Hyperimmunized egg products for treating or preventing alcoholic liver disease and graft-versus-host disease - Patents.com

Abstract

In one aspect, the disclosure relates to a method of preventing or treating alcoholic liver disease or graft-versus-host disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-laying animal, thereby preventing or treating alcoholic liver disease or graft-versus-host disease in the subject, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies against an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, and Enterococcus faecium. The disclosure also relates to hyperimmunized eggs and egg products produced by an animal that has been hyperimmunized with an antigen selected from the group consisting of Enterococcus faecalis, isolated Enterococcus faecalis cytolysin toxin, and Enterococcus faecium. Methods for preparing hyperimmunized eggs and egg products are also disclosed.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 175,603, filed April 16, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] Sequence Listing The sequence listing associated with this application has been submitted in electronic format via EFS-Web and is hereby incorporated by reference in its entirety. The name of the text file containing the sequence listing is 131331_00320_Sequence_Listing. The size of the text file is 1004 bytes and the text file was created on April 14, 2022. [Background technology]

[0003] Alcoholic liver disease (ALD) encompasses liver manifestations due to excessive alcohol consumption, including fatty liver, alcoholic hepatitis, and chronic hepatitis with liver fibrosis or cirrhosis. The most severe form of alcoholic liver disease is alcoholic hepatitis, with mortality rates ranging from 20% to 40% at 1-6 months, and as many as 75% of patients die within 90 days of diagnosis of severe alcoholic hepatitis. See Duan et al., 2019, Nature 575: 505-511. Corticosteroid therapy is only marginally effective. Early liver transplantation is the only curative therapy, but is only offered in selected centers and to a limited group of patients. See Duan et al., cited above.

[0004] Graft-versus-host disease (GVHD) affects the skin, liver, and gastrointestinal tract and occurs when donor T cells recognize recipient tissues as foreign, causing tissue inflammation and damage. Prevention and treatment strategies for GVHD are an active area of ​​research. See Garrett, 2020, New England Journal of Medicine 382(11): 1064-1066. Thus, there is a need for improved methods of treating or preventing ALD and GVHD. Summary of the Invention

[0005] In certain aspects, the disclosure relates to a method of preventing or treating alcoholic liver disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-laying animal, thereby preventing or treating alcoholic liver disease in the subject, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies against an antigen selected from the group consisting of Enterococcus faecalis and Enterococcus faecalis cytolysin toxin.

[0006] In certain aspects, the disclosure relates to a method of preventing or treating graft-versus-host disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-laying animal, thereby preventing or treating graft-versus-host disease in the subject, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies against an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, and Enterococcus faecium.

[0007] In certain embodiments, the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies against Enterococcus faecalis and a therapeutically effective amount of one or more antibodies against Enterococcus faecalis cytolysin toxin. In certain embodiments, the method further comprises hyperimmunizing the egg-laying animal with an antigen selected from the group consisting of Enterococcus faecalis, isolated Enterococcus faecalis cytolysin toxin, and Enterococcus faecium. In certain embodiments, the method further comprises collecting hyperimmunized eggs from the egg-laying animal that has been hyperimmunized, and preparing a hyperimmunized egg product from the hyperimmunized eggs. In certain embodiments, the hyperimmunized egg product comprises at least 20% more by weight of IgY antibodies specific for an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, and Enterococcus faecium compared to a control egg product obtained from an egg-laying animal that has not been hyperimmunized. In certain embodiments, the hyperimmunized egg product is administered to a subject 1-4 times per day. In certain embodiments, the hyperimmunized egg product is administered orally or intravenously. In certain embodiments, administration of the hyperimmunized egg product to a subject reduces levels of Enterococcus faecalis in the subject compared to a subject not administered the hyperimmunized egg product. In certain embodiments, administration of the hyperimmunized egg product to a subject reduces liver damage in the subject compared to a subject not administered the hyperimmunized egg product. In certain embodiments, the subject is a human.

[0008] In certain aspects, the disclosure relates to hyperimmunized eggs produced by an animal that has been hyperimmunized with an antigen selected from the group consisting of Enterococcus faecalis, isolated Enterococcus faecalis cytolysin toxin, and Enterococcus faecium, wherein the hyperimmunized eggs have increased levels of antibodies against the antigen compared to eggs from animals that have not been hyperimmunized. In certain embodiments, the animal has been hyperimmunized with Enterococcus faecalis and isolated Enterococcus faecalis cytolysin toxin, and wherein the levels of antibodies against Enterococcus faecalis and isolated Enterococcus faecalis cytolysin toxin are increased compared to eggs from animals that have not been hyperimmunized.

[0009] In certain aspects, the disclosure relates to a hyperimmunized egg product obtained from the hyperimmunized eggs described herein. In certain embodiments, the hyperimmunized egg product is a whole egg. In certain embodiments, the hyperimmunized egg product is an egg yolk. In certain embodiments, the hyperimmunized egg product is a purified or partially purified IgY antibody against Enterococcus faecalis cytolysin toxin. In certain embodiments, the hyperimmunized egg product is a purified or partially purified IgY antibody against Enterococcus faecalis. In certain embodiments, the hyperimmunized egg product consists of a purified or partially purified IgY antibody against Enterococcus faecalis and a purified or partially purified IgY antibody against Enterococcus faecalis cytolysin toxin.

[0010] In certain aspects, the present disclosure relates to a pharmaceutical composition comprising the hyperimmunized egg product of any one of claims 14 to 19 and a pharma- ceutically acceptable carrier. In certain embodiments, the composition comprises 3 to 10 grams of whole egg. In certain embodiments, the composition comprises 1 to 3 grams of egg yolk. In certain embodiments, the composition comprises 0.05 to 1 gram of purified or partially purified IgY. In certain embodiments, the pharmaceutical composition is formulated for oral administration. In certain embodiments, the hyperimmunized egg product is formulated in a nanoparticle or emulsion. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0011] In certain aspects, the disclosure provides a method of preparing a hyperimmunized egg product, comprising: i) hyperimmunizing an egg-laying animal with an antigen selected from the group consisting of Enterococcus faecalis, isolated Enterococcus faecalis cytolysin toxin, and Enterococcus faecium; and ii) preparing the hyperimmunized egg product from one or more eggs produced by the animal. In certain embodiments, the antigen is selected from the group consisting of isolated Enterococcus faecalis cytolysin toxin and Enterococcus faecium. In certain embodiments, the antigen is selected from the group consisting of Enterococcus faecalis and isolated Enterococcus faecalis cytolysin toxin. In certain embodiments, the antigen comprises Enterococcus faecalis and isolated Enterococcus faecalis cytolysin toxin. In certain embodiments, the egg-laying animal is a chicken. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows an outline of an experiment evaluating anti-Enterococcus faecalis and anti-cytolysin IgY antibodies in a mouse model of alcoholic liver disease. [Diagram 2]Purity of anti-Enterococcus faecalis and anti-cytolysin IgY antibodies analyzed by SDS-PAGE electrophoresis. IgY has a molecular weight of 180 KDa and is composed of two subunits, a heavy chain of 67 KDa and a light chain of 23 KDa. Lane 1 is a molecular weight ladder, lane 2 is an IgY standard, and lanes 3 and 4 are 1 μg and 2 μg of total soluble protein from egg yolks of chickens hyperimmunized with Enterococcus faecalis and cytolysin, respectively. [Figure 3A] FIG. 13 shows Enterococcus faecalis specific activity of IgY from chickens immunized with Enterococcus faecalis plus cytolysin. [Figure 3B] Figure 1 shows the cytolysin specific activity of IgY from chickens immunized with Enterococcus faecalis + cytolysin. IgY from non-immunized hens was used as control IgY. Specific activity is expressed by antibody reactivity and titer. [Figure 4] Figure 1. Inhibitory effect of anti-Enterococcus faecalis and anti-cytolysin IgY on the growth of E. faecalis. Whole E. faecalis bacteria, untreated and treated with 15 mg / ml IgY, were incubated for 24 hours at 37°C. Cultures were serially diluted to 104 and plated on BHI agar plates. After overnight incubation, colonies were counted using ImageJ Count. [Diagram 5] Quantification of cfu / ml in untreated and anti-Enterococcus faecalis + anti-cytolysin IgY treated E. faecalis cultures. Percent inhibition was calculated. [Figure 6]Figures 6A-6E show the effect of anti-Enterococcus faecalis plus anti-cytolysin IgY administration in gnotobiotic mice subjected to a chronic-plus-binge ethanol feeding model. C57BL / 6 germ-free mice were colonized with feces from cytolysin-positive human patients with alcoholic hepatitis and subjected to a chronic-plus-binge ethanol feeding model (n=5 per group) and fed vehicle, control IgY and anti-Enterococcus faecalis plus anti-cytolysin IgY in a liquid diet. (A) Food intake. (B) Body weight. (C) Liver weight. (D) Serum levels of alanine aminotransferase (ALT). (E) Liver triglyceride (TAG, triacylglycerol) content. Results are expressed as mean ± sem. P values ​​were determined by one-way ANOVA with Tukey's post-hoc test. *P<0.05, **P<0.01, ***P<0.001 [Figure 7] Figure 1 shows the effect of anti-Enterococcus faecalis + anti-cytolysin IgY administration on the liver of gnotobiotic mice subjected to the chronic + excessive ethanol feeding model. C57BL / 6 germ-free mice were colonized with feces from cytolysin-positive patients with alcoholic hepatitis and subjected to the chronic excessive ethanol feeding model (n=5 per group) and given vehicle, control IgY or anti-Enterococcus faecalis + anti-cytolysin IgY in a liquid diet. Representative liver sections after hematoxylin and eosin staining are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The term "hyperimmunization" refers to repeated exposure to one or more antigens such that an immune response is mounted and sustained beyond the natural unexposed state.

[0014] "Hyperimmune state" refers to the mounting immune response in an egg-laying animal that has been hyperimmunized.

[0015] As used herein, the term "egg" refers to a whole egg (table, hyperimmunized or otherwise). As used herein, the term "egg product" refers to a whole egg or any product or fraction derived from a whole egg. In a particular embodiment, the egg product is an egg yolk, e.g., egg yolk powder. In another embodiment, the egg product is an egg white, e.g., egg white powder. In another embodiment, the egg product is derived from a whole egg, e.g., whole egg powder (e.g., spray dried whole egg powder).

[0016] The term "control eggs" refers to eggs obtained from egg-laying animals that are not maintained in a hyperimmunized state, i.e., animals that are not hyperimmunized. The term "control egg product" refers to control eggs or egg products obtained from control eggs.

[0017] The term "hyperimmunized eggs" refers to whole eggs obtained from egg-laying animals that are maintained in a hyperimmune state, ie, that are hyperimmunized.

[0018] The term "hyperimmunized egg product" refers to hyperimmunized eggs or any product derived from hyperimmunized eggs. In certain embodiments, the hyperimmunized egg product is a concentrate. As used herein, the term "concentrate" refers to a hyperimmunized egg product that has been at least partially purified such that the concentration of antibodies in the concentrate is higher than the concentration of antibodies in hyperimmunized eggs.

[0019] The term "egg powder" refers to whole eggs that have been dried. In some embodiments, the egg powder is spray dried.

[0020] The term "oviparous animal" means any oviparous animal, including any animal that lays eggs, such as birds, fish and reptiles.

[0021] The term "bird" refers to animals that are members of the class Aves, including, but not limited to, chickens, turkeys, geese, ducks, pheasants, quail, pigeons, and ostriches.

[0022] The term "supranormal level" refers to a level that exceeds the level found in the eggs of an egg-laying animal that has not been hyperimmunized. For example, a supranormal level of an antibody to a particular antigen is a level of antibody that exceeds the level found in the eggs of an egg-laying animal that has not been hyperimmunized with the particular antigen.

[0023] The term "administering" refers to any method of providing a substance to a subject, including oral, intranasal, parenteral (intravenous, intramuscular, or subcutaneous), rectal, topical, or ocular.

[0024] The term "antigen" refers to a substance capable of inducing a humoral antibody and / or cell-mediated immune response, but not immune tolerance. The term refers to the ability to stimulate an immune response and react with its products, e.g., antibodies.

[0025] As used herein, an "antibody" is a protein that comprises at least one complementarity determining region that binds to a specific target antigen, such as an Enterococcus faecalis antigen, an isolated Enterococcus faecalis cytolysin toxin or an Enterococcus faecium antigen disclosed herein. For example, an antibody can comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. In certain embodiments, the antibody is a polyclonal antibody. As used herein, the term "polyclonal antibody" refers to a population of antibody molecules that can immunoreact with different epitopes on a particular antigen. In certain embodiments, the antibody is an IgY antibody.

[0026] As used herein, the terms "alcoholic liver disease", "ALD", "alcohol-associated liver disease" and "alcohol-related liver disease" refer to liver manifestations of excessive alcohol consumption, including fatty liver (steatosis), alcoholic hepatitis and chronic hepatitis with liver fibrosis or cirrhosis. As used herein, "alcohol-related disorder" refers to diseases and disorders associated with alcohol consumption, including, but not limited to, alcohol-induced psychotic disorder with delusions, alcohol abuse, excessive drinking, binge drinking, problem drinking, alcoholism, alcohol withdrawal, alcoholism delirium, alcohol withdrawal delirium, alcohol-induced persistent dementia, alcohol-induced persistent amnesic disorder, alcoholism, alcohol-induced psychotic disorder with hallucinations, alcohol-induced mood disorder, alcohol-induced or related bipolar disorder, alcohol-induced or related post-traumatic stress disorder, alcohol-induced anxiety disorder, alcohol-induced sexual dysfunction, alcohol-induced sleep disorder and alcohol-related disorder not otherwise specified (NOS).

[0027] As used herein, the term "alcohol abuser" refers to a subject who meets the DSM IV criteria for alcohol abuse (i.e., "recurrent use despite recurrent adverse consequences"), but is not dependent on alcohol.

[0028] As used herein, the term "heavy drinker" refers to men who consume more than 21 standard drinks per week and women who consume more than 14 standard drinks per week. A standard drink is 0.5 ounces (15 ml) of absolute alcohol, which is equivalent to 10 ounces (300 ml) of beer, 4 ounces (120 ml) of wine, or 1 ounce (30 ml) of 100-proof liquor. These individuals are not dependent on alcohol, but may or may not meet DSM IV criteria for alcohol abuse.

[0029] As used herein, the term "heavy drinker" refers to men who consume more than 14 standard drinks per week and women who consume more than 7 standard drinks per week. These individuals are not dependent on alcohol, but may or may not meet DSM IV criteria for alcohol abuse.

[0030] As used herein, the term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of a hyperimmunized egg product that, when administered to a subject, is sufficient to prevent or treat a disorder, such as alcoholic liver disease (ALD) or graft-versus-host disease (GVHD). An effective amount can vary depending on, for example, the age, weight, and / or health of the subject being treated.

[0031] As used herein, the term "isolated" refers to a biological compound (e.g., a protein) that is purified or partially purified from the cells in which it is produced. For example, an isolated Enterococcus faecalis cytolysin toxin is purified or partially purified from the Enterococcus faecalis cells that produced it. In some embodiments, an isolated Enterococcus faecalis cytolysin toxin is a recombinant Enterococcus faecalis cytolysin toxin, e.g., an Enterococcus faecalis cytolysin toxin produced in a cell other than an Enterococcus faecalis cell.

[0032] Alcoholic Liver Disease (ALD) 80% of alcohol is detoxified through the liver. Chronic alcohol consumption leads to the secretion of pro-inflammatory cytokines (e.g., TNF-alpha, interleukin 6 and interleukin 8), oxidative stress, lipid peroxidation and acetaldehyde toxicity. These factors cause hepatocyte inflammation, apoptosis and ultimately fibrosis, leading to alcoholic liver disease (ALD). ALD includes fatty liver (steatosis), alcoholic hepatitis and chronic hepatitis with hepatic fibrosis or cirrhosis.

[0033] Fatty liver or steatosis is the accumulation of fatty acids within liver cells. Heavy alcohol consumption causes the development of large fat globules (macrovesicular steatosis) throughout the liver. Alcohol is metabolized by alcohol dehydrogenase (ADH) to acetaldehyde, which is then further metabolized by aldehyde dehydrogenase (ALDH) to acetic acid, which is finally oxidized to carbon dioxide and water. This process generates NADH and increases the NADH / NAD+ ratio. Higher NADH concentrations induce fatty acid synthesis, while low NAD levels result in decreased fatty acid oxidation. Higher levels of fatty acids then signal liver cells to synthesize it into glycerol to form triglycerides. The accumulation of these triglycerides results in fatty liver.

[0034] Alcoholic hepatitis is characterized by hepatocyte inflammation and appears to predispose the liver to fibrosis. Proinflammatory cytokines (TNF-alpha, IL6, and IL8) are thought to be essential in the initiation and perpetuation of liver injury by inducing apoptosis and necrosis. One possible mechanism for increased activity of TNF-alpha is increased intestinal permeability due to liver disease. This promotes absorption of endotoxin produced in the gut into the portal circulation. Hepatic Kupffer cells then phagocytose endotoxin, stimulating the release of TNF-a. TNF-a then triggers apoptotic pathways through activation of caspases, resulting in hepatocyte death.

[0035] Cirrhosis is the later stage of severe liver disease characterized by inflammation (swelling), fibrosis (hardening of cells) and damaged membranes that prevent the detoxification of chemicals in the body, eventually leading to scarring and necrosis (cell death). Acetaldehyde may contribute to alcohol-induced fibrosis by stimulating collagen deposition by hepatic stellate cells. The generation of oxidants from NADPH oxidase and / or cytochrome P-450 2E1 and the formation of acetaldehyde-protein adducts damage cell membranes. Symptoms include jaundice (yellowing), hepatomegaly, and pain and tenderness due to structural changes in the damaged liver structure. If alcohol use is not completely eliminated, cirrhosis eventually leads to liver failure.

[0036] Chronic liver disease due to alcohol use disorders is a significant cause of worldwide morbidity and mortality burden. The most severe form of alcohol-related liver disease is alcoholic hepatitis (AH), with mortality rates ranging from 20% to 40% at 1-6 months. Recently, it has been demonstrated that a proportion of alcoholic hepatitis patients (approximately 30%) who harbor Enterococcus faecalis, which produces a toxin called cytolysin, develop a more severe form of the disease. E. faecalis is present at low levels in the microbiome of non-alcohol drinkers, approximately 0.1-0.5% of the individual's microbiome, but in patients with alcoholic liver disease (ALD), its abundance expands significantly to approximately 5%. Seventy-five percent of alcoholic hepatitis patients whose microbiomes are enriched with cytolysin-positive E. faecalis die within 90 days of diagnosis of severe alcoholic hepatitis (Maddrey, et al., 1978, Gastroenterology 75, 193-199). Standard treatment with corticosteroids is only marginally effective (Thursz, et al., 2015, N. Engl. J. Med. 372, 1619-1628). Early liver transplantation and alcohol abstinence are the only effective treatments, but are available only to a limited group of patients (Mathurin, et al., 2012, Management of alcoholic hepatitis. J. Hepatol. 56, S39-S45).

[0037] Duan et al. (2019, Nature 575:505-511) demonstrated the efficacy of bacteriophages targeting cytolysin-positive E. faecalis in blocking disease progression in humanized mice colonized with bacteria present in the feces of patients with alcoholic hepatitis. Treatment with bacteriophages that lyse cytolysin-positive E. faecalis demonstrated a reduction in hepatic cytolysins in these humanized mice, blocking the progression of ethanol-induced liver disease.

[0038] Graft-versus-host disease (GVHD) GVHD is a major complication occurring after solid organ and stem cell transplants, e.g., bone marrow transplants. The donor's immune system white blood cells remaining in the donated tissue (graft) recognize the recipient (host) as foreign. White blood cells present in the transplanted tissue then attack the recipient's body's cells, causing GVHD. See Hoffmann et al., 2002, J. Exp. Med. 196(3): 389-399. GVHD can occur in acute and chronic forms. The acute form of the disease is usually observed within the first 100 days after transplantation and represents a major challenge to transplantation due to the associated morbidity and mortality. Chronic forms of GVHD usually occur after 100 days. The emergence of moderate to severe cases of chronic GVHD adversely affects long-term survival.

[0039] Acute GVHD (aGVHD) affects the skin, liver and gastrointestinal tract and occurs when donor T cells recognize recipient tissues as foreign, causing tissue inflammation and damage. Specifically, aGVHD is initiated by alloreactive donor T cells that recognize MHC class I and II molecules on the surface of host cells and the peptides presented by them. Infiltration of several target organs, such as the intestine, liver and skin, by donor leukocytes, including T cells, is thought to be one of the key processes in the early stages of aGVHD. Affected tissues are further damaged by activation and proliferation of donor T cells, which leads to the secretion of proinflammatory cytokines and the recruitment of additional inflammatory effector cells to these sites. See Hoffmann et al., cited above.

[0040] Enterococcus faecalis and cytolysin and ALD The gut microbiota promotes alcoholic liver disease in mice, but little is known about the microbial factors involved in this process. Recently, cytolysin, a two-subunit exotoxin secreted by Enterococcus faecalis, was identified as a cause of hepatocyte death and liver injury. See Duan et al., 2019, Nature 575: 505-511, which is incorporated herein by reference in its entirety. Enterococcus faecalis is a Gram-positive, non-spore-forming commensal bacterium that inhabits the gastrointestinal tract of humans and other mammals.

[0041] Highly virulent E. faecalis strains express a pore-forming exotoxin called cytolysin that lyses both bacterial and eukaryotic cells in response to quorum signals. Compared to nonalcoholic individuals or patients with alcohol use disorder, patients with alcoholic hepatitis have increased numbers of E. faecalis in their feces. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of alcoholic liver disease and mortality in patients with alcoholic hepatitis. Furthermore, bacteriophages targeting cytolytic E. faecalis reduce cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. See Duan et al., cited above.

[0042] A functional cytolysin toxin is expressed by the gene cy1L L and cy1L S The E. faecalis cytolysin component, Cy1L, is composed of oligopeptides of large and small subunits encoded by, respectively. L and Cy1L Sare classified as type A pore-forming antibiotics and more recently as two-component class II antibiotics. Antibiotics are complex polycyclic antimicrobial peptides that are ribosomally synthesized by Gram-positive bacteria and are characterized by the presence of lanthionine and methyllanthionine bridges between dehydrated serine and threonine residues and cysteine ​​thiols. Antibiotics have a great diversity of structures and functions, but they all undergo extensive post-translational modifications and are characterized by having either antibiotic or morphogenic activity. Cytolysins appear to be unique among antibiotics in that they can lyse other bacteria as well as red blood cells and other eukaryotic cells. See Van Tyne et al., 2013, Toxins 5(5): 895-911.

[0043] Cy1L L and the amino acid sequence of Cy1Ls is provided below: Cy1L L TTPVCAVAATAAASSAACGWVGGGIFTGVTVVVSLKHC (SEQ ID NO: 1) CylL S TTPACFTIGLGVGALFSAKFC (SEQ ID NO: 2)

[0044] Enterococcus and GVHD There is growing evidence that a patient's gut microbiota may play a role in susceptibility to GVHD, particularly acute GVHD resulting from allogeneic hematopoietic cell transplantation. See Garrett, 2020, New England Journal of Medicine 382(11): 1064-1066, which is incorporated herein by reference in its entirety. Some enterococci species in the gut (e.g., Enterococcus faecalis and Enterococcus faecium) are opportunistic pathogens that frequently cause life-threatening bloodstream infections in patients undergoing allogeneic hematopoietic cell transplantation. Studies have shown that enterococci, particularly E. faecium, predominate in the fecal community of a significant subgroup of patients undergoing allogeneic hematopoietic cell transplantation, and that this predominance of enterococci correlates with decreased overall survival and increased mortality from GVHD. See Stein-Thoeringer et al., 2019, Science 366: 1143-9, which is incorporated by reference in its entirety. Stein-Thoeringer et al. observed that the dietary carbohydrate lactose induces enterococcal proliferation in a mouse model of GVHD, and that a lactose-free diet reduces enterococcal proliferation and T cell-induced inflammation in GVHD.

[0045] Hyperimmunized egg products In certain aspects, the disclosure relates to a method of preparing a hyperimmunized egg product, comprising: i) hyperimmunizing an egg-laying animal with an antigen selected from the group consisting of Enterococcus faecalis, isolated Enterococcus faecalis cytolysin toxin, and Enterococcus faecium; and ii) preparing the hyperimmunized egg product from one or more eggs produced by the animal. In some embodiments, the antigen is selected from the group consisting of Enterococcus faecalis and isolated Enterococcus faecalis cytolysin toxin. In some embodiments, the antigen comprises Enterococcus faecalis and isolated Enterococcus faecalis cytolysin toxin.

[0046] Egg-laying animals produce antibodies in the blood and eggs that are specific to certain immunogens. For example, various genera of birds, such as chickens (Gallus domesticus), turkeys, and ducks, produce antibodies against antigens associated with avian diseases. LeBacq-Verheyden et al. (Immunology 27:683 (1974)) and Leslie, GA et al. (J. Med. 130:1337 (1969)) quantitatively analyzed immunoglobulins in chickens. Polson, A. et al. (Immunological Communications 9:495-514 (1980)) immunized hens against several proteins and natural mixtures of proteins and detected IgY antibodies in the egg yolk. Fertel, R. et al. (Biochemical and Biophysical Research Communications 102:1028-1033 (1981)) immunized hens against prostaglandins and detected antibodies in their egg yolks. Jensenius et al. (Journal of Immunological Methods 46:63-68 (1981)) provide a method for isolating egg yolk IgG for use in immunodiagnosis. Polson et al. (Immunological Communications 9:475-493 (1980)) describe antibodies isolated from the egg yolks of hens immunized with various plant viruses.

[0047] U.S. Patent No. 4,748,018 discloses a method for passive immunization of a mammal, which comprises parenteral administration of purified antibodies obtained from eggs of birds immunized against a corresponding antigen, the mammal acquiring immunity against the eggs. U.S. Patent No. 5,772,999, assigned to DCV-Biologics, discloses a method for preventing, combating or reducing chronic gastrointestinal disorders or nonsteroidal anti-inflammatory drug-induced (NSAID-induced) gastrointestinal damage in a subject by administering to the subject hyperimmunized eggs and / or milk or fractions thereof.

[0048] Immunized eggs are, for example, eggs from birds that have been immunized with a specific antigen or mixture of antigens. Hyperimmunized eggs are, for example, eggs from birds that have been brought to a specific immunized state by regular booster administration of antigens. Regardless of the type of antigen administered by the bird manufacturer, hyperimmunized eggs have been found to have various beneficial factors, including chronic gastrointestinal disorders, treatment of NSAID-induced gastrointestinal damage (see U.S. Pat. No. 5,772,999), and anti-inflammatory effects due to the presence of anti-inflammatory compositions (see U.S. Patent Application Publication No. US2004 / 0156857), as described above.

[0049] One advantage of the hyperimmunized egg product is that it has higher and more consistent levels of antibodies (e.g., IgY antibodies) against Enterococcus faecium, Enterococcus faecalis, or Enterococcus faecalis cytolysin toxins compared to control egg products or egg products from chickens immunized with Enterococcus faecium, Enterococcus faecalis, or Enterococcus faecalis cytolysin toxins using standard immunization techniques. In some embodiments, the hyperimmunized egg product has higher levels of IgY antibodies against Enterococcus faecalis and / or isolated Enterococcus faecalis cytolysin toxins compared to chickens immunized with Enterococcus faecalis and / or Enterococcus faecalis cytolysin toxins using standard immunization techniques. Typically, standard immunization consists of an initial immunization followed by one or two booster immunizations at 30 day intervals. In some embodiments, hyperimmunization comprises at least 4, 5, 6, 7, 8, 9, or 10 immunizations with Enterococcus faecium, Enterococcus faecalis, and / or an isolated Enterococcus faecalis cytolysin toxin described herein. In some embodiments, hyperimmunization comprises immunizing an egg-laying animal with an Enterococcus faecium, Enterococcus faecalis, and / or an isolated Enterococcus faecalis cytolysin toxin described herein less than 30 days, less than 25 days, less than 20 days, less than 15 days, less than 10 days, or less than 5 days apart.

[0050] The hyperimmunized egg product may be produced by any egg-laying animal. Preferably, the animal is a member of the class Aves, or in other words, a bird. Within the class Aves, domesticated poultry are preferred, although other members of this class, such as turkeys, ducks, and geese, are suitable sources of hyperimmunized egg products. In a particular embodiment, the egg-laying animal is a chicken.

[0051] This special hyperimmunized state is preferably achieved by administering a primary immunization followed by regular boosters with a sufficiently high dose of the specific antigen or mixture of antigens. The booster dosage may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the dosage required to produce the primary immunity of the egg-laying animal. Any of these percentages can be used to define a range of booster dosages. For example, in some embodiments, the booster dosage is 20%-80%, 30%-70% or 50%-100% of the dosage required to produce the primary immunity of the egg-laying animal. In certain embodiments, the booster dosage is 50% of the primary immunization dosage. With knowledge of the requirements for generating and maintaining a hyperimmunized state, it is within the skill of the art to vary the amount of antigen administered depending on the genus and strain of egg-laying animal utilized to maintain the animal in a hyperimmunized state.

[0052] The hyperimmune state can be brought about by a single antigen or a combination of antigens. Hyperimmunization can be achieved by multiple exposures to multiple antigens or multiple exposures to a single antigen.

[0053] Antigens for hyperimmunization In some embodiments, the antigens for hyperimmunization include one or more of Enterococcus faecium, Enterococcus faecalis, and Enterococcus faecalis cytolysin toxin. In some embodiments, the egg-laying animals are hyperimmunized with Enterococcus faecalis alone, i.e., no additional antigens are used for hyperimmunization. In some embodiments, the egg-laying animals are hyperimmunized with isolated Enterococcus faecalis cytolysin toxin alone. In some embodiments, the egg-laying animals are hyperimmunized with Enterococcus faecium alone.

[0054] In some embodiments, a hyperimmunized egg product comprising antibodies against Enterococcus faecalis is prepared by hyperimmunizing an egg-laying animal with Enterococcus faecalis. In some embodiments, a hyperimmunized egg product comprising antibodies against Enterococcus faecalis cytolysin toxin is prepared by hyperimmunizing an egg-laying animal with Enterococcus faecalis cytolysin toxin. In some embodiments, a hyperimmunized egg product comprising antibodies against Enterococcus faecium is prepared by hyperimmunizing an egg-laying animal with Enterococcus faecium.

[0055] Any combination of antigens disclosed herein can be used to hyperimmunize egg-laying animals. For example, in some embodiments, egg-laying animals are hyperimmunized with Enterococcus faecium, Enterococcus faecalis, and isolated Enterococcus faecalis cytolysin toxins. In some embodiments, egg-laying animals are hyperimmunized with Enterococcus faecium and Enterococcus faecalis. In some embodiments, egg-laying animals are hyperimmunized with Enterococcus faecium and isolated Enterococcus faecalis cytolysin toxins. In some embodiments, egg-laying animals are hyperimmunized with Enterococcus faecium and isolated Enterococcus faecalis cytolysin toxins. In some embodiments, egg-laying animals are hyperimmunized with Enterococcus faecalis and isolated Enterococcus faecalis cytolysin toxins.

[0056] Hyperimmunization Procedure The following list of steps is an example of a preferred procedure used to render egg-laying animals hyperimmune so that the resulting hyperimmune eggs or egg products can be administered to birds: 1. Select one or more antigens. 2. Induce an immune response in egg-laying animals by primary immunization. 3. Administer a booster vaccine of appropriate dosages of one or more antigens to induce and maintain a hyperimmune state.

[0057] Step 1: The key to this step is that the antigen must be capable of inducing an immune and hyperimmune state in the egg-laying animal. In some embodiments, the egg-laying animal is immunized with Enterococcus faecalis. In some embodiments, the egg-laying animal is immunized with Enterococcus faecalis cytolysin toxin. In some embodiments, the egg-laying animal is hyperimmunized with Enterococcus faecium. In some embodiments, the egg-laying animal is hyperimmunized with two or more of Enterococcus faecium, Enterococcus faecalis, and Enterococcus faecalis cytolysin toxin.

[0058] Step 2: For Enterococcus faecalis and Enterococcus faecium, the vaccine can be either a killed or live attenuated vaccine. The vaccine can be administered by any method that elicits an immune response. Immunization is preferably accomplished by administering the vaccine via intramuscular injection. The preferred muscle for injection in birds is the pectoral muscle. The dosage is preferably 0.05 to 5 milligrams of immunogenic vaccine. Other methods of administration that may be used include intravenous injection, intraperitoneal injection, intradermal, rectal suppository, aerosol or oral administration.

[0059] Many methods known to those skilled in the art of immunology can be used to determine whether a vaccine has induced an immune response in an egg-laying animal. Examples of these include enzyme-linked immunosorbent assays (ELISAs), tests for the presence of antibodies against the stimulating antigen, and tests designed to evaluate the ability of immune cells from the host to respond to the antigen. The minimum dosage of antigen required to induce an immune response depends on the vaccination procedure used, including the type of adjuvant used and the formulation of the antigen, as well as the type of egg-laying animal used as the host.

[0060] Step 3: A hyperimmune state is preferably induced and maintained in the target animal by repeated booster administration of appropriate dosages at regular time intervals. The time intervals are preferably 2-8 weeks apart over a period of 6-12 months. However, it is essential that the booster administration does not induce immune tolerance. Such processes are well known in the art. Methods for preparing hyperimmunized egg products are described, for example, in U.S. Patent No. 6,803,035, which is incorporated herein by reference in its entirety.

[0061] In certain embodiments, the antigens (e.g., Enterococcus faecium, Enterococcus faecalis and / or Enterococcus faecalis cytolysin toxin) are formulated into a Freund's vaccine. For the initial vaccination, the egg-laying animals are administered two 0.5 ml doses of each antigen. Two weeks later, the egg-laying animals are administered one 0.5 ml dose of each antigen as a booster vaccination. An additional booster vaccination is administered four weeks after the initial vaccination. The vaccine can be administered into mammary tissue.

[0062] Other hyperimmunization maintenance procedures or combinations of procedures can be used, for example, intramuscular injection for primary immunization and intravenous injection for booster injection. Further procedures include simultaneous administration of microencapsulated and liquid antigens, or intramuscular injection for primary immunization and booster dosing by oral or parenteral administration by microencapsulation means. Several combinations of primary and hyperimmunization are known to those skilled in the art.

[0063] In some embodiments, the hyperimmunized egg product comprises an antibody to Enterococcus faecium. In certain embodiments, the hyperimmunized egg product comprises an antibody to Enterococcus faecalis. In some embodiments, the hyperimmunized egg product comprises an antibody to Enterococcus faecalis cytolysin toxin. The antibody to the Enterococcus faecalis cytolysin toxin can bind to and neutralize the toxin. In some embodiments, the hyperimmunized egg product comprises an antibody to Enterococcus faecalis cytolysin toxin and an antibody to at least one additional Enterococcus faecalis antigen that is not an Enterococcus faecalis cytolysin toxin.

[0064] The antibody may be an IgA, IgM or IgY antibody. In certain embodiments, the antibody is an IgY antibody.

[0065] The hyperimmunized eggs or hyperimmunized egg products may contain increased levels of antibodies (e.g., IgY antibodies) specific for a particular antigen disclosed herein compared to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with the particular antigen. For example, in some embodiments, the hyperimmunized eggs or hyperimmunized egg products contain increased levels of antibodies specific for Enterococcus faecalis compared to a control egg or egg product obtained from an egg-laying animal that has not been hyperimmunized. In some embodiments, the hyperimmunized eggs or hyperimmunized egg products contain increased levels of antibodies specific for Enterococcus faecalis cytolysin toxin compared to a control egg or egg product obtained from an egg-laying animal that has not been hyperimmunized. In some embodiments, the hyperimmunized eggs or hyperimmunized egg products contain increased levels of antibodies specific for Enterococcus faecium compared to a control egg or egg product obtained from an egg-laying animal that has not been hyperimmunized.

[0066] In some embodiments, a hyperimmunized egg or egg product contains at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more by weight of antibodies (e.g., IgY antibodies) specific for a particular antigen disclosed herein (e.g., Enterococcus faecium, Enterococcus faecalis, or Enterococcus faecalis cytolysin toxin) compared to a control egg or egg product obtained from a laying animal that has not been hyperimmunized with the particular antigen. For example, in some embodiments, a hyperimmunized egg or hyperimmunized egg product contains at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies (e.g., IgY antibodies) specific for Enterococcus faecalis compared to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with Enterococcus faecalis. In some embodiments, a hyperimmunized egg or hyperimmunized egg product contains at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies (e.g., IgY antibodies) specific for Enterococcus faecalis cytolysin toxin compared to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with Enterococcus faecalis cytolysin toxin. In some embodiments, the hyperimmunized egg or hyperimmunized egg product contains at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% or more antibodies (e.g., IgY antibodies) specific for Enterococcus faecium compared to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with Enterococcus faecium.

[0067] A hyperimmunized egg or egg product can contain increased levels of two or more antibodies (e.g., IgY antibodies), each specific for a different antigen disclosed herein, compared to a control egg or egg product obtained from a non-hyperimmunized egg-laying animal. For example, in some embodiments, a hyperimmunized egg or egg product contains increased levels of one or more antibodies specific for Enterococcus faecalis cytolysin toxin and one or more antibodies specific for Enterococcus faecalis (e.g., one or more antibodies specific for an Enterococcus faecalis antigen other than Enterococcus faecalis cytolysin toxin) compared to a control egg or egg product obtained from a non-hyperimmunized egg-laying animal. In some embodiments, a hyperimmunized egg or egg product contains increased levels of antibodies specific for Enterococcus faecalis cytolysin toxin and increased levels of antibodies specific for Enterococcus faecium compared to a control egg or egg product obtained from a non-hyperimmunized egg-laying animal. In some embodiments, the hyperimmunized eggs or hyperimmunized egg products contain increased levels of antibodies specific for Enterococcus faecium and increased levels of antibodies specific for Enterococcus faecalis compared to control eggs or egg products obtained from egg-laying animals that are not hyperimmunized.

[0068] For example, in some embodiments, a hyperimmunized egg or hyperimmunized egg product contains at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies to Enterococcus faecalis and at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies to Enterococcus faecalis cytolysin toxin compared to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with Enterococcus faecalis or Enterococcus faecalis cytolysin toxin. In some embodiments, the hyperimmunized egg or hyperimmunized egg product comprises at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies to Enterococcus faecalis and at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies to Enterococcus faecium compared to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with Enterococcus faecalis or Enterococcus faecium. In some embodiments, the hyperimmunized egg or hyperimmunized egg product comprises at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies to Enterococcus faecalis cytolysin toxin and at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies to Enterococcus faecium compared to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with Enterococcus faecium or Enterococcus faecium.In some embodiments, the hyperimmunized egg or hyperimmunized egg product has at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% or more reduced levels of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin or Enterococcus faecium compared to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with Enterococcus faecalis, Enterococcus faecalis cytolysin toxin or Enterococcus faecium. the antibodies against Enterococcus faecalis, at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% or more of antibodies against Enterococcus faecalis cytolysin toxins, and at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% or more of antibodies against Enterococcus faecium.

[0069] Comparison of antibody titers in hyperimmunized and control egg products can be determined by methods known in the art. For example, in one embodiment, eggs are collected and antibody titers are monitored at regular intervals by ELISA. To determine antibody titers, total IgY is extracted from eggs using Pierce™ Chicken IgY Purification Kit (Thermo Fisher Scientific, Waltham, WA). Briefly, 2 mL of eggs are mixed with 5 volumes of delipidation reagent and IgY is purified according to the manufacturer's instructions. Spray-dried egg powder samples are reconstituted to 1 mg / mL in sterile PBS and filtered through a 0.22 μm membrane filter. Specific antibody titers in isolated IgY or egg powder samples are measured by ELISA. Flat-bottom 96-well microtiter plates (Corning® Costar®, Corning, NY) are coated with antigen (e.g., Enterococcus faecalis antigen, isolated Enterococcus faecalis cytolysin toxin, or Enterococcus faecium antigen) at 10 μg / mL (100 μL / well) and incubated overnight at 4°C. Plates are washed twice with PBS containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO), blocked with 100 μL / well of PBS containing 1% bovine serum albumin (BSA), and incubated for 1 h at room temperature. IgY samples serially diluted (in PBS with 0.1% BSA) from egg powder samples are added to the plate in triplicate wells (100 μL / well) and incubated for 2 h at room temperature with constant shaking. Plates were then washed with PBS-T, treated with peroxidase-conjugated rabbit anti-chicken IgY (IgG) antibody (1:500; Sigma), incubated for 30 min, and then developed with 0.01% tetramethylbenzidine substrate (Sigma) in 0.05 M phosphate-citrate buffer, pH 5.0, for 10 min. Bound antibody was detected by measuring the optical density at 450 nm (OD450) using a microplate reader (Bio-Rad, Hercules, CA).The antibody titer can be expressed by the maximum dilution of the egg product that still contains detectable antibody as measured by optical density as described above. For example, an antibody titer of 1000 indicates that a 1000-fold dilution of the egg product contains detectable antibody, but higher dilutions do not contain detectable antibody. In some embodiments, the antibody titer (e.g., titer of antibodies to Enterococcus faecalis cytolysin toxin, Enterococcus faecalis and / or Enterococcus faecium) in the hyperimmunized egg product is at least 1000, at least 2000, at least 4000, at least 8000, at least 16,000, at least 32,000, at least 64,000, at least 100, at least 15,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, at least 100,000, at least 15 ...5,000, at least 100,000, at least 15,000, at least 15,0 In certain embodiments, the antibody titer in the hyperimmunized egg product (e.g., titer of antibodies to Enterococcus faecalis cytolysin toxin, Enterococcus faecalis and / or Enterococcus faecium) is at least 100,000, at least 128,000, at least 250,000, at least 500,000, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 million. In a more specific embodiment, the titer of antibodies to Enterococcus faecalis cytolysin toxin and the titer of antibodies to Enterococcus faecalis in the hyperimmunized egg product is at least 16,000.

[0070] In some embodiments, the hyperimmunized egg or egg product comprises at least 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05% or 0.1% by weight of IgY antibodies against a specific antigen (e.g., an Enterococcus faecium antigen, an Enterococcus faecalis antigen or an Enterococcus faecalis cytolysin toxin). Typically, a whole chicken egg weighs about 60 grams excluding the shell, the yolk weighs about 20 grams and the egg white weighs about 40 grams. In some embodiments, 3 grams of egg yolk contains about 20-30 milligrams of total IgY, so that a whole egg contains about 150-200 mg of total IgY. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25% or 30% of the total IgY in the hyperimmunized egg or egg product is specific for one of the antigens used for hyperimmunization (e.g., Enterococcus faecium, Enterococcus faecalis or isolated Enterococcus faecalis cytolysin toxin).

[0071] Compositions and Administration In certain aspects, the disclosure relates to a method of preventing or treating alcoholic liver disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-laying animal, thereby preventing or treating alcoholic liver disease in the subject, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies against an antigen selected from the group consisting of Enterococcus faecalis and Enterococcus faecalis cytolysin toxin. In some embodiments, the hyperimmunized egg product comprises one or more antibodies against Enterococcus faecalis cytolysin toxin and a therapeutically effective amount of one or more antibodies against Enterococcus faecalis. In some embodiments, the antibodies against Enterococcus faecalis comprise antibodies against one or more Enterococcus faecalis antigens that are not Enterococcus faecalis cytolysin toxins.

[0072] In certain aspects, the disclosure relates to a method of preventing or treating graft-versus-host disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-laying animal, thereby preventing or treating graft-versus-host disease in the subject, the hyperimmunized egg product comprising a therapeutically effective amount of one or more antibodies against an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, and Enterococcus faecium. Once the egg-laying animals are sufficiently hyperimmunized, eggs from these animals are preferably collected and processed to produce an administrable form of the hyperimmunized egg product. The hyperimmunized egg product may be prepared by dehydration, spray drying, or freeze drying of whole eggs, egg yolks, or purified IgY fractions. The dried hyperimmunized egg product may be mixed with an agent, such as silicone or a silicone derivative that improves flow properties. The dried hyperimmunized egg product may include a desiccant. The hyperimmunized egg product may be stored at ambient temperature or refrigerated, for example at 4° C.

[0073] In some embodiments, administration of the hyperimmunized egg product to a subject reduces the level of Enterococcus faecalis in the subject, e.g., compared to the level of Enterococcus faecalis in the subject prior to administration of the hyperimmunized egg product, or compared to a subject not receiving the hyperimmunized egg product. In some embodiments, administration of the hyperimmunized egg product to a subject reduces liver damage in the subject (e.g., reduces serum levels of alanine aminotransferase (ALT)), e.g., compared to the level of liver damage prior to administration of the hyperimmunized egg product, or compared to a subject not receiving the hyperimmunized egg product.

[0074] In some embodiments, the hyperimmunized egg product is encapsulated. Methods for encapsulating antibodies and other proteins are known in the art and are described, for example, in U.S. Pat. No. 7,105,158. Materials that are biodegradable and non-antigenic can be used as the encapsulation material. Encapsulation materials include, but are not limited to, albumin, PLGA, globulins, natural and synthetic polymers, and thermoplastic polymers. Any biocompatible and biodegradable polymer can be used for encapsulation. Many available crosslinking agents, such as glutaraldehyde, can be used to crosslink the encapsulation material. Additionally, the pharma- ceutical delivery material can contain microspheres of encapsulated drugs, whereby the microspheres are crosslinked with different concentrations of crosslinking agents, resulting in a long-term continuous release of the antibody.

[0075] In some embodiments, the hyperimmunized egg product is in the form of microparticles or nanoparticles, such as encapsulated microparticles or encapsulated nanoparticles. The microparticles and nanoparticles can have any shape. Typically, the microparticles and nanoparticles are spherical. Other suitable shapes include, but are not limited to, flakes, triangles, ellipses, rods, polygons, needles, tubes, cubes, and rectangular structures. In certain embodiments, the microparticles have a diameter of less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 microns. Any of these values ​​can be used to define a range of microparticle diameters. For example, the microparticles may have a diameter of about 0.1 to about 10 microns, about 0.1 to about 1 micron, or about 0.1 to about 2 microns. In other embodiments, larger microparticles or particles may be used. For example, the microparticles may have a diameter in the range of 10 microns to 1000 microns. In certain embodiments, the nanoparticles have a diameter of less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, or 10 nm. Any of these values ​​can be used to define a range of nanoparticle diameters. For example, the nanoparticles can have a diameter of about 10 to about 1000 nm, about 100 to about 1000 nm, or about 10 to about 100 nm.

[0076] There are several processes that can encapsulate microparticles or nanoparticles, including, for example, multi-layer microencapsulation, hot melt encapsulation, phase separation encapsulation, spontaneous emulsification, solvent evaporation microencapsulation, solvent removal microencapsulation and coacervation.These methods are known in the art.A detailed description of this method is discussed in Mathiowitz et al., "Microencapsulation", in Encyclopedia of Controlled Drug Delivery, vol. 2, pp. 495-546, 1999, John Wiley & Sons, Inc.. New York, NY, which is incorporated herein by reference in its entirety.

[0077] In some embodiments, IgY antibodies specific for an antigen disclosed herein (e.g., Enterococcus faecium, Enterococcus faecalis, or Enterococcus faecalis cytolysin toxin) are administered to a subject in a concentrated form. For example, in some embodiments, the IgY antibodies are purified and concentrated prior to administration to a subject. Methods for purifying and concentrating IgY antibodies from egg products are known in the art and are described, for example, in U.S. Pat. No. 5,367,054, which is incorporated herein by reference in its entirety. In some embodiments, the hyperimmunized egg product comprises or consists of a purified or partially purified IgY antibody against Enterococcus faecalis cytolysin toxin. In some embodiments, the hyperimmunized egg product comprises or consists of a purified or partially purified IgY antibody against Enterococcus faecalis. In some embodiments, the hyperimmunized egg product comprises or consists of a purified or partially purified IgY antibody against Enterococcus faecium. In some embodiments, the hyperimmunized egg product comprises or consists of purified or partially purified IgY antibodies against Enterococcus faecalis and purified or partially purified IgY antibodies against Enterococcus faecalis cytolysin toxin.

[0078] In some embodiments, the hyperimmunized egg products described herein are used to treat alcoholic liver disease in subjects who already have the disease. For example, in some embodiments, the subject has alcoholic liver disease at the time of administration of the hyperimmunized egg product. An increased proportion of Enterococcus species has been observed in patients with alcoholic liver disease (e.g., alcoholic hepatitis) compared to patients without alcohol use disorder. For example, in patients with alcoholic hepatitis, 5.59% of fecal bacteria were Enterococcus species compared to nearly none (0.023%) in control subjects without alcohol use disorder. See Duan et al., 2019, Nature 575: 505-511. Thus, in some embodiments, at least 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5% of fecal bacteria in the subject are Enterococcus species at the time of administration of the hyperimmunized egg product. It has been shown that fecal samples from patients with alcoholic hepatitis have about 2,700 times more E. faecalis than samples from control subjects without alcohol use disorder, as measured by quantitative PCR (qPCR), and about 80% of patients with alcoholic hepatitis are positive for E. faecalis in feces.See Duan et al., cited above.Thus, in some embodiments, subjects have at least 100 times, 500 times, 1000 times, 1500 times, 2000 times, or 2500 times more E. faecalis in fecal samples compared to subjects without alcoholic liver disease.

[0079] In some embodiments, the hyperimmunized egg products described herein are used to prevent the development of alcoholic liver disease in a subject. For example, in some embodiments, the subject does not have alcoholic liver disease at the time of administration of the hyperimmunized egg product. In some embodiments, the subject has an alcohol-related disorder other than alcoholic liver disease at the time of administration of the hyperimmunized egg product. In some embodiments, the subject is an alcohol abuser, an excessive drinker, or a heavy drinker. In certain embodiments, the subject to whom the hyperimmunized egg product is administered is a human.

[0080] In some embodiments, the hyperimmunized egg products described herein are used to treat GVHD in subjects who have already developed the disease. For example, in some embodiments, the subject has GVHD upon administration of the hyperimmunized egg product. As discussed above, increased fecal levels of Enterococcus species (e.g., E. faecium) have been observed in patients with GVHD. See Garrett, cited above. Thus, in some embodiments, subjects with GVHD show increased fecal levels of one or more Enterococcus species (e.g., E. faecalis or E. faecium) compared to healthy subjects (e.g., subjects without GVHD) upon administration of the hyperimmunized egg product. In some embodiments, the subject with GVHD has received an organ transplant, stem cell transplant, bone marrow transplant, or allogeneic hematopoietic cell transplant. In some embodiments, the subject has acute GVHD. In some embodiments, the subject has chronic GVHD.

[0081] In some embodiments, the hyperimmunized egg products described herein are used to prevent the development of GVHD in a subject. For example, in some embodiments, the subject does not have GVHD at the time of administration of the hyperimmunized egg product. In some embodiments, the subject has undergone a transplant (e.g., an organ transplant, a stem cell transplant, a bone marrow transplant, or an allogeneic hematopoietic cell transplant) and has not yet developed GVHD. In certain embodiments, the subject to whom the hyperimmunized egg product is administered is a human.

[0082] The hyperimmunized egg product of the present invention is administered to a subject (e.g., a human) by any means that treats or prevents alcoholic liver disease or GVHD in the subject. In certain embodiments, administration is by oral administration. Eggs and egg yolks are natural food ingredients and are non-toxic and safe. In other embodiments, the hyperimmunized egg product may be administered by injection, for example, intravenous, subcutaneous or intramuscular injection. In certain embodiments, the hyperimmunized egg product is purified or partially purified IgY administered by intravenous injection.

[0083] Any of a number of known pharmaceutical carriers can be used to prepare injectable or other administrable formulations, including phosphate buffered saline, saline, ethanol, propylene glycol, etc. In some embodiments, the hyperimmunized egg products are administered via drinking water.

[0084] In certain embodiments, the hyperimmunized egg product is administered as a composition that includes one or more additional compounds, such as nutrients or probiotics. For example, in one embodiment, the hyperimmunized egg product of the present invention is incorporated into a dietary supplement. One method of preparing the egg of the present invention to be incorporated into a dietary supplement includes drying the egg into egg powder. Although various methods are known for drying eggs, spray drying is a preferred method. The process of spray drying eggs is well known in the art. In some embodiments, the composition is an aqueous solution that includes the hyperimmunized egg product.

[0085] In certain embodiments, whole eggs are divided into separate fractions, such as egg yolk and egg white. For example, it is commonly known in the art that IgY antibodies are found in egg yolk. Thus, one skilled in the art will clearly recognize that separation of egg yolk can provide more effective fractions or remove undesirable components, and also allows for other modes of administration, such as parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, oral or topical administration of hyperimmunized egg products. Such further separation will provide the ability to create encapsulated products and compositions that include the eggs or fractions thereof.

[0086] The hyperimmunized egg product is preferably administered to the subject in an amount that is immunologically effective in treating or preventing alcoholic liver disease or GVHD. The dosage and duration of administration will depend on the particular condition of the subject. In some embodiments, the hyperimmunized egg product is administered to the subject for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 60, 90, 180, or 365 days. The hyperimmunized egg product may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times per day. Any of these values ​​may be used to define a range of times the hyperimmunized egg product may be administered to the subject per day. For example, in some embodiments, the hyperimmunized egg product is administered to the subject 1-2 times per day, 1-3 times per day, or 1-4 times per day. In some embodiments, the hyperimmunized egg product is administered to the subject at least twice per day. In some embodiments, the hyperimmunized egg product is administered to the subject at least once per day. In some embodiments, the hyperimmunized egg product is administered to the subject daily. In some embodiments, the hyperimmunized egg product is administered to the subject once every two days. In some embodiments, the hyperimmunized egg product is administered to the subject once every three days. In some embodiments, the hyperimmunized egg product is administered to the subject once per week. In certain embodiments, the hyperimmunized egg product is administered to the subject once per day for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive days.

[0087] In some embodiments, a daily amount of hyperimmune whole eggs (or a hyperimmune egg product containing the equivalent of one to a few hyperimmune whole eggs) can be administered to a subject depending on the particular circumstances of the condition. More effective fractions can be isolated and concentrated from hundreds of eggs by methods well known in the art. In certain embodiments, an effective amount of hyperimmune egg product administered to a subject (e.g., a human) is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 20 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams per day. For example, in some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams of whole egg are administered to the subject per day. In some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 grams of dried egg yolk or dried whole egg are administered to the subject per day. Any of these values ​​can be used to define a range of effective amounts of hyperimmunized egg product administered to a mammal. For example, in some embodiments, an effective amount of hyperimmunized egg product is between 0.1 and 10 grams, between 0.5 and 6 grams, or between 1 and 5 grams per day. In certain embodiments, 3 grams of egg yolk is administered to a subject (e.g., a human) per day.

[0088] In certain embodiments, the composition comprises at least 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% w / w of hyperimmunized egg product. Any of these values ​​can be used to define a range of concentrations of hyperimmunized egg product in the composition. For example, in some embodiments, the composition comprises between 0.01% and 50%, between 0.1% and 50%, or between 1% and 50% w / w of hyperimmunized egg product.

[0089] [Example] [Example 1] Preparation and evaluation of anti-Enterococcus faecalis and anti-cytolysin IgY antibodies The current study was designed to evaluate the efficacy of hyperimmune eggs containing E. faecalis cytolysin toxins and targeted IgY antibodies against E. faecalis when orally administered to a humanized mouse ALD model colonized with bacteria derived from the feces of patients with alcoholic hepatitis. A schematic diagram of the experiment is provided in Figure 1.

[0090] method Preparation of hyperimmune egg powder A 2 mL primary culture of Enterococcus faecalis was started from a glycerol stock. A large batch culture was developed by inoculating a 500 mL culture bottle with the primary culture at 0.2% and grown overnight at 37 °C with shaking at 180 rpm. Bacteria were pelleted by centrifugation at 6000 rpm at 4 °C. The pellet was washed once with ice-cold PBS. The pellet was again resuspended in ice-cold PBS, transferred to a glass tube, and sonicated at the highest setting for 10 cycles, 30 min each cycle. The resulting lysate was centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatant was collected. Total protein was quantified by BCA protein assay.

[0091] Preparation of recombinant cytolysin Pure recombinant E. faecalis cytolysin toxin was prepared by Genscript USA, Piscataway, NJ.

[0092] Hyperimmunization Protocol A mixture of E. faecalis bacterial lysate and E. faecalis cytolysin toxin was diluted in PBS to 2 mg / mL and 1 mg / ml, respectively, and used as an inoculum. Separate chickens were immunized with each of the antigens using a 1:1 mixture of the inoculum and Freund's complete adjuvant. Immunizations were performed on days 0, 4, 7, 14, and 28, and eggs were collected.

[0093] Antibody extraction for titer and neutralization assessment IgY antibodies were extracted from hyperimmunized eggs using a water dilution method: 100 μL of egg yolk was diluted with 10 ml of water, mixed thoroughly, and centrifuged to pellet lipids while extracting antibodies and other soluble egg proteins into the aqueous phase, which was used to assess titers by ELISA as described below.

[0094] For neutralization assays, IgY was extracted from hyperimmunized egg yolks by water dilution and NaCl precipitation. Four to five egg yolks were diluted in a 1:10 ratio with water acidified to pH 5 using 0.5 M HCl and frozen overnight. The diluted egg yolks were thawed at 37°C and centrifuged at 6000 rpm for 20 min to separate lipids. The supernatant was filtered, mixed with 8.8% NaCl at pH 4, and precipitated for 2 h. IgY was separated from the precipitate by centrifugation and excess salt was removed by dialysis in PBS. Total IgY was quantified by A280 (absorbance at 280 nm) values ​​using a NanoDrop™ One Microvolume UV-Vis spectrophotometer (Thermo Scientific). The purity of IgY was analyzed on SDS-PAGE gels.

[0095] Detection of antibody specificity and titer by ELISA Antibody specificity and titer were measured by ELISA. Briefly, to measure anti-E. faecalis antibody titer, 96-well plates were coated with 1 mg / mL E. faecalis lysate in carbonate-bicarbonate buffer (pH 9.3) and incubated overnight at 4°C. Plates were washed once with PBS and blocked with 1% BSA in PBS for 1 h at 37°C. Plates were washed once with PBS containing 0.05 wt% Tween-20 (PBST) and treated with primary anti-E. faecalis antibodies serially diluted on the plate for 1 h at 37°C. Plates were washed and treated with goat anti-chicken HRP secondary antibody. TMB substrate was added to develop the signal and the reaction was stopped by HCl. Plates were read at 450 nm in a plate reader. To measure anti-cytolysin antibody titer, another 96-well plate was coated with 0.5 mg / ml cytolysin and a similar experiment was performed using anti-cytolysin antibody.

[0096] Testing the neutralizing capacity of IgY Enterococcus faecalis was cultured to an OD of 1.5 in BHI broth. 600 Culture overnight and dilute 1:50 in broth until OD 600 The culture was incubated for an additional 10 min in BHI broth until the β- 3 The cultures were further diluted to 10 cfu / ml and treated with antibodies at 15 mg / ml. After 24 hours of incubation at 37°C, the cultures were 4 2-fold serial dilutions were plated on sterile plates and incubated at 37° C. for 12 hours. cfu / mL was quantified by counting the number of colonies using the particle function in ImageJ Count. Percent inhibition of bacterial colony formation was calculated.

[0097] result Total soluble proteins including IgY from hyperimmunized chicken egg yolk were extracted by water dilution and NaCl method. The purity of IgY was analyzed by SDS-PAGE electrophoresis under reducing conditions. IgY has a molecular weight of 180KD and is composed of two subunits, a heavy chain of 67kDa and a light chain of 23KD. The electrophoretic pattern of IgY extracted from hyperimmunized egg yolk was similar to that of the IgY standard. See Figure 2.

[0098] Assessment of target-specific antibody titers The specific activity of IgY antibodies against E. faecalis and cytolysin antigens was measured by indirect ELISA. The ELISA results showed positive binding of IgY to specific antigens with a high titer of 1:32,000 when compared to the control IgY. See Figures 3A and 3B.

[0099] Target Neutralization and Evaluation The growth inhibitory effect of IgY antibodies against E. faecalis was investigated. Whole cell E. faecalis bacteria were incubated with 15 mg / ml IgY and cfu analysis was performed after 24 hours of incubation. Treatment with 15 mg / ml IgY inhibited the growth of E. faecalis by more than 70%, as visualized by a reduction in the number of colonies on the agar plates. See Figure 4.

[0100] The growth inhibitory effect of IgY against E. faecalis was quantified and the percent inhibition was plotted, see Figure 5. Treatment of E. faecalis with anti-E. faecalis + cytolysin IgY antibody at a concentration of 15 mg / ml resulted in 75% inhibition of E. faecalis growth, indicating successful generation of neutralizing antibodies against E. faecalis, see Figure 5.

[0101] [Example 2] Effect of oral administration of anti-Enterococcus faecalis and anti-cytolysin IgY antibodies in a mouse model of alcoholic liver disease method Preparation of egg powder for preclinical studies The egg white and egg yolk were homogenized and lyophilized to produce egg powder. The egg powder was stored at 4° C. The lyophilized egg powder was analyzed for antibody activity as described above in Example 1 and then evaluated in a humanized model of alcoholic liver disease (ALD) as described below.

[0102] Stool samples from cytolysin-positive human patients with alcoholic hepatitis were used for fecal transplantation in germ-free mice. Mice were gavaged with 100 μl stool samples (1 g stool dissolved in 30 ml Luria-Bertani (LB) medium containing 15% glycerol under anaerobic conditions) starting from 5–6 weeks of age and repeated 2 weeks later. Two weeks after the second gavage, mice were fed with ethanol or a control (isocaloric) diet as described in Duan et al., 2019, Nature 575: 505-511. Mice were periodically evaluated for serum ALT, liver pathology and other parameters, such as weight gain, liver weight and food intake.

[0103] Alcoholic Liver Disease (ALD) Model Male and female C57BL / 6 germ-free mice were housed as described in Duan et al., 2019, Nature 575: 505-511, and fecal transplants with stool samples from one cytolysin-positive human patient with alcoholic hepatitis were performed at 5–6 weeks of age and repeated 2 weeks later. Mice were fed a Lieber-DeCarli diet, with caloric intake from ethanol being 0% on days 1–5 and 36% from day 6 to the end of the study period. On day 16, mice were gavaged with a single dose of ethanol (5 g / kg body weight) in the early morning and sacrificed 9 h later. Pair-fed control mice were fed an isocaloric glucose-replaced diet.

[0104] Egg powder containing control IgY (15 mg / day) or anti-E. faecalis + anti-cytolysin IgY (1, 5 or 15 mg per day) from eggs of non-immunized hens was added to the liquid diet (containing fat, carbohydrate, protein and ethanol / isocaloric glucose) at the start of alcohol administration. See Table 1 below for the weight of the dry egg powder. Diet was changed every 3 days. Mice were randomly assigned to groups at the start of the study. There were no deaths in any group during the ethanol feeding period.

[0105] [Table 1]

[0106] biochemical analysis Serum levels of alanine aminotransferase (ALT; a test for liver injury) were measured using the Infinity ALT kit (Thermo Scientific). Liver triglyceride levels were measured using a triglyceride liquid reagent kit (Pointe Scientific).

[0107] Staining procedure Formalin-fixed tissue samples were embedded in paraffin (Paraplast plus, McCornick) and stained with hematoxylin-eosin (Surgipath).

[0108] statistical analysis Results are expressed as mean ± sem. The number of biological replicates was n = 5 in each ethanol-containing diet group. Significance was assessed using one-way analysis of variance (ANOVA) with Tukey's post-hoc test. P values ​​< 0.05 were considered statistically significant. Statistical analysis was performed using R statistical software, R version 1.3.1093, 2020 R Foundation for Statistical Computing and GraphPad Prism v8.4.3.

[0109] result To determine the effect of anti-E. faecalis + anti-cytolysin IgY in a mouse model of ethanol-induced liver disease, germ-free C57BL / 6 mice were colonized with feces from a cytolysin-positive patient with alcoholic hepatitis and subjected to a chronic excessive ethanol feeding model. Food intake, body weight, and liver weight were not significantly different in the ethanol-fed groups. See Figures 6A-6C. There was a trend toward reduced body weight in the anti-E. faecalis + anti-cytolysin IgY-treated mouse group, which is related to the reduced body weight at the start of the study. Mice administered 5 mg or 15 mg of anti-E. faecalis + anti-cytolysin IgY showed significantly less liver damage as indicated by reduced serum levels of alanine aminotransferase (ALT) compared to mice administered control IgY after chronic ethanol feeding (see Figure 6D). No significant differences were observed in liver triglycerides, but there was a trend toward reduced hepatic steatosis in anti-E. faecalis + anti-cytolysin IgY-treated mice (see FIG. 6E). Histological analysis showed that anti-E. faecalis + anti-cytolysin IgY-treated mice exhibited significantly lower ethanol-induced liver injury and hepatic steatosis (see FIG. 7).

Claims

1. 1. A composition for preventing or treating alcoholic liver disease in a subject in need thereof, the composition comprising a hyperimmunized egg product obtained from an egg-laying animal, the hyperimmunized egg product comprising a therapeutically effective amount of one or more antibodies against an antigen selected from the group consisting of Enterococcus faecalis and Enterococcus faecalis cytolysin toxin.

2. 1. A composition for preventing or treating graft-versus-host disease in a subject in need thereof, the composition comprising a hyperimmunized egg product obtained from an egg-laying animal, the hyperimmunized egg product comprising a therapeutically effective amount of one or more antibodies against an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, and Enterococcus faecium.

3. 3. The composition of claim 1 or 2, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies against Enterococcus faecalis and a therapeutically effective amount of one or more antibodies against Enterococcus faecalis cytolysin toxin.

4. 3. The composition of claim 1 or 2, wherein the hyperimmunized egg product contains at least 20% by weight more IgY antibodies specific for an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin and Enterococcus faecium compared to a control egg product obtained from a non-hyperimmunized egg-laying animal.

5. 3. The composition of claim 1 or 2, wherein the hyperimmunized egg product is administered to the subject 1 to 4 times per day.

6. 3. The composition of claim 1 or 2, wherein the hyperimmunized egg product is administered orally or intravenously.

7. 3. The composition of claim 1 or 2, wherein administration of the hyperimmunized egg product to a subject reduces levels of Enterococcus faecalis in the subject compared to a subject not administered the hyperimmunized egg product.

8. 10. The composition of claim 1, wherein administration of the hyperimmunized egg product to a subject reduces liver damage in the subject compared to a subject not receiving the hyperimmunized egg product.

9. The composition of claim 1 or 2, wherein the subject is a human.

10. A hyperimmunized egg produced by an animal that has been hyperimmunized with an antigen selected from the group consisting of Enterococcus faecalis expressing a cytolysin toxin and isolated Enterococcus faecalis cytolysin toxin, wherein the level of antibodies against the antigen in the hyperimmunized egg is increased compared to eggs from an animal that is not hyperimmunized.

11. 11. The hyperimmunized egg of claim 10, wherein the animal has been hyperimmunized with Enterococcus faecalis and the isolated Enterococcus faecalis cytolysin toxin and the level of antibodies against Enterococcus faecalis and the isolated Enterococcus faecalis cytolysin toxin is increased compared to eggs from animals that have not been hyperimmunized.

12. The hyperimmunized egg of claim 10 or 11, wherein the animal has been further hyperimmunized with Enterococcus faecium.

13. 12. A hyperimmunized egg product obtained from the hyperimmunized egg of claim 10 or 11.

14. 14. The hyperimmunized egg product of claim 13, wherein the hyperimmunized egg product is a whole egg.

15. 14. The hyperimmunized egg product of claim 13, wherein the hyperimmunized egg product is an egg yolk.

16. 14. The hyperimmunized egg product of claim 13, wherein the hyperimmunized egg product is a purified or partially purified IgY antibody against Enterococcus faecalis cytolysin toxin.

17. 14. The hyperimmunized egg product of claim 13, wherein the hyperimmunized egg product is a purified or partially purified IgY antibody against Enterococcus faecalis.

18. 14. The hyperimmunized egg product of claim 13, wherein the hyperimmunized egg product consists of purified or partially purified IgY antibodies against Enterococcus faecalis and purified or partially purified IgY antibodies against Enterococcus faecalis cytolysin toxin.

19. 14. A pharmaceutical composition comprising the hyperimmunized egg product of claim 13 and a pharma- ceutically acceptable carrier.

20. 20. The pharmaceutical composition of claim 19, comprising 3 to 10 grams of whole egg.

21. 20. The pharmaceutical composition of claim 19, comprising 1 to 3 grams of egg yolk.

22. 20. The pharmaceutical composition of claim 19, comprising 0.05 to 1 gram of purified or partially purified IgY.

23. 20. The pharmaceutical composition of claim 19, formulated for oral administration.

24. 24. The pharmaceutical composition of claim 23, wherein the hyperimmunized egg product is formulated in a nanoparticle or an emulsion.

25. 20. The pharmaceutical composition of claim 19, formulated for intravenous administration.

26. 1. A method for preparing a hyperimmunized egg product, comprising: i) hyperimmunizing an egg-laying animal with an antigen selected from the group consisting of Enterococcus faecalis expressing a cytolysin toxin and an isolated Enterococcus faecalis cytolysin toxin; and ii) preparing the hyperimmunized egg product from one or more eggs produced by the animal.

27. The method of claim 26, wherein the egg-laying animal is further hyperimmunized with Enterococcus faecium.

28. 27. The method of claim 26, wherein the antigen comprises Enterococcus faecalis expressing a cytolysin toxin and an isolated Enterococcus faecalis cytolysin toxin.

29. 27. The method of claim 26, wherein the egg-laying animal is a chicken.

Images