Composition for immune enhancement

Abstract

The present invention relates to an immunostimulatory composition comprising various lipopolysaccharides and carbomers derived from hafnia albeit. The immunostimulatory composition according to the present invention possesses unique physical properties (pH, viscosity, particle size) in the final formulation, exhibits excellent ability to promote antigen-specific antibody formation, and can effectively induce cellular and humoral immunity within the individual. Therefore, the immunostimulatory composition of the present invention can be usefully utilized in various fields, such as the prevention of various infectious diseases caused by pathogens, immunostimulation, and vaccine adjuvantage.

Description

Technology Field

[0001] The present invention relates to an immunostimulating composition comprising a lipopolysaccharide derived from hafnia albei and a carbomer, having a pH of 6.5 to 7.5, a viscosity of 1.95 to 2.15 cp, or a particle size of 95 nm to 140 nm. Background Technology

[0003] Vaccines are classified according to the type of antigen into live-attenuated vaccines, killed / inactivated vaccines, subunit vaccines, virus-like particles vaccines, and DNA / RNA-based vaccines. Live-attenuated vaccines generally contain components capable of self-replication within the human body and acting as natural adjuvants, allowing them to simultaneously induce humoral and cellular immune responses to establish a highly effective defense. However, due to concerns regarding side effects caused by mutations, they have transitioned to killed / inactivated vaccines; a disadvantage is that their efficacy is relatively low because they cannot self-replicate within the human body and do not follow normal infection pathways. Subunit vaccines, which use only a portion of the pathogen as an antigen (recombinant protein, peptide, etc.), allow for rapid and effective mass production during an epidemic; however, a disadvantage is that they do not possess the microbe-associated molecular patterns (MAMPs; a preferred term over the existing pathogen-associated molecular patterns (PAMPs)) inherent in the microorganism, resulting in significantly lower efficacy when the antigen is administered alone. Therefore, the use of adjuvants is essential to enhance the immune response of inactivated / suppressed vaccines and subtype vaccines.

[0004] An adjuvant is a major component of a vaccine that plays a role in inducing the host's immune response to an antigen upon in vivo vaccination. It can effectively increase the in vivo immune response that is insufficient when using the antigen alone; consequently, the use of adjuvants can enhance the immune response of immunocompromised groups, such as infants or the elderly, and can induce specific immune responses (humoral or cellular immune responses) depending on the mixture of immune-inducing substances. Furthermore, through the activation of the immune response resulting from the use of adjuvants, the amount of antigen used per vaccination can be reduced or the number of vaccinations can be minimized.

[0005] According to recent research trends, adjuvants containing a mixture of various immunomodulators are being developed. Research is being conducted on discovering new Toll-like receptor agonists (TLR1 / 2 agonist: PAM3CSK4, TLR3 agonist: Poly(I:C), TLR4 agonist: Lipopolysaccharide, etc.) to activate necessary immune responses (humoral or cellular, or simultaneous humoral / cellular activation) based on the infection mechanism of pathogens, or on various formulations (emulsion, liposome, gel type, etc.) to delay antigen release at the vaccination site and improve the recruiting and antigen absorption capabilities of immune cells. The problem to be solved

[0007] The inventors have completed the present invention by confirming that an immune-enhancing composition comprising lipopolysaccharides and carbomers derived from Hafnia albei bacteria, satisfying specific pH, viscosity, and particle size, has a significant immune-enhancing effect.

[0008] Therefore, the object of the present invention is to provide a composition comprising the following components: a) Hafnia Albay ( Hafnia alvei) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm.

[0009] Another object of the present invention is to provide a method for inducing an immune response in an individual, comprising the step of administering the composition to an individual in need thereof. means of solving the problem

[0011] To achieve the above objective, the present invention provides an immune-enhancing composition comprising the following components: a) hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) pH 6.5 to pH 7.5.

[0012] In addition, the present invention provides an immune-enhancing composition comprising the following components: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp.

[0013] In addition, the present invention provides an immune-enhancing composition comprising the following components: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) particle size of 95 nm to 140 nm.

[0014] The present invention also provides a vaccine adjuvant composition comprising the following components: a) hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm.

[0015] The present invention also provides a vaccine composition comprising the following components: a) Hafnia albei ( Hafnia alvei) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm.

[0016] The present invention also provides a pharmaceutical composition for the prevention or treatment of infectious diseases comprising the following components: a) hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm.

[0017] In addition, the present invention provides a health functional food composition for immune enhancement comprising the following components: a) Hafnia Albay ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm.

[0018] The present invention also provides an immune-enhancing feed additive composition comprising the following components: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm.

[0019] In addition, the present invention provides a method for inducing an immune response in an individual, comprising the step of administering the immune-enhancing composition to an individual in need thereof. Effects of the invention

[0021] It has been experimentally confirmed that the immunostimulatory composition according to the present invention possesses unique physical properties regarding pH, viscosity, and particle size of the final formulation, exhibits excellent antigen-specific antibody formation ability, and effectively induces cellular and humoral immunity. Therefore, the immunostimulatory composition and manufacturing technology of the present invention can be usefully utilized in various fields such as the prevention and treatment of pathogen-infected diseases, immune enhancement, and vaccine adjuvantage. Brief explanation of the drawing

[0023] Figure 1A shows various concentrations of Carbopol 971P and Hafnia albei ( H. alvei This figure shows the results confirming the ability to form antigen-specific antibodies (Total IgG) following vaccination with an adjuvant-mixed vaccine containing strain BA2000346) LPS (*: P < 0.05). Fig. 1B shows various concentrations of Carbopol 974P and Hafnia albei ( H. alvei This figure shows the results confirming the antigen-specific antibody Total IgG formation ability following vaccination with an adjuvant mixture vaccine containing strain BA2000346) LPS (*: P < 0.05). Figure 2A shows various concentrations of Carbopol 971P and Hafnia albeit in immune cells of C57BL / 6 mice ( H. alvei This figure shows the results of comparing the ratios of CD4+ and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 following vaccination with a combined immunoadjuvant vaccine containing LPS (*: P < 0.05). Figure 2B shows various concentrations of Carbopol 974P and Hafnia albeit in immune cells of C57BL / 6 mice ( H. alveiThis figure shows the results of comparing the ratios of CD4+ and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 following vaccination with a combined immunoadjuvant vaccine containing LPS (*: P < 0.05). Figure 3A shows various concentrations of Carbopol 971P and Hafnia albei ( H. alvei This figure shows the results of comparing the effect of inducing antigen-specific cellular immune responses (ELISpot) following vaccination with an adjuvant-mixed vaccine containing strain BA2000346) LPS (*: P < 0.05). Figure 3B shows various concentrations of Carbopol 974P and Hafnia albei ( H. alvei This figure shows the results of comparing the effect of inducing antigen-specific cellular immune responses (ELISpot) following vaccination with an adjuvant-mixed vaccine containing strain BA2000346) LPS (*: P < 0.05). Figure 4A shows 0.01% (w / v) of Carbopol 971P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results of comparing the antigen-specific antibody (Total IgG) formation ability following vaccination with an adjuvant mixed vaccine containing LPS (20 ug / ml) derived from strain BA2000346, 9-126A-1, ATCC 51815). P < 0.05). Fig. 4B shows 0.01% (w / v) of Carbopol 974P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results of comparing the antigen-specific antibody (Total IgG) formation ability following vaccination with an adjuvant mixed vaccine containing LPS (20 ug / ml) derived from strain BA2000346, 9-126A-1, ATCC 51815). P < 0.05). Figure 5A shows Carbopol 971P at a concentration of 0.1% (w / v) and various hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results comparing the antigen-specific antibody (Total IgG) formation ability following vaccination with an adjuvant mixed vaccine containing LPS (20 ug / ml) (*: P < 0.05). Fig. 5B shows Carbopol 974P at a concentration of 0.1% (w / v) and various hafnia albeit in C57BL / 6 mice ( Hafnia alvei This figure shows the results comparing the antigen-specific antibody (Total IgG) formation ability following vaccination with an adjuvant mixed vaccine containing LPS (20 ug / ml) (*: P < 0.05). Figure 6A shows 0.1% (w / v) of Carbopol 971P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results comparing the antigen-specific antibody (Total IgG) formation ability following vaccination with an adjuvant mixed vaccine containing LPS (100 ug / ml) (*: P < 0.05). Fig. 6B shows 0.1% (w / v) of Carbopol 974P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results comparing the antigen-specific antibody (Total IgG) formation ability following vaccination with an adjuvant mixed vaccine containing LPS (100 ug / ml) (*: P < 0.05). Fig. 7A shows 0.01% (w / v) Carbopol 971P and various Hafnia albeit ( H. alveiThis figure shows the results of comparing the ratios of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in C57BL / 6 mouse immune cells vaccinated with an adjuvant-mixed vaccine containing LPS (20 µg / ml) (*: P < 0.05). Fig. 7B shows 0.01% (w / v) Carbopol 974P and various Hafnia albeit ( H. alvei This figure shows the results of comparing the ratios of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in C57BL / 6 mouse immune cells vaccinated with an adjuvant-mixed vaccine containing LPS (20 µg / ml) (*: P < 0.05). Fig. 8A shows 0.1% (w / v) Carbopol 971P and various Hafnia albeit ( H. alvei This figure shows the results of comparing the ratios of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in C57BL / 6 mouse immune cells vaccinated with an adjuvant-mixed vaccine containing LPS (20 µg / ml) (*: P < 0.05). Fig. 8B shows 0.1% (w / v) Carbopol 974P and various Hafnia albeit ( H. alvei This figure shows the results of comparing the ratios of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in C57BL / 6 mouse immune cells vaccinated with an adjuvant-mixed vaccine containing LPS (20 µg / ml) (*: P < 0.05). Fig. 9A shows 0.1% (w / v) Carbopol 971P and various Hafnia albeit ( H. alvei This figure shows the results of comparing the ratios of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in C57BL / 6 mouse immune cells vaccinated with an adjuvant-mixed vaccine containing LPS (100 µg / ml) (*: P < 0.05). Fig. 9B shows 0.1% (w / v) Carbopol 974P and various Hafnia albeit ( H. alvei This figure shows the results of comparing the ratios of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in C57BL / 6 mouse immune cells vaccinated with an adjuvant-mixed vaccine containing LPS (100 µg / ml) (*: P < 0.05). Fig. 10A shows 0.01% (w / v) Carbopol 971P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results confirming the effect of inducing an antigen-specific cellular immune response (ELISpot) following the administration of a vaccine mixed with an adjuvant containing LPS (20 ug / ml) (*: P < 0.05). Fig. 10B shows 0.01% (w / v) Carbopol 974P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results confirming the effect of inducing an antigen-specific cellular immune response (ELISpot) following the administration of a vaccine mixed with an adjuvant containing LPS (20 ug / ml) (*: P < 0.05). Fig. 11A shows 0.1% (w / v) Carbopol 971P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results confirming the effect of inducing an antigen-specific cellular immune response (ELISpot) following the administration of a vaccine mixed with an adjuvant containing LPS (20 ug / ml) (*: P < 0.05). Fig. 11B shows 0.1% (w / v) Carbopol 974P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results confirming the effect of inducing an antigen-specific cellular immune response (ELISpot) following the administration of a vaccine mixed with an adjuvant containing LPS (20 ug / ml) (*: P < 0.05). Fig. 12A shows 0.1% (w / v) Carbopol 971P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results confirming the effect of inducing an antigen-specific cellular immune response (ELISpot) following the administration of a vaccine mixed with an adjuvant containing LPS (100 ug / ml) (*: P < 0.05). Fig. 12B shows 0.1% (w / v) Carbopol 974P and various Hafnia albeit in C57BL / 6 mice ( H. alvei This figure shows the results confirming the effect of inducing an antigen-specific cellular immune response (ELISpot) following the administration of a vaccine mixed with an adjuvant containing LPS (100 ug / ml) (*: P < 0.05). Specific details for implementing the invention

[0024] The present invention is Hafnia Albay ( Hafnia alveiThe present invention relates to the use of an immunostimulatory composition comprising a lipopolysaccharide derived from ) and a carbomer, having characteristics of pH 6.5 to pH 7.5, viscosity 1.95 to 2.15 cp, or particle size 95 nm to 140 nm. The immunostimulatory composition according to the present invention has unique physical characteristics in terms of pH, viscosity, and particle size of the final formulation, excellent antigen-specific antibody formation ability, and can effectively induce cellular and humoral immunity.

[0026] The present invention will be described in detail below.

[0028] According to an embodiment of the present invention, the present invention provides an immune-enhancing composition comprising the following components:

[0029] a) Hafnia Albay ( Hafnia alvei ) derived lipopolysaccharide;

[0030] b) Carbomer; and

[0031] c) pH 6.5 to pH 7.5.

[0032] The present invention also provides an immune-enhancing composition comprising the following components:

[0033] a) Hafnia Albay( Hafnia alvei ) derived lipopolysaccharide;

[0034] b) Carbomer; and

[0035] c) Viscosity 1.95 to 2.15 cp.

[0036] The present invention also provides an immune-enhancing composition comprising the following components:

[0037] a) Hafnia Albay ( Hafnia alvei ) derived lipopolysaccharide;

[0038] b) Carbomer; and

[0039] c) Particle size of 95 nm to 140 nm.

[0040] 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 an immune response and obtaining an immunostimulating effect by increasing the activity of immune cells. For example, phagocytes play a major role in the immune response; phagocytosis, which is the primary role of macrophages, involves absorbing microorganisms and other pyrogenic particles, and also stimulates 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), as well as cytotoxic and inflammatory substances such as nitric oxide (NO). Therefore, increasing macrophage activity can be a means for immunostimulation. Since infections and diseases primarily occur when immune function is compromised, various studies are being conducted to enhance these immune responses through immune-boosting substances when the body's immune system function is impaired.

[0041] The immunoadjuvant composition of the present invention can be used as an adjuvant or a vaccine adjuvant. An adjuvant generally refers to any substance that increases the humoral or cellular immune response to an antigen. Adjuvants slow the release of antigens from the injection site and stimulate the immune system. Traditional vaccines generally consist of crude preparations of inactivated, killed, or modified live pathogenic microorganisms; however, since the immunity induced by these is insufficient, the immune response can be further promoted through the addition of 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 enables more economical vaccine manufacturing.

[0042] In the present invention, the immunity increased by the administration of an immune-enhancing composition is preferably innate immunity or adaptive immunity, and more preferably may refer to both innate immunity and adaptive immunity.

[0043] In the present invention, lipopolysaccharide (LPS) is a substance present in the outer membrane of Gram-negative bacteria, and is a powerful inflammatory agent capable of inducing the expression of various types of inflammatory cytokines (TNF-α, IL-1β, IL-6, etc.) by activating the NK-κB signaling pathway through the recognition of lipid A of LPS by Toll-like receptor 4.

[0044] In a specific embodiment of the present invention, the lipopolysaccharide (LPS) derived from hafnia albey may include all of the lipopolysaccharide of hafnia albey or a part thereof, and may include all of their natural, chemically modified, and synthetic forms. Since various strains belonging to hafnia albey may share common or similar LPS structures and activities, LPS derived from various hafnia albey strains can be combined with carbomer to induce an excellent immunostimulatory effect.

[0045] Therefore, the Hafnia Albay of the present invention Hafnia alvei BAA-2768(ATCC, USA), Hafnia alvei 51815(ATCC, USA) and Hafnia alvei Known readily available strains such as 25927 (ATCC, USA), as well as isolated and identified Hafnia albei strains, such as Hafnia albei BA2000346 (Deposit No.: KCTC15966BP) or Hafnia alveiIt may include strains such as 9-126A-1 (Deposit No.: KCTC16220BP) without limitation, and preferably may be strain Hafnia albey BA2000346 (Deposit No.: KCTC15966BP). In addition, examples of known and easily available Hafnia albey strains include Hafnia alvei ATCC 13337, Hafnia alvei ATCC 51873, Hafnia alvei ATCC 51873D-5, Hafnia alvei ATCC 29926, Hafnia alvei ATCC BAA-2768, Hafnia alvei ATCC 51815, Hafnia alvei ATCC 9760, Hafnia alvei ATCC 11604, Hafnia alvei ATCC 23280 and Hafnia alvei There is ATCC 29927.

[0046] In one embodiment of the present invention, lipopolysaccharides derived from the Hafnia albey strain BA2000346 (Deposit No.: KCTC15966BP), the 9-126A-1 strain (Deposit No.: KCTC16220BP), and the ATCC 51815 strain were used. The Hafnia albey strain BA2000346 was isolated from roe deer feces and was deposited on July 18, 2024, at the National Institute of Biological Resources located at 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, and was assigned accession number KCTC15966BP. Additionally, the Hafnia albey strain 9-126A-1 was deposited on January 7, 2025, at the National Institute of Biological Resources located at 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, and was assigned accession number KCTC16220BP.

[0047] In the present invention, the carbomer is a polymer of polymerized 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 may be used as the material that links the polymer chains together. Many acrylic acid units combine to form a long chain structure, and a three-dimensional network structure is formed through a crosslinking agent. This structure allows for the formation of a gel by absorbing water and expanding, and can be controlled to various concentrations and viscosities. The carbomer is [C₃H₄O₂] n It can be expressed by the chemical formula, and n can be selected within the range of 1,000 to 1,000,000. For example, low molecular weight carbomer can be selected in the range of 1,000 to 10,000, medium molecular weight carbomer in the range of 10,000 to 100,000, and high molecular weight carbomer in the range of 100,000 to 1,000,000. Alternatively, the above carbomer may be selected according to 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 according to the purpose, and preferably, for example, a carbomer having a viscosity of 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 synthetic polymers synthesized from acrylic acid without limitation, for example, Carbopol® 940P, 934P, 971P, and 974P, and in an embodiment of the present invention, Carbopol 971P and Carbopol 974P were used.

[0048] Hafnia Albay of the present invention Hafnia alveiLipopolysaccharides derived from hafnia albey can possess desirable physical properties as an immunostimulatory composition compared to lipopolysaccharides derived from hafnia albey alone when mixed with carbomer. For example, in a mixed composition, lipopolysaccharides derived from hafnia albey are stabilized together with carbomer, allowing for the sustained expression of an immunostimulatory effect. Carbomer helps the lipopolysaccharides derived from hafnia albey to be released slowly, thereby inducing an immune response more effectively. Furthermore, the lipopolysaccharide composition derived from hafnia albey mixed with carbomer exhibits high viscosity and stability, enabling its development into various administration forms (e.g., injectables, gel-type injectables, etc.). This facilitates storage and handling and can enhance vaccine efficacy. Additionally, the mixed composition allows for precise control of the concentration of lipopolysaccharides derived from hafnia albey, thereby optimizing the immune response while minimizing side effects. Moreover, carbomer can reduce skin irritation, thereby lowering the likelihood of adverse effects. Furthermore, in terms of sustained efficacy, lipopolysaccharides derived from hafnia albeit mixed with carbomer are slowly released and can maintain an immune response for an extended period. This ensures the long-term effectiveness of the vaccine. In addition, the mixed composition ensures stability and consistency during the manufacturing process, is suitable for mass production, and is easy to commercialize in various adjuvant forms.

[0049] In addition, the carbomer may be included in the immune-enhancing composition at 0.005 to 0.25% (w / v), preferably at 0.007 to 0.15%, and more preferably at 0.01 to 0.1% (w / v). If the carbomer exceeds 0.4% (w / v), the manufacturing time for the oil-in-water emulsion may be prolonged and incomplete dissolution may occur, which may make it unsuitable for the manufacturing process.

[0050] In the present invention, the lipopolysaccharide derived from hafnia albei may be included in the immunostimulatory composition at a concentration of 10 to 200 μg / ml, preferably 15 to 150 μg / ml, and more preferably 20 to 100 μg / ml. If the lipopolysaccharide derived from hafnia albei exceeds 200 μg / ml, safety as an immunostimulator may be reduced.

[0051] In a specific embodiment of the present invention, the lipopolysaccharide derived from hafnia albei and the carbomer are preferably formulated as an oil-in-water emulsion. In the present invention, an "oil-in-water (O / W)" emulsion refers to one liquid (oil phase) being dispersed within another liquid (water phase). The oil-in-water emulsion may include oil and a surfactant.

[0052] In a preferred embodiment of the present invention, the immune-enhancing composition may further include squalene, and the oil-in-water emulsion may be a squalene-based oil-in-water emulsion, but may be applied without limitation as long as the purpose of the present invention is achieved.

[0053] In an embodiment of the present invention, the oil-in-water emulsion is prepared by separately preparing an oil phase containing squalene and a water phase, then mixing the two phases and finally preparing an O / W emulsion formulation through high-speed mixing and high-pressure crushing processes. That is, the composition of the present invention can be prepared through a conventional method for preparing a squalene-based oil-in-water emulsion form: 1) a step of high-speed mixing the oil phase containing squalene and the water phase; and 2) a step of high-pressure crushing the prepared mixture; thereby preparing an oil-in-water emulsion form, and additionally, a pH adjuster may be added to adjust the pH of the formulation. The pH adjuster to be used in the present invention may include, without limitation, pH adjusters widely known in the art as long as they do not impede the oil-in-water emulsion form of the present invention, such as Sodium Metabisulfite, Sodium Thiosulfate, Aminomethyl Propanediol, Disodium Phosphate, Acetic Acid, Triethanolamine, Sodium Phosphate, Potassium Phosphate, etc., but is not limited thereto.

[0054] In a preferred embodiment of the present invention, lipopolysaccharides derived from hafnia albey and carbomer may be included in the immunostimulatory composition at 1 to 200 μg / ml and 0.005 to 0.25% (w / v), respectively. In a preferred embodiment of the present invention, 20 or 100 μg / ml of lipopolysaccharides derived from hafnia albey, which has manufacturing process advantages, exhibits low toxicity, and can most balancedly induce antibody formation and cellular immunity suitable as an immunostimulator; and 0.01 or 0.1% (w / v) of Carbopol 974P were used.

[0055] In the most preferred embodiment of the present invention, the immunostimulatory composition of the present invention may comprise 20 μg / ml of lipopolysaccharide derived from hafnia albey and 0.1% (w / v) of Carbopol 974P and be formulated as an oil-in-water emulsion. Additionally, the immunostimulatory composition of the present invention may comprise 20 μg / ml of lipopolysaccharide derived from hafnia albey and 0.01% (w / v) of Carbopol 974P and be formulated as an oil-in-water emulsion. Additionally, the immunostimulatory composition of the present invention may comprise 100 μg / ml of lipopolysaccharide derived from hafnia albey and 0.01% (w / v) of Carbopol 974P and be formulated as an oil-in-water emulsion.

[0056] In a preferred embodiment of the present invention, the immune-enhancing composition of the present invention may exhibit a uniform viscosity value of 1.95 to 2.15 cp, and preferably may have a viscosity of 2.00 to 2.11 cp. The viscosity may refer to a viscosity value measured using the Brookfield viscosity analysis method after stabilization at 20°C to 25°C.

[0057] In addition, the immune-enhancing composition of the present invention may have a pH of 6.5 to 7.5, and preferably a pH of 6.7 to 7.2.

[0058] In addition, the immune-enhancing composition of the present invention may have a particle size of 95 to 140 nm, and preferably a uniform particle size of 98.6 to 131.9 nm.

[0059] In the most preferred embodiment of the present invention, the immunostimulating composition of the present invention may have a viscosity of 1.95 to 2.15 cp, a pH of 6.5 to 7.5, and a particle size of 95 to 140 nm. More preferably, the immunostimulating composition of the present invention may have a viscosity of 2.00 to 2.11 cp, a pH of 6.7 to 7.2, and a particle size of 98.6 to 131.9 nm.

[0061] According to another aspect of the present invention, the present invention provides a vaccine adjuvant composition comprising the following components; or a vaccine composition;

[0062] a) Hafnia Albay ( Hafnia alvei ) derived lipopolysaccharide;

[0063] b) Carbomer; and

[0064] c) Viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5, or particle size 95 nm to 140 nm.

[0065] In the present invention, a vaccine adjuvant refers to a drug that enhances a specific immune response to a vaccine or inoculated material. The vaccine adjuvant may be defined as any substance or preparation that acts to accelerate, prolong, or enhance the quality of specific immune reactivity to an immunogenic substance.

[0066] The above vaccine adjuvant may be included in the vaccine and administered as a single dose or multiple doses, and since the desired immune-enhancing effect can be achieved with only a single dose, it may preferably be for single administration, and in the case of multiple administration, it may be for two doses.

[0067] The composition and vaccine adjuvant of the present invention may further include one or more known active ingredients having an immune-enhancing effect, and more preferably, a stimulating 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 acid DMXAA), methoxyvone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3',6'-dihydroxyflavone, 7, It may further include one or more selected from the group consisting of 2'-dihydroxyflavone (7,2'-dihydroxyflavone), daidzein, formononetin, retusin 7-methyl ether, and xanthone.

[0068] 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), and more preferably in a ratio of 1:1 (v / v).

[0069] The antigen included in the vaccine composition of the present invention may be a protein of a pathogen, a recombinant protein, a glycoprotein, a peptide, a polysaccharide, a lipopolysaccharide, a polynucleotide, a hormone, a tumor antigen, an allergen, a cell, a bacterium, a fungus, a virus, a parasite, or a combination thereof.

[0070] The amount of antigen used to induce an immune response will vary considerably depending on the antigen used, the target, and the desired level of response, and can be determined by a person skilled in the art. Hafnia Albay of the present invention ( Hafnia alvei The combination of lipopolysaccharides and carbomer derived from ) can induce a superior immune response when administered with the same amount of antigen compared to conventional adjuvants, and this is a superior effect compared to the immune-enhancing effect of individual components.

[0071] In the present invention, Hafnia Albay ( Hafnia alvei The mixing ratio of an adjuvant containing a lipopolysaccharide derived from ) and a carbomer to an antigen may be 1:0.01 to 100 (v / v), preferably 1:0.1 to 10 (v / v), more preferably 1:0.1, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5 (v / v).

[0073] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of infectious diseases comprising the following components:

[0074] a) Hafnia Albay ( Hafnia alvei ) derived lipopolysaccharide;

[0075] b) Carbomer; and

[0076] c) Viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5, or particle size 95 nm to 140 nm.

[0077] 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 may be sepsis, septic shock, SARS-CoV-2 infection, 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, the common cold, influenza, airway 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, urinary tractitis, cystitis, vaginitis, cervicitis, salpingitis, infectious erythema, bacterial dysentery, abscess and ulcer, bacteremia, diarrhea, dysentery, gastroenteritis, gastroenteritis, genitourinary abscess, infection of an open wound or wound, purulent inflammation, abscess, boil, pyoderma, impetigo, folliculitis, cellulitis, postoperative wound infection, skin laceration 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, pleuritis, abdominal abscess, liver abscess, cholecystitis, splenic abscess, pericarditis, myocarditis, placentaitis, amniotic fluid, mastitis, puerperal fever, toxic shock syndrome, Lyme disease, gas gangrene,It may be one or more selected from the group consisting of atherosclerosis, Mycobacterium avium syndrome (MAC), enterohemorrhagic Escherichia coli (EHEC) infection, enteropathogenic Escherichia coli (EPEC) infection, enteroinvasive Escherichia coli (EIEC) infection, methicillin-resistant Staphylococcus aureus (MRSA) infection, vancomycin-resistant Staphylococcus aureus (VRSA) infection, and listerosis.

[0078] The composition according to the present invention may include a pharmaceutically effective amount of microbial cells and an excipient or diluent.

[0079] In the above, the term "pharmaceuticalally effective amount" refers to an amount sufficient to exert an immune-enhancing effect. The term "pharmaceuticalally acceptable" refers to a composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness.

[0080] In addition, the composition according to the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, 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, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. When formulating the above composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa paste, laurin, glycerogelatin, etc. may be used.

[0081] The composition of the present invention may 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 is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art and may 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, body weight, general health condition, gender and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and concurrently used drugs. For a desirable effect, the composition of the present invention may be administered in an amount of 1 to 10,000 mg / kg / day, preferably 1 to 200 mg / kg / day, and may be administered once a day or divided into several doses.

[0083] According to another aspect of the present invention, the present invention provides an immune-enhancing food and a health functional food composition comprising the following components:

[0084] a) Hafnia Albay ( Hafnia alvei ) derived lipopolysaccharide;

[0085] b) Carbomer; and

[0086] c) Viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5, or particle size 95 nm to 140 nm.

[0087] In addition, the composition of the present invention may be a health functional food composition for immune enhancement or a food composition. 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 may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing 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 conventional methods.

[0088] In this specification, the term "food" refers to a natural or processed product containing one or more nutrients, preferably one that has undergone a certain degree of processing to become ready for direct consumption, in the conventional sense, and the term "food composition" is intended to include all of the following: food, food additives, health functional foods, and beverages.

[0089] There are no special limitations on the types of food or health functional food of the present invention, and examples include ramen, other noodles, beverages, tea, drinks, alcoholic beverages, various soups, meat, sausages, bread, chocolate, candies, confectionery, pizza, chewing gum, ice cream, and dairy products, or vitamin complexes.

[0090] In particular, the lipopolysaccharide and carbomer derived from hafnia albei according to the present invention may be added to various food products intended for immune enhancement to exhibit function, and may be used in combination with other appropriate auxiliary ingredients and known additives that are typically contained in health functional foods, at the choice of a person skilled in the art. The known additives include other microorganisms that can be used together with the strain according to the present invention.

[0091] Accordingly, the food composition of the present invention may preferably be a health functional food. The above-mentioned health functional food refers to a food manufactured or processed by methods such as extraction, concentration, purification, or mixing of specific components contained in food raw materials for the purpose of health assistance, and refers to a food designed and processed to sufficiently exert biological regulatory functions on the body, such as biological defense, regulation of biological rhythms, and prevention and recovery from disease, through the above-mentioned components, and refers to a food capable of performing functions related to immune enhancement, etc.

[0093] According to another aspect of the present invention, the present invention provides an immune-enhancing feed additive composition comprising the following components:

[0094] a) Hafnia Albay ( Hafnia alvei ) derived lipopolysaccharide;

[0095] b) Carbomer; and

[0096] c) Viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5, or particle size 95 nm to 140 nm.

[0097] The above immune-enhancing feed additive composition may be a feed additive or a compound feed.

[0098] In the present invention, “feed additive” refers to a substance added in trace amounts to feed for nutritional or specific purposes, and the immune enhancer of the present invention may be applied in an amount of 0.01 to 99 weight percent of the total weight of the feed additive. The feed composition of the present invention may additionally include other nutritional components within the range that a person skilled in the art can expect.

[0099] Hafnia Albay of the present invention Hafnia alveiLipopolysaccharides and carbomers derived from ) can be incorporated into commercially conventional animal feed compositions and supplied, for example, to cattle, pigs, sheep, poultry, etc. To this end, the strain of the present invention and its culture medium can be mixed with conventional animal feed components and, if necessary, processed into a purified form. Conventional animal feed components are, for example, corn, barley, oats, soybeans, fish meal, bran, soybean oil, minerals, trace elements, amino acids, and vitamins.

[0101] According to another aspect of the present invention, the present invention provides a method for inducing an immune response in an individual, comprising the step of administering the immune-enhancing composition to an individual in need thereof.

[0102] The routes of administration of the immunoadjuvant composition are not limited thereto but include parenteral, oral, palatal-nasal, intrabronchial, topical, intramuscular, and in ova. The composition may be administered using any suitable device, including syringes, droppers, needleless injection devices, patches, etc. The route and device selected for use may be appropriately adjusted depending on the composition of the immunoadjuvant, the antigen, and the target.

[0103] The above-mentioned individuals may include any animal for which administration of the immune-enhancing composition is desirable without limitation, and may include all mammals and non-mammalians.

[0105] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0107] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples. The carbomers used in the following examples are Carbopol 971P and 974P. In addition to this, [C₃H₄O₂] n Results of substantially equivalent or similar trends to the examples below can be obtained by using any carbomer selected from carbomers that can be represented by the chemical formula (n is 1,000 - 1,000,000). In addition, the Hafnia albei bacteria used in the examples below H. alvei 51815(ATCC, USA), H. alvei BA2000346 strain (Deposit No.: KCTC15966BP) or H. alvei It is 9-126A-1 (Deposit No.: KCTC16220BP). In addition to this, any H. alvei It is expected that using strain-derived lipopolysaccharides will yield results that are substantially equivalent to or similar to the examples below.

[0109] 1. varsity H. alvei LPS preparation

[0110] By isolating the dominant bacterial species from the feces of a male roe deer rescued on November 11, 2019, in Jiam-ri, Sabuk-myeon, Chuncheon-si, Gangwon-do, and through a method of subculture on blood medium Hafnia alvei BA2000346 and Hafnia alvei 9-126A-1 was isolated and identified. The isolated and identified Hafnia alvei The strain BA2000346 was deposited as a patent with the Korean Collection for Type Cultures (KCTC) on July 18, 2024, and was assigned accession number KCTC15966BP. In addition Hafnia alveiThe 9-126A-1 strain was deposited with the Korean Collection for Type Cultures (KCTC) on January 7, 2025, and was assigned accession number KCTC16220BP.

[0111] The above H.alvei LPS derived from BA2000346 (hereinafter, H. alvei BP2000346 LPS) and Hafnia alvei 9-126A-1 derived LPS (hereinafter, H.alvei 9-126A-1 LPS or H.alvei BA2000103 LPS) was used in the manufacture of the following mixed vaccine.

[0112] also H. alvei The ATCC 51815 strain was obtained from the American Type Culture Collection (ATCC), and its LPS (hereinafter, H.alvei ATCC 51815 LPS) was used in the manufacture of a mixed vaccine.

[0114] Example 2. สายดี่มื่อมี่ 971P, 974P and H. alvei LPS 다이 연이증강제 제형의 타이스타이스트

[0115] 2.1 카보한 971P and 974P mixed density by 장하다 and 지도 에서

[0116] To determine the solubility of aqueous solutions according to the mixing concentration of Carbopol, two types of Carbopol (971P, 974P) were mixed at various concentrations (0.1%, 0.2%, 0.3%, 0.4%, 0.5% (w / v)) and the dissolution time, dissolution state (appearance), and viscosity were measured. The results of the solubility and viscosity measurements are shown in Table 1 below.

[0118] Carbopol 971P composition (w / v) 0.1% 0.2% 0.3% 0.4% 0.5% 용해지지 3 3 5 - - 에도 4.28 8.74 - - - pH 6.79 3.39 3.26 - - 성상 투명한 안상 투명한 안상 용해되나 지이 에이 시 기포 에성 용해 안됐이,침전 용해 안됐이,침전 Carbopol 974P composition (w / v) 0.1% 0.2% 0.3% 0.4% 0.5% 용해지지 2 2 4 - - 에도 2.53 4.43 - - - pH 6.84 6.37 5.88 성상 Completely dissolved (clear liquid) Completely dissolved (clear liquid) It dissolves, but bubbles are generated during suction and discharge. Insoluble, precipitate Insoluble, precipitate

[0119] As can be seen in Table 1, solubility decreased as the mixed concentration of Carbopol 971P and 974P increased, confirming that the mixed concentration of Carbopol significantly affects viscosity, pH, and properties. Regardless of the type of Carbopol 974P or 971P, dissolution time increased significantly at concentrations of 0.3% or higher, and complete dissolution did not occur. In particular, for both Carbopol 971P and 974P, the pH of the aqueous solution became strongly acidic as the mixed concentration increased, It was confirmed that 0.3% (w / v) Carbopol 971P has a pH of 3.26 and 0.3% (w / v) Carbopol 974P has a pH of 5.88. By comprehensively considering these solubility, viscosity, and pH, the maximum mixing concentration of carbomer used in the adjuvant was set to 0.2% (w / v).

[0121] 2.2 Carbopol 971P, 974P and various concentrations H. alvei pH measurement of LPS mixed adjuvant

[0122] Carbomer and various H. alvei To determine the pH of the adjuvant formulation according to LPS mixing, two types of Carbopol (971P, 974P) and H. alvei BA2000346 LPS, H. alvei 9-126A-1 LPS, H. alvei ATCC 51815 LPS was mixed at various concentrations to prepare an oil-in-water emulsion containing squalene oil. The above-mentioned adjuvant is an oil-in-water (O / W) emulsion formulation based on squalene. Specifically, an oil phase and a water phase containing squalene were prepared separately, then the two phases were mixed and subjected to high-speed mixing and high-pressure crushing processes to finally produce an O / W emulsion formulation.

[0123] The pH measurement results of the manufactured adjuvants are shown in Tables 2 and 3 below.

[0124] Carbopol 971P H. alvei LPS strain Carbopol concentration (w / v) LPS concentration (ug / ml) pH - 0.1% - 6.96 - 0.2% - 7.01 BA2000346 0.01% 20 6.91 0.01% 100 6.86 0.1% 20 6.87 0.1% 100 6.91 0.1% 200 6.86 0.1% 400 6.88 0.2% 20 6.90 0.2% 100 6.93 0.2% 200 6.98 0.2% 400 6.91 9-126A-1 0.01% 20 6.85 0.1% 20 6.88 0.1% 100 6.86 0.2% 400 6.94 ATCC51815 0.01% 20 6.89 0.1% 20 6.90 0.1% 100 6.92 0.2% 400 6.94

[0125] Carbopol 974P H. alvei LPS strain Carbopol concentration (w / v) LPS concentration (ug / ml) pH - 0.1% - 6.81 - 0.2% - 6.87 BA2000346 LPS 0.01% 20 6.85 0.01% 100 6.72 0.1% 20 6.94 0.1% 100 6.94 0.1% 200 6.94 0.1% 400 6.93 0.2% 20 6.87 0.2% 100 6.86 0.2% 200 6.87 0.2% 400 6.78 9-126A-1 LPS 0.01% 20 7.19 0.01% 100 6.90 0.1% 20 7.01 0.1% 100 6.99 0.2% 400 6.86 ATCC 51815 LPS 0.01% 20 7.09 0.01% 100 6.94 0.1% 20 7.07 0.1% 100 7.06 0.2% 400 6.84

[0126] As shown in Tables 2 and 3, two types of Carbopol (971P, 974P) and three types H. alvei It was confirmed that the O / W formulations of adjuvants prepared by mixing bacterial-derived LPS at various concentrations had a pH range of 6.7 to 7.2 for all formulations.

[0128] 2.3 Carbopol 971P, 974P and various concentrations H. alvei Viscosity measurement of LPS mixed adjuvant

[0129] Carbomer and various H. alvei To determine the viscosity of the adjuvant formulation according to the LPS mixing concentration, two types of Carbopol (971P, 974P) and H. alvei BA2000346 LPS was mixed at various concentrations to prepare squalene oil-containing oil-in-water emulsions using the same method as in Example 2.2. Viscosity measurements were performed using the Brookfield viscosity analysis method after stabilization at 25°C for 1 minute using a spindle. The viscosity measurement results of the prepared adjuvants are shown in Table 4 below.

[0131] Carbopol Carbopol concentration (w / v) LPS concentration (ug / ml) Viscosity (cP) 971P 0.1% 20 2.00 0.1% 400 2.01 0.2% 20 2.11 0.2% 400 2.08 974P 0.1% 20 2.00 0.1% 400 2.03 0.2% 20 2.08 0.2% 400 2.10

[0132] As shown in Table 4 above, the concentrations of the two types of Carbopol (971P, 974P) were varied from 0.1 to 0.2%, and H. alvei It was confirmed that all immunoadjuvants prepared by mixing different amounts of derived LPS ranging from 20 to 400 ug / ml exhibited similar viscosity ranges and showed viscosity values ​​ranging from a minimum of 2.00 to a maximum of 2.11 CP.

[0134] 2.4 Carbopol 971P, 974P and various concentrations H. alvei Particle size measurement of LPS mixed adjuvant

[0135] Carbomer and various H. alvei To determine the particle size of adjuvant formulations according to LPS mixing concentration, two types of Carbopol (971P, 974P) and three types H. alvei LPS was mixed at various concentrations to prepare a squalene oil-containing oil-in-water emulsion in the same manner as in Example 2.2. The results of measuring the particle size of the adjuvant are shown in Tables 5 and 6 below.

[0137] Carbopol 971P H. alvei LPS strain Carbopol concentration (w / v) LPS concentration (ug / ml) Particle size (nm) - 0.1% - 111.0±1.6 - 0.2% - 117.9±1.3 BA2000346 0.01% 20 111.6±1.3 0.01% 100 117.5±1.0 0.1% 20 113.4±0.6 0.1% 100 126.5±0.6 0.1% 200 124.8±1.0 0.1% 400 128.5±1.7 0.2% 20 118.8±0.6 0.2% 100 114.8±2.6 0.2% 200 122.9±0.8 0.2% 400 131.9±1.3 9-126A-1 0.01% 20 109.5±0.0 0.1% 20 107.0±0.6 0.1% 100 112.0±0.7 0.2% 400 112.5±4.7 ATCC51815 0.01% 20 115.2±1.5 0.2% 20 114.4±1.3 0.1% 100 116.5±1.6 0.1% 400 107.5±0.3

[0138] Carbopol 974P H. alvei LPS strain Carbopol concentration (w / v) LPS concentration (ug / ml) Particle size (nm) - 0.1% - 104.8±1.8 - 0.2% - 104.5±1.1 BA2000346 0.01% 20 98.6±2.3 0.01% 100 99.4±1.6 0.1% 20 107.1±0.3 0.1% 100 111.2±0.6 0.1% 200 122.6±0.1 0.1% 400 131.0±1.3 0.2% 20 111.6±0.6 0.2% 100 114.5±0.4 0.2% 200 119.3±0.8 0.2% 400 123.1±2.2 9-126A-1 0.01% 20 101.8±3.1 0.01% 100 104.7±2.3 0.1% 20 100.7±1.8 0.1% 100 106.4±1.6 0.2% 400 110.8±1.2 ATCC 51815 0.01% 20 117.2±1.6 0.01% 100 111.3±2.0 0.1% 20 104.4±0.4 0.1% 100 107.3±0.4 0.2% 400 114.1±0.9

[0139] As shown in Tables 5 and 6 above, the concentrations of the two types of Carbopol (971P, 974P) were varied from 0.01 to 0.2%, and H. alvei It was confirmed that immunoadjuvants prepared by mixing derived LPS at concentrations ranging from 20 to 400 ug / ml exhibited a uniform range of particle sizes from a minimum of 98.6 nm to a maximum of 131.9 nm, despite various concentration combinations.

[0141] The results of the pH, viscosity, and particle size analysis as described above are carbomer and H. alvei This indicates that an adjuvant with uniform physical properties can be produced by combining derived LPS at the appropriate concentration to prepare a squalene oil-containing O / W emulsion.

[0144] Example 3. Carbopol of various concentrations (971P or 974P) and H. alvei Immunogenicity Evaluation of a Combination Vaccine Containing a Strain BA2000346 LPS-Based Adjuvant

[0145] 3.1 Preparation of Immunostimulant Experimental Group and Mouse Model

[0146] By varying the type and mixing concentration of Carbopol, various concentrations H. alvei An adjuvant was prepared by mixing strain BA2000346 LPS, and its effect was confirmed. Specifically, Carbopol 971P and 974P were varied at different concentrations of 0.01%, 0.1%, 0.2%, and w / v, and H. alveiSqualene oil-containing oil-in-water emulsions containing strain BA2000346 LPS were prepared at different concentrations of 20 μg / ml and 100 μg / ml using the same method as in Example 2.2. As the antigen, ovalbumin (OVA) (Invivogen, Cat. No.: vac-pova), which is commonly used in the development of adjuvants, was used at a concentration of 10 μg / head and mixed with the adjuvant at a ratio of 1:1 (v / v) at the time of final vaccination. The adjuvant experimental groups are shown in Table 7 below.

[0147] Experiments were conducted to evaluate the immunogenicity and safety of the protein antigen and adjuvant combination vaccine in a mouse model (IACUC approval number: EU-24-021). Specifically, 6-week-old C57BL / 6 (female) mice were admitted in groups of four and stabilized for one week. The adjuvant combination vaccine according to Table 7 was administered intramuscularly twice at 2-week intervals, with 50 μl administered to each thigh for a total of 100 μl. Mouse body weight was measured once a week (DPV 0-28), and blood samples (DPV 13, 28) and serum isolation were performed at 2 weeks after the first and second vaccinations to measure antigen-specific antibody titers. The in vivo safety of the adjuvant was confirmed by examining abnormal findings at the injection site during necropsy (DPV 28).

[0149] Carbopol experimental group candidate substances 971P Group 1 PBS (negative control group) Group 2 Antigen (Immunostimulant efficacy comparison control group, antigen administered alone) Group 3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion Group 4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion Group 5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion Group 6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion Group 7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion Group 8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 974P Group 1 PBS (negative control group) Group 2 Antigen (Immunostimulant efficacy comparison control group, antigen administered alone) Group 3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion Group 4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion Group 5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion Group 6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion Group 7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion Group 8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion

[0151] 3.2 Comparative Evaluation of Antigen-Specific Antibody Formation Following Combination Vaccination

[0152] Antigen-specific antibody formation following the administration of the mixed vaccine in mice was evaluated using ELISA (Enzyme-linked immunosorbent assay). Specifically, the antigens used in the mixed vaccine were mixed in ELISA coating buffer (0.1M Sodium Carbonate, pH 9.5) to a concentration of 1 μg / well, and then 100 μl was dispensed into 96-well Immunoplates (Nunc, Cat. No.: 439454) and incubated overnight at 4°C. After washing four times with PBS-Tween 20 (0.05%), 200 μl of blocking solution (1% Bovine serum albumin in PBS) was added, and the plates were incubated overnight at 4°C. 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 the plates were incubated at 37°C for 2 hours. Subsequently, the sample was 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 sample was washed four times again with PBS-Tween 20 (0.05%). 100 μl of 1-Step Ultra TMB-ELISA Substrate Solution (ThermoFisher, Cat No.: 34029) was added 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 evaluation of antigen-specific Total IgG antibody formation following the administration of the mixed vaccine 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 Tables 8 and 9.

[0154] Carbopol Group Ratio (based on the control group vaccinated only with antigen) 971P G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 6.79 G4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 6.72 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 6.68 G6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 7.89 G7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 5.05 G8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 7.57

[0155] Carbopol Group Ratio (based on the control group vaccinated only with antigen) 974P G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 14.39 G4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 15.70 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 13.81 G6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 15.06 G7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 15.00 G8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 15.30

[0156] As shown in Fig. 1, Table 8, and Table 9, Carbopol 971P, 974P, and H. alvei Antibody formation increased in the strain BA2000346 LPS-based adjuvant O / W emulsion mixed formulation groups (Groups 3–8) compared to the antigen-alone group (Group 2), and higher antibody titers were observed in the Carbopol 974P-based adjuvant groups compared to Carbopol 971P. In particular, 0.01% Carbopol 974P and 100 µg / ml H. alvei The highest antibody formation effect, approximately 15.70 times higher than the antigen-alone group (Group 2), was confirmed in the O / W inoculation group containing strain BA2000346 LPS (Group 4). P < 0.05).

[0158] 3.3 Comparative Evaluation of Antigen-Specific Cellular Immune Responses Following Combination Vaccination (Intracellular Cytokine Staining Assay)

[0159] Splenocytes, a mouse immune cell, were placed in a 96-well cell culture plate (NUNC, Cat. No.: 167008) at a volume of 2.0 x 10 6Cells were seeded to form cells / well. The seeded cells were treated with OVA protein (Invivogen, Cat. No.: vac-pova) at a concentration of 50 μg / ml and cultured for 15 hours in a 37°C, 5% CO2 incubator. Subsequently, Brefeldin A (1 / 1,000 dilution) was applied 10 hours prior to antibody staining. Anti-mouse CD16 / CD32 was applied at a concentration of 20 μg / ml and incubated at room temperature for 20 minutes. After washing twice with 1% BSA (in DPBS), immune cell surface factor (CD4, CD8) binding antibodies diluted in 1% BSA (in DPBS) were applied to the cells and incubated 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), cells were treated with Fixation / Permeabilization solution (BD Bioscience, Cat. No.: 555028) and incubated at 4°C for 20 minutes. After removing the supernatant, the cells were washed twice with Perm / Wash buffer (BD Bioscience, Cat. No.: 555028). After washing, cytokine (IL-2, IFN-γ, TNF-α) binding antibodies were added and incubated at 4°C in a dark room 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).

[0160] Figure 2 shows the results of confirming the ratio of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in C57BL / 6 mouse immune cells vaccinated with an adjuvant using intracellular cytokine staining (ICS) experiments. In addition, the ratio of CD4+ and CD8+ T cells derived from C57BL / 6 splenocytes expressing antigen-specific cytokines was calculated compared to the antigen-only group, and the results are shown in Tables 10 to 13.

[0162] Carbopol Group Ratio (based on the control group vaccinated only with antigen) CD4+ T cell IFN-γ TNF-α IL-2 971P G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 5.15 5.67 2.63 G4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 13.66 19.72 6.10 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 1.36 3.91 2.38 G6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 7.00 12.28 4.84 G7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 2.21 3.32 1.42 G8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 5.04 9.33 3.19

[0163] Carbopol Group Ratio (based on the control group vaccinated only with antigen) CD4+ T cell IFN-γ TNF-α IL-2 974P G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 1.95 17.96 9.07 G4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 8.90 101.81 36.67 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 2.14 25.97 13.07 G6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 3.24 45.99 17.67 G7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 2.88 43.02 10.05 G8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 4.25 55.30 21.87

[0164] Carbopol Group Ratio (based on the control group vaccinated only with antigen) CD8+ T cell IFN-γ TNF-α IL-2 971P G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 9.19 16.10 0.83 G4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 6.14 11.08 8.63 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 3.41 4.90 7.02 G6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 5.14 19.08 3.14 G7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 0.45 0.43 3.97 G8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 3.47 8.43 0.78

[0165] Carbopol Group Ratio (based on the control group vaccinated only with antigen) CD8+ T cell IFN-γ TNF-α IL-2 974P G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 27.49 6.03 0.00 G4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 69.78 18.58 4.50 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 9.69 3.72 0.43 G6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 34.94 20.22 0.00 G7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 15.67 7.81 0.00 G8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 15.94 6.96 0.00

[0166] As shown in FIG. 2 and Tables 10 to 13, Carbopol 971P, 974P and H. alvei It was confirmed that the proportion of CD4+ T and CD8+ T cells expressing IFN-γ(Th1), TNF-α(Th1), and IL-2(Th1) increased in the strain A2000346 LPS-based adjuvant O / W emulsion formulation mixed groups (Groups 3–8) compared to the antigen-alone group (Group 2). P < 0.05). Various Carbopol and H. alvei Among strain BA2000346 LPS mixed concentrations, particularly 0.01% Carbopol 974P, H. alvei It was confirmed that in the 100 µg / ml LPS-O / W emulsion group (Group 4), the proportions of CD4+ T cells expressing IFN-γ(Th1), TNF-α(Th1), and IL-2(Th1) were approximately 8.90-101.81-36.67-fold higher, and the proportions of CD8+ T cells expressing cytokines were 69.78-18.58-4.5-fold higher, respectively, compared to the antigen-alone group (Group 2).P < 0.05).

[0168] 3.4 Comparative Evaluation of Antigen-Specific Cellular Immune Responses (ELISpot) Following Combination Vaccination

[0169] To evaluate antigen-specific cellular immune responses, C57BL / 6 mouse splenocytes were necrolytic and extracted at week 2 (DPV 28) after the second dose of the adjuvant-mixed vaccine. The extracted mouse splenocytes were plated at 2.5 x 10⁶ IFN-γ ELISpot plates (Mabtech, Cat. No.: 3321-4HPT-10). 5 Cells were seeded to a cell / well ratio. To confirm the activation of antigen-specific cellular immune responses, OVA protein (Invivogen, Cat. No.: vac-pova) was added at a concentration of 10 μg / ml, and the cells were cultured for 20 hours in a 37°C, 5% CO2 incubator. After washing the cultured cells 5 times with PBS, 100 µl of Biotinylated Detection mAb, diluted to 1 / 1,000 in PBS (with 0.5% Fetal Bovine Serum), was added to each well and stored at room temperature in the dark for 2 hours. Subsequently, the cells were washed 5 times with PBS, 100 µl of Streptavidin-HRP, diluted to 1 / 1,000 in PBS (with 0.5% Fetal Bovine Serum), was added to each well and stored at room temperature in the dark for 1 hour. Washed 5 times with PBS, added 100 µl of TMB substrate per well, and reacted until fewer than 5 spots appeared in the negative control well (reaction time: 30 min). Washed with double distilled water to stop color development, dried the ELISpot plate in the dark, and analyzed using an ELISpot Reader (Cellular Technology Limited, BioSpot S5).

[0170] Figure 3 shows the results of confirming antigen-specific IFN-γ formation ability in splenocytes of C57BL / 6 mice through the ELISpot experiment, and Tables 14 and 15 show the results of calculating antigen-specific IFN-γ formation ability compared to the antigen-only inoculation group.

[0172] Carbopol Group Ratio (based on the control group vaccinated only with antigen) 971P G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 107.28 G4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 70.08 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 31.72 G6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 40.81 G7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 7.17 G8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 43.11

[0173] Carbopol Group Ratio (based on the control group vaccinated only with antigen) 974P G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 354.57 G4 Antigen / 0.01% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 571.00 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 135.71 G6 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 288.57 G7 Antigen / 0.2% 카보폴, 20 μg / ml H. alvei LPS - O / W emulsion 177.00 G8 Antigen / 0.2% 카보폴, 100 μg / ml H. alvei LPS - O / W emulsion 183.86

[0174] As shown in Fig. 3, Table 14, and Table 15, Carbopol 971P, 974P, and H. alvei Spot formation ability increased in the strain BA2000346 LPS-based adjuvant O / W emulsion mixed formulation groups (Groups 3–8) compared to the antigen-alone inoculation group (Group 2). In particular, 0.01% Carbopol 974P, 100 µg / ml H. alvei The spot-forming ability was highest in the strain BA2000346 LPS - O / W inoculated group (Group 4), approximately 571 times higher compared to the antigen-only inoculated group (Group 2). P < 0.05).

[0176] Example 4. Carbopol of various concentrations (971P or 974P) and H. alvei (BA2000346, 9-126A-1, ATCC 51815) Evaluation of Immunogenicity of a Combination Vaccine Containing a Bacterial-Derived LPS-Based Adjuvant

[0177] 4.1 Preparation of Immunostimulant Experimental Group and Mice

[0178] Various immune boosters H. alvei To compare the efficacy of adjuvants according to bacterial-derived LPS mixtures, two types of Carbopol (971P, 974P) and at various concentrations (0.01%, 0.1%, w / v) were H. alveiStrain BA2000346, 9-126A-1, ATCC 51815 LPS (20 μg / ml, 100 μg / ml) was prepared as an oil-in-water emulsion containing squalene oil using the same method as in Example 2.2. As the antigen, ovalbumin (OVA) (Invivogen, Cat. No.: vac-pova), which is commonly used in the development of adjuvants, was used at a concentration of 10 μg / head and mixed with the adjuvant at a ratio of 1:1 (v / v) at the time of final vaccination. The adjuvant experimental groups are shown in Tables 16, 17, and 18 below.

[0179] Experiments were 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-24-021). Specifically, 6-week-old C57BL / 6 (female) mice were admitted in groups of four and stabilized for one week. The adjuvant combination vaccine was administered intramuscularly twice at 2-week intervals, with 50 μl in each thigh of the mice for a total of 100 μl. Mouse body weight was measured once a week (DPV 0-28), and blood samples (DPV 13, 28) and serum isolation were performed at 2 weeks after the first and second vaccinations to measure antigen-specific antibody titers. The in vivo safety of the adjuvant was confirmed by examining abnormal findings at the injection site during necropsy (DPV 28).

[0181] Carbopol experimental group candidate substances 971P Group 1 PBS (negative control group) Group 2 Antigen (Immunostimulant efficacy comparison control group, antigen administered alone) Group 3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion Group 4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion Group 5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 974P Group 1 PBS (negative control group) Group 2 Antigen (Immunostimulant efficacy comparison control group, antigen administered alone) Group 3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion Group 4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion Group 5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion

[0182] Carbopol experimental group candidate substances 971P Group 1 PBS (negative control group) Group 2 Antigen (Immunostimulant efficacy comparison control group, antigen administered alone) Group 3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion Group 4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion Group 5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 974P Group 1 PBS (negative control group) Group 2 Antigen (Immunostimulant efficacy comparison control group, antigen administered alone) Group 3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion Group 4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion Group 5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion

[0183] Carbopol experimental group candidate substances 971P Group 1 PBS (negative control group) Group 2 Antigen (Immunostimulant efficacy comparison control group, antigen administered alone) Group 3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion Group 4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion Group 5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 974P Group 1 PBS (negative control group) Group 2 Antigen (Immunostimulant efficacy comparison control group, antigen administered alone) Group 3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion Group 4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion Group 5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC51815 LPS - O / W emulsion

[0184] 4.2 Comparative Evaluation of Antigen-Specific Antibody Formation Following Combination Vaccination

[0185] Antigen-specific antibody formation following the administration of the mixed vaccine in mice was evaluated using ELISA (Enzyme-linked immunosorbent assay). Specifically, the antigens used in the mixed vaccine were mixed in ELISA coating buffer (0.1M Sodium Carbonate, pH 9.5) to a concentration of 1 μg / well, and then 100 μl was dispensed into 96-well Immunoplates (Nunc, Cat. No.: 439454) and incubated overnight at 4°C. After washing four times with PBS-Tween 20 (0.05%), 200 μl of blocking solution (1% Bovine serum albumin in PBS) was added, and the plates were incubated overnight at 4°C. 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 the plates were incubated at 37°C for 2 hours. Subsequently, the sample was 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 sample was washed four times again with PBS-Tween 20 (0.05%). 100 μl of 1-Step Ultra TMB-ELISA Substrate Solution (ThermoFisher, Cat No.: 34029) was added 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 antigen-specific Total IgG antibody formation following the administration of the mixed vaccine are shown in Figures 4, 5, and 6. In addition, the antibody formation rate compared to the antigen-only vaccination group was calculated, and the results are shown in Tables 19 and 20.

[0187] Carbopol Carbopol and LPS mixed concentration Group Ratio (based on the control group vaccinated only with antigen) 971P 0.01% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 6.79 G4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 6.29 G5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 6.66 0.1% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 6.68 G4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 4.80 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 5.97 0.1% Carbopol, 100 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion 7.89 G4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 8.24 G5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 7.57

[0188] Carbopol Carbopol and LPS mixed concentration Group Ratio (based on the control group vaccinated only with antigen) 974P 0.01% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 14.39 G4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 12.97 G5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 14.45 0.1% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 13.81 G4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 15.34 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 13.71 0.1% Carbopol, 100 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion 15.06 G4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 14.63 G5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 16.28

[0189] As shown in FIGS. 4, 5, 6 and Tables 19 and 20, Carbopol 971P, 974P and various H. alvei Antibody formation increased in the bacterial-derived LPS-based adjuvant O / W emulsion mixed groups (Groups 3–5) compared to the antigen-alone group (Group 2), and higher antibody titers were observed in the Carbopol 974P-based adjuvant groups compared to Carbopol 971P. In particular, 0.1% Carbopol 974P, 100 µg / ml H. alvei It was highest in the strain ATCC 51815 LPS - O / W inoculated group (Group 5), at approximately 16.28 times compared to the antigen-only inoculated group (Group 2). P < 0.05).

[0191] 4.3 Comparative Evaluation of Antigen-Specific Cellular Immune Responses Following Combination Vaccination (Intracellular Cytokine Staining Assay)

[0192] Splenocytes, a mouse immune cell, were placed in a 96-well cell culture plate (NUNC, Cat. No.: 167008) at a volume of 2.0 x 10 6Cells were seeded to form cells / well. The seeded cells were treated with OVA protein (Invivogen, Cat. No.: vac-pova) at a concentration of 50 μg / ml and cultured for 15 hours in a 37°C, 5% CO2 incubator. Subsequently, Brefeldin A (1 / 1,000 dilution) was applied 10 hours prior to antibody staining. Anti-mouse CD16 / CD32 was applied at a concentration of 20 μg / ml and incubated at room temperature for 20 minutes. After washing twice with 1% BSA (in DPBS), immune cell surface factor (CD4, CD8) binding antibodies diluted in 1% BSA (in DPBS) were applied to the cells and incubated 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), cells were treated with Fixation / Permeabilization solution (BD Bioscience, Cat. No.: 555028) and incubated at 4°C for 20 minutes. After removing the supernatant, the cells were washed twice with Perm / Wash buffer (BD Bioscience, Cat. No.: 555028). After washing, cytokine (IL-2, IFN-γ, TNF-α) binding antibodies were added and incubated at 4°C in a dark room 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).

[0193] Figures 7, 8, and 9 show the results of confirming the ratio of CD4+ T and CD8+ T cells expressing antigen-specific cytokines IFN-γ, TNF-α, and IL-2 in C57BL / 6 mouse immune cells vaccinated with an adjuvant using intracellular cytokine staining (ICS) experiments. In addition, the ratio of CD4+ and CD8+ T cells derived from C57BL / 6 splenocytes expressing antigen-specific cytokines was calculated compared to the antigen-only group, and the results are shown in Tables 21 to 24.

[0195] Carbopol Carbopol and LPS mixed concentration Group Ratio (based on the control group vaccinated only with antigen) CD4+ T cell IFN-γ TNF-α IL-2 971P 0.01% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 5.15 5.67 2.63 G4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 3.58 6.71 3.83 G5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 6.91 10.05 3.55 0.1% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 1.36 3.91 2.38 G4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 1.72 2.65 2.39 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 3.11 5.13 2.03 0.1% Carbopol, 100 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion 7.00 12.28 4.84 G4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 3.32 4.62 2.69 G5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 2.14 4.50 2.53

[0197] Carbopol Carbopol and LPS mixed concentration Group Ratio (based on the control group vaccinated only with antigen) CD4+ T cell 974P 0.01% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 1.95 17.96 9.07 G4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 2.37 28.04 14.67 G5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 2.48 20.93 9.07 0.1% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 2.14 25.97 13.07 G4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 1.89 18.87 13.27 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 3.84 53.36 20.47 0.1% Carbopol, 100 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion 3.24 45.99 17.67 G4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 4.91 54.01 14.53 G5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 4.82 85.44 28.09

[0198] Carbopol Carbopol and LPS mixed concentration Group Ratio (based on the control group vaccinated only with antigen) CD8+ T cell IFN-γ TNF-α IL-2 971P 0.01% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 9.19 16.10 10.41 G4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 3.24 3.90 3.12 G5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 7.99 20.63 5.55 0.1% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 3.41 4.90 3.78 G4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 3.11 8.20 3.35 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 3.24 7.00 2.05 0.1% Carbopol, 100 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion 5.14 19.08 4.79 G4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 10.44 20.85 8.29 G5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 1.53 4.04 2.24

[0199] Carbopol Carbopol and LPS mixed concentration Group Ratio (based on the control group vaccinated only with antigen) CD8+ T cell IFN-γ TNF-α IL-2 974P 0.01% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 27.49 6.03 0.00 G4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 23.90 4.76 3.96 G5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 38.09 12.01 3.68 0.1% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 9.69 3.72 0.43 G4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 14.71 3.99 0.47 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 14.53 4.20 2.08 0.1% Carbopol, 100 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 1.00 1.00 G3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion 34.94 20.22 0.00 G4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 20.40 7.54 0.00 G5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC51815 LPS - O / W emulsion 21.13 11.15 1.78

[0200] As shown in FIGS. 7, 8, 9, and Tables 21 to 24, Carbopol 971P, 974P, and various H. alvei It was confirmed that the proportion of CD4+ T and CD8+ T cells expressing IFN-γ(Th1), TNF-α(Th1), and IL-2(Th1) increased in the bacterial-derived LPS-based adjuvant O / W emulsion formulation mixed groups (Groups 3–5) compared to the antigen-alone group (Group 2). P < 0.05).

[0201] Various Carbopol and H. alvei Among the LPS mixed concentrations, 0.1% Carbopol 974P as shown in Table 22 and Fig. 9B, H. alvei In the strain ATCC 51815 100 ug / ml LPS-O / W emulsion group (Group 5), the proportions of CD4+ T cells expressing IFN-γ(Th1), TNF-α(Th1), and IL-2(Th1) were approximately 4.82-fold, 85.44-fold, and 20.89-fold higher, respectively, compared to the antigen-alone group (Group 2). P< 0.05). Also, 0.01% Carbopol 974P in Table 24 and Fig. 7, H. alvei strain ATCC 51815 20 µg / ml It was confirmed that the proportion of CD8+ T cells expressing IFN-γ(Th1), TNF-α(Th1), and IL-2(Th1) in the LPS-O / W emulsion group (Group 5) was approximately 38.09-fold, 12.01-fold, and 3.68-fold higher, respectively, compared to the antigen-alone group (Group 2). P < 0.05).

[0203] 4.4 Comparative Evaluation of Antigen-Specific Cellular Immune Responses (ELISpot) Following Combination Vaccination

[0204] To evaluate antigen-specific cellular immune responses, C57BL / 6 mouse splenocytes were necrolytic and extracted at week 2 (DPV 28) after the second dose of the adjuvant-mixed vaccine. The extracted mouse splenocytes were plated at 2.5 x 10⁶ IFN-γ ELISpot plates (Mabtech, Cat. No.: 3321-4HPT-10). 5Cells were seeded to a cell / well ratio. To confirm the activation of antigen-specific cellular immune responses, OVA protein (Invivogen, Cat. No.: vac-pova) was added at a concentration of 10 μg / ml, and the cells were cultured for 20 hours in a 37°C, 5% CO2 incubator. After washing the cultured cells 5 times with PBS, 100 µl of Biotinylated Detection mAb, diluted to 1 / 1,000 in PBS (with 0.5% Fetal Bovine Serum), was added to each well and stored at room temperature in the dark for 2 hours. Subsequently, the cells were washed 5 times with PBS, 100 µl of Streptavidin-HRP, diluted to 1 / 1,000 in PBS (with 0.5% Fetal Bovine Serum), was added to each well and stored at room temperature in the dark for 1 hour. Washed 5 times with PBS, added 100 µl of TMB substrate per well, and reacted until fewer than 5 spots appeared in the negative control well (reaction time: 30 min). Washed with double distilled water to stop color development, dried the ELISpot plate in the dark, and analyzed using an ELISpot Reader (Cellular Technology Limited, BioSpot S5).

[0205] The results of confirming antigen-specific IFN-γ formation ability in splenocytes of C57BL / 6 mice through the ELISpot experiment are shown in Figures 10, 11, and 12, and the results of calculating antigen-specific IFN-γ formation ability compared to the antigen-only inoculation group are shown in Tables 25 and 26.

[0207] Carbopol Carbopol and LPS mixed concentration Group Ratio (based on the control group vaccinated only with antigen) 971P 0.01% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 107.28 G4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 48.42 G5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 48.64 0.1% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 31.72 G4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 35.28 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 12.17 0.1% Carbopol, 100 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion 40.81 G4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 72.25 G5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 11.06

[0208] Carbopol Carbopol and LPS mixed concentration Group Ratio (based on the control group vaccinated only with antigen) 974P 0.01% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 354.57 G4 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 310.43 G5 Antigen / 0.01% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 639.86 0.1% Carbopol, 20 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei BA2000346 LPS - O / W emulsion 135.71 G4 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 385.57 G5 Antigen / 0.1% 카보폴, 20 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 187.29 0.1% Carbopol, 100 ug / ml LPS G2 Antigen administered alone (control group for comparison of immune adjuvant efficacy) 1.00 G3 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei BA2000346 LPS - O / W emulsion 288.57 G4 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei 9-126A-1 LPS - O / W emulsion 394.71 G5 Antigen / 0.1% 카보폴, 100 μg / ml H. alvei ATCC 51815 LPS - O / W emulsion 401.14

[0209] As shown in Figs. 10, 11, 12 and Tables 25 and 26, Carbopol 971P, 974P and various H. alveiSpot formation ability increased in the bacterial-derived LPS-based adjuvant O / W emulsion formulation mixed group (Groups 3–5) compared to the single inoculation group (Group 2).

[0210] In particular, 0.01% Carbopol 974P, 20 ug / ml H. alvei The spot-forming ability was highest in the strain ATCC 51815 LPS - O / W inoculated group (Group 5), approximately 639.86 times higher compared to the antigen-only inoculated group (Group 2). P < 0.05).

[0212] In summary, the inventors of the present invention have various carbomers and various Hafnia alvei An immunostimulatory composition containing a bacterial-derived lipopolysaccharide has been developed. The immunostimulatory composition according to the present invention possesses unique physical properties regarding pH, viscosity, and particle size of the final formulation, and it has been experimentally confirmed to exhibit excellent antigen-specific antibody formation ability within the individual and effectively induce cellular and humoral immunity. Therefore, the immunostimulatory composition and manufacturing technology of the present invention can be usefully utilized in various fields, such as the prevention of infectious diseases caused by pathogens (viruses, bacteria, etc.), immunostimulation, vaccine adjuvantage, and anticancer treatment.

[0214] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0216] Name of depositing institution: Biological Resource Center Trustee Number: KCTC15966BP Date of Deposit: 2024-07-18 Name of depositing institution: Biological Resource Center Trustee Number: KCTC16220BP Date of Deposit: 2025-01-07

Claims

Claim 1 Immunostimulative composition comprising the following composition: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) pH 6.5 to pH 7.

5. Claim 2 Immunostimulative composition comprising the following composition: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp. Claim 3 Immunostimulative composition comprising the following composition: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) particle size of 95 nm to 140 nm. Claim 4 An immune-enhancing composition according to any one of claims 1 to 3, wherein the carbomer is included in an amount of 0.005 to 0.25% (w / v). Claim 5 An immune-enhancing composition according to any one of claims 1 to 3, wherein the lipopolysaccharide derived from hafnia albei is included in an amount of 10 to 200 μg / ml. Claim 6 An immune-enhancing composition according to any one of claims 1 to 3, wherein the lipopolysaccharide derived from hafnia albei and the carbomer are formulated as an oil-in-water emulsion. Claim 7 An immune-enhancing composition according to any one of claims 1 to 3, wherein the immune-enhancing composition further comprises squalene. Claim 8 Vaccine adjuvant composition comprising the following composition: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm. Claim 9 Vaccine composition comprising the following components: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm. Claim 10 Pharmaceutical composition for the prevention or treatment of infectious diseases comprising the following composition: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm. Claim 11 A composition for the prevention or treatment of an infectious disease according to claim 10, wherein the infectious disease is caused by one or more infections selected from the group consisting of parasites, viruses, bacteria, and fungi. Claim 12 Immunostimulating health functional food composition comprising the following composition: a) Hafnia Albay ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm. Claim 13 Immunostimulant feed additive composition comprising the following composition: a) Hafnia albei ( Hafnia alvei ) derived lipopolysaccharide; b) carbomer; and c) viscosity 1.95 to 2.15 cp, pH 6.5 to pH 7.5 or particle size 95 nm to 140 nm. Claim 14 A method for inducing an immune response in an individual, comprising the step of administering an immune-enhancing composition of any one of claims 1 to 3 to an individual in need thereof.

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