Method for inducing trained immunity

Abstract

A method for inducing trained immunity in a subject in need thereof is provided. The method include: administering to the subject an effective amount of a recombinant lipidated FLIPr (rLF) protein. The rLF protein is administered without a specific antigen.

Description

TITLEMETHOD FOR INDUCING TRAINED IMMUNITYCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 754,076, filed on February 5th, 2025. The content of the application is incorporated herein by reference.

[0002] The present application hereby incorporates by reference the entire contents of the text file named “NHR-P0042-PUS-Sequencing Listing.xml” in XML format. The text file containing the Sequencing Listing of the present application was created on January 28, 2026 and is 4,873 bytes in size.BACKGROUND OF THE INVENTION

[0003] 1. FIELD OF THE INVENTION

[0004] The present disclosure relates to a method for inducing trained immunity, and more particularly, to a method for inducing trained immunity by an effective amount of a recombinant lipidated FLIPr (rLF) protein.

[0005] 2. DESCRIPTION OF THE PRIOR ART

[0006] Conventional vaccine strategies are mainly developed to stimulate adaptive immunity, relying on the selection of specific antigens to elicit antigen-specific antibodies or T-cell responses. The disadvantages and limitations of the conventional vaccine strategies include the restriction of the protective effect. The protective effect is typically restricted to the specific pathogen from which the antigen is derived, offering limited cross-protection against heterologous strains or emerging pathogens. For example, the efficacy of seasonal influenza vaccines significantly diminishes when there is a mismatch between the vaccine strains and the circulating viruses. Furthermore, developing effective adaptive vaccines against certain pathogens, such as multi-drug resistant bacteria or rapidly mutating viruses, remains a persistent challenge globally.

[0007] Recently, trained immunity, where innate immune cells undergo epigenetic and metabolic reprogramming after an initial stimulus, leading to an enhanced response toward subsequent and unrelated triggers, is introduced. P-glucan is a well-studied inducer of trained immunity, operating through the Dectin- 1 / Raf-l signaling axis and primarily relies on Type I interferon dependent pathways. However, P-glucan and other known inducers may not provideoptimal protection against all types of infections or diseases, and specific mechanisms of action of these trained immunity inducers often limit the clinical application.

[0008] In view of the foregoing, it is necessary to provide a novel trained immunity inducer capable of inducing trained immunity through distinct biological pathways, to provide an immediate, a broad-spectrum, and a long-lasting protection against heterologous pathogens without the requirement for co-administration with specific antigens.SUMMARY OF THE INVENTION

[0009] To solve the aforementioned problems, the present disclosure provides a method for inducing trained immunity in a subject in need thereof, including: administering to the subject an effective amount of a recombinant lipidated FLIPr (rLF) protein. The rLF protein is administered without a specific antigen.

[0010] These and other objectives of the present disclosure will no doubt become understandable to those of ordinary skill in the art after reading the following detailed description of the preferred embodiment that is illustrated in the various figures and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] FIG. 1 is a schematic diagram of mechanism of trained immunity induced by recombinant lipidated FLIPr (rLF) according to an embodiment of the present disclosure.

[0013] FIG. 2 A and FIG. 2B illustrate that rLF training increases IL-6 and TNF-a production upon LPS restimulation in mouse white blood cells (WBCs) and human peripheral blood mononuclear cells (PBMCs) according to an embodiment of the present disclosure. Panel (1) of FIG. 2A is a schematic representation of the in vitro training model. Mouse WBCs (panel (2) of FIG. 2A and panel (3) of FIG. 2A) and human PBMCs (panel (4) of FIG. 2A; panel (5) of FIG.2 A; panel (6) of FIG. 2 A; panel (1) of FIG. 2B; panel (2) of FIG. 2B; and panel (3) of FIG. 2B) are trained with various doses of recombinant LF (rLF) for one day and rested for three days. Cells are then stimulated with or without lipopolysaccharide (LPS), and supernatants are collected 24 hours later to measure IL-6 (panel (2) of FIG. 2 A; panel (4) of FIG. 2 A; panel (5) ofFIG. 2 A; and panel (6) of FIG. 2 A) and TNF-a (panel (3) of FIG. 2 A; panel (1) of FIG. 2B; panel (2) of FIG. 2B; and panel (3) of FIG. 2B) levels by ELISA. Data are shown as mean ± SEM. Statistical significance is determined by one-way ANOVA with Dunnett' s post hoc test versus0.0001; ***P< 0.001; **P<0.01.

[0014] FIG. 3 illustrates rLF training promotes proinflammatory mediators release and recruits innate immune cells upon LPS restimulation in mice according to an embodiment of the present disclosure. BALB / c mice (n=7) are intranasally administered rLF (5 pg / dose) or PBS once weekly for three consecutive weeks, followed by LPS stimulation (10 pg) at week 3. Bronchoalveolar lavage fluid (BALF) is collected 3.5 hours after LPS stimulation. A schematic of the in vivo training model is shown (panel (1) of FIG. 3). Levels of IL-6, TNF-a, and RANTES in BALF are measured by ELISA (panel (2) of FIG. 3). Macrophages, neutrophils, monocytes, and NK cells are analyzed by flow cytometry (panel (3) of FIG. 3). Statistical significance is determined using an unpaired t test versus the PBS group. ****P < 0.0001; ***P<0.001; **P<0.01; *P<0.05.

[0015] FIG. 4 illustrates rLF training promotes progenitor cell expansion in mice according to an embodiment of the present disclosure. BALB / c mice (n=5-6) are intranasally administered rLF (5 pg / dose) or PBS once weekly for three consecutive weeks. Bone marrow is collected at week 3. The experimental scheme is shown (panel (1) of FIG. 4). The hematopoietic stem cell pool (panel (2) of FIG. 4) and progenitor populations (panel (3) of FIG. 4) in the bone marrow are analyzed by flow cytometry. Statistical significance is determined using an unpaired t test versus the PBS group. ***P < 0.001; **P < 0.01; *P < 0.05.

[0016] FIG. 5A and FIG. 5B illustrates training in severe combined immunodeficient (SCID) and gene knockout mice reveals distinct profiles of proinflammatory cytokine release and innate immune cell recruitment upon LPS restimulation according to an embodiment of the present disclosure. SCID (n=ll) (panel (1) of FIG. 5 A), toll-like receptor 2 knockout (TLR-2 knockout) (n=6-7) (panel (2) of FIG. 5A), type I interferon receptor knockout (IFNAR-knockout) (n=8) (panel (3) of FIG. 5A), IFNyR-knockout (n=12) (panel (1) of FIG. 5B), and signal transducer and activator of transcription 1 -knockout (S TATI -knockout) (n=6) (panel (2) of FIG. 5B) mice are intranasally administered rLF (5 pg / dose) or PBS once weekly for three consecutive weeks, followed by LPS stimulation (10 pg) at week 3. Bronchoalveolar lavage fluid (BALF) is collected 3.5 hours after LPS stimulation. Levels of IL-6, TNF-a, and RANTES in BALF are measured by ELISA. Macrophages, neutrophils, monocytes, and NK cells are analyzed by flowcytometry. Statistical significance is determined using an unpaired t test versus the PBS group. ****p<0.0001; ***P<0.001; **P<0.01; *P<0.05.

[0017] FIG. 6A and FIG. 6B illustrate rLF training enhances resistance of mice to virus infection according to an embodiment of the present disclosure, (panel (1) of FIG. 6A) BALB / c mice are intranasally administered rLF (5 pg / dose) or PBS once weekly for three consecutive weeks. H7N9 virus is intranasally administered at week 3. (panel (2) of FIG. 6A) Viral loads (n=8) in the lungs are assessed on day 3 post-infection. Statistical significance is determined using an unpaired t test versus the PBS group. *P < 0.05. (panel (3) of FIG. 6A and panel (4) of FIG. 6A) A subset of mice (n=10) is monitored for body weight changes (panel (3) of FIG. 6A) and survival (panel (4) of FIG. 6A) over 14 days post-challenge, (panel (5) of FIG. 6A and panel (6) of FIG. 6A) Mice (n=12) are challenged at week 8, and body weight (panel (5) of FIG. 6A) and survival (panel (6) of FIG. 6A) are monitored for 14 days, (panel (7) of FIG. 6A and panel (8) of FIG. 6 A) Mice (n=12) are challenged at week 11, and body weight (panel (7) of FIG. 6 A) and survival (panel (8) of FIG. 6A) are monitored for 14 days, (panel (1) of FIG. 6B and panel (2) of FIG. 6B) To assess the role of neutrophils, rLF -trained mice (n=8) are treated with anti-Ly6G-depleting antibodies or isotype controls one day prior to challenge. Body weight (panel (1) of FIG. 6B) and survival (panel (2) of FIG. 6B) are monitored, (panel (3) of FIG. 6B and panel (4) of FIG. 6B) To assess the role of macrophages / monocytes, rLF-trained mice (n=8 or 10) are treated with clodronate-encapsulated liposomes or control liposomes one day prior to challenge. Body weight (panel (3) of FIG. 6B) and survival (panel (4) of FIG. 6B) are monitored. Mice trained with rLF or PBS alone are included as reference groups.

[0018] FIG. 7A and FIG. 7B illustrate rLF training alters the transcriptional profile in neutrophils according to an embodiment of the present disclosure, (panel (1) of FIG. 7A) BALB / c mice are intranasally administered rLF (5 pg / dose) or PBS once weekly for three consecutive weeks, followed by LPS stimulation (10 pg) at week 3. Peripheral blood is collected 3.5 hours after LPS stimulation, and neutrophils are enriched by density gradient centrifugation for transcriptomic profiling. A schematic of the in vivo training model is shown, (panel (2) of FIG. 7A) Volcano plot showing -logw(p. adjust) versus log2(fold change) for all detected genes, (panel (3) of FIG. 7A) Top 20 pathways are identified by gene ontology enrichment analysis in rLF-trained mice compared with PBS-trained mice, (panel (1) of FIG. 7B; panel (2) of FIG. 7B; and panel (3) of FIG. 7B) Gene set enrichment analysis for genes related to neutrophil chemotaxis (G0:0030593), regulation of leukocyte degranulation (G0:0043300), and production of molecular mediator involved in inflammatory response (G0:0002532).

[0019] FIG. 8 illustrates rLF training increases histone 3 lysine 27 acetylation (H3K27ac) enrichment in neutrophils according to an embodiment of the present disclosure, (panel (1) of FIG. 8) BALB / c mice are intranasally administered rLF (5 pg / dose) or PBS once weekly for three consecutive weeks. Peripheral blood is collected at week 3, and neutrophils are enriched by density gradient centrifugation for CUT&Tag sequencing. A schematic of the in vivo training model is shown, (panel (2) of FIG. 8; panel (3) of FIG. 8; and panel (3) of FIG. 8) Heatmap showing changes in H3K27ac levels in rLF-trained neutrophils at loci associated with neutrophil migration and activation genes.

[0020] FIG. 9 illustrates rLF training increases antiviral activity in H1N1 according to an embodiment of the present disclosure.

[0021] FIG. 10 illustrates rLF training increases antibacterial activity according to an embodiment of the present disclosure.

[0022] FIG. 11 illustrates rLF training increases antitumor activity according to an embodiment of the present disclosure.

[0023] FIG. 12A and FIG. 12B illustrate rLF training improves allergic airway inflammation according to an embodiment of the present disclosure.DETAILED DESCRIPTION

[0024] The following embodiments are provided to illustrate the present disclosure in detail. A person having ordinary skills in the art can easily understand the advantages and effects of the present disclosure after reading the disclosure of this specification, and also can implement or apply in other different embodiments. Therefore, it is possible to modify and / or alter the following embodiments for carrying out this disclosure without contravening its scope for different aspects and applications, and any element or method within the scope of the present disclosure disclosed herein can combine with any other element or method disclosed in any embodiments of the present disclosure.

[0025] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

[0026] As used herein, the singular forms “a,” “an,” and “the” include plural referents, unless expressly and unequivocally limited to one referent. For example, “an experiment” means one experiment or more than one experiment, e.g., a plurality of experiments. The term “or” is used interchangeably with the term “and / or” unless the context clearly indicates otherwise.

[0027] As used herein, the term “comprising,” “comprises” “include,” “including,” “have,” “having,” “contain,” “containing,” and any other variations thereof are intended to cover a non-exclusive inclusion. For example, when describing an object “comprises” a limitation, unless otherwise specified, it may additionally include other ingredients, elements, components, structures, regions, parts, domains, loci, sequences, steps, or connections, etc., and should not exclude other limitations.

[0028] As used herein, the term “administering” or “administration” refers to the placement of an active agent into a subject by a method or route which results in at least partial localization of the active agent at a desired site to produce a desired effect. The active agent described herein may be administered by any appropriate route known in the art.

[0029] As used herein, the term “effective amount” refers to the amount of an active agent or a pharmaceutical composition that is sufficient to bring about an effect on treating, preventing, or ameliorating a disorder, disease, or condition of a subject in need thereof. The effective amount may vary by a person ordinarily skilled in the art, depending on excipient usage, routes of administration, the possibility of co-usage with other therapeutic treatment, or the condition to be treated, but the present disclosure is not limited thereto.

[0030] As used herein, the term “active agent” refers to a substance, which is administered, alone or in combination with one or more biologically or pharmaceutically acceptable carrier, to a subject for preventing, ameliorating or treating one or more symptoms of a disorder, disease, or condition. In at least one embodiment of the present disclosure, the “active agent” may be rLF protein or a variant thereof. The variant of the rLF protein is functionally equivalent to the rLF protein.

[0031] As used herein, the term “biologically or pharmaceutically acceptable carrier” maybe any and all solvents, dispersion media, diluents, excipients, antibacterial and antifungal agents,isotonic agents, absorption delaying agents, buffering agents, humectants, or other components that are suitable for the formulation and administration of the pharmaceutical composition described in the present disclosure. The carrier may be compatible with the active ingredients and may not produce an adverse biological reaction when administered. The term “pharmaceutically acceptable” indicates that the carrier is approved by a regulatory authority such as the U.S. Food and Drug Administration, or is listed in the United States Pharmacopeia or other recognized pharmacopeia for use in animals, particularly in humans. Examples of biologically or pharmaceutically acceptable carriers include, but are not limited to, saline, sterile water, aqueous dextrose, glycerol, ethanol, propylene glycol, and combinations thereof. Solid carriers may include starch, lactose, sucrose, gelatin, or talc. The choice of carrier will depend on the intended mode of administration, such as oral, intramuscular, subcutaneous, or intravenous routes. Suitable examples and formulations may be found in standard references such as Remingtons ’ Pharmaceutical Sciences.

[0032] The numeral ranges used herein are inclusive and combinable, any numeral value that falls within the numeral scope herein could be taken as a maximum or minimum value to derive the sub-ranges therefrom. For example, it should be understood that the numeral range “0.05 pg / mL to 15 pg / mL” comprises any sub-ranges between the minimum value of 0.05 pg / mL to the maximum value of 15 pg / mL, such as the sub-ranges from 0.05 pg / mL to 5 pg / mL, from 1.0 pg / mL to 15 pg / mL, from 0.5 pg / mL to 8 pg / mL and so on. In addition, a plurality of numeral values used herein can be optionally selected as maximum and minimum values to derive numerical ranges. For instance, the numerical ranges of 0.75 pg / mL to 5 pg / mL, 0.75 pg / mL to 10 pg / mL, and 5 pg / mL to 10 pg / mL can be derived from the numeral values of 0.75 pg / mL, 5 pg / mL, and 10 pg / mL.

[0033] As used herein, the term “about” generally referring to the numerical value meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from a given value or range. Such variations in the numerical value may occur by, e.g., the experimental error, the typical error in measuring or handling procedure for making compounds, compositions, concentrates, or formulations, the differences in the source, manufacture, or purity of starting materials or ingredients used in the present disclosure, or like considerations. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of time periods, temperatures, operating conditions, ratios of amounts, and the likes disclosed herein should be understood asmodified in all instances by the term “about.”

[0034] As used herein, “subject” is used to mean any vertebrate including, but not limited to, humans, or non-human mammals such as deer, mule, elk, or mule deer. However, advantageously, the subject is a mammal such as a human, or an animal mammal such as a domesticated mammal, e.g., a dog, a cat, a horse, a rat, a mouse, or the like.

[0035] As used herein, the term related to the percentage of sequence identity, such as “at least 90% identical to SEQ ID NO: 1,” refers to the extent to which sequences are identical on an amino acid-by-amino acid basis over a window of comparison. The percentage of sequence identity may be calculated by (1) comparing two optimally aligned sequences over the comparison window; (2) determining the number of positions where the identical amino acid base (e.g., glutamine, valine, and histidine) shows in both sequences to find the number of matched positions; and (3) dividing the number of matched positions by the total number of positions in the comparison window and then multiplying the result thereof by 100 to yield the percentage of sequence identity. In at least one embodiment, included herein are amino acids having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the specific sequences (e.g., SEQ ID NO: 1) recited in the Sequence Listing, and these variants maintain at least one biological activity or function of the specific sequences.

[0036] As used herein, an “amino acid” refers to any monomer unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term “amino acid” includes the following twenty natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid or aspartate (Asp or D), cysteine (CyS or C), glutamine (Gin or Q), glutamic acid or glutamate (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V).

[0037] The term “functionally equivalent” is used to describe a specific rLF protein variant having the substitution or mutation that is considered to occur at the amino acid position in the other rLF protein, the rLF protein variant according to the sequence alignment, or a reference sequence, which has the same functional or structural role in the trained immunity inducer. The equivalent positions may be defined according to homologues, conserved motifs, user-defined, orderived, consensus sequence.

[0038] As used herein, the term “consisting essentially of’ is used to define the scope of an amino acid sequence, occupying a middle ground between closed terminology such as “consisting of’ and open terminology such as “comprising.” When used in the context of an amino acid sequence (e.g., SEQ ID NO: 4), the recitation “rLF protein consisting essentially of SEQ ID NO: 4” means that the rLF protein consists of a variant of SEQ ID NO: 4 that may optionally have other amino acid residues or modifications of SEQ ID NO: 4 but do not materially affect the function of the rLF protein, i.e., the variant of SEQ ID NO: 4 is functionally equivalent to the rLF protein. The “function” of the rLF protein refers to its biological activity in inducing trained immunity, and the said “other amino acid residues” and “modifications” can be achieved by a person ordinarily skilled in the art based on the present disclosure. In some embodiments of the present application, a protein consisting essentially of SEQ ID NO: 4 may include, for example, additional amino acids, signal peptides, or chemical modifications, provided that such additions do not significantly diminish or alter the protein’s ability to induce trained immunity as described herein.

[0039] In some embodiments of the present disclosure, immune responses have traditionally been divided into two arms: innate and adaptive immunity. Innate immunity is primarily mediated by various cells, including neutrophils, monocytes, and macrophages. These cells respond rapidly and non-specifically to pathogens. In contrast, adaptive immunity involves specialized T and B lymphocytes that acquire high antigen specificity and generate long-term immunological memory. Consequently, the concept of vaccines has historically been closely associated with the memory of adaptive immunity. However, the notion that only adaptive immunity can generate immunological memory has been challenged in recent years. Emerging studies suggest that innate immune cells can also undergo long-lasting functional reprogramming that enhances innate immune cells responses to subsequent infections. This phenomenon, known as trained immunity, represents a form of innate immune memory.

[0040] In some embodiments of the present disclosure, toll-like receptor (TLR) agonists have also been identified as important inducers of trained immunity, acting through metabolic reprogramming and epigenetic modifications. Formyl peptide receptor-like 1 inhibitor protein (FLIPr) is an immune evasion protein produced by Staphylococcus aureus. A recombinant lipoprotein platform is utilized to produce recombinant lipidated FLIPr (rLF) using an Escherichia coli-based system. As bacterial-derived lipoproteins acting as a TLR agonist, rLFmay serve as an inducer of trained immunity.

[0041] In some embodiments of the present disclosure, rLF may induce trained immunity and confer protection against viral infection, infectious pathogens, and cancer, and rLF not only enhances cytokine production and immune cell recruitment upon secondary stimulation but also promotes expansion of hematopoietic progenitors in the bone marrow. Mechanistically, rLF-induced trained immunity depends on TLR-2 and the IFN-y / STATl signaling axis. Importantly, rLF confers protection against lethal H7N9 infection in a neutrophil-dependent manner and induces transcriptomic and epigenetic reprogramming in neutrophils that reinforces migration, effector functions, and host protection (FIG. 1). Therefore, the central trained immunity is supported by progenitor cell expansion, e.g., hematopoietic progenitor cells expansion. Together, rLF may be identified as a novel inducer of trained immunity and highlight rLF’s potential as an immunomodulatory agent to enhance host defense against viral infection.

[0042] In at least one embodiment of the present disclosure, the rLF protein may be administered as the sole immunologically active agent to the subject.

[0043] In at least one embodiment of the present disclosure, the rLF protein may include an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4.

[0044] In some embodiments of the present disclosure, the rLF protein may include an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 4, such as about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.

[0045] In at least one embodiment of the present disclosure, the rLF protein may include the amino acid sequence consisting essentially of SEQ ID NO: 4. In some embodiments of the present disclosure, the rLF protein may include the amino acid sequence of SEQ ID NO: 4.

[0046] In at least one embodiment of the present disclosure, the rLF protein may be lipidated at an N-terminal cysteine residue.

[0047] In at least one embodiment of the present disclosure, the rLF protein may induce epigenetic reprogramming of innate immune cells in the subject.

[0048] In at least one embodiment of the present disclosure, the epigenetic reprogrammingmay include increasing histone 3 lysine 27 acetylation (H3K27ac) levels in the innate immune cells. The innate immune cells may include neutrophils.

[0049] In at least one embodiment of the present disclosure, the inducing of trained immunity may be mediated through a TLR-2 signaling pathway and an IFN-y / STATl signaling axis, and independent via type II interferon signaling.

[0050] In at least one embodiment of the present disclosure, the method may further include preventing, treating, or alleviating an infectious disease caused by a pathogen in the subject. In some embodiments of the present disclosure, the preventing, treating, or alleviating may be mediated by a neutrophil-dependent mechanism. In some embodiments of the present disclosure, the pathogen may be heterologous to the rLF protein. In some embodiments of the present disclosure, the pathogen may be a virus or a bacterium. In some embodiments of the present disclosure, the bacterium may be selected from the group consisting of Streptococcus pneumoniae, Acinetobacter baumannii, and combinations thereof. In some embodiments of the present disclosure, the pathogen may be an antibiotic-resistant pathogen. In some embodiments of the present disclosure, the virus may be an influenza virus.

[0051] In at least one embodiment of the present disclosure, the method may further include preventing, treating, or alleviating a cancer in the subject.

[0052] In at least one embodiment of the present disclosure, the method may further include preventing, treating, or alleviating an inflammation.

[0053] In at least one embodiment of the present disclosure, the administering may be performed via a mucosal route. In some embodiments of the present disclosure, the mucosal route may be intranasal administration.

[0054] In at least one embodiment of the present disclosure, the trained immunity may persist for at least 4 weeks after the administering.

[0055] In some embodiments of the present disclosure, the trained immunity may persist for about at least 4 weeks after the administering, such as about 4 weeks, 5 weeks, or 6 weeks after the administering.

[0056] In some embodiments of the present disclosure, the rLF protein may induce trained immunity. By quickly awakening innate immunity, the rLF protein may fight against various infectious pathogens such as viruses and bacteria without the need for the presence of specific antigens. In addition, the trained immunity may also inhibit the growth of tumor cells.

[0057] Also provided herein is a use of the rLF protein for manufacture of a medicament for inducing trained immunity in a subject in need thereof.

[0058] Also provided herein is a pharmaceutical composition for use in inducing trained immunity in a subject in need thereof, including the rLF protein. The pharmaceutical composition may further include a biologically or pharmaceutically carrier.Example

[0059] Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed to limit the scope of the present disclosure.Materials and methodsPreparation of animal model

[0060] BALB / c mice are purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). SCID mice (CB17 / Icr-Prkdcscid / IcrIcoCrlBltw) are purchased from Lasco Co., Ltd. (Taipei, Taiwan). The IFNAR-knockout mice and IFNyR-knockout mice are provided by Dr. Guann-Yi Yu. STAT 1 -knockout mice are provided by Dr. Chien-Kuo Lee. TLR-2 knockout mice are provided by Dr. Shih-Jen Liu. All mice are housed at the Laboratory Animal Center of the National Health Research Institutes (NHRI). All animal studies are approved and are performed in compliance with the guidelines of the Animal Committee of the NHRI (approval no. 112077 and no.111086).Preparation of virus and recombinant proteins

[0061] The H7N9 influenza virus (A / Guangdong / 17SF003 / 2016; CBER-RG7D) is propagated in Madin-Darby canine kidney (MDCK) cells. All procedures involving H7N9 virus are conducted in a biosafety level 2 (BSL-2) or animal biosafety Level 2 (ABSL-2) laboratory.

[0062] Recombinant lipidated FLIPr (rLF) is prepared as follow. First, construction of expression vectors is conducted. Based on the amino acid sequence of FLIPr (accession number BAB57318), the DNA sequence encoding FLIPr is optimized for Escherichia coli codon usage and fully synthesized by Genomics Co. (New Taipei City, Taiwan). The FLIPr gene is cloned into the Ndel and Xhol sites of the expression vector pET- 22b(+) (Novagen, Madison, WI) to produce the plasmid pF. To clone and express LF, the DI domain and the lipid signal peptide of the lipoprotein Ag47319 are cloned into the Ndel and BamHI sites of the pET-22b(+) expression vector (Novagen, Madison, WI, USA) to obtain the plasmid pLipo. The forward primer (5'-ACTGCGGGATCCTTTTTTAGCTATGAATGG-3') (SEQ ID NO: 1) and the reverse primer (5'-GTGGTGCTCGAGATCCCAATAAATGCTATC-3') (SEQ ID NO: 2) are used to amplify the FLIPr gene. The PCR product is cloned into the BamHI and Xhol sites of the pLipo plasmid to produce the plasmid pLF. As a result, the C-termini of rF and rLF contained a hexahistidine tag (His-tag).

[0063] Second, production and purification of recombinant proteins are conducted. To express recombinant FLIPr (rF), the E. coli BL21(DE3) strain is transformed with pF. The transformed cells are cultured with LB broth at 23 °C overnight. The overnight culture is scaled up 25 times the original volume in a 2 L shake flask and incubated at 37 °C until the OD6oo reached 0.4. Then, the culture is cooled to 20 °C, and protein expression is induced (OD60o = 0.9) by adding 1 mM IPTG, followed by incubation at 20 °C for 24 h. rF is purified by disrupting the harvested cells in a French press (Constant Systems, Daventry, UK) at 27 Kpsi in homogenization buffer [20 mM Tris (pH 8.0), 40 mM sucrose, 400 mM NaCl and 10% glycerol]. The cell lysate is clarified by centrifugation at 119000 * g for 40 min at 4 °C. Most of the rF is solubilized. The extracted fraction is loaded onto immobilized metal affinity chromatography (IMAC) columns (2.5 cm i.d. x 10.0 cm) (BIO-RAD, Hercules, CA, USA) containing 20 ml of Ni-NTA resin (Qiagen, San Diego, CA) to purify the rF. The column is washed with a 40-fold column volume of the same homogenization buffer and followed by a 50-fold column volume of the same buffer containing 10 mM imidazole. Then, a 100-fold column volume of 10 mM Na2HPC>4 buffer (pH 9.6) containing 0.1% Triton X-114 is washed to remove the lipopolysaccharide. Next, the column is washed with the same buffer without 0.1% Triton X-114 to remove the residual detergent, and the rF is eluted with elution buffer [10 mM Na2HPO4 (pH 9.6), 10 mM imidazole]. The eluted rF is dialyzed to 10 mM Na2HPO4 (pH 9.6) three times for at least 6 h each time.

[0064] To express the rLF, the E. coli C43(DE3) strain is transformed with pLE Thetransformed cells are cultured with LB broth at 37 °C overnight. The overnight culture is scaled up by 50 times the original volume in a 2L shake flask and incubated at 37 °C until the OD6oo reached 0.6. Protein expression is induced (OD60o = 0.6) by adding 1 mM IPTG, followed by incubation at 20 °C for 24 h. rLF is purified by disrupting the harvested cells in a French press (Constant Systems, Daventry, UK) at 27 Kpsi in homogenization buffer [20 mM Tris (pH 8.0), 40 mM sucrose, 400 mM NaCl and 10% glycerol]. The cell lysate is clarified by centrifugation at 119000 * g for 40 min at 4 °C. Most of the rLF is present in inclusion bodies. rLF is then solubilized with extraction buffer [10 mM Na2HPO4 (pH 9.0) and 1% Triton X-100], The extracted fraction is loaded onto immobilized metal affinity chromatography (IMAC) columns (2.5 cm i.d. x 10.0 cm) (BIO-RAD, Hercules, CA) containing 20 ml of Ni-NTA resin (Qiagen) to purify rLF. The column is washed with the extraction buffer and the same buffer containing 20 mM imidazole. Then, the rLF is eluted with homogenization buffer containing 500 mM imidazole. The eluted rLF is dialyzed to 20 mM Tris (pH 8.0) three times for at least 6h each time. After dialysis, the rLF is loaded onto a 20-ml Q Sepharose fast flow column (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The column is washed with dialysis buffer containing 200 mM NaCl and then washed with a 100-fold column volume of dialysis buffer containing 0.1% Triton X-114 to remove the lipopolysaccharide. Next, the column is washed without 0.1% Triton X-114 to remove the residual detergent, and rLF is eluted with elution buffer [10 mM Na2HPO4 (pH 9.0), 300 mM NaCl and 8M urea]. The eluted rLF is dialyzed to 10 mM Na2HPO4(pH 9.0) three times for at least 6 h each time.

[0065] The endotoxin levels of the purified rF and rLF are determined using the limulus amebocyte lysate (LAL) assay (Associates of Cape Cod, Inc., Cape Cod, MA), and the resulting endotoxin levels are <30 EU / mg. After dialysis, the rF and rLF are lyophilized and stored at -20 °C. The fractions from each step are analyzed by SDS-PAGE and immunoblotted with anti-FLIPr (homemade) and anti -His tag antibodies with 1:1000 dilution (Bio-Rad, Cat#MCA1396G, clone ADI.1.10). The N-terminal fragments of rLF are obtained and identified after trypsin digestion of rLF. The identification of the lipid moiety in rLF is performed on a Waters® MALDI micro MX™ mass spectrometer.

[0066] The amino acid sequences of FLIPr and recombinant lipidated FLIPr (rLF) are shown below.Table 1. The amino acid sequences of FLIPr and recombinant lipidated FLIPr (rLF).<<<<<<<<<<Preparation and training of murine leukocytes and human peripheral blood mononuclear cells (hPBMCs)

[0067] Murine primary leukocytes are isolated from peripheral blood. Briefly, venous blood is mixed with an equal volume of 3% dextran T-500 (Sigma-Aldrich, St. Louis, MO) in 0.9% NaCl (Sigma- Aldrich) and allowed to sediment for 30 min at room temperature. The leukocyte-rich plasma is collected, centrifuged at 300 x g for 10 min at 4 °C, and the supernatant discarded. Cell pellets are resuspended in 20 mL of cold 0.2% NaCl for red blood cell lysis, followed by restoration of isotonicity with 20 mL of ice-cold 1.6% NaCl. Cells are centrifuged at 300 x g for 6 min at 4 °C and resuspended in ice-cold PBS.

[0068] Human peripheral blood mononuclear cells (hPBMCs) are purchased from AllCells, LLC (Alameda, CA). Donor lots include lot 3138260 (male, 53 years, blood type AB+), lot 3134483 (female, 22 years, blood type O), and lot 3134490 (male, 28 years, blood type A+). Donors provide informed consent under AllCells’ Institutional Review Board (IRB)-approved protocols, and de-identified PBMCs are supplied. All experiments involving human PBMCs are approved by the Institutional Review Board of the NHRI, Taiwan (approval no. EC1130803-W).

[0069] Murine leukocytes or hPBMCs are seeded in 96-well plates in LCM medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 pg / mL streptomycin, 100 U / mL penicillin, and 10 mM HEPES, pH 7.4). Cells are trained with rLF for 24 h, washed, and cultured for an additional 3 days to establish a resting state. Subsequently, cells are stimulated with LPS for 24 h, and IL-6 and TNF-a levels in culture supernatants are quantified by anti-mouse IL-6 and anti-mouse TNF-a ELISA kit (Invitrogen, Carlsbad, CA).Murine immunization and sample collection

[0070] BALB / c and SCID mice (female, 6-8 weeks of age) and knockout mice (TLR2 / , IFNAR / ", IFNGR / , or STAT17"; 6-8 weeks of age, with approximately equal numbers of males and females) are intranasally (i.n.) administered with 5 pg rLF diluted in PBS or PBS alone three times per week for 3 weeks. One week after the final administration, mice are challenged i.n. with 10 pg LPS (Sigma-Aldrich) diluted in PBS. At 3.5 h post-challenge, mice are humanely euthanized by CO2 asphyxiation. Bronchoalveolar lavage fluid (BALF) is collected, and cells are recovered by centrifugation for subsequent flow cytometric analysis of immune cell composition. Cytokine and chemokine concentrations in BALF are measured using commercial ELISA kits (Invitrogen). Peripheral blood neutrophils are isolated by density-gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich). Purified neutrophils are subsequently subjected to RNA-seq analysis. In certain experiments, BALB / c mice are sacrificed one week after the last rLF administration, reflecting the post-training resting phase in the absence of LPS challenge. Bone marrow cells are harvested for analysis of myelopoiesis by flow cytometry, and peripheral blood neutrophils are isolated for epigenetic profiling using CUT&Tag to examine H3K27ac enrichment.Multiparameter flow cytometry

[0071] For cell-surface marker analysis, freshly processed BALF cells are stained ex vivo. Cells recovered by centrifugation are first incubated with anti-mouse CD 16 / 32 antibody (clone 93) (10 min, on ice) to block Fc receptor-mediated nonspecific binding. Cell viability is assessed using the Zombie Yellow Fixable Viability Kit (BioLegend, San Diego, CA). Leukocytes are identified by anti-mouse CD45-PE / Cyanine7 antibody (clone 30-F11), and B and T cells are excluded by staining with anti-mouse CD19-Brilliant Violet 570™ (clone 6D5) and anti -mouse CD3e-Brilliant Violet 510™ (clone 145-2C11), respectively. NK cells are identified using anti-mouse NK1.1-FITC (clone PK136). The remaining CD19 CD3e NK1.1 population is further subdivided into myeloid subsets: monocytes as CDllb+Ly6C+Ly6G" [anti-CDllb-Alexa Fluor® 700 (clone MI / 70), anti-Ly-6C-Brilliant Violet 785™ (clone HK1.4), anti-Ly-6G-Pacific Blue™ (clone 1A8)]; neutrophils as CDllb+Ly6C+Ly6G+(same antibody panel as monocytes); macrophages as F4 / 80+[anti -F 4 / 80-PE (clone BM8)]. Flow cytometric acquisition is performed on a Cytek Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA).

[0072] For Bone marrow cell staining and progenitor analysis, bone marrow cells are flushed from femurs and tibias, subjected to red blood cell lysis, and washed with PBS containing 0.5% BSA (Sigma-Aldrich). To block nonspecific Fc receptor binding, cells (except those analyzed for myeloid progenitors) are incubated with anti-CD 16 / 32 (clone 93) for 10 min on ice. For lineage exclusion, cells are stained with the following biotin-conjugated antibodies: anti-Terll9 (clone TER-119), anti-CDllb (clone MI / 70), anti-CD5 (clone 53-7.3), anti-CD4 (clone RM4-5), anti-CD8a (clone 53-6.7), anti-CD45R / B220 (clone RA3-6B2), anti-Ly6G / C (clone RB6-8C5), anti-CD127 (clone A7R34). Staining is followed by Streptavidin-APC-Cy7. Additional antibodies used for progenitor analysis include anti-c-Kit-APC (clone 2B8), anti-Sca-l-PE-Cy7 (clone D7), anti-CD 150-Alexa Fluor® 488 (clone TC15-12F12.2), anti-CD48-PerCP / Cyanine5.5 (clone HM48-1), anti-CD34-FITC (clone SA376A4), and anti-CD16 / 32-AF700 (clone S17011E) (all at 1:100 dilution, 30 min, 4 °C). Above antibody are all from BioLegend.

[0073] Hematopoietic stem and progenitor cells (HSPCs) are defined as follows: HSCs (Lineage c-Kit+Sca-l+), LT-HSCs (LKS+CD150+CD48 ), ST-HSCs (LKS+CD150 CD48 ), MPPs2 (LKS+CD150+CD48+), and MPPs3 (LKS+CD150 CD48+). Myeloid progenitors are defined as CMPs (Lin c-Kit+Sca-l CD34+CD16 / 32int), GMPs (Lin c-Kit+Sca-l CD34+CD16 / 32hi), and MEPs (Lin-c-Kit+Sca-l CD34-CD16 / 32-). Flow cytometric acquisition is performed on an Attune NxT cytometer (Thermo Fisher Scientific, Waltham, MA). All data are analyzed using FlowJo software (vl0.8, BD Biosciences, San Jose, CA).Influenza virus infection model

[0074] For the influenza virus infection model, BALB / c mice receive PBS or 5 pg rLF i.n. three times per week for 3 weeks. At 1, 3, 6, or 9 weeks after the final administration, mice are challenged i.n. with a 2-fold 50% minimum lethal dose (MLD50) of H7N9 in a 20 pL volume under isoflurane anesthesia and monitored daily for weight and survival. Mice that lost >20% of their initial body weight are humanely euthanized and recorded as dead according to institutional guidelines. For viral load analysis, whole lungs are harvested at day 3 post-infection, homogenized in PBS using a gentleMACS® Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), and centrifuged at 600 x g for 10 min at 4 °C. Supernatants are used for TCIDso assay.Cell depletion experiments

[0075] For macrophage depletion, mice are intraperitoneally injected with Clodrosome® liposomal clodronate (2 mg per mouse; Encapsula NanoSciences, LLC, Brentwood, TN) 3 days prior to infection. Control animals receive an equivalent dose of Encapsome® control liposomes (Encapsula NanoSciences). For neutrophil depletion, mice are intraperitoneally injected with InVivoMab™ anti-mouse Ly6G antibody (clone 1A8; 250 pg per mouse; Bio X Cell, Lebanon, NH) 1 day before infection and again 3 days after infection. Control animals are administered an equivalent dose of InVivoMab™ rat IgG2a isotype control antibody (Bio X Cell).Virus titration and lung viral load analysis (TCIDso assay)

[0076] H7N9 viral titers are determined by tissue culture infectious dose 50 (TCIDso) assay using MDCK cells. MDCK cells are seeded in 96-well plates (3 x io4cells / well) and cultured overnight in DMEM supplemented with 5% heat-inactivated FBS. After washing with PBS, cells are maintained in infection medium (DMEM containing 2 pg / mL TPCK-trypsin, without serum). Serial 10-fold dilutions of lung homogenates from infected mice are prepared in infection medium and inoculated onto MDCK monolayers (100 pL / well, six replicates per dilution). Plates are incubated at 37 °C for 5 to 7 days and monitored for cytopathic effect (CPE). Viral titers are calculated by the Reed-Muench method and expressed as TCIDso per mL of lung homogenate.RNA sequencing and analysis

[0077] Total RNA from peripheral blood neutrophils is extracted with TRIzol Reagent (Invitrogen), and 1 pg RNA per sample is used for library preparation with the KAPA mRNA HyperPrep Kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. The quality of amplified libraries is assessed using the QseplOO DNA / RNA Analyzer (BiOptic Inc., New Taipei City, Taiwan) and Qubit® 2.0 Fluorometer (Thermo Fisher Scientific). Libraries are sequenced on an Illumina NovaSeq X platform to generate 150-bp paired-end reads. Raw sequencing reads are assessed for quality using FastQC and trimmed with Trimmomatic to remove low-quality bases and adapter sequences, then aligned to the mouse reference genome (GRCm39) using HISAT2. Gene-level read counts are obtained with featureCounts, normalized using DESeq2 or edgeR, and differentially expressed genes (DEGs) are identified with DESeq2. Functional annotation is carried out using clusterProfiler, and enrichment results are visualized as volcano plots, top-ranked GO pathways, and gene set enrichment analysis (GSEA) of selectedGO terms.CUT&Tag analysis

[0078] Peripheral blood neutrophils (1 x 105cells per sample) are subjected to CUT&Tag using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (#TD904, Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions. For histone modification profiling, cells are incubated with an anti-H3K27Ac antibody (clone D5E4; Cell Signaling Technology, Danvers, MA) as the primary antibody. Library quality is assessed using the QseplOO system (BiOptic), and paired-end sequencing (2 x 150 bp) is performed on an Illumina NovaSeq X platform (Illumina, Inc., San Diego, CA). Adapter sequences are trimmed, and reads with Phred quality scores > 25 and lengths > 100 bp are retained using fastp (vl.0.0). High-quality reads are aligned to the mouse reference genome (GRCm39, UCSC) using Bowtie2 (v2.5.3). Peak calling is conducted using MACS2 (v2.2.9.1) with a q-value cutoff of 0.1, and peaks are annotated with ChlPseeker (vl.34.1) using GENCODE M37 annotation. For motif analysis, 500-bp regions centered on peak summits are analyzed with MEME-ChIP (v5.5.8) against the HOCOMOCO Mouse vll FULL database. Differential peak analysis between groups is performed with edgeR (v3.40.2), and significantly enriched regions are defined as log2(fold change) > 1 or < -1 with p < 0.05.

[0079] For visualization of key loci, read counts at H3K27ac peaks assigned to selected genes are obtained from featureCounts and normalized by library size. Values are transformed into z-scores across samples, and heatmaps are generated.Statistical analyses

[0080] Statistical analyses are performed using GraphPad Prism 10.4.2 (GraphPad Software, San Diego, CA). Two-group comparisons use an unpaired t-test, while multiple-group comparisons versus the PBS group use one-way ANOVA with Dunnett’s post hoc test. Differences with p < 0.05 are considered significant.ResultsrLF training enhances IL-6 and TNF-a production upon LPS restimulation in mouse and human immune cells

[0081] To assess whether rLF, a bacterial-derived recombinant lipoprotein engaging TLR signaling, can induce trained immunity response, mouse white blood cells (WBCs) and human peripheral blood mononuclear cells (PBMCs) are trained in vitro. Cells are primed with PBS or varying concentrations of rLF (0.08 to 10 pg / mL) for 24 hours, washed, rested for 3 days, and then restimulated with lipopolysaccharide (LPS) (panel (1) of FIG. 2A). In the absence of LPS, IL-6 (panel (2) of FIG. 2A) and TNF-a (panel (3) of FIG. 2A) levels remained at basal levels. However, upon LPS restimulation, rLF-trained mouse WBCs exhibited significantly elevated levels of IL-6 and TNF-a compared to PBS-trained controls. Similarly, rLF training enhanced IL-6 (panel (4) of FIG. 2 A; panel (5) of FIG. 2 A; and panel (6) of FIG. 2 A) and TNF-a (panel (1) of FIG. 2B; panel (2) of FIG. 2B; and panel (3) of FIG. 2B) secretion in PBMCs from three independent human donors. These results demonstrate that rLF induces a trained immunity-like phenotype in both murine and human innate immune cells.Administration of rLF enhances inflammatory mediator production and innate immune cell recruitment upon LPS restimulation, and promotes progenitor cell expansion

[0082] The rLF-induced trained immunity in vivo is further assessed. Mice are intranasally administered rLF once weekly for three consecutive weeks (week 0, 1, and 2), followed by LPS stimulation at week 3. Bronchoalveolar lavage fluid (BALF) is collected 3.5 hours after LPS stimulation (panel (1) of FIG. 3). PBS-treated mice served as controls. LPS stimulation leads to significantly increased levels of IL-6, TNF-a, and RANTES in the BALF of rLF-trained mice compared to PBS-treated controls (panel (2) of FIG. 3). Additionally, the numbers of macrophages, neutrophils, monocytes, and NK cells are increased in the rLF-trained group (panel (3) of FIG. 3). These results suggest that rLF induces trained immunity in vivo.

[0083] Trained immunity can occur at both central and peripheral levels, involving hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and tissue-resident immune cells, respectively. Whether rLF training modulates hematopoietic progenitor cells in the bone marrow is investigated. Mice are intranasally administered rLF once weekly for three consecutive weeks (weeks 0, 1, and 2), and bone marrow cell profiles are analyzed at week 3 (panel (1) of FIG. 4). Intranasal administration of rLF induces a significant increase in the number of hematopoietic stem cells (HSCs, Lineage-cKit+Scal+), long-term HSCs (LT-HSCs, LKS+CD150+CD48-), short-term HSCs (ST-HSCs, LKS+CD150-CD48-), and multipotent progenitors 2 (MPPs2, LKS+CD150+CD48+). There is no statistical difference in multipotentprogenitors 3 (MPPs3, LKS+CD150-CD48+) (panel (2) of FIG. 4). These results suggest that rLF induces expansion of the HSC pool as previously classified. The effect of rLF on myeloid progenitors is next determined. While the number of common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte-erythrocyte progenitors (MEPs) is significantly increased in rLF-trained mice compared to PBS-trained mice (panel (3) of FIG.4). These results suggest that rLF elicits expansion of myeloid progenitors.TLR-2 and IFN-y / STATl signaling mediate rLF-induced trained immunity

[0084] Trained immunity is an immunological process characterized by functional changes in innate immune cells upon secondary stimulation. It is demonstrated that rLF enhances the production of inflammatory mediators and promotes the recruitment of innate immune cells following LPS restimulation (FIG. 3). The underlying mechanisms and signaling pathways responsible for these effects are next investigated. All mice are trained with rLF or PBS as described in panel (1) of FIG. 3.

[0085] As shown in panel (1) of FIG. 5A, significantly elevated levels of IL-6, TNF-a, and RANTES are detected in the BALF of rLF-trained severe combined immunodeficiency (SCID) mice compared to PBS-treated controls. In addition, neutrophils, monocytes, and NK cells are significantly increased in the rLF-trained group compared to PBS-treated controls. SCID mice lack functional T and B cells. These results suggest that the enhanced production of inflammatory mediators and the recruitment of innate immune cells following LPS restimulation occur independently of adaptive immune cells (T and B cells).

[0086] Bacterial-derived lipoproteins have been shown to act as TLR-2 agonists. To examine the role of TLR-2 in the induction of trained immunity, TLR-2 knockout mice (panel (2) of FIG.5A) are trained with rLF or PBS. No significant increases in IL-6, TNF-a, or RANTES levels, nor in the numbers of macrophages, neutrophils, and NK cells, compared rLF-trained mice to PBS-treated controls. These findings suggest that rLF-induced trained immunity is dependent on TLR-2 signaling pathways.

[0087] It has been shown that type I interferon signaling is required for induction of trained immunity. To investigate whether interferon signaling pathways are involved in rLF induced trained immunity, IFNAR-knockout (panel (3) of FIG. 5A), IFNyR-knockout (panel (1) of FIG.5B), and STAT1 knockout (panel (2) of FIG. 5B) mice are trained with rLF or PBS. InIFNAR-knockout mice, rLF training leads to significant increases in IL-6, TNF-a, and RANTES levels, as well as in the numbers of macrophages, neutrophils, monocytes and NK cells, compared to PBS-treated controls. In contrast, training rLF with IFNyR-knockout and STAT1 knockout mice does not result in increased production of inflammatory mediators or recruitment of innate immune cells. These results suggest that rLF-induced trained immunity depends on type II interferon (interferon-y) / STATl axis, but not type I interferon, signaling pathways.rLF-induced trained immunity confers neutrophil-dependent protection against H7N9 infection

[0088] Given the capacity of rLF to induce trained immunity in mice, whether rLF training confers protection against H7N9 infection is next investigated. To this end, mice receive intranasal administrations of rLF or PBS control at weeks 0, 1, and 2, followed by an intranasal H7N9 challenge 1 week after the final rLF administration (week 3). Mice are sacrificed on day 3 post-infection to assess viral loads in the lungs. A subset of mice is monitored for body weight changes over 14 days post-challenge (panel (1) of FIG. 6A). Mice trained with rLF showed significantly lower viral loads in the lungs compared to PBS-treated controls (panel (2) of FIG.6A). Consistent with the viral load findings, 7 out of 10 rLF-trained mice recover their body weight by days 6-9 post-challenge, whereas PBS-treated mice experience a decline to less than 80% of their initial body weight within 6 days (panel (3) of FIG. 6A). Consequently, the overall survival rate of rLF-trained mice (70%) is higher than that of the PBS group (0%) (panel (4) of FIG. 6A).

[0089] To assess the duration of rLF-induced protective effects, mice are trained with rLF or PBS as described in panel (1) of FIG. 6A and subsequently challenged with H7N9 virus at either 6 (week 8) or 9 (week 11) weeks after the last rLF administration. When challenge at week 8, rLF-trained mice remaine protected against H7N9 infection. Specifically, 7 out of 12 mice recover their body weight by days 6 to 8 post-challenge (panel (5) of FIG. 6A). The overall survival rate of rLF-trained mice (58.3%) is higher than that of the PBS group (16.7%) (panel (6) of FIG. 6A). In contrast, at week 11, there are no significant differences between the rLF-trained and PBS-treated groups in terms of body weight changes (panel (7) of FIG. 6A) or survival rates (panel (8) of FIG. 6A). These results suggest that the protective effects of rLF-induced trained immunity persist for at least 6 weeks after the last rLF administration.

[0090] To further identify the cell subsets critical for rLF-mediated protection, neutrophils and macrophages / monocytes are selectively depleted in rLF-trained mice one day prior to H7N9challenge using anti-Ly6G-depleting antibodies and clodronate-encapsulated liposomes, respectively. As shown in panel (1) of FIG. 6B, 5 out of 8 mice in both the rLF -trained group and the rLF-trained group treated with isotype control antibodies recovered their body weight by days 6 to 7 post-challenge. In contrast, only 2 out of 8 mice in the rLF-trained group treat with anti-Ly6G antibodies show recovery. Correspondingly, the overall survival rates are 62.5% for both the rLF-trained and isotype control groups, but dropped to 25% in the anti-Ly6G-treated group (panel (2) of FIG. 6B). In the macrophage / monocyte depletion experiment, comparable trends in body weight recovery (panel (3) of FIG. 6B) and overall survival (panel (4) of FIG. 6B) are observed among the rLF-trained, control liposome-treated, and clodronate-treated groups. These findings suggest that neutrophils, but not macrophages or monocytes, are essential for rLF-mediated protection against H7N9 infection.rLF training induces transcriptomic changes and epigenetic reprogramming

[0091] In light of the critical role of neutrophils in protection against H7N9 infection, transcriptomic analysis of these cells is performed. Groups of mice receive intranasal administration of rLF or PBS once weekly for three consecutive weeks (weeks 0, 1, and 2), followed by LPS stimulation at week 3. Peripheral blood is collected 3.5 hours after LPS stimulation, and neutrophils are enriched by density gradient centrifugation for transcriptomic profiling (panel (1) of FIG. 7A). Neutrophils from rLF-trained mice display an altered transcriptomic profile compared with those from PBS-trained mice, with 1,726 genes upregulated and 1,194 genes downregulated (panel (2) of FIG. 7A). Gene ontology enrichment analysis reveals that the majority of the top 20 biological process pathways are strongly associated with chemotaxis or activation of innate immune cells (9 of 20, red), while 3 are related to cytokine production involved in inflammatory responses (blue) (panel (3) of FIG. 7A). Gene set enrichment analysis (GSEA) of representative pathways, including neutrophil chemotaxis (G0:0030593), regulation of leukocyte degranulation (G0:0043300), and production of molecular mediators involved in the inflammatory response (G0:0002532), is shown FIG. 7B.

[0092] Because 9 of the top 20 enriched innate immune pathways are related to chemotaxis or activation, these processes are next focused. To investigate whether the observed transcriptional changes are accompanied by epigenetic modifications, H3K27ac levels, a marker of active chromatin regions, in neutrophils after training with rLF or PBS using CUT&Tag sequencing are assessed (panel (1) of FIG. 8). Prominent H3K27ac gains are observed at Cxcr2, Fprl, and Fpr2, indicating transcriptional priming of G protein, coupled receptor (GPCR)-driven chemotaxis(panel (2) of FIG. 8). Downstream, enrichment at Gnaq (Gaq) and Gngl2 (Gy l 2) supports engagement of heterotrimeric G-protein modules, while parallel activation of the Ras-RAF-MEK-MAPK axis31-33 is reflected by increased acetylation at Mapkl4 (p38) and Mapk3 (ERK1) (panel (3) of FIG. 8). In addition, increased H3K27ac levels at Itgal and Itgb21 (LFA-1), together with Rassf5, Syk, Pyk2, and Vav3, suggest priming of the P2 integrin-cytoskeleton module underlying adhesion and polarization (panel (4) of FIG. 8). Collectively, these findings highlight that rLF training establishes an epigenetic landscape integrating GPCR, MAPK, and integrin pathways, thereby reinforcing cytoskeletal remodeling, neutrophil polarization, and sustained migratory responses.rLF training increases antiviral activity in H1N1

[0093] The BALB / C mice receive intranasal rLF for three consecutive weeks (at weeks -2, -1, and 0). On day 7 after the last administration, mice are challenged with H1N1 to evaluate the protective efficacy of rLF -trained mice against pathogens (panel (1) of FIG. 9). After H1N1 challenge, there is a noticeable delay in weight loss and a significant delay in time to death in rLF-trained mice (panel (2) of FIG. 9 and panel (3) of FIG. 9).rLF training increases antibacterial activity

[0094] The BALB / C mice receive intranasal rLF for three consecutive weeks (at weeks -2, -1, and 0). On day 7 after the last administration, mice are challenged with Streptococcus pneumoniae or Acinetobacter baumannii to evaluate the protective efficacy of rLF-trained mice against pathogens (panel (1) of FIG. 10). In the Streptococcus pneumoniae or Acinetobacter baumannii challenge model, rLF-trained mice demonstrate significant improvements in survival rates and delays in time to death (panel (2) of FIG. 10; panel (3) of FIG. 10; and panel (4) of FIG.10).

[0095] Therefore, the results demonstrate that mice trained with rLF show protective responses against assaults from non-specific pathogens, at least in the cases of influenza, Streptococcus pneumoniae, and Acinetobacter baumannii.rLF training increases antitumor activity

[0096] C57BL / 6 mice receive intranasal rLF for three consecutive weeks (at weeks -2, -1, and0). On day 7 after the last administration (at week 1), mice are intravenously challenged with B16F10 tumor cells. Two weeks after tumor challenge, mice are sacrificed and the tumor nodules in the lung are counted (panel (1) of FIG. 11). The results demonstrate that mice trained with rLF enhance antitumor activity responses (panel (2) of FIG. 11).rLF training improves allergic airway inflammation

[0097] C57BL / 6 mice receive intranasal administration of rLF for three consecutive weeks (at weeks -2, -1, and 0). The House Dust Mite (HDM) extract combines Dermatophagoides pteronyssinus and Dermatophagoides farinae. On day 7 after the final administration, airway inflammation is subsequently induced. After induction, bronchoalveolar lavage fluid (BALF), serum immunoglobulin levels, and airway responsiveness are assessed (FIG. 12A). As demonstrated in the experimental results, mice trained with rLF exhibit reduced airway inflammatory responses, as evidenced by decreased IgE levels and reduced leukocyte infiltration, including eosinophil accumulation, in the lung, together with improved airway responsiveness (panel (1) of FIG. 12B; panel (2) of FIG. 12B; panel (3) of FIG. 12B; panel (4) of FIG. 12B; and panel (5) of FIG. 12B). The results demonstrate that mice trained with rLF exhibit enhanced resistance to airway inflammatory responses.

[0098] Trained immunity has emerged as a paradigm in which innate immune cells acquire long-lasting functional adaptations following an initial stimulus. Recombinant lipidated FLIPr (rLF) is identified as a novel inducer of trained immunity with molecular features that distinguish it from previously characterized stimuli. P-glucans, the prototypical inducers, act through the Dectin- 1 / Raf-l signaling axis and promote type I interferon-dependent pathways. By contrast, it is demonstrated that rLF, a bacterial-derived recombinant lipoprotein, reproduces key hallmarks of trained immunity through a distinct mechanism. Specifically, the requirement of TLR-2 signaling for rLF-induced effects aligns with prior work establishing bacterial lipoproteins as potent TLR-2 agonists, leading to trained immunity mediated via type II interferon. Together, these findings reveal divergent receptor- and cytokine-dependent routes to trained immunity and underscore the heterogeneity of innate immune memory programs shaped by different microbial ligands.

[0099] A second major finding is that rLF induces transcriptomic and epigenetic reprogramming in neutrophils that reinforces their migratory and effector capacities. Transcriptome analysis reveals robust induction of genes involved in chemotaxis, degranulation,and inflammatory mediator production, while CUT&Tag profiling identified increased H3K27ac deposition at loci encoding GPCRs, MAPK signaling molecules, and P2 integrin-cytoskeleton modules. Enriched H3K27ac modification represents an active chromatin state that drives transcription. These molecular signatures are consistent with functional data showing rapid neutrophil recruitment to the lungs following LPS challenge. Together, these results indicate that rLF not only primes neutrophils at the transcriptional level but also establishes an epigenetic landscape that facilitates polarization, adhesion, and migration upon secondary stimulation. This neutrophil-centered program provides a mechanistic basis for the enhanced protection observed in vivo.

[0100] Importantly, rLF-induced protection persisted for at least six weeks, despite the inherently short lifespan of neutrophils is observed. This temporal discrepancy suggests that rLF elicits central trained immunity through the reprogramming of hematopoietic progenitor cells. Supporting this notion, it is found that rLF promotes expansion of progenitor populations in the bone marrow, providing a sustained cellular reservoir for the generation of trained neutrophils. These findings align with reports that central trained immunity underlies the HSCs expansion of other stimuli, such as P-glucan or BCG vaccination, and extend this concept to rLF as a novel inducer of progenitor-level reprogramming.

[0101] In conclusion, rLF is a novel inducer of trained immunity that protects against viral infection via neutrophil-dependent mechanisms. By integrating TLR-2 and IFN-y / STATl signaling with transcriptional and epigenetic reprogramming, rLF establishes a primed innate immune state that enhances host defense. Beyond its role in trained immunity, rLF also functions as an adjuvant to boost mucosal and systemic responses across various vaccine platforms. Together, these findings broaden the spectrum of trained immunity inducers and highlight rLF as a promising candidate for immunomodulatory strategies, with potential applications in both infectious and non-infectious diseases, including cancer and inflammatory disorders.

[0102] The foregoing outlines the features of several embodiments, enabling those skilled in the art to fully appreciate the aspects of the present disclosure. Those skilled in the art should recognize that the present disclosure provides a foundation for designing or modifying other processes and structures to achieve substantially the same functions and / or substantially the same results as those of the embodiments introduced herein. Furthermore, such equivalent arrangements do not deviate from the spirit and scope of the present disclosure, and various changes, substitutions, and alterations may be made without so departing.

Claims

CLAIMSWhat is claimed is:

1. A method for inducing trained immunity in a subject in need thereof, comprising:administering to the subject an effective amount of a recombinant lipidated FLIPr (rLF) protein, wherein the rLF protein is administered without a specific antigen.

2. The method of claim 1, wherein the rLF protein is administered as the sole immunologically active agent to the subject.

3. The method of claim 1, wherein the rLF protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4.

4. The method of claim 3, wherein the rLF protein comprises the amino acid sequence consisting essentially of SEQ ID NO: 4.

5. The method of claim 3, wherein the rLF protein is lipidated at an N-terminal cysteine residue.

6. The method of claim 1, wherein the rLF protein induces epigenetic reprogramming of innate immune cells in the subject.

7. The method of claim 6, wherein the epigenetic reprogramming comprises increasing histone 3 lysine 27 acetylation (H3K27ac) levels in the innate immune cells, and wherein the innate immune cells comprise neutrophils.

8. The method of claim 1, wherein the inducing of trained immunity is mediated through a TLR-2 signaling pathway and an IFN-y / STATl signaling axis, and is independent via type II interferon signaling.

9. The method of claim 1, further comprising preventing, treating, or alleviating an infectious disease caused by a pathogen in the subject.

10. The method of claim 9, wherein the preventing, treating, or alleviating is mediated by a neutrophil-dependent mechanism.

11. The method of claim 9, wherein the pathogen is heterologous to the rLF protein.Page 1 of 2912. The method of claim 9, wherein the pathogen is a virus or a bacterium.

13. The method of claim 12, wherein the bacterium is selected from the group consisting of Streptococcus pneumoniae, Acinetobacter baumannii, and combinations thereof.

14. The method of claim 12, wherein the pathogen is an antibiotic-resistant pathogen.

15. The method of claim 12, wherein the virus is an influenza virus.

16. The method of claim 1, further comprising preventing, treating, or alleviating a cancer in the subject.

17. The method of claim 1, further comprising preventing, treating, or alleviating an inflammation.

18. The method of claim 1, wherein the administering is performed via a mucosal route.

19. The method of claim 18, wherein the mucosal route is intranasal administration.

20. The method of claim 1, wherein the trained immunity persists for at least 4 weeks after the administering.

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