# Patent

## Title
Interleukin-10 peptides and antibodies thereof for inhibiting adverse effects of protozoan infection

## Abstract

The present disclosure is directed to interleukin-10 (IL-10) peptides and isolated antibodies that specifically bind to the IL-10 peptides. The IL-10 peptides and the isolated antibodies may be administered alone or as an animal feed additive to treat gastrointestinal protozoan infection in animals.

## Background
INCORPORATION OF SEQUENCE LISTING
A paper copy of the Sequence Listing and a computer readable form of the sequence containing the file name “28243-164 (P120128U501)_ST25.txt”, which is 3810 bytes in size (as measured in MS-DOS) are provided herein and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOS: 1-20.
BACKGROUND OF THE DISCLOSURE
The present disclosure relates generally to interleukin-10 (IL-10) peptides and to isolated antibodies that specifically bind to the IL-10 peptides. The present disclosure further relates to animal feed additives comprising the IL-10 peptides and/or the isolated antibodies. The present disclosure further relates to methods for treating gastrointestinal protozoan infection, and in particular, maintaining growth in an animal infected with a protozoan infection by administering the IL-10 peptides and the isolated antibodies that specifically bind to the IL-10 peptides.
Coccidiosis is a common parasitic protozoan infection of livestock and poultry species, and is capable of infecting both invertebrates and vertebrates, including humans. Coccidiosis in chickens is caused by infection of the epithelial cells lining the alimentary tract and the cells of associated glands by the parasitic protozoa of the genus Eimeria. The Eimeria genus includes at least seventeen species capable of infecting birds, most notably E. tenella, E. necatrix, E. maxima, E. brunette, and E. acervulina. 
The life cycle of Eimeria takes about four to seven days to complete and begins when active oocysts are ingested by a host. The parasite obtains nutrients from the host and is prolific in nature, although the parasite will generally stop multiplying before causing the death of the host. Coccidiosis disrupts the digestive tract and enteric flora of an animal. Symptoms of the disease include weight loss, growth suppression, diarrhea, bloody diarrhea, anorexia, necrotizing enterocolitis, and sometimes death. Coccidiosis in poultry alone has a severe economical effect. Specifically it is estimated to cost the poultry industry one billion dollars a year in reduced animal performance.
Coccidiosis has generally been treated using anti-Coccidial drugs in animal feed and administering vaccinations using an attenuated Coccidiosis vaccine. Due to the potentially adverse effects on humans and animals, the use of anti-Coccidial drugs and antibiotics is being phased out in many countries including European countries and Japan. In addition, the increased emergence of drug resistant strains and the increased costs of developing new drugs have led to an interest in developing alternative methods for controlling Coccidiosis. Using vaccines for treating Coccidiosis, for example, has many disadvantages, including risk that the vaccine will not generate a sufficient amount of antibodies for treating the infection. Additionally, vaccination requires several weeks to produce antibodies, leaving a several week period in which the immunized birds may become infected with Coccidiosis. Attenuated vaccines may also negatively impact animal growth, resulting in suppression of animal performance.
In light of the current problems associated with controlling Coccidiosis using drugs and/or vaccines, there exists a need for a feed additive capable of treating Coccidiosis and other gastrointestinal protozoan infections, while maintaining growth in an animal. Additionally, there exists a need for a natural feed additive, acceptable by regulatory authorities throughout the world, and which comports with global trends aimed at eliminating drugs and antibiotics from animal feed and the resulting animal products.
SUMMARY OF THE DISCLOSURE
The present disclosure relates generally to IL-10 peptides. More particularly, the present invention relates to IL-10 peptides having an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. It has been found that these IL-10 peptides, and the isolated antibodies that specifically bind to these peptides, may be used for treating gastrointestinal protozoan infection in animals. Particularly, in one embodiment, the IL-10 peptides and/or the isolated antibodies that specifically bind to the IL-10 peptides may be administered to an animal infected with a gastrointestinal protozoa in an amount effective to maintain the growth of the animal.
In one aspect, the present disclosure is directed to an interleukin-10 peptide including an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
In another aspect, the present disclosure is directed to an isolated antibody that specifically binds to an interleukin-10 peptide including an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
In another aspect, the present disclosure is directed to a method for treating a gastrointestinal protozoan infection in an animal. The method includes administering an interleukin-10 peptide including an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
In another aspect, the present disclosure is directed to a method for treating a gastrointestinal protozoan infection in an animal. The method includes administering to the animal an isolated antibody that specifically binds to an interleukin-10 peptide including an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
In another aspect, the present disclosure is directed to a method of generating an antibody that specifically binds to an interleukin-10 peptide including an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. The method includes administering the interleukin-10 peptide to an animal.
In another aspect, the present disclosure is directed to an animal feed additive including at least one of an interleukin-10 peptide, an isolated antibody that specifically binds to the interleukin-10 peptide, and combinations thereof. The interleukin-10 peptide includes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
In another aspect, the present disclosure is directed to an animal feed comprising from about 0.05% by weight to about 1% by weight of an animal feed additive. In one embodiment, the animal feed additive is a dried egg powder. The animal feed additive includes at least one of an interleukin-10 peptide, an isolated antibody that specifically binds to the interleukin-10 peptide, and combinations thereof. The interleukin-10 peptide includes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.


DETAILED DESCRIPTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable materials and methods are described below.
In accordance with the present disclosure, IL-10 peptides and isolated antibodies that specifically bind to IL-10 peptides, animal feed additives including the IL-10 peptides and the isolated antibodies that specifically bind to IL-10 peptides, and methods for treating gastrointestinal protozoan infection and maintaining growth in an animal infected with a protozoan infection by administering the IL-10 peptides and/or antibodies which specifically bind to IL-10 peptides have been discovered.
Significantly, the IL-10 peptides and isolated antibodies that specifically bind to IL-10 peptides have been found to prevent growth suppression effects typically associated with Coccidiosis infection. Using the IL-10 peptides, isolated antibodies that specifically bind to the IL-10 peptides, and/or animal feed additives containing the IL-10 peptides, antibodies, and combinations thereof avoids significant concerns associated with anti-Coccidial drugs and Coccidial vaccines, including safety concerns, drug resistance concerns, and cost or drug development concerns. Additionally, concerns associated with administering Coccidiosis vaccines, including growth suppression effects and ineffectiveness due to both insufficient antibody production and periods of time between administration of the vaccine and antibody production may be avoided.
Interleukin-10 (IL-10) Peptide and Antibodies Thereof
As noted above, the present disclosure is generally directed to IL-10 peptides and antibodies that specifically bind to the IL-10 peptides for used in animals to treat gastrointestinal protozoan infection. The term “peptide” includes the peptide as well as pharmaceutically acceptable salts of the peptide. “Amino acid residue” means the individual amino acid units incorporated into the peptides of the disclosure. As used herein, the term “amino acid” means a naturally occurring or synthetic amino acid, as well as amino acid analogs, stereoisomers, and amino acid mimetics that function similarly to the naturally occurring amino acids.
As used herein, the term “antibody”, or “immunoglobulin”, encompasses naturally occurring antibodies, such as polyclonal and monoclonal antibodies, as well as artificial or synthetic antibodies or genetically engineered forms of antibodies, including single chain antibodies, chimeric, and bifunctional antibodies, as well as fragments thereof.
The term “isolated antibody” as used herein, refers to an antibody that is substantially free of other naturally associated molecules, or substantially free of antibodies having different antigenic specificities.
The IL-10 peptide of the present disclosure includes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 (see Table 1). In particularly suitable embodiments, the IL-10 peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10.







TABLE 1







Sequence ID NO. and Corresponding


Amino Acid Sequence.










SEQ ID NO:
AMINO ACID SEQUENCE






SEQ ID NO: 1
DDELNIQL


 



SEQ ID NO: 2
DQMGDLL


 



SEQ ID NO: 3
DQLHSLL


 



SEQ ID NO: 4
VLPRAMQT


 



SEQ ID NO: 5
VMPKAESD


 



SEQ ID NO: 6
VMPQAENH


 



SEQ ID NO: 7
EKMDENGI


 



SEQ ID NO: 8
SKLQERGV


 



SEQ ID NO: 9
SELQERGV


 



SEQ ID NO: 10
EPTCLHFS


 



SEQ ID NO: 11
ENSCIHFP


 



SEQ ID NO: 12
DSSCIHLP


 



SEQ ID NO: 13
DQLNSML


 



SEQ ID NO: 14
VMPQAENH


 



SEQ ID NO: 15
NMLQERGV


 



SEQ ID NO: 16
DSSCTHFP


 



SEQ ID NO: 17
DDLEIGL


 



SEQ ID NO: 18
VLPTAIADMTEE


 



SEQ ID NO: 19
TQMEGKGP


 



SEQ ID NO: 20
NQCCRFV









SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10 are amino acid sequences corresponding to four peptides which stimulate IL-10 cytokine production in avian animals, such as chickens, quail, and turkeys. It has been surprisingly found that the IL-10 peptides of the present disclosure prevent growth suppression effects typically associated with gastrointestinal protozoan infection.
The present disclosure is further directed to antibodies that specifically bind to the IL-10 peptides (also referred to herein as “anti-IL-10 antibody”). These antibodies have surprisingly been found to prevent gastrointestinal protozoan growth suppression effects associated with gastrointestinal protozoan infection when isolated and administered thereto. The antibodies of the present disclosure specifically bind to IL-10 peptides including amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments, the isolated antibodies specifically bind to IL-10 peptides having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10.
Methods of Generating the Antibodies
The present disclosure is further directed to generating antibodies that specifically bind to the IL-10 peptides. In one embodiment, an antibody is generated by administering the IL-10 peptides described above to an animal. Suitable animals to administer the IL-10 peptides for generating the antibodies may be, for example, avians. Suitable avians may be, for example, chickens, turkeys, ducks, quail, and pheasant. Additional animals include livestock animals such as cows, pigs, sheep, and fish.
Suitable methods for administering the IL-10 peptides to the animal may be, for example by injection or oral administration. Injection and oral admission may include use of an adjuvant such as, for example, Freunds Complete adjuvant and Cholera toxin. Administration may further include conjugation of the IL-10 peptide to a carrier protein such as, for example, bovine gamma globulin.
In one embodiment, antibodies to the IL-10 peptides are generated by an animal (referred to herein as the “producer animal”). When the animal is an avian animal, as know by those skilled in the art, the antibodies generated are passed to the egg, and may specifically be concentrated in the egg yolk of the avian producer animal. Alternatively, antibodies of the present disclosure may be isolated from the animal such as from serum.
The antibodies that specifically bind to IL-10 peptides may be isolated and purified from animal serum or egg using any suitable method known in the art. Such methods include affinity chromatography, as well as other suitable methods for antibody isolation and purification known in the art and described in U.S. Pat. No. 6,608,172 and De Meulenaer et al., “Isolation and Purification of Chicken Egg Yolk Immunoglobulins: A Review,” Food and Agricultural Immunology, Vol. 13(4), 2001, hereby incorporated by reference to the extent that they are consistent herewith. In one particularly suitable embodiment, the animal is an avian animal such as a chicken, turkey, duck, or quail, and the antibody can be isolated from the egg yolk of the egg of the avian animal.
In one embodiment, the egg yolk or serum including the antibodies are further dried to form a powder including the antibodies. The whole egg, egg yolk or parts of the egg may be spray dried. Serum may be separated from whole blood according to conventional methods known by those skilled in the art. Spray drying of egg and serum may be performed using conventionally known spray drying methods and commercially available spray drying equipment. Dry egg and serum powders may also be prepared by lyophilization. The dried egg, egg yolk or serum powder may then be introduced into animal feeds as a feed additive to transfer antibodies to an animal.
Animal Feed Additive
The present disclosure is further generally directed to animal feed additives including the IL-10 peptides or isolated antibodies which specifically bind to IL-10 peptides or combinations thereof.
As used herein, the term “feed” broadly refers to any kind of material, liquid or solid that is used for nourishing an animal, and for sustaining normal or accelerated growth of an animal including newborns or young and developing animals. The term includes any compound, preparation, mixture, or composition suitable for intake by an animal. Preferably, the feed is suitable for intake by livestock animals such as cows, pigs, sheep, and fish, as well as avian animals such as quail, ducks, turkeys, and chickens. The term “feed additive” as used herein refers to components typically included in small quantities for the purpose of fortifying basic feed with nutrients, stimulants, medicine, or to promote feed intake or alter metabolism. Feed additives may include pre-mixes of biological compositions, or in the present disclosure, pre-mixes of IL-10 peptide or isolated antibody that specifically binds to IL-10 peptide or combinations thereof.
In one embodiment, the present disclosure is directed to an animal feed additive including IL-10 peptides including an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. Particularly, the feed additive may include IL-10 peptides having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10, and combinations thereof.
In another embodiment, the present disclosure is directed to an animal feed additive including isolated antibodies that specifically bind to the IL-10 peptide including an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In particularly suitable embodiments, the feed additive includes isolated antibodies that specifically bind to IL-10 peptides having an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and combinations thereof.
The IL-10 peptides and/or isolated antibodies which specifically bind to IL-10 peptides may be added to an animal feed as a feed additive or mixed into an animal feed by any method known in the art for mixing feed additives and animal feed. In one embodiment, the IL-10 peptide or isolated antibody which specifically binds to IL-10 peptide may be directly added to the animal feed or mixed with the animal feed just prior to feeding the animal.
The amount of the IL-10 peptide or isolated antibody that specifically binds to IL-10 peptide added and/or mixed with the animal feed depends on the feeding regimen and the type of feed for the animal, and may be determined by those skilled in the art. Typically, the amounts of IL-10 peptides and/or isolated antibodies to IL-10 peptide to be used in an animal feed are summarized in Table 2 below. Antibody prepared using other sources may be calculated as equivalents using Table 2.







TABLE 2







Dose of Anti-IL-10 Antibody in Animal Feed (mg/Kg diet)


prepared using egg yolk antibody.











Source
Low Dose
High Dose















Affinity purified anti-peptide
0.0015
0.5



Anti-peptide IgY
0.015
50



Dry Immune Yolk
0.8
4000



Dried Immune Whole Egg
1.5
7500







Range in doses shown are based on the amount of epitope specific antibody in total IgY (1 to 10%), the amount of IgY in egg (5-10 mg/Kg of feed), antibody losses due to drying storage and gastrointestinal degradation.






The animal feed of the present disclosure may further include optional ingredients including vitamins, minerals, antibiotics, lipids, carbohydrates, proteins, antioxidants, and amino acids.
Suitable vitamins which may be included in the animal feed of the present disclosure include Vitamin A, Vitamin B, Vitamin D, Vitamin E, and Vitamin K.
Suitable minerals which may be included in the animal feed of the present disclosure may include calcium, phosphorus, sodium, potassium, magnesium, chlorine, cobalt, iodine, iron, manganese, copper, molybdenum, zinc and selenium. Common mineral supplements used in poultry feed, for example, include limestone, bone meal, oyster shell, sodium chloride, dicalcium phosphate, manganese sulphate, potassium iodide, and superphosphate.
In some embodiments, one or more antibiotics may be included in the animal feed along with the feed additive. Suitable antibiotics included in the feed may be, for example, penicillin, streptomycin, tetracyclines, and aureomycin.
Suitable lipids included in the animal feed may be, for example, any oil seed, oil and lipid derived from plants or animals. Sources of oilseeds, oils and lipids which may be used in the animal feed include corn, soybean, cotton, lupin, peanut, sunflower, canola, sesame seed oil, olive oil, copra and coconut oil, palm kernels and palm oil, casein, butterfat, lard, fish oils, linseed and oil, tuna oil, tallow and yellow grease, and mixtures thereof.
Suitable carbohydrates which may be included in the animal feed may include starch, cellulose, pentosans, other complex carbohydrates, corn, milo, barley, rye, oats, wheat, wheat middlings, and various grain-by-products.
Suitable sources of protein which may be included in the feed may be, for example, protein obtained from meat meal or fish meal, liquid or powdered egg, fish solubles, whey, milk protein, rice, milo, millet, corn, oats, barley, wheat, rye, wheat bran and/or middlings, soybeans, sesame seeds, peas and beans, sunflower seeds, wheat germ, alfalfa seed, flaxseed, yeast, earthworms, and fish.
Suitable amino acids which may be included in the feed, in addition to the IL-10 peptides disclosed herein, may be, for example, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HC1, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cystein ethyl HC1, and analogs, and salts thereof.
Suitable antioxidants which may be included in the feed include beta-carotene, Vitamin E, Vitamin C, and tocopherol, or synthetic antioxidants.
Preferably, the animal feed including the feed additive of either IL-10 peptide and/or isolated antibody is a feed for livestock. Preferably, the animal administered the feed composition is selected from the group consisting of cows, pigs, sheep, fish, as well as avian animals such as quail, ducks, turkeys, and chickens.
Methods of Use
The methods of the present disclosure are generally directed to methods for treating gastrointestinal protozoan infection in an animal, and in particular, maintaining growth in an animal infected with a gastrointestinal protozoan infection. In one embodiment, the methods involve injecting or orally administering an IL-10 peptide to an animal, thereby producing antibodies within the animal that specifically bind to the IL-10 peptide. IL-10 cytokine production is associated with down regulation of inflammation, and the IL-10 cytokine functions as an essential immunoregulator of the intestinal tract.
In some embodiments, the methods involve injecting or orally administered an antibody to the IL-10 peptide to an animal. The term “animal”, as used herein to describe animals administered an IL-10 peptide or isolated antibody to the IL-10 peptide in accordance with the present disclosure, includes any animal, including a livestock animal, more preferably an animal selected from the group consisting of cows, pigs, sheep, fish, as well as avian animals such as quail, ducks, turkeys, pheasants, and chickens.
It has unexpectedly been found that antibodies that specifically bind to IL-10 peptides treat gastrointestinal protozoan infection in an animal and, additionally, prevent growth suppression typical of gastrointestinal protozoan-infected animals. This is an unexpected surprise, as it is generally known that Coccidiosis infection causes inflammation, which is linked to growth suppression, and that antibody to cytokine IL-10 increases the immune system's inflammatory response, which should thereby lead to increased growth suppression.
Gastrointestinal protozoa include parasites from the kingdom Protozoa. In a suitable embodiment, the protozoa treated by the presently disclosed methods may be from Apicomplexa. Suitable Apicomplexa may be, for example, Coccidiasina. In a particularly suitable embodiment, the protozoa is Eimeriorina such as, for example, Eimeriidae and Cryptosporidiidae. In a particularly suitable embodiment, the protozoa is selected from the group consisting of Cryptosporidium, Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria brunetti. 
In one aspect, the present disclosure is directed to methods for preventing gastrointestinal protozoan infection and maintaining growth in an animal infected with protozoan infection or at risk of protozoan infection by administering isolated antibodies that specifically bind to IL-10 peptides including amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. For example, the methods may include administering isolated antibodies that specifically bind to IL-10 peptides having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and combinations thereof. As used herein “at risk of” refers to having little resistance to a certain condition or disease (i.e., protozoan infection), including being genetically predisposed, having a family history of, and/or having symptoms of the condition or disease, and being exposed to other animals that have been exposed to or have the condition or disease.
Additionally, the IL-10 peptides and/or isolated antibodies may further be used as a feed additive for animal feed. The animal feed may be administered to an animal to treat protozoan infection in the gastrointestinal tract of an animal.
The disclosure will be more fully understood upon consideration of the following non-limiting Examples.
EXAMPLES
Example 1
Detection of Antibody Production
In this Example, the concentration of anti-IL-10 antibody production contained within the egg yolk of IL-10 peptide-administered producer hens was determined by using Enzyme-linked immunosorbent assay (ELISA) techniques.
Specifically, each of four IL-10 peptides selected from the group consisting of IL-10 Peptide #1 (SEQ ID NO: 1), IL-10 Peptide #2 (SEQ ID NO: 4), IL-10 Peptide #3 (SEQ ID NO: 7), and IL-10 Peptide #4 (SEQ ID NO: 10) was conjugated to hen ovalbumin (OVA, Sigma, St. Louis, Mo.) for ELISA using glutaraldehyde procedure. A 96-well Nunc immunosorbent F-series microplate (Sigma, St. Louis, Mo.) was coated with 100 μg/plate of peptide-specific OVA conjugate in sodium carbonate coating buffer having a pH of 9.6. The plate was allowed to coat overnight (100 μl/well) at 4° C. Dry egg yolk samples containing antibody to IL-10 Peptide #1, #2, #3, or #4 were diluted 1:10 in acidic PBS having a pH of 4 and allowed to incubate overnight at 4° C. After overnight incubation, the plate was washed 6 times with PBS/0.5% Tween solution, blocked with non-protein blocking buffer (175 μl/well, Pierce Scientific, Rockford, Ill.), and allowed to incubate at room temperature for at least 1 hour. The plate was washed 6 times and then samples of either FCA control or antibody were added at a concentration of 100 μl/well in duplicate at 10× serial dilutions starting at 1:1000. Primary antibodies were incubated for 1 hour, the plate was washed 6 times, and then secondary antibody (HRP-conjugated goat anti-chicken antibody, Bethyl Labs, Montgomery, Tex.) was diluted in blocking buffer 1:5000 and added at a concentration of 100 μl/well. Secondary antibody was incubated for 30 minutes, and then substrate solution containing 19.74 ml 0.05M sodium acetate, 100 μA 20 mg/mL 3,3′,5,5′ Tetramethyl Benzidine (TMB), 128 μl 0.5M H2O2 was added at a concentration of 125 μl/well and allowed to incubate until sufficient color development during the linear phase of development (blue color indicates primary antibody presence). A stop solution (0.5M sulfuric acid) was added to produce a yellow stable color and the plate was read at 450 nm on a Biotek EL800 plate reader. Duplicate optical densities were averaged and blocking buffer background was subtracted to produce a final optical density. Optical density of antibody to IL-10 peptides #1-4 and FCA control were compared to determine specificity and dose level used in the final chick experiment (see Table 3).







TABLE 3







Optical Densities of Anti-IL-10 Peptides.













Dilution
IL-10 #1
IL-10 #2
IL-10 #3
IL-10 #4

















10
1.0585
0.935
0.968
0.8822



100
1.049
1.006
0.973
0.9



1000
0.9705
1.033
1.001
0.678



10000
0.4795
0.6775
0.593
0.224



100000
0.075
0.129
0.115
0.059



1000000
0.028
0.03
0.037
0.03



10000000
0.059
0.016
0.027
0.028



100000000
0.022
0.0055
0.0181
0.033



FCA
0.562

0.499
0.60










Example 2
Chicks Fed Antibody to IL-10 Peptide
In this Example, the use of isolated anti-IL-10 antibody to prevent growth suppression due to Coccidiosis infection was determined
Specifically, Single Comb White Leghorn laying hens were injected with one of four IL-10 peptides: IL-10 peptide #1 (SEQ ID NO: 1), IL-10 peptide #2 (SEQ ID NO: 4), IL-10 peptide #3 (SEQ ID NO: 7), or IL-10 peptide #4 (SEQ ID NO: 10). Egg antibodies directed against the four hydrophilic, antigenic and accessible peptides of IL-10 were then produced in the laying hens. More specifically, the egg antibodies were directed against IL-10 peptide #1 (SEQ ID NO: 1), IL-10 peptide #2 (SEQ ID NO: 4), IL-10 peptide #3 (SEQ ID NO: 7), or IL-10 peptide #4 (SEQ ID NO: 10). Egg yolks were collected from the producer hens, lyophilized, and the egg powder was added to feed at a concentration of 3.41 grams of egg powder/Kg feed. Heavy breed broiler chicks were then divided into five groups of at least 40 chicks/group. Four of the groups were fed egg antibody specific to IL-10 Peptides #1-4. The fifth group, or the control group, was fed egg antibody or egg antibody collected from hen injected with Freund's complete adjuvant and no IL-10 peptides. Three days following egg antibody feeding, half of all five groups of chicks were either subjected to oral gavage with saline and half were subjected to a 10× dose of live attenuated Coccidiosis mixture, the mixture consisting of the following Eimeria species: Eimeria acervulina, Eimeria tenella, Eimeria maxima, and Eimeria brunetti (ADVENT® Coccidiosis Vaccine control, Novus Intl., St. Charles, Mo.). The chicks were then monitored for 7 days, or from day 3 to day 10, for weight gain post-Coccidiosis infection.
As shown in Table 4, demonstrating the composite results of the study carried out twice, the Coccidiosis-exposed chicks in each of the four groups fed antibody to IL-10 Peptides #1-4 demonstrated greater weight gain (in grams) than the control fed Coccidiosis-exposed chicks. Of the four groups of Coccidiosis-exposed chicks fed antibody to IL-10 peptides, chicks fed antibody to IL-10 Peptide #2 demonstrated the greatest weight gain as compared to the other three groups of chicks fed antibody to IL-10 peptides #1, #3, and #4. Additionally, weight gain for chicks fed antibody to IL-10 Peptide #2 approached that found in non-Coccidiosis exposed control chicks. Chicks not exposed to Coccidiosis and fed antibody to IL-10 peptides in all four groups showed greater weight gain than the control fed chicks not exposed to Coccidiosis.







TABLE 4







Effect of Feeding Anti-IL-10 on Growth Response 


after 7 Days (in grams)









Antibody
No Coccidiosis
Coccidiosis












Control
192
174


Anti-IL-10 #1
193
177


Anti-IL-10 #2
195
185


Anti-IL-10 #3
200
180


Anti-IL-10 #4
196
175









Table 5 demonstrates the average rate of weight gain (in grams) in a two day period for Coccidiosis-exposed chicks fed control antibody versus antibody to IL-10 peptide #2. Table 3 also demonstrates the average total rate of weight gain (in grams) in a 7-day period for Coccidiosis-exposed chicks fed control antibody versus antibody to IL-10 peptide #2. As shown in Table 3, Coccidiosis-exposed chicks fed antibody to IL-10 peptide #2 demonstrated a greater weight gain (in grams) as compared to Coccidiosis-exposed control-fed chicks.







TABLE 5







Effect of Feeding Anti-IL-10 on Growth Response after 2 Days 


and after 7 Days (in grams)












Average Rate of Weight 
Average Rate of Weight



Antibody
Gain in 2 Day Period
Gain in 7 Day Period















FCA Control
43.1
173.7778



Anti-IL-10 #2
48.4
195.1333










These results suggest that the chicks infected with Coccidiosis and fed control antibody had suppressed growth rates as compared to chicks not infected with Coccidiosis and fed control antibody. Thus, these results suggest that Coccidiosis infection contributes to growth suppression in chicks infected with Coccidiosis. Further, these results suggest that isolated antibodies to IL-10 peptides #1-4 prevent growth suppression caused by Coccidiosis infection. These results also suggest that feeding isolated anti-IL-10 peptides to healthy broiler chicks does not adversely affect growth, but may actually improve growth in healthy chicks.
Example 3
Hens Injected with IL-10 Peptides and Passive Transfer of Antibody to IL-10 Peptides to Hatched Chicks
In this Example, the use of antibodies, specific to IL-10 peptides, which were passively transferred to hatched chicks to prevent growth suppression due to Coccidiosis, was determined.
Single Comb White Leghorn laying hens were divided into five groups. Four groups were injected with four IL-10 peptides: IL-10 peptide #1 (SEQ ID NO: 1), IL-10 peptide #2 (SEQ ID NO: 4), IL-10 peptide #3 (SEQ ID NO: 7), and IL-10 peptide #4 (SEQ ID NO: 10). The fifth group, or control group, was injected with Freund's complete adjuvant alone. Twenty-one days after the first injections, hens were inseminated, fertile eggs incubated, and chicks were hatched with the circulating antibodies. Three days after the chicks hatched, the chicks were injected with a 10-dose of live attenuated Coccidiosis mixture, the mixture consisting of the following Eimeria species: Eimeria acervulina, Eimeria tenella, Eimeria maxima, and Eimeria brunetti (ADVENT® Coccidiosis Vaccine control, Novus Intl., St. Charles, Mo.). Growth was determined for 7 days, or from day 3 to day 10, following injection with the Coccidiosis vaccine.
As shown in Table 6, the Coccidiosis-exposed chicks with circulating antibody to IL-10 peptides showed greater weight gain as compared to Coccidiosis-exposed chicks with circulating control antibody. Coccidiosis-exposed chicks without circulating antibody to IL-10 peptides showed less weight gain as compared to non-Coccidiosis exposed chicks without circulating antibody to IL-10 peptides.







TABLE 6







Effect of Coccidiosis and Circulating Anti-IL-10 


on Growth (in grams)









Antibody
Control
Anti-IL-10





No Coccidiosis
95(2)
97(2)


Coccidiosis
87(4)
96(3)









Table 7 shows the average weight gain (in grams) for Coccidiosis-exposed chicks with circulating antibody to IL-10 Peptide #4 as well as non-Coccidiosis exposed chicks with circulating antibody to IL-10 Peptide #4. Table 5 also shows the average weight gain (in grams) for Coccidiosis-exposed chicks with circulating control antibody as well as non-Coccidiosis exposed chicks with circulating control antibody. Table 5 demonstrates that Coccidiosis-exposed chicks with circulating antibody to IL-10 peptide #4 had a greater weight gain as compared to the chicks with circulating control antibody, both exposed and not exposed to Coccidiosis. Table 5 further demonstrates that the Coccidiosis-exposed chicks with circulating control antibody gained less weight than non-Coccidiosis exposed chicks with circulating control antibody. Table 5 further demonstrates that non-Coccidiosis exposed chicks with circulating antibody to IL-10 peptide #4 had a greater weight gain than non-Coccidiosis-exposed chicks with the circulating control antibody.







TABLE 7







Effect of Circulating Anti-IL-10 #4 on Growth Response 


Following Coccidiosis Exposure (in grams)









Antibody
Coccidiosis
No Coccidiosis












FCA Control
87.083
94.583


Anti-IL-10 #4
96.071
97.266









These results suggest that Coccidiosis infection contributes to growth suppression in chicks infected with Coccidiosis. These results also suggest that administering IL-10 peptides to hens causes the hens to passively transfer the antibodies that specifically bind to IL-10 peptides to their chicks. Furthermore, these results suggest the passively transferred antibodies that specifically bind to IL-10 peptides prevent growth suppression caused by Coccidiosis infection. Lastly, these results suggest that administering IL-10 peptides to animals does not adversely affect growth, but may actually improve growth in healthy chicks.
In view of the above, it will be seen that the several advantages of the disclosure are achieved and other advantageous results attained. As various changes could be made in the above peptides and methods without departing from the scope of the disclosure, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
When introducing elements of the present disclosure or the various versions, embodiment(s) or aspects thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

## Claims

1. An isolated interleukin-10 peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

2. The isolated interleukin-10 peptide of claim 1 consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10.

3. A method for treating a Coccidiosis gastrointestinal protozoan infection in an animal, the method comprising administering the interleukin-10 peptide of claim 1.

4. The method of claim 3 wherein the interleukin-10 peptide is administered to the animal by injection or oral delivery.

5. The method of claim 3 wherein the animal is selected from the group consisting of a cow, a pig, a sheep, a fish, a quail, a duck, a turkey, a pheasant and a chicken.

6. The method of claim 3 wherein the animal is an avian animal selected from the group consisting of a chicken, turkey, duck, pheasant and quail.

7. The method of claim 3 wherein the Coccidiosis gastrointestinal protozoan infection is caused by an Apicomplexa protozoa.

8. The method of claim 7 wherein the protozoa is selected from the group consisting of Cryptosporidium, Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria brunetti.

9. The method of claim 3 wherein treating the Coccidiosis gastrointestinal protozoan infection comprises maintaining growth of the animal.

10. An animal feed additive comprising at least one of an isolated interleukin-10 peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

11. The animal feed additive of claim 10 wherein the interleukin-10 peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10.

12. An animal feed comprising from about 0.15 mg/kg to about 50 mg/kg of the animal feed additive of claim 10.