USE OF A HYPERIMMUNIZED EGG PRODUCT FOR THE TREATMENT OR PREVENTION OF ALCOHOLIC LIVER DISEASE AND GRAFT-VERSUS-HOST DISEASE, HYPERIMMUNIZED EGG, HYPERIMMUNIZED EGG PRODUCT, PHARMACEUTICAL COMPOSITION AND METHOD FOR PREPARING A HYPERIMMUNIZED EGG PRODUCT

Abstract

Hyperimmunized egg product for the treatment or prevention of alcoholic liver disease and graft-versus-host disease. In one aspect, the present invention is directed to a method for preventing or treating alcoholic liver disease or graft-versus-host disease in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-producing animal, thereby preventing or treating alcoholic liver disease or graft-versus-host disease in the individual, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, and Enterococcus faecium.The present invention is also directed 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, cytolysin toxin isolated from Enterococcus faecalis, and Enterococcus faecium. Methods for preparing hyperimmunized eggs and egg products are also described.

Description

1 / 43 “USE OF A HYPERIMMUNIZED EGG PRODUCT FOR THE TREATMENT OR PREVENTION OF ALCOHOLIC LIVER DISEASE AND GRAFT-VERSUS-HOST DISEASE, HYPERIMMUNIZED EGG, HYPERIMMUNIZED EGG PRODUCT, PHARMACEUTICAL COMPOSITION AND METHOD FOR PREPARING A HYPERIMMUNIZED EGG PRODUCT” RELATED REQUEST

[001] This application claims priority to US Provisional Patent Application No. 63 / 175,603 filed on April 16, 2021, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[002] The Sequence Listing associated with this application is deposited electronically via EFS-Web and incorporated herein by reference in the descriptive report 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 1,004 bytes, and the text file was created on April 14, 2022. FOUNDATION

[003] Alcoholic liver disease (ALD) encompasses the hepatic manifestations of excessive alcohol consumption, including fatty liver, alcoholic hepatitis, and chronic hepatitis with hepatic fibrosis or cirrhosis. The most severe form of alcoholic liver disease is alcoholic hepatitis; mortality ranges from 20% to 40% in 1-6 months, and up to 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 it is offered only in select centers and to a limited group of patients. See Duan et al., cited above.

[004] Graft-versus-host disease (GVHD) affects the skin, liver, and gastrointestinal tract, and occurs when donor T cells recognize the tissue Petition 870250024929, dated 03 / 28 / 2025, page 57 / 105 2 / 43 receptors are perceived as foreign and trigger inflammation and tissue damage. Preventive and therapeutic strategies for GVHD are active areas of research. See Garrett, 2020, New England Journal of Medicine 382(11): 1064-1066. Therefore, there is a need for improved methods to treat or prevent ALD and GVHD. SUMMARY OF THE INVENTION

[005] In certain respects, the description refers to a method for preventing or treating alcoholic liver disease in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-producing animal, thereby preventing or treating alcoholic liver disease in the individual, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to an antigen selected from the group consisting of Enterococcus faecalis and Enterococcus faecalis cytolysin toxin.

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

[007] In certain embodiments, the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to Enterococcus faecalis, and a therapeutically effective amount of one or more antibodies to Enterococcus faecalis cytolysin toxin. In certain embodiments, the method further comprises hyperimmunizing the egg-producing animal with an antigen. Petition 870250024929, dated 03 / 28 / 2025, page 58 / 105 3 / 43 selected from the group consisting of Enterococcus faecalis, cytolysin toxin isolated from Enterococcus faecalis, and Enterococcus faecium. In certain embodiments, the method further comprises collecting a hyperimmunized egg from an egg-laying animal that has been hyperimmunized, and preparing a hyperimmunized egg product from the hyperimmunized egg. In certain embodiments, the hyperimmunized egg product comprises at least 20% more by weight of an IgY antibody specific for an antigen selected from the group consisting of Enterococcus faecalis, cytolysin toxin from Enterococcus faecalis, 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 the individual 1 to 4 times per day. In certain modalities, the hyperimmunized egg product is administered orally or intravenously.In certain embodiments, administration of the hyperimmunized egg product to an individual reduces the level of Enterococcus faecalis in that individual compared to an individual who was not administered the hyperimmunized egg product. In certain embodiments, administration of the hyperimmunized egg product to an individual reduces liver damage in that individual compared to an individual who was not administered the hyperimmunized egg product. In certain embodiments, the individual is a human being.

[008] In certain respects, the description refers to a hyperimmunized egg produced by an animal that has been hyperimmunized with a selected antigen from the group consisting of Enterococcus faecalis, cytolysin toxin isolated from Enterococcus faecalis, and Enterococcus faecium, in which the antibody level to the antigen in the hyperimmunized egg is increased relative to an egg from an animal that has not been hyperimmunized. In certain embodiments, the animal has been hyperimmunized with Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis, and in which the antibody level to Enterococcus faecalis and cytolysin toxin isolated from Petition 870250024929, dated 03 / 28 / 2025, p. 59 / 105 4 / 43 Enterococcus faecalis are increased compared to an egg from an animal that has not been hyperimmunized.

[009] In certain aspects, the description refers to a hyperimmunized egg product obtained from a hyperimmunized egg described herein. In certain embodiments, the hyperimmunized egg product is a whole egg. In certain embodiments, the hyperimmunized egg product is egg yolk. In certain embodiments, the hyperimmunized egg product is a purified or partially purified IgY antibody to Enterococcus faecalis cytolysin toxin. In certain embodiments, the hyperimmunized egg product is a purified or partially purified IgY antibody to Enterococcus faecalis. In certain embodiments, the hyperimmunized egg product consists of purified or partially purified IgY antibody to Enterococcus faecalis and purified or partially purified IgY antibody to Enterococcus faecalis cytolysin toxin.

[010] In certain aspects, the description refers to a pharmaceutical composition comprising the hyperimmunized egg product of any of claims 14 to 19 and a pharmaceutically 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 nanoparticles or in an emulsion. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.

[011] In certain respects, the description refers to a method of preparing a hyperimmunized egg product comprising: i) hyperimmunizing an egg-producing animal with an antigen selected from the group consisting of Petition 870250024929, dated 03 / 28 / 2025, pp. 60 / 105 5 / 43 i) preparing a hyperimmunized egg product from one or more eggs produced by the animal. In certain embodiments, the antigen is selected from the group consisting of Enterococcus faecalis, cytolysin toxin isolated from Enterococcus faecalis, and Enterococcus faecium; and ii) preparing a hyperimmunized egg product from one or more eggs produced by the animal. In certain embodiments, the antigen is selected from the group consisting of Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis. In certain embodiments, the antigen comprises Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis. In certain embodiments, the egg-producing animal is a chicken. BRIEF DESCRIPTION OF THE FIGURES

[012] Figure 1 shows a schematic of an experiment evaluating anti-Enterococcus faecalis and anti-cytolysin IgY antibodies in a Mouse Model of Alcoholic Liver Disease.

[013] Figure 2 shows the 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, lanes 3 and 4 are 1 pg and 2 pg, respectively, of total soluble egg yolk protein from chickens hyperimmunized with Enterococcus faecalis and cytolysin.

[014] Figure 3A shows the specific Enterococcus faecalis IgY activity of chickens immunized with Enterococcus faecalis + cytolysin. Figure 3B shows the specific cytolysin IgY activity of chickens immunized with Enterococcus faecalis + cytolysin. IgY from non-immunized laying hens was used as a control IgY. Specific activity is represented by antibody reactivity and titer.

[015] Figure 4 shows the inhibitory effects of anti-Enterococcus faecalis and Petition 870250024929, dated 03 / 28 / 2025, page 61 / 105 6 / 43 anticytolysin IgY on the growth of E. faecalis. Whole, untreated E. faecalis bacteria with 15 mg / ml IgY were incubated for 24 hours at 37°C. Cultures were serially diluted and diluted 10⁴ times and plated on BHI agar plates. After overnight incubation, colonies were counted using ImageJ Count.

[016] Figure 5 shows the quantification of cfu / ml in untreated and treated E. faecalis culture with anti-Enterococcus faecalis + anticytolysin IgY. The percentage of inhibition was calculated.

[017] Figures 6A-6E show the effects of administering anti-Enterococcus faecalis + anti-cytolysin IgY in gnotobiotic mice subjected to a chronic and compulsive ethanol feeding model. Germ-free C57BL / 6 mice were colonized with feces from a cytolysin-positive human patient with alcoholic hepatitis and subjected to a chronic and compulsive ethanol feeding model (n=5 per group), and received vehicle, control IgY, and anti-Enterococcus faecalis + anti-cytolysin IgY in the liquid diet. (A) Food intake. (B) Body weight. (C) Liver weight. (D) Serum alanine aminotransferase (ALT) level. (E) Liver triglyceride (TAG, triacylglycerol) content. Results 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.

[018] Figure 7 shows the effects of administering anti-Enterococcus faecalis + anti-cytolysin IgY in the livers of gnotobiotic mice subjected to a chronic and compulsive ethanol feeding model. Germ-free C57BL / 6 mice were colonized with feces from a cytolysin-positive patient with alcoholic hepatitis and subjected to a chronic and compulsive ethanol feeding model (n=5 per group), and received vehicle, control IgY, or anti-Enterococcus faecalis + anti-cytolysin IgY in the liquid diet. Representative sections of the liver are shown after staining with hematoxylin and eosin. Petition 870250024929, dated 03 / 28 / 2025, page 62 / 105 7 / 43 DETAILED DESCRIPTION

[019] The term hyperimmunization means repeated exposure to one or more antigens such that an immune response is elevated and maintained above the natural unexposed state.

[020] A hyperimmune state refers to an elevated immune response in an egg-producing animal that has been hyperimmunized.

[021] The term egg, as used herein, refers to a whole egg (table, hyperimmunized or other). The term egg product, as used herein, refers to a whole egg or to any product or fraction obtained from a whole egg. In one particular embodiment, the egg product is an egg yolk, for example, powdered egg yolk. In another embodiment, the egg product is an egg white, for example, powdered egg white. In yet another embodiment, the egg product is obtained from a whole egg, for example, powdered whole egg (for example, spray-dried powdered whole egg).

[022] The term control egg refers to an egg obtained from an egg producer that is not kept in a hyperimmunized state, i.e., an animal that has not been hyperimmunized. The term control egg product refers to a control egg or an egg product obtained from a control egg.

[023] The term hyperimmunized egg refers to a whole egg obtained from an egg-producing animal maintained in a hyperimmune state, that is, an egg-producing animal that has been hyperimmunized.

[024] The term hyperimmunized egg product refers to a hyperimmunized egg or any product obtained from a hyperimmunized egg. In certain embodiments, the hyperimmunized egg product is a concentrate. As used herein, the term concentrate refers to a hyperimmunized egg product that is at least partially purified so that the antibody concentration in the concentrate is higher than the antibody concentration in an egg. Petition 870250024929, dated 03 / 28 / 2025, p. 63 / 105 8 / 43 hyperimmunized.

[025] The term powdered egg refers to a whole egg that has been dried. In some embodiments, the powdered egg is spray-dried.

[026] The term egg-producing animal means any oviparous animal and includes any animal that lays eggs, such as birds, fish and reptiles.

[027] The term avian refers to an animal that is a member of the class 4ves. Birds include, but are not limited to, chickens, turkeys, geese, ducks, pheasants, quails, pigeons, and ostriches.

[028] The term supranormal levels means levels higher than those found in eggs from egg-producing animals that are not hyperimmunized. For example, supranormal levels of an antibody to a particular antigen are levels of the antibody higher than those found in eggs from egg-producing animals that are not hyperimmunized to the particular antigen.

[029] The term administer means any method of delivering a substance to an individual, including by oral, intranasal, parenteral (intravenous, intramuscular or subcutaneous), rectal, topical or intraocular routes.

[030] The term antigen refers to a substance that is capable of inducing a humoral antibody and / or cell-mediated immune response rather than immunological tolerance. The term means the ability to stimulate an immune response as well as react with its products, for example, an antibody.

[031] As used herein, an antibody is a protein that includes at least one complementarity-determining region that binds to a specific target antigen, for example, an Enterococcus faecalis antigen, cytolysin toxin isolated from Enterococcus faecalis, or an Enterococcus faecium antigen described herein. For example, an antibody may include a variable heavy chain (H) region (hereinafter abbreviated as VH) and a variable light chain (L) region (hereinafter abbreviated as VL). In another example, an antibody includes two variable regions of Petition 870250024929, dated 03 / 28 / 2025, page 64 / 105 9 / 43 heavy chain (H) and two variable light chain (L) regions. In a particular embodiment, the antibody is a polyclonal antibody. The term polyclonal antibody, as used herein, refers to a population of antibody molecules that are capable of immunoreacting with different epitopes on a particular antigen. In a particular embodiment, the antibody is an IgY antibody.

[032] As used herein, the terms alcoholic liver disease, ALD, alcohol-associated liver disease, and alcohol-related liver disease refer to the hepatic manifestations of excessive alcohol consumption, including fatty liver (steatosis), alcoholic hepatitis, and chronic hepatitis with hepatic fibrosis or cirrhosis.Alcohol-related disorders, as used herein, refer to diseases and disorders related to alcohol consumption and include, but are not limited to, alcohol-induced psychotic disorder with delusions; alcohol abuse; excessive alcohol consumption; heavy alcohol consumption; problems with alcohol consumption; alcohol intoxication; alcohol withdrawal; delirium of alcohol intoxication; delirium of alcohol withdrawal; alcohol-induced persistent dementia; alcohol-induced persistent amnestic disorder; alcohol dependence; alcohol-induced psychotic disorder with hallucinations; alcohol-induced mood disorder; alcohol-induced or associated bipolar disorder; alcohol-induced or associated post-traumatic stress disorder; alcohol-induced anxiety disorder; alcohol-induced sexual dysfunction; alcohol-induced sleep disorder; and alcohol-related disorder not otherwise specified (NOS).

[033] The term alcohol abuser, as used herein, refers to an individual who meets the DSM IV criteria for alcohol abuse (i.e., repeated use despite recurring adverse consequences) but is not alcohol dependent.

[034] The term binge drinker, as used herein, refers to men who drink 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. Petition 870250024929, dated 03 / 28 / 2025, pp. 65 / 105 10 / 43 (15 ml) of absolute alcohol, 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 alcohol dependent, but may or may not meet the DSM-IV criteria for alcohol abuse.

[035] The term heavy drinker, as used herein, 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 alcohol dependent, but may or may not meet the DSM IV criteria for alcohol abuse.

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

[037] The term isolate, as used herein, refers to a biological compound (e.g., a protein) that is purified or partially purified from the cell in which it was produced. For example, a cytolysin toxin isolated from Enterococcus faecalis is purified or partially purified from the Enterococcus faecalis cell in which it was produced. In some embodiments, the cytolysin toxin isolated from Enterococcus faecalis is a recombinant cytolysin toxin from Enterococcus faecalis, for example, a cytolysin toxin from Enterococcus faecalis that is produced in a cell other than an Enterococcus faecalis cell. Alcoholic liver disease (ALD)

[038] Eighty percent of alcohol passes through the liver to be detoxified. Chronic alcohol consumption results in 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 inflammation, apoptosis, and eventually fibrosis of liver cells, leading to alcoholic liver disease. Petition 870250024929, dated 03 / 28 / 2025, page 66 / 105 11 / 43 (ALD). ALD includes fatty liver disease (steatosis), alcoholic hepatitis, and chronic hepatitis with hepatic fibrosis or cirrhosis.

[039] Fatty liver, or steatosis, is the accumulation of fatty acids in liver cells. Heavy alcohol consumption causes the development of large fat globules (macrovesicular steatosis) throughout the liver. Alcohol is metabolized by alcohol dehydrogenase (ADH) into acetaldehyde, then metabolized by aldehyde dehydrogenase (ALDH) into acetic acid, which is finally oxidized into carbon dioxide and water. This process generates NADH and increases the NADH / NAD+ ratio. A higher concentration of NADH induces fatty acid synthesis, while a reduced level of NAD results in decreased fatty acid oxidation. Subsequently, the higher levels of fatty acids signal liver cells to combine them with glycerol to form triglycerides. These triglycerides accumulate, resulting in fatty liver.

[040] Alcoholic hepatitis is characterized by inflammation of hepatocytes, which appears to predispose the liver to fibrosis. Inflammatory cytokines (TNF-alpha, IL6, and IL8) are believed to be essential in initiating and perpetuating liver injury by inducing apoptosis and necrosis. A possible mechanism for the increased TNF-α activity is increased intestinal permeability due to liver disease. This facilitates the absorption of endotoxin produced by the intestine into the portal circulation. The Kupffer cells of the liver then phagocytose the endotoxin, stimulating the release of TNF-α. TNF-α then triggers apoptotic pathways through the activation of caspases, resulting in the death of liver cells.

[041] Cirrhosis is a late stage of severe liver disease, marked by inflammation (swelling), fibrosis (cell hardening), and damaged membranes, preventing the detoxification of chemicals in the body, resulting in scarring and necrosis (cell death). Acetaldehyde may be responsible for alcohol-induced fibrosis, stimulating collagen deposition by stellate cells. Petition 870250024929, dated 03 / 28 / 2025, page 67 / 105 12 / 43 liver damage. The production of oxidants derived from NADPH oxidase and / or cytochrome P450 2E1 and the formation of acetaldehyde-protein adducts damage the cell membrane. Symptoms include jaundice (yellowing), enlarged liver, and pain and tenderness due to structural changes in the damaged liver architecture. Without complete abstinence from alcohol use, cirrhosis will eventually lead to liver failure.

[042] Chronic liver disease due to alcohol use disorder contributes significantly to the global burden of disease and mortality. The most severe form of alcohol-related liver disease is alcoholic hepatitis (AH); mortality ranges from 20% to 40% in 1-6 months. It has recently been shown that the disease is more severe in a subset of patients with alcoholic hepatitis (approximately 30%) who harbor Enterococcus faecalis, which produces a toxin called cytolysin. Although E. faecalis is present in the microbiome of non-alcoholic individuals at a low level, at approximately 0.1-0.5% of the individual's microbiome, in patients with alcoholic liver disease (ALD) its abundance is greatly expanded to approximately 5%. Seventy-five percent of patients with alcoholic hepatitis whose microbiome is abundant in cytolysin-positive E. faecalis die within 90 days of diagnosis of severe alcoholic hepatitis (Maddrey, et al., 1978, Gastroenterology 75, 193-199).Standard of care corticosteroid therapy 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 therapies, but are available only to a limited group of patients (Mathurin, et al., 2012, Management of alcoholic hepatitis. J. Hepatol. 56, S39-S45).

[043] Duan et al. (2019, Nature 575: 505-511) demonstrated the efficacy of bacteriophages targeting cytolysin-positive E. faecalis in humanized mice colonized with bacteria from feces of patients with alcoholic hepatitis, in preventing disease progression. It was shown that the Petition 870250024929, dated 03 / 28 / 2025, p. 68 / 105 13 / 43 Treatment with bacteriophages that lyse cytolysin-positive E. faecalis decreases cytolysin in the liver and prevents the progression of ethanol-induced liver disease in these humanized mice. Graft-versus-host disease (GVHD)

[044] GVHD is a significant complication following solid organ and stem cell transplantation, such as those occurring in bone marrow transplants. The donor's immune system white blood cells that remain within the donor tissue (the graft) recognize the recipient (the host) as foreign. The white blood cells present in the transplanted tissue attack the recipient's body cells, leading to 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 post-transplant and represents a major challenge for transplants due to associated morbidity and mortality. The chronic form of GVHD usually occurs after 100 days. The appearance of moderate to severe cases of chronic GVHD negatively influences long-term survival.

[045] Acute GVHD (aGVHD) affects the skin, liver, and gastrointestinal tract and occurs when donor T cells recognize the recipient tissue as foreign and trigger inflammation and tissue damage. Specifically, aGVHD is initiated by alloreactive donor T cells that recognize MHC class I and II molecules on the surface of host cells, as well as peptides presented by them. Infiltration of various target organs, such as the intestine, liver, and skin, by donor leukocytes, including T cells, is believed to be one of the key processes in the initial phase of aGVHD. Activation and expansion of donor T cells, leading to the secretion of pro-inflammatory cytokines and the recruitment of additional inflammatory effector cells to these sites, further damage the affected tissues. See Hoffmann et al., cited above. Petition 870250024929, dated 03 / 28 / 2025, page 69 / 105 14 / 43 Enterococcus faecalis and Cytolysin and ALD

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

[047] Highly virulent strains of E. faecalis express a pore-forming exotoxin called cytolysin, which lyses bacterial and eukaryotic cells in response to quorum signals. Compared with non-alcoholic individuals or patients with alcohol use disorder, patients with alcoholic hepatitis exhibit increased fecal counts of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis has correlated with the severity of alcoholic liver disease and mortality in patients with alcoholic hepatitis. Furthermore, bacteriophages targeting cytolytic E. faecalis decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. See Duan et al., cited above.

[048] Functional cytolysin toxin consists of large and small subunit oligopeptides, encoded by the cy1LL and cy1LS genes, respectively. The E. faecalis cytolysin components Cy1Ll and Cy1Ls have been classified as Type A pore-forming antibiotics and, more recently, as two-component Class II antibiotics. The antibiotics are complex polycyclic antimicrobial peptides, which 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. Petition 870250024929, dated 03 / 28 / 2025, pp. 70 / 105 15 / 43 Antibiotics have extremely varied structures and functions, but all are characterized by undergoing extensive post-translational modifications and possessing antibiotic or morphogenic activities. Cytolysin appears to be unique among antibiotics, as it can lyse other bacteria as well as erythrocytes and other eukaryotic cells. See Van Tyne et al., 2013, Toxins 5(5): 895-911.

[049] The amino acid sequences of Cy1 Ll and Cy1 Ls are provided below: Cy1LL TTPVCAVAATAAASSAACGWVGGGIFTGVTVVVSLKHC (SEQ ID NO: 1) CylLs TTPACFTIGLGVGALFSAKFC (SEQ ID NO: 2) Enterococcus and GVHD

[050] 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 stem cell transplantation. See Garrett, 2020, New England Journal of Medicine 382(11): 1064-1066, which is incorporated here by reference in its entirety. Some enterococci species in the gut (e.g., Enterococcus faecalis and Enterococcus faecium) are opportunistic pathogens that frequently cause potentially fatal bloodstream infections in patients undergoing allogeneic hematopoietic stem cell transplantation. Studies have shown that enterococci, particularly E. faecium, dominate fecal communities in a considerable subgroup of patients undergoing allogeneic hematopoietic stem cell transplantation, and that this enterococcal dominance has correlated with decreased overall survival and increased mortality from GVHD. See Stein-Thoeringer et al., 2019, Science 366: 1143-9, which are incorporated here by reference in their entirety. Stein-Thoeringer et al. observed that the dietary sugar, lactose, drives enterococcal growth in a mouse model of GVHD, and that a lactose-free diet attenuated enterococcal expansion and T-cell-induced inflammation in GVHD. Petition 870250024929, dated 03 / 28 / 2025, page 71 / 105 16 / 43 Hyperimmunized egg product

[051] In certain aspects, the present invention relates to a method of preparing a hyperimmunized egg product comprising: i) hyperimmunizing an egg-producing animal with an antigen selected from the group consisting of Enterococcus faecalis, cytolysin toxin isolated from Enterococcus faecalis, and Enterococcus faecium; and ii) preparing a 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 cytolysin toxin isolated from Enterococcus faecalis. In some embodiments, the antigen comprises Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis.

[052] Egg-producing animals produce antibodies in their blood and eggs that are specific to particular immunogens. For example, several genera of the class Aves, 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 chicken immunoglobulins. Polson, A., et al. (Immunological Communications 9:495-514(1980)) immunized laying hens against various proteins and natural protein mixtures, and detected IgY antibodies in egg yolks. Fertel, R., et al. (Biochemical and Biophysical Research Communications 102:1028-1033 (1981)) immunized laying hens against prostaglandins and detected antibodies in the egg yolk. Jensenius et al. (Journal of Immunological Methods 46:63-68 (1981)) provide a method for isolating IgG from egg yolk for use in immunodiagnostics.Polson et al. (Immunological Communications 9:475-493 (1980)) describe antibodies isolated from the yolk of laying hens that were immunized with a variety of plant viruses.

[053] US Patent No. 4,748,018 describes a method of immunization Petition 870250024929, dated 03 / 28 / 2025, p. 72 / 105 17 / 43 passive in a mammal comprising the parenteral administration of purified antibody obtained from the eggs of a bird that has been immunized against the corresponding antigen, and in which the mammal has acquired immunity to the eggs. US Patent No. 5,772,999, assigned to DCV-Biologics, describes a method for preventing, combating, or reducing chronic gastrointestinal disorders or gastrointestinal damage induced by nonsteroidal anti-inflammatory drugs (NSAID-induced) in an individual by administering hyperimmunized egg and / or milk or fractions thereof to the individual.

[054] An immunized egg is an egg that comes from a bird that has been immunized with, for example, a specific antigen or a mixture of antigens. A hyperimmunized egg is an egg that comes from a bird that has been brought to a specific state of immunization through, for example, periodic booster administrations of antigens. Hyperimmunized eggs, regardless of the type of antigen administered to their poultry producer, have been found to exhibit several beneficial factors, including, as mentioned above, the treatment of chronic gastrointestinal disorders, NSAID-induced gastrointestinal damage (see US Patent No. 5,772,999), and anti-inflammatory effects due to the presence of an anti-inflammatory composition (see US Application Publication No. US 2004 / 0156857).

[055] One of the advantages of hyperimmunized egg product is that it would have a higher and more consistent level of antibodies (e.g., IgY antibodies) against Enterococcus faecium, Enterococcus faecalis, or Enterococcus faecalis cytolysin toxin compared to a control egg product or an egg product from a hen that was immunized with Enterococcus faecium, Enterococcus faecalis, or Enterococcus faecalis cytolysin toxin using standard immunization techniques. In some embodiments, the hyperimmunized egg product has higher levels of IgY antibodies to Enterococcus faecalis and / or Enterococcus faecalis cytolysin toxin isolated from relative to a hen that was immunized Petition 870250024929, dated 03 / 28 / 2025, page 73 / 105 18 / 43 with Enterococcus faecalis and / or Enterococcus faecalis cytolysin toxin 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 Enterococcus faecalis cytolysin toxin as described herein. In some embodiments, hyperimmunization involves immunizing an egg-producing animal with Enterococcus faecium, Enterococcus faecalis and / or cytolysin toxin isolated from Enterococcus faecalis described herein at intervals of 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.

[056] Hyperimmunized egg product can be produced by any egg-laying animal. It is preferable that the animal be a member of class 4ves or, in other words, a bird. Within class 4ves, domesticated birds are preferred, but other members of this class, such as turkeys, ducks, and geese, are a suitable source of hyperimmune egg product. In one particular embodiment, the egg-laying animal is a chicken.

[057] This special state of hyperimmunization is preferably achieved through the administration of an initial immunization, followed by periodic boosters with sufficiently high doses of specific antigens or antigen mixtures. The booster dosage may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the dosage required to produce primary immunization of the egg-laying animal. Any of these percentages may be used to define a booster immunization dosage range. For example, in some embodiments, the booster dosage is 20% to 80%, 30% to 70%, or 50% to 100% of the dosage required to produce primary immunization of the egg-laying animal. In one particular embodiment, the booster immunization dosage is 50% Petition 870250024929, dated 03 / 28 / 2025, pp. 74 / 105 19 / 43 of the primary immunization dosage. Having knowledge of the requirements to develop and maintain a hyperimmune state, it is within the knowledge of those skilled in the art to vary the amount of antigen administered, depending on the sex of the egg-producing animal and the strain used, in order to maintain the animal in a hyperimmune state.

[058] The hyperimmune state can be produced by a single antigen or by a combination of antigens. Hyperimmunization can be achieved by multiple exposures to multiple antigens, or multiple exposures to a single antigen. Antigens for hyperimmunization

[059] In some embodiments, the antigens for hyperimmunization comprise one or more of Enterococcus faecium, Enterococcus faecalis, and Enterococcus faecalis cytolysin toxin. In some embodiments, the egg-producing animal is hyperimmunized only with Enterococcus faecalis, i.e., no additional antigen is used for hyperimmunization. In some embodiments, the egg-producing animal is hyperimmunized only with Enterococcus faecalis cytolysin toxin isolated from Enterococcus faecalis. In some embodiments, the egg-producing animal is hyperimmunized only with Enterococcus faecium.

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

[061] Any combination of the antigens described here can be used for Petition 870250024929, dated 03 / 28 / 2025, pp. 75 / 105 20 / 43 hyperimmunize an egg-producing animal. For example, in some embodiments, the egg-producing animal is hyperimmunized with Enterococcus faecium, Enterococcus faecalis, and cytolysin toxin isolated from Enterococcus faecalis. In some embodiments, the egg-producing animal is hyperimmunized with Enterococcus faecium and Enterococcus faecalis. In some embodiments, the egg-producing animal is hyperimmunized with Enterococcus faecium and cytolysin toxin isolated from Enterococcus faecalis. In some modalities, the egg-producing animal is hyperimmunized with Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis. Enterococcus faecalis. Hyperimmunization procedure

[062] The following list of steps is an example of a preferred procedure used to bring an egg-producing animal to a heightened state of immunity from which the resulting hyperimmune egg or egg product can be fed to a bird: 1. Select one or more antigens. 2. To induce an immune response in the egg-producing animal through primary immunization. 3. Administer booster vaccines of one or more antigens at appropriate dosages to induce and maintain a hyperimmune state.

[063] Step 1: The critical point in this step is that the antigens must be capable of inducing immune and hyperimmune states in the egg-producing animal. In some embodiments, the egg-producing animal is immunized with Enterococcus faecalis. In some embodiments, the egg-producing animal is immunized with Enterococcus faecalis cytolysin toxin. In some embodiments, the egg-producing animal is hyperimmunized with Enterococcus faecium. In some embodiments, the egg-producing animal is hyperimmunized with two or more of Enterococcus faecium, Enterococcus faecalis, and Enterococcus cytolysin toxin. Petition 870250024929, dated 03 / 28 / 2025, pp. 76 / 105 21 / 43 faecalis.

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

[065] It can be determined whether the vaccine triggered an immune response in the egg-laying animal through various methods known to those skilled in the art of immunology. Examples of these include enzyme-linked immunosorbent assays (ELISA), tests for the presence of antibodies against stimulating antigens, and tests designed to assess the ability of the host's immune cells to respond to the antigen. The minimum antigen dosage required to induce an immune response depends on the vaccination procedure used, including the type of adjuvants and antigen formulation used, as well as the type of egg-laying animal used as the host.

[066] Step 3: The hyperimmune state is preferably induced and maintained in the target animal through repeated booster administrations of an appropriate dosage at fixed time intervals. The time intervals are preferably 2 to 8 weeks over a period of 6 to 12 months. However, it is essential that the booster administrations do not lead to immunological tolerance. Such processes are well known in the art. Methods of preparing the hyperimmunized egg product are described, for example, in US Patent No. 6,803,035, which is incorporated herein by reference in its entirety.

[067] In a particular embodiment, an antigen (for example, Petition 870250024929, dated 03 / 28 / 2025, page 77 / 105 22 / 43 Enterococcus faecium, Enterococcus faecalis and / or Enterococcus faecalis cytolysin toxin) is formulated into a Freund vaccine. In the first vaccination, the egg-laying animal receives two 0.5 ml doses of each antigen. Two weeks later, a 0.5 ml dose of each antigen is administered to the egg-laying animal as a booster vaccination. An additional booster vaccination is given 4 weeks after the first vaccination. The vaccines can be administered into mammary tissue.

[068] It is possible to use other procedures for maintaining hyperimmunization or a combination of procedures, such as, for example, intramuscular injection for primary immunization and intravenous injection for booster injections. Other procedures include the simultaneous administration of microencapsulated antigen and liquid, or intramuscular injection for primary immunization, and booster doses by oral administration or parenteral administration by means of microencapsulation. Several combinations of primary hyperimmunization are known to those skilled in the art.

[069] In some embodiments, the hyperimmunized egg product comprises antibodies to Enterococcus faecium. In certain embodiments, the hyperimmunized egg product comprises antibodies to Enterococcus faecalis. In some embodiments, the hyperimmunized egg product comprises antibodies to Enterococcus faecalis cytolysin toxin. Antibodies against Enterococcus faecalis cytolysin toxin can bind to and neutralize the toxin. In some embodiments, the hyperimmunized egg product comprises antibodies to Enterococcus faecalis cytolysin toxin and antibodies to at least one additional Enterococcus faecalis antigen other than Enterococcus faecalis cytolysin toxin.

[070] The antibody can be an IgA, IgM, or IgY antibody. In one particular embodiment, the antibody is an IgY antibody. Petition 870250024929, dated 03 / 28 / 2025, page 78 / 105 23 / 43

[071] The hyperimmunized egg or hyperimmunized egg product may contain an increased level of an antibody (e.g., an IgY antibody) specific to a particular antigen described herein relative to a control egg or control egg product obtained from an egg-laying animal that is not hyperimmunized with the particular antigen. For example, in some embodiments, the hyperimmunized egg or hyperimmunized egg product contains an increased level of an antibody that is specific to Enterococcus faecalis relative to a control egg or egg product obtained from an egg-laying animal that has not been hyperimmunized. In some embodiments, the hyperimmunized egg or hyperimmunized egg product contains an increased level of an antibody that is specific to Enterococcus faecalis cytolysin toxin relative to a control egg or egg product obtained from an egg-laying animal that has not been hyperimmunized.In some embodiments, the hyperimmunized egg or hyperimmunized egg product contains an increased level of an antibody that is specific to Enterococcus faecium compared to a control egg or egg product obtained from an egg-producing animal that has not been hyperimmunized.

[072] In some embodiments, the hyperimmunized egg or egg product comprises at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies (e.g., IgY antibody) specific to a particular antigen described herein (e.g., Enterococcus faecium, Enterococcus faecalis or Enterococcus faecalis cytolysin toxin) by weight relative to a control egg or control egg product obtained from an egg-producing animal that is not hyperimmunized with the particular antigen. For example, 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 antibody (e.g., IgY antibody) specific for Enterococcus faecalis compared to a control egg or control egg product obtained from a Petition 870250024929, dated 03 / 28 / 2025, pp. 79 / 105 24 / 43 egg-producing animal that has not been hyperimmunized with Enterococcus faecalis. 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 antibody (e.g., IgY antibody) specific for Enterococcus faecalis cytolysin toxin relative to a control egg or control egg product obtained from an egg-producing animal that has not been hyperimmunized with 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 antibody (e.g., IgY antibody) specific for Enterococcus faecium compared to a control egg or control egg product obtained from an egg-producing animal that has not been hyperimmunized with Enterococcus faecium.

[073] Hyperimmunized eggs or egg products may contain an increased level of two or more antibodies (e.g., IgY antibodies), each of which is specific for a different antigen described herein, relative to a control egg or egg product obtained from an egg-producing animal that is not hyperimmunized. For example, in some embodiments, the hyperimmunized egg or hyperimmunized egg product contains an increased level of one or more antibodies that are specific for Enterococcus faecalis cytolysin toxin and one or more antibodies that are specific for Enterococcus faecalis (e.g., one or more antibodies that are specific for an Enterococcus faecalis antigen other than Enterococcus faecalis cytolysin toxin) relative to a control egg or egg product obtained from an egg-producing animal that has not been hyperimmunized.In some embodiments, the hyperimmunized egg or hyperimmunized egg product contains an increased level of an antibody that is specific to Enterococcus faecalis cytolysin toxin and an increased level of an antibody that is specific to Enterococcus faecium compared to a control egg. Petition 870250024929, dated 03 / 28 / 2025, pages 80 / 105 25 / 43 Egg product obtained from an egg-laying animal that has not been hyperimmunized. In some embodiments, the hyperimmunized egg or hyperimmunized egg product contains an increased level of an antibody that is specific for Enterococcus faecium and an increased level of an antibody that is specific for Enterococcus faecalis compared to a control egg or egg product obtained from an egg-laying animal that has not been hyperimmunized.

[074] For example, 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 faecalis cytolysin toxin relative to a control egg or control egg product obtained from an egg-producing 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 relative to a control egg or control egg product obtained from an egg-producing 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 in relation to a control egg or control egg product obtained from an egg-laying animal that has not been hyperimmunized with cytolysin toxin of Enterococcus faecalis or Enterococcus faecium. In some Petition 870250024929, dated 03 / 28 / 2025, pp. 81 / 105 26 / 43 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, 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 relative to a control egg or product of Control egg obtained from an egg-laying animal that has not been hyperimmunized with Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, or Enterococcus faecium.

[075] Comparisons of antibody titers in hyperimmunized egg products 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 by ELISA at regular intervals. To determine antibody titers, total IgY is extracted from eggs using the Pierce™ Chicken IgY Purification Kit (Thermo Fisher Scientific, Waltham, MA). Briefly, 2 mL of egg are mixed with five volumes of delipidation reagent and the IgY is purified following the manufacturer's instructions. Spray-dried egg powder samples are reconstituted in sterile PBS at 1 mg / mL and filtered through a 0.22 µm membrane filter. Specific antibody titers in isolated IgY samples or egg powder are measured by ELISA.96-well flat-bottom microtiter plates (Corning® Costar®, Corning, NY) are coated with antigens (e.g., an Enterococcus faecalis antigen, cytolysin toxin isolated from Enterococcus faecalis, or an Enterococcus faecium antigen) at 10 pg / mL (100 pL / well) and incubated overnight at 4°C. The plates are washed twice with PBS containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO) and blocked with 100 pL / well of PBS containing 1% Bovine Serum Albumin (BSA) and incubated for 1 h at room temperature. Petition 870250024929, dated 03 / 28 / 2025, pp. 82 / 105 27 / 43 ambient. Serially diluted IgY samples (in PBS with 0.1% BSA) from egg powder samples are added to plates in triplicate wells (100 pL / well) and incubated for 2 h at room temperature with constant shaking. The plates are then washed with PBS-T and treated with peroxidase-conjugated rabbit anti-line IgY (IgG) antibody (1:500; Sigma), incubated for 30 minutes, followed by color development for 10 minutes with 0.01% tetramethylbenzidine substrate (Sigma) in 0.05 M phosphate-citrate buffer, pH 5.0. Bound antibodies are detected by measuring the optical density at 450 nm (0D450) using a microplate reader (Bio-Rad, Hercules, CA). Antibody titers can be expressed by the highest dilution of the egg product that still contains detectable antibodies, as measured by optical density, as described above. For example, an antibody titer of 1,000 would indicate that a dilution of 1.000 times the amount of egg product contains detectable antibodies, but higher dilutions contain no detectable antibodies. In some embodiments, the antibody titer (e.g., antibody titer to Enterococcus faecalis cytolysin toxin, Enterococcus faecalis, and / or Enterococcus faecium) in the hyperimmunized egg product is at least 1,000, at least 2,000, at least 4,000, at least 8,000, at least 16,000, at least 32,000, at least 64,000, at least 100,000, at least 128,000, at least 250,000, at least 500,000, or at least 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 11 million, 12 million, 13 million, 14 million, 15 million, 16 millions, 17 million, 18 million, 19 million or 20 million.In one particular embodiment, the antibody titer (e.g., antibody titer to Enterococcus faecalis cytolysin toxin, Enterococcus faecalis, and / or Enterococcus faecium) in the hyperimmunized egg product is at least 16,000. In another particular embodiment, the antibody titer to Enterococcus faecalis cytolysin toxin and the antibody titer to Enterococcus faecalis in the egg product... Petition 870250024929, dated 03 / 28 / 2025, pp. 83 / 105 28 / 43 hyperimmunized is at least 16,000.

[076] 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 an IgY antibody to a specific antigen (e.g., an Enterococcus faecium antigen, an Enterococcus faecalis antigen, or Enterococcus faecalis cytolysin toxin). Typically, a whole chicken egg weighs approximately 60 grams without the shell, with the egg yolk weighing approximately 20 grams and the egg white weighing approximately 40 grams. In some embodiments, 3 grams of egg yolk contain approximately 2030 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 to one of the antigens used for hyperimmunization (e.g., Enterococcus faecium, Enterococcus faecalis, or cytolysin toxin isolated from Enterococcus faecalis). Compositions and Administration

[077] In certain aspects, the present invention relates to a method for preventing or treating alcoholic liver disease in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-producing animal, thereby preventing or treating alcoholic liver disease in the individual, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to an antigen selected from the group consisting of Enterococcus faecalis and Enterococcus faecalis cytolysin toxin. In some embodiments, the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to Enterococcus faecalis cytolysin toxin and one or more antibodies to Enterococcus faecalis. In Petition 870250024929, dated 03 / 28 / 2025, pp. 84 / 105 29 / 43 In some forms, antibodies to Enterococcus faecalis include antibodies to one or more Enterococcus faecalis antigens that are not Enterococcus faecalis cytolysin toxin.

[078] In certain aspects, the present invention relates to a method for preventing or treating graft-versus-host disease in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a hyperimmunized egg product obtained from an egg-producing animal, thereby preventing or treating graft-versus-host disease in the individual, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, and Enterococcus faecium. Once the egg-producing animals have been sufficiently hyperimmunized, it is preferable that the eggs from these animals be collected and processed to produce a hyperimmunized egg product in an administrable form.Hyperimmunized egg product can be prepared by dehydration, spray drying or freeze-drying of whole egg, yolk or purified IgY fraction. The dried hyperimmunized egg product can be mixed with an agent such as silicon or silicon derivatives that improve flow properties. The dried hyperimmunized egg product may include a desiccant. The hyperimmunized egg product can be stored at room temperature or refrigerated, for example, at 4°C.

[079] In some embodiments, administration of the hyperimmunized egg product to the individual reduces the level of Enterococcus faecalis in the individual, for example, relative to the level of Enterococcus faecalis in the individual before administration of the hyperimmunized egg product, or relative to an individual who was not administered the hyperimmunized egg product. In some embodiments, administration of the hyperimmunized egg product to the individual reduces liver damage. Petition 870250024929, dated 03 / 28 / 2025, pages 85 / 105 30 / 43 in the individual (for example, reduces serum alanine aminotransferase (ALT) levels), for example, relative to the level of liver damage before administration of the hyperimmunized egg product, or relative to an individual who was not administered the hyperimmunized egg product.

[080] In some embodiments, the hyperimmunized egg product is encapsulated. Methods of encapsulating antibodies and other proteins are known in the art and are described, for example, in US Patent No. 7,105,158. Materials that are biodegradable and non-antigenic can be used as the encapsulating material. Encapsulating materials include, but are not limited to, albumin, PLGA, globulin, natural and synthetic polymers, and thermoplastic polymers. Any polymer that is biocompatible and bioerodible can be used for encapsulation. Various available crosslinking agents, such as glutaraldehyde, can be used to crosslink the encapsulating material. In addition, the pharmaceutically administered material may contain encapsulated drug microspheres, wherein the microspheres have different concentrations of crosslinking agent used, thus creating a prolonged continuous release of the antibody.

[081] In some embodiments, the hyperimmunized egg product is in the form of a microparticle or nanoparticle, for example, an encapsulated microparticle or encapsulated nanoparticle. Microparticles and nanoparticles can have any shape. Typically, microparticles and nanoparticles are spherical. Other suitable shapes include, but are not limited to, flakes, triangles, ovals, rods, polygons, needles, tubes, cubes, and cuboid structures. In certain embodiments, the microparticles have a diameter smaller 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 micron. Any of these values ​​can be used to define a range for the microparticle diameter. For example, the diameter of the microparticle can be from about 0.1 to about 10 microns, from about 0.1 to about 1 micron, or from about 0.1 to about 2 microns. In Petition 870250024929, dated 03 / 28 / 2025, pp. 86 / 105 31 / 43 Other embodiments, microparticles, or larger particles can be used. For example, microparticles can have a diameter ranging from 10 microns to 1,000 microns. In certain embodiments, nanoparticles have diameters less than 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, or 10 nm. Any of these values ​​can be used to define a range for the nanoparticle diameter. For example, the nanoparticle diameter can be from about 10 to about 1,000 nm, from about 100 to about 1,000 nm, or from about 10 to about 100 nm.

[082] There are several processes by which microparticles or nanoparticles can be encapsulated, including, for example, multi-wall microencapsulation, hot melt encapsulation, phase separation encapsulation, spontaneous emulsion, solvent evaporation microencapsulation, solvent removal microencapsulation, and coacervation. These methods are known in the art. Detailed descriptions of the methods are 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 are hereby incorporated by reference in their entirety.

[083] In some embodiments, the IgY antibody specific for an antigen described herein (e.g., Enterococcus faecium, Enterococcus faecalis, or Enterococcus faecalis cytolysin toxin) is administered to the individual in a concentrated form. For example, in some embodiments, the IgY antibody is purified and concentrated before administration to the individual. Methods for purifying and concentrating IgY antibodies from egg products are known in the art and are described, for example, in U.S. Patent No. 5,367,054, which is incorporated herein by reference in its entirety. In some embodiments, the hyperimmunized egg product comprises or consists of purified or partially purified IgY antibody for Enterococcus faecalis cytolysin toxin. In some embodiments, the hyperimmunized egg product comprises or consists of antibody Petition 870250024929, dated 03 / 28 / 2025, pp. 87 / 105 32 / 43 Purified or partially purified IgY antibody for Enterococcus faecalis. In some embodiments, the hyperimmunized egg product comprises or consists of purified or partially purified IgY antibody for Enterococcus faecium. In some embodiments, the hyperimmunized egg product comprises or consists of purified or partially purified IgY antibody for Enterococcus faecalis and purified or partially purified IgY antibody for Enterococcus faecalis cytolysin toxin.

[084] In some embodiments, the hyperimmunized egg products described herein are used to treat alcoholic liver disease in an individual who has already developed the disease. For example, in some embodiments, the individual has alcoholic liver disease at the time of administration of the hyperimmunized egg product. An increased proportion of Enterococcus spp. in patients with alcoholic liver disease (e.g., alcoholic hepatitis) compared to patients without an alcohol use disorder has been observed. For example, in patients with alcoholic hepatitis, 5.59% of fecal bacteria were Enterococcus spp. compared with almost none (0.023%) in control individuals without an 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 the fecal bacteria in the individual are Enterococcus spp. at the time of administration of the hyperimmunized egg product.It has been shown that fecal samples from patients with alcoholic hepatitis contain approximately 2,700 times more E. faecalis than samples from control individuals without an alcohol use disorder, as measured by quantitative PCR (qPCR), and about 80% of patients with alcoholic hepatitis are positive for E. faecalis in their stool. See Duan et al., cited above. Thus, in some forms, the individual has at least 100 times, 500 times, 1,000 times, 1,500 times, 2,000 times, or 2,500 times more E. faecalis in a fecal sample compared to an individual without alcoholic liver disease. Petition 870250024929, dated 03 / 28 / 2025, pages 88 / 105 33 / 43

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

[086] In some embodiments, the hyperimmunized egg products described herein are used to treat GVHD in an individual who has already developed the disease. For example, in some embodiments, the individual has GVHD at the time of administration of the hyperimmunized egg product. As discussed above, an increase in fecal levels of Enterococcus spp. (e.g., E. faecium) in patients with GVHD has been observed. See Garrett, cited above. Thus, in some embodiments, the individual with GVHD has increased fecal levels of one or more Enterococcus spp. (e.g., E. faecalis or E. faecium) compared to a healthy individual (e.g., an individual who does not have GVHD) at the time of administration of the hyperimmunized egg product. In some embodiments, the individual with GVHD has received an organ transplant, a stem cell transplant, a bone marrow transplant, or an allogeneic hematopoietic stem cell transplant. In some cases, the individual has acute GVHD.In some conditions, the individual has chronic GVHD.

[087] In some embodiments, the hyperimmunized egg products described herein are used to prevent the development of GVHD in an individual. For example, in some embodiments, the individual does not have GVHD at the time of administration of the hyperimmunized egg product. In some embodiments, the Petition 870250024929, dated 03 / 28 / 2025, pp. 89 / 105 34 / 43 individuals received a transplant (e.g., an organ transplant, a stem cell transplant, a bone marrow transplant, or an allogeneic hematopoietic cell transplant) but have not yet developed GVHD. In one particular embodiment, the individual to whom the hyperimmunized egg product is administered is a human being.

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

[089] Any of several known pharmaceutical vehicles may be used in the preparation of an injectable or otherwise administrable preparation, including phosphate-buffered saline, saline solution, ethanol, propylene glycol and the like. In some embodiments, the hyperimmunized egg product is administered via drinking water.

[090] In certain embodiments, the hyperimmunized egg product is administered as a composition comprising one or more additional compounds, for example, a nutrient or probiotic. For example, in one embodiment, the hyperimmunized egg product of the invention is integrated into a dietary supplement. One method for preparing the egg of the invention for incorporation into a dietary supplement involves drying the egg into an egg powder. Although several methods for drying eggs are known, spray drying is the preferred method. The spray drying process of eggs Petition 870250024929, dated 03 / 28 / 2025, pp. 90 / 105 35 / 43 is well known in the art. In some embodiments, the composition is an aqueous solution comprising the hyperimmunized egg product.

[091] In certain embodiments, whole eggs are divided into separate fractions, such as egg yolks and egg whites. For example, it is generally known in the art that the IgY antibody is found in egg yolks. In this way, those skilled in the art would clearly recognize that separating egg yolks could provide more potent fractions or eliminate undesirable components, and would allow other modes of administration, such as administration of hyperimmunized egg product via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, oral, or topical routes. This further separation will provide the ability to produce encapsulated products and compositions comprising said egg or fraction thereof.

[092] The hyperimmune egg product is preferably administered to the individual in an amount that is immunologically effective in the treatment or prevention of alcoholic liver disease or GVHD. The dosage and duration of administration will depend on the individual's particular condition. In some embodiments, the hyperimmunized egg product is administered to the individual 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 individual 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 for the number of times the hyperimmunized egg product may be administered to the individual per day. For example, in some modalities, the hyperimmunized egg product is administered to the individual 1-2 times a day, 1-3 times a day, or 1-4 times a day.In some modalities, the hyperimmunized egg product is administered to the individual at least twice a day. In some modalities, the hyperimmunized egg product is administered to the individual at least once a day. In some modalities, the egg product... Petition 870250024929, dated 03 / 28 / 2025, pp. 91 / 105 In some embodiments, the hyperimmunized egg product is administered to the individual once every two days. In some embodiments, the hyperimmunized egg product is administered to the individual once every three days. In some embodiments, the hyperimmunized egg product is administered to the individual once a week. In one particular embodiment, the hyperimmunized egg product is administered to the individual once a day for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive days.

[093] In some embodiments, daily amounts ranging from less than one to several whole hyperimmune eggs (or hyperimmune egg product containing the equivalent of less than one to several whole hyperimmune eggs) may be administered to the individual, depending on the particular circumstance of the condition. More potent fractions may be separated and concentrated by methods well known in the art, from several hundred eggs. In certain embodiments, the effective amount of hyperimmunized egg product administered to an individual (e.g., a human being) 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 modalities, 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 per day of whole egg are administered to the individual.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 per day of egg yolk are administered to the individual. 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 per day of dehydrated egg yolk or dehydrated whole egg are administered to the individual. Any of these values ​​can be used to define a range for the effective amount of hyperimmunized egg product administered to the mammal. For example, in some modalities, the 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 one particular modality, 3 grams of... Petition 870250024929, dated 03 / 28 / 2025, pp. 92-105 37 / 43 egg yolks are administered to the individual (e.g., a human being) per day.

[094] 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 ​​may be used to define a range for the concentration of hyperimmunized egg product in the composition. For example, in some forms, the composition comprises between 0.01% and 50%, between 0.1% and 50%, or between 1% and 50% w / w of hyperimmunized egg product. EXAMPLES Example 1. Preparation and evaluation of anti-Enterococcus faecalis and anti-cytolysin IgY antibodies.

[095] The present study was designed to evaluate the efficacy of hyperimmune eggs containing IgY antibodies directed against E. faecalis cytolysin toxin and E. faecalis when administered orally to a humanized mouse ALD model colonized with bacteria from the feces of patients with alcoholic hepatitis. A scheme of the experiment is provided in Figure 1. Methods Preparation of hyperimmune egg powder

[096] Two (2) mL of primary Enterococcus faecalis culture were started from glycerol stocks. A large batch culture was developed by inoculating with the 0.2% primary culture in a 500 mL culture flask and growing overnight at 37°C with shaking at 180 rpm. The bacteria were pelleted by centrifugation at 6,000 rpm at 4°C. The pellet was washed once with ice-cold PBS. The pellet was resuspended in ice-cold PBS, transferred to a glass tube, and sonicated for 10 cycles at the highest setting for 30 minutes per cycle. The resulting lysate was centrifuged at 10,000 rpm at 4°C for 15 minutes and the Petition 870250024929, dated 03 / 28 / 2025, pp. 93 / 105 38 / 43 of the supernatant was collected. Total protein was quantified using the BCA protein assay. Recombinant cytolysin preparation

[097] Pure recombinant cytolysin toxin from E. faecalis was prepared by Genscript USA, Piscataway, NJ. Hyperimmunization Protocol

[098] A mixture of E. faecalis bacterial lysate and E. faecalis cytolysin toxin diluted to 2 mg / mL and 1 mg / mL in PBS, respectively, was used as the 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 at 0, 4, 7, 14, and 28 days, and eggs were collected. Antibody Extraction for Titer Evaluation and Neutralization

[099] IgY antibody was extracted from hyperimmunized eggs using a water dilution method. 100 pL of yolk were diluted in 10 ml of water, well mixed, and centrifuged to pelletize the lipids while the antibodies and other soluble egg proteins were extracted into the aqueous phase. The aqueous phase was used to assess titer determination by ELISA as described below.

[0100] For neutralization assays, IgY was extracted from hyperimmunized egg yolks by dilution in water and precipitation with NaCl. 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 yolk was thawed at 37°C and centrifuged at 6,000 rpm for 20 minutes to separate the lipids. The supernatant was filtered and mixed with 8.8% NaCl at pH 4 and allowed to precipitate 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 values ​​(absorption at 280 nm) using a NanoDrop™ One Microvolume UV-Vis spectrophotometer (Thermo Scientific). The purity of IgY was analyzed using an SDS-PAGE gel. Petition 870250024929, dated 03 / 28 / 2025, pp. 94-105 39 / 43 Detection of antibody specificity and titer by ELISA.

[0101] Antibody specificity and titer were determined by ELISA. Briefly, to determine the anti-E. faecalis antibody titer, a 96-well plate was coated with 1 mg / mL of E. faecalis lysate in carbonate-bicarbonate buffer (pH 9.3) and incubated overnight at 4°C. The plate was washed once with PBS and blocked with 1% BSA in PBS for 1 ha at 37°C. The plate was washed once with PBS containing 0.05% by weight Tween-20 (PBST) and treated with serially diluted primary anti-E. faecalis antibody on the plate for 1 ha at 37°C. The plate was washed and treated with secondary HRP anti-goat line antibody. TMB substrate was added to develop the signal and the reaction was stopped by HCl. The plate was read at 450 nm in a plate reader. To determine the anti-cytolysin antibody titer, another 96-well plate was coated with 0.5 mg / ml of cytolysin and a similar experiment was performed using anti-cytolysin antibodies. Test for IgY neutralization skills

[0102] Enterococcus faecalis was cultured overnight in BHI broth to an OD600 of 1.5 and diluted 1:50 in the broth and incubated to an ODgqq of 0.3. The culture was further diluted to 103 cfu / ml in BHI broth and treated with antibodies at 15 mg / ml. After 24 hours of incubation at 37°C, the cultures were serially diluted 104 times and plated on sterile plates and incubated at 37°C for 12 hours. Cfu / mL was quantified by counting the number of colonies using the ImageJ Count particle function. The percentage of inhibition of bacterial colony formation was calculated. Results

[0103] Total soluble proteins including IgY from hyperimmunized chicken egg yolks were extracted by dilution in water and the NaCl method. The purity of IgY was analyzed by SDS-PAGE electrophoresis under reducing conditions. IgY has a molecular weight of 180 kDa and is composed of two subunits, a heavy chain Petition 870250024929, dated 03 / 28 / 2025, pp. 95 / 105 40 / 43 of 67 kDa and a light chain of 23 kDa. The electrophoretic pattern of IgY extracted from hyperimmunized egg yolks was similar to the IgY pattern. See Figure 2. Target-Specific Antibody Titer Assessment

[0104] The specific activity of IgY antibody against E. faecalis and cytolysin antigens was measured by indirect ELISA. ELISA results indicated positive binding of IgY to specific antigens when compared to control IgY with a high titer of 1:32,000. See Figures 3A and 3B. Neutralization and Target Assessment

[0105] The inhibitory effects of IgY antibody growth on E. faecalis were investigated. Whole E. faecalis bacterial cells were incubated with 15 mg / ml IgY, and cfu analyses were performed after 24 hours of incubation. Treatment with 15 mg / ml IgY inhibited E. faecalis growth by >70%, as visualized by the reduction in the number of colonies on the agar plate. See Figure 4.

[0106] The inhibitory effect of IgY growth on E. faecalis was quantified, and the percentage of inhibition was plotted. See Figure 5. Treatment of E. faecalis with anti-E. faecalis IgY antibody + cytolysin at a concentration of 15 mg / ml resulted in 75% inhibition of E. faecalis growth, indicating successful generation of neutralizing antibodies for E. faecalis. See Figure 5. Example 2. Effect of oral feeding of anti-Enterococcus faecalis and anti-cytolysin IgY antibodies in a mouse model of alcoholic liver disease. Methods Preparation of powdered eggs for preclinical studies.

[0107] Egg whites and yolks were homogenized and lyophilized to create an 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 the humanized model of alcoholic liver disease (ALD) described Petition 870250024929, dated 03 / 28 / 2025, pp. 96 / 105 41 / 43 below.

[0108] Stool samples from human patients positive for cytolysin with alcoholic hepatitis were used for fecal transplantation in germ-free mice. Mice were fed 100 μL of stool samples (1 g of feces dissolved in 30 ml of Luria-Bertani (LB) medium containing 15% glycerol under anaerobic conditions), starting at 5–6 weeks of age and repeated two weeks later. Two weeks after the second gavage, mice were placed on either an ethanol or control (isocaloric) diet, as described by Duan et al., 2019, Nature 575: 505–511. Periodically, mice were assessed for serum ALT, liver pathology, and other parameters such as weight gain, liver weight, and food intake. Alcoholic liver disease (ALD) model

[0109] Male and female germ-free C57BL / 6 mice were bred and fecal transplantation with stool samples from a cytolysin-positive human patient with alcoholic hepatitis was performed at 5–6 weeks of age and repeated 2 weeks later, as described by Duan et al., 2019, Nature 575: 505511. Mice were fed a Lieber-DeCarli diet, and caloric intake from ethanol was 0% on days 1–5 and 36% from day 6 until the end of the study period. On day 16, mice were fed a single dose of ethanol (5 g / kg body weight) early in the morning and sacrificed 9 hours later. Control mice fed in pairs received a diet with an isocaloric dextrose replacement.

[0110] Egg powder containing control IgY (15 mg / day) from eggs of non-immunized laying hens, or anti-E. faecalis + anticytolysin IgY (1, 5 or 15 mg per day) was added to the liquid diet (containing fat, carbohydrates, protein and isocaloric ethanol / dextrose) at the start of alcohol administration. See Table 1 below for the weight of the dry egg powder. The diets were changed every three days. Petition 870250024929, dated 03 / 28 / 2025, pp. 97 / 105 42 / 43 The mice were randomly assigned to groups at the start of the study. There was no mortality in any of the groups during the ethanol feeding period. Table 1. IgY doses for the mouse ALD model. Treatment group IgY dose (mg) Whole egg powder dose (g) Anti-E. faecalis + anticytolysin IgY 1 0.18 Anti-E. faecalis + anticytolysin IgY 5 0.88 Anti-E. faecalis + anticytolysin IgY 15 2.63 control IgY 15 2.24 Biochemical Analyses

[0111] Serum alanine aminotransferase (ALT; a test for liver damage) levels were measured using an Infinity ALT kit (Thermo Scientific). Liver triglyceride levels were measured using the Liquid Triglyceride Reagent Kit (Pointe Scientific). Coloring Procedures

[0112] Formalin-fixed tissue samples were embedded in paraffin (Paraplast plus, McCornick) and stained with hematoxylin-eosin (Surgipath). Statistical Analyses

[0113] Results are expressed as mean ± sem. The numbers for biological replicates were n = 5 in each ethanol-containing diet group. Significance was assessed using one-way analysis of variance (ANOVA) with Tukey's post-hoc test. A P-value < 0.05 was considered statistically significant. Statistical analyses were performed using the statistical software R, version R 1.3.1093, 2020, R Foundation for Statistical Computing and GraphPad Prism v8.4.3. Results

[0114] To determine the effect of anti-E. faecalis + anticytolysin IgY in a mouse model of ethanol-induced liver disease, germ-free C57BL / 6 mice were colonized with feces from a patient positive for Petition 870250024929, dated 03 / 28 / 2025, pages 98 / 105 43 / 43 cytolysin with alcoholic hepatitis and subjected to the ethanol feeding model with chronic binge eating. 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 lower body weight in mice treated with anti-E. faecalis + anticytolysin IgY, which is related to lower body weight at baseline. Mice receiving 5 mg or 15 mg of anti-E. faecalis + anticytolysin IgY showed significantly less liver damage, as indicated by a decreased serum alanine aminotransferase (ALT) level compared to mice receiving control IgY after chronic ethanol feeding (see Figure 6D). No significant differences were observed in liver triglycerides, although there was a trend toward less hepatic steatosis in mice treated with anti-E. faecalis + anticytolysin IgY (see Figure 6E).Histological analyses showed that mice treated with anti-E. faecalis + anti-cytolysin IgY exhibited significantly less ethanol-induced liver damage and hepatic steatosis (see Figure 7). Petition 870250024929, dated 03 / 28 / 2025, pp. 99 / 105

Claims

1 / 5 CLAIMS 1. Use of a hyperimmunized egg product obtained from an egg-producing animal, CHARACTERIZED in that it is for the preparation of a medicament for the prevention or treatment of alcoholic liver disease in an individual in need thereof, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to an antigen selected from the group consisting of Enterococcus faecalis and Enterococcus faecalis cytolysin toxin, wherein the hyperimmunized egg product comprises at least 0.0001% by weight of IgY antibody to the antigen, and wherein the hyperimmunized egg product is prepared by administering at least 4 immunizations of the antigen to the egg-producing animal at intervals of less than 30 days.

2. Use of a hyperimmunized egg product obtained from an egg-laying animal, CHARACTERIZED in that it is for the preparation of a medicament for the prevention or treatment of graft-versus-host disease in an individual in need thereof, wherein the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to an antigen selected from the group consisting of Enterococcus faecalis, Enterococcus faecalis cytolysin toxin, and Enterococcus faecium, wherein the hyperimmunized egg product comprises at least 0.0001% by weight of IgY antibody to the antigen, and wherein the hyperimmunized egg product is prepared by administering at least 4 immunizations of the antigen to the egg-laying animal at intervals of less than 30 days.

3. Use, according to claim 1 or 2, CHARACTERIZED in that the hyperimmunized egg product comprises a therapeutically effective amount of one or more antibodies to Enterococcus faecalis, and a therapeutically effective amount of one or more antibodies to cytolysin toxin of Enterococcus faecalis.

4. Use, according to any one of claims 1 to 3, CHARACTERIZED in that the hyperimmunized egg product comprises at least 20% more by weight of an IgY antibody specific to 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-producing animal that has not been hyperimmunized.

5. Use, according to any one of claims 1 to 4, CHARACTERIZED in that the hyperimmunized egg product is formulated to be administered to the individual 1 to 4 times per day.

6. Use, according to any one of claims 1 to 5, CHARACTERIZED by the fact that the hyperimmunized egg product is formulated to be administered orally.

7. Use, according to any of claims 1 to 6, CHARACTERIZED in that administration of the hyperimmunized egg product to the individual reduces the level of Enterococcus faecalis in the individual relative to an individual who was not administered the hyperimmunized egg product.

8. Use, according to any of claims 1 and 3 to 7, CHARACTERIZED by the fact that administration of the hyperimmunized egg product to the individual reduces liver injury in the individual compared to an individual who was not administered the hyperimmunized egg product.

9. Use, according to any of claims 1 to 8, CHARACTERIZED by the fact that the individual is a human being.

10. Hyperimmunized egg CHARACTERIZED by the fact that it is produced by an animal that has been hyperimmunized with an antigen selected from the group consisting of Enterococcus faecalis, cytolysin toxin isolated from Enterococcus faecalis, and Enterococcus faecium, in which the level of antibodies to the antigen in Petition 870250112386, dated 08 / 12 / 2025, page. 15 / 23 3 / 5 hyperimmunized egg is increased relative to an egg from an animal that has not been hyperimmunized, and wherein the antibody titer to Enterococcus faecalis, Enterococcus faecalis cytolysin toxin or Enterococcus faecium in the hyperimmunized egg product is at least 8,000, wherein the hyperimmunized egg product comprises at least 0.0001% by weight of IgY antibody to the antigen, and wherein the hyperimmunized egg is prepared by administering at least 4 immunizations of the antigen to the egg-producing animal at intervals of less than 30 days.

11. Hyperimmunized egg, according to claim 10, CHARACTERIZED in that the animal has been hyperimmunized with Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis, and in that the level of antibodies to Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis is increased relative to an egg from an animal that has not been hyperimmunized.

12. Hyperimmunized egg product CHARACTERIZED in that it is obtained from hyperimmunized eggs as defined in claim 10 or 11.

13. Hyperimmunized egg product, according to claim 12, CHARACTERIZED in that the hyperimmunized egg product is a whole egg.

14. Hyperimmunized egg product, according to claim 12, CHARACTERIZED in that the hyperimmunized egg product is egg yolk.

15. Hyperimmunized egg product, according to claim 12, CHARACTERIZED in that the hyperimmunized egg product is a purified or partially purified IgY antibody to Enterococcus faecalis cytolysin toxin.

16. Hyperimmunized egg product, according to claim 12, CHARACTERIZED in that the hyperimmunized egg product is a purified or partially purified IgY antibody to Enterococcus faecalis.

17. Hyperimmunized egg product, according to claim 12, CHARACTERIZED in that the hyperimmunized egg product consists of Petition 870250112386, dated 08 / 12 / 2025, page 16 / 23 4 / 5 purified or partially purified IgY antibody to Enterococcus faecalis and purified or partially purified IgY antibody to Enterococcus faecalis cytolysin toxin.

18. Pharmaceutical composition CHARACTERIZED in that it comprises the hyperimmunized egg product, as defined in any one of claims 12 to 17, and a pharmaceutically acceptable vehicle, wherein the pharmaceutical composition comprises 0.01% to 99% w / w of the hyperimmunized egg product.

19. Pharmaceutical composition, according to claim 18, CHARACTERIZED in that it is formulated for oral administration.

20. Pharmaceutical composition, according to claim 19, CHARACTERIZED in that the hyperimmunized egg product is formulated in nanoparticles or in an emulsion.

21. Method for preparing a hyperimmunized egg product, CHARACTERIZED in that it comprises: i) hyperimmunizing an egg-producing animal with an antigen selected from the group consisting of Enterococcus faecalis, cytolysin toxin isolated from Enterococcus faecalis, and Enterococcus faecium, wherein the hyperimmunization comprises administering at least 4 immunizations of the antigen to the egg-producing animal at intervals of less than 30 days; and ii) preparing a hyperimmunized egg product from one or more eggs produced by the animal, wherein the hyperimmunized egg product comprises at least 0.0001% by weight of IgY antibody to the antigen.

22. Method according to claim 21, CHARACTERIZED in that the antigen is selected from the group consisting of cytolysin toxin isolated from Enterococcus faecalis and Enterococcus faecium. Petition 870250112386, dated 08 / 12 / 2025, page 17 / 23 5 / 5 23. Method according to claim 21, CHARACTERIZED in that the antigen is selected from the group consisting of Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis.

24. Method according to claim 21, CHARACTERIZED in that the antigen comprises Enterococcus faecalis and cytolysin toxin isolated from Enterococcus faecalis.

25. Method, according to any one of claims 21 to 24, CHARACTERIZED in that the egg-producing animal is a hen. Petition 870250112386, dated 08 / 12 / 2025, pp. 18 / 23

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