β-Glucan Particles for Trained Immunity

Abstract

An effective amount of (1→3)-β-D-glucan particles derived from Saccharomyces cerevisiae for use in a method for programming hematopoietic stem and progenitor cells (HSPCs) to promote the proliferation of multipotent progenitor cells (MPPs) that are favorably inclined towards the bone marrow hematopoiesis of interest.

Description

Technical Field

[0001] The present invention relates to whole β-glucan particles for use in inducing trained immunity in a subject. More specifically, the present invention relates to the use of whole β-glucan particles for programming hematopoietic stem and progenitor cells (HSPCs) to effect the proliferation of multipotent progenitor cells (MPPs) that are advantageous for bone marrow hematopoiesis.

Background Art

[0002] In vertebrates, the function of the immune system depends on two major pillars, the innate immune system and the adaptive immune system, in fighting infections and pathogens. The innate immune system is the first line of defense against pathogens, while the adaptive immune system provides a coordinated and specific response. The adaptive immune system has long been known to exhibit an immunological memory function. For example, the adaptive immune system is most sought after in the vaccination process.

[0003] Until 2011, patrol immune cells belonging to the innate immune system, such as monocytes and macrophages, were thought to lack immunological memory. However, since then, several studies have revealed that innate immune cells exposed to pathogens and danger signals can exhibit characteristics of immunological memory upon subsequent reinfection. This phenomenon is called "trained immunity". Since patrol innate immune cells provide the body's first immune response against invading organisms, targeting and "priming" these innate immune cells can significantly enhance the body's ability to enhance immune function and resistance to infection.

[0004] Exposure to microbial stimuli has been shown to metabolically and epigenetically change cells. Innate immune genes (pro-inflammatory cytokines, chemokines) are epigenetically primed at the chromatin level, leading to changes (often, enhancement) in responses and accelerating kinetics upon maturation and rechallenge (Netea, M.G. et al., Defining trained immunity and its role in health and disease. Nat Rev Immuno, 2020).

[0005] Trained immunity can be effectively induced by prior exposure to β-glucan derived from the cell walls of pathogenic yeasts such as Candida albicans, and has previously been shown to be mechanistically induced by long-term epigenetic and metabolic reprogramming in blood-derived monocytes. Recent findings have established that trained immunity acts at the whole-organism level, targeting the hematopoietic stem and progenitor cell (HSPC) compartment of the bone marrow, thereby conferring sustained protection against various pathogens.

[0006] Much of this research has been demonstrated using fungal β-glucan derived from C. albicans (Quintin, J., et al Candida albicans infection affords protection against reinfection via functional reprogramming of monocytes. βCell Host Microbe, 12 223-232, 2012; Cheng, S.C. et al. mTOR and HI-1 alpha-mediated aerobic glycolysis as metabolic basis for trained immunity. Science 345,(2014); Garcia-Valtanen, P., et al., Evaluation of trained immunity by beta-1, 3(d)-glucan on murine monocytes in vitro and duration of response in vivo. Immunol Cell Biol 95, 601-610,(2017)). More recent studies demonstrating in vivo training by bone marrow HSPC reprogramming and bone marrow hematopoiesis have been demonstrated after intraperitoneal (IP) injection of β-glucan from the macrofungus (mushroom) Trametes versicolor or C. albicans (C.albicans-glucan) (Moorlag, S. et al. beta-Glucan Induces Protective Trained Immunity against Mycobacterium tuberculosis Infection: A Key Role for IL-1. βCell Rep 31,(2020); Mitroulis, I. et al. Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity. Cell 172, 147-161(2018).

[0007] The anti-tuberculosis vaccine Bacillus Calmette-Guerin (BCG) has also been demonstrated to induce natural training. Mice injected intravenously or subcutaneously with BCG show an increase in the number and proportion of Lineage - c-Kit + Sca-1 + (LKS+) cells, as well as an increase in the proliferation of progenitor cells biased towards lymphoid cells, namely MPP4, in exchange for a decrease in the proliferation of progenitor cells biased towards myeloid cells, called MPP3 (Kaufmann, E. et al. BCG Educates Hematopoietic Stem Cells to Generate Protective Innate Immunity against Tuberculosis. Cell 172, 176-190(2018)(Kaufmann et al., 2018)). Similarly, intraperitoneal injection of β-glucan peptide derived from Trametes versicolor has been shown to induce a significant increase in hematopoietic ability in LKS+ cells and MPP3 (Mitroulis et al.,2018).

[0008] β-Glucan is a heterogeneous family of structural carbohydrates with many biological activities (Camilli, G., Taboure, G. & Quintin, J. The Complexity of Fugal beta-Glucan in Health and Disease: Effects on the Mononuclear Phagocyte System. Front Immunol 9, 673, (2018)). More common yeast β-glucans, especially food-grade baking and brewing yeasts, contain more branched β-glucans with a larger molecular weight (MW) that are said to have various health effects. Wellmune is a common and well-tolerated nutraceutical, beverage, and supplement ingredient derived from the cell wall of baker's yeast (Saccharomyces cerevisiae). This is a whole glucan particle (WGP) with a backbone of glucose molecules linked via unique chemical bonds (β1,3 chains with glucose chains linked via β1,6 bonds). In clinical trials, its ability to enhance human immune function and its protective effect against various respiratory infections have been demonstrated.

[0009] To date, conventional strategies for improving immune function and resistance to infections have relied on vaccination, which targets the so-called adaptive immune system and promotes immune memory through the stimulation of disease-specific adaptive T and B cells. Therefore, there is great interest in showing how common nutraceuticals derived from yeast can enhance immunity against various sources of infection, including bacteria and viruses.

[0010] However, prior to the present invention, such an analysis of trained immunity using Wellmune has not been performed. Specifically, oral administration of Wellmune, or any other β-glucan variant, to animal models to reconstitute the innate immune memory function in mature innate immune cells has never been carried out nor published.

[0011] Elna De Marco et al (Mol Nutr Food Res, 2021, 65) discuss the general state of the art related to glucans and immunity. Immune training in the publication is used as a general term and there is no disclosure regarding the training of innate immunity or the programming of hematopoietic stem and progenitor cells in the bone marrow.

[0012] The present invention is for solving such problems of the prior art.

Summary of the Invention

[0013] The inventors have found that oral administration of whole β-glucan particles derived from Saccharomyces cerevisiae (baker's yeast) (referred to herein as "β-glucan of the present invention"), specifically (1→3)-β-D glucan, when administered orally by a dietary supplement, induces changes in myeloid progenitor cells, i.e., hematopoietic stem and progenitor cells (HSPC) in the bone marrow, leads to the proliferation of the progenitor cell population at the bone marrow level, and improves the function of mature immune cells derived from these progenitor cells for the first time. The changes include skewing of myeloid progenitor cells, i.e., myeloid progenitor cells preferentially inclined to MMP3. Bone marrow cells include, among others, neutrophils, monocytes, and macrophages, all of which together constitute an important arm of the immune system responsible for innate immunity.

[0014] Without being bound by theory, oral administration of the β-glucan of the present invention is thought to induce immunometabolic changes in myeloid progenitor cells and thus increase the level of inflammatory cytokine production. This inflammation-promoting microenvironment is thought to promote the proliferation of multipotent progenitor cells (MPP) preferentially inclined to bone marrow hematopoiesis. These innate immune cells (e.g., monocytes or neutrophils) are trained, or "primed," to differentiate into mature innate immune cells (e.g., macrophages) with trained immune characteristics. Therefore, it is established that long-term trained immunity can be promoted through reprogramming of hematopoietic progenitor cells by administration.

[0015] In this field, such an effect after oral administration is surprising since oral administration was thought not to induce such changes. In fact, it was expected that the intestinal assimilation mechanism would either degrade the active ingredient of the β-glucan of the present invention, induce major structural changes in the molecule, or simply break the molecule into fragments. These results suggest the ability of WGP particles to interact with the mucosal immune system and induce central innate immune memory.

[0016] The inventors compared the β-glucan of the present invention with other β-glucans and defined the structural and signaling requirements for training monocytes in mice both in vitro and in vivo. The inventors' research shows that the β-glucan of the present invention can induce a training effect on human innate immune cells such as monocytes at the bone marrow level not only in the laboratory but also in injected mice. The inventors also showed that the β-glucan of the present invention is more efficient than other β-glucans in inducing trained immune properties in human macrophages derived from in vitro-trained monocytes (Figs. 1-5). Furthermore, in the soluble and non-particulate form of β-glucan, the same effect was not observed.

[0017] The inventors also showed that intraperitoneal (IP) injection of the β-glucan of the present invention in mice induces stronger bone marrow changes than other β-glucan variants. The inventors hypothesized that oral administration of the β-glucan of the present invention would induce stronger hematopoietic skewing than other β-glucan variants.

[0018] The inventors further showed that feeding a yeast-derived WGP-containing diet for 4 weeks results in the proliferation of progenitor cells (MPP3) committed to the bone marrow immune system, and as a result, an increase in progenitor cells (CMP, GMP) committed to the more mature myeloid lineage. Consistent with these bone marrow changes, an increase in responsiveness to activation was observed in mature macrophages derived from these bone marrow-derived macrophages (Fig. 16).

[0019] The present invention provides an effective amount of Saccharomyces cerevisiae-derived whole β-glucan particles, namely "β-glucan of the present invention", for use in a method of priming hematopoietic stem and progenitor cells (HSPCs) of a subject. The HSPCs in the subject promote the proliferation of multipotent progenitor cells (MPPs) that are favorably inclined towards bone marrow hematopoiesis.

[0020] One aspect of the present invention provides an effective amount of Saccharomyces cerevisiae-derived β-glucan particles for use in a method for inducing trained innate immunity in a subject. Thus, glucan is used to bring about an enhanced immune response against invading pathogens. The glucan is whole glucan particles.

[0021] One aspect of the present invention provides an effective amount of Saccharomyces cerevisiae-derived β-glucan particles for use in a method for generating trained innate immune cells in a subject. The trained innate immune cells can be mature innate immune cells such as monocytes (plural) and / or macrophages. This glucan is whole glucan particles.

[0022] In one embodiment of any aspect of the present invention, the effective amount of yeast-derived β-glucan particles can be formulated for oral administration or intraperitoneal injection (IP), preferably for oral administration.

[0023] The β-glucan particles of the present invention are (1→3)-β-D-glucan derived from Saccharomyces cerevisiae. This may be derived from any strain of Saccharomyces cerevisiae.

[0024] Generally, an effective amount of glucan prevents, delays the onset of, or reduces the severity of a disease or infection in the subject.

[0025] Preferably, the β-glucan is administered at the early stage of a disease or infection, or when the subject is suspected of having a disease or infection.

[0026] Preferably, the subject is one or more selected from the group consisting of an athlete, a subject experiencing or suffering from stress, an immunocompromised subject, a subject over 65 years old, preferably over 75 years old, a subject under 16 years old, a subject having or recovering from cancer, and a subject having innate immune deficiency.

[0027] In certain embodiments, the subject is an athlete, generally a high-performance athlete. Such athletes are more prone to upper respiratory tract infections in certain situations, such as after a marathon.

[0028] The subject may be a healthy subject. The subject may be of any age, preferably a subject over 65 years old, preferably over 75 years old, or a subject under 16 years old.

[0029] The subject may be an obese subject. The subject may be an obese subject with diabetes.

[0030] The subject may be suffering from post-infection immunodysregulation (e.g., post-COVID-19) that exhausts adaptive immune memory cells and dysregulates innate immune cells. (By inducing trained immunity, a regulated innate immune response can be restored and protection against severe infections can be provided).

[0031] In one embodiment, the β-glucan of the present invention is administered / ingested to the subject in a periodic cycle. This cycle can be a period of 3 to 4 weeks. Administration may be daily, e.g., once or twice a day, for a period of 3 to 4 weeks, after which administration is interrupted or stopped.

[0032] In one embodiment, the glucan of the present invention is a β-1,6 branched β-1,3 glucan (or "β-(1,3 / 1,6)").

[0033] Preferably, the (1→3)-β-D-glucan is a β-1,6 branched β-1,3 glucan and has the following, usually repeating, structure:

Chemical formula

[0034] In one embodiment, the 1,6-linked side chains of the glucan of the present invention are in the range of 3 to 8 glucose molecules, such as 3 to 6 glucose molecules, or 4 to 5 glucose molecules. In one embodiment, a maximum of 8 glucose molecules are present.

[0035] The glucan of the present invention may have a degree of branching of 3 to 5%, such as 4%.

[0036] In one embodiment, the glucan of the present invention is whole glucan particles (WGP).

[0037] In one embodiment, the WGP has a size of about 1 to about 6 microns (i.e., μm), or about 2 to about 5 microns, or about 3 to about 4 microns.

[0038] In one embodiment, the WGP is insoluble.

[0039] Generally, the glucan is derived from or obtained from the cell wall of baker's yeast.

[0040] In one embodiment, the β-glucan of the present invention is a β-glucan preparation derived from a strain of Saccharomyces cerevisiae.

[0041] In one embodiment, the glucan of the present invention is provided as a health supplement or nutritional supplement containing the glucan of the present invention. The nutritional supplement may be a nutritional supplement fortified with the glucan of the present invention. The supplements may include, but are not limited to, tablets, capsules, gummies, and powders, beverages / drinks, and energy bars.

[0042] In one embodiment, the nutritional supplement contains 75% or more of β1,3 / 1,6 glucan on a dry weight basis.

[0043] In one embodiment, the supplement is a wellmune supplement or any other S. cerevisiae-derived β-glucan preparation.

[0044] The supplement contains 80% or more of β-glucan. The β-glucan may be 82% - 90%, or 82% - 84%.

[0045] Generally, the supplement contains 75% or more of β1,3 / 1,6 glucan, more than 3.5% protein, more than 10% fat, more than 3% ash, more than 8% moisture, more than 0.1 mg / kg mercury, more than 0.5 mg / kg lead, more than 1.0 mg / kg arsenic, and more than 1.0 mg / kg cadmium on a dry weight basis.

[0046] A method for programming hematopoietic stem and progenitor cells (HSPCs) of a subject, the method comprising administering the glucan of the present invention to the subject, wherein the HSPCs in the subject promote the proliferation of multipotent progenitor cells (MPPs) that are favorably inclined towards bone marrow hematopoiesis.

[0047] A method for inducing trained innate immunity in a subject, the method comprising administering the glucan of the present invention to the subject.

[0048] A method for training natural immune cells in a subject, the method comprising administering the glucan of the present invention to the subject. The trained natural immune cells can be mature natural immune cells such as monocytes and / or macrophages.

[0049] Definitions and general preferences All publications, patents, patent applications, and other references mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated as being incorporated by reference and the entire contents thereof were cited.

[0050] As used herein, unless otherwise indicated, the following terms are intended to have the following meanings in addition to the broader (or narrower) meanings the terms may have in the art.

[0051] Except where the context requires otherwise, the use of the singular form in this specification is to be construed as including the plural form, and vice versa. The term "a" or "an" used in connection with an item is to be construed as referring to one or more of that item. Thus, the terms "one (a or an)", "one or more", and "at least one" are used interchangeably herein.

[0052] As used herein, the term "comprise", or variations thereof such as "comprises" or "comprising", are to be interpreted as indicating the inclusion of all recited integers (e.g., features, elements, characteristics, properties, method / process steps or limitations) or groups of integers (e.g., features, elements, characteristics, properties, method / process steps or limitations), but not the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprising" is inclusive or open-ended and does not exclude additional unrecited integers or method / process steps.

[0053] As used herein, the terms "disease" or "condition" are used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, disease, condition, or syndrome in which physiological function is impaired, regardless of the nature of the causative agent (or whether the etiological basis of the disease has actually been established). Thus, it encompasses conditions resulting from infection, trauma, injury, surgery, radiation ablation, poisoning, or nutritional disorders.

[0054] As used herein, the terms "treat" or "treatment" refer to an intervention (e.g., administration of an agent to a subject) that cures, ameliorates, or alleviates a condition or symptoms of a disease, or eliminates (or reduces the effect of) its cause(s). In this context, the term is used synonymously with the term "therapy". Treatment can be manifested by a permanent or temporary improvement in the condition of the subject. In this context, treatment includes limiting and / or reversing the progression of a disease.

[0055] As used herein, the terms "prevent" or "preventing" refer to an intervention (e.g., administration of an agent to a subject) that prevents a condition in a subject, or delays its onset or progression, or prevents or delays the severity of the condition, or reduces (or eradicates) its incidence within the population being treated.

[0056] As used herein, the term "composition" is meant to mean something made by human hands and should be understood to exclude compositions that occur in nature. Compositions can be formulated in unit dosage forms, i.e., in the form of individual portions containing a unit dose or multiple or sub-unit amounts of a unit dose.

[0057] The term "symptom" is defined as an indicator of a disease, illness, injury, or something being wrong somewhere in the body.

[0058] As used herein, the term "effective amount or therapeutically effective amount" as applied to the glucans of the present invention defines an amount that can be administered to a subject without undue toxicity, irritation, allergic reaction, or other problems or complications and that provides a reasonable benefit / risk ratio, but is sufficient to provide the desired effect. This amount will vary for each subject depending on the age and general condition of the individual, the mode of administration, and other factors. Accordingly, it is not possible to specify an exact effective amount, but one of ordinary skill in the art will be able to determine the appropriate "effective" amount in any individual case using routine experimentation and general knowledge of the background. The therapeutic result need not be complete cure. The therapeutic result may be a permanent or temporary improvement in the condition of the subject.

[0059] The term "subject" means a human or an animal, more typically a mammal. In one aspect, the subject is a human.

[0060] "β-Glucan" is a polysaccharide found inside the cell walls of bacteria and fungi. β-Glucan is a polymer of glucose (D-glucose) linked together by a 1→3 linear β-glycosidic chain core, with their lengths and branching structures differing from each other. The branches derived from the glycosidic chain core are different, and the two main branching groups are 1→4 or 1→6 glycosidic chains. In the present invention, the β-glucan is derived from Saccharomyces cerevisiae. Saccharomyces cerevisiae may be any suitable Saccharomyces cerevisiae strain.

[0061] As used herein, the term "(1→3)-β-D-glucan" refers to a glucan containing D-glucose units having β-1,3 linkages.

[0062] "β-1,6-branched β-1,3 glucan (or "β-(1,3 / 1,6)") is composed of a backbone of glucose molecules linked through a unique chemical bond in which glucose chains are attached to β1,3 chains via β1,6 linkages.

[0063] As used herein, the term "trained immunity" refers to the immunological memory properties of innate immune cells mediated by epigenetic and / or metabolic reprogramming. Trained immunity usually results in protection against infection and enhancement of the immune response during infection, in the absence of antibodies.

[0064] As used herein, the term "immune memory" or "immunological memory" refers to the ability of the immune system to rapidly and specifically recognize pathogenic components recognized as non-self and initiate an immune response based on past exposure. Immune memory can be classified into conventional adaptive immune memory, which induces antibody and cellular responses via lymphocytes depending on the recognition of specific antigens, or innate immune memory / trained immunity, in which specific stimuli such as β-glucan prime innate immune cells, causing different responses to a wide range of pathogenic components.

[0065] "Innate immune response" is the first line of defense against invading pathogens and is activated as a physical and / or chemical barrier against infectious agents. Innate immune cells include natural killer cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. The innate immune system functions to activate the adaptive immune system through antigen presentation.

[0066] As used herein, the term "hematopoietic stem and progenitor cells (HSPC)" refers to a population of progenitor cells that have the ability to self-renew and undergo multi-lineage differentiation. Functionally distinct subsets of hematopoietic progenitor cells include multipotent progenitor cells 2-4 (MPP2, MPP3, and MPP4). MPP3 is related to the myeloid cell lineage. MPP4 is lymphocyte-related. In the bone marrow (BM), HSPC ensures the homeostasis of blood cells.

[0067] As used herein, the term "trained monocytes" refers to monocytes that have immune memory mediated by epigenetic and / or metabolic reprogramming. For example, in this context, "trained monocytes" are monocytes trained by exposure to β-glucan in the laboratory or monocytes derived from the bone marrow of animals / subjects exposed to β-glucan. Trained monocytes typically show an increased pro-inflammatory cytokine response to restimulation with TLR ligands (Saeed et al., Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity, Science 2014).

[0068] As used herein, the term "trained macrophages" refers to macrophages (sentinel cells of the innate immune system) derived from the bone marrow (via monocytes) or from animals / subjects exposed to β-glucan / zymosan or other inducers of trained immunity, and having enhanced functions mediated by epigenetic and / or metabolic reprogramming.

[0069] As used herein, the term "hematopoiesis" refers to the formation of blood cell components. All cellular blood components are derived from hematopoietic stem cells (HSCs). HSCs are stem cells that give rise to other blood cells. In human adults, hematopoiesis occurs in the red bone marrow of many bones.

[0070] As used herein, the term "myeloid cells" refers to blood cells that arise from progenitor cells of granulocytes, monocytes, erythrocytes, or platelets. This progenitor cell can be a myeloid progenitor cell or a progenitor cell, such as a common myeloid progenitor cell (CMP). The progenitor cell can specifically be of the myeloblast lineage, i.e., derived from myelocytes and monocytes.

[0071] As used herein, the term "myeloid hematopoiesis" refers to the process by which mature innate immune cells develop from myeloid progenitor cells.

[0072] As used herein, the term "expansion of multipotent progenitor cells (MPPs)" refers to an increase in the relative proportion and / or absolute number of the MPP pool of cells in an animal / subject, usually in response to treatment.

[0073] As used herein, the term "whole glucan particles" refers to β-glucans isolated from glucans that contain cell walls and substantially retain the in vivo form of glucans. WGPs are insoluble and preferably spherical. The WGPs of the present invention are generally pure glucan particles, e.g., 80% or more, 90% or more, 95%, 98%, or 100% β-glucan. In one embodiment, the WGPs exhibit a high water retention capacity as indicated by their viscosity in an aqueous solution. For example, WGPs of about 2 - 4 microns containing 5.5 grams of glucan per deciliter have a viscosity of about 1000 centipoise. Methods for measuring viscosity are well known in the art (e.g., US4992540).

[0074] Methods for preparing WGP are well known in the art. Exemplary methods are disclosed in the publications that disclose Wellmune® herein. In one embodiment, the WGP is produced by one or more of these methods. Generally, this method involves extracting the alkali-soluble components from the glucan-containing cell wall without the previous description of the cell wall to produce whole glucan particles that retain the in vivo glucan form. In this way, the β-glucan layer of the cell wall is isolated and remains intact to form so-called "ghost cells", which are called dispersed whole glucan particles (dWGP).

[0075] For example, Saccharomyces cerevisiae is selectively grown to obtain a pure culture and grown in a stainless steel fermentation vessel. After fermentation, the cells are lysed by holding them at 45-55 °C for about 24 hours. After autolysis, the cell wall is separated from the soluble yeast extract using a continuous centrifuge. The recovered yeast cell wall is further treated with a series of alkali washes and hot water washes (70-90 °C) to remove the mannosylated proteins and any residual cell lipids of the cell wall. The chitin of the cell wall is removed in the subsequent acidification step, and the remaining purified β-1,3 / 1,6 glucan slurry is washed with hot water, concentrated, and the pH is adjusted as necessary. The resulting product is flash pasteurized and spray dried. By this method, a glucan that retains its yeast cell-like macrostructure is obtained.

[0076] Another method involves culturing a Saccharomyces cerevisiae yeast strain in a medium, recovering all yeast cells from the medium, and contacting the all yeast cells with an aqueous hydroxide solution having a pH of about 4.0 to about 12.5 or a normality of 0.75 to about 1.5 and a temperature of about 25 °C to about 100 °C for a time sufficient to extract proteins from the all yeast cells to form water-insoluble whole glucan particles containing less than 1% by weight of protein, wherein the glucan particles substantially retain the three-dimensional structure of the in vivo glucan and generally consist essentially of a glucan having β(1-6) and β(1-3) linkages (US4810646).

[0077] It is not a linear soluble β-glucan (low molecular weight, e.g., 25 KDa or less) as disclosed in the prior art. In other words, it is intact.

[0078] As used herein, the term "β-glucan preparation" refers to a β-glucan isolated from a fungal species and prepared in a specific manner different from other β-glucans isolated from the same or other species.

[0079] As used herein, thymosan is a β-glucan preparation derived from S. cerevisiae for isolating particulate β-glucan-enriched cell walls (i.e., ghost yeast cells). Its physical and chemical properties are described in De Graaff et al., Cancer Immunology, Immunotherapy (2021) 70:547-561. Depleted thymosan is a preparation of thymosan in which the outer mannan layer is removed, concentrated for β-glucan content, and further purified to remove contaminating ligands. This is described in Gantner et al., J. Exp. Med, Volume 197, Number 9, May 5, 2003, 1107-1117 and in Ikeda et al, Biol Pharm Bull. 2008 31(1):13-8.

[0080] As used herein, the term "obese subject" refers to an individual, usually a human, with a BMI of less than 30, a fasting blood glucose level of less than 6 mmol / l, and an HbA1C of less than 42 mmol / mol. Fasting blood glucose and HbA1C are calculated using methods well known in the art.

[0081] As used herein, the term "obese and diabetic subject" refers to an individual, usually a human, with a BMI of less than 30, a fasting blood glucose level of less than 7 mmol / l, and a fasting HbA1C of 48 mmol / mol or more. Fasting blood glucose and HbA1C are calculated using methods well known in the art.

[0082] The present invention will be described with reference to the following drawings.

Brief Description of the Drawings

[0083]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8A

Figure 8B

Figure 8C

Figure 8D

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21-1

Figure 21-2

DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention provides an effective amount of whole Saccharomyces cerevisiae (1→3)-β-D-glucan particles for use in a method of programming hematopoietic stem and progenitor cells (HSPCs) in a subject. The HSPCs in the subject result in the expansion of multipotent progenitor cells (MPPs) that favorably act on post-exposure bone marrow hematopoiesis, i.e., the production of innate immune cells, particularly monocytes. The mature cells derived from this expanded population have altered functions.

[0085] Therefore, the subject produces more MPP3, i.e., myeloid progenitor cells. Generally, the ratio of MPP4 to MPP3 in the subject is about 60:40 (Figure 12G). However, administration of the glucan of the present invention results in a larger MPP3 population in the subject's HSPC and increases this ratio to favor MPP3. The ratio can be 50 or 40:60 or 30:70. It will be recognized that this ratio may depend on the condition, dosage, and / or subject.

[0086] Without being bound by theory, β-glucan induces its effects via the phagocytic C-type lectin receptor dectin-1 (encoded by Clec7a) (Brown, G. D. et al. Dectin-1 mediates the biological effects of beta-glucans. J Exp Med 197, (2003)), which induces metabolic and epigenetic changes in innate immune cells, particularly monocytes, which is thought to promote the increased macrophage response to restimulation, i.e., trained immunity.

[0087] Training of innate immune cells by epigenetic modification enhances the induction of innate immune genes and causes a quantitatively greater and earlier cytokine response (i.e., a systemic boost in immune signaling). It results in enhanced induction of chemokines by monocyte-derived macrophages. It enhances the induction of the pro-inflammatory cytokine (TNF). It results in an increase in the anti-inflammatory immunomodulatory factor (IL-10).

[0088] The inventors have shown that all fungal particles are recognized by dectin-1, internalized by phagocytic synapses, thereby inducing an optimal antimicrobial response. Dectin-1 has a signaling function and has been shown to activate the expression of NFκB and pro-inflammatory genes via SYK / CARD10. Different β-glucans can differentially bind to dectin-1 depending on the form of presentation. Soluble low molecular weight (MW) β-glucans can bind to dectin-1 and induce NFκB, but recognition of larger β-glucan chains presented on intact fungal particles such as the β-glucan of the present invention is required to induce the localization of surface dectin-1 receptors and the formation of phagocytic synapses associated with antibacterial activities such as ROS.

[0089] Since β-glucan is a widely used and approved food ingredient, it is particularly suitable as a dietary supplement. When a subject ingests the β-glucan of the present invention, for example, as a dietary supplement or food, natural immune cell production with MPP3 and immune memory becomes dominant in the subject's HSPC, thereby enhancing the subject's response to infection by pathogens.

[0090] Interestingly, a major feature of trained natural immune cells is that their recall ability is not related to the nature of the training stimulus (i.e., trained natural immune cells can efficiently respond to restimulation by a variety of non-specific ligands).

[0091] The β-glucan of the present invention can be formulated as a composition comprising the β-glucan of the present invention. In one embodiment, the β-glucan of the present invention is dispersed.

[0092] The β-glucan of the present invention can be used as a health supplement or a nutritional supplement. The supplement can be selected from the group including "shots", i.e., beverages containing a small amount of beverage portion, bakery products, dairy products, snack products, powder products, powdered milk, confectionery, yogurt, breakfast cereals, bread products, nutritional supplements, and sports nutritional supplements. The nutritional supplement may be a powdered nutritional supplement added to a food or beverage product.

[0093] The β-glucan of the present invention can also be formulated as a capsule or a tablet.

[0094] Notably, the β-glucan of the present invention is the whole glucan particle (WGP) of Saccharomyces cerevisiae. In one example, the whole glucan particle is a β-glucan sphere derived from yeast (Saccharomyces cerevisiae) cells further purified based on alkali solubility, and substantially retains the in vivo glucan form / three-dimensional structure. The WGP is not soluble. Methods for preparing WGP are well known in the art. Exemplary methods are disclosed in the publication that discloses WelMune® within this specification, and the following research (Li B, et al. Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88- and Syk kinase-dependent pathways. Clin Immunol. 2007 Aug;124(2):170-81). The WGP differs from other β-glucan preparations in terms of the purity of β-glucan, but importantly, it maintains the shape of the intact yeast cell wall as a non-viable ghost form. The WGP of the present invention is commercially available for research purposes and is known to activate the conventional innate immune signaling pathway (https: / / www.invivogen.com / wgp-dispersible; Goodridge HS. et al., 2011. Activation of the innate immune receptor Dectin-1 upon formation of a ‘phagocytic synapse’. Nature. 472(7344):471-5.), but it is not described for activating trained immunity, especially when administered orally.

[0095] In one embodiment, the particles have a particle size, i.e., diameter, of about 1 micron to about 5 microns (or μm), about 2 microns to about 4.5 microns, about 2.5 microns to about 4 microns, or about 3 to about 3.5 microns. This may be an average diameter. At least 80% to 99%, or more than 85% or 90% of the particles may have a diameter within this range. The particle size is measured using a laser diffraction particle size analyzer, and such methods are well known in the art.

[0096] The glucan of the present invention can be a preparation containing a plurality of β-glucans. The WGP in the preparation has an average diameter, or particle size distribution, with an average value of 1 to 6 microns, preferably 2 to 4 microns.

[0097] The composition of the present invention may have a plurality of total β-glucan particles derived from different S. cerevisiae strains or preparations.

[0098] The glucan is derived from, or isolated from, the cell wall of baker's yeast (Saccharomyces cerevisiae).

[0099] Notably, the glucan of the present invention is Wellmune®.

[0100] Wellmune® is a component of health foods, beverages, and dietary supplements. Wellmune® is an insoluble large whole glucan particle (WGP) derived from the cell wall of baker's yeast (Saccharomyces cerevisiae), and the β-1,3 / 1,6-glucan is composed of a backbone of glucose molecules linked via the unique chemical bond of β1,3 chains linked to glucose chains via β1,6 bonds. The Wellmune® glucan may be similar to those disclosed in US4810646, US4992540, US5037972, US5082936, US5028703, US5250436, and US5506124, each of which is incorporated herein by reference.

[0101] Those skilled in the art will recognize that the appropriate dosage of the glucan of the present invention or the food supplement containing glucan to be administered can be determined without undue experimentation. The amount and frequency are those most suitable for the purpose. The frequency of application or administration can vary significantly depending on the needs of each subject, ranging from once a month to ten times a day, preferably once a week to four times a day, more preferably three times a week to three times a day, and even more preferably once or twice a day for application or administration.

[0102] In one embodiment, the daily dose is about 250 mg (which is 0.003 w / w% of the dietary intake). In one embodiment, the daily dose can range from 250 mg (0.003 w / w% of the dietary intake) to 5 g (0.05 w / w% of the dietary intake) per day. In one embodiment, the daily dose is 500 mg to 4 g per day, or 1 g to 3 g per day.

[0103] In one aspect of the present invention, an effective amount of the β-D-glucan of the present invention is provided to prevent a disease or infection in the above-mentioned subject, delay its onset, or reduce its severity. Notably, the subject can be a subject who is susceptible to infection after a specific event or at a specific time. For example, the subject can be a high-performance athlete who is susceptible to upper respiratory tract (URT) infections. By using the glucan of the present invention as a supplement, the incidence of URT during the time window when the athlete's vulnerability is increased (e.g., after a marathon) can be reduced. The athlete's innate immune system is considered to be trained to effectively resist these infections.

[0104] In one embodiment, the β-glucan of the present invention is co-administered or combined with one or more antigens to induce antigen-specific immune memory.

[0105] In one aspect, the present invention provides a Saccharomyces cerevisiae-derived β-glucan of the present invention or its use in an effective amount for treating post-infection immunomodulatory deficiency in a subject. The subject is suffering from post-infection immunomodulatory deficiency, such as post-COVID-19 (SARS-CoV-2 infection). This exhausts adaptive immune memory cells and causes dysregulation of innate immune cells. By inducing trained immunity, a regulated innate immune response can be restored and protection against severe infections can be provided. Immunodeficiency has been discussed in relation to SARS-CoV-2 infection by Phetsouphanh et al, (Nature Immunology, vol 23, February 2022, 210-216).

[0106] A further aspect of the present invention provides a composition such as a dietary supplement containing two or more β-glucans derived from different strains of Saccharomyces cerevisiae. In other words, the composition contains a plurality of types of β-glucans each derived from a different strain of Saccharomyces cerevisiae.

[0107] It will be understood that the features of the embodiments of the present invention can be combined in any combination.

[0108] The present invention will be described below with reference to specific examples. These examples are merely illustrative and are shown for explanatory purposes only and are not intended to limit the exclusive scope claimed or the invention described in any way. These examples constitute the best mode contemplated at present for carrying out the present invention.

Examples

[0109] Overview of the method The inventors investigated the ability of the food-grade yeast WGP of the present invention to induce responses trained in human monocyte-derived macrophages, in parallel with a panel of various β-glucans, using a HEK-based reporter cell assay to examine dectin-1 signaling. The inventors also examined the ability of this WGP to promote hematopoiesis, which has been shown to be associated with the long-term memory effect of trained immunity.

[0110] In the first part of the tests shown as Figures 1 to 5, the in vitro innate immune training of human monocytes is described. Leukocytes were isolated from healthy anonymous donors, and monocytes were isolated. These progenitor cells were treated with a training substance, e.g., S. cerevisiae Wellmune® dispersed (dWGP) or a control for 24 hours, rested, and matured into macrophages. After this resting period, the mature macrophages were restimulated with a common innate immune activator, mainly the Gram-negative component LPS, and cytokine production was measured using TNF ELISA of the supernatant from these cultures. Training was indicated by an enhanced response to LPS restimulation, measured by an increase in TNF production in cells trained with dWGP compared to the untrained control.

[0111] In the second part of the test, bone marrow was collected from all mice and grown in the laboratory to mature macrophages (BMDM) using L929-conditioned medium. At the time of reaching the mature state (6 days after differentiation), the BMDM were stimulated with various doses of microbial agonists (e.g., lipopolysaccharide (LPS) of Escherichia coli cells, or heat-killed Mycobacterium tuberculosis (Mtb) that activates BMDM to produce pro-inflammatory cytokines (e.g., TNF)). This serves as a readout of innate immune function enhanced by trained immunity.

[0112] The third part of the study (Figures 15 to 21) was conducted to further confirm the first and second parts and showed the following.

[0113] Oral administration of yeast WGP by gavage enhances the responsiveness to activation in mature macrophages derived from these bone marrow (BMDM).

[0114] Feeding a yeast WGP-containing diet for 4 weeks resulted in the proliferation of bone marrow myeloid / natural immunity-committed progenitor cells and an increase in more mature, generally myeloid-committed progenitor cells, consistent with enhanced responsiveness to activation in mature macrophages derived from these bone marrow (BMDM). Feeding a yeast WGP-containing diet also altered the composition of natural immune cells in the intestine of obese mice and restored the defective natural response.

[0115] In this study, β-glucan with the trade name WelMune was used.

[0116] This is important because the changes previously observed in the bone marrow of mice after feeding a yeast WGP diet were related to a low abundance HSPC progenitor cell population. This new data examines more committed progenitor cells further downstream in the myeloid lineage and supports the idea that small changes previously seen in the low abundance HSPC progenitor cell population lead to larger, more significant changes in more committed cells. The mature cells obtained from these new tests (4 weeks after WGP feeding) also showed improved responsiveness to restimulation when measuring the pro-inflammatory cytokines TNF and IL6, beyond the framework of previous observations limited to TNF only. This new data also shows a comparison with fiber-matched diets and normal mouse diets in controlling the effects of the fiber in the WelMune diet.

[0117] Materials and Methods Detailed methods: Cell isolation and culture reagents hDectin-1b-HEK293 NFκB-SEAP reporter cells were obtained from Invivogen and cultured according to the manufacturer's instructions. Puromycin and HEK-Blue™ CLR Selection (Invivogen) were used as selective antibiotics, and the cells were cultured using DMEM (4.5 g / L glucose), 10% (v / v) fetal bovine serum (FBS), 100 U / ml penicillin, 100 μg / ml streptomycin, 100 μg / ml normocin, and 2 mM L-glutamine. Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from the Irish Blood Transfusion Services (Dublin, Ireland) using density gradient centrifugation with Lymphoprep (Stem Cell Technologies) followed by erythrocyte lysis using lysis buffer Hybri-Max™ (Sigma-Aldrich). PBMCs were resuspended in differentiation medium (cRMPI, 10% FBS, 10 ng / mL M-CSF) and monocytes enriched by attachment to plastic were used for subsequent training assays or stimulation. The supply of human blood products from IBTS was approved by clinical indemnity. Bone marrow-derived macrophages (BMDMs) were isolated by flushing the tibias and femurs collected from both hindlimbs of C57BL / 6J male mice generated and maintained at the Comparative Medicine Unit, Trinity College Dublin (Dublin, Ireland) with DMEM (Gibco). The resulting suspension of bone marrow cells was filtered through a 40 μm nylon mesh cell strainer (Biolegend). After washing, the pellet was resuspended and treated with erythrocyte (RBC) lysis buffer Hybri-Max™ (Sigma-Aldrich) for 2 minutes to lyse erythrocytes. After washing and counting, the cells were resuspended in DMEM, 10% FBS, 20% L929-conditioned medium, seeded, and differentiated into BMDMs over 1 week. Mature BMDMs were scraped off, the cells were reseeded at the required density in DMEM, 10% FBS, 5% L929-conditioned medium, and left to stand overnight before stimulation.

[0118] Preparation of β-glucan and training stimulus β All glucan particles derived from Saccharomyces cerevisiae were provided by Sonja Nodland, Kerry Health & Nutrition, Minnesota, USA. The whole glucan particles include Wellmune® whole glucan particles (WGP) (which yield non-aggregated ghost yeast cells) with cell wall β-glucan preserved, or a soluble preparation obtained from heat-treated WGP. In all experiments, the WGP powder was resuspended in PBS and sonicated to dissociate the aggregates, yielding a dispersion of “single cell” ghost particles, i.e., dispersed WGP (dWGP). The soluble preparation consists of a heterogeneous mixture of soluble β-glucans of various molecular weights, but this formulation is referred to as soluble WGP (sWGP) to emphasize that dWGP and sWGP are composed of β-glucans derived from the same source. Dr. Nodland also provided β-glucan fractions of different molecular weights isolated from sWGP by size exclusion chromatography followed by filtration (a filtration step of 1.5 - 100 nm). These fractions were as follows: F1: <100 kDa, F2: 100 - 400 kDa, F3: 400 - 800 kDa, and F4: >800 kDa. The sonication procedure for generating dWGP from WGP powder was as follows: The WGP was weighed and dissolved in sterile endotoxin-free water (Invitrogen) to obtain 10 - 15 mL of 25 mg / mL WGP. After leaving this solution at room temperature overnight (8 - 16 hours), it was sonicated with a 150VT ultrasonic homogenizer equipped with a 5 / 32” microtip. The solution was sonicated for 5 minutes at an output of 50% and a time pulse rate of 50% while immersing the tip approximately 5 mm below the liquid surface. Since heat is generated by sonication, the tube containing the solution was maintained in ice. Following this sonication step, the dWGP was pelleted by centrifugation (1,000 G, 10 minutes, room temperature), the water was carefully decanted off, and it was replaced with an aqueous solution of 0.2 M NaOH of a volume to give 25 mg / mL WGP. After 20 minutes, the dWGP was washed three times with sterile water using the same pelleting, decanting, and solvent replacement conditions as described.Finally, the last two washes were performed to replace the sterile water with sterile PBS and stored at 4°C for up to 6 months. Since dWGP settles out of solution, it was also necessary to vortex it before each use. Whole glucan particles without sonication (uWGP) were used in some experiments and resuspended directly in PBS without sonication. Particles derived from Saccharomyces cerevisiae-β-glucan, crude thymosan, and depleted thymosan were obtained from Invivogen.

[0119] In some experiments, 3-μm aminated polystyrene particles (AM-PS, Magsphere) were conjugated with sWGP based on a previously published method (Tam J. M. et al. Use of fungal derived polysaccharide-conjugated particles to probe Dectin-1 responses in innate immunity. Integr Biol (Camb) 4, 220-227 (2011)). After washing 2 mg of AM-PS three times with anhydrous DMSO (Sigma) using a centrifugal filter containing a 0.65-μm PVDF membrane (Ultrafree, Millipore), it was incubated with 250 μL of 2 M 1,1'-carbonyldiimidazole (CDI, Sigma) and newly dissolved in anhydrous DMSO with shaking for 1 h at room temperature. The particles were then washed twice with anhydrous DMSO to remove excess CDI (using the centrifugal filter again), and then incubated with 250 μL of 0.1 mg / mL sWGP dissolved in anhydrous DMSO with shaking for 1 h at room temperature. After this conjugation step, the sWGP was recovered by centrifugation using a centrifugal filter for blocking the particles, and the efficiency of conjugation was evaluated by measuring the loss of sWGP using a filtered sWGP solution without AM-PS or CDI as a control. sWGP was measured using the phenol-sulfuric acid method based on the reported protocol (Masuko, T. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem. 339, 69-72 (2005)). After adding 130 μL of concentrated sulfuric acid (Sigma) to 50 μL of the sWGP sample on a 96-well plate, 30 μL of a sterile aqueous solution of 5% w / v phenol (Sigma) was added, and then the solution was immediately pipetted to mix well. The plate was placed on a hot plate at 90 °C for 5 min and then cooled to room temperature in a water bath. The absorbance at 492 nm was measured, and the concentration of sWGP in each sample was determined using a standard curve of known amounts of sWGP, thereby quantifying the amount of sWGP conjugated to AM-PS.

[0120] Other β-glucans containing the following were obtained from Invivogen: β-glucan peptide (BGP) (a high molecular weight polysaccharide extracted from the macrofungus Trametes versicolor), thymosan (a cell wall preparation derived from Saccharomyces cerevisiae), schizophyllan (a high molecular weight β-glucan derived from the fungus Schizophyllum commune), curdlan (a β-1,3-linked glucan derived from the bacterium Alcaligenes faecalis), and pustulan (an intermediate molecular weight β-1,6-linked glucan derived from the algal lichen Lasalia pstulata). All β-glucans were used at concentrations in the range of 1, 10, or 100 μg / mL. Heat-killed Candida albicans (HKCA) was also obtained from Invivogen and used at a concentration of 1×10 4~6 cells / mL.

[0121] Activation / restimulation of macrophages Ultra-pure lipopolysaccharide (LPS) derived from E. coli O111:B4 was obtained from Invivogen and used at concentrations of 1, 10, and 100 ng / mL to induce resistance in human monocytes, or in most experiments at 10 ng / mL or 10 - 100 ng / mL to restimulate trained macrophages. Pam3CSK4, a synthetic triacylated lipopeptide TLR2 / TLR1 agonist, was obtained from Invivogen and used at 100 μg / mL for restimulation. HKCA was used at 1×10 6Trained macrophages at cells / mL were restimulated. Trained monocyte-derived macrophages were restimulated with inactivated irradiated Mycobacterium tuberculosis (iMtb) obtained from the American Type Culture Collection (ATCC) (Manassas, VA), prepared according to the manufacturer's instructions, and used at 500 μg / mL. Bone marrow-derived macrophages (BMDMs) from mice trained in vivo were stimulated with heat-killed Mycobacterium tuberculosis (hk-Mtb) from Invivogen and used at a concentration of 500 - 1000 μg / mL. Trained monocyte-derived macrophages were also infected with the viable Mycobacterium tuberculosis (Mtb) strain H37Ra obtained from ATCC and grown to the logarithmic phase in Middlebrook 7H9 medium. On the day of infection, the bacteria in the logarithmic phase were pelleted by centrifugation and resuspended in DMEM. The bacterial pellet was dissociated by passing it through a syringe equipped with a 25G needle several times. A single-cell suspension was separated by centrifuging the bacterial suspension at 800 rpm for 3 minutes. The supernatant of this spin was quantified by spectrophotometry (OD 600nm ) and used to infect macrophages. Macrophages were infected at a multiplicity of infection (MOI) of 5 bacilli per cell for 3 hours (as previously described in Hackett EE, et al., Mycobacterium tuberculosis Limits Host Glycolysis and IL-1β by Restroction of PFK-M via MicroRNA-21 Cell Rep. 2020 Jan 7;30(1):124-136), centrifuged at 13,000 rpm for 10 minutes for further purification to pellet extracellular bacteria. After washing the bacteria-free medium with DMEM, it was returned to the macrophages to remove extracellular bacteria, and the cultures were grown for 72 hours after infection.

[0122] Training assay and lead-out To perform a human monocyte training assay, PBMCs isolated from human blood were seeded in 96-well plates (100,000 cells per well in 180 μL of RPMI, 10% FBS, 50 ng / mL of M-CSF), and 20 μL of the training stimulant was added immediately. The cells were incubated overnight at 37 °C. The medium was removed, and the cells were washed very gently twice with 100 μL of warm PBS to remove the training stimulant, and 200 μL of fresh medium was added. The cells were washed and given fresh medium every 2 - 3 days, and on day 5, the cells were restimulated with fresh medium for the time indicated. In the training inhibition assay, the PBMC cells were incubated with the desired inhibitor for the time indicated before adding the training stimulant. Unless otherwise indicated, the inhibitor concentrations were as follows: 1 mM of 5'-methylthioadenosine, excess sWGP (10 - 100 μg / mL), 10 - 100 μg / mL of laminarin (Invivogen), 4, 10 - 30 μM of picetanol, 5 μM of BAY-11-087, 1, 2.5, 5, 10 mM of 2-deoxyglucose, 1, 10, 100 nM of rapamycin, 0.1, 1, 10 μM of wortmannin, 2, 10 - 100 μM of cytochalasin D, and 1, 5 - 10 μM of bafilomycin A1. Unless otherwise indicated, all inhibitors were obtained from Sigma-Aldrich. As a readout of training, TNF secretion from the trained cells was measured by ELISA of the supernatant (human TNF ELISA kit, Invitrogen). Alternatively, the production of CXCL8 or IL-10 was measured by ELISA. To perform a metabolic analysis of the trained cells, the lactate concentration in the supernatant was measured using a colorimetric lactate assay kit (MAK064, Sigma-Aldrich). The medium removed from the trained monocytes after 24 hours was also analyzed for TNF production using ELISA to evaluate the effect of the training stimulant on monocyte activation, or used to measure extracellular lactate production. In some experiments, RNA was isolated from the trained monocytes using an RNeasy kit (Qiagen).To analyze gene expression, cDNA was prepared using the High-Capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer's instructions, and individual mRNAs were monitored using the human TaqMan assays (Applied Biosystems) listed below: 18s (Hs03003631_g1), hexokinase-2 (HK2, Hs00606086_m1), GLUT-1 transporter (SLC2A1, Hs00892681_m1), lactate dehydrogenase A (LdhA, Hs01378790_g1), and PKM-2 (Hs00987255_m1). The AB7900HT platform (Applied Biosystems) was used for all PCRs and performed in triplicate in FAST mode. Changes in expression were calculated by the threshold change (ΔΔCT) method using 18S as the endogenous control and normalized to the results obtained in untreated cells. In experiments where trained macrophages were infected with Mycobacterium tuberculosis (Mtb), baseline growth was evaluated by lysing in 0.1% Triton-X for 10 minutes at the 3-hour time point. Serial dilutions were plated in triplicate on 7H10 Middlebrook agar, and after plating, incubated at 37°C for 14 - 21 days before colony-forming units were counted. For subsequent growth measurements, this lysate was combined with pelleted extracellular bacteria obtained by centrifugation of the supernatant, and the fold change in bacterial colony-forming units (CFU) was expressed relative to the baseline time point. To perform the supernatant transfer experiment, supernatants from human monocytes were collected 24 hours after training, administered to naive untrained monocytes along with monocytes trained with dWGP, matured for 5 days, and then restimulated with LPS to evaluate whether soluble factors induced by the training stimulus enhanced responsiveness to the restimulation.

[0123] The BMDM training assay was performed as follows: Six days after isolation, the BMDMs were stimulated with a training stimulus and then allowed to recover and mature for an additional six days, with the medium changed every three days in DMEM, 10% FBS, and 5 - 7% L929-conditioned medium. Twelve days after isolation, the cells were restimulated with LPS. The TNF production in the supernatant was analyzed using the Invitrogen mouse TNF ELISA kit according to the manufacturer's instructions, or the lactate production was measured as described above.

[0124] QuantiBlue assay h dectin 1b-HEK293 NFκB-SEAP reporter cells (Invivogen) were cultured according to the manufacturer's instructions. Reporter cell assays were performed by seeding 50,000 cells per well in 180 μL in a 96-well flat-bottom plate and incubating the cells overnight with 20 μL of the indicated agonist. SEAP activity was measured using Quanti-Blue (Invivogen) according to the manufacturer's instructions. Briefly, 20 μL of the supernatant was added to 180 μL of the QUANTI-Blue solution and incubated at 37°C for 15 minutes. Next, the optical density at 620 nm was measured using a plate reader.

[0125] Animal experiments To perform in vivo induction of trained immunity, C57BL / 6J-OlaHsd male mice were generated and maintained in the Comparative Medicine Research Facility at Trinity College Dublin (Dublin, Ireland). Mice were fed, bred, and maintained under specific pathogen-free conditions with free access to diet and water. Mice aged 8–12 weeks were used. All experiments were performed with the approval of the Irish Medicines Board and the Trinity College Dublin Animal Research Ethics Committee. Trained immunity was induced in mice by a single intraperitoneal injection of either 200 μL of PBS or 200 μL of dWGP resuspended in PBS at 1 mg / ml or 2 mg / ml as a control. To perform in vivo induction of trained immunity by oral supplementation, mice were divided into four main experimental groups. The control group was fed a standard diet with some modifications containing 25 g / kg of inulin (2.5% inulin per kg of diet) as a fiber source (see D11112201). The other three groups of mice were fed diets supplemented with 0.003% (0.03 g per kg of diet), 0.025% (0.25 g per kg of diet), or 0.05% (0.5 g per kg of diet) dietary fiber dWGP, respectively, and the amount of inulin was proportionally decreased to balance the amount of dWGP fiber added to the nutraceutical diet. After acclimating all groups of mice to the inulin-enriched standard diet for 2 weeks, they were switched to the dWGP-supplemented diet for 1–3 weeks. At the end of the experiment, animals were euthanized by CO2 inhalation and tissues were harvested for analysis. Bone marrow cells were harvested by flushing the tibia and femur collected from both legs with DMEM (Gibco). Myeloid progenitor cells were either stained for flow cytometry analysis as described below or used to generate BMDMs as described above. After lysis, washing, and counting of red blood cells, 3 million cells were retained for the following flow cytometry analysis, and the remaining cells were resuspended in DMEM, 10% FBS, 20% L929 conditioned medium, seeded, and differentiated into BMDMs over 1 week. To isolate splenocytes, the spleen was carefully excised and harvested after euthanasia and maintained on ice in RPMI / FBS 0.1% medium. The spleen was then minced into small pieces, gently disrupted through a 70-μm nylon mesh cell strainer, and diluted with PBS.After washing, the pellets were resuspended and treated with red blood cell (RBC) lysis buffer Hybri-Max™ (Sigma-Aldrich) for 2 minutes. The splenocytes were then washed again and resuspended in DMEM, 10% FBS, 5% L929 conditioned medium and seeded. Cells were stimulated with LPS (10 ng / mL) or heat-killed Mycobacterium tuberculosis H37Ra (hk-Mtb, 500 - 1000 μg / mL). To isolate immune cells from the intestine and other tissues, mice were euthanized and dissected after washing to remove feces and tissue pieces were isolated. After collagenase digestion, a single cell suspension was prepared and centrifuged using a 40 / 80% Percoll gradient. The isolated immune cells were stained for flow cytometry analysis as described below. For experiments using labeled Wellmune WGP, fluorescent DTAF was conjugated to WGP as described in Geller et al, Nat Commun. 2022 Sep 9;13(1):759. In the obesity modeling experiments, a diet-induced obesity model was used. 8-week-old male C57 / BL6 mice were fed a high-fat diet (research diet) containing 40% kCal from palmitate or a control standard-fat diet (10% fat), or a modified high-fat diet supplemented with 0.05 w / w% Wellmune. Mice were fed for 12 weeks, body weight was monitored weekly, body fat accumulation at the time of sacrifice was monitored, and induction of obesity was confirmed by measuring metabolic regulation using a glucose tolerance test. Addition of Wellmune WGP did not result in changes in either induction of obesity or metabolic dysregulation (data not shown).

[0126] Multivariate flow cytometry analysis of human monocytes To analyze the activity of phospho-S6 ribosomal protein after β-glucan treatment, PBMCs were isolated as described above and 2×10 6Resuspended in RPMI-1640 medium supplemented with 10% human AB serum (Sigma-Aldrich) per mL. First, the cells were incubated with the metabolic inhibitor for 15 minutes as shown above. Then, the cells were stimulated with either LPS (10 ng / mL, Invivogen), sWGP (10 μg / mL), uWGP (10 μg / mL), or dWGP (10 μg / mL) for 30 minutes, 1 hour, 2 hours, 6 hours, or 24 hours. After the incubation time, the cells were quickly vortexed to resuspend and washed with flow buffer [PBS-1X (Gibco) supplemented with 5% heat-inactivated FBS (Gibco) and 0.1% sodium azide (Sigma)], and the following flow cytometry staining protocol was applied to all samples with a maximum of 100,000 cells per sample. The cells were stained with the fixable viability stain ZombieAqua™ (Biolegend) at a concentration of 1:500 for 15 minutes. Subsequently, the samples were washed with flow buffer and incubated with anti-Dectin-1-PE (clone 15E2, Biolegend) antibody for Dectin-1 surface staining, then washed and fixed with IC Fixation Buffer (Invitrogen) for 20 minutes. Alternatively, the cells were incubated with anti-CD14-APC (clone M5E2, Biolegend), anti-CD16-PE-Cy7 (clone 3G8, Biolegend), anti-HLA-DR-BB515 (clone G46-6, BD Bioscience) at a concentration of 1:100 in flow buffer at 4°C for 30 minutes. Then, all the cells were washed with flow buffer and resuspended with IC Fixation Buffer (Invitrogen) for 20 minutes for fixation. Next, the cells were washed with flow buffer and incubated with Perm / Wash Buffer (BD Biosciences) containing anti-phospho-S6-PE (Ser235 / 236) (clone D57.2.2E, Cell Signaling) at 1:200 for 30 minutes at 4°C for intracellular pS6 staining, and finally washed again with flow buffer. Compensation controls were obtained after staining UltraComp eBeads™ compensation beads (Invitrogen) with the appropriate antibodies.Cells were acquired using a BD FACS Canto II flow cytometer equipped with FACSDiva software. Data analysis and flow cytometry plots were performed using FlowJo software v.7.6 (TreeStar).

[0127] To isolate human monocyte subsets, PBMCs were isolated from peripheral blood buffy coats as described above. After isolation, PBMCs were resuspended in flow buffer and the following flow cytometry staining protocol was applied to all samples containing up to 30,000,000 cells per donor. Cells were stained with the viability stain propidium iodide (Biolegend) at a concentration of 1:500 for 15 minutes. Samples were then washed with flow buffer and incubated with anti-CD14-APC (clone M5E2, Biolegend), anti-CD16-PE-Cy7 (clone 3G8, Biolegend), anti-HLA-DR-BB515 (clone G46-6, BD Bioscience), anti-CCR2-APC-Cy7 (Biolegend) at a concentration of 1:100 in flow buffer at 4°C for 30 minutes. All cells were then washed and resuspended in flow buffer. Compensation controls were obtained after staining UltraComp eBeads™ compensation beads (Invitrogen) with the appropriate antibodies. Cells were acquired and sorted using a BD FACSAria Fusion Cell Sorter equipped with FACSDiva software. Data analysis and flow cytometry plots were performed using FlowJo software v.7.6 (TreeStar). After sorting, the monocyte subsets were seeded at 1×10 6 / mL in RPMI-1640 medium supplemented with 10% human AB serum (Sigma-Aldrich) in separate 96-well plates, allowed to settle for 24 hours, and then stimulated and trained according to the protocol described above.

[0128] Multivariable flow cytometry analysis of mouse bone marrow cells To analyze the HSPC population in mouse bone marrow after in vivo induction of trained immunity, the isolated bone marrow cells were resuspended in flow buffer [PBS-1X (Gibco) supplemented with 5% heat-inactivated FBS (Gibco) and 0.1% sodium azide (Sigma)], and the following flow cytometry staining protocol was applied to all bone marrow samples containing up to 3,000,000 cells per sample. Cells were stained with the fixable viability stain ZombieAqua™ (Biolegend) at a concentration of 1:500 for 15 minutes. The samples were then washed with flow buffer and incubated with anti-CD16 / 32 (Biolegend) at a concentration of 1:100 in flow buffer for 20 minutes at 4°C. Next, the following antibodies were used to stain Lin-, c-Kit+, Sca-1+ cells (LKS), hematopoietic stem cells (HSC), and multipotent progenitor cells (MPP): anti-Ter-119, anti-CD11b (clone M1 / 70), anti-CD5 (clone 53-7.3), anti-CD4 (clone RM4-5), anti-CD8a (clone 53-6.7), anti-CD45R+ (clone RA3-6B3), anti-Ly6G / C+ (clone RB6-8C5), all biotin conjugates (all from Biolegend) were added at a concentration of 1:50 for 30 minutes at 4°C. The cells were then washed with flow buffer. Streptavidin-APC-Cy7 (Biolegend), anti-c-Kit-APC (clone 2B8, Biolegend), anti-Sca-1-PE-Cy7 (clone D7, eBioscience), anti-CD150-eFluor450 (clone mShad150, eBioscience), anti-CD48-PerCP-eFluor710 (clone HM48-1, BD Bioscience), anti-CD34-FITC (clone RAM34, eBioscience), anti-Flt3-PE (clone A2F10.1, Biolegend) were added and incubated for 30 minutes at 4°C. For Figures 15 - 17, LKS+ HSPC were enriched using Milteny cKit+ beads on a MACS column. Mature myeloid progenitor cells (MP, CMP, GMP, and CLP) in Figure 17 were measured using cKIT-negative cells. LKS+ cells were stained as outlined above.The FMO (Fluorescence Minus One) control was performed using 1,000,000 cells obtained by mixing equal volumes of samples from different experimental conditions and stained with appropriate antibodies. An example of gating is shown in Figure 21. Subsequently, all cells were washed with flow buffer and resuspended in IC Fixation Buffer (Invitrogen). Compensation controls were obtained after staining UltraComp eBeads™ compensation beads (Invitrogen) with appropriate antibodies. Cells were acquired on a BD FACSCanto II flow cytometer equipped with FACSDiva software. Data analysis and flow cytometry plots were performed using FlowJo software v.7.6 (TreeStar).

[0129] Experimental design, statistical analysis and presentation The data shown represent the mean data of experiments performed on human monocytes / macrophages derived from the indicated number of independent donors, the indicated number of animals per group for in vitro tests, or independent BMDM preparations for mouse in vitro tests. The data were analyzed in Graph Pad Prism or Excel and graphed with annotations in Adobe Illustrator to generate the figures. Analysis of variance was performed in multivariate experiments using post hoc tests to show significant differences between each treatment group or each condition, and these were shown in the figures.

[0130] Results and conclusions In a preliminary screening, the inventors first tested various commonly available β-glucans for their ability to induce canonical dectin-1 / NFκB signaling using reporter cells overexpressing HEK-dectin-1b. Well-characterized dectin-1 activators, including S. cerevisiae zymosan (a cell wall β-glucan preparation) and β-glucan peptide (BGP) from T. versicolor, induced NFκB signaling in a dose-dependent manner, similar to β-glucans of similar concentrations from fungal (schizophyllan), bacterial (curdlan), and lichen (pustulan) sources (Figure 1A). Wellmune® Dispersible (the particulate form of S. cerevisiae β-glucan in which the β-glucan layer of the yeast cell wall is isolated and forms “ghost cells” in an intact state), referred to herein as dispersed whole glucan particles (dWGP), also induced NFκB activation via dectin-1b. However, the soluble form derived from the same S. cerevisiae β-glucan (prepared by thermal degradation of dWGP, which results in a heterogeneous mixture of β-glucan chains of various molecular weights and lacks the intact single-cell structure of dWGP, referred to herein as soluble WGP (sWGP)) did not induce NFκB activation via dectin-1b.

[0131] Next, the ability of these β-glucans to induce monocyte training was tested by exposing freshly isolated human monocytes to the same β-glucan concentration for 24 hours. After washing and maturing into human monocyte-derived macrophages (hMDMs) for 5 days, the cells were restimulated with the TLR4 ligand lipopolysaccharide (LPS), and extracellular TNF production was measured as a readout of reactivation (Dominguez-Andres, J. et al. In vitro induction of trained immunity in adherent human monocytes. STAR Protoc 2, (2021)). When both BGP and LPS were used as their respective positive and negative controls for macrophage restimulation (Figure 1B, left and right ends), an over- and under-response of the dose-dependent TNF response indicating both training and tolerance was observed. A tendency for some training to occur at higher concentrations with other β-glucans containing curdlan was observed, but both thymosan and heat-killed C. albicans (HKCA) induced a high restimulation response at lower concentrations tested. Significant training was observed in monocytes treated with higher concentrations of dWGP (10 - 100 μg / mL), but the soluble form of this β-glucan (sWGP) did not enhance the response compared to control-treated cells. Interestingly, the ability of the various β-glucans tested to induce training did not appear to be related to their ability to induce standard dectin-1b NFκB signaling (Figure 1A and Figure 2A). Therefore, the inventors decided to use high molecular weight soluble synthetic peptides, BGP, as well as ghost yeast cell particles dWGP and its soluble form sWGP in future tests to clarify the requirements for training.

[0132] As suggested by the reporter assays with dectin-1b, the ability of various β-glucan preparations to promote a trained response is not related to their ability to induce pro-inflammatory signaling during the training phase. BGP treatment at the monocyte stage (24 hours after training) induced little TNF production, and in monocytes treated with dWGP, more modest levels were observed (right axis of Figure 1C) even though both have the ability to promote similar macrophage training (left axis of Figure 1C and Figure 2B). LPS, which induces a resistant response in macrophages at the concentrations (plural possible) used, stimulated significant TNF production at the monocyte stage (Figure 2C). In contrast, BGP treatment at the monocyte stage increased the survival rate of the resulting macrophages (5 days after treatment, Figure 2D), while dWGP, sWGP, or LPS treatment had no effect on macrophage survival.

[0133] Therefore, the inventors confirmed that dWGP β-glucan induces unique properties in trained cells and found that supernatants from monocytes trained with dWGP for 24 hours were unable to induce a high restimulation response to LPS at maturity when transferred to untrained monocytes (Figure 2E), demonstrating that training is not mediated by soluble factors. However, inhibition of intracellular DNA methyltransferase by pretreatment of monocytes with 5'-methylthioadenosine (MTA) prior to training with β-glucan was able to prevent the increased responsiveness of β-glucan-trained macrophages to both BGP and dWGP (Figure 1D). This confirmed that these diverse β-glucans train monocytes to increase their responsiveness to restimulation as a result of epigenetic modification of cell fate.

[0134] Training of innate immune cells by epigenetic modification enhances the induction of innate immune genes and quantitatively elicits a larger and earlier cytokine response. Training of monocytes induced by dWGP resulted in an increase in TNF production 3 hours after stimulation and a significant increase 6 hours after stimulation compared to untrained macrophages, whereas macrophages tolerized with LPS showed a decrease in the TNF response to restimulation (Figure 3A). Similar results were observed in macrophages trained with BGP (Figure 4A). Although training has been shown to induce the production of pro-inflammatory cytokines, training induced by β-glucan results in a systemic boost of immune signaling, and the induction of chemokines by macrophages (CXCL8), pro-inflammatory cytokines (TNF) and anti-inflammatory immunomodulatory cytokines (IL-10) were enhanced by both dWGP and BGP training compared to controls (Figure 3B, Figure 4B). This indicates that trained cells maintain regulation of their activation while responding more rapidly and efficiently.

[0135] Since trained innate immune cells can efficiently respond to restimulation by a variety of non-specific ligands, a major feature of trained innate immune cells may be that their recall ability is not related to the nature of the training stimulus. The inventors confirmed this in macrophages trained with dWGP, and an increase in TNF production was observed in macrophages restimulated not only with LPS as described above, but also with the TLR2 bacterial lipopeptide ligand PAM3CSK4, or in macrophages treated with heat-inactivated fungus Candida albicans (HKCA, Figure 3C). Similarly, when trained macrophages were challenged with irradiated Mycobacterium tuberculosis H37Rv (iMtb), the production of various cytokines (TNF, IL-1β, and IL-10) increased 24 hours after treatment, but LPS tolerance suppressed these responses (Figure 3D). The concept of enhancing the early innate immune response without causing prolonged inflammation with dysregulation is attractive in enhancing the response to infectious diseases such as those where early containment failure can lead to disease exacerbation (e.g., COVID-19 and tuberculosis (TB)). Therefore, the inventors tested the ability of macrophages trained with dWGP to contain intracellular infection. The inventors found that containment was superior in macrophages trained with dWGP compared to untrained macrophages after infection with Mtb H37Ra (72h) (Figure 3E). Interestingly, macrophages trained with dWGP infected with Mtb produce more TNF immediately after infection (Figure 3F, 3 hours), but the levels normalize at a time point after mycobacterial growth is blocked (72 hours), supporting the concept that macrophages trained with dWGP have superior natural antimicrobial power.

[0136] After characterizing the enhanced response to restimulation in macrophages trained with dWGP, the inventors sought to determine how β-glucan particles such as dWGP induce the training process in monocytes. Since both BGP and dWGP induced NFκB activation mediated by dectin-1 at comparable levels, the inventors investigated whether dectin-1 is required for the training downstream of these diverse β-glucans. It has previously been reported that soluble β-glucan blocks the activation of dectin-1 signaling by larger β-glucan particles by occupying sites on the receptor and preventing binding and subsequent signaling. Therefore, the inventors used Wellmune® Soluble (referred to herein as sWGP), a heterogeneous mixture of high and low molecular weight soluble β-glucans released from the particle structure, and laminarin, a soluble low molecular weight β-glucan derived from large algae. Both compounds blocked dectin-1b-mediated NFκB activation in reporter cells induced by both BGP and dWGP, and in both cases sWGP was slightly more effective (Figure 5A). Pretreatment of monocytes with sWGP prior to training blocked the enhancement of the restimulation effect seen with multiple concentrations of dWGP (Figure 6A), and similar results were obtained when laminarin and sWGP were compared in parallel in cells trained with both BGP and dWGP (Figure 5B), with laminarin being more effective at all doses tested.

[0137] Therefore, blocking the occupancy of the dectin-1 receptor may prevent the training of monocytes induced by higher molecular weight β-glucans. Although standard dectin-1 signaling uses the adapter protein SYK and there is a SYK-independent pathway, β-glucan-induced NFκB activation is SYK-dependent. The inventors confirmed this in reporter cells by blocking NFκB activation induced by dWGP and BGP by pretreatment with increasing concentrations of the SYK kinase inhibitor, piceatannol (PIC, Figure 5C). Similarly, pretreatment with PIC suppressed the training of monocytes induced by both BGP and dWGP as measured by the LPS restimulation response (Figure 6B), but was more effective at higher concentrations than that observed for NFκB activation. Similar results were observed in trained macrophages when NFκB activation was blocked downstream of dWGP using the IKKb inhibitor BAY11-7082 and TNF production induced by restimulation with LPS was abolished (Figure 6C). These data, combined with previous observations that various β-glucans can activate NFκB but not all induce monocyte training, suggest a model in which early NFκB activation is necessary but not fully sufficient to reprogram the myeloid response. Indeed, blocking early NFκB activation increased initial TNF production (Figure 5D), suggesting a branch in the β-glucan / dectin-1 response associated with training, despite impaired training in dWGP-treated cells. Therefore, the inventors attempted to identify additional cellular pathways and processes affected by the training β-glucan inducer.

[0138] β-Glucan derived from C. albicans has been shown to induce significant metabolic reprogramming in trained monocytes related to epigenetic modifications required for enhanced macrophage responsiveness. In particular, it has been revealed that the upregulation of cytosolic glycolysis induced by HIF1 is an important signal activated via the dectin-1 / PI3K / mTOR pathway. The inventors measured the ability of various β-glucans to induce glycolysis in trained monocytes by measuring extracellular lactate production over time. The results show that dWGP is a potent inducer of this process that is maintained as monocytes differentiate over time (up to 6 days post-treatment), whereas similar soluble β-glucan preparations (BGP, sWGP) are not (Figures 7A and 8A). Blocking the switch to glycolysis by targeting the gatekeeper enzyme hexokinase with 2DG abolished the ability of dWGP to increase the response to LPS restimulation (Figure 7B). The inventors found that monocytes treated with dWGP induced phosphorylation of the mTOR substrate S6, which indicates mTOR activation, in a rapamycin-dependent manner (gating strategy shown in Figures 7C-E, 8B). LPS also promotes this phenotype, but treatment with soluble sWGP does not. This upregulated mTOR activity is particularly prominent at early time points after treatment (2 hours, Figure 8C), but is maintained up to 24 hours after treatment when untreated monocytes upregulate mTOR as part of the normal differentiation process (Figure 7F). Pretreating macrophages trained with dWGP with rapamycin to block mTOR activity restricts the trained response to LPS restimulation, indicating that this early mTOR activity promotes β-glucan training (Figure 7G). mTOR activation downstream of dectin-1 supports reprogramming of the glycolytic system in trained monocytes by enhancing the expression of rate-limiting glycolytic genes such as SLCA1, HK2, PKM2, and LDHA, which were observed 3 days after training in cells trained with β-glucan (Figure 7H).Previous data using C. albicans β-glucan indicated that this glycolytic pathway was induced by PI3K signaling and, indeed, addition of wortmannin during the training phase abolished the trained response induced by WGP (Figure 7I). Interestingly, pretreatment of monocytes trained with dWGP with PIC was able to block conventional pro-inflammatory signaling and TNF production by monocytes (Figure 8D), yet did not affect the upregulation of mTOR (Figure 7J), suggesting that β-glucan-induced dectin-1 signaling leading to SYK activation was not required for mTOR activation. This data indicates a divergence from conventional dectin-1 signaling and suggests that the unique properties of particulate β-glucan may support an augmented metabolic response required for training.

[0139] Previous comparisons of soluble yeast β-glucan and particulate yeast β-glucan have shown that the particulate structure induces an antibacterial response that includes phagocytosis via the binding of dectin-1 clusters at the cell surface. The inventors measured the surface expression of dectin-1 on the cell surface by flow cytometry and found that dWGP tended to cause a loss of surface expression of dectin-1 immediately after treatment (15 minutes), which was not seen in soluble BGP β-glucan (Figures 9A and 10A). This effect was blunted by pretreatment with cytochalasin-D (cytD), an inhibitor of actin polymerization, indicating that this loss of dectin-1 surface expression by dWGP is phagocytosis-dependent (Figure 9B). Similarly, pretreatment with cytD before training with dWGP affected the ability of untrained cells to respond to LPS but selectively blocked the training effect observed after LPS restimulation in macrophages treated with dWGP (Figure 9C). Pretreatment with cytD also blocked the mTOR activation of monocytes induced by dWGP (Figure 9D). These data support the role of phagocytosis and internalization of the dWGP-dectin-1 receptor complex in inducing mTOR and subsequent metabolic responses. β-Glucan phagocytosis induced by the internalization of bound dectin-1 clusters results in phagosome-lysosome fusion. Lysosomes are also the sites of mTOR activation reported in many other situations, where the inventors hypothesized that β-glucan phagocytosis selectively induced by particles delivers the dectin-1 complex to lysosomes and activates the signaling mechanism necessary for mTOR activation. Therefore, when the inventors used bafilomycin A1 (which blocks the lysosomal membrane VATPase pump required for acidification) to prevent lysosome maturation, a significant decrease in monocyte mTOR activation by dWGP was observed, similar to the experiment using pretreatment with cytD (Figure 9E).

[0140] Since soluble β-glucan does not similarly induce mTOR activation, lactate production, or training, the inventors tested whether other forms of yeast β-glucan particles could induce metabolic signaling and training. Preparation of Wellmune® dispersed yeast ghost cells consisting of intact β-glucan cell walls can result in gross aggregation of particles as clusters. Disrupting these by sonication yields single, dispersible particles called dWGP (illustrated in Figure 10C). The unsonicated Wellmune® dispersed preparation (uWGP) induced NFκB activation in dectin-1b reporter cells (Figure 10D) and could induce significant early monocyte proinflammatory signaling as measured by TNF production 24 hours after treatment, to the same extent as that observed with dWGP (Figure 9F). However, uWGP treatment could not induce monocyte training as seen with dWGP. Indeed, while training with dWGP induced characteristically enhanced TNF production in response to LPS restimulation, macrophages treated with uWGP appeared to be dysfunctional in terms of their TNF response to restimulation (Figure 9G). Similarly, uWGP treatment of monocytes did not result in loss of cell surface dectin-1 expression equivalent to that of dWGP (Figure 9H), suggesting that these crude β-glucan clusters are too large to induce efficient phagocytosis. As a result, monocytes treated with uWGP did not upregulate extracellular lactate production (Figure 9I), underscoring the importance of internalization and phagocytosis of the dectin-1 / β-glucan complex in inducing training-related metabolic reprogramming.

[0141] The insolubility of intact particulate β-glucan is determined by the length and linkage of the glucose chains. Wellmune® dispersed particles (dWGP) are heterogeneous complexes of multiple cross-linked chain lengths with different molecular weights. These data suggest that the size and storage of β-glucan particles are important properties that control recognition, uptake, internalization, and engagement with intracellular metabolic machinery. Therefore, the inventors attempted to determine the optimal chain length required for training. Wellmune® soluble (sWGP) cannot induce training, but is the chemically identical form of β-glucan in dWGP where the β-glucan chains are released from yeast ghost cells to confer solubility. This represents a mixture of soluble non-particulate forms of glucan chains and heterogeneous molecular weights. Therefore, the inventors separated specific molecular weight fractions of the sWGP preparation such that each fraction represented pure β-glucan of various chain lengths defined by their molecular weight, and compared these in signaling assays to dWGP and sWGP formulations. Some of these fractions, particularly the low molecular weight fraction (F1, >100 kDa) induced NFκB activation in dectin-1 reporter cells, but none of these induced training to the same extent as dWGP (Figure 9J). Notably, these defined molecular weight soluble yeast-derived β-glucans enhanced the response to restimulation with LPS when compared to the sWGP preparation (Figure 9K), and together with the finding of improved signaling capacity in dectin-1b reporter cells, show that determining optimal training via dectin-1 is not the chain length and molecular weight of the β-glucan, but rather the overall solubility determined by the extraction method.

[0142] Soluble yeast-derived β-glucan can induce NFκB activation, but unlike dWGP particles, it does not induce training. Therefore, the inventors hypothesized that the recognition of particles is an important step in the training of monocytes and ensures the proper activation of metabolic reprogramming. To test this, the inventors bound well-mune soluble yeast β-glucan (sWGP) to 3 μm amino-functionalized polystyrene particles (AM-PS) as shown in the attached Figure 10E by the loss of sWGP in the bound preparation. Human monocytes were then stimulated with the resulting particles or controls, equal amounts of sWGP, AM-PS alone, or dWGP. Comparing the increase in extracellular lactate production after 3 days indicating upregulation of glycolysis, it was found that neither sWGP alone nor AM-PS particles alone changed cell metabolism (Figure 10L). However, sWGP bound to AM-PS increased the ability to induce lactate production, although to a lower extent than pure dWGP particles (Figure 9L). These data indicate that while β-glucan is required to activate conventional dectin-1 signaling, the recognition of β-glucan presented on microbial-sized particles is required to induce metabolic reprogramming and the cell's investment in the trained phenotype.

[0143] Many of the documents related to training use in vitro stimulation of monocytes. Therefore, to establish the relevance of the human β-glucan / dectin-1 pathway in which various monocyte subsets exist, the inventors examined the expression of dectin-1 across these cell populations. Compared with CCR2, a marker of inflammatory, migratory monocytes (Figure 11A), dectin-1 was present on classical inflammatory and intermediate macrophages gated for CD14 / CD16 expression, although the overall expression level was low. Dectin-1 was also present, although in lower amounts, on non-inflammatory CD14− / CD16+ monocytes (Figure 12A). When measured by TNF production 24 hours after stimulation, all three subsets responded to the initial treatment with WGP (Figure 11B), but only classical and intermediate monocytes showed enhanced LPS responses indicative of training upon restimulation (Figure 12B). Since training was not predicted by the presence or absence of dectin-1 on monocytes, the inventors examined other cell substrates for training in vivo.

[0144] HSPCs have been found to be sensitive to systemic delivery of training stimuli by various methods, including intravenous delivery of BCG, intraperitoneal injection of β-glucan from C. albicans, or induction of NLRP3 inflammation by a Western-type diet-induced hypercholesterolemia. In particular, the resulting inflammation leads to an increase in the total bone marrow HSPC number, accompanied by an increased skewing of the ratio of multipotent progenitor cells (MPP) towards MPP3, which commits to the myeloid lineage from the more dominant lymphoid MPP4 cells.

[0145] The inventors determined whether mouse bone marrow-derived macrophages are suitable for training. Training with dWGP, similar to BGP, resulted in enhanced LPS restimulation responses in BMDMs trained 5 days prior to restimulation (Figure 12C). This correlated with increased production of extracellular lactate by these cells (Figure 11C), supporting a model in which WGP reprograms mature mouse macrophages by metabolic reprogramming in vitro. The inventors examined whether a similar long-term immune training could be observed in vivo by examining HSPC progenitor cells in mouse bone marrow. Importantly, since mouse HSPCs express low levels of Clec7a mRNA, changes in the bone marrow compartment after systemic delivery of the training substance by intraperitoneal delivery were examined.

[0146] The inventors found that intraperitoneal injection of WGP, in a similar manner as previously observed with injection of BGP, led to a decrease in total bone marrow c-Lin - , ckit + , Sca-1 +(LKS+) HSPC cells were increased (Figure 12D), and it was found that this proliferation was particularly strong with low amounts of dWGPβ glucan. The absolute numbers of LT-HSPC and ST-HSPC were significantly increased (a representative flow chart is shown in Figure 11D along with quantification), which indicates an increase in the metabolic and proliferative activities required to give the turnover of HSPC, although the overall frequency of each subset did not change significantly, and a significant proliferation of more committed lineage MPPs (Figure 12E - F). However, when examining the more abundant subset of MPP cells (gating is shown in Figure 11E along with quantification), the inventors observed a significant expansion of MPP3 cells committed to the myeloid lineage in exchange for lymphoid progenitor cell MPP4, as observed with equivalent high concentrations of BGP in mice injected with both concentrations of dWGP employed (Figure 12). There was no significant change in the abundance of megakaryocyte-related progenitor cell MPP2 between each treatment. In addition to the increase in myelopoiesis, the training stimulus has been shown to reprogram HSPCs to enhance their activity at maturity, consistent with what has been observed with the training of peripheral monocytes. Spleens were isolated immediately after sacrifice to prepare splenic macrophages, which were stimulated with the TLR4 ligand LPS or heat-killed Mtb (hk-Mtb) at various concentrations. Splenic macrophages from animals trained in vivo with BGP or low concentrations of dWGP showed a significant enhancement of TNF production compared to splenocytes from control-injected mice (Figure 12H). These data indicate that the intraperitoneal delivery of particulate β-glucan can result in the reprogramming of the bone marrow of mice, quantitatively increasing the turnover of bone marrow cells and qualitatively changing their phenotype, such that the resulting mature cells show a high response to activation.

[0147] One of the major problems in the field of trained immunity is the lifespan of the trained effect. Therefore, the inventors conducted a time-course analysis of the effect of intraperitoneal injection of dWGP on myelopoiesis. Consistent with the previous results, when 0.2 mg of dWGP was injected into C57 / BL6-JOlaHsd mice, a significant increase in the percentage (%) of MPP3 cells was observed in exchange for MPP4 cells one week after injection (Figure 13A). This was not seen one day after dWGP injection, was maintained until two weeks after injection, but disappeared by three weeks. Importantly, splenocytes extracted from these mice showed enhanced response to LPS stimulation, which was particularly prominent and maintained for three weeks after injection (Figure 13B). These results are consistent with previous studies on β-glucan injection, which showed dynamic remodeling of myeloid progenitor cells over time.

[0148] β-Glucan represents a major class of indigestible dietary fiber present in many foods and supplements. Yeast β-glucan is particularly well-tolerated and safe. The inventors analyzed whether dietary intake of Wellmune (registered trademark) dispersed type (dWGP) could promote a training effect in vivo. A dose equivalent to the intraperitoneal injection amount (0.2 mg of dWGP) was administered by forced oral gavage (OG). Bone marrow was collected one week after OG, and HSPC subsets were examined. An increase in the total HSPC number similar to that observed in previous intraperitoneal (IP) injections was not observed by OG administration (data not shown), but the ratio of MPP3 cells committed to the myeloid lineage increased in mice administered dWGP via both the IP and OG routes (Figure 13C). When splenocytes from these mice were stimulated with LPS, an increase in TNF production was observed in mice administered dWGP by forced oral gavage (Figure 13D). These qualitative and quantitative differences in the bone marrow effect are likely the result of different administration routes of the training substance, but this data supports the concept that β-glucan intake can enhance innate immune function.

[0149] To examine this in a more relevant context, the inventors planned a study in which increasing doses of dWGP added as a well - known commercial ingredient were fed to groups of mice for up to 3 weeks in parallel with a control diet to which an inert indigestible dietary fiber (inulin) was equally added to match dietary energy and fiber intake. Again, in mice fed dWGP, there was no increase in the total bone marrow LKS+ cell number as observed in the previous OG test (Figure 14C). Nevertheless, dynamic changes were observed in the LKS population over time. In mice after 1 week of feeding, the frequency of LT - HSCs increased, and then, by 3 weeks after feeding, this population decreased as a result compared to the control, and in parallel, the percentage of more committed MPP cells at this same time point increased (Figures 14D - E). Most importantly, the ratio of MPP3 committed to the myeloid lineage to MPP4 committed to the lymphoid lineage was significantly increased in mice fed a dWGP - containing diet compared to the control diet, particularly 3 weeks after the start of feeding (Figure 13E). Significantly, mature BMDMs derived from the bone marrow of these mice showed differences in functional responses to stimulation. BMDMs prepared from mice after 1 week of feeding showed enhanced TNF production in response to stimulation with LPS or hk - Mtb, which was not evident in BMDMs obtained from mice fed for 3 weeks (Figures 13F - G). In particular, cytokine production was enhanced 6 hours after stimulation in mice fed the Wellmune® dispersed form and became less clear 24 hours after stimulation (Figures 14G - H), consistent with a training reprogramming effect enhancing the dynamics of innate immunity. Notably, the differences in cytokine production were more evident in mice given the maximum amount of dWGP, whereas the effect on HSPC proliferation was more evident in mice given a lower amount of dWGP, suggesting that the qualitative and quantitative effects on training - induced bone marrow reprogramming can be decoupled and that these can be differentially involved in the oral intake of training reagents. In conclusion, the Wellmune® dispersed form is a yeast - derived β - glucan particle that can induce a trained response in innate immune cells (both monocytes and bone marrow progenitor cells).

[0150] To do this, the inventors designed control diets containing inert indigestible dietary fiber (inulin) to match calorie consumption and dietary fiber effects, and various mouse feeds (Carpenter, K. C., et al., Baker’s yeast beta - glucan supplementation increases monocytes and cytokines post - exercise:implications for infection risk?Br J Nutr 109, 478 - 486,(2013)) containing increasing amounts of dWGP in increasing doses, present as the composition “Wellmune”. C57 / BL6OwaJ mice were fed these Wellmune - containing diets for up to 4 weeks and the bone marrow populations were examined. Unlike BGP - or dWGP - injected mice, the inventors did not observe an increase in total bone marrow LKS+ cell numbers in mice fed WGP (Figure 12A).

[0151] However, when the inventors examined the relative frequency of specific HSPC populations, they observed an increase in the frequency of LT-HSCs in mice after one week of feeding, after which this population was ultimately decreased compared to controls three weeks after feeding, and the percentage of more committed MPP cells increased at the same time point (Figure 12B). This data indicates that the dramatic expansion of the HSPC population that occurs after an inflammatory challenge (intraperitoneal injection) does not occur in mice fed the training substance, but that there is still a dynamic remodeling of HSPC progenitor cells in these mice. The ratio of MPP3 committed to the myeloid lineage to MPP4 committed to the lymphoid lineage did not change significantly (Figures 12C, 13B), but when the inventors prepared mature BMDMs from these bone marrows, differences in functional responses to stimulation were observed. BMDMs prepared from mice after one week of feeding showed enhanced TNF production in response to stimulation with LPS or hk-Mtb, which was less evident in BMDMs obtained from mice fed for three weeks (Figures 12D - E). In particular, cytokine production was enhanced 6 hours after stimulation and less pronounced 24 hours after stimulation (Figures 13C - D), consistent with a reprogramming effect of training that enhances the dynamics of innate immunity. Notably, the differences in cytokine production were more evident in mice given the maximum amount of WGP, whereas the modest effect on HSPC expansion was more evident in mice given a lesser amount of WGP, suggesting that the qualitative and quantitative effects on bone marrow reprogramming by training can be decoupled and that they may be differentially involved in the oral ingestion of the training reagent. In conclusion, WGP is a yeast-derived β-glucan particle that can induce a trained response in innate immune cells (both monocytes and bone marrow progenitor cells).

[0152] Further feeding trials were conducted in C57 / BL6 mice fed a control diet (Wellmune®-free, i.e., without dispersed Wellmune® or dWGP) or a high Wellmune® diet (0.05% dWGP) for 0 - 12 weeks. Mice were sacrificed and bone marrow was harvested. The HSPC cell population was measured by flow cytometry (Figures 15A - B), and after 4 weeks of WGP feeding, a significant increase in the percentage (%) of the MPP3 population committed to the myeloid lineage was shown. However, this decreased to control levels after 12 weeks of feeding. Furthermore, these bone marrow cells were matured into BMDMs and restimulated with TLR ligands. BMDMs from Wellmune®-fed mice showed an increase in TNF production after restimulation in mice fed Wellmune® for 4 and 8 weeks, but not in mice fed Wellmune® for 12 weeks. This data is consistent with previous data showing that Wellmune® feeding does not induce an increase in HSPC numbers, but affects the frequency and turnover of HSPC cells in the bone marrow and optimally induces skewing of MPP3 committed to the myeloid lineage over a 3 - 4 week period, demonstrating central training in vivo. This effect is not maintained after 8 weeks of feeding.

[0153] Therefore, mice fed Wellmune® via the diet exhibit features of trained immunity, accompanied by time-dependent reprogramming of the bone marrow HSPC population and enhanced innate immune function of mature macrophages derived from these cells.

[0154] Administration of WGP by forced oral gavage results in enhanced responsiveness to activation in mature macrophages derived from these bone marrows (BMDMs).

[0155] The inventors have previously demonstrated that IP injection of WGP induces a significant increase in the number of LKS+ HSPC cells in the mouse bone marrow, as well as an increase in the proportion of the MPP3 subset committed to the myeloid lineage. However, administration of an equal dose of WGP by forced oral gavage does not result in the same increase in the number of LKS+ HSPC cells observed with intraperitoneal injection (Figure 16A). However, within the LKS+ population, the proportion of the MPP3 subset committed to the myeloid lineage is increased in both WGP-injected mice and mice administered WGP by forced oral gavage (Figure 16B). The inventors show below, beyond previous data, that this bone marrow containing these expanded MPP3 populations, when grown to mature macrophages (bone marrow-derived macrophages), exhibits enhanced function. When stimulated with low concentrations of the TLR4 ligand LPS, production of the moderately pro-inflammatory cytokine TNF is observed in BMDMs of control mice. However, this is enhanced and accelerated in BMDMs derived from mice administered WGP by IP (Figure 16C), consistent with enhanced function as a result of trained immunity. BMDMs derived from mice administered WGP by forced oral gavage showed an increase in the level of TNF production compared to control BMDMs, although at a slower rate than that observed in trained mice by intraperitoneal administration (Figure 16C). These data indicate that oral administration of WGP results in some of the features associated with trained immunity and does not share all of the features observed with intraperitoneal injection, which is consistent with delivery via the more permissive environment of the gut, but shows increased sensitivity to activation at low doses.

[0156] Feeding a diet containing Wellmune WGP for 4 weeks resulted in an increase in precursors committed to a more mature, common myeloid lineage, consistent with enhanced responsiveness to activation in mature macrophages derived from these bone marrow (BMDM). Our previous data support that there is rapid proliferation of early immune cell precursors after feeding WGP, which begins to contract and return to baseline levels despite continued feeding. To determine whether feeding WGP results in a long-term response in more committed, more mature precursors, we conducted a new feeding trial focusing this time on 4 weeks of addition of the highest concentration of WGP. Since inulin was used as a control fiber in previous feeding studies, we compared the same amount of WGP with the same amount of inulin (with respect to fiber content), including an additional control diet without inulin, to confirm that some of the previously observed dose-dependent effects were not due to inulin. As before, no increase in total LKS+ precursor cell numbers was measured (Figure 17A). Consistent with previous results, an increase in the proportion of MPP3 cells committed to the myeloid lineage in the LKS+ population was measured (Figure 17B), indicating increased myelopoiesis. To determine whether the change in this low-abundance population resulted in an increase in cells further downstream in the hematopoietic tree, we measured myeloid lineage precursors in the LKS(-) fraction of bone marrow cells. We observed a significant increase in total myeloid lineage precursors, as well as an increase in GMP and CMP, which are granulocyte and macrophage precursors, along with a significant decrease in CLP, a lymphocyte lineage precursor (Figure 17C). Mature BMDM derived from WGP-supplemented mice showed enhanced production of TNF and IL6 after LPS stimulation (Figure 17D), supporting the idea, consistent with innate immune memory, that reprogramming at the level of early precursors recovers to baseline levels after 4 weeks of feeding, but functional changes persist in more mature cells. Importantly, all of these trained-immunity-like effects were limited to the Wellmune WGP-supplemented group, and the behavior of mice fed inulin was similar to that of mice fed a control diet.

[0157] Oral administration of Wellmune WGP β-glucan alters the immune composition of the intestinal mucosa.

[0158] Tests from the intraperitoneal administration of β-glucan or the intravenous administration of BCG to induce trained immunity suggest that local innate immune cells induce cytokines that affect bone marrow hematopoiesis. Oral administration of Wellmune WGP β-glucan, both by forced gavage and addition to the mouse diet, results in bone marrow hematopoiesis and supports the innate immune response in progeny macrophages, so the effect on intestinal immune cells was examined. Mice fed a diet supplemented with Wellmune WGP β-glucan (WGP) for 4 weeks showed no change in total CD45-positive immune cells in the small intestine compared to mice fed a standard diet (SFD, Figure 18A, right panel). However, when the proportion of monocytes was measured, this was significantly enhanced in mice given WGP (Figure 18A, left panel). This suggests that monocytes migrate to that site after WGP delivery to the intestine, which indicates an inflammatory response. To determine whether these cells contain β-glucan, the inventors adopted a strategy of fluorescently labeling Wellmune WGP β-glucan using DTAP. This was the one adopted in a recent report examining the intraperitoneal administration of WGPβ-glucan, which showed the accumulation of WGPβ-glucan-positive macrophages in the pancreas. The inventors also observed this in preliminary experiments after intraperitoneal administration (Figure 18B, blue dots). Interestingly, this was not seen when the labeled WGP β-glucan was administered by forced gavage. However, at this time point (3 days after administration), WGPβ-glucan-positive macrophages were measured in the lamina propria of the colon. This data supports that β-glucan is sensed by innate immune cells that reside in or are mobilized to the intestinal tract where an inflammatory response that can affect bone marrow hematopoiesis is initiated.

[0159] Applicability of WGP to restore defective innate immune function in a model of obesity. Dysregulation of inflammation is now recognized not only to lead to the development of obesity-related diseases such as diabetes, but also to increase the risk of cancer and severe infections. Using a diet-induced obesity model in which C57 / BL6 mice are fed a high-fat diet (HFD) for 12 weeks, obesity and related metabolic dysfunctions are modeled in mice. Bone marrow-derived macrophages (BMDMs) obtained from these HFD mice showed changes in cytokine patterns induced by LPS (Figure 19A) compared to mice fed a standard chow diet (SFD) without obesity, with a significant increase in the pro-inflammatory TNF and a decrease in the immunomodulatory IL-10 production. This is consistent with the loss of immunomodulatory responses and chronic inflammation underlying the obese state. However, when Wellmune WGP β-glucan (HFD-WGP) is added to the HFD, the BMDM cytokine response shows a more controlled response similar to that of SFD mice (Figure 19A), with a smaller increase in TNF and IL-10 levels compared to BMDMs from HFD-fed mice. Since obese individuals are more susceptible to infections and it has become clear that diabetes is a risk factor for TB, the inventors tested the response to infection with the causative bacterium Mycobacterium tuberculosis (Mtb) in these mouse-derived BMDMs. Consistent with the loss of innate immune function, intracellular Mtb could not be contained in macrophages from HFD-fed mice and was found at significantly higher levels than those observed in BMDMs from SFD-fed mice at a later time point (72 h) after infection (Figure 19B). However, HFD mice supplemented with the β-glucan of Wellmune WGP showed improved containment of Mtb, as seen in macrophages from SFD-fed mice. These data support the idea that obesity promotes a state of dysregulated inflammation that affects host defense functions. In this dysregulated state, oral supplementation with WGP β-glucan may correct at least some of these immunodeficiencies and restore normal innate immune responses.

[0160] Other yeast-derived β-glucan particles can also induce trained immunity in vitro To test whether the trained immune responses reported in well - munched WGP are specific to this molecule or are conserved features of β - glucan particles, the inventors used Thymosan, a generally available research - grade β - glucan (a cell wall preparation rich in β - glucan from Saccharomyces cerevisiae). Figure 20A shows that crude Thymosan (obtained from Invivogen) activates not only dectin - 1, a β - glucan receptor, but also other innate immune receptors including TLR2 and 4. However, a purer version of this, called Thymosan - depleted (ZYM - d), from which contaminating non - β - glucan ligands have been removed, does not induce TLR2 / 4. While crude Thymosan results in a tolerance - like phenotype in the training assay, pure ZYM - d induces a trained immune response more similar to that observed with dWGP (Figure 20B). In human monocytes and mouse macrophages (Figures 20C - D), pure ZYM - d induced a trained response, and an enhanced kinetics of TNF production was observed upon restimulation with LPS.

[0161] Discussion The inventors have revealed that the recognition of intact fungal particles and subsequent internalization via phagocytic synapses are closely related to the metabolic investment into a trained phenotype via the binding of mTOR by target cells. In these studies, particles rich in artificial β-glucan that bind to dectin-1 in this way were used, which avoids inappropriate and wasteful resources in the long-term memory response to soluble ligands or non-viable pathogens, and rather represents a conserved pathway of the innate immune system to promote a trained phenotype only when necessary in response to intact fungal particles. This includes reprogramming cellular metabolism to adopt a glycolytic profile only when lysosomal mTOR is involved without hindrance following phagocytosis of intact particles. This deep involvement in glycolysis is associated with epigenetic changes that result in an accelerated response to the trained phenotype and non-specific restimulation. After observing this pathway in monocytes, which are short-lived unless they migrate to tissues as macrophages, the inventors also observed that particles rich in artificial β-glucan (dWGP) not only affect myeloid progenitor cells in the bone marrow, but also have the ability to enhance the functional responses of mature macrophages. These functional responses include cytokine responses. Therefore, by utilizing the physical and chemical requirements for an optimal trained response, it is possible to improve innate immune function for therapeutic approaches such as vaccination or immunotherapy, or to promote an increase in resistance to new pathogens without existing acquired memory such as the current spread of COVID-19. In the post-COVID world, long-term immunomodulatory deficiencies have been reported, and the inventors have hypothesized that β-glucan training to produce a balanced natural response can prevent an increase in the severity of other infectious diseases.

[0162] Wellmune® (dWGP) itself represents a mannan-rich artificial β-glucan preparation from which contaminating TLR2 ligands and other mannans have been removed. In C. albicans, it has been observed that the mannan in the outer cell wall blocks the interaction of β-glucan with the innate immune system and induces phagocytosis, thus revealing that mannan is an immune evasion strategy to prevent host education by β-glucan exposure. Crude timosan represents a particulate S. cerevisiae yeast cell wall preparation containing β-glucan masked by a mannan layer, but it cannot induce the same level of trained immunity in vitro; instead, it promotes an acute inflammatory response. Removal of these mannans confers the ability to metabolically reprogram and train cells with depleted timosan preparations. Therefore, the finding that Wellmune WGP and other yeast-derived β-glucan particle preparations constitute potent inducers of trained immunity may actually be an artificial observation that does not occur in nature due to masking by mannan. However, this is knowledge that can be utilized to enhance immune function.

[0163] This study has revealed that what affects the interaction with the innate immune system is not the molecular weight of the β-glucan chains in the mixture, but ultimately the physical properties and the way these ligands are presented to the innate immune cells, i.e., their insoluble properties determined by the extraction process that preserves the intact yeast cell wall with high purity. Soluble yeast β-glucan is used in both immunotherapeutic approaches and nutritional supplementation strategies. These data suggest that Wellmune® soluble form cannot train, but instead blocks receptor occupancy by high molecular weight particulate β-glucan, while other soluble β-glucans, such as the BGP from Trametes versicolor, can induce trained responses both in vitro and in vivo. As shown in the training model using the enhanced growth factor signaling associated with differentiation recently demonstrated for C. albicans (β-1,3)-glucan or other yeast β-glucan preparations, the underlying mechanism can be accompanied by an increase in proliferation without significant glycolytic activity that epigenetically changes cell fate (Figure 12A), but still remains unclear.

[0164] As used herein, WGP is sold as Wellmune® dispersed form and has been studied for its effects on circulating cytokine levels and LPS-induced monocyte production after oral consumption in elite marathon runners as well as in immunocompromised populations. The inventors' discovery that oral consumption of WGP has biological and training-like effects in mice, consistent with in vitro training assays in human monocytes, opens up new avenues for the commercial and therapeutic use of this group of molecules. Notably, there was little effect on murine hematopoiesis via two different pathways examined. Herein, the inventors show that WGP affects hematopoiesis by both quantitatively increasing HSPC and qualitatively skewing towards myeloid hematopoiesis.

[0165] Macrophages trained with WGP show both increased clearance of Mtb and upregulation of glycolytic metabolism. Therefore, metabolically reprogrammed trained macrophages are thought to be metabolically primed to be more hostile to intracellular pathogen replication. Indeed, macrophages trained with WGP produce more pro-inflammatory cytokines at early time points after infection with Mtb, but cytokine levels normalize by the time macrophages eliminate more Mtb, supporting the concept that training epigenetically primes and does more than just change cytokine kinetics. In the studies of the present inventors, mTOR activation is positioned as the center of the training induced by β-glucan microparticles. The present inventors have demonstrated that mTOR activation is upregulated by dWGP in a rapamycin-sensitive form. S6K itself is an important ribosomal protein that induces protein synthesis activated by mTOR in response to changes in lysosomal activity, and its activation during the training phase induced by dWGP suggests that large-scale proteome and metabolic changes can occur and persist throughout maturation in trained macrophages.

[0166] In conclusion, the present inventors have identified a new immunomodulatory role of yeast-derived microparticle β-glucan by inducing metabolic reprogramming in target cells required for trained immunity. This may impact strategies using these and similar β-glucans to boost innate immune function and may be useful in promoting innate immune resistance to infections.

Claims

1. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use in a method for programming bone marrow hematopoietic stem and progenitor cells (HSPCs) to promote the proliferation of target bone marrow hematopoietic hematopoiesis-oriented pluripotent progenitor cells (MPPs), wherein the β-glucan is in the form of substantially spherical whole glucan particles, and the β-glucan is administered orally.

2. A sufficient amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 1, wherein bone marrow hematopoiesis produces trained innate immune cells.

3. The effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 2, wherein the immune cells are monocytes and / or monocyte-derived macrophages.

4. An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 1, for preventing, delaying the onset of, or reducing the severity of a disease or infection in the subject.

5. An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 1, wherein the subject is in the early stages of a disease or infection, at risk of a disease or infection, or suspected to have a disease or infection.

6. An effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 1, wherein the subject is one or more selected from the group including healthy subjects, athletes, subjects experiencing or suffering from stress, immunocompromised subjects, obese subjects, diabetic subjects, subjects over 65 years of age, preferably over 75 years of age, subjects under 16 years of age, subjects with or recovered from cancer, and subjects with innate immune deficiency.

7. The subject is a healthy subject, and the effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 6.

8. An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 6, wherein the subject is obese.

9. The subject having post-infectious disease immunodysregulation, the subject, in an effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 1.

10. The effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 1, wherein the infection is SARS-CoV-2 infection.

11. The effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan according to claim 1, wherein the (1→3)-β-D-glucan is β-1,6-branched β-1,3-glucan (or "β-(1,3 / 1,6)").

12. The (1→3)-β-D-glucan is a β-1,6-branched β-1,3-glucan with the following structure: 【Chemistry 2】 An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 11, comprising the above.

13. The WGP has a size of about 1 to about 6 microns (i.e., μm), or about 2 to about 5 microns, or about 3 to about 4 microns, and is an effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 1.

14. Provided as a nutritional supplement containing the (1→3)-β-D-glucan derived from Saccharomyces cerevisiae, an effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 1.

15. The nutritional supplement contains 75% or more β1,3 / 1,6 glucan on a dry weight basis, wherein the effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 14.

16. The Saccharomyces cerevisiae-derived (1→3)-β-D-glucan is Wellmune®, wherein the effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 1.

17. The β-glucan is administered on a cyclical schedule in an effective amount of Saccharomyces cerevisiae-derived β-glucan for use according to claim 1.

18. The cyclical schedule is a block of 3 to 4 weeks, wherein an effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 17.

19. The cyclical schedule is a four-week block, wherein an effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 18.

20. An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use in a method for inducing trained innate immunity in a subject, wherein the β-glucan is in the form of substantially spherical whole glucan particles, and the β-glucan is administered orally.

21. An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use in a method for producing an enhanced immune response to an invading pathogen within a subject, wherein the β-glucan is in the form of substantially spherical whole glucan particles, and the β-glucan is administered orally.

22. An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use according to claim 21, wherein the β-glucan is administered in the early stages of a disease or infection when the subject is at risk of disease or infection, when the subject is suspected of having a disease or infection, and / or when the subject exhibits symptoms of a disease or infection.

23. An effective amount of (1→3)-β-D-glucan derived from Saccharomyces cerevisiae for use in treating post-infection immunomodulation in the target area.

24. The effective amount of Saccharomyces cerevisiae-derived (1→3)-β-D-glucan for use according to claim 23, wherein the infection is SARS-CoV-2 infection.

25. A composition comprising multiple types of β-glucans, each derived from a different strain of Saccharomyces cerevisiae.

Images