KR20140060053A - Immunity reinforcement agent for influenza vaccine - Google Patents

Abstract

The present invention relates to an adjuvant composition for an influenza vaccine and a pharmaceutical composition of an influenza vaccine comprising the same. The deacylated non-toxic LOS and Alum used together as an adjuvant exhibits superior immunostimulation effects in an immune reaction to the influenza and has little toxicity, and therefore, has significantly excellent safety. Accordingly, the adjuvant composition and the pharmaceutical composition of the present invention can be valuably used as an influenza vaccine.

Description

[0001] Immunity reinforcement agent for influenza vaccine [

The present invention relates to an immunostimulant composition for influenza vaccine and an influenza vaccine pharmaceutical composition comprising the same.

The influenza that causes the flu in humans has been causing significant damage every year since the Spanish flu in 1919. In addition, avian influenza, which infects chickens, ducks and migratory birds, has been damaging human and poultry industries worldwide for several years. Influenza is easily developed. In 2009, a new strain of influenza occurred, affecting about 480,000 people worldwide, of whom 6,200 people died. In 2010, 79 people died from swine flu in India.

Influenza viruses are 0.1 nm diameter particles with epidermis and RNA inside. These influenza viruses are classified as A, B, and C, and it is known to cause A and B viruses in humans. Type B is weak and only one kind exists, but there are various kinds of A type according to the type of H antigen and N antigen on the virus surface. H1, H2, H3 and N1, N2 are the types of antigens that cause common human illness. The H and N antigens that appear in algae usually do not cause disease in humans, but if they mutate in the virus or exchange genes with the kinds of antigens that cause diseases in humans, they can easily turn into diseases that can cause illness in humans. This new influenza virus, which does not have immunity to humans, can cause a pandemic that sweeps the world.

By the term "adjuvant" is meant any substance that generally increases body fluids or cellular immune responses to an antigen. Immunostimulators are used to achieve two purposes. They 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. Impurities associated with these cultures of pathogenic microorganisms may act as immunity enhancers to improve the immune response. However, immunity caused by vaccines using homologous agents of pathogenic microorganisms or purified protein subunits as antigens is often poor. Therefore, the addition of some exogenous substances such as immunostimulants is necessary. In addition, synthetic and subunit vaccines are expensive to manufacture. The addition of immunostimulants makes it possible to use less antigen in order to stimulate a similar immune response, thereby reducing the production cost of the vaccine.

Alum used in 'Gardasil', a vaccine for human cervical cancer, which is one of immunity enhancer, is also used for diphtheria, tetanus and hepatitis B vaccine, which has been reported to increase antigen stability and induce cytokine release , The vaccine is not freeze-dried or frozen, is not effective against all antigens, and promotes only the humoral immune response.

To date, a number of immunomodulators such as metal oxides, inorganic chelates of salts, gelatin, various paraffin-type oils, synthetic resins, alginates, mucoids and polysaccharide compounds, kagenates and blood-derived materials (for example, fibrin clot) Enhancers have been proposed, but none have been ideally suited for all vaccines.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to develop immunity enhancers for influenza vaccine. As a result, it was confirmed that the immune response of the influenza vaccine can be greatly improved when the deacylated LPS lipid A derived from Escherichia coli is combined with deacylated non-toxic LOS (Lipooligosaccharide) and Alum in combination as an immunostimulating agent. Completed.

It is therefore an object of the present invention to provide an immunostimulant composition for an influenza vaccine.

It is another object of the present invention to provide an influenza vaccine pharmaceutical composition.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

In accordance with one aspect of the present invention, the present invention provides a method of preparing a liposomal composition comprising: (i) Lipooligosaccharide Lipooligosaccharide (LPS) by deacylation of Lipopolysaccharide (LPS) lipid A isolated from E. coli; And (ii) an aluminum hydroxide as an active ingredient.

The present inventors have made efforts to develop immunity enhancers for influenza vaccine. As a result, it was confirmed that the immune response of the influenza vaccine can be greatly improved when the deacylated LPS lipid A derived from Escherichia coli is combined with deacylated non-toxic LOS (Lipooligosaccharide) and Alum in combination as an immunostimulating agent. Completed.

The term "immunopotentiator" as used herein generally refers to any substance that increases body fluids or cellular immune response to an antigen.

The greatest feature of the immunostimulant composition of the present invention is that a combination of deacylated non-toxic LOS (Lipooligosaccharide) and aluminum hydroxide is used together as an active ingredient of the immunostimulating agent.

As used herein, the term "LOS" is a modification of LPS (lipopolysaccharide), meaning it has a shorter molecular weight than a natural occurring LPS. The pre-deacylated LOS preferably has a molecular weight of 5,000-10,000 Da. The term "deacylated LOS" means that in this LOS the fatty acids bound to the glucosamine of lipid A by the -C (O) O- bond are removed and the toxicity is greatly reduced compared to LPS. Fatty acids in lipid A glucosamine are linked via -C (O) O- and -C (O) NH- bonds. The deacylated LOS of the present invention shows that the fatty acid bound by -C (O) O- bond is removed by deacylation of lipid A.

The deacylated non-toxic LOS can be prepared by various methods and can be prepared according to the methods disclosed in Korean Patent Registration No. 0456681, WO 2004/039413, Korean Patent Registration No. 0740237 and WO 2006/121232. For example, LPS can be deoxidized by treatment with a strong base (e.g., 2 N NaOH) to deacylate and remove some fatty acids from lipid A.

According to a preferred embodiment of the present invention, the deacylated non-toxic LOS used as an immunostimulating agent in the present invention is unoxidized by alkali treatment of LPS and deacylation. Preferred examples of the alkali are NaOH, KOH, Ba (OH) 2, CsOH, Sr (OH) 2, Ca (OH) 2, LiOH, the RbOH comprising a Mg (OH) _2, more preferably NaOH, KOH (OH) ₂ , Ca (OH) ₂ , LiOH and Mg (OH) ₂ , more preferably NaOH, KOH and Mg (OH) ₂ , and most preferably NaOH.

The degree of toxicity of LPS can be assayed through a variety of methods known in the art. For example, toxicity can be analyzed by measuring the amount of TNF-alpha (tumor necrosis factor-alpha) secreted in LPS treated THP-1 (Acute monocytic leukemia). The dedifferentating non-toxic LOS of the present invention induces a relatively small amount of TNF-a secretion compared to conventional LPS.

Another feature of the deacylated non-toxic LOS of the present invention is that the molecular weight is small compared to the native LPS. Preferably, the de-acylating non-toxic LOS used in the present invention is 1,500-10,000 Da, more preferably 2,000-5,000 Da, even more preferably 2,000-4,000 Da, even more preferably 3,000-4,000 Da, Most preferably from 3,200 to 3,7000 Da. Such a molecular weight can be measured using a conventional method in the art, for example, MALDI-MASS.

According to a preferred embodiment of the present invention, the Escherichia coli is E. coli EG0021 (KCCM 10374). That is, the deacylated non-toxic LOS of the present invention is due to the deacylation of LPS lipid A isolated from E. coli EG0021 (KCCM 10374).

In the present invention, the aluminum hydroxide is used interchangeably with Alum.

According to a preferred embodiment of the present invention, the influenza virus includes influenza viruses capable of infecting mammals or birds, for example, birds, people, dogs, horses, pigs, cats and the like, no.

According to a more preferred embodiment of the present invention, the influenza virus is a type A influenza virus, a type B influenza virus or a type C influenza virus, preferably an influenza A virus, more preferably a H1N1 influenza virus, (H1N1), A / WSN / 33 (H1N1), A / Bervig-Mission / 1/18 (rvH1N1), A / Singapore / 6/86 (H1N1) A / California / 07/09, A / Brisbane / 59/2007 or A / New Caledonia / 20/99 and most preferably A / California / A / Brisbane / 59/2007 or A / New Caledonia / 20/99. The influenza viruses can cause flu, cold, sore throat, bronchitis and pneumonia, and can cause bird flu, swine flu or goat flu in particular.

According to a preferred embodiment of the present invention, the composition ratio of desalted non-toxic LOS and Alum in the immunostimulant composition of the present invention is 1: 45-55, preferably 1: 47-52, more preferably 1: 49-51.

According to a preferred embodiment of the present invention, the immunostimulant composition of the present invention may comprise a pharmaceutically acceptable carrier, and a description thereof is given below.

The combination of deacylated non-toxic LOS and Alum used in the present invention is highly suitable for influenza vaccine compositions because the immunostimulatory efficacy against influenza virus antigens is superior as well as other toxicities compared to other immunoadjuvants Do. The superior immune response promoting effect of the deacylated non-toxic LOS and Alum combinations is demonstrated in the following examples.

According to another aspect of the present invention, the present invention provides a composition comprising (a) (i) an immunostimulant composition for an influenza vaccine of the present invention; And influenza virus antigens; And,

(b) a pharmaceutically acceptable carrier.

Since the pharmaceutical composition of the present invention uses the above-described immunostimulant composition as an active ingredient, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.

As used herein, the term "vaccine" is used in its broadest sense to refer to a composition that positively affects the immune response of the subject. The vaccine composition provides the subject with an enhanced systemic or localized immune response, such as induction of a cellular immune response, e. G., CTL or humoral immune response, e.

According to a preferred embodiment of the present invention, the antigen of influenza virus contained in the influenza vaccine pharmaceutical composition includes influenza virus itself and various influenza virus-derived antigens conventionally known. The antigen refers to an antigen component capable of causing an immune function among components of the virus, and is preferably hemaglutinin (HA), neuraminidase (NA) or a fragment thereof.

As demonstrated in the following examples, vaccines comprising the immunostimulants of the invention can induce an excellent immune response with small doses (0.05 ug) of antigen (see FIG. 11).

According to a preferred embodiment of the present invention, the vaccine of the present invention may be an attenuated live vaccine or a sadox vaccine, a subunit vaccine, a synthetic vaccine or a genetic enginerring vaccine .

The pharmaceutically acceptable carriers contained in the vaccine pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, , Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, But is not limited thereto. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, the pharmaceutical composition may be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, have.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The oral dosage amount of the pharmaceutical composition of the present invention is preferably 0.0001-1000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides an influenza vaccine-resistant immunostimulant composition comprising deoxylating non-toxic LOS and Alum as an active ingredient by deacylation of LPS lipid A isolated from E. coli, and an influenza vaccine comprising the same.

(ii) Deacidification as an immune enhancer When non-toxic LOS and Alum are used in combination, they exert excellent immunostimulatory effect on influenza immune response and have excellent safety because they have little toxicity.

(iii) Therefore, the present invention can be usefully used as an influenza vaccine.

FIG. 1 is a graph showing the effect of IFN-.alpha. And IFN-.beta. Secretion by CIA05. FIG.
Figure 2 is a graph showing influenza antigen specific blood IgG antibody titers measured in various immunostimulant systems and mice immunized with influenza antigen: (A) 2 weeks after the first inoculation, (B) 2 weeks after the second inoculation result.
Figure 3 is a graph showing the serum HI antibody titer for influenza virus strains.
Figure 4 is a graph showing induction of IFN-y production in the spleen of mice immunized with influenza vaccine: (A) Results after 2 weeks of the first inoculation and (B) after 2 weeks of the second inoculation.
Figure 5 is a graph showing induction of IL4, IL5 and IL10 production in the spleen of mice immunized with influenza vaccine.
Figure 6 is a graph showing induction of T cell subset dependent IFN-y production in the spleen of mice immunized with influenza vaccine.
FIG. 7 is a photograph of an ELISPOT assay showing induction of influenza antigen-specific IgG antibody-secreting B cell production in lung tissue cells of a mouse immunized with influenza vaccine.
Figure 8 is a graph showing the cross reactivity of the vaccine induced serum IgG antibody against the H1N1 virus strain.
Figure 9 is a graph showing A / California / 04/09 (H1N1) virus specific serum total IgG and IgG isotype antibody titers in influenza antigens and mice immunized with various immune enhancer systems.
Figure 10 is a graph showing A / California / 07/09 (H1N1) virus specific serum total IgG and IgG isotype antibody titers in influenza antigens and mice immunized with various immune enhancer systems.
Figure 11 is a graph showing the serum HI antibody titer for the A / California / 04/09 (H1N1) virus strain.
Figure 12 is a graph showing the serum HI antibody titer for the A / California / 07/09 (H1N1) virus strain.
Figure 13 is a graph showing the induction of production of IFN-y and IL4 cytokines in the spleen of mice immunized twice with influenza vaccine (in the first experiment).
Figure 14 is a graph showing the induction of production of IFN-y and IL4 cytokines in the spleen of mice immunized twice with influenza vaccine (in the second experiment).
Figure 15 is a graph showing induction of T cell subset dependent IFN-y production in the spleen of mice immunized twice with influenza vaccine
16 is an ELISPOT assay showing the induction of production of influenza antigen-specific IgG antibody-secreting B cells in lung tissue cells of mice immunized twice with influenza vaccine.
17 is a graph showing the cross reactivity of the vaccine induced serum IgG antibody against the H1N1 virus strain.
Figure 18 is a graph showing the survival rate (A) and weight change (B) of influenza virus antigens and mice immunized with various immune enhancer systems.
Figure 19 is a graph showing the survival rate (A) and weight change (B) of influenza virus antigens and mice immunized with various immune enhancer systems.
Figure 20 is a graph showing induction of humoral immune responses in influenza virus antigens and mice immunized twice with various immune enhancer systems.
Figure 21 is a graph showing induction of humoral immune responses in influenza virus antigens and mice immunized twice with various immune enhancer systems.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1. Preparation of immunity enhancer for influenza vaccine

The present inventors have conducted studies to select optimal immunity enhancer system for influenza vaccine using the following immune enhancer candidate substances.

The CIA02 is a DNA fragment derived from Escherichia coli and disclosed in Korean Patent Publication No. 10-2003-0090897. CIA05 is a non-toxic lipopolysaccharide disclosed in Korean Patent No. 0740237, WO 2006/121232, Korean Patent Registration No. 0456681 and WO 2004/039413. The manufacturing process of CIA05 will be briefly described as follows. LPS purification was carried out according to the method described in the patent from E. coli of Deposit No. KCCM 10374, a strain (E. coli EG0021) with LPS having a very short chain of lipopolysaccharide chains. The molecular weight of the purified LPS was measured by MALDI-MASS (Shimadz, Axima-LNR V 2.3.5 (Mode Liner, Power: 106)) and the molecular weight of the purified LPS was found to be about 3500 Da. To purify the purified LPS according to the protocol disclosed in the patent, the purified LPS was mixed with 3 mg / ml of 2 N NaOH in a 1: 1 volume, followed by 10 minutes at 60 ° C The mixture was shaken and deacylated for 140 minutes, titrated to pH 7.0 with about 1/5 of 1 N acetic acid at an initial volume of 0.2 N NaOH, pH titrated and ethanol precipitation to obtain CIA05, a non-toxic lipopolysaccharide.

Example 2. Assessment of immune enhancing activity of influenza vaccine in vivo

≪ 2-1 > Evaluation of activity promoting secretion of type-I interferon of CIA05

IFN-α and IFN-β secretion activity of CIA05, a basic substance of influenza vaccine-resistant immunity enhancer system, was analyzed. For this purpose, bone marrow cells were isolated from C57BL / 6 mice and differentiated into immature dendritic cells. MPL and CIA05 were treated with different concentrations (0.001, 0.01, 0.1 and 1 μg / ml) for 24 hours. The supernatant was separated and the concentration of cytokine secreted from matured cells was measured by ELISA. The separated supernatant and IFN-α and IFN-β standard products were added to each plate coated with the antibody against each cytokine, and 100 μl each of the supernatant was reacted at room temperature for one hour. After completion of the reaction, the plate was washed with 0.5% -PBST solution, and the secondary antibody was added thereto, followed by washing at room temperature for one hour and washing. HRP solution, reacted, washed, developed with TMB substrate solution, terminated and absorbance was measured at OD ₄₅₀ . The TLR4 agonist adjuvant MPL, a major component of GSK's immunostimulant system AS04, was used as a control to assess the secretion performance of IFN-α and IFN-β induced by CIA05. The experimental results are shown in Fig.

As shown in Fig. 1, IFN- [alpha] and IFN- [beta] secretion were increased in dendritic cells derived from mouse bone marrow, depending on CIA05 concentration, which was 3 times higher than that of MPL (Fig. 1).

<2-2> Mouse immunization

In order to analyze the in vivo efficacy of the immune enhancer, 0.2 μg of A / California / 07/09 split vaccine antigen was mixed with an immunostimulant and immunized by intramuscular injection into the thigh of the mouse. Mouse immunization was performed twice at 3-week intervals, and blood, lung and spleen tissues were collected from the mice 2 weeks after each inoculation day. Mice were anesthetized with ketamine and rum punch and cardiac blood collection was performed. The blood was left at 4 ° C for 12 hours or more, centrifuged, and the serum was separated and stored at -70 ° C. Lung and spleen tissues were separated into single cells and used in the experiment. Table 2 shows animal experimental groups for in vivo immunogenicity evaluation of influenza vaccine containing an immunostimulant.

<2-3> Influenza antigen-specific blood IgG antibody titers

In order to analyze the humoral immune response enhancing effect of influenza vaccine using the immunostimulant system, the serum of the immunized mice was separated and the antigen specific IgG antibody titer of the whole blood was measured by the ELISA method. ELISA was performed by coating the vaccine antigen with 1 μg / ml and high-titer serum was included in each plate to correct deviations between ELISA plates. The plate coated with the vaccine antigen was blocked with 1% BSA-PBS solution for 1 hour at room temperature, washed, and 100 쨉 l of 2-fold serial diluted mouse serum was added thereto, followed by reaction at 37 캜 for 2 hours. After completion of the reaction, the plate was washed with 0.5% -PBST solution and HRP-conjugated secondary antibody was added thereto, followed by reaction at room temperature for one hour. After washing, TMB substrate solution was used for color development for 5 minutes. The reaction was terminated with 1 N HCl solution and absorbance was measured at OD ₄₅₀ . The serum dilution factor representing OD 0.1 was determined as the IgG antibody and the antibody titer per mouse was calculated as the geometric mean. The experimental results are shown in Fig.

As shown in FIG. 2, the combination of the immunostimulant with the antigens showed that the combination of CIA07 and alum exhibited antibody titer 4.5 times higher than that of the antigen alone (p <0.01; A). In the case of two injections, the total IgG antibody titer of the blood was increased by 10 times as compared with the single injection, and the combination of CIA05 and alum (CIA06) was 2.8 times higher than that of the antigen alone (Fig. 2B). &Lt; tb &gt;&lt; TABLE &gt;

<2-4> HI titers of immunized mouse sera

In order to measure the HI titer of the immunized mouse antibody, the serum of the immunized mouse was reacted at 56 ° C for 30 minutes to heat-inactivate the serum. The serum was reacted with the 25% kaolin solution at room temperature for 20 minutes to give a nonspecific reaction . The serum was reacted with 4 HAU (hemagglutination unit) A / California / 07/09 virus. 1% turkey RBC solution was added and incubated for 3 hours to measure the HI titer of immunized mouse serum. The experimental results are shown in Fig.

As shown in FIG. 3, when the vaccine was administered twice, the HI titer was significantly increased 4-fold (p <0.01) in the combination administration of CIA05 and alum compared to the antigen alone group (p <0.01) The HI titer was 3.3 times higher than that of the antigen-treated group (FIG. 3).

<2-5> Influenza antigen-specific cytokine induction activity assay

To analyze influenza antigen-specific cytokine-inducing activity, mice were immunized twice at intervals of 3 weeks, and splenocytes of mice were isolated two weeks after each immunization. The concentration of IFN-γ, IL-4, IL-5, IL-10 and IL-17 was analyzed by ELISA method after spleen cells were stimulated with 2 μg of influenza antigen and cultured for 3 days. 100 쨉 l of each separated supernatant and each cytokine standard was added to each plate coated with an antibody against each cytokine, and reacted at room temperature for one hour. After completion of the reaction, the plate was washed with 0.5% -PBST solution, and the secondary antibody was added thereto, followed by washing at room temperature for one hour and washing. HRP solution, reacted, washed, developed with TMB substrate solution, terminated and absorbance was measured at OD ₄₅₀ . The results of the analysis are shown in FIGS.

As shown in FIG. 4, the IFN-γ cytokine concentration was found to be 1.5- to 1.5-fold higher in the CIA07 group, CIA02 and alum mixture group, and CIA05 and alum mixture group than the antigen- (A). The IFN-γ level, which was secreted antigen-specific, could be evaluated in experiments in which IFN-γ concentration was observed after two immunizations. IFN- [gamma] secretion was observed in the immunized group of CIA02 with the influenza antigen more than twice as high as that of the other immunostimulants. In the CIA05 group, IFN- gamma secretion was observed. IFN-γ was secreted at a level of about 10 ng / ㎖ in the mixed agent of CIA05 and alum at a level two times higher than that of the CIA05 group and four times higher than that of the antigen alone group (B). These results indicate that the combination of CIA02 or CIA05 and alum has the potential to induce influenza antigen-specific Th1 responses as compared to other immune enhancer systems.

As shown in FIG. 5, the cytokines IL-4, IL-5 and IL-10, which induce the Th2 response, were analyzed. As a result, low levels of cytokine secretion were observed in a single immunization, The level of each cytokine was higher in the antigen alone group than in the immunotherapy combination group. In the case of IL-4 and IL-10, a higher level of cytokine was secreted in the mixture of antigen alone and CIA02 and alum than in the other experimental groups. After one immunization, the antigen alone showed the ability to induce Th2 response, which is consistent with the high IgG level in the mouse serum alone group. IL-4, IL-5, and IL-10 were secreted at a high level in the antigen-only group after two immunizations. In contrast, when CIA02 and CIA05 were used as immunopotentiating agents and CIA07, a combination of these two immunostimulants, the secretion of IL-4, IL-5 and IL-10 was more than 2 times lower than that of the antigen alone group. In the case of a system in which alum was mixed with these immunostimulants, secretion of cytokines was increased, but secretion of each cytokine was lower than that of the antigen alone. The above results indicate that the immunological enhancer system that induces an appropriate level of Th2-type cytokine is selected and applied to an influenza vaccine, whereby the Th2-type cytokines IL-4, IL-5 and IL-10 And lower the risk of cytokine storm, a side effect of vaccine administration.

As shown in FIG. 5, the secretion level of IL-17 was high due to the mixed preparation of CIA07 and alum at the time of two injections by CIA06 in one injection, and it was confirmed that the two types of immunostimulants were capable of inducing the Th17 response .

<2-6> T cell subset-dependent IFN-γ secretion activity

BALB / c mice were immunized twice at 3-week intervals to analyze the effect of influenza antigen-specific IFN-γ secretion activated by the immune system enhancer system on T cell subset-dependent expression, and after 2 weeks of the last immunization, Cells were isolated. The splenocytes were treated with anti-mouse CD4 antibody or anti-mouse CD8 antibody, stimulated with 2 μg of influenza antigen, and cultured for 3 days. After the supernatant was separated, IFN-γ concentration was analyzed by ELISA to compare IFN-γ secretion by CD8 ⁺ T cells and CD4 ⁺ T cells. 100 μl of each separated supernatant and IFN-γ standard was added to each plate coated with anti-IFN-γ antibody, followed by reaction at room temperature for 1 hour. After completion of the reaction, the plate was washed with 0.5% -PBST solution, and the secondary antibody was added thereto, followed by washing at room temperature for one hour and washing. HRP solution, reacted, washed, developed with TMB substrate solution, terminated and absorbance was measured at OD ₄₅₀ . The experimental results are shown in Fig.

As shown in FIG. 6, the level of IFN-.gamma. Secretion was highest in the group treated with CIA02, and this pattern was also observed when CD4 ⁺ or CD8 ⁺ T cells were blocked. When CD4 surface markers were blocked, cytokines were secreted about 50% lower than when CD8 ⁺ T cells were blocked. This pattern was similar in most immunomodulatory groups. These results indicate that IFN-γ cytokines secreted by the immunostimulant system are secreted by all CD4 ⁺ / CD8 ⁺ T cells, whereas CD4 ⁺ and CD8 ⁺ The blocking level of IFN-γ cytokine was significantly higher than that of the other immunoenhancer system in the case of blocking the CD4 / CD8 block. It looked. Based on this, it was confirmed that when CIA07 and alum were combined, IFN-γ cytokine secretion was induced by another kind of T cell other than CD4 ⁺ or CD8 ⁺ T cells (FIG. 6).

<2-7> Analysis of B cell production ability with antigen-specific antibody-releasing ability in lung tissue cells

In order to analyze B cells secreting antigen-specific antibodies present in lung tissue cells, mice were immunized by intramuscular injection once with A / California / 07/09 split vaccine antigen, and after 2 weeks, mouse lung tissue cells were isolated And analyzed by ELISPOT. Lung tissue cells were serially diluted in two steps from 1 ㅧ ¹⁰⁶ cells, planted in an ELISPOT plate coated with anti-mouse IgG antibody, stimulated with 2 쨉 g of antigen, and cultured for 1 day. After removing the cells, the cells were treated with anti-IgG antibody, reacted for 2 hours at room temperature, washed, reacted with streptavidin-HRP solution at room temperature for 1 hour, and then added with a coloring solution. B cell spots secreting the detected antigen - specific antibody were analyzed by ELISPOT reader. The experimental results are shown in Fig.

As shown in FIG. 7, a B cell spot that secretes an antigen-specific antibody was detected in a mixture of CIA05 and alum and a mixture of CIA07 and alum. From the above results, it was found that the immunostimulant system administered in combination with the influenza antigen can induce the production of B cells secreting antigen-specific antibodies in the lung tissue by intramuscular injection (Fig. 7).

<2-8> Assessment of Cross-Reaction Enhancement Effect on Influenza Virus by Immune Enhancer System

A / California / 07/09 The cross reactivity of IgG antibody in blood of mice immunized with split vaccine antigen was evaluated by ELISA method. Cross reactivity was assessed using H1N1 virus strain A / Brisbane / 59/2007 and A / New Caledonia / 20/99, and each virus was coated with 16 HAU / well and subjected to ELISA. Each virus-coated plate was blocked with 1% BSA-PBS solution for 1 hour at room temperature, washed, and 100 μl of double-serial diluted mouse serum was added thereto, followed by reaction at 37 ° C for 2 hours. After completion of the reaction, the plate was washed with 0.5% -PBST solution and HRP-conjugated secondary antibody was added thereto, followed by reaction at room temperature for one hour. After washing, TMB substrate solution was used for color development for 5 minutes. The reaction was terminated with 1 N HCl solution and the absorbance at OD ₄₅₀ was measured. The serum dilution factor representing OD 0.1 was determined as the IgG antibody and the antibody titer per mouse was calculated as the geometric mean. A / California / 07/09 The reactivity of whole IgG antibodies against two H1N1 influenza virus strains and vaccine antigen strains by using sera from mice administered two times at 3-week intervals with split vaccine antigen and each immunostimulant system And the results are shown in FIG.

As shown in FIG. 8, all the IgG titers of the A / New Caledonia / 20/99 virus strains were similar to those of the vaccine antigens in all the experimental strains, and the A / Brisbane / 59 / Caledonia / 20/99 showed lower total IgG titers. Although there was a difference in the level of total IgG antibody titer according to the virus strain, when CIA05 and alum were applied in the immune system, the highest antibody titer was shown in all virus strains tested (Fig. 8).

Example 3: Antigen reduction effect of influenza vaccine using an immune enhancer and evaluation of immune enhancing activity in vivo

<3-1> Antibody-inducing activity assay of influenza virus of immunopotentiator

Mice were immunized by mixing immunostimulants (alum, CIA05 and CIA06) with A / California / 07/09 split vaccine antigens to analyze the in vivo efficacy of the immune enhancer. Two experiments were conducted to see the effect of antigen dose reduction. In the first experiment, 0.2, 0.1, 0.05, and 0.02 μg of antigens were mixed with the immunostimulant, respectively, and injected intramuscularly into the thigh of the mouse. Mouse immunization was performed twice at 3-week intervals, and blood, lung tissue and spleen tissue were collected from the mice 2 weeks after the last inoculation day. Antibody titers against the A / California / 04/09 (H1N1) virus strain were tested using sera isolated from the first experiment. The experimental results are shown in Fig.

As shown in FIG. 9, the virus-specific total IgG, IgG1 and IgG2a antibody levels in the mouse serum were significantly higher in all experimental groups administered with the immunopotentiating agent than in the antigen-alone administration group. When CIA06 was used, antibody titer was higher at all doses than alum. Antibody titers were dependent on the antigenic dose in the antigen-alone group, but antibody titers in the group administered with the immunomodulating agents such as alum and CIA06 showed high antibody titer regardless of the antigen dose. Antigens of 0.2 μg and 1 / There was no significant difference between groups at 0.02 μg at 10th level. When combined with an immunostimulant, the effect of decreasing the antigen dose in the serum antibody titer was clearly evident, indicating that CIA06 was the most effective (Fig. 9).

The present inventors conducted a second experiment to repeat the above experiment. Influenza vaccine antigens 0.1, 0.05 and 0.01 μg were mixed with each immunostimulant and immunized. Mouse immunization was performed twice at intervals of two weeks, and blood, lung tissue and spleen tissues were collected from the mice two weeks after the last inoculation day. The experimental results are shown in Fig.

As shown in FIG. 10, the antibody titers to the A / California / 07/09 virus strains were measured using the sera isolated from the second experiment. As a result, only a high level of IgG, IgG1 and IgG2a antibody titers And there was no difference according to the antigen dose. In the case of CIA06, antibody titers were similar to those of alum group. However, in the experiment group in which CIA06 and 0.01 μg of antigen were mixed, total IgG antibody titer was 1.6 times higher than alum, and CIA06 and 0.05 μg of antigen IgG2a antibody titer was 4.5 times higher than that of alum. As a result of the above antibody titers, it was confirmed that virus-specific antibody production was increased due to CIA06 when the antigen dose was reduced to 1/2 or 1/4 through two experiments (FIG. 10).

<3-2> Analysis of HI titers of immunized mouse sera

The HI antibody titer in immunized mouse serum was analyzed by decreasing the antigen dose. In the first experiment, mice were immunized with 0.2, 0.1, 0.05, and 0.02 μg of antigen, pretreated with immunized mouse serum, and then measured for HI titer against A / California / 04/09 virus of 4 HAU. The experimental results are shown in Fig.

As shown in Fig. 11, in the first experiment, the HI level in the mouse serum immunized with the antigen only was very low. In addition, no activity was observed in the group to which 0.02 μg of the antigen was applied. On the other hand, HI titer was measured regardless of the antigen dose of 0.2-0.05 μg in the group administered with the immunostimulant, and the HI titer of the CIA06 group was higher than that of the alum group. From the above results, it can be seen that when CIA06 is used, the HI level is maintained at a high level even when the antigen dose is reduced to 0.05 μg (FIG. 11).

To repeat the experiment, the inventors performed a second experiment by immunizing mice with 0.1, 0.05 and 0.01 μg of antigen. Mouse serum was reacted with 8 HAU A / California / 07/09 virus suspension to determine HI titer. The experimental results are shown in Fig.

As shown in FIG. 12, in the second experiment, the highest HI titer was shown in mice administered with 0.1 μg or 0.05 μg of antigen and CIA06 (FIG. 12).

When CIA06 was used as an immunostimulating agent, the highest HI titer was shown in the second experiment when 0.1 μg of antigen was used, but the highest HI titer was shown in the first experiment when 0.05 μg was used. In the HI assay, the A / California / 04/09 virus strain, which is not identical to the virus strain used in the first experiment in the first experiment but the same strain in the same year, was used. In the second experiment, A / California / 07/09. Based on the above results, when the vaccine is developed by applying CIA06 as an immunity enhancer, the optimal antigenic capacity to exhibit a high HI titer for the pandemic strain is estimated to be 0.05 μg / dose.

Example 4. Analysis of cell-mediated immunity induction activity against influenza antigen

<4-1> Influenza antigen-specific cytokine induction activity assay

For analysis of influenza antigen-specific cytokine secretion, mice were immunized twice with influenza vaccine antigen and immunostimulant, and then splenocytes were isolated from mice two weeks after the final immunization. The splenocytes were stimulated with 2 μg of influenza antigen, cultured for 3 days, and then the supernatant was separated to analyze the IFN-γ and IL-4 cytokine-inducing activity by ELISA. 100 μl of each separated supernatant and each cytokine standard was added to a plate coated with an antibody against cytokine, and reacted at room temperature for one hour. After completion of the reaction, the plate was washed with 0.5% -PBST solution, and the secondary antibody was added thereto, followed by washing at room temperature for one hour and washing. HRP solution, reacted, washed, developed with TMB substrate solution, terminated and absorbance was measured at OD ₄₅₀ . Similar to the analysis of the antibody-induced immunity induction activity, the experiment was carried out twice. Experimental results are shown in Figs.

As shown in FIGS. 13 and 14, in the case of antigen-alone group and alum-treated group, it was observed that IFN-.gamma. Secretion was almost the same when stimulated with antigen and stimulated, and was secreted to basal level. In CIA06 group, IFN-y secretion level was significantly increased. In both the first and second experiments, IFN-γ secretion was activated dependent on the antigen concentration. When IL-4 was measured, high level of IL-4 secretion was observed regardless of the antigen concentration in the antigen-only group. In the case of administration of alum, CIA05 or CIA06, the level of IL-4 secretion was 50% IL-4 &lt; / RTI &gt; When CIA06 was applied to influenza vaccine antigens, it was confirmed that not only the typical Th2 type but also the Th1 type cytokine secretion response was induced (FIGS. 13 and 14).

<4-2> T cell subset-dependent IFN-γ secretion activity

CIA06-induced influenza antigen-specific IFN-y secretion was identified as a T cell subset-dependent phenomenon. BALB / c mice were immunized twice with 0.2 μg of influenza vaccine antigen and immunostimulant, and splenocytes were separated from the mice 2 weeks after the last immunization. Separated cells were treated with anti-mouse CD4 antibody or anti-mouse CD8 antibody and stimulated with 2 μg of influenza antigen for 3 days. IFN-γ levels in the supernatants were analyzed by ELISA to compare IFN-γ secretion by CD8 ⁺ T cells and CD4 ⁺ T cells, respectively. 100 μl of each separated supernatant and IFN-γ standard was added to each plate coated with anti-IFN-γ antibody, followed by reaction at room temperature for 1 hour. After completion of the reaction, the plate was washed with 0.5% -PBST solution, and the secondary antibody was added thereto, followed by washing at room temperature for one hour and washing. HRP solution, reacted, washed, developed with TMB substrate solution, terminated and absorbance was measured at OD ₄₅₀ . As a result of the analysis, the influenza vaccine using CIA06 activated CD4 ⁺ / CD8 ⁺ T cells as shown in Fig.

<4-3> Analysis of B Cell Production Ability with Antigen-Specific Antibody Releasing Capability in Lung Tissue Cells

The degree of B cell production with antigen - specific antibody secretion in lung tissue was analyzed. A / California / 07/09 split immunized with 0.2 μg of split vaccine antigen two times, and after 2 weeks, mouse lung tissue cells were extracted and the B cells secreting antigen-specific IgG antibody were detected by ELISPOT . The mouse lung tissue cells were separated and serially diluted in two steps from 1 ㅧ ¹⁰⁶ cells. The cells were seeded on an anti-mouse IgG coated ELISPOT plate, stimulated with 2 쨉 g of antigen, and cultured for 1 day. After removing the cells, the cells were treated with anti-IgG antibody, reacted for 2 hours at room temperature, washed, reacted with streptavidin-HRP solution at room temperature for 1 hour, and then added with a coloring solution. B cell spots secreting the detected antigen - specific antibody were analyzed by ELISPOT reader. The experimental results are shown in Fig.

As shown in FIG. 16, when the antigen alone and CIA05 were mixed with the antigen, the same level of B cells was detected. When alum was used, 1.5 times more B cells were detected than the antigen alone. When CIA06 was used, the levels of B cells were increased by 2-fold and 1.5-fold, respectively, compared with CIA05 and alum groups. These results indicate that synergy can be obtained when CIA05 and alum are administered together. When CIA06 is used as an immunostimulant, antigen-specific antibody-secreting B cells are generated in the lung tissue by intramuscular injection. This enhances the protective ability of the influenza virus through the respiratory tract after immunization. (Fig. 16). In addition, the present invention is not limited thereto.

<4-4> Assessment of the cross-reactivity enhancement effect of influenza virus in the immune system

Cross reactivity to A / Brisbane / 59/2007 and A / New Caledonia / 20/99 virus strains was measured by ELISA using sera from mice immunized with various doses of A / California / 07/09 split vaccine antigen . Each virus was blocked with 1% BSA-PBS solution in a plate coated with 16 HAU / well for 1 hour at room temperature, washed, and 100 ㎕ of double-serially diluted mouse serum was added thereto, followed by reaction at 37 캜 for 2 hours. After completion of the reaction, the plate was washed with 0.5% -PBST solution and HRP-conjugated secondary antibody was added thereto, followed by reaction at room temperature for one hour. After washing, TMB substrate solution was used for color development for 5 minutes. The reaction was terminated with 1 N HCl solution and the absorbance at OD ₄₅₀ was measured. The serum dilution factor representing OD 0.1 was determined as the IgG antibody and the antibody titer per mouse was calculated as the geometric mean. The experimental results are shown in Fig.

As shown in FIG. 17, virus-specific IgG in serum reacted with A / New Caledonia / 20/99 similar to A / California / 04/09. A / Brisbane / 59/2007 showed a slight decrease in response but showed the same pattern of total IgG titers in the same pattern for the three virus types. In the case of alone, the antibody titer was dependent on the vaccine antigen used, and when CIA06 was used, the antibody titer was at least 3 to 100 times higher than that of the antigen alone, and the cross-reaction enhancing effect was the most excellent in CIA06 compared to alum (Fig. 17).

Example 5 Evaluation of the effect of enhancing the immunity enhancer against influenza virus in the mouse model

Experiments were conducted twice to evaluate the effect of influenza vaccine against influenza virus using an immunopotentiator. In the first experiment, 0.02, 0.2 and 1 μg of A / California / 07/09 split vaccine antigen were mixed with antigen alone or immunostimulant to immunize mice. Vaccine antigen and immunostimulant were mixed and immunized twice at intervals of 2 weeks. After 4 weeks, mice were infected with 10 LD _{50 of} A / California / 04/09 and mouse adapted influenza virus strains. The survival rate and body weight change of the mice were examined for 2 weeks. The experimental results are shown in Fig.

As shown in FIG. 18, when the survival rate was observed for 2 weeks after the virus infection, 100% of the negative control group died at 6 days after infection, but survival rate was 20% when immunized with CIA06 alone. The survival rate of 0.02 μg and 0.2 μg respectively showed survival rates of 60% and 80%, respectively. Immunization of alum with CIA05 and CIA06 resulted in a 100% survival rate, confirming the superior efficacy of infection defense (Fig. 18A).

The present inventors conducted a second experiment to repeat the above experiment. 0.05 and 0.01 μg of influenza vaccine antigen were mixed with antigen alone or with an immunostimulant to immunize mice. Vaccine antigen and immunostimulant were mixed and immunized twice at intervals of 2 weeks. After 4 weeks, mice were infected with 10 LD _{50 of} A / California / 04/09 and mouse adapted influenza virus strains. The survival rate and body weight change of the mice were examined for 2 weeks. The experimental results are shown in Fig.

As shown in FIG. 19, when the survival rate was observed for 2 weeks after the virus infection, 100% of the negative control group died at 7 days after infection, but when immunized with 0.05 μg and 0.01 μg of vaccine antigen, 60% and 40% Survival rate. Immunization of alum with CIA05 and CIA06 in 0.05 μg of vaccine antigen resulted in a 100% survival rate. In the case of vaccine antigen, 0.01 μg of alum was mixed with 80% of CIA05 and CIA06, %, Indicating that the antiviral defense efficacy by CIA05 and CIA06 was excellent even when the antigen dose was reduced to 0.01 μg (FIG. 19A).

In addition, the body weight change of the mouse for 2 weeks after the virus infection was observed, and the weight change range of the mouse was the largest at 6-8 days after the infection in the first experiment, and recovered thereafter. When 0.2 μg of vaccine antigen and CIA06 were mixed, there was almost no change in body weight during the experiment, but when immunized with only 0.2 μg of vaccine antigen, the body weight decreased by about 15% on the 6th day after virus infection compared with the case of CIA06 Was gradually recovered. When 1.0 μg of vaccine antigen and CIA06 were mixed, body weight was reduced by 10% after 7 days of infection compared with 0.2 μg of vaccine antigen and CIA06. Alum or CIA05 were mixed and immunized, weight loss was observed compared to the CIA06 group (Fig. 18B).

As a result of observing the weight change of the mice in the second experiment, the weight change range of the mice was the largest at 6 to 8 days after the infection and recovered thereafter. When CIA06 was added to 0.05 μg of vaccine antigen, the weight change was the smallest during the experiment. In addition, CIA05 and alum mixed with 0.05 and 0.01 μg of antigen, respectively, showed 10% weight loss on the 8th and 9th days after viral infection (Fig. 19B). In Fig.

In order to observe antibody immunity formation by immunization twice with vaccine antigen and immunostimulant, two sera were isolated from virus infection two weeks after secondary immunization of mice in two infectious defense tests, and total IgG and IgG isotype Ab Histological, immunohistochemical and immunohistochemical studies were performed to measure the HI and micro-neutralization titers. Experimental results are shown in Figs. 20 and 21.

As shown in FIG. 20, when the vaccine antigen 0.2 .mu.g and 1 .mu.g were mixed with CIA06 in the first experiment, not only the total IgG, IgG1 and IgG2a antibody titers were measured, but also the HI titer and the MN inversion Respectively. The results of the second experiment also showed that when the vaccine antigen 0.05 μg and 0.01 μg were mixed with CIA06, the total IgG, IgG1 and IgG2a antibody titer was measured at a high level, and the HI titer and the MN titer were also high (Fig. 21).

Based on the experimental results, it was found that the correlation between the level of antibody immunity and the protection effect of infection was high.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

(i) deacylation by deacylation of LPS (lipopolysaccharide) lipid A isolated from E. coli; non-toxic LOS (Lipooligosaccharide); And (ii) an aluminum hydroxide as an active ingredient.
The method of claim 1, wherein the LPS separated from the Escherichia coli has a molecular chain length of 5,000-10,000 Da, which is shorter than that of the natural LPS derived from Escherichia coli, and the deacylated non-toxic LOS is obtained by deacylation of LPS lipid A Wherein the composition has a molecular weight of 2,000-5,000 Da.
2. The immunostimulant composition according to claim 1, wherein the Escherichia coli is KCCM 10374.
2. The composition of claim 1, wherein said influenza virus is type A influenza virus.
(a) (i) an immunostimulant composition according to any one of claims 1 to 4; And influenza virus antigens; And,
(b) a pharmaceutically acceptable carrier.

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