Composition for enhancing immunity, comprising hafnia alvei-derived lipopolysaccharide and carbomer

Abstract

The present invention relates to a composition for enhancing immunity, comprising a Hafnia alvei-derived lipopolysaccharide and a carbomer. The composition for enhancing immunity, according to the present invention, has an excellent antigen-specific antibody formation promoting ability, and can effectively induce cellular and humoral immunity in an individual. Therefore, the composition for enhancing immunity of the present invention can be usefully utilized in various fields such as prevention of various infectious diseases caused by pathogens, immunity enhancement, vaccine assistance, and the like.

Description

Immunoenhancing composition comprising lipopolysaccharide and carbomer derived from Hafnia albaei

[0001] The present invention relates to an immune-enhancing composition comprising a lipopolysaccharide derived from Hafnia albaei and a carbomer.

[0002]

[0003] Vaccines are classified into live-attenuated vaccines, killed / inactivated vaccines, subunit vaccines, virus-like particle vaccines, and DNA / RNA-based vaccines depending on the type of antigen. Live-attenuated vaccines are generally capable of self-replication in the human body and contain components that act as natural immune adjuvants, so they can induce both humoral and cellular immune responses simultaneously, establishing a highly effective defense. However, due to concerns about side effects caused by mutations, they were switched to killed / inactivated vaccines. However, because they cannot self-replicate in the human body and do not go through the normal infection route, their efficacy is relatively low. Subunit vaccines, which use only a part of the pathogen as an antigen (recombinant protein, peptide, etc.), can be mass-produced quickly and effectively in the event of an epidemic. However, because they do not contain the microbe-associated molecular patterns (MAMPs; a preferred term compared to the existing pathogen-associated molecular patterns (PAMPs)) inherent in the microorganism, the vaccine efficacy is significantly lower when administered as the antigen alone. Therefore, the use of adjuvants is essential to enhance the immune response of inactivated / killed vaccines and subtype vaccines.

[0004] Adjuvants are a key component of vaccines. They stimulate the host's immune response to antigens during in vivo vaccination. They can effectively enhance the in vivo immune response, which is insufficient when administered alone. Therefore, adjuvants can enhance the immune response in immunocompromised populations, such as infants and the elderly. Furthermore, adjuvants can be combined with other immune-inducing substances to induce specific immune responses (humoral or cellular). Furthermore, the immune response activation achieved through the use of adjuvants can reduce the amount of antigen used per dose or minimize the number of vaccines administered.

[0005] Recent research trends indicate that adjuvants are being developed by combining various immunomodulators. Based on the pathogen's infection mechanism, new Toll-like receptor agonists (TLR1 / 2 agonist: PAM3CSK4, TLR3 agonist: Poly(I:C), TLR4 agonist: Lipopolysaccharide, etc.) are being developed to activate the necessary immune response (humoral or cellular, or simultaneous humoral / cellular activation). Furthermore, research is being conducted on various formulations (emulsion, liposome, gel type, etc.) to delay antigen release at the vaccination site and enhance immune cell recruitment and antigen absorption.

[0006]

[0007] The present inventors studied a new immune-enhancing agent and completed the present invention by confirming that an immune-enhancing composition containing a lipopolysaccharide derived from Hafnia albaei and a carbomer has a remarkable immune-enhancing effect.

[0008] Accordingly, the purpose of the present invention is to provide an immune-enhancing composition comprising a lipopolysaccharide derived from Hafnia albaei and a carbomer, a vaccine composition comprising the same, and a method for producing the same.

[0009]

[0010] To achieve the above purpose, the present invention provides an immune-enhancing composition comprising a lipopolysaccharide derived from Hafnia alvei and a carbomer.

[0011] The present invention also provides a vaccine adjuvant composition comprising a lipopolysaccharide derived from Hafnia alvei and a carbomer.

[0012] The present invention also provides a vaccine composition comprising a lipopolysaccharide derived from Hafnia alvei, a carbomer, and an antigen.

[0013] The present invention also provides a composition for preventing or treating infectious diseases, comprising a lipopolysaccharide and an antigen derived from Hafnia alvei.

[0014] In addition, the present invention provides a health functional food composition for enhancing immunity, comprising a lipopolysaccharide and carbomer derived from Hafnia alvei.

[0015] The present invention also provides an immune-enhancing feed additive composition comprising a lipopolysaccharide and carbomer derived from Hafnia alvei.

[0016] The present invention also provides a method for inducing an immune response in a subject, comprising administering the immune-enhancing composition to a subject in need thereof.

[0017] The present invention also provides a method for producing an immune-enhancing composition, comprising the step of mixing a lipopolysaccharide derived from Hafnia alvei and a carbomer.

[0018]

[0019] The immune-enhancing composition according to the present invention exhibits excellent antigen-specific antibody formation promotion capabilities and can effectively induce cellular and humoral immunity within an individual. Therefore, the immune-enhancing composition of the present invention can be usefully utilized in various fields, including prevention of various infectious diseases caused by pathogens, immune enhancement, and vaccine adjuvantage.

[0020]

[0021] Figure 1 is a diagram showing the results of confirming the antigen-specific antibody Total IgG formation ability according to the administration of a mixed vaccine containing various concentrations of carbopol and Hafnia alvei LPS as an immune adjuvant in BALB / c mice (*:P< 0.05).

[0022] Figure 2 is a diagram showing the results of confirming the proportion of CD4+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in immune cells of BALB / c mice vaccinated with a combination vaccine containing various concentrations of carbopol and Hafnia alvei LPS as an immune adjuvant (*: P < 0.05).

[0023] Figure 3 shows the results of confirming the antigen-specific Total IgG, the proportion of CD4+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in immune cells, and the cellular immune response induction effect (ELISpot) following administration of a combined vaccine containing various concentrations of Hafnia alvei LPS based on 0.1% (w / v) carbopol as an immune adjuvant in BALB / c mice. (*:P< 0.05).

[0024] Figure 4 is a diagram showing the results of confirming the ability to form antigen-specific antibodies Total IgG, IgG1, and IgG2c following administration of a combination vaccine containing carbopol or Hafnia alvei LPS as an immune inducer and carbopol / Hafnia alvei LPS as an immune enhancer in C57BL / 6 mice (*:P< 0.05).

[0025] Figure 5A is a diagram showing the results of comparing the antigen-specific antibody Total IgG, IgG1, and IgG2c formation ability following administration of a combined vaccine containing carbopol and Hafnia alvei LPS as an immune adjuvant in C57BL / 6 mice with the existing O / W emulsion immune adjuvant AddaS03 and AddaVaX experimental groups (*: P< 0.05).

[0026] Figure 5B is a diagram showing the results of comparing the antigen-specific antibody Total IgG, IgG1, and IgG2c formation ability of a combined vaccine containing carbopol and Hafnia alvei LPS as an immune adjuvant in BALB / c mice with the existing O / W emulsion immune adjuvant AddaS03 and AddaVaX experimental groups (*:P< 0.05).

[0027] Figure 6A is a diagram showing the results of comparing the proportion of CD4+, CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in immune cells of C57BL / 6 mice after administration of a combined vaccine containing carbopol and Hafnia alvei LPS as an immune adjuvant with the experimental groups of existing O / W emulsion immune adjuvants, AddaS03 and AddaVaX (*: P< 0.05).

[0028] Figure 6B is a diagram showing the results of comparing the proportion of CD4+T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in the immune cells of BALB / c mice after administration of a combined vaccine containing carbopol and Hafnia alvei LPS as an immune adjuvant with the experimental groups using the existing O / W emulsion immune adjuvants AddaS03 and AddaVaX (*: P < 0.05).

[0029] Figure 7A is a diagram showing the results of comparing the antigen-specific cellular immune response following administration of a combined vaccine containing carbopol and Hafnia alvei LPS as an adjuvant in C57BL / 6 mice with the existing O / W emulsion adjuvant, AddaS03 and AddaVaX experimental groups (*: P < 0.05).

[0030] Figure 7B is a diagram showing the results of comparing the antigen-specific cellular immune response following administration of a combined vaccine containing carbopol and Hafnia alvei LPS as an adjuvant in BALB / c mice with the experimental groups using existing O / W emulsion adjuvants, AddaS03 and AddaVaX (*: P < 0.05).

[0031]

[0032] The present invention relates to an immune-enhancing composition comprising a lipopolysaccharide derived from Hafnia alvei and a carbomer, a vaccine adjuvant comprising the same, and a vaccine composition. The immune-enhancing composition comprising a lipopolysaccharide derived from Hafnia alvei and a carbomer of the present invention has excellent antigen-specific antibody formation ability and can effectively induce both cellular and humoral immunity.

[0033] The present invention is described in detail below.

[0034] In the present invention, immunostimulation is one of the important therapeutic strategies for reinforcing the body's defense mechanisms against various diseases such as infectious diseases, cancer, and inflammatory diseases, and refers to stimulating the immune response and obtaining an immune-enhancing effect by increasing the activity of immune cells. For example, phagocytes play a key role in the immune response. The main role of macrophages, phagocytosis, is to absorb microorganisms and other pyrogenic particles, and also stimulate the immune response by secreting a number of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-12 (IL-12), and cytotoxic and inflammatory substances such as nitric oxide (NO). Therefore, increasing macrophage activity can be one means for immune enhancement. Since the occurrence of infections and diseases mainly occurs when the immune function is weakened, various studies are being conducted to enhance these immune responses through immune-enhancing substances when the function of the body's immune system is weakened.

[0035] The immunostimulating composition of the present invention can be used as an immunostimulant or vaccine adjuvant. An adjuvant generally refers to any substance that enhances the humoral or cellular immune response to an antigen. An adjuvant slows the release of an antigen from the injection site and stimulates the immune system. Traditional vaccines are typically composed of crude preparations of live pathogenic microorganisms that have been inactivated, killed, or modified. However, the immunity induced by these is insufficient. Therefore, the immune response can be further stimulated by adding exogenous substances such as adjuvants. The addition of an adjuvant allows for the use of a smaller amount of antigen to induce a similar immune response, which allows for more economical vaccine production.

[0036] In the present invention, the immunity increased by administration of the composition for enhancing immunity is preferably innate immunity or adaptive immunity, and more preferably may refer to both innate immunity and adaptive immunity.

[0037] In the present invention, lipopolysaccharide (LPS) is a substance present in the outer cell membrane of Gram-negative bacteria, and is a potent inflammatory substance that can induce the expression of various types of inflammatory cytokines (TNF-α, IL-1β, IL-6, etc.) by activating the NK-κB signaling pathway by recognizing the lipid A of LPS by Toll-like receptor 4.

[0038] In a specific embodiment of the present invention, the lipopolysaccharide (LPS) derived from Hafnia albeit may include all of the lipopolysaccharides of Hafnia albeit or a part thereof, and may include all of their natural forms, chemically modified forms, and synthetic forms. Since various strains belonging to Hafnia albeit may share common or similar LPS structures and activities, LPS derived from various Hafnia albeit strains may be combined with carbomer to induce excellent immune-enhancing effects.

[0039] Therefore, the Hafnia alvei of the present invention may include, without limitation, not only known easily available strains such as Hafnia alvei BAA-2768 (ATCC, USA), Hafnia alvei 51815 (ATCC, USA), and Hafnia alvei 25927 (ATCC, USA), but also Hafnia alvei strains that have been isolated or identified, such as Hafnia alvei BA2000346 strain (Accession No.: KCTC15966BP) or Hafnia alvei BA2000103 strain, and preferably Hafnia alvei BA2000346 strain (Accession No.: KCTC15966BP).

[0040] Therefore, in one embodiment of the present invention, a lipopolysaccharide derived from the Hafnia albei BA2000346 strain (accession number: KCTC15966BP) was used. The Hafnia albei BA2000346 strain was isolated from deer feces and was patent deposited at the Biological Resource Center located at 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do on November 3, 2023, and was assigned the domestic accession number KCTC 19127P, and was assigned the international accession number KCTC15966BP in accordance with the Budapest Treaty.

[0041] In the present invention, carbomer is a polymer formed by polymerizing acrylic acid, and can be used interchangeably with carbopol or carboxyvinyl polymer. Acrylic acid molecules are used as the basic unit, and polyalkenyl ethers or divinyl glycol can be used as a material that allows the polymer chains to be linked to each other. Many acrylic acid units are combined to form a long chain form, and a three-dimensional network structure is formed through a cross-linker. This structure allows it to absorb water, swell, and form a gel, and can be adjusted to various concentrations and viscosities. Carbomer can be expressed by the chemical formula [C3H4O2]n, where n can be selected within the range of 1,000 to 1,000,000. For example, a low molecular weight carbomer may be selected where n is in the range of 1,000 to 10,000, a medium molecular weight carbomer may be selected where n is in the range of 10,000 to 100,000, and a high molecular weight carbomer may be selected where n is in the range of 100,000 to 1,000,000. Alternatively, the carbomer may be selected based on viscosity, for example, various carbomers having a viscosity of at least 4,000 to a maximum of 77,000 cP when measured at 0.5 wt% at pH 7.5 may be selected depending on the purpose, and preferably, a carbomer having a viscosity of, for example, 4,000 to 60,000 cP or about 4,000 to 40,000 cP, specifically, a carbomer having a viscosity of 29,400 to 39,400 cP may be preferred. That is, in the present invention, the carbomer may include, without limitation, a synthetic polymer synthesized from acrylic acid, and may be, for example, Carbopol® 940P, 934P, 971P, 974P, and in one embodiment of the present invention, Carbopol 974P was used.

[0042] The Hafnia alvei-derived lipopolysaccharide of the present invention, when mixed with carbomer and used as a composition, can have desirable properties as an immune-enhancing composition compared to the Hafnia alvei-derived lipopolysaccharide alone. For example, in the mixed composition, the Hafnia alvei-derived lipopolysaccharide can be stabilized together with carbomer, thereby enabling a sustained immune-enhancing effect. The carbomer can help the Hafnia alvei-derived lipopolysaccharide to be slowly released, thereby inducing an immune response more effectively. In addition, the Hafnia alvei-derived lipopolysaccharide composition mixed with carbomer has high viscosity and stability, and can be developed into various dosage forms (e.g., injections, gel-type injections, etc.). This allows for easy storage and handling, and can enhance the efficacy of the vaccine. Furthermore, the mixed composition can precisely control the concentration of Hafnia alvei-derived lipopolysaccharide, thereby optimizing the immune response while minimizing side effects. Furthermore, carbomer can reduce skin irritation, thereby reducing the likelihood of side effects. Furthermore, in terms of sustained efficacy, Hafnia alvei-derived lipopolysaccharide mixed with carbomer is slowly released and can sustain an immune response for a long period of time. This ensures the long-term efficacy of the vaccine. Furthermore, the mixed composition ensures stability and consistency during the manufacturing process, is suitable for mass production, and is easily commercialized in the form of various adjuvants.

[0043]

[0044] In a specific embodiment of the present invention, the Hafnia albaei-derived lipopolysaccharide and carbomer are preferably formulated as an oil-in-water emulsion. In the present invention, an "oil-in-water (O / W)" emulsion refers to one in which one liquid (oil phase) is dispersed within another liquid (water phase). An oil-in-water emulsion may include oil and a surfactant.

[0045] In a preferred embodiment of the present invention, the composition for enhancing immunity may further include squalene, and the oil-in-water emulsion may be a squalene-based oil-in-water emulsion, but these may be applied without limitation as long as the purpose of the present invention can be achieved.

[0046] In the present invention, the Hafnia albay-derived lipopolysaccharide may be included in the immune-enhancing composition at 1 to 100 μg / ml, preferably 1 to 50 μg / ml, more preferably 1 to 30 μg / ml, and most preferably 5 to 20 μg / ml. If the Hafnia albay-derived lipopolysaccharide exceeds 100 μg / ml, safety may be reduced.

[0047] In addition, the carbomer may be included in the immune-enhancing composition at 0.005 to 0.5% (w / v), preferably 0.01 to 0.5%, and more preferably 0.05 to 0.3% (w / v). If the carbomer is included in an amount exceeding 0.5% (w / v), for example, 1.0% (w / v), the manufacturing time may be prolonged when manufacturing an oil-in-water emulsion, and incomplete dissolution may occur, which may not be suitable for the manufacturing process.

[0048] In the present invention, the lipopolysaccharide derived from Hafnia albay and the carbomer can be included in the composition for enhancing immunity at 1 to 100 μg / ml and 0.005 to 0.5% (w / v), respectively, and in a preferred embodiment of the present invention, 5 or 20 μg / ml of the lipopolysaccharide derived from Hafnia albay and 0.1% (w / v) of the carbomer were used, which have advantages in the manufacturing process, exhibit low toxicity, and can induce antibody formation and cellular immunity suitable as an immunostimulant in the most balanced manner.

[0049] According to another aspect of the present invention, there is provided an immune-enhancing vaccine adjuvant composition comprising a lipopolysaccharide derived from Hafnia alvei and a carbomer. The present invention also provides a vaccine composition comprising a lipopolysaccharide derived from Hafnia alvei, a carbomer, and an antigen.

[0050] In the present invention, a vaccine adjuvant refers to a drug that enhances a specific immune response to a vaccine or inoculation. The vaccine adjuvant may be defined as any substance or agent that acts to accelerate, prolong, or improve the quality of a specific immune response to an immunogenic substance.

[0051] The above vaccine adjuvant may be included in the vaccine and administered in a single or multiple doses. Since the desired immune enhancement effect can be achieved with just a single dose, it may preferably be administered in a single dose, and in the case of multiple doses, it may be administered in two doses.

[0052] The composition of the present invention, the vaccine adjuvant, may further include one or more known effective ingredients having an immune-enhancing effect, more preferably, a stinging agent, such as cGAMP, 3'3'-cGAMP, c-di-GAMP, c-di-AMP, 2'3'-cGAMP, 10-(carboxymethyl)9(10H)acridone(CMA), 6-dimethylxanthenone-4-acetic acid (5,6-Dimethylxanthenone-4-acetic acidㅡ DMXAA), methoxyvone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3',6'-dihydroxyflavone, 7, It may further include at least one selected from the group consisting of 2'-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether, and xanthone.

[0053] The above vaccine adjuvant composition can be mixed with the vaccine in a ratio of 1:0.01 to 100 (v / v), preferably in a ratio of 1:0.1 to 10 (v / v), more preferably in a ratio of 1:1 (v / v).

[0054] The antigen included in the vaccine composition of the present invention may be a pathogen protein, recombinant protein, glycoprotein, peptide, polysaccharide, lipopolysaccharide, polynucleotide, hormone, tumor antigen, allergen, cell, bacterium, fungus, virus, parasite, or a combination thereof.

[0055] The amount of antigen used to induce an immune response will vary considerably depending on the antigen used, the target, and the desired response level, and can be determined by those skilled in the art. The combination of Hafnia alvei-derived lipopolysaccharide and carbomer of the present invention can induce a superior immune response when administered with the same amount of antigen compared to conventional immunostimulants, and this effect is superior to the immune-enhancing effect of the individual components.

[0056] In the present invention, the mixing ratio of the immune enhancer including lipopolysaccharide and carbomer derived from Hafnia alvei and the antigen may be 1:0.01 to 100 (v / v), preferably 1:0.1 to 10 (v / v), and more preferably 1:0.1, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5 (v / v).

[0057] The present invention also provides a method for inducing an immune response in a subject, comprising administering the immunostimulating composition to the subject in need thereof. Routes of administration of the immunostimulating composition include, but are not limited to, parenteral, oral, palatal, intratracheal, topical, intramuscular, and intraovarian. The composition may be administered using any suitable device, including a syringe, dropper, needle-free injection device, patch, or the like. The route and device selected for use may be appropriately adjusted depending on the composition of the immunostimulating composition, antigen, and target.

[0058] The above-mentioned subject may include, without limitation, any animal to which administration of the immunostimulating composition is desirable, and may include all mammals and non-mammals.

[0059]

[0060] According to another aspect of the present invention, the present invention provides a composition for preventing or treating an infectious disease, comprising a lipopolysaccharide derived from Hafnia albaei and a carbomer.

[0061] In a specific embodiment of the present invention, the infectious disease may be caused by one or more infections selected from the group consisting of parasites, viruses, bacteria and fungi, and more preferably, the infectious disease is sepsis, septic shock, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome (MERS), salmonellosis, food poisoning, typhoid fever, paratyphoid fever, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), pneumonia, pulmonary tuberculosis, tuberculosis, cold, influenza, respiratory tract infection, rhinitis, nasopharyngitis, otitis media, bronchitis, lymphadenitis, parotitis, lymphadenitis, cheilitis, stomatitis, arthritis, myositis, dermatitis, vasculitis, Gingivitis, periodontitis, keratitis, conjunctivitis, wound infection, peritonitis, hepatitis, osteomyelitis, cellulitis, meningitis, encephalitis, brain abscess, encephalomyelitis, meningitis, osteomyelitis, nephritis, carditis, endocarditis, enteritis, gastritis, esophagitis, duodenitis, colitis, uritis, cystitis, vaginitis, cervicitis, salpingitis, erythema infectiosum, shigellosis, abscesses and ulcers, bacteremia, diarrhea, dysentery, gastroenteritis, gastroenteritis, urogenital abscess, infection of open wounds or wounds, purulent inflammation, abscesses, boils, pyoderma, impetigo, folliculitis, cellulitis, postoperative wound infection, skin scald syndrome, skin burn syndrome, thrombotic thrombocytopenia, hemolytic uremic syndrome, renal failure, pyelonephritis, glomerulonephritis, nervous system abscess, otitis media, Sinusitis, pharyngitis, tonsillitis, mastoiditis, cellulitis, odontogenic infection, dacryocystitis, pleurisy, abdominal abscess, liver abscess, cholecystitis, splenic abscess, pericarditis, myocarditis, placentitis, amniotic sac, mastitis, puerperal fever, toxic shock syndrome, Lyme disease, gas gangrene,It may be at least one selected from the group consisting of atherosclerosis, Mycobacterium avium syndrome (MAC), enterohemorrhagic Escherichia coli (EHEC) infection, enteropathogenic Escherichia coli (EPEC) infection, enteroinvasive Escherichia coli infection (EIEC), methicillin-resistant Staphylococcus aureus (MRSA) infection, vancomycin-resistant Staphylococcus aureus (VRSA) infection, and listerosis.

[0062] The composition according to the present invention may contain a pharmaceutically effective amount of the fungus and an excipient or diluent.

[0063] The pharmaceutically effective amount described above refers to an amount sufficient to exert an immune-enhancing effect. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions, such as gastrointestinal upset or dizziness, or similar reactions.

[0064] In addition, the composition according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. Suitable formulations known in the art are preferably those disclosed in the literature (Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA). Carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, etc. When formulating the composition, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0065] The composition of the present invention can be administered in a therapeutically effective amount, which is the amount of the active ingredient or composition that induces a biological or medical response in a tissue system, animal, or human as conceived by a researcher, veterinarian, physician, or other clinician. It will be apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by those skilled in the art, and can be adjusted according to various factors including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, and concurrently used drugs. For a desirable effect, the composition of the present invention can be administered in an amount of 1 to 10,000 mg / kg / day, preferably 1 to 200 mg / kg / day, and can be administered once a day or divided into several times.

[0066]

[0067] In addition, the composition of the present invention may be a health functional food composition or a food composition for enhancing immunity. When the composition of the present invention is a food composition, the type of food is not particularly limited and may include all foods in the conventional sense. Non-limiting examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. When the composition is used as a food additive, the composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method.

[0068] In this specification, food means a natural or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and has a conventional meaning, and the food composition is intended to include all foods, food additives, health functional foods, and beverages.

[0069] There is no particular limitation on the type of food or health functional food of the present invention, and examples thereof include ramen, other noodles, beverages, tea, drinks, alcoholic beverages, various soups, meat, sausage, bread, chocolate, candy, confectionery, pizza, gum, dairy products including ice cream, or vitamin complexes.

[0070] In particular, the lipopolysaccharide and carbomer derived from Hafnia albaei according to the present invention can be added to various foods for the purpose of enhancing immunity and can be used in combination with other appropriate auxiliary ingredients and known additives that can be conventionally included in health functional foods, according to the selection of a person skilled in the art. The known additives also include other microorganisms that can be used together with the strain according to the present invention.

[0071] Therefore, the food composition of the present invention may preferably be a health functional food. The health functional food refers to a food manufactured or processed using a specific ingredient as a raw material or a specific ingredient contained in a food raw material by methods such as extraction, concentration, purification, and mixing for the purpose of health supplementation, and refers to a food designed and processed so that the ingredient can sufficiently exert a bioregulatory function such as biological defense, biological rhythm regulation, disease prevention and recovery, etc. on the body, and refers to a food that can perform functions related to immune enhancement, etc.

[0072]

[0073] The present invention also relates to a feed additive composition for enhancing immunity, comprising a lipopolysaccharide derived from Hafnia alvei and a carbomer. The feed additive composition for enhancing immunity may be a feed additive or a compound feed.

[0074] In the present invention, "feed additive" refers to a substance added in small amounts to feed for nutritional or specific purposes, and the immune booster of the present invention may be applied in an amount of 0.01 to 99 wt% of the total weight of the feed additive. The feed composition of the present invention may additionally include other nutritional ingredients within a range that can be expected by those skilled in the art.

[0075] The Hafnia alvei-derived lipopolysaccharide and carbomer of the present invention can be incorporated into commercially available animal feed compositions and fed to, for example, cattle, pigs, sheep, poultry, etc. For this purpose, the strain of the present invention and its culture solution can be mixed with conventional animal feed components and, if necessary, processed into a purified form. Conventional animal feed components include, for example, corn, barley, oats, soybeans, fish meal, bran, soybean oil, minerals, trace elements, amino acids, and vitamins.

[0076]

[0077] The present invention also provides a method for preparing an immune-enhancing composition, comprising the step of mixing a lipopolysaccharide derived from Hafnia alvei and a carbomer. The mixing step may further include mixing squalene into an oil-in-water emulsion formulation. Methods for mixing and preparing an oil-in-water emulsion are well known to those skilled in the art, and generally, the method may include mixing a squalene oil phase with a surfactant and homogenizing using a homogenizer. In an oil-in-water emulsion, the oil and the emulsifier must be present in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.

[0078]

[0079] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0080]

[0081] Hereinafter, the present invention will be described in more detail through examples. These examples are only for the purpose of illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not construed as being limited by these examples. The carbomer used in the examples below is Carbopol 974P. In addition, it is expected that results having a tendency substantially equivalent to or similar to those of the examples below can be obtained by using any carbomer selected from the carbomers that can be expressed by the chemical formula [C3H4O2]n (where n is 1,000 to 1,000,000).

[0082]

[0083] Example 1. Immunogenicity evaluation of a combination vaccine containing various concentrations of carbopol and H. alvei LPS mixed adjuvant.

[0084] 1.1H. alveiLPS manufacturing

[0085] On November 11, 2019, a dominant bacterium was isolated from the feces of a male roe deer rescued in Jiam-ri, Sabuk-myeon, Chuncheon-si, Gangwon-do, and Hafnia alveiBA2000346 was isolated and identified through subculture on blood medium. The isolated and identified strain was patented and deposited with the Korean Collection for Type Cultures (KCTC) on November 3, 2023, and was assigned the domestic accession number KCTC19127P and the international accession number KCTC15966BP under the Budapest Treaty. The Hafnia alveiBA2000346-derived LPS (hereinafter referred to as H. alveiLPS) was used in the preparation of the following combination vaccine.

[0086]

[0087] 1.2 Preparing the Mouse

[0088] To determine the efficacy of an immunostimulant according to the concentration of the mixture of the immune-inducing substance Carbopol and H. alvei LPS, various concentrations of H. alvei LPS (20 μg / ml, 100 μg / ml) and Carbopol (0.01%, 0.1%) were mixed in an oil-in-water emulsion containing squalene oil, and the effects were determined. 6-week-old BALB / c (Female) were used in the experiment, and the immunostimulant experimental groups are shown in Table 1 below.

[0089] Experimental group candidate substance Group 1 PBS (negative control) Group 2 Vehicle (0.1% carbopol, 100 μg / ml H. alveiLPS - O / W emulsion) Group 3 Antigen (control group for comparison of immune adjuvant efficacy, antigen administered alone) Group 4 Antigen / 0.01% carbopol, 20 μg / ml H. alveiLPS - O / W emulsion Group 5 Antigen / 0.01% carbopol, 100 μg / ml H. alveiLPS - O / W emulsion Group 6 Antigen / 0.1% carbopol, 20 μg / ml H. alveiLPS - O / W emulsion Group 7 Antigen / 0.1% carbopol, 100 μg / ml H. alveiLPS - O / W emulsion

[0090] The antigen was ovalbumin (OVA) (Invivogen, Cat. No.: vac-pova), which is commonly used in the development of immune boosters, at a concentration of 10 μg / head, and was mixed with the immune booster at a ratio of 1:1 (v / v) for the final vaccination.

[0091] An experiment was conducted to evaluate the immunogenicity and safety of the above-mentioned protein antigen and adjuvant combination vaccine in a mouse model (IACUC Approval Number: EU-22-014). Specifically, 6-week-old BALB / c (female) mice (4 per group) were admitted and stabilized for 1 week. The adjuvant combination vaccine was administered intramuscularly twice, 50 μl each, into both thighs of the mice at 2-week intervals (100 μl total). The body weights of the mice were measured twice a week (DPV 0-28), and blood samples were collected (DPV 13, 28) and serum was isolated two weeks after the first and second vaccinations to measure antigen-specific antibodies. The in vivo safety of the adjuvant was confirmed by examining abnormal findings at the injection site during necropsy (DPV 28).

[0092]

[0093] 1.3 Evaluation of antigen-specific antibody formation following administration of a combination vaccine containing carbopol and H. alvei LPS-based adjuvants

[0094] The formation of antigen-specific antibodies in mice following combined vaccine administration was evaluated using an enzyme-linked immunosorbent assay (ELISA). Specifically, the antigens used in the combined vaccine were mixed in ELISA coating buffer (0.1 M sodium carbonate, pH 9.5) at a concentration of 1 μg / well, and 100 μl was dispensed into each 96-well immunoplate (Nunc, Cat. No.: 439454) and incubated overnight at 4°C. The cultured cells were washed four times with PBS-Tween 20 (0.05%), and 200 μl blocking solution (1% bovine serum albumin in PBS) was added and incubated overnight at 4°C. The collected mouse serum was prepared by diluting it in PBS-Tween 20 (0.05%). 100 μl of the diluted serum was added to each well and incubated at 37°C for 2 hours. Afterwards, the cells were washed four times with PBS-Tween 20 (0.05%), and 100 μl of diluted anti-mouse IgG H+L (ThermoFisher, Cat No.: 62-6520, 1 / 5,000) was added and incubated at 37°C for 1 hour. After incubation, the cells were washed four times with PBS-Tween 20 (0.05%). 100 μl of 1-Step Ultra TMB-ELISA Substrate Solution (ThermoFisher, Cat No.: 34029) was added to the washed cells and incubated at room temperature for 30 minutes. To evaluate antibody formation, the TMB reaction was stopped by adding 100 μl of 2N H2SO4 (DAEJUNG, Cat. No.: 7691-3700), and the absorbance was measured at 450 nm. The results of the antigen-specific total IgG antibody formation evaluation according to the combined vaccine administration are shown in Figure 1. In addition, the antibody formation rate compared to the antigen-only vaccination group was calculated, and the results are shown in Table 2.

[0095]

[0096] Group Ratio (based on control antigen-only vaccination group) G3 Antigen administration alone (control group for comparison of immune booster efficacy) 1.00 G4 Antigen / 0.01% carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 10.80 G5 Antigen / 0.01% carbopol, 100 μg / ml H. alvei LPS - O / W emulsion 10.85 G6 Antigen / 0.1% carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 13.15 G7 Antigen / 0.1% carbopol, 100 μg / ml H. alvei LPS - O / W emulsion 13.43

[0097]

[0098] As shown in Figure 1, the group containing the carbopol and H. alvei LPS-based immune-enhancing O / W emulsion formulation mixture (Groups 4 to 7) showed increased antibody formation compared to the group vaccinated with only the antigen (Group 3).

[0099] As shown in Table 2, the antibody titer of antigen-specific Total IgG was approximately 13.43 times higher in the 0.1% carbopol, 100 μg / mlH. alveiLPS - O / W emulsion (Group 7) than in the antigen-only vaccination group (Group 3) (*: P < 0.05).

[0100]

[0101] 1.4. Evaluation of Antigen-Specific Cellular Immune Responses Following Administration of a Combination Vaccine Containing Carbopol and H. alvei LPS-Based Adjuvants (Intracellular Cytokine Staining Assay)

[0102] Spleen cells, which are mouse immune cells, were seeded at 2.0 x 10 in a 96-well cell culture plate (NUNC, Cat. No.: 167008). 6Cells were seeded so that 100 cells / well were obtained. The seeded cells were treated with OVA protein (Invivogen, Cat. No.: vac-pova) at a concentration of 50 μg / ml and cultured in a 5% CO2 incubator at 37°C for 15 hours. Brefeldin A (1 / 1,000 dilution) was treated 10 hours before antibody staining. Anti-mouse CD16 / CD32 was treated at a concentration of 20 μg / ml and reacted for 20 minutes at room temperature. After washing twice with 1% BSA (in DPBS), the cells were treated with antibodies conjugated to immune cell surface factors (CD4, CD8) diluted in 1% BSA (in DPBS) and reacted for 30 minutes at room temperature. After washing twice with DPBS, Live / Dead cell stain (Invitrogen, Cat. No.: L34967) was reacted for 20 minutes at room temperature. After washing with 1% BSA (in DPBS), cells were treated with Fixation / Permeabilization solution (BD Bioscience, Cat. No.: 555028) and incubated at 4°C for 20 minutes. After removing the supernatant, cells were washed twice with Perm / Wash buffer (BD Bioscience, Cat. No.: 555028). After washing, cells were treated with cytokine (IL-2, IFN-γ, TNF-α)-conjugated antibodies and incubated at 4°C in the dark for 30 minutes. After washing twice with Perm / Wash buffer, cells were resuspended in 1% BSA (in DPBS) and the proportion of immune cells was analyzed using a flow cytometer (BD Bioscience, Cat. No.: FACSCelesta). The results of the intracellular cytokine staining (ICS) experiment to determine the proportion of CD4+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in the immune cells of BALB / c mice vaccinated with a vaccine containing an immune adjuvant are shown in Figure 2.Additionally, the proportion of CD4+ T cells expressing antigen-specific cytokines was calculated compared to the antigen-only vaccination group, and the results are shown in Table 3.

[0103] Group Ratio (based on control antigen only vaccination group) CD4+ T cell IFN-γ TNF-α IL-2 G3 Antigen only administration (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G4 Antigen / 0.01% carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 3.4 72.2 72.29 G5 Antigen / 0.01% carbopol, 100 μg / ml H. alvei LPS - O / W emulsion 18.3 26.3 74.6 1 G6 Antigen / 0.1% carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 20.7 5.9 0 4.08 G7 Antigen / 0.1% carbopol, 100 μg / ml H. alveiLPS - O / W emulsion17.016.354.96

[0104]

[0105] As shown in Fig. 2 and Table 3, the adjuvant H. alveiLPS and carbopol-based O / W emulsion formulation mixture groups (Groups 4 to 7) showed an increase in the proportion of CD4+ T cells expressing IFN-γ (Th1), TNF-α (Th1), and IL-2 (Th1) compared to the antigen-only group (Group 3) (*: P < 0.05). In particular, the 0.1% carbopol, 20 μg / ml H. alveiLPS - O / W emulsion formulation mixture group (Group 6) showed 20.75-fold, 5.90-fold, and 4.08-fold higher expression of IFN-γ (Th1), TNF-α (Th1), and IL-2 (Th1), respectively, than the antigen-only group (Group 3).

[0106]

[0107] 1.5 Additional confirmation of the immune-enhancing effect of combined vaccine administration containing carbopol and H. alvei LPS-based immune-enhancing agents.

[0108] Through the above Examples 1.3 and 1.4, it was confirmed that excellent antibody formation effects and cellular immunity induction effects were shown depending on the mixed concentration of the immune-inducing substances Carbopol and H. alvei LPS, and in particular, it was confirmed that the combination of 0.1% (w / v) Carbopol and H. alvei LPS evenly increased antibody formation and cellular immunity induction. Therefore, the mixed concentration of 0.1% (w / v) Carbopol and H. alvei LPS was further expanded to confirm the immune enhancement effect in the same manner as in Examples 1.3 and 1.4. In addition, the antigen-specific cellular immune response was compared using ELISpot. The ELISpot experiment was performed on BALB / c mice in the same manner as in Comparative Example 1.4 below.

[0109] The experimental groups used in additional experiments are shown in Table 4, and the results confirming the effects of inducing antibody formation and cellular immune response in each experimental group are shown in Figure 3.

[0110] Experimental group candidate substance Group 1 PBS (negative control) Group 2 Vehicle (0.1% carbopol, 100 μg / ml H. alveiLPS - O / W emulsion) Group 3 Antigen (control group for comparison of immune adjuvant efficacy, antigen administered alone) Group 4 Antigen / 0.1% carbopol, 5 μg / ml H. alveiLPS - O / W emulsion Group 5 Antigen / 0.1% carbopol, 20 μg / ml H. alveiLPS - O / W emulsion Group 6 Antigen / 0.1% carbopol, 100 μg / ml H. alveiLPS - O / W emulsion

[0111]

[0112] As shown in Fig. 3, the adjuvant containing 0.1% carbopol and 5 μg / ml, 20 μg / ml, or 100 μg / ml H. alveiLPS showed superior antibody formation induction and cellular immune response activation effects than the antigen-only group (Group 3). Therefore, it was confirmed that the combination of 0.1% carbopol and 5 μg / ml, 20 μg / ml, or 100 μg / ml H. alveiLPS can simultaneously activate the antibody and cellular immune responses aimed at the present invention. However, since the use of H. alveiLPS exceeding 100 μg / ml may induce toxicity, 0.1% carbopol and 20 μg / ml H. alveiLPS was selected as a combination that satisfies higher safety and was used in the following experiments.

[0113]

[0114] Example 2. Immunogenicity evaluation of a combination vaccine containing carbopol and H. alvei LPS as adjuvants.

[0115] 2.1 Preparing the Mouse

[0116] To determine whether mixing the immune-inducing substance Carbopol and H. alvei LPS enhances the efficacy of the immunostimulant, H. alvei LPS and Carbopol were mixed and prepared as an oil-in-water emulsion containing squalene oil, and its effect was determined. 6-week-old C57BL / 6 (Female) mice were used, and the immunostimulant experimental groups are shown in Table 5 below. Carbopol 974P was used as the carbopol.

[0117]

[0118] Experimental group candidate substance Group 1 PBS (negative control) Group 2 Vehicle (0.1% carbopol, 100 μg / ml H. alveiLPS - O / W emulsion) Group 3 Antigen (control group for comparison of immune adjuvant efficacy, antigen administered alone) Group 4 Antigen / 0.1% carbopol - O / W emulsion Group 5 Antigen / 20 μg / ml H. alveiLPS - O / W emulsion Group 6 Antigen / 0.1% carbopol, 20 μg / ml H. alveiLPS - O / W emulsion

[0119] The antigen was ovalbumin (OVA) (Invivogen, Cat. No.: vac-pova), which is commonly used in the development of immune boosters, at a concentration of 10 μg / head, and was mixed with the immune booster at a ratio of 1:1 (v / v) for the final vaccination.

[0120] An experiment was conducted to evaluate the immunogenicity and safety of the above-mentioned protein antigen and adjuvant combination vaccine in a mouse model (IACUC Approval Number: EU-22-014). Six-week-old C57BL / 6 (female) mice were housed in groups of four per group and stabilized for one week. The adjuvant combination vaccine was administered intramuscularly twice at two-week intervals, with 50 μl each in each thigh, for a total of 100 μl. The body weights of the mice (DPV 0-28) were measured twice a week, and blood samples were collected weekly (DPV 7, 13, 21, 28) and serum was isolated to measure antigen-specific antibodies. The in vivo safety of the adjuvant was confirmed by examining abnormal findings at the injection site during necropsy (DPV 28).

[0121]

[0122] 2.2 Evaluation of antigen-specific antibody formation following administration of a combination vaccine containing carbopol and H. alvei LPS-based adjuvants

[0123] There are four subclasses of immunoglobulin G (IgG) in mice: IgG1, IgG2a (IgG2c in C57BL / 6 mice), IgG2b, and IgG3. To analyze the antigen-specific antibody formation ability following vaccination, total IgG antibody production is analyzed for total IgG, cellular immune response (Helper T cell, Th1) activation is analyzed for IgG2a or IgG2c, and humoral immune response (Helper T cell, Th2) activation is analyzed for IgG1. Therefore, antigen-specific antibody formation following combined vaccine administration in mice was evaluated for total IgG, IgG1, and IgG2c using ELISA (Enzyme-linked immunosorbent assay). Specifically, the antigens used in the combined vaccine were mixed in ELISA coating buffer (0.1 M sodium carbonate, pH 9.5) to a concentration of 1 μg / well, and 100 μl was dispensed into each 96-well immunoplate (Nunc, Cat. No.: 439454) and incubated overnight at 4°C. The cultured cells were washed four times with PBS-Tween 20 (0.05%), and 200 μl blocking solution (1% bovine serum albumin in PBS) was added and incubated overnight at 4°C. The collected mouse serum was prepared by diluting it in PBS-Tween 20 (0.05%). 100 μl of the diluted serum was added to each well and incubated at 37°C for 2 hours. After washing four times with PBS-Tween 20 (0.05%), the cells were incubated with anti-mouse IgG H+L (ThermoFisher, Cat No.: 62-6520, 1 / 5,000), anti-mouse IgG1 (ThermoFisher, Cat No.: PA1-74421, 1 / 5,000), anti-mouse IgG2c (Abcam, Cat No.: ab97255, 1 / 5,000) was diluted and added 100 μl each and incubated at 37°C for 1 hour. After incubation, the cells were washed four times with PBS-Tween 20 (0.05%). 100 μl of 1-Step Ultra TMB-ELISA Substrate Solution (ThermoFisher, Cat. No.: 34029) was added to the washed cells and incubated at room temperature for 30 minutes. To evaluate antibody formation, 100 μl of 2N H2SO4 (DAEJUNG, Cat. No.: 7691-3700) was added to stop the TMB reaction, and the absorbance was measured at 450 nm. The results of the antigen-specific antibody formation evaluation according to the combined vaccine administration are shown in Figure 4. In addition, the antibody formation rate compared to the antigen-only vaccination group was calculated, and the results are shown in Table 6.

[0124] Group Ratio (based on control group antigen-only vaccination) Total IgGIgG1 (Th2) IgG2c (Th1) G3 Antigen (control group for comparison of immune booster efficacy, antigen-only administration) 1.00 1.00 1.00 G4 Antigen / 0.1% Carbopol - O / W emulsion 2.90 12.7 13.45 G5 Antigen / 20 μg / ml H. alvei LPS - O / W emulsion 4.01 19.48 0.79 G6 Antigen / 0.1% Carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 4.79 25.5 6 5.38

[0125] As shown in Figure 4, the group of mixed O / W emulsion formulations of carbopol and H. alvei LPS-based immune booster (Groups 4 to 7) showed increased antibody formation compared to the group vaccinated with only the antigen (Group 3).

[0126] As shown in Table 6, the antibody titer of antigen-specific Total IgG increased 2.90-fold in the 0.1% carbopol - O / W emulsion group (Group 4) compared to the antigen-only group (Group 3), and 4.01-fold and 4.79-fold in the 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) and the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 6), respectively. In addition, the antibody titer of IgG1 (Th2) increased 12.71-fold in the 0.1% carbopol - O / W emulsion group (Group 4), and 12.71-fold in the 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5), and 0.1% carbopol, 20 μg / mlH. In the alveiLPS - O / W emulsion group (Group 6), the levels increased by 19.48 times and 25.56 times, respectively (*:P< 0.05).

[0127] In particular, the antibody titer of antigen-specific IgG2c (Th1) increased 5.38-fold (*: P < 0.05) in Group 6, the immunoadjuvant experimental group of the present invention, compared to the antigen-only vaccinated group (Group 3), showing a very significant increase compared to the H. alveiLPS-only experimental group (Group 5), which showed no increase, or the carbopol-only experimental group (Group 4), which showed only a 3.45-fold increase in effect. When developing an immunoadjuvant, IgG2c is known to be an antibody produced when the Th1 response, a cellular immune response, is activated, and thus serves as a very important indicator for selecting an immunoadjuvant. Therefore, it was confirmed that the above experimental group, which can significantly increase the expression of IgG2c, is a candidate group that can show the best effect as an immunoadjuvant.

[0128] Comparative Example 1. Comparison of the effects of carbopol and H. alvei LPS-based immunostimulants compared to existing immunostimulants.

[0129] 1.1. Preparing the Mouse

[0130] An experiment was conducted to compare the effects of vaccination with an adjuvant containing carbopol and H. alvei LPS as immune-inducing substances with those of existing commercially available adjuvants, AddaS03 (InvivoGen, cat code: vac-as03-10) and AddaVaX (InvivoGen, cat code: vac-adx-10). The adjuvant containing carbopol and H. alvei LPS as immune-inducing substances was prepared as an oil-in-water emulsion containing squalene oil and used in the experiment. The mice used in the experiment were 6-week-old C57BL / 6 (Female) and BALB / c (Female), and the adjuvant experimental groups are shown in Table 7 below. The antigen was ovalbumin (OVA) (Invivogen, Cat. No.: vac-pova), which is commonly used in the development of immune boosters, at a concentration of 10 μg / head, and was mixed with the immune booster at a ratio of 1:1 (v / v) for the final vaccination.

[0131] Experimental group candidate substance Group 1 PBS (negative control group) Group 2 Antigen (control group for comparison of immune adjuvant efficacy, antigen administered alone) Group 3 Antigen / AddaS0 Group 3 Antigen / AddaVax Group 5 Antigen / 0.1% carbopol, 20 μg / ml H. alvei LPS - O / W emulsion

[0132] An experiment was conducted to evaluate the immunogenicity and safety of the above-mentioned protein antigen and adjuvant combination vaccine in a mouse model (IACUC Approval Number: EU-22-014). Specifically, 6-week-old C57BL / 6 (Female) and BALB / c (Female) mice (4 per group) were admitted to a laboratory and stabilized for 1 week. The adjuvant combination vaccine was administered intramuscularly twice at 2-week intervals, with a total of 100 μl (50 μl each) in both thighs of the mice. The body weights (DPV 0-28) of the mice were measured twice a week, and blood samples were collected (DPV 13, 28) and serum was isolated two weeks after the first and second vaccinations to measure antigen-specific antibodies. The in vivo safety of the adjuvant was confirmed by examining abnormal findings at the injection site during necropsy (DPV 28).

[0133]

[0134] 1.2. Comparative evaluation of antigen-specific antibody formation according to combined vaccine administration

[0135] The formation of antigen-specific antibodies in mice following combined vaccine administration was evaluated using an enzyme-linked immunosorbent assay (ELISA). Specifically, the antigens used in the combined vaccine were mixed in ELISA coating buffer (0.1 M sodium carbonate, pH 9.5) at a concentration of 1 μg / well, and 100 μl was dispensed into each 96-well immunoplate (Nunc, Cat. No.: 439454) and incubated overnight at 4°C. The immunoplate was washed four times with PBS-Tween 20 (0.05%), and 200 μl blocking solution (1% bovine serum albumin in PBS) was added and incubated overnight at 4°C. The collected mouse serum was prepared by diluting it in PBS-Tween 20 (0.05%). 100 μl of the diluted serum was added to each well and incubated at 37°C for 2 hours. Afterwards, the cells were washed four times with PBS-Tween 20 (0.05%), and 100 μl each of diluted anti-mouse IgG H+L (ThermoFisher, Cat No.: 62-6520, 1 / 5,000), anti-mouse IgG1 (ThermoFisher, Cat No.: PA1-74421, 1 / 5,000), and anti-mouse IgG2c (Abcam, Cat No.: ab97255, 1 / 5,000) were added and incubated at 37°C for 1 hour. After incubation, the cells were washed four times with PBS-Tween 20 (0.05%). 100 μl of 1-Step Ultra TMB-ELISA Substrate Solution (ThermoFisher, Cat No.: 34029) was added to the washed cells, and incubated at room temperature for 30 minutes. To evaluate antibody formation, the TMB reaction was stopped by adding 100 μl of 2N H2SO4 (DAEJUNG, Cat. No.: 7691-3700), and the absorbance was measured at 450 nm.

[0136]

[0137] The results of evaluating antigen-specific antibody formation following combined vaccine administration in C57BL / 6 (female) mice are shown in Figure 5A. Furthermore, the antibody formation rate compared to the antigen-only group was calculated, and the results are shown in Table 8.

[0138] C57BL / 6 Group Ratio (based on control antigen-only vaccination group) Total IgGIgG1 (Th2) IgG2c (Th1) G2 Antigen-only administration Comparison of immune-boosting efficacy Control group 1.00 1.00 1.00 G3 Antigen / AddaS 0 3 4.8 1 2 4.1 3 1.5 G4 Antigen / AddaVax 5.0 4 16.9 7 0.75 G5 Antigen / 0.1% Carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 4.7 8 25.5 6 5.38

[0139] As shown in Figure 5A, it was confirmed that antibody formation increased in all groups administered the immune booster O / W emulsion formulation mixture (Groups 3 to 5) compared to the antigen-only group (Group 2) (*: P < 0.05).

[0140] As shown in Table 8, the antibody titers of antigen-specific Total IgG were 5.04- and 4.78-fold higher in the AddaVax (Group 4) and 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion groups (Group 5), respectively, compared to the antigen-only group (Group 2). In addition, the antibody titers of IgG1 (Th2) were increased 24.13- and 25.56-fold in the AddaS03 (Group 3) and 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion groups (Group 5), respectively (*: P < 0.05).

[0141] Meanwhile, the antibody titer of antigen-specific IgG2c (Th1) increased approximately 5.38-fold in the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) compared to the antigen-only group (Group 2), and it was confirmed that it effectively activated IgG2c antibody formation compared to other O / W emulsion immune boosters (*:P< 0.05).

[0142]

[0143] The results of the antigen-specific antibody formation evaluation following combined vaccine administration in BALB / c (female) mice are shown in Figure 5B. In addition, the antibody formation rate compared to the antigen-only vaccination group was calculated, and the results are shown in Table 9.

[0144]

[0145] BALB / c Group Ratio (based on control antigen-only vaccination group) Total IgGIgG1 (Th2) IgG2c (Th1) G2Antigen (control group for comparison of immune adjuvant efficacy, antigen-only administration) 1.00 1.00 1.00 G3Antigen / AddaS 03 10.97 28.36 0.98 G4Antigen / AddaVax 10.03 27.06 1.11 G5Antigen / 0.1% Carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 13.15 31.2 12.04

[0146] As shown in Figure 5B, it was confirmed that antibody formation increased in the immune booster O / W emulsion formulation mixture group (Groups 3 to 5) compared to the antigen-only group (Group 2) (*: P < 0.05).

[0147] As shown in Table 9, the antibody titer of antigen-specific Total IgG was approximately 13.15 times higher in the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) than in the antigen-only group (Group 2). In addition, the antibody titer of IgG1 (Th2) increased approximately 31.21 times in the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) (*: P < 0.05). In particular, the antibody titer of antigen-specific IgG2c (Th1) increased approximately 2.04 times in the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) than in the antigen-only group (Group 2) (*: P < 0.05).

[0148]

[0149] 1.3. Comparative Evaluation of Antigen-Specific Cellular Immune Responses (Intracellular Cytokine Staining Assay) Following Combined Vaccine Administration

[0150] Spleen cells, which are mouse immune cells, were seeded at 2.0 x 10 in a 96-well cell culture plate (NUNC, Cat. No.: 167008). 6Cells were seeded so that 10 cells / well. The seeded cells were treated with OVA protein (Invivogen, Cat. No.: vac-pova) at a concentration of 50 μg / ml and cultured in a 5% CO2 incubator at 37°C for 15 hours. Brefeldin A (1 / 1,000 dilution) was treated 10 hours before antibody staining. Anti-mouse CD16 / CD32 was treated at a concentration of 20 μg / ml and reacted at room temperature for 20 minutes. After washing twice with 1% BSA (in DPBS), the cells were treated with immune cell surface factor (CD4, CD8)-conjugated antibodies diluted in 1% BSA (in DPBS) and reacted at room temperature for 30 minutes. After washing twice with DPBS, Live / Dead cell staining (Invitrogen, Cat. No.: L34967) was performed at room temperature for 20 minutes. After washing with 1% BSA (in DPBS), the cells were treated with Fixation / Permeabilization solution (BD Bioscience, Cat. No.: 555028) and incubated at 4°C for 20 minutes. The supernatant was removed and washed twice with Perm / Wash buffer (BD Bioscience, Cat. No.: 555028). After washing, the cells were treated with cytokine (IL-2, IFN-γ, TNF-α)-conjugated antibodies and incubated at 4°C in the dark for 30 minutes. After washing twice with Perm / Wash buffer, the cells were resuspended in 1% BSA (in DPBS) and the immune cell ratio was analyzed using a flow cytometer (BD Bioscience, Cat. No.: FACSCelesta).

[0151] The results of an intracellular cytokine staining (ICS) experiment to determine the proportion of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in the immune cells of C57BL / 6 mice vaccinated with a vaccine containing an immune adjuvant are shown in Figure 6A. In addition, the proportion of CD4+ and CD8+ T cells derived from C57BL / 6 splenocytes expressing antigen-specific cytokines was calculated compared to the antigen-only vaccinated group, and the results are shown in Tables 10 and 11.

[0152]

[0153] C57BL / 6 Group Ratio (based on control antigen-only vaccination group) CD4+ T cell IFN-γ TNF-α IL-2 G2 Antigen administration alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / AddaS 0 38.30 5.33 6.44 G4 Antigen / Addavax 10.98 5.95 7.70 G5 Antigen / 0.1% Carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 98.47 10.5 18.33

[0154] C57BL / 6 Group Ratio (based on control antigen-only vaccination group) CD8+ T cell IFN-γ TNF-α IL-2 G2 Antigen administration alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / AddaS 03 15.95 3.5 20.00 G4 Antigen / Addavax 52.94 7.7 34.5 1 G5 Antigen / 0.1% Carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 103.12 13.5 01.67

[0155] As shown in Figure 6A, it was confirmed that the ratio of CD4+, CD8+ T cells expressing TNF-α (Th1), IFN-γ (Th1), and IL-2 (Th1) increased in the immune-enhancing O / W emulsion formulation mixture group (Groups 3 to 5) (*: P < 0.05).

[0156] As shown in Table 10, the proportion of cytokine-expressing CD4+ T cells in the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) was 98.47 times, 10.51 times, and 8.33 times higher than that in the antigen-only group (Group 2). (*:P< 0.05)

[0157] In addition, as shown in Table 11, the proportion of cytokine-expressing CD8+ T cells in the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) was 103.12 times, 13.50 times, and 1.67 times higher, respectively, than in the antigen-only group (Group 2) in comparison with the antigen-only group (Group 2) (*: P < 0.05).

[0158]

[0159] The same Intracellular Cytokine Staining (ICS) experiment was performed in BALB / c mice vaccinated with a vaccine containing an immunoadjuvant, and the results are shown in Figure 6B. In addition, the percentage of CD4+ T cells derived from BALB / c splenocytes was calculated and shown in Table 12.

[0160]

[0161] BALB / c Group Ratio (based on control antigen-only vaccination group) CD4+ T cell IFN-γ TNF-α IL-2 G2 Antigen administration alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / AddaS 0 3 2.8 1 3.4 5 2.9 2 G4 Antigen / Addavax 1.8 6 6 1 6 5.6 7 G5 Antigen / 0.1% Carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 20.7 5 5.9 4.0 8

[0162] As shown in Figure 6B, it was confirmed that the proportion of CD4+ T cells expressing TNF-α (Th1), IFN-γ (Th1), and IL-2 (Th1) increased in the immune-enhancing O / W emulsion formulation mixture group (Groups 3 to 5) (*: P < 0.05).

[0163] As shown in Table 12, the proportion of cytokine-expressing CD4+ T cells in the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) was 20.75 times, 5.90 times, and 4.08 times higher, respectively, than in the antigen-only group (Group 2) compared to the antigen-only group (Group 2) (*: P < 0.05).

[0164]

[0165] 1.4 Comparative evaluation of antigen-specific cellular immune responses (ELISpot) according to combined vaccine administration

[0166] To evaluate antigen-specific cellular immune responses, splenocytes from C57BL / 6 and BALB / c mice were harvested and necropsied 2 weeks after the second dose of the adjuvanted vaccine (DPV 28). The harvested mouse splenocytes were plated at a density of 2.5 × 10 on an IFN-γ ELISpot plate (Mabtech, Cat. No.: 3321-4HPT-10). 5Cells were seeded so that 10 cells / well was obtained. To confirm the activation of antigen-specific cellular immune response, OVA protein (Invivogen, Cat. No.: vac-pova) was treated at a concentration of 10 μg / ml and cultured in a 5% CO2 incubator at 37°C for 20 hours. The cultured cells were washed five times with PBS, and 100 μl of biotinylated detection mAb was diluted 1 / 1,000 in PBS (with 0.5% fetal bovine serum) and added per well. The cells were stored at room temperature, protected from light, for 2 hours. After washing five times with PBS, 100 μl of streptavidin-HRP was diluted 1 / 1,000 in PBS (with 0.5% fetal bovine serum) and added per well. The cells were stored at room temperature, protected from light, for 1 hour. After washing five times with PBS, 100 μl of TMB substrate was added per well and reacted until fewer than five spots appeared in the negative control well (reaction time: 30 min). Color development was stopped by washing with double-distilled water, and the ELISpot plate was dried in a light-protected manner and analyzed using an ELISpot Reader (Cellular Technology Limited, BioSpot S5).

[0167] The results of confirming the antigen-specific IFN-γ formation ability through ELISpot experiment in spleen cells of C57BL / 6 mice are shown in Figure 7A, and the results of calculating the antigen-specific IFN-γ formation ability compared to the antigen-only vaccination group are shown in Table 13.

[0168] C57BL / 6 Group Ratio (based on control antigen-only vaccination group) G2Antigen administration alone (control group for comparison of immune-enhancing efficacy) 1.00 G3Antigen / AddaS 0.3 0.50 G4Antigen / Addavax 3.00 G5Antigen / 0.1% Carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 9.17

[0169] As shown in Fig. 7A and Table 13, the spot-forming ability of the C57BL / 6 mice in the adjuvant O / W emulsion formulation mixture group (Groups 3 to 5) increased compared to the antigen-only group. In particular, the spot-forming ability increased approximately 9.17-fold in the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) compared to the antigen-only group (Group 2).

[0170]

[0171] The results of confirming the antigen-specific IFN-γ formation ability through ELISpot experiment in spleen cells of BALB / c mice are shown in Figure 7B, and the results of calculating the IFN-γ formation ability compared to the control group administered antigen alone are shown in Table 14.

[0172]

[0173] BALB / c Group Ratio (based on control antigen-only vaccination group) G2 Antigen administration alone (control group for comparison of immune booster efficacy) 1.00 (spot count was 0, but was marked as 0.1 for ratio calculation) G3 Antigen / AddaS 0 3 7 0.00 G4 Antigen / Addavax 3 0.00 G5 Antigen / 0.1% Carbopol, 20 μg / ml H. alvei LPS - O / W emulsion 5 50.00

[0174] As shown in Figure 7B and Table 14, the 0.1% carbopol, 20 μg / mlH. alveiLPS - O / W emulsion group (Group 5) was approximately 550 times higher than the antigen-only group (Group 2) (*: P < 0.05).

[0175]

[0176] In summary, the present inventors have developed an immune-enhancing composition comprising carbomer (carbopol) and lipopolysaccharide derived from Hafnia alvei. The immune-enhancing composition according to the present invention has been experimentally confirmed to exhibit excellent antigen-specific antibody formation ability in a subject and to effectively induce cellular and humoral immunity. Therefore, the immune-enhancing composition of the present invention can be usefully utilized in various fields, including prevention of pathogen (viral, bacterial, etc.) infections, immune enhancement, vaccine adjuvant, and anticancer treatment.

[0177]

[0178] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0179]

[0180] Accession number

[0181] Name of depositor: Biological Resource Center

[0182] Accession number: KCTC19127P

[0183] Date of acceptance: 20231103

[0184]

[0185] Accession number

[0186] Name of depositor: Biological Resource Center

[0187] Accession number: KCTC15966BP

[0188] Date of acceptance: 20240718

[0189]

Claims

1. An immune-enhancing composition comprising lipopolysaccharide and carbomer derived from Hafnia alvei.

2. An immune-enhancing composition according to claim 1, wherein the hafnia albaei-derived lipopolysaccharide and carbomer are formulated as an oil-in-water emulsion.

3. In the second paragraph, the composition for enhancing immunity further comprises squalene.

4. An immune-enhancing composition according to claim 1, wherein the lipopolysaccharide derived from Hafnia albaei is contained in an amount of 1 to 100 μg / ml.

5. An immune-enhancing composition according to claim 1, wherein the carbomer is contained in an amount of 0.005 to 0.5% (w / v).

6. A vaccine adjuvant composition comprising a lipopolysaccharide derived from Hafnia alvei and a carbomer.

7. A vaccine composition comprising a lipopolysaccharide, carbomer and antigen derived from Hafnia alvei.

8. A composition for preventing or treating infectious diseases, comprising lipopolysaccharide, carbomer and antigen derived from Hafnia alvei.

9. A composition for preventing or treating an infectious disease, wherein the infectious disease is caused by one or more types of infection selected from the group consisting of parasites, viruses, bacteria, and fungi.

10. A health functional food composition for enhancing immunity, comprising lipopolysaccharide and carbomer derived from Hafnia alvei.

11. A feed additive composition for enhancing immunity, comprising lipopolysaccharide and carbomer derived from Hafnia alvei.

12. A method for inducing an immune response in a subject, comprising administering to a subject in need thereof an immune-enhancing composition according to any one of claims 1 to 5.

13. A method for producing an immune-enhancing composition, comprising the step of mixing a lipopolysaccharide derived from Hafnia alvei and a carbomer.

14. A method for producing an immune-enhancing composition in claim 13, wherein the mixing step further includes squalene and mixes the composition into an oil-in-water emulsion formulation.

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