can 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 (eg cheese or yogurt).

Mastitis can be treated by the use of certain antibiotics, for example by intramammary or systemic administration. The target location of antibacterial treatment in clinical mastitis can vary by different pathogens. (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 E. coli (eg, 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 can be selected from, for example, beta-lactams (amoxicillin, cefthioflu, cepapilin, cloxacillin, hetacillin, and penicillin), and lincosamides (pilrimycin). Some intramammary antibiotics have been withdrawn from the US market, but no new intramammary antibiotics have been derived since 2006.

There is a tendency to reduce the use of antibiotics in order to reduce the likelihood of developing resistance to antibiotics in pathogens. Reduces the severity and duration of infection, sharply reduces SCC, reduces the number of days of lost milk, reduces the use of antibiotics, improves the effectiveness of antibiotics, and / or of antibiotic treatment There is a need for alternative mastitis treatments to reduce the duration.

Surprisingly, a composition for milk-producing animals containing an effective amount of a chimeric polypeptide containing somatostatin-14, a spacer polypeptide, and a substantially inert and truncated chloramphenicol acetyltransferase carrier polypeptide. Administration increases milk production while reducing the severity and duration of mastitis, sharply reducing SCC, reducing the number of days of lost milk, reducing antibiotic use, and antibiotics. It has been found to improve efficacy and / or reduce the duration of antibiotic treatment.

(Somatostatin)
Many studies have shown that somatostatin-immunized animals have an average daily weight gain of 10 to 20%, a 9% decrease in appetite, and an 11% increase in food utilization efficiency. .. Animals immunized with somatostatin, and their offspring, are of the correct size, and the distribution of animal body weight among muscle, bone, and fat is the same as in control animals (see Reichlin, 1987). .. Therefore, somatostatin immunity provides a useful and safe way to improve the productivity of targeted animals. This is because its use of recombinant growth hormone has increased concerns about hormones in milk or meat derived from treated animals, the safety of the animals themselves, or the increase in hormones in the ecosystem, especially in the groundwater supply. Especially when compared to use. Another disadvantage of recombinant growth hormone is the increased incidence of mastitis in milk-producing animals.

Somatostatin (also known as growth hormone inhibitory hormone, or GHIH) is a peptide hormone produced in the hypothalamus and certain parts of the digestive tract. Somatostatin is generally involved in the regulation of the endocrine system through interaction with G protein-coupled somatostatin receptors. This somatostatin-based signaling cascade causes many effects that spread throughout the body.

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 that are inhibited by somatostatin include insulin, glucagon, secretin, gastrin, pepsin, maletin, and the like. (Patel YC and Srikant CB Somatostatin and its receptors Adv Mol Cell Endocrinol, 1999. 3 43-73). Somatostatin's ability to control so many factors / hormones required for growth and food use makes somatostatin a central target for controlling animal growth in the livestock sector. That is, inhibition of somatostatin leads to increased levels of growth hormone present in the target animal, thereby having improved abilities such as producing milk and providing a larger amount of meat. The result is an animal.

Animal immunity to somatostatin has been recognized as a means of neutralizing somatostatin in target animals, thereby removing the usual inhibitory effect of somatostatin on various aspects of animal productivity (eg, 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 direct use of anabolic hormones (eg, growth hormone) in animals and allow small changes in the concentration of endogenous anabolic factors. It provides ecologically pure food.

(Composition)
In some embodiments, treating or preventing mastitis in animals, including polypeptide conjugates and pharmaceutically acceptable diluents or adjuvants, reducing the severity of mastitis, and / or of mastitis. A composition for reducing the duration is provided that comprises somatostatin-14 in which the polypeptide conjugate is covalently linked to the inactive chloramphenicole acetyltransferase (CAT) enzyme by a linker polypeptide. NS.

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

In some embodiments, after administration of the composition to the animal, the animal exhibits a reduction in the onset, duration, and / or severity of mastitis as compared to a control animal that has not received the composition. ..

After administration of the composition to the animals, the animals show a reduction in the onset, duration, and / or severity of mastitis and significant milk production compared to control animals not receiving the composition. Also shows an increase. In some embodiments, milk production is increased within 4 days after administration of the composition. In some embodiments, milk production peaks within 8-14 days after administration of the composition. In some embodiments, increased milk production lasts for at least 21 days.

In some embodiments, treating, preventing, reducing the severity of mastitis, and / or reducing the duration of mastitis in animals, including administering to the animal an effective amount of the composition. A method is provided in which the composition comprises a polypeptide conjugate comprising somatostatin-14 covalently attached to an inactive chloramphenicol acetyltransferase (CAT) enzyme by a linker polypeptide. NS.

In some embodiments, methods for treating mastitis in milk-producing animals are provided. Treating milk-producing animals with the compositions of the present disclosure results in a reduced incidence of mastitis, a reduced severity, and / or a reduced duration, and at the same time a sharp increase in milk production. The method for treatment is to administer an effective amount of a composition comprising a polypeptide conjugate containing somatostatin-14 covalently attached to the chloramphenicol acetyltransferase (CAT) enzyme, which is inactive by the linker polypeptide, to the animal. This may be included, and milk production is significantly increased as compared to control animals not receiving the vaccine. In some embodiments, the animal is a dairy cow, beef cattle, goat, sheep, or sow. In some embodiments, milk production is increased within 4 days after administration of the composition. In some embodiments, milk production peaks within 8-14 days after administration of the composition. In some embodiments, increased milk production lasts for at least 21 days.

The polypeptide conjugates provided herein may exhibit improved antigenicity of somatostatin. The polypeptide conjugates of the present invention contain somatostatin-14 fused to a substantially inactive chloramphenicol acetyltransferase protein via a functionally optimized linker. The polypeptide conjugates provided herein provide a highly effective and low cost material for use in the livestock sector 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: 1-30.

The present invention also provides a production and purification method for producing the polypeptide conjugate of the present invention which is endotoxin-free and in a highly functional state. Endotoxin-free polypeptides offer considerable advantages for use in certain target animals, and small amounts of endotoxin, which are typically thought to be advantageous for eliciting an immune response, are in fact a significant functional disadvantage. Can cause. This is even more so when the polypeptides of the invention are used to immunize dairy cows bred and raised in the United States. In addition, polypeptide conjugates may be required in lower effective doses and / or less frequent doses are used to treat / immunize the target animal. This reduction in the required amount also provides a consequential reduction in endotoxin in the compositions of the present invention. The combination of endotoxin-free isolation and less effective amounts of the polypeptide conjugates of the invention allows for the substantially endotoxin-free vaccines used herein.

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) (other nucleic acid sequences can be used to encode SEQ ID NO: 1, however, SEQ ID NO: 15 is for exemplary purposes. Please note that it is provided in).

Somatostatin-14 is known to have a strong inhibitory effect on many hormones involved in growth and food use in animals. As previously described in US Pat. Nos. 6,316,004, 7,722,881, 7,943,143, 8,367,073, and 10,441,652 (incorporated herein by reference), the chimeric forms of somatostatin-14 and somatostatin It can be used in animal immunization for increased daily weight, and optionally milk production. It should be noted that treatment of target animals with anti-somatostatin antibodies has proven to be expensive and functionally non-dramatic, thereby reducing direct antibody treatment as impractical. .. Muromtsev G.S., et al., 1990, Basics of agricultural biotechnology, Agropromizdat, Moscow, pp 102-106. One aspect of the invention is the notion that the anti-somatostatin antibody formed by the composition, and the methods described herein, weaken most, but not completely eliminate, the inhibitory effect of somatostatin on the target animal. based on. This process results in a naturally balanced increase in growth and productivity in immunized target animals.

Surprisingly, as disclosed herein, a composition comprising a polypeptide conjugate comprising somatostatin and an inactivated CAT carrier polypeptide reduces the severity and / or duration of mastitis. , It has been found to reduce non-productive days in milk-producing animals (eg, dairy cows).

Therefore, aspects of the invention facilitate somatostatin-based immunization by providing highly immunogenic substances for use in immunization of target animals. These somatostatin-based immune compounds were optimized for expression and antigenicity. In some embodiments, somatostatin-14 is expressed as a codon-optimized, CAT-deficient somatostatin chimeric polypeptide. These substances treat bovine mastitis, including the use of antibiotics, many of which are pathogen-specific, can stimulate the development of antibiotic-resistant pathogenic microorganisms, and require removal from the product. Providing unexpected improvements beyond other methods.

Embodiments of the invention also provide novel adjuvant compositions for improved induction of humoral immunity in a target subject. These adjuvant compositions provide a significant improvement over conventional materials for the induction of humoral responses and are safe. The adjuvant compositions herein are used with recombinant protein-based antigens to produce the vaccines of the invention. As a result, the vaccines of the present invention are useful in the treatment of numerous diseases or conditions.

In embodiments herein, all components of the adjuvant composition are not of animal origin, thereby removing secondary contamination from potentially contaminated adjuvant components. For example, embodiments herein can utilize Tween 80, which is not of animal origin. Surprisingly, non-animal Tween 80 showed significantly better results in the use of the vaccines herein compared to animal-derived Tween 80s, with animal contamination of the vaccine (eg, bovine spongiform). Exclude the possibility of encephalopathy (BSE)). In addition, non-animal Tween 80 exhibits a better ability to emulsify compared to animal-derived Tween 80, providing additional unexpected benefits for use according to embodiments herein.

The adjuvant embodiments herein also do not include others such as benzene and carcinogenic compounds. These embodiments provide safety benefits not available in most conventional adjuvant compositions. For example, embodiments herein utilize carbopole 974P, or benzene-free polycyclic acids.

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 carbopole 974P in water or physiological saline. The squalene base is prepared from a combination of squalene, non-animal Tween 80, and Span 85. In some embodiments, the squalene base is MF59 (Chiron Corp., Emeryville, CA). Arabinogalactan is dissolved in PBS or saline. The adjuvant composition is combined with the chimeric polypeptide of the invention to produce the vaccine of the invention.

In still other embodiments, the immunological adjuvant comprises a carbopol-based, squalene-based, and tragacanthin solution. More specifically, the carbopol base is prepared using carbopole 974P in water or physiological saline. The squalene base is prepared from a combination of squalene, non-animal Tween 80, and Span 85. Purified tragacantin is dissolved in PBS or saline. The adjuvant composition is combined with the chimeric polypeptide of the invention to produce the vaccine of the invention.

Specific adjuvant combinations and concentrations are shown in the examples below. The adjuvant according to the present invention is safe and effective for human use, avoids animal products, avoids petroleum hydrocarbons, and avoids carcinogenic compounds.

(Vector and host cell)
The present invention also relates to vectors containing the polynucleotide molecules of the invention, and host cells transformed with such vectors. Any polynucleotide molecule of the invention may generally be placed in a vector for the growth host of interest, including selectable markers and origins of replication. Host cells are genetically modified to contain these vectors, thereby expressing the polypeptides of the invention. In general, the vectors herein include polynucleotide molecules of the invention operably linked to suitable transcriptional or translational control sequences (eg, sequences of bacterial or viral host cells). Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosome binding sites, and suitable sequences that control transcription and translation. When the control sequences herein are functionally related to the chimeric polypeptides encoding the polynucleotides of the invention, the nucleotide sequences are operably linked.

Typical vehicles include plasmids, yeast shuttle vectors, baculoviruses, inactivated adenoviruses and the like. In one embodiment, the vehicle is a modified pET30b CatSom plasmid. Target host cells for use herein include bacterial hosts (eg, E. coli), yeast, SF-9 insect cells, mammalian cells, plant cells and the like.

In one embodiment, the control sequence comprises a T7lac, CAT, Trp, or T5 promoter for expression of the chimeric polypeptide of the invention in E. coli or other bacteria. These control sequences are known in the art and are used under appropriate and known conditions.

If the recombinant green plant cell is utilized for expression, a system developed by Planet Biotechnology and others can be utilized.

The various plasmids of the invention have been constructed for the expression of the chimeric polypeptides of the invention through the use of target control sequences. An exemplary plasmid can include the T7lac promoter.

Host cells for expression of the target chimeric polypeptide include prokaryotes, yeasts, and higher eukaryotic cells. Exemplary prokaryotic hosts include bacteria of the genus Escherichia, Bacillus, and Salmonella, as well as the genus Pseudomonas and Streptomyces. In a typical embodiment, the host cell belongs to the genus Escherichia and may be Escherichia coli (E. Coli).

As shown in the examples below, the constructs of the invention are provided for optimal CAT deletion recombinant protein expression under a variety of circumstances. These constructs are particularly effective for expression in prokaryotic hosts, especially bacteria of the genus Escherichia. It should be noted that various plant expression systems can also be used in the present invention, typically using Agrobacterium trameficies.

(Purification of endotoxin-free fusion protein)
Aspects of the invention are endotoxin-free, codon-optimized, for use in vaccination of animals, especially for vaccination of livestock, which are dairy cows bred and raised in the United States in some cases. Includes the use of CAT-deficient recombinant proteins. Endotoxin-free substances are of particular importance to cattle bred and raised in the United States (eg, Drackley, JK 2004. Physiological adaptations in transition dairy cows. Pp 74-87 in Proc. Minnesota Dairy Herd Health Conf., See St Paul, MN. University of Minnesota, St. Paul). Also, endotoxin-free compositions are increasingly important, as the methods considered herein involve repeated vaccinations in animals.

In one embodiment, the chimeric immunologically recombinant protein-containing protein of the invention is prepared by transforming a target cell with a suitable recombinant protein-containing vehicle. As mentioned above, vehicles for use herein include known plasmid and vector systems suitable for expression in selected target cells.

In aspects of the invention, the chimeric immunologically recombinant protein-containing protein is expressed in the target host cell. Expression of the chimeric protein is carried out using a target control sequence. In some embodiments, the chimeric polypeptide has been optimized for expression in E. coli (especially with respect to the linker sequences disclosed herein).

The chimeric protein can then be purified according to known protein purification techniques, including, for example, lysozyme lysis, fractional centrifugation of inclusion bodies, sieving chromatography and the like. The refolding method can be carried out in guanidine chloride and urea at an alkaline pH after dialysis and lyophilization.

In one embodiment, E. coli uses a codon-optimized CAT-deficient recombinant protein-containing plasmid that has an appropriate E. coli-based control sequence for expression. , Transformed. 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, producing 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 can be chimeric proteins (data not shown).

In some embodiments herein, the chimeric proteins of the invention are purified from host cells transformed in a substantially endotoxin-free state. The perception that multiple exposures to endotoxin, especially endotoxin, in some animals, especially dairy cows, results in substantially impaired animals (mastitis and endotoxin shock in dairy cows bred and raised in the United States). It was an unexpected and surprising result obtained by the present inventor. This recognition resulted in attempts to eliminate or reduce the dose of endotoxin or the number of exposures in dairy cow vaccination. It should be noted that this endotoxin-based effect is less pronounced in cattle bred and raised in Russia and other countries because dairy cows are offspring of different bovine species (). Holstein Association, 1 Holstein Place, Brattleboro, Vermont 05302-0808). This finding in US dairy cows generally shows that some vaccines help maximize the immune response of animals, similar to dairy cows when vaccinated with somatostatin in some other European markets. It is the exact opposite of the expectation that it should contain small amounts of endotoxin (see US Pat. No. 6,316,004).

As such, some embodiments herein include the production of substantially endotoxin-free chimeric proteins for use in vaccines, and in particular in vaccines used in the livestock industry and the livestock industry in the United States. I am aiming. 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 invention are substantially endotoxin-free. be.

In one embodiment, the IP recovered from the lysed host cells is washed multiple times using a endotoxin-free wash (ie, endotoxin-free water or solution). The recovered IP precipitate may, in some cases, be washed to below approximately 1 EU / ml endotoxin levels (endotoxin tests include commercially available test kits from MP Biochemicals, Charles River, etc., Can be done using one or more known assays). In some embodiments, the lavage fluid is endotoxin-free and one or more proteolytic protein inhibitors (eg, phenylmethanesulfonyl fluoride (PMSF), 4- (2-aminoethyl) -benzenesulfonyl fluoride (eg, phenylmethanesulfonyl fluoride (PMSF), 4- (2-aminoethyl) -benzenesulfonyl fluoride). AEBSF) etc.) are included. 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.

In some embodiments, substantially endotoxin-free precipitates are treated with a protein unfolding solution at pH 12.5 containing urea with arginine, glycerol, and / or urea with reduced molar concentrations. It is possible to refold in a protein refolding solution containing. The purified chimeric protein concentrate may be varied from 0.5 to 4, or between 1 and 3 mg / ml, typically from about 1.4 to 1.8 mg / ml. In some cases, substantially endotoxin-free chimeric proteins are provided in vaccine formulations at doses of about 0.5 to 5 mg / 2 ml, more typically 1 to 4 mg / 2 ml.

Other methods of endotoxin removal are envisioned within the scope of the invention and may include, for example, commercially available ion exchange endotoxin removal columns, hydrophobic columns, etc. (see, eg, Mustang E or G Columns (Millipore)). can.

(Improved immune response adjuvant)
Embodiments of the invention are humoral immunity, an embodiment of immunity mediated by macromolecules (eg, secreted antibodies and antibacterial peptides) found in extracellular fluid (cell-mediated immunity, ie phagocytic cells, antigen-specific). For improved induction of cytotoxic T cells, and immune aspects associated with cytokine responses), eg, the adjuvants disclosed in US Pat. No. 8,367,073 are provided / used. These adjuvants provide a significant improvement over conventional substances in inducing a humoral response. Although the adjuvants herein can be used with a number of vaccines, examples show for use with the polypeptides of the invention for vaccination in producing animals (eg, dairy cows).

In some embodiments, all components of the adjuvant need not be of animal origin, thereby excluding potential secondary contamination of vaccinated animals from the potentially contaminated adjuvant composition. For example, embodiments herein can utilize Tween 80, which is not of animal origin. This is especially important if the target animal is a dairy cow, as there are concerns about bovine spongiform encephalopathy (BSE) or other bovine diseases. It should be noted that these concerns are equally relevant for human treatment, where non-animal-derived adjuvants provide significant safety benefits. In addition, the adjuvant embodiments herein do not include others such as benzene and carcinogenic compounds. These embodiments provide safety benefits not available with most conventional adjuvant compounds. For example, embodiments herein can utilize Carbopol® 974P, or benzene-free polybasic acids.

In one embodiment, the immunological adjuvant may include an oil-in-water emulsion in combination with the selected antigen mixed in the emulsion premix.

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. Typical oil-water emulsions are about 8-10% mineral oil (v / v), 0.003-0.004% Tween 80 (v / v), 0.007-0.008 Span 85 (v / v), and 0.04-0.06%. Includes polymer (w / v).

The exemplary premix emulsion of the present invention is composed of a polymeric polymer, a surfactant, and an emulsifier on an approximately 50% oil-water base. Polymer polymers for use herein include acrylic acid crosslinked with allyl ether of pentaerythritol. In some cases, the polymeric polymer has a Brookfield RVT viscosity between about 29,000 and 40,000, such as Carbopol® 974P (Noveon, Inc).

(How to get an optimized immune response in dairy cows)
According to the compositions and methods of the invention, the immunological compositions described herein (endotoxin-free, codon-optimized, CAT-deficient somatostatin constructs) are combined with the novel adjuvants described above. , Provide the vaccine of the present invention. In one embodiment, about 1 to about 4 mg / 2 ml, of which 5% to 25% (v / v) is an adjuvant, more typically 10% to 20% is an adjuvant, most typical. Is administered an endotoxin-free, codon-optimized, CAT-deficient somatostatin construct at a total dose (about 20% is an adjuvant). Other conventional adjuvants may be used with the codon-optimized, CAT-deficient somatostatin constructs of the present invention.

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

The preparation is preferably less than 12 times, more preferably less than 6 times, and in some cases only 1 time, 2 times, 3 times, 4 times, 5 times, or 6 times, intramuscularly or subcutaneously. You may. If multiple injections are required in the target animal, an interval of 14 to 28 days is typical before the next injection. As mentioned above, embodiments herein avoid the use of recombinant hormone treatments on target animals, which is of great benefit in the livestock sector (recombinant growth hormone is an early onset of puberty in girls, And related to various environmental concerns that fertilizers from treated cattle have a detrimental effect on both the surface and groundwater environment).

The sterile compositions of the present invention can be administered by a subcutaneous or intramuscular route. Other sites may be utilized, but typically the site of administration is the neck or tail of the target animal. It should be noted that the site should be used so that side effects do not interfere with the animal's ability to move, eat, drink, etc.

As mentioned above, the vaccines herein, using the adjuvants described herein and typically in an endotoxin-free state, provide a significant improvement in milk production in dairy cows. However, these treatments are not associated with increased feed consumption.

Example 1: Construction of a CAT-deficient somatostatin fusion protein
Production of the CAT-deficient somatostatin fusion protein was performed according to the method provided in US Pat. No. 7,722,881 (incorporated herein by reference). Site-specific 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.

Spacers in the same pET30b-Cat-Som (with His substitution) were codon-optimized for expression by E. coli in the absence of co-expressed tRNA molecules.

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

Example 2: CAT-deficient somatostatin fusion protein may be expressed at high levels Codon-optimized CAT-deficient somatostatin constructs, such as those described in Example 1, are described in US Pat. No. 7,722,881. Was used to express the fusion protein in BL21 (DE3) cells. Transformed cells were grown in LB and induced with 0.4 mM IPTG for approximately 3 hours. 1 ml cells from the culture with a density of OD 0.7 were pelleted and heated at 70 ° C. for 10 minutes in 100 μl SDS sample buffer. 40 μl of cell extract of the sample was loaded lane by lane for SDS PAGE.

As shown in FIG. 2, a band of 28 KD, which corresponds to the expected size of the codon-optimized CAT-deficient somatostatin fusion protein, appears in lane 1 (LB + IPTG, reduction), after induction with IPTG. And lane 3 (LB + IPTG). No expression was observed in control lane 2 (LB, reduction) and lane 4 (LB). As expected, there was no difference in the size of the fusion proteins when performed in the standard or reduced state.

Example 3: Composition Containing CAT-Deleted Somatostatin for Use as a Vaccine (Example 3A) The fusion protein of the aforementioned Example according to the present invention (eg, endotoxin of Example 2 (SEQ ID NO: 13)). An exemplary vaccine comprising a composition comprising a free fusion protein (eg, disclosed in US Pat. No. 10,441,652).
Reagent solution:
1. Carbo pole base
Dissolve 0.5 grams of Carbopol 974P in water or physiological saline.
b. Mix and boil to dissolve. Then autoclave.
c. Store at 4 ° C.
2. Squalene base
Mix 58.1 ml squalene, 4.6 ml non-animal Tween 80, and 5.2 ml Span 85.
b. Filter the mixture to 0.2μ.
c. Store at 4 ° C.
3. Tragacantin solution
Extract the tragacant rubber with methanol.
b. Recover the insoluble fraction in methanol.
c. Dry at room temperature.
d. Store dry at room temperature.
e. Add 1 gram of dry tragacantin to water or saline.
f. Mix and boil to dissolve. Then autoclave.
g. Store at 4 ° C.

Vaccine preparation
1. Prepare vaccine antigen at 5 mg / ml or less in saline or PBS.
2. Add 6.79 ml of squalene base to the mixing bottle.
3. Add 10 ml of carbopole base to squalene base (CS).
4. Mix well.
5. Add 10 ml of tragacantin solution to the CS solution.
6. Mix well.
7. Add undiluted or diluted vaccine antigen 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.

(Example 3B) A second exemplary vaccine according to the invention can be prepared using the fusion protein of Example 2 (SEQ ID NO: 13) (eg, disclosed in US Pat. No. 10,441,652). NS).
Reagent solution:
1. Carbo pole base
Dissolve 0.5 grams of Carbopol 974P in water or physiological saline.
b. Mix and boil to dissolve. Then autoclave.
c. Store at 4 ° C.
2. Squalene base
Mix 58.1 ml squalene, 4.6 ml non-animal Tween 80, and 5.2 ml Span 85 and filter to 0.2 μ.
b. Store at 4 ° C.
3. Arabinogalactan solution
Add 1 to 10 grams of arabinogalactan to PBS or saline.
b. Mix and boil to dissolve. Then autoclave.
c. Store at 4 ° C.

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

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

Example 4: Endotoxin-free chimeric peptides / adjuvants result in increased milk production Random pool dairy cows (Holstein Crosses bred and raised in the United States), as disclosed in US Pat. No. 7,722,881. Were confirmed, and each was 31 to 65 days after delivery (3rd to 5th breastfeeding). Each cow was examined by a veterinarian and determined to be in optimal health.

The average cow weight in this study ranged from about 1000 to 1200 lbs. Six lactating cows were treated with a dose of 1.96 mg / chimeric protein / 2 ml in JH14. Instead, nine cows underwent conventional rBST treatment. Treatment and milk production studies were focused on milk-producing dairy farms and conducted on a large scale.

Vaccination was given on day 0. Anti-SST serum antibody and IGF-1 serum levels were tested at 4 weeks and milk production and overall animal health confirmation were performed on a regular basis.

Six cows vaccinated using the compositions of the invention described herein had a normal appearance and were free of endotoxin reactions and food withdrawal. All 6 cows had a positive serological response to SST with an average titer of 1:14. Milk production in 6 cows was obtained with only one vaccination (see Figure 3A), showing an average yield increase of 23.7%. Nine animals treated using conventional rBST injections on days 0 and 14 had an overall average of 2% increase in milk production (see Figure 3B).

The data in this example show a dramatic increase in efficacy with respect to the use of endotoxin-free constructs in combination with the adjuvants of the invention in dairy cows. These results were dramatically improved towards animal health and fertility compared to cattle injected twice with rBST.

Example 5: Multiple vaccinations with chimeric peptides / adjuvants increase milk production 92 clinically healthy dairy cows (single and multiple calvings) as described in US Pat. No. 10,441,652. Girilander and Girilander / Holstein hybrids) were confirmed, each 90 to 120 days after delivery. Cows were divided into four treatment groups: a single dose, a double dose, a quadruple dose, and a saline control group. Treatment and milk production studies were focused on milk-producing dairy farms and conducted on a large scale.

Cows were intramuscularly vaccinated in the neck area using alternating right, left, and 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 after the start of the study.

Treated cows showed increased milk production compared to pre-vaccination production, with the increase starting by 4 days post-vaccination and lasting for 21 days, with maximum production being vaccinated. It was 8 to 14 days after inoculation. This trend was repeated for each of the three vaccinations, as shown in FIGS. 4A and 4B. Compared to control cows, vaccinated cows demonstrated a 5-20% increase in milk production and an average increase in milk production of 13.6% during the post-vaccination period. Using the LSmeans model, there were highly statistical differences in milk production between treatment weeks.

There were no significant differences between treatment groups in terms of site response, body score, overall health, mastitis, or food problems. Vaccination (even at quadruple dose) was well tolerated in cattle and had no side effects compared to controls.

The data in this example show that multiple repeated injections of the compounds of the invention are highly effective with respect to the rapid increase in milk production, and the increase in production is maintained over a long period of time using multiple injections. Shows a surprising finding that it is possible to do. Multiple or repeated injections at regular intervals over a significant or indefinite period are expected to continue to cause improved milk production. This represents the disclosure of a vaccine that is very useful in the long-term improvement of milk production.

Example 6: Clinical Study of Mastitis A clinical study of mastitis was conducted in a Brazilian dairy cow herd for a period of 63 days. In this study, mastitis was unique in the herd used. All cows were previously 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 induce mastitis-prone breast tissue, where inflammation increases somatic cells (SCC) in the milk. Other side effects of rBST include hoof disorders, including loss of muscle mass, increased bone growth, and uneven hoof growth. In this herd, mastitis was chronic and was caused by Escherichia coli.

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

Cows were monitored for signs of mastitis and excluded from milk production if there was a clinical observation of SCC> 400000, swelling of the udder and / or secretion from the udder. Cows found to have mastitis received antibiotic infusions and were returned to production based on clinical symptoms, less than 350,000 SCCs, visual health, and ease of exercise.

The individual cows of the control, 1x and 4x vaccine groups that were excluded due to mastitis during the study are shown in Table 2.

As shown in FIG. 5 and Table 3, the number of days of lost milk was significantly reduced in the 1x and 4x groups compared to the control group. Surprisingly, in cattle with mastitis treated with the 1x or 4x vaccine test composition, the symptoms of mastitis were less severe and the mastitis resolved very quickly. Very surprisingly, the symptoms of mastitis in 2 of 3 cows receiving the 4x dose vaccine test composition responded very rapidly to antibiotic treatment and were returned to the herd after only 4 days. rice field. The number of days of milk lost is calculated in Table 3. Cows receiving the 1x or 4x vaccine test composition had a significant reduction in lost milk days, a significant reduction in% lost milk days, and a significant reduction in% lost milk days compared to control cows. It showed a significant reduction in the weight of lost milk (Kg) (Kg milk lost).

In addition, cows treated with the 1x or 4x vaccine test composition show increased milk production, as previously demonstrated, and side effects associated with rBST (eg, loss of muscle mass, bone growth). No increase or hoof disorder) was shown. The composition of milk in cows receiving the 1x or 4x vaccine test composition was the same as in control cows with no increased SCC (signs of mastitis).

Claims (21)

A vaccine composition for reducing the severity and / or duration of mammitis, a somatostatin-14 polypeptide having the amino acid sequence of SEQ ID NO: 1, and SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 1. Severity of mammitis in milk-producing animals, including a chimeric polypeptide comprising a chloramphenicol acetyltransferase polypeptide having a sequence selected from the group consisting of 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. And / or a composition that reduces the duration. The composition according to claim 1, wherein the milk-producing animal is selected from the group consisting of dairy cows, beef cattle, buffaloes, goats, sheep, camels, yaks, horses, reindeer, donkeys, and sows. The composition according to claim 1, wherein the composition is at least 80% endotoxin-free. The composition according to claim 1, wherein the chimeric polypeptide has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 13. The composition according to claim 1, wherein the chimeric polypeptide has the amino acid sequence of SEQ ID NO: 13. The composition according to claim 1, which is formulated for use as a vaccine. The composition of claim 1, wherein the chloramphenicol acetyltransferase polypeptide lacks enzymatic activity. The composition according to claim 1, wherein the somatostatin-14 polypeptide and the inert chloramphenicol acetyltransferase polypeptide are linked by a spacer. The spacers are 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: The composition according to claim 8, comprising a sequence selected from the group consisting of 25. The composition according to claim 1, further comprising an adjuvant. The composition of claim 10, further comprising one or more antigens selected from the group consisting of native polypeptides, recombinant polypeptides, synthetic polypeptides, polysaccharides, and glycoproteins. A composition for reducing the severity and / or duration of mammitis in milk-producing animals, comprising a somatostatin-14 polypeptide having the amino acid sequence of SEQ ID NO: 1 and a chloramphenicol acetyltransferase polypeptide. A composition comprising a chimeric polypeptide, wherein the chloramphenicol acetyltransferase polypeptide is number 192 with respect to the wild-type chloramphenicol acetyltransferase polypeptide represented by residues 1-210 of SEQ ID NO: 14. A composition that has been modified at a residue, residue 193, or both residues 192 and 193, and the modification comprises the independent substitution of one or both histidines with glycine or alanine. The composition according to claim 12, wherein the modification comprises the substitution of histidine at residue 193 with glycine. The composition according to claim 12, wherein the modification comprises the substitution of histidine at residue 193 with alanine. The composition according to claim 12, wherein the modification comprises the substitution of histidine at residue 192 with glycine and the substitution of histidine at residue 193 with glycine. The composition according to claim 12, wherein the chloramphenicol acetyltransferase polypeptide has 10 amino acids truncated at the C-terminal to the wild-type polypeptide. The composition according to claim 12, wherein the composition is substantially endotoxin-free. The composition according to claim 12, wherein the composition is at least 80% endotoxin-free. Animals have reduced severity and / or duration of mastitis, sharply reduced SCC, reduced days of lost milk, reduced antibiotic use, antibiotic efficacy compared to untreated control animals The composition of claim 12, which exhibits improved sex and / or reduced duration of antibiotic treatment. Antibiotics are selected from the group consisting of beta-lactams and lincosamides, in some cases the beta-lactams are amoxicillin, cefthioflu, cepapirin, cloxacillin, hetacillin, or penicillin, and in some cases the lincosamides are pyrrimycin. The composition according to claim 19. Milk-producing animals show a significant increase in milk production and no loss of muscle mass, increased bone growth, injection site reactions, or hoof disorders within 4 days of the first vaccination, claim 12. The composition according to.

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