EA042022B1 - IMMUNE STRENGTHENING COMPOSITION CONTAINING IMMUNOMODULATOR AND CATIONIC LIPOSOME AND VACCINE COMPOSITION CONTAINING THEM - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to immune enhancing compositions containing an immunomodulator having a novel structure. In particular, the present invention relates to immune enhancing compositions containing a reduced toxicity lipopolysaccharide (LPS) analogue and a cationic liposome, and uses thereof.

State of the art

Liposomes have advantages in that they exhibit excellent biocompatibility, are easy to prepare, and are capable of delivering water-soluble and lipid-soluble drugs, therefore, such liposomes are being actively researched as a drug delivery system having fewer side effects in vivo. Conventional liposomes have disadvantages in that they cause aggregation, fusion, hydrolysis of phospholipids, oxidation, leakage of encapsulated drugs, and the like. when storing liposomes in the form of a dispersed phase in an aqueous solution, since the system itself is physically and chemically unstable. Thus, their disadvantage is that it is necessary to ensure the stability of the liposomes themselves when stored for a long time. In order to overcome these shortcomings, research has been actively conducted to improve the stability of liposomes by storing them as a fine powder in order to overcome the physicochemical instability of liposomes. However, a freeze-drying process is used to convert the liposomes into a fine powder. In such a process, when the liposomes are used by themselves, the stability and uniformity of the liposomes are deteriorated, and thus their physical and chemical properties are changed. As a result, unfortunately, the function of liposomes as a drug delivery system and their immune activity deteriorates, and toxicity occurs. Therefore, research is under way on compositions that are advantageous for manufacturing, storage, and delivery while maximizing the benefits of liposomes by adding cryoprotectants and the like. in order to prevent their deformation during the freeze-drying process.

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and contributes to the activation of various immune cells, especially the innate immune system. LPS activates antigen-presenting cells through cytokine secretion by cells of the immune system, expression of costimulatory molecules on antigen-presenting cells, and induction of antigen presentation, and links innate immunity to adaptive immunity (Akira S, Uematsu S, Takeuchi O., Cell 124: 783-801 (2006) Schnare M, Barton GM, Holt AC, Takeda K, Akira S et al., Nat Immunol 2: 947-950 (2001)).

The LPS is composed of three domains, namely an amphiphilic domain (lipid A), a central oligosaccharide (OS) and an O antigen (or O antigenic polysaccharide). Lipid A is known to play a role in the endotoxin activity of LPS and to exert immunostimulatory effects through signaling through TLR4 (Toll-like receptor-4) in various immune cell types (Raetz CR, Whitfield C, Annu Rev Biochem. 71: 635-700 ( 2002)). Lipid A derivatives that exhibit reduced toxicity have been targets for the development of immunoadjuvants for human vaccines. Monophosphoryl lipid A (MPL) is a non-toxic derivative of LPS isolated from a crude strain of Salmonella minnesota. In addition, combinations of aluminum salts with MPL have been approved as immunoadjuvants for vaccines against HBV (hepatitis B virus) and HPV (human papillomavirus).

Since the 1950s, LPS has been known to have anti-cancer activity, but it is not suitable for use due to toxicity that can cause death from sepsis even when contaminated at the nanogram (ng) level. Therefore, studies aimed at reducing the toxicity of LPS have been constantly carried out, and the toxicity of LPS has been reduced, in particular, by removal of polysaccharide chains or deacylation of lipid A (Katz SS et al., J Biol Chem. Dec 17, 274 (51): 36579-84 , 1999). In particular, MPL obtained by phosphorylation of lipid A obtained by removing the polysaccharide chain of LPS has been developed as an immune anti-cancer agent devoid of LPS toxicity, but its effects are known to be insufficient.

As a result of efforts to develop LPS analogs capable of exhibiting excellent immunostimulatory activity while reducing the toxicity that caused problems with conventional LPS and overcoming the physical and chemical instability of liposomes, the present inventors have discovered an EG-immunomodulator (EG-IM) with a novel structure and reduced toxicity by isolating and purifying LOS lacking an O antigen site from an E. coli strain occurring in the human intestine and deacylating it, and found that a vaccine composition containing this immunomodulator and a cationic liposome exhibits excellent immunostimulatory activity. Based on this, the present invention has been accomplished.

The essence of the invention

Technical problem

Thus, one of the objectives of the present invention is to provide an immune enhancing composition comprising (a) an immunomodulator represented by the following formula 1:

- 1 042022

GlcN HEP KDO GlcNAc Acyl glc HEP HEP KDO GlcNAc Acyl

[Formula 1] where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind, and (b) a cationic liposome.

Another object of the present invention is to provide a vaccine composition containing (a) an antigen and (b) said immune enhancing composition as an active ingredient.

Another object of the present invention is a method for preparing said composition for enhancing immunity, comprising (a) preparing a solution containing an immunomodulator represented by formula 1

GlcN

glc

HEP

HEP

HEP

KDO GlcNAc Acyl KDO GlcNAc Acyl

[Formula 1] where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind; and (b) dissolving lipids in an organic solvent to obtain a mixed lipid solution; (c) lyophilizing the mixed lipid solution from step (b) to remove the organic solvent; and (d) rehydrating the substance obtained in step (c) and then mixing this substance with the solution from step (a), thereby obtaining an immune enhancing composition.

Another object of the present invention is a method for producing said vaccine composition, comprising (a) preparing a solution containing an immunomodulator represented by formula 1

where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind; and (b) dissolving lipids in an organic solvent to obtain a mixed lipid solution; (c) lyophilizing the mixed lipid solution from step (b) to remove the organic solvent; (d) rehydration of the substance obtained in step (c), thereby obtaining liposomes; and (e) adding the solution from step (a) and the antigen to the liposomes from step (d), followed by re-lyophilization.

Technical solution

To achieve the above objects, the present invention provides an immune enhancing composition comprising (a) an immunomodulator represented by the following formula 1; and (b) a cationic liposome

- 2 042022

[Formula 1] where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A to F are positions to which phosphate can bind.

The present invention also provides a vaccine composition containing (a) an antigen; and (b) said immune enhancing composition as an active ingredient.

The present invention also provides a method for preparing said composition for enhancing immunity, comprising (a) preparing a solution containing an immunomodulator represented by formula 1

GlcN HEP KDO GlcNAc Acyl glc HEP HEP KDO GlcNAc Acyl

[Formula 1] where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind; and (b) dissolving lipids in an organic solvent to obtain a mixed lipid solution; (c) lyophilizing the mixed lipid solution from step (b) to remove the organic solvent; and (d) rehydrating the substance obtained in step (c) and then mixing this substance with the solution from step (a), thereby obtaining an immune enhancing composition.

The present invention also provides a method for producing said vaccine composition, comprising (a) preparing a solution containing an immunomodulator represented by formula 1

[Formula 1] where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind; and (b) dissolving lipids in an organic solvent to obtain a mixed lipid solution; (c) lyophilizing the mixed lipid solution from step (b) to remove the organic solvent; (d) rehydration of the substance obtained in step (c), thereby obtaining liposomes; and (e) adding the solution from step (a) and the antigen to the liposomes from step (d), followed by re-lyophilization.

Brief description of the figures

In FIG. 1A shows the results of the identification of the extracted LOS by electrophoresis and silver staining, and FIG. 1B is a result showing that the size of LOS-based EG-IM extracted after removal of the O-acyl chain of lipid A is reduced when LOS is treated with alkali, where M is a marker, lane 1-LOS extracted prior to deacylation, and lane 2 is an immunomodulator. (EG-IM).

In FIG. 2 shows the structure of an EG-immunomodulator (EG-IM) of the present invention, where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are the positions that can bind phosphate.

In FIG. 3 shows liposome size distribution diagrams in the lipid dissolution method, and FIG. 4 shows liposome size distribution diagrams in the simple mixing glycolipid-liposome method.

In FIG. 5 is a graph of the effect of liposomal EG-IM on immune cell proliferation.

In FIG. 6 is a graph of the effect of liposomal EG-IM on TNF-α secretion.

- 3 042022

In FIG. 7 is a graph of the decrease in cytotoxicity with liposomal EG-IM.

In FIG. 8 is a result indicating that the formation of the vaccine containing liposomal EG-IM occurs by adsorption by electrostatic attraction, where LP means liposomes, S-supernatant, and P-precipitate.

In FIG. 9 is a graph of immune cell recruitment by liposomal EG-IM.

In FIG. 10 shows the results of analysis of the uptake effect of a vaccine containing liposomal EG-IM by flow cytometry, and FIG. 11 - the effect of absorption by monocytes, and in FIG. 11A shows the results of analysis of fluorescently labeled gE protein-specific cells in dLN, FIG. 11B shows results of analysis of fluorescently labeled gE protein-specific cells in muscle tissue, FIG. 11C shows results of measurement of fluorescently labeled gE protein-specific monocytes in dLN, and FIG. 11D are measurements of fluorescently labeled gE protein-specific monocytes in muscle tissue.

In FIG. 12 shows the depot effect of a vaccine containing liposomal EG-IM.

In FIG. 13 shows antibody titers specific for Zika virus antigen in Zika virus vaccine containing liposomal EG-IM.

In FIG. 14A shows JEV-specific antibody titers from Japanese encephalitis (JEV) vaccine containing liposomal EG-IM, and FIG. 14B is the level of secretion of the cytokine IFN-γ after administration of the Japanese encephalitis vaccine.

In FIG. 15A shows the titers of TB-specific antibodies in a vaccine against tuberculosis (TB) containing liposomal EG-IM, FIG. 15B shows the level of secretion of the cytokine IFN-γ after the administration of the tuberculosis vaccine, and in FIG. 15C - results of the analysis of bactericidal action in the tuberculosis vaccine containing liposomal EG-IM.

In FIG. 16A shows the titers of aP-specific antibodies in a pertussis vaccine containing liposomal EG-IM, and FIG. 16B - results of analysis of CD4+ T cell proliferation under the influence of pertussis vaccine.

In FIG. 17A shows the level of secretion of the cytokine IFN-γ after pertussis vaccine administration, with light bars representing spleen cell results and dark bars representing lung cell results. In FIG. 17B and 17C show the ability to induce IFN-γ and IL-17, respectively, upon administration of pertussis vaccine.

In FIG. 18 shows the level of secretion of the cytokine IFN-γ after administration of a varicella-zoster virus vaccine containing liposomal EG-IM.

In FIG. 19-21 show the results of an analysis of synergistic effects when adding lipid ingredients to a varicella zoster virus vaccine containing liposomal EG-IM, FIG. 19 shows the results with the addition of cholesterol, FIG. 20 shows the results with the addition of squalene, and FIG. 21 - results when adding tricaprin.

In FIG. 22 shows the synergistic effect of adding a saponin-based immunoadjuvant ingredient to a varicella-zoster vaccine containing liposomal EG-IM.

In FIG. 23 shows an analysis of immunogenicity depending on the final formula of the varicella zoster virus vaccine containing liposomal EG-IM. In FIG. 23A shows the level of secretion of the cytokine IL-5, and FIG. 23B and 23C - the level of secretion of the cytokine IFN-γ, and NP-A means DDA/DOPC, NP-B-DOTAP/DMPC, NP-C-DOTAP/DMPC/squalene, NP-D-DOTAP/DMPC/tricaprin, + means simple mixed form, and / means mixed and lyophilized form.

Disclosure of the essence of the invention

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as known to those skilled in the art to which the present invention pertains. In general, the nomenclature used here and the experimental methods described below are well known in the art and are commonly used.

In one embodiment of the present invention, liposomal EG-IM is obtained by mixing a mixed lipid solution containing a mixture of DMPC and DOTAP with an EG-immunomodulator (EG-IM) solution, obtaining a mixed lipid solution containing EG-IM, followed by rehydration, and the effect of liposomal EG-IM for immune cell proliferation and reduced cytotoxicity was tested in a mouse macrophage cell line.

Accordingly, in one aspect, the present invention is directed to immune enhancing compositions comprising (a) an immunomodulator represented by the following formula 1; and (b) a cationic liposome

- 4 042022

where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind.

In another aspect, the present invention is directed to a process for the preparation of immune enhancing compositions, wherein the immune enhancing compositions may be prepared by the following steps:

(a) preparing a solution containing an immunomodulator represented by formula 1

where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind;

(b) dissolving lipids in an organic solvent to obtain a mixed lipid solution;

(c) lyophilizing the mixed lipid solution from step (b) to remove the organic solvent; and (d) rehydrating the substance obtained in step (c) and then mixing this substance with the solution from step (a), thereby obtaining an immune enhancing composition.

The EG-immunomodulator (EG-IM) of the present invention may be represented by the following formula 1:

where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind.

In the present invention, the term LOS (lipooligosaccharide) refers to variants of LPS (lipopolysaccharide) with a shorter saccharide chain than natural LPS, so they have a lower molecular weight. Before deacylation, the LOS preferably has a molecular weight of 3000 to 10000 Da, more preferably 3000 to 4000 Da. The term "deacylated LOS" refers to a LOS in which the fatty acid linked to the glucosamine of lipid A via a -C(O)O- bond has been removed and toxicity is greatly reduced compared to LOS. The fatty acid binds to the glucosamine of lipid A via -C(O)O- and -C(O)NH- bonds. The deacylated LOS of the present invention is a LOS in which the -C(O)O- linked fatty acid has been removed by lipid A deacylation.

EG-IM can be obtained in various ways, but can be obtained in accordance with the methods disclosed in the previous patents of the present inventors, namely Korean Patent No. 0456681; WO2004/039413; Korean Patent No. 0740237; and WO 2006/121232. For example, LPS is deacylated by treatment with a strong base (eg, 0.2N NaOH) to remove some of the fatty acids from lipid A and thereby detoxify it.

According to the present invention, EG-IM can be linked to 2-6 phosphate groups, preferably 3-4 phosphate groups, without limitation. In addition, the number and position of phosphate groups in formula 1 may be as given below in table. 1.

- 5 042022

Table 1

Example Number of phosphate groups Positions of phosphate groups Example Number of phosphate groups Positions of phosphate groups Example 1 2 A, B Example 25 3 A, E, F Example 2 2 A, C Example 26 3 B, C, D Example 3 2 A, D Example 27 3 ALL Example 4 2 A, E Example 28 3 B, C, F Example 5 2 A, F Example 29 3 B, D, E Example 6 2 c, c Example 30 4 B, D, F Example 7 2 B, D Example 31 3 B, E, F Example 8 2 B, E Example 32 3 C, D, E Example 9 2 B, F Example 33 3 C, D, F Example 10 2 C, D Example 34 3 C, E, F Example 11 2 C, E Example 35 3 D, E, F Example 12 2 C, F Example 36 4 A, B, C, D Example 13 2 D, E Example 37 4 AND ALL Example 14 2 D,F Example 38 4 A, B, C, F Example 15 2 E, F Example 39 4 A, B, D, E Example 16 3 A, B, C Example 40 4 A, B, D, F Example 17 3 A, B, D Example 41 4 A, B, E, F Example 18 3 A, B, E Example 42 4 A, C, D, E Example 19 3 A, B, F Example 43 4 A, C, D, F Example 20 3 A, C, D Example 44 4 A, D, E, F Example 21 3 A, C, E Example 45 5 A, B, C, D, E Example 22 3 A, C, F Example 46 5 A, B, C, D, F Example 23 3 A, D, E Example 47 6 A, B, C, D, E, F Example 24 3 A, D, F

The phosphate binds at positions selected from the group consisting of AB, AC, AD, AE, AF, BC, BD, BE, BF, CD, CE, CF, DE, DF, EF, ABC, ABD, ABE, ABF, ACD , ACE, ACF, ADE, ADF, AEF, BCD, ALL, BCF, BDE, BDF, BEF, CDE, CDF, CEF, DEF, ABCD, ABCE, ABCF, ABDE, ABDF, ABEF, ACDE, ACDF, ADEF, BCDE , BCDF, BCEF, BDEF, CDEF, ABCDE, ABCEF and ABCDEF in formula 1.

According to the present invention, the saccharides in formula 1 are selected from the group consisting of hexose, hexosamine, N-acetylhexosamine, heptose and Kdo (2-keto-3-deoxyoctonate).

In the present invention, the term hexose means a monosaccharide containing 6 carbon atoms in a molecule, and examples thereof include, without limitation, ketohexoses (psychose, fructose, sorbose, tagatose), aldohexoses (allose, altrose, glucose, mannose, gulose, idose, galactose, talose) and deoxyhexoses (fucose, fuculose, rhamnose).

According to the present invention, the hexose is represented by aldohexose, and in certain examples, aldohexose is represented by glucose or galactose.

In the present invention, the term heptose means a monosaccharide containing 7 carbon atoms per molecule, which are subdivided into aldoheptoses (position 1) and ketoheptoses (position 2) according to the position of the functional groups (aldehyde and keto groups). An example of aldoheptose is L-glycero-D-mannoheptose, without limitation. An example of ketoheptose is sedoheptulose and mannoheptulose, without limitation.

According to the present invention, hexosamine is glucosamine, galactosamine, or mannosamine, and in certain instances, hexosamine is glucosamine.

According to the present invention, N-acetylhexosamine is N-acetylglucosamine, N-acetylgalactosamine or N-acetylmannosamine, and in certain examples, N-acetylhexosamine is N-acetylglucosamine.

EG-IM of the present invention contains less saccharides than wild-type LPS. According to the present invention, EG-IM may contain from 5 to 7 saccharides. In certain examples, EGIM contains 6 or 7 sugars, without limitation.

The EG-IM of the present invention does not contain O-linked fatty acids.

The EG-IM of the present invention is characterized in that it is significantly less toxic because the fatty acid (eg C14 fatty acid) has been removed (deacylated) from lipid A. The fatty acid binds to glucosamine in lipid A via a -C(O)O- or -C(O)NH- bond. In the present invention, deacylation means the removal of a fatty acid linked through a -C(O)O- bond.

Deacylation can be carried out by treating the LOS with an alkali, and the alkalis include NaOH, KOH, Ba(OH) ₂ , CsOH, Sr(OH) ₂ , Ca(OH)2, LiOH, RbOH and Mg(OH)2, more preferably NaOH, KOH , Ba(OH) ₂ , Ca(OH) ₂ , LiOH and Mg(OH) ₂ , even more preferably NaOH, KOH and Mg(OH) ₂ , most preferably NaOH.

According to the present invention, the EG-IM of the present invention is from E. coli and the E. coli is Escherichia coli EG0024 (accession number KCTC 12948BP). This strain was depot

- 6 042022 registered on November 19, 2015 with deposit number KCTC 12948BP in the Korean Type Culture Collection of the Korea Research Institute of Biology and Biotechnology.

The EG-IM of the present invention is particularly suitable for the vaccine compositions of the present invention as it exhibits excellent immunostimulatory effects and less toxicity compared to conventional immunoadjuvants. The EG-IM of the present invention is less toxic than MPL (monophosphoryl lipid A) obtained by phosphorylation of lipid A obtained by removing the polysaccharide chain from LPS to eliminate the toxicity of LPS.

The cationic liposome of the present invention is a cationic lipid selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), 1,2-dioleoyl-3trimethylammonium propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol ( DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 14:1), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 16:0-18:1), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 18: 1), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 18:0), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 16:0), 1,2-dimyristoyl -sn-glycero-3-ethylphosphocholine (ethyl-RS 14:0), 1,2dilauroyl-sn-glycero-3-ethylphosphocholine (ethyl-PC 12:0), N1-[2-((1S)-1-[ (3-aminopropyl)amino]-4-[di(3aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]benzamide (MVL5), 1,2-dimyristoyl-3 dimethylammonium propane (DAP 14:0 ), 1,2-dipalmitoyl-3-di methylammonium-propane (DAP 16:0), 1,2distearoyl-3-dimethylammonium-propane (DAP 18:0), N-(4)-carboxybenzyl)-N,N-dimethyl-2,3bis(oleoyloxy)propane-1 -aminium (DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (TAP 18:0), 1,2-dipalmitoyl-3-trimethylammonium-propane (TAP 16:0), 1,2-dimyristoyl-3-trimethylammonium- propane (TAP 14:0); and N4-cholesteryl-stermine (GL67). The cationic liposome may be used alone or in combination with a neutral lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC ), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), phosphoethanolamine (PE) and phosphatidylcholine (PC). In one embodiment of the present invention, a combination of DOTAP and DMPC (DOTAP:DMPC), a combination of DDA and DOPC (DDA:DOPC) and a combination of DDA and DMPC (DDA:DMPC) are mainly used.

In the present invention, the term immunostimulation refers to the induction of an initial immune response or enhancement of a normal immune response to an antigen to a measurable extent.

In another embodiment of the present invention, liposomal EG-IM is prepared by mixing a mixed lipid solution containing a mixture of DMPC and DOTAP with an EG-immunomodulator (EG-IM) solution, obtaining a mixed lipid solution containing EG-IM, followed by rehydration, and then preparing a vaccine, by adding an antigen to liposomal EG-IM, and to establish the effectiveness of vaccines, vaccines obtained by mixing liposomal EG-IM with recombinant Zika virus envelope protein, Japanese encephalitis virus (JEV) antigen, recombinant tuberculosis antigens, namely Ag85A, ESAT-6 and HspX , pertussis vaccine cell-free antigen (aP), and recombinant varicella-zoster virus (VZV) gE antigen, are injected into mice, their antibody titers are measured, and cytokine assays are performed. As a result, each of the vaccines was found to effectively induce excellent antibody-mediated immunity and/or cellular immunity.

Accordingly, in another aspect, the present invention is directed to vaccine compositions containing an antigen and an immune enhancing composition as an active ingredient.

In another aspect, the present invention is directed to a method for preparing vaccine compositions, wherein the vaccine compositions may be prepared by the following steps:

(a) preparing a solution containing an immunomodulator represented by the following formula 1

GlcN

glc

HEP

HEP

HEP

KDO

KDO

GlcNAc H Acyl

GlcNAc - Acyl

[Formula 1] where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind;

(b) dissolving lipids in an organic solvent to obtain a mixed lipid solution;

(c) lyophilizing the mixed lipid solution from step (b) to remove the organic solvent; and (d) rehydrating the substance obtained in step (c), thereby obtaining liposomes; and (e) adding the solution from step (a) and the antigen to the liposomes from step (d), followed by repeat

- 7 042022 lyophilization.

In the present invention, the term antigen refers to substances that induce an immune response in a recipient. Thus, any substances exhibiting such immune response inducing activity can be used in the present invention without limitation.

The antigen of the present invention may be a peptide, protein, nucleic acid, saccharide, pathogen, attenuated pathogen, inactivated pathogen, virus, virus-like particles (VLP), cells, or cell fragments.

In the present invention, the antigen may be Zika virus antigen, Japanese encephalitis virus antigen, Mycobacterium tuberculosis antigen, whooping cough antigen, varicella zoster virus antigen, Haemophilus influenzae type B (HTB) antigen, Middle East respiratory syndrome (MERS) virus antigen, Pseudomonas aeruginosa antigen, anthrax, hepatitis A virus (HAV) antigen, hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) antigen, human immunodeficiency virus (HIV) antigen, Herpes simplex virus (HSV) antigen, Neisseria meningitidis antigen, Corynebacterium diphtheria antigen , Bordetella pertussis antigen, Clostridium tetani antigen, human papillomavirus (HPV) antigen, enterococcal antigen, Staphylococcus aureus antigen, Klebsiella pneumoniae antigen, Acinetobacter baumannii antigen, Enterobacter antigen, Helicobacter pylori antigen, malaria antigen, dengue virus antigen, Orientia tsutsugamushi antigen, bunyavirus antigen for severe fever with thrombocytopenia syndrome Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Antigen, Influenza Virus Antigen, Ebola Virus Antigen, and Diplococcus pneumoniae Antigen. In one embodiment of the present invention, the immunogenicity enhancing effect of the present invention is identified by a Zika virus antigen, a Japanese encephalitis virus antigen, a Mycobacterium tuberculosis antigen, a pertussis antigen, and a varicella-zoster virus antigen.

The varicella zoster virus antigen of the present invention may include at least one selected from the group consisting of gB (gpII), gC (gpV), gE (gpI), gH (gpIII), gI (gpIV), gK and gL ( gpVI).

The vaccines of the present invention may be used to prevent or treat diseases, and the vaccine may be in the form of an inactivated vaccine, an attenuated vaccine, a subunit vaccine, a conjugate vaccine, a recombinant vaccine, a monovalent vaccine, a polyvalent vaccine, or a mixed vaccine.

In addition, the vaccine of the present invention may be a Zika virus vaccine, a Japanese encephalitis vaccine, a tuberculosis vaccine, a pertussis vaccine, a varicella-zoster virus (VZV) vaccine, a Haemophilus influenzae type B vaccine, a MERS vaccine, a Pseudomonas aeruginosa, cancer vaccine, anthrax vaccine, HAV vaccine, HBV vaccine, HCV vaccine, HIV vaccine, meningococcal vaccine, diphtheria vaccine, tetanus vaccine, multidrug-resistant bacteria vaccine, enterococcal vaccine , Staphylococcus aureus vaccine, Klebsiella pneumoniae vaccine, Acinetobacter baumannii vaccine, Enterobacter vaccine, Helicobacter pylori vaccine, Malaria vaccine, Dengue virus vaccine, Orientia tsutsugamushi vaccine, Bunyavirus severe fever with thrombocytopenia syndrome (SFTS) vaccine , wack severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola virus vaccine, influenza virus vaccine, or Diplococcus pneumoniae vaccine.

The cancer vaccine may be selected from the group consisting of fibrosarcoma, bladder cancer, pituitary adenoma, glioma, brain tumors, nasopharyngeal cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colon cancer liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, ureteral cancer, renal cell carcinoma, prostate cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasmacytic tumors, leukemia, cancer in children, skin cancer, ovarian cancer and cervical cancer, without limitation.

The vaccine of the present invention may be a simple mixture type formulation or a lyophilized form.

The vaccine of the present invention may contain the antigen described above, the immunomodulator represented by formula 1, and the cationic liposome in one container (all-in-one type vaccine). Based on this feature, the vaccine can be prepared in a ready-to-use form. Preferably, the varicella-zoster virus vaccine, ie the Herpes zoster or varicella vaccine, can be prepared in a ready-to-use form.

In addition, the vaccine of the present invention may further contain a lipid ingredient accepted in the field, and in one embodiment of the present invention, vaccines additionally containing cholesterol, squalene or tricaprin were prepared, and the effect of enhancing their immunogenicity was found (Fig. 19-21).

In addition, the vaccine of the present invention may further contain saponin, Quil A, QS21, etc. as an immunoadjuvant acceptable in this field. In one incarnation of the infusion

- 8 042022 vaccines containing Quil A, a substance derived from saponin, were obtained, and the effect of enhancing immunogenicity was found (Fig. 22).

The vaccine compositions of the present invention may contain a pharmaceutically acceptable carrier, as well as ingredients that are commonly used as a prescription substance, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, without limitation. The vaccine compositions of the present invention may also contain lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives, and the like, in addition to the ingredients described above. Suitable pharmaceutically acceptable carriers and formulations are detailed in Remington's Pharmaceutical Sciences (19th ed., 1995).

Vaccine compositions of the present invention can be administered orally or parenterally. In the case of parenteral administration, the vaccine compositions may be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, transdermally, and the like.

Suitable dosages of the vaccine compositions of the present invention may be administered in various ways based on such factors as formulation, route of administration, and age, body weight, sex, pathology, diet, administration time, route of administration, rate of elimination, and patient responsiveness.

The vaccine compositions of the present invention may be prepared in single dose form or alternatively formulated into multiple dose vials when formulated using a pharmaceutically acceptable carrier and/or excipient in a manner that can be readily performed by those of ordinary skill in the art to to which the present invention relates. Presently, dosage forms may be in the form of solutions, suspensions or emulsions in an oily or aqueous medium, or alternatively in the form of an extract, powder, granules, tablets or capsules, and may additionally contain a dispersant or stabilizer.

Examples

Hereinafter, the present invention will be described in more detail in the following examples. However, it should be clear to those skilled in the art that these examples are provided to illustrate the present invention only and should not be construed as limiting the scope of the present invention.

Preparative Example 1 Preparation of an immunomodulator (EG-IM).

1. Obtaining dried cells of the strain.

E. coli was cultured with shaking at 80 rpm or less in 30 g/l of TSB medium (tryptic soy broth, Difco) at 37° C. for 20 h, and cells were harvested by centrifuge. The collected cells were mixed with ethanol and centrifuged to give a pellet. Acetone was then added to the resulting precipitate, thoroughly mixed, and then centrifuged to obtain a precipitate. Ethyl ether was added to the resulting precipitate, thoroughly mixed, and then centrifuged to give a precipitate. The resulting precipitate was dried in an oven at 60° C. to obtain dried bacterial cells.

2. Extraction of LOS.

After weighing the dried bacterial cells, 7.5 ml of PCP (phenol, chloroform, petroleum ether) extraction solution per 1 g of mass was added to isolate LOS from the bacterial cells. From the LOS extract thus obtained, the organic solvent was removed at high temperature on a rotary evaporator. The resulting extract was centrifuged at high temperature to give a pellet, which was washed with ethyl ether. Then purified water was added there to form a precipitate. The precipitate formed was centrifuged and separated from the supernatant, and the resulting precipitate was washed with ethanol and collected. The precipitate was thoroughly dried in a drying oven at high temperature and then dissolved in purified water to extract the LOS.

3. Elimination of LOS toxicity.

After determining the content of the LOS extract, the concentration of the LOS extract was adjusted to 3 mg/ml, and the LOS extract was mixed with 0.2 N. NaOH at a volume ratio of 1:1. The reaction was carried out in a water bath with a constant temperature at 60°C for 120 min with stirring on a vibrating stirrer for 5 s every 10 min. Then 1N acetic acid was added there in an amount of about 1/5 of the initial amount of 0.2 N NaOH. Then received immunomodulator EG-IM by precipitation with ethanol.

4. Definition and identification of LOS and EG-IM.

LOS and EG-IM content was measured by KDO (2-keto-3-dihydroxyoctonate) analysis with 2thiobarbituric acid, their concentrations were measured, and LOS and EG-IM were separated by size by SDS-PAGE and identified by silver staining, which is shown in FIG. . 1. In FIG. 1A shows the results of the identification of the extracted LOS by electrophoresis and silver staining. In FIG. 1B is a result showing that the size of LOS-based EG-IM extracted from lipid A degradation is reduced when LOS is treated with alkali, where M is a marker (SeeBlue® Plus 2 prestained standard, Invitrogen, LC5952), lane 1 - LOS,

- 9 042022 extracted before deacylation, and lane 2 - deacylated LOS (EG-IM).

Example 1 Structural Analysis of an Immunomodulator (EG-IM).

The purified sample was appropriately diluted with purified water. A CU18 reverse phase column (Acquity BEH300 C18 1.7 µm, 2.1x150 mm) was installed on the instrument (UPLC, Waters) and then the sample was separated in a concentration gradient from 35 to 95% of mobile phase A (50 mM ammonium formate, pH 4, 5) and mobile phase B (100% acetonitrile). Analysis was carried out by MS and MS/MS on a mass spectrometer (Velos Pro, Thermo). Analyzed molecules with molecular weights from 100 to 3000 wt/charge. After MS analysis, identified molecules with a molecular weight of 50 to 2000 w/charge were analyzed again. A molecule with a molecular weight of 2372 w/charge was identified as the main ingredient, and in FIG. 2 is a schematic representation of the structure obtained from the analysis of each peak.

Preparation 2 Preparation of liposomes containing EG-IM (liposomal EG-IM).

1. Preparation of EG-IM solution and MPL solution.

(1) Preparation of a 0.3% (v/v) solution of triethylamine containing EG-IM A 0.3% (v/v) solution of triethylamine containing 1 mg/ml of EG-IM was prepared and stored at - 20°C.

(2) Preparation of 10% (w/v) sucrose solution containing EG-IM A 10% (w/v) sucrose solution containing 1 mg/mL of EG-IM was prepared.

(3) Preparation of a 0.3% (v/v) triethylamine solution containing MPL.

To a vial containing 5 mg MPL was added 5 ml of 0.3% (v/v) triethylamine, vigorously mixed with a vibrator and sonicated for 10 min to obtain a homogeneous solution of MPL. The resulting solution in 1 mg/ml was added in portions into silicone tubes and stored at -20°C.

2. Obtaining liposomal EG-IM.

(1) Dissolution of lipids and preparation of samples by mixing with glycolipid.

A 1 mg/ml solution of DMPC or DOTAP was prepared using tert-butyl alcohol as a solvent and then mixed with a vibratory mixer. 1 ml of the prepared DMPC solution and 1 ml of the DOTAP solution were filled into vials to form a mixed lipid solution in which DMPC and DOTAP were mixed in a 1:1 (v/v) ratio.

The mixed lipid solution was mixed with 0.3% (v/v) triethylamine containing EG-IM to give mixed lipid solutions containing 6 mol%, 3 mol%, or 1 mol% EG-IM, as well as mixed the lipid solution was mixed with 0.3% (v/v) triethylamine containing MPL to give mixed lipid solutions containing 6 mol%, 3 mol% or 1 mol% MPL. Then, the vials containing mixed lipid solutions were first frozen in a -70°C freezer for 1 hour, and then lyophilized for 12 hours or more to obtain lipid mixture lozenges.

(2) Obtaining liposomes through the process of rehydration.

In the plain lipid mixture vials and the lipid mixture vials containing EG-IM or MPL, 1 ml of a 10% (w/v) sucrose solution was added in the presence of a lipid mixture pellet, and then mixed with a vibrator to obtain liposomes.

(3) Preparation of samples by simply mixing glycolipids with liposomes 10% (w/v) sucrose solutions containing EG-IM were mixed with a solution of liposomes at an appropriate dosage, obtaining samples containing simple mixtures of liposomes with 6 mol%, 3 mol.% or 1 mol.% EG-IM.

0.3% (v/v) triethylamine solutions containing MPL were mixed with a solution of liposomes, obtaining samples containing simple mixtures of liposomes with 6 mol.%, 3 mol.% or 1 mol.% MPL.

Example 2 Physical Properties Analysis of Liposomal EG-IM.

1. Analysis of the sizes of liposomes.

(1) Analysis of the degree of change in the size of liposomes.

1 ml of the sample with lipid dissolution or 1 ml of the sample obtained by simply mixing the glycolipid from Preparation 2-(3) with liposomes was added to the cuvettes, and the hydrodynamic dimensions were measured using a dynamic light scattering apparatus (ZSP Nano, Malvern Instruments) (Table .2).

table 2

Liposomes size (d, nm) Type of glycolipid Method of Incorporating Glycolipid Glycolipid content 6 mol.% 3 mol.% 1 mol% MPL lipid dissolution 355.4±7.1 309.2±3.7 367.9 ± 16.1 easy mixing with liposomes 1538.7±457.1 1206.1±244.2 694.9±33.2 EG-IM lipid dissolution 439.3±6.1 386.1 ± 1.2 372.7 ± 6.0 easy mixing with liposomes 829.8 ± 11.1 414.0±43.7 565.7±28.9

As a result, it has been observed that MPL causes dimensional expansion when simply mixed with

- 10 042022 liposomes. On the other hand, EG-IM increased the dimensions slightly, but they were less than 1 µm when produced by a simple mixing method at a high content, and the content and method of production did not affect the size change. Thus, liposomal EG-IM (containing EG-IM liposomes) can be obtained in various forms such as encapsulation or adsorption forms due to a small degree of size changes after production, and can also be obtained in the nano range from 300 to 600 nm (<1 μm) .

(2) Liposome size uniformity analysis.

1 ml of the sample with lipid dissolution or 1 ml of the sample obtained by simply mixing the glycolipid from Preparation 2-(3) with liposomes was added to the cuvettes, and the hydrodynamic dimensions and their distribution were analyzed on a dynamic light scattering apparatus (ZSP Nano, Malvern Instruments ) (Table 3, figs. 3 and 4). In FIG. 3 shows liposome size distribution diagrams in the lipid dissolution method, and FIG. 4 shows liposome size distribution diagrams in the simple mixing glycolipid-liposome method.

Table 3

PDI liposomes Type of glycolipid Method of Incorporating Glycolipid Glycolipid content 6 mol.% 3 mol.% 1 mol% MPL lipid dissolution 0.405 ± 0.080 0.412±0.030 0.353±0.011 easy mixing with liposomes 0.887±0.098 0.793 ± 0.052 0.552±0.020 EG-IM lipid dissolution 0.271±0.209 0.405±0.019 0.407 ± 0.020 easy mixing with liposomes 0.227±0.031 0.402±0.133 0.471±0.016

As a result, it turned out that the dispersion of MPL increases depending on the concentration. On the other hand, the dispersion of EG-IM remained the same regardless of content.

By incorporating the glycolipid in a lipid dissolving manner, liposomes are formed in which the glycolipid is encapsulated (FIG. 3). It turned out that in the case of MPL, the size distribution of the obtained liposomes was non-uniform and micelles of the MPL itself were formed (Fig. 3A, 3C and 3E). On the other hand, in the case of EG-IM, the liposome preparations appeared to be homogeneous, forming a single peak (FIGS. 3B, 3D and 3F). Thus, it appeared that EG-IM could be uniformly encapsulated in liposomes.

By simply mixing liposomes with glycolipid, liposomes are formed in which the glycolipid is adsorbed on the surface of the liposomes (Fig. 4). In the case of MPL, it turned out that the size distribution of the obtained liposomes was non-uniform (Table 3) and micelles of the MPL itself were formed (Fig. 4A, 4C and 4E). On the other hand, in the case of EG-IM, the liposome preparations appeared to be homogeneous, forming a single peak (FIGS. 4B, 4D and 4F). Thus, it turned out that EG-IM can also be uniformly adsorbed on liposomes.

2. Analysis of the surface charge of liposomes.

1 ml of the sample with lipid dissolution or 1 ml of the sample obtained by simply mixing the glycolipid from Preparative Example 2-(3) with liposomes was added to the cuvettes for measuring the surface charge ((ζ-potential), and the surface charge was measured using a dynamic scattering apparatus. light (ZSP Nano, Malvern Instruments) (Table 4).

Table 4

Surface charge of liposomes (mV) Type of glycolipid Method of Incorporating Glycolipid Glycolipid content 6 mol.% 3 mol.% 1 mol% MPL lipid dissolution 23.7±0.6 56.2±0.6 57.0±3.0 easy mixing with liposomes 48.5 ± 1.4 56.9±3.4 57.8 ± 1.6 EG-IM lipid dissolution 54.7 ± 1.3 59.2 ± 1.0 55.2±2.7 easy mixing with liposomes 57.5 ± 1.3 56.5 ± 2.3 55.1±2.6

As a result, in the case of EG-IM, the surface charge remained the same at 55 mV, regardless of content. On the other hand, in the case of MPL, the surface charge was drastically reduced when mixed at a high content of 6 mol%.

Example 3 Analysis of the Mechanism of Liposomal EG-IM.

1. Effect of liposomal EG-IM on immune cell proliferation and reduction of cytotoxicity (1) Effect of liposomal EG-IM on immune cell proliferation.

Mouse macrophage cell line (J774A.1) cells were treated with EG-IM (0.025, 0.05, 0.1, 0.5, or 1.0 µg/mL) and various concentrations of liposomes (1.25, 2.5, 5 , 25 or 50 µg/mL cationic lipids)

- 11 042022 or a mixture thereof. 24 hours after the cells were treated with the test reagent, the cells were treated with a CCK-8 solution and left to react for 1 hour. After the completion of the reaction, the cultured cells were collected, the absorption at 450 nm was measured, and the cell viability in each experimental group was calculated, taking as 100% the absorption in the group treated with culture solution (negative control group).

The NP group received liposome treatment only, the NP + EG-IM group received treatment with a simple mixture of EG-IM and liquid liposomes, and the NP/EG-IM group received treatment with a preparation obtained by lyophilization of a mixture of liposomes and EG-IM.

NP-A is DDA:DOPC liposomes, NP-B is DOTAP:DMPC liposomes, NP-C is DOTAP:DMPC:squalene liposomes, and NP-D is DOTAP:DMPC:tricaprin liposomes.

As a result, it turned out that when EG-IM was mixed with various concentrations of cationic liposomes, the growth of immune cells was enhanced (Fig. 5).

(2) Effect of liposomal EG-IM on TNF-α secretion.

Mouse macrophage cell line (J774A.1) cells were treated with EG-IM (0.025, 0.05, 0.1, 0.5, or 1.0 µg/mL) and various concentrations of liposomes (1.25, 2.5, 5 , 25 or 50 µg/ml of cationic lipids) or a mixture thereof. The cells were treated with the test reagent and cultured for 24 h, and then the level of secretion of the cytokine TNF-α in the cell culture medium was measured by sandwich ELISA.

The NP group received treatment with liposomes alone, the NP + EG-IM group received treatment with a simple mixture of EG-IM and liquid liposomes, and the NP/EG-IM group received treatment with a preparation obtained by lyophilization of a mixture of liposomes and EG-IM.

NP-A is DDA:DOPC liposomes, NP-B is DOTAP:DMPC liposomes, NP-C is DOTAP:DMPC:squalene liposomes, and NP-D is DOTAP:DMPC:tricaprin liposomes.

As a result, it turned out that when EG-IM was mixed with different concentrations of cationic liposomes, active secretion of TNF-α was induced (Fig. 6).

(3) Effect of liposomal EG-IM on attenuation of cytotoxicity.

It is well known that cationic liposomes cause cytotoxicity. Whether the resulting liposomal EG-IM would attenuate the cytotoxicity of cationic liposomes by EG-IM was determined. Cytotoxicity of liposomal EG-IM was measured in human fibroblast MRC-5 cells. The cytotoxicity of the obtained liposomes and cationic liposomes composed of DDA:DMPC was determined depending on the presence or absence of cholesterol and the homogenization method, as well as the presence or absence of the immunostimulatory substance, EG-IM. MRC-5 cells were stimulated by treatment with each substance at various concentrations, and cytotoxicity was assessed by the MTT assay.

A is cholesterol-free liposomes, B is cholesterol-containing liposomes, group 1 with extruder homogenized liposomes, and group 2 with sonicated liposomes.

As a result, it appeared that cytotoxicity, which was manifested in a DDA:DMPC concentration-dependent manner, was attenuated by the mixture with EG-IM (FIG. 7).

2. The mechanism of formation of a vaccine containing liposomal EG-IM: adsorption due to electrostatic attraction.

Liposomes (LP), VZV recombinant gE protein and EG-IM were mixed and centrifuged (4°C, 100,000xg, 1 h) to determine if the mechanism of vaccine formation with liposomal EG-IM adsorption was due to electrostatic attraction. After separation of the supernatant (S) and precipitate (P), the precipitate was completely suspended by sonication in the same volume of PBS as the supernatant. Samples with the same volume were subjected to electrophoresis and then stained with silver.

As a result, when only the VZV recombinant gE protein was subjected to ultracentrifugation, the recombinant gE protein was detected in the supernatant. However, in the case of the liposomal EG-IM vaccine (LP:gE:EG-IM mixture), the recombinant gE protein was detected in the pellet (FIG. 8). We believe that the recombinant gE protein is found in the sediment because the recombinant gE protein is adsorbed on liposomes.

3. Immunological effects of vaccines containing liposomal EG-IM.

(1) Immune cell recruiting effect of liposomal EG-IM.

Liposomal EG-IM was administered intramuscularly to 6-week-old female BALB/c mice and cells were isolated from muscle tissue at the injection site 3 and 24 hours after administration. To characterize immune cells among isolated cells, cells were immunostained with Ab against CD11b (FITC), Ab against CD11c (FITC), Ab against Ly6C (PE), Ab against Ly6G (PE), Ab against F4/80 (PE), Ab against MHCII (PE) and Ab against CD3 (PE) and analyzed by flow cytometry.

As a result, it turned out that the migration of immune cells CD11b ⁺ (monocytes, granulocytes), DCs, monocytes, macrophages and granulocytes to the injection sites was enhanced by liposomal EG-IM (Fig. 9). This means that liposomal EG-IM initiates immune cell recruitment, that liposomal EG-IM can induce innate immunity, and that liposomal EG-IM uptake can be increased.

- 12 042022

EG-IM immune cells.

(2) Capture effect in vaccines containing liposomal EG-IM.

The gE protein was labeled with a fluorescent dye (A1exa647), dendritic cells (DC) were treated with this gE protein, and those DCs in which fluorescence was detected were analyzed by flow cytometry.

As a result, fluorescent DCs were not observed when gE protein or EG-IM were used alone, but fluorescent DCs appeared when liposomal EG-IM was used as an adjuvant system (FIG. 10). This means that the vaccine containing liposomal EG-IM increases the efficiency of antigen uptake by DC cells.

In addition, recombinant VZV gE protein was labeled with a fluorescent dye (Alexa647) and then mixed with liposomal EG-IM to prepare a vaccine. The resulting vaccine was administered to mice in the deltoid muscle of the thigh, and 4 hours and 24 hours after administration, staining with a fluorescent marker was performed on the surface of immune cells in the draining lymph nodes (dLN) and muscle tissue, and then analyzed by flow cytometry. The results of the analysis are shown in Fig. 11. In FIG. 11A shows the results of analysis of fluorescently labeled gE protein-specific cells in dLN, FIG. 11B shows results of analysis of fluorescently labeled gE protein-specific cells in muscle tissue, FIG. 11C shows results of measurement of fluorescently labeled gE protein-specific monocytes in dLN, and FIG. 11D are measurements of fluorescently labeled gE protein-specific monocytes in muscle tissue.

As a result, when liposomal EG-IM was used as an adjuvant system, cells were recruited to dLN and muscle tissue and vaccine uptake by monocytes was actively induced compared to when recombinant VZV gE protein or EG-IM was used alone (Fig. 11).

(3) Depot effect of a vaccine containing liposomal EG-IM.

The gE protein was labeled with a fluorescent dye (Alexa647) and mixed with liposomal EG-IM to prepare a vaccine. At 30 min and 24 h after administration of the resulting vaccine to the deltoid muscle of the thigh, in vivo fluorescence images were taken (A) and the fluorescence was measured in the lymph nodes isolated from the mice (B).

As a result, fluorescence was detected at the injection site up to 24 hours, both when the gE protein (antigen) was administered alone and when the vaccine containing liposomal EG-IM (VAC) was administered. Fluorescence analysis was performed for each lymph node. As a result, fluorescence was detected with relatively high intensity in the lumbar and popliteal nodes. With the introduction of the antigen alone, the fluorescence intensity decreased by half after 24 hours, but with the administration of the vaccine containing liposomal EG-IM, the fluorescence intensity did not decrease (Fig. 12). This means that the vaccine containing liposomal EG-IM enhances the depot effect.

Example 4 Immunogenicity Assay of Liposomal EG-IM in Zika Vaccine.

Recombinant Zika virus envelope protein was used to identify the immunogenicity enhancing effect of liposomal EG-IM in Zika vaccine. As various immunoadjuvants, 6-week-old BALB/c mice (SLC, Japan) were intramuscularly injected twice with an interval of two weeks with a mixture of recombinant Zika virus envelope glycoprotein and alum (aluminum hydroxide; Brenntag, Germany), EG-IM, or liposomal EG-IM . Regarding the amount administered, recombinant Zika virus envelope glycoprotein was administered at 2 μg/mouse, alum was administered at 25 μg/mouse, EG-IM was administered at 0.5 μg/mouse, and liposomal EG-IM was administered at 25 μg/mouse. Two weeks after the last injection, the mice were anesthetized, the spleen tissues were removed and divided into individual cells. The isolated spleen cells were stimulated with 5 μg/ml antigen and cultured for 72 hours. The level of secreted cytokine IFN-γ in the cell culture medium was then analyzed using an ELISA kit (R&D Systems, Millipore).

As a result, IFN-γ secretion was increased in the liposomal EG-IM experimental group compared to the alum alone or liposomes alone experimental group (FIG. 13). Thus, the Zika vaccine containing liposomal EG-IM appeared to have an excellent ability to induce cellular immunity.

Example 5 Immunogenicity Assay of Liposomal EG-IM in a Japanese Encephalitis Virus Vaccine.

Japanese encephalitis virus (JEV) antigen was used to identify the immunogenicity enhancing effect of liposomal EG-IM in Japanese encephalitis virus (JEV) vaccine formulation. As immunoadjuvants, 6-week-old BALB/c mice (SLC, Japan) were intramuscularly injected twice with an interval of two weeks with a mixture of inactivated JEV antigen and EG-IM or liposomal EG-IM. Regarding the amount administered, inactivated JEV antigen was administered at 0.5 μg/mouse, EG-IM was administered at 0.5 μg/mouse, and liposomal EG-IM at 25 μg/mouse.

1. Measurement of the titer of antibodies specific to the JEV antigen.

4 weeks after the last injection, mice were anesthetized and cardiac blood was taken from them.

- 13 042022 for the preparation of blood samples. To determine the titer of antibodies specific to the JEV antigen in serum after immunization, the method of enzyme-linked immunosorbent assay was used to the end point of dilution. JEV antigen was diluted to a concentration of 1 μg/ml, fixed in a 96-well plate at 100 μl/well (4°C, overnight) and blocked with 300 μl of 1% BSA (bovine serum albumin) (room temperature, 1 h). After blocking, it was washed three times with PBS containing 0.05% Tween-20, and the serum obtained after immunization was diluted in 10-fold serial dilutions and 100 μl of each reaction serum dilution was added (37°C, 2 h). Horseradish peroxidase-conjugated anti-mouse IgG (Jackson, 115-035-003), horseradish peroxidase-conjugated anti-mouse IgG1 (Serotec, STAR132P), and horseradish peroxidase-conjugated anti-mouse IgG2a (Serotec , STAR133P) and run the reaction, then add TMB (tetramethylbenzidine, BD BioScience, #55555214) and stop the reaction with 1N H ₂ SO ₄ . The determination results were obtained by measuring the absorbance at 450 nm and calculating the titers of antibodies of the IgG, IgG1 and IgG2a types in the blood after immunization.

As a result, the liposomal EG-IM treatment group had elevated levels of JEV antigen-specific IgG type, IgG1 type, and IgG2a type antibodies compared to the EG-IM alone or liposome-only treatment groups (FIG. 14A). Thus, the Japanese encephalitis vaccine of the present invention containing liposomal EG-IM exhibits excellent immune action, that is, effectiveness as a vaccine.

2. Analysis of cytokines.

14 weeks after the last injection, mice were anesthetized, spleen tissues were removed and separated into individual cells, and then these cells were stimulated with 1 μg/ml of recombinant JEV glycoprotein. After 24 hours, cells secreting the cytokine IFN-γ were analyzed by ELISpot ELISA (Millipore).

As a result, in the experimental group with liposomal EG-IM, the number of cells secreting the cytokine IFN-γ increased compared to the experimental group with only liposomes (Fig. 14B). Thus, the Japanese encephalitis vaccine containing liposomal EG-IM has an excellent ability to induce cellular immunity.

Example 6 Immunogenicity Assay of Liposomal EG-IM in a Tuberculosis Vaccine.

The recombinant tuberculosis antigens Ag85A, ESAT-6, or HspX alone or a mixture of these three antigens were used to identify the immunogenicity enhancement effect of liposomal EG-IM in the tuberculosis (TB) vaccine formulation. As immunoadjuvants, 6-week-old BALB/c mice (SLC, Japan) were injected intramuscularly with a mixture of recombinant tuberculosis antigens with EG-IM or with liposomal EG-IM three times with an interval of two weeks. Regarding the amount administered, recombinant tuberculosis antigens were administered at 5 μg/mouse, EG-IM was administered at 0.5 μg/mouse, and liposomal EG-IM at 250 μg/mouse.

1. Measurement of the titer of antibodies specific to TB antigens.

3 weeks after the last injection, mice were anesthetized and cardiac blood was taken from them for the preparation of blood samples. To determine the titer of antibodies specific to TB antigens in serum after immunization, the method of enzyme immunoassay was used to the end point of dilution. The results of the determination were obtained by measuring the absorbance at 450 nm and calculating the titers of antibodies of the IgG type in the blood after immunization.

As a result, when using Ag85A, ESAT-6, or HspX alone or as a mixture of these three antigens, levels of IgG antibodies specific to recombinant TB antigens were increased in the experimental group with liposomal EG-IM compared with the experimental group with EGIM alone (Fig. 15A). Thus, the tuberculosis vaccine of the present invention containing liposomal EG-IM exhibits an excellent immune effect, that is, efficacy as a vaccine.

2. Analysis of cytokines.

3 weeks after the last injection, mice were anesthetized, spleen tissues were removed and separated into individual cells, and then these cells were stimulated with 1 μg/ml of recombinant TB gE protein and cultured for 24 hours. Cells secreting the cytokine IFN-γ were then analyzed ELISpot ELISA method (Millipore).

As a result, in all cases where Ag85A, ESAT-6, or HspX antigen was used, the liposomal EG-IM treatment group had an increased number of cells secreting the cytokine IFN-γ compared to the EG-IM alone treatment group (Fig. 15B) . Thus, the tuberculosis vaccine of the present invention containing liposomal EG-IM exhibits the ability to induce cellular immunity.

3. Analysis of bactericidal action.

Spleen cells from immunized mice were co-cultured with BCG-infected macrophages. After 7 days of culture, cells were isolated, lysed and plated on Bacto Middlebrook 7H11 agar plates (Difco) containing Middlebrook OADC enrichment (Difco). After cultivation at 37°C for 4 weeks, the number of bacterial colonies formed was counted.

- 14 042022

As a result, the liposomal EG-IM test group showed an excellent bactericidal effect on BCG compared to the EG-IM alone test group (FIG. 15C).

Example 7 Immunogenicity Assay of Liposomal EG-IM in Pertussis Vaccine.

Cell-free pertussis vaccine antigen (aP) was used to identify the immunogenicity enhancing effect of liposomal EG-IM in the pertussis vaccine formulation. As immunoadjuvants, 6-week-old BALB/c mice (SLC, Japan) were intramuscularly injected twice with an interval of two weeks with a mixture of cell-free pertussis vaccine antigen (aP) with EG-IM or with liposomal EG-IM. Regarding the amount administered, the aP antigen was administered at 5 μg/mouse, liposomes were administered at 25 μg/mouse, EG-IM at 0.5 μg/mouse, and liposomal EG-IM at 25 μg/mouse.

1. Measurement of the titer of antibodies specific to the aP antigen.

2 weeks after the last injection, mice were anesthetized and cardiac blood was taken from them for blood sample preparation. To determine the titer of antibodies specific to the aP antigen in serum after immunization, the method of enzyme immunoassay was used to the end point of dilution. The determination results were obtained by measuring the absorbance at 450 nm and calculating the titer of IgG type antibodies in the blood after immunization.

As a result, the liposomal EG-IM treatment group had increased production of aP antigen-specific IgG antibodies compared to the EG-IM alone or liposome-only treatment groups (FIG. 16A). Thus, the pertussis vaccine of the present invention containing liposomal EG-IM exhibits excellent immune action, that is, effectiveness as a vaccine.

2. Analysis of the proliferation of CD4+ T cells.

2 weeks after the last injection, mice were anesthetized and spleen tissues were removed, stimulated with 5 μg of aP antigen and cultured for 0, 4 and 8 days. The proliferation of CD4+ T cells was then determined by flow cytometry.

As a result, the liposomal EG-IM treatment group showed the highest proliferation efficiency of CD4+ T cells compared to the EG-IM alone or liposome-only treatment groups (FIG. 16B).

3. Analysis of cytokines.

2 weeks after the last injection, mice were anesthetized, spleen and lung tissues were removed and separated into individual cells, and then these cells were stimulated with 5 μg/ml aP antigen and cultured for 24 hours. Cells secreting the cytokine IFN-γ were then analyzed. ELISpot ELISA method (Millipore). Light bars represent spleen cell results and dark bars represent lung cell results. In addition, spleen cells were stimulated with 5 μg/ml aP antigen and cultured for 72 hours, and then IFN-γ cytokine secretion was analyzed by sandwich ELISA (R&D Systems).

As a result, the number of IFN-γ secreting cells increased in the liposomal EG-IM treatment group compared to the EG-IM alone or liposomes alone treatment group (FIG. 17A). In addition, the liposomal EG-IM test group showed superior secretion of the cytokines IFN-γ and IL-17 (FIGS. 17B and 17C). In addition, the pertussis vaccine of the present invention containing liposomal EG-IM exhibits an excellent ability to induce cellular immunity.

Example 8 Immunogenicity Assay of Liposomal EG-IM in Varicella Vaccine.

Recombinant varicella zoster virus (VZV) gE antigen was used to identify the immunogenicity enhancing effect of liposomal EG-IM in the varicella zoster virus vaccine. 6-week-old C57BL/6 mice (SLC, Japan) were injected subcutaneously with attenuated VZV to obtain a priming model. Four weeks later, 6-week-old BALB/c mice (SLC, Japan) were intramuscularly injected twice with a two-week interval with a varicella-zoster virus (VZV) vaccine obtained by mixing recombinant VZV gE antigen with alum, EG-IM, or liposomal EGIM as immunoadjuvants. In terms of administration amount, recombinant VZV gE antigen was administered at 5 μg/mouse, EG-IM was administered at 2 or 3 μg/mouse, and liposomal EG-IM at 50 μg/mouse. As a control group, the commercially available Zostavax vaccine (Merck, USA) was diluted to 1/10 of the human dose and administered once to mice. In addition, a comparison was made under conditions with a change in the composition of liposomes, which are an ingredient of liposomal EG-IM, to the ratio of DOTAP:DMPC=1:1 or DODAP:DMPC=1:2.

1. Analysis of cytokines.

4 weeks after the last injection, mice were anesthetized, spleen tissues were removed and separated into individual cells, and then these cells were stimulated with 1000 PFU/ml Zostavax (attenuated virus) or 4 μg/ml gE peptide mixture and cultured for 72 h. Then analyzed the secretion of the cytokine IFN-γ in cell culture using an ELISA kit (R&D Systems). Spleen cells were also stimulated separately with 1000 PFU/ml Zostavax (attenuated virus) or 4 μg/ml gE peptide mixture. After 24 hours, cells secreting the cytokine IFN

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Claims (15)

γ by ELISpot ELISA method (Millipore). As a result, IFN-γ secretion was increased in the liposomal EG-IM test group compared to the EG-IM alone test group and also compared to Zostavax (Merck, USA), a commercially available vaccine (FIG. 18). Thus, the varicella-zoster vaccine of the present invention containing liposomal EG-IM exhibits an excellent ability to induce cellular immunity. 2. Analysis of the synergistic effect when added to the composition of lipids. Cholesterol (chol), squalene, or tricaprin was further added to the formulation of liposomal EG-IM as lipid ingredients in order to ascertain whether a synergistic effect would occur. Each of the vaccines was administered intramuscularly twice with an interval of 2 weeks, and 2 or 4 weeks after the last injection, mice were anesthetized and spleen tissues were removed, stimulated with 10 μg/ml recombinant gE VZV and cultured for 72 hours. Then, the secretion of the cytokine IFN was analyzed -γ in cell culture using an ELISA kit (R&D Systems). Also, spleen cells were separately stimulated with 4 μg/ml gE peptide mixture and cultured for 24 hours. Cells secreting the cytokine IFN-γ were then analyzed using an ELISpot kit (Millipore). As a result, IFN-γ secretion was increased with liposomal EG-IM supplemented with cholesterol (FIG. 19), squalene (FIG. 20), or tricaprin (FIG. 21). Thus, it appeared that a vaccine additionally containing cholesterol, squalene or tricaprin as additional lipid ingredients in liposomal EG-IM showed a better ability to induce cellular immunity compared to when only liposomal EG-IM was used. 3. Analysis of the synergistic effect when adding an immunoadjuvant. It was tested whether there would be a synergistic effect with the further addition of Quil A, a saponin-based substance, as an additional adjuvant to liposomal EG-IM. Each vaccine was administered intramuscularly twice with an interval of 2 weeks. 4 weeks after the last injection, the mice were anesthetized, the spleen tissues were removed and divided into individual cells. Cells were stimulated with 4 μg/ml VZV gE recombinant peptide mixture or 1000 PFU/ml Zostavax and cultured for 24 hours. Cells secreting IFN-γ cytokine were then analyzed using an ELISpot kit (Millipore). As a result, IFN-γ secretion was increased with liposomal EG-IM supplemented with a saponin derivative, substance Quil A (FIG. 22). Thus, it appeared that a vaccine additionally containing a saponin-derived substance as an additional immunity enhancer along with liposomal EG-IM showed a better ability to induce cellular immunity compared to when EG-IM alone was used. 4. Analysis of the synergistic effect depending on the composition. In order to determine the differences in immunogenicity depending on the final formula of the varicella-zoster virus (VZV) vaccine, experiments were carried out on a simple mixed vaccine (+) and a mixed and lyophilized vaccine (/). Each vaccine composition was administered intramuscularly to 7-week-old BALB/c mice (SLC, Japan) twice with an interval of two weeks. 2 weeks after the last injection, the mice were anesthetized, the spleen tissues were removed and divided into individual cells. Cells were stimulated with 5 μg/ml of VZV recombinant gE antigen and cultured for 72 hours. The secretion levels of cytokines IL-5 and IFN-γ in the cell culture medium were then analyzed by sandwich ELISA (R&D Systems; DY485, DY405). In FIG. 23 NP-A means DOTAP/DOPC, NP-B means DOTAP/DMPC, NP-C DOTAP/DMPC/squalene, NP-D means DOTAP/DMPC/tricaprin, + means simple mixed form, and / means mixed and lyophilized form. As a result, the form obtained by lyophilization of liposomal EG-IM with antigen enhanced the secretion of IFN-γ to the greatest extent, but almost did not increase the secretion of IL-5 (Fig. 23). Thus, it turned out that the form obtained by lyophilization of liposomal EG-IM with antigen exhibits an excellent ability to induce Th1-type cellular immunity. Industrial Applicability The present invention overcomes the physicochemical instability of liposomes and therefore provides advantages in terms of production, delivery and storage, and is advantageous as an immune delivery system due to improved stability. In addition, the present invention contains the entire antigen, the immunomodulator and the cationic liposome, so that the immune enhancing effect is better than when only the immunomodulator is used. CLAIM 1. An immune enhancing composition comprising (a) an immunomodulator represented by the following formula 1: - 16 042022 where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind; and (b) a cationic liposome. 2. Composition according to claim 1, wherein the phosphate binds at positions selected from the group consisting of AB, AC, AD, AE, AF, BC, BD, BE, BF, CD, CE, CF, DE, DF, EF , ABC, ABD, ABE, ABF, ACD, ACE, ACF, ADE, ADF, AEF, BCD, ALL, BCF, BDE, BDF, BEF, CDE, CDF, CEF, DEF, ABCD, ABCE, ABCF, ABDE, ABDF , ABEF, ACDE, ACDF, ADEF, BCDE, BCDF, BCEF, BDEF, CDEF, ABCDE, ABCEF and ABCDEF in formula 1. 3. Composition according to claim 1, wherein the cationic liposome is a cationic lipid selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), 1,2-dioleoyl-3trimethylammonium propane (DOTAP), 3b-[Na(N,N-dimethylaminoethane) carbamoyl]cholesterol (DC-Chol), 1,2dioleoyloxy-3-dimethylammonium propane (DODAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 14:1), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 16:0-18:1), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (ethyl -RS 18:1), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 18:0), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 16:0) , 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 14:0), 1,2dilauroyl-sn-glycero-3-ethylphosphocholine (ethyl-RS 12:0), N1-[2-(( 1 S)-1-[(3-aminopropyl)amino]-4-[di(3aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]benzamide (MVL5), 1,2-dimyristoyl-3dimethylammonium- propane (DAP 14:0), 1,2-dipalmitoyl-3- dimethylammonium-propane (DAP 16:0), 1,2distearoyl-3-dimethylammonium-propane (DAP 18:0), N-(4)-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane -1-aminium (DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (TAP 18:0), 1,2-dipalmitoyl-3-trimethylammonium-propane (TAP 16:0), 1,2-dimyristoyl-3- trimethylammonium-propane (TAP 14:0) and N4-cholesteryl-spermine (GL67). 4. The composition of claim 3, wherein the cationic liposome further comprises a neutral lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2dilinoleyl-sn-glycero-3-phosphocholine (DLPC), phosphoethanolamine (PE), and phosphatidylcholine (PC). 5. Vaccine composition comprising (a) an antigen; (b) an immune enhancing composition according to claim 1 as an active ingredient. 6. The vaccine composition of claim 5, wherein the antigen is selected from the group consisting of a peptide, protein, nucleic acid, sugar, pathogen, attenuated pathogen, inactivated pathogen, virus, virus-like particles (VLP), cells, or cell fragments. 7. The vaccine composition of claim 5, wherein the antigen is selected from the group consisting of Zika virus antigen, Japanese encephalitis virus antigen, Mycobacterium tuberculosis antigen, pertussis antigen, varicella zoster virus antigen, Haemophilus influenzae type B (HIB) antigen, virus antigen Middle East respiratory syndrome (MERS), Pseudomonas aeruginosa antigen, Anthrax antigen, Hepatitis A virus (HAV) antigen, Hepatitis B virus (HBV) antigen, Hepatitis C virus (HCV) antigen, Human immunodeficiency virus (HIV) antigen, Herpes virus antigen simplex (HSV), Neisseria meningitidis antigen, Corynebacterium diphtheria antigen, Bordetella pertussis antigen, Clostridium tetani antigen, human papillomavirus (HPV) antigen, Enterococcus antigen, Staphylococcus aureus antigen, Klebsiella pneumoniae antigen, Acinetobacter baumannii antigen, Enterobacter antigen, Helicobacter pylori antigen , malaria antigen, dengue virus antigen, Orientia tsutsugamushi antigen , bunyavirus severe fever with thrombocytopenia syndrome (SFTS) antigen, severe acute respiratory syndrome coronavirus (SARS-CoV) antigen, influenza virus antigen, Ebola virus antigen, and Diplococcus pneumoniae antigen. 8. The vaccine composition of claim 5, wherein the vaccine is an inactivated vaccine, an attenuated vaccine, a subunit vaccine, a conjugate vaccine, a recombinant vaccine, a monovalent vaccine, a polyvalent vaccine, or a mixed vaccine. 9. The vaccine composition according to claim 5, wherein the vaccine is selected from the group consisting of Zika virus vaccine, Japanese encephalitis vaccine, tuberculosis vaccine, pertussis vaccine, varicella-zoster virus (VZV) vaccine, Haemophilus influenzae type vaccine. IN, - 17 042022 MERS vaccines, Pseudomonas aeruginosa vaccines, cancer vaccines, anthrax vaccines, HAV vaccines, HBV vaccines, HCV vaccines, HIV vaccines, meningococcal vaccines, diphtheria vaccines, tetanus vaccines, multidrug-resistant bacteria, enterococcal vaccines, Staphylococcus aureus vaccines, Klebsiella pneumoniae vaccines, Acinetobacter baumannii vaccines, Enterobacter vaccines, Helicobacter pylori vaccines, malaria vaccines, dengue virus vaccines, Orientia tsutsugamushi vaccines, vaccines against severe fever bunyavirus with thrombocytopenia syndrome (SFTS), severe acute respiratory syndrome coronavirus (SARS-CoV) vaccine, Ebola virus vaccine, influenza virus vaccine, and Diplococcus pneumoniae vaccine. 10. The vaccine composition according to claim 5, wherein the vaccine is lyophilized. 11. The vaccine composition according to claim 9, wherein the varicella-zoster virus (VZV) vaccine is in a ready-to-use form. 12. The vaccine composition according to claim 5, wherein the vaccine additionally contains cholesterol, squalene or tricaprin. 13. The vaccine composition according to claim 5, wherein the vaccine further comprises saponin, Quil A or QS21 as a saponin derivative substance. 14. The method of obtaining the composition for enhancing immunity according to claim 1, including (a) preparing a solution containing an immunomodulator represented by the following formula 1 where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind; (b) dissolving lipids in an organic solvent to obtain a mixed lipid solution; (c) freeze-drying the mixed lipid solution from step (b) to remove the organic solvent; (d) rehydrating the substance obtained in step (c) and then mixing this substance with the solution from step (a), thereby obtaining an immune enhancing composition. 15. A method for producing a vaccine composition according to claim 5, including (a) preparing a solution containing an immunomodulator represented by the following formula 1 where Glc is glucose, GlcN is glucosamine, HEP is heptose, KDO is 2-keto-3-deoxyoctonate, GlcNAc is Nacetylglucosamine, and A-F are positions to which phosphate can bind; (b) dissolving lipids in an organic solvent to obtain a mixed lipid solution; (c) freeze-drying the mixed lipid solution from step (b) to remove the organic solvent; (d) rehydration of the substance obtained in step (c), thereby obtaining liposomes; (e) adding the solution from step (a) and the antigen to the liposomes from step (d), followed by re-lyophilization. -