VACCINE COMPOSITION

Abstract

A vaccine composition for administration to the oral cavity of a human or an animal, the vaccine composition containing at least one antigen derived from an infectious disease, and at least one selected from the group consisting of a toll-like receptor 4 (TLR4) agonist, a toll-like receptor 2/6 (TLR2/6) agonist, and cyclic dinucleotide, or a derivative or salt thereof.

Claims

1. A vaccine composition for administration to the oral cavity of a human or an animal, the vaccine composition comprising: at least one antigen derived from an infectious disease, and a toll-like receptor 2/6 (TLR2/6) agonist comprising Pam.sub.2CSK.sub.4 and salts thereof.

2. The vaccine composition according to claim 1, wherein: the vaccine composition induces a mucosal immune response and a systemic immune response, the mucosal immune response is antigen-specific IgA antibody production, and the systemic immune response is antigen-specific IgG antibody production and antigen-specific cell-mediated immune production.

3. The vaccine composition according to claim 1, wherein the antigen derived from an infectious disease is an influenza virus-derived antigen.

4. The vaccine composition according to claim 3, wherein the influenza virus-derived antigen is hemagglutinin protein.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0075] FIG. 1 is a graph showing the results of mouse serum influenza HA-specific IgG titer in Examples 1 to 6 and Comparative Examples 1 to 9.

[0076] FIG. 2 is a graph showing the results of mouse nasal wash influenza HA-specific IgA titer in Examples 1 to 6 and Comparative Examples 1 to 9.

[0077] FIG. 3 is a graph showing the results of mouse serum influenza HA-specific IgG titer in Examples 7 to 9 and Comparative Examples 10 to 12.

[0078] FIG. 4 is a graph showing the results of mouse nasal wash influenza HA-specific IgA titer in Examples 7 to 9 and Comparative Examples 10 to 12.

[0079] FIG. 5 is a graph showing the survival rates of mice infected with influenza virus in Example 10, Comparative Examples 13 and 14.

[0080] FIG. 6 is a graph showing the results of mouse serum influenza HA-specific IgG titer in Examples 11, Comparative Examples 1 and 14.

[0081] FIG. 7 is a graph showing the results of mouse nasal wash influenza HA-specific IgA titer in Examples 11, Comparative Examples 1 and 14.

[0082] FIG. 8 is a graph showing the results of mouse serum influenza HA-specific IgG titer in Examples 12 and 13, Comparative Examples 15, 16 and 1.

[0083] FIG. 9 is a graph showing the results of mouse nasal wash influenza HA-specific IgA titer in Examples 12 and 13, Comparative Examples 15, 16 and 1.

[0084] FIG. 10 is a graph showing the results of mouse serum influenza HA-specific IgG titer in Examples 12, 14 and 15, Comparative Examples 16 and 1.

[0085] FIG. 11 is a graph showing the results of mouse nasal wash influenza HA-specific IgA titer in Examples 12, 14 and 15, Comparative Examples 16 and 1.

[0086] FIG. 12 is a graph showing the results of mouse serum influenza HA-specific IgG titer in Examples 16 and 17, Comparative Examples 17 and 18.

[0087] FIG. 13 is a graph showing the results of mouse nasal wash influenza HA-specific IgA titer in Examples 16 and 17, Comparative Examples 17 and 18.

DESCRIPTION OF EMBODIMENTS

[0088] Hereinafter, the present invention is described further in detail with reference to the following examples, but is not limited thereto.

Example 1

[0089] Phosphate buffer (Nacalai Tesque, Inc.) was added to 76.3 L (236 g/mL) of an influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University) and 30 L (1 mg/mL) of a solution containing E. Coli lipopolysaccharide (Nacalai Tesque, Inc.) to prepare 120 L of a vaccine composition.

[0090] Five mice (7-week old, C57BL/6 female mice, Japan SLC, Inc.) prepared in advance were anaesthetized (Somnopentyl, Kyoritsu Seiyaku Corporation), and 20 L of the prepared vaccine composition was sublingually administered to each of the mice.

[0091] 1 week later from the administration, the mice were anaesthetized again and 20 L of the prepared vaccine composition was sublingually administered to each of the mice.

[0092] Another week later from the second administration, the serum and nasal wash were collected from the mice, and the serum influenza HA-specific IgG titer and nasal wash influenza HA-specific IgA titer were measured by ELISA. The measurement method is described later in detail.

Examples 2 to 6

[0093] Vaccine compositions were prepared in the same manner as Example 1, except that, in place of the E. coli lipopolysaccharide, Pantoea lipopolysaccharide (macrophi inc.) was used in Example 2, glucopyranosyl lipid (MPLAs, InvivoGen) was used in Example 3, FSL-1 (InvivoGen) was used in Example 4, Pam.sub.2CSK.sub.4 (InvivoGen) was used in Example 5 and c-di-GMP (Cyclic diguanosine monophosphate, Biolog Inc.) was used in Example 6. The test was carried out by the same procedure as Example 1 in the doses shown in Table 1.

Comparative Example 1

[0094] Five mice (7-week old, C57BL/6 female mice, Japan SLC, Inc.) prepared in advance were anaesthetized, 120 L of phosphate buffer (Nacalai Tesque, Inc.) was prepared and 20 L thereof was sublingually administered to each of the mice. The test was carried out by the same subsequent procedure as Example 1.

Comparative Example 2

[0095] In place of the phosphate buffer, phosphate buffer (Nacalai Tesque, Inc.) was added to 76.3 L (236 g/mL) of an influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University) to prepare 120 L of a vaccine composition. The test was carried out by the same subsequent procedure as Example 1 in the dose shown in Table 1.

Comparative Examples 3 to 9

[0096] Vaccine compositions were prepared in the same manner as Comparative Example 2, except that, in addition to the influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University), peptidoglycan (PGN derived from Salmonella, InvivoGen) was used in Comparative Example 3, zymosan (Nacalai Tesque, Inc.) was used in Comparative Example 4, Pam.sub.3CSK.sub.4 (InvivoGen) was used in Comparative Example 5, Poly (I:C) (InvivoGen) was used in Comparative Example 6, flagellin (InvivoGen) was used in Comparative Example 7, imiquimod (InvivoGen) was used in Comparative Example 8 and CpG (InvivoGen) was used in Comparative Example 9. The test was carried out by the same procedure as Example 1 in the doses shown in Table 1.

TABLE-US-00001 TABLE 1 Antigen Adjuvant Dose (g/ Substance Dose (g/ No. body/time) Ligand name body/time) Example 1 3 TLR4 E. coli 5 LPS Example 2 3 TLR4 Pantoea 5 LPS Example 3 3 TLR4 GLA 5 Example 4 3 TLR2/6 FSL-1 5 Example 5 3 TLR2/6 Pam.sub.2CSK.sub.4 5 Example 6 3 unclear c-di-GMP 138 Comparative Example 1 Comparative 3 Example 2 Comparative 3 TLR2 PGN 100 Example 3 Comparative 3 TLR2/ zymosan 5 Example 4 Dectin1 Comparative 3 TLR2/1 Pam.sub.3CSK.sub.4 5 Example 5 Comparative 3 TLR3 poly I:C 5 Example 6 Comparative 3 TLR5 flagelin 5 Example 7 Comparative 3 TLR7 imiquimod 5 Example 8 Comparative 3 TLR9 CpG 5 Example 9

Examples 7 to 9

[0097] In addition to 19 L (236 g/mL) of the influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University), phosphate buffer (Nacalai Tesque, Inc.) was added to 30 L (1 mg/mL) of glucopyranosyl lipid (MPLAs, InvivoGen) to prepare 120 L of a vaccine composition in Example 7. 20 L thereof was sublingually administered to the mice (7-week old BALE/c female mice, Japan SLC, Inc.) under anaesthesia. In place of the glucopyranosyl lipid used in Example 7, FSL-1 (InvivoGen) was used in Example 8, and c-di-GMP (Cyclic diguanosine monophosphate, Biolog Inc.) was used in Example 9 to prepare vaccine compositions. The doses are as shown in Table 2. The test was carried out by the same procedure as Example 1 after the preparation, except that the mouse type was different.

Comparative Example 10

[0098] Phosphate buffer (Nacalai Tesque, Inc.) was added to 19 L (236 g/mL) of the influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University) to prepare 120 L of a vaccine composition. The dose was as shown in Table 2. The test was carried out by the same procedure as Example 1 after the preparation, except that BALE/c mice were used.

Comparative Examples 11 and 12

[0099] In addition to 76.3 L (236 g/mL) of the influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University), in Comparative Example 11, phosphate buffer (Nacalai Tesque, Inc.) was added to 30 L (20 mg/mL) of peptidoglycan (PGN derived from Salmonella, InvivoGen) to prepare 120 L of a vaccine composition. In place of the peptidoglycan used in Comparative Example 11, Pam.sub.3CSK.sub.4 (InvivoGen) was used in Comparative Example 12 to prepare a vaccine composition. The doses were as shown in Table 2. The test was carried out by the same procedure as Example 1 after the preparation, except that BALB/c mice were used.

TABLE-US-00002 TABLE 2 Antigen Adjuvant Dose (g/ Substance Dose (g/ No. Type body/time) Ligand name body/time) Example 7 A/IvPR8/ 0.75 TLR4 GLA 5 34(H1N1) Example 8 A/IvPR8/ 0.75 TLR2/6 FSL-1 5 34(H1N1) Example 9 A/IvPR8/ 0.75 unclear c-di-GMP 138 34(H1N1) Comparative A/IvPR8/ 0.75 Example 10 34(H1N1) Comparative A/IvPR8/ 0.75 TLR2 PGN 100 Example 11 34(H1N1) Comparative A/IvPR8/ 0.75 TLR2/1 Pam.sub.3CSK.sub.4 5 Example 12 34(H1N1)

Example 10

[0100] Phosphate buffer (Nacalai Tesque, Inc.) and 60 L (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 152.6 L (236 g/mL) of the influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University), The Research Foundation for Microbial Diseases of Osaka University) to prepare 240 L of a vaccine composition.

[0101] Ten mice (7-week old, BALE/c female mice, Japan SLC, Inc.) prepared in advance were anaesthetized and 20 L of the prepared vaccine composition was sublingually administered to each of the mice.

[0102] 1 week later from the administration, the mice were anaesthetized again and 20 L of the prepared vaccine composition was sublingually administered to each of the mice.

[0103] Another week later from the second administration, the mice were infected with the lethal dose of influenza virus (A/IvPR8/34(H1N1) and monitored for the following 2 weeks to measure the survival rates. The measurement method is described later in detail.

Comparative Examples 13 and 14

[0104] In Comparative Example 13, a solution containing only the influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University) was prepared, and in Comparative Example 14, a phosphate buffer was prepared. The test was carried out by the same procedure as Example 10 in the doses shown in Table 3.

TABLE-US-00003 TABLE 3 Antigen Adjuvant Dose (g/ Substance Dose (g/ No. Type body/time) Ligand name body/time) Example 10 A/IvPR8/ 3 TLR4 Pantoea 5 34(H1N1) LPS Comparative A/IvPR8/ 3 Example 13 34(H1N1) Comparative Example 14

Example 11

[0105] Phosphate buffer (Nacalai Tesque, Inc.) and 30 L (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 10.1 L (1776 g/mL) of an influenza HA antigen-containing inactivated whole virus solution (A/California/7/2009(H1N1), The Research Foundation for Microbial Diseases of Osaka University) to prepare 120 L of a vaccine composition.

[0106] The test was carried out by the same procedure as Example 1 in the dose shown in Table 4.

Comparative Example 14

[0107] In Comparative Example 14, a solution containing only the influenza HA antigen-containing inactivated whole virus solution (A/California/7/2009 (H1N1)) was prepared. The test was carried out by the same procedure as Example 1 in the dose shown in Table 4.

TABLE-US-00004 TABLE 4 Antigen Adjuvant Dose (g/ Substance Dose (g/ No. Type body/time) Ligand name body/time) Example 11 Inactivated whole virus 3 TLR4 Pantoea 5 (A/California/7/2009(H1N1)) LPS Comparative Inactivated whole virus 3 Example 14 (A/California/7/2009(H1N1)) Comparative Example 1

Example 12

[0108] Phosphate buffer (Nacalai Tesque, Inc.) and 30 L (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 36 L (500 g/mL) of an influenza HA antigen-containing solution (A/California/7/2009(H1N1), The Research Foundation for Microbial Diseases of Osaka University) to prepare 120 L of a vaccine composition.

[0109] The test was carried out by the same procedure as Example 1 in the dose shown in Table 5.

Example 13

[0110] In place of the lipopolysaccharide derived from Pantoea used in Example 12, c-di-GMP (Cyclic diguanosine monophosphate, Biolog Inc.) was used in Example 13 to prepare a vaccine composition in the same manner as Example 12. The test was carried out by the same procedure as Example 1 in the dose shown in Table 5.

Comparative Example 15

[0111] In place of the lipopolysaccharide derived from Pantoea used in Example 12, imiquimod (InvivoGen) was used in Comparative Example 15 and the test was carried out by the same procedure as Example 1 in the dose shown in Table 5.

Comparative Example 16

[0112] In place of the lipopolysaccharide derived from Pantoea used in Example 12, a solution containing only the influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University) was prepared without adding any other components in Comparative Example 16. The test was carried out by the same procedure as Example 1 in the dose shown in Table 5.

TABLE-US-00005 TABLE 5 Antigen Adjuvant Dose (g/ Substance Dose (g/ No. Type body/time) Ligand name body/time) Example 12 A/California/7/2009 3 TLR4 Pantoea 5 (H1N1) LPS Example 13 A/California/7/2009 3 unclear c-di-GMP 5 (H1N1) Comparative A/California/7/2009 3 TLR7 imiquimod 5 Example 15 (H1N1) Comparative A/California/7/2009 3 Example 16 (H1N1) Comparative Example 1

Example 14

[0113] Phosphate buffer (Nacalai Tesque, Inc.) and 30 L (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 33.7 L (534 g/mL) of an influenza HA antigen-containing solution (A/Victria/361/2009(H3N2), The Research Foundation for Microbial Diseases of Osaka University) to prepare 120 L of a vaccine composition. The test was carried out by the same procedure as Example 1 in the dose shown in Table 6.

Example 15

[0114] Phosphate buffer (Nacalai Tesque, Inc.) and 30 L (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 47.4 L (380 g/mL) of the influenza HA antigen-containing solution (B/Brisbane/60/2008, The Research Foundation for Microbial Diseases of Osaka University) to prepare 120 L of a vaccine composition. The test was carried out by the same procedure as Example 1 in the dose shown in Table 6.

TABLE-US-00006 TABLE 6 Antigen Adjuvant Dose (g/ Substance Dose (g/ No. Type body/time) Ligand name body/time) Example 12 A/California/7/2009 3 TLR4 Pantoea 5 (H1N1) LPS Example 14 A/Victoria/361/2009 3 TLR4 Pantoea 5 (H3N2) LPS Example 15 B/Brisbane/60/2008 3 TLR4 Pantoea 5 LPS Comparative A/California/7/2009 3 Example 16 (H1N1) Comparative Example 1

Example 16

[0115] A vaccine composition was prepared in the same manner as Example 12. More specifically, phosphate buffer (Nacalai Tesque, Inc.) and 30 L (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 36 L (500 g/mL) of an influenza HA antigen-containing solution (A/California/7/2009(H1N1), The Research Foundation for Microbial Diseases of Osaka University) to prepare 120 L of a vaccine composition.

[0116] The test was carried out by the same procedure as Example 1 in the dose shown in Table 7, except that BALB/c mice were used.

Example 17

[0117] Phosphate buffer (Nacalai Tesque, Inc.) and 30 L (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 18 L (1 mg/mL) of OVA (Ovalbumin, Sigma-Aldrich Co. LLC.) to prepare 120 L of a vaccine composition.

[0118] The test was carried out by the same procedure as Example 1 in the dose shown in Table 7, except that BALB/c mice were used.

Comparative Example 17

[0119] In place of the lipopolysaccharide derived from Pantoea used in Example 16, a solution containing only the influenza HA antigen-containing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University) was prepared without adding any other components in Comparative Example 17. The test was carried out by the same procedure as Example 1 in the dose shown in Table 7, except that BALB/c mice were used.

Comparative Example 18

[0120] In place of the lipopolysaccharide derived from Pantoea used in Example 17, a solution containing only OVA (Ovalbumin, Sigma-Aldrich Co. LLC.) was prepared without adding any other components in Comparative Example 18. The test was carried out by the same procedure as Example 1 in the dose shown in Table 7, except that BALE/c mice were used.

TABLE-US-00007 TABLE 7 Antigen Adjuvant Dose (g/ Substance Dose (g/ No. Type body/time) Ligand name body/time) Example 16 A/California/7/2009 3 TLR4 Pantoea 5 (H1N1) LPS Example 17 Ovalbumin (OVA) 3 TLR4 Pantoea 5 LPS Comparative A/California/7/2009 3 Example 17 (H1N1) Comparative Ovalbumin (OVA) 3 Example 18

Test Method

[0121] The systemic immune response was evaluated by measuring the mouse serum influenza HA- or OVA-specific IgG titer. The mucosal immune response was evaluated by measuring the mouse nasal wash influenza HA- or OVA-specific IgA titer. Each of the evaluation method is described below. Each of the evaluation results are shown in FIGS. 1 to 13 (except FIG. 5).

[0122] A virus infection experiment was meanwhile carried out to reveal the vaccine efficacy by infecting the mice with the influenza virus. The evaluation results are shown in FIG. 5.

Measurement Method of Mouse Serum Influenza HA-Specific IgG Titer (ELISA)

[0123] 100 L of each influenza HA (e.g., A/IvPR8/34 (H1N1) influenza HA antigen solution for measuring an A/IvPR8/34(H1N1)-specific IgG antibody titer) diluted with carbonate buffer or an OVA-containing solution (5 g/mL) was added to an ELISA 96-well plate and allowed to stand overnight.

[0124] The wells were washed 3 times with a wash (Tween20-containing PBS) prepared in advance, and 200 L of a blocking solution wherein a blocking agent (Block Ace, DS Pharma Biomedical Co., Ltd.) was diluted to 4 g/100 mL with purified water was added to each well, which was allowed to stand at room temperature for 2 hours. Subsequently, the wells were washed 3 times with the wash (Tween20-containing PBS).

[0125] The serum collected from the mice in advance was centrifuged at 4 C. at 3000 G for 10 minutes, 300 L of phosphate buffer (Nacalai Tesque, Inc.) was added to 20 L of the supernatant to prepare a diluted serum solution.

[0126] Using a solution wherein a blocking agent (Block Ace, DS Pharma Biomedical Co., Ltd.) wad diluted to 0.4 g/100 mL with phosphate buffer (Nacalai Tesque, Inc.), the above diluted serum solution was diluted two-fold in 16 steps, and 50 L of the solution was added and allowed to stand at room temperature for 2 hours.

[0127] The wells were washed 3 times with a wash (Tween20-containing PBS), an HRP-labeled anti-mouse IgG antibody (Goat-anti-mouse IgG Fc HRP, BETHYL Laboratories, Inc.) was diluted 10000-fold with the solution wherein a blocking agent (Block Ace, DS Pharma Biomedical Co., Ltd.) was diluted to 0.4 g/100 mL with phosphate buffer (Nacalai Tesque, Inc.), and 100 L each was added and allowed to stand at room temperature for 1 hour.

[0128] Subsequently, the wells were washed 3 times with the wash (Tween20-containing PBS) and 100 L each of a TMB solution (ELISA POD TMB kit, Nacalai Tesque, Inc.) was added. 100 L of a 1M sulfuric acid solution was added thereto and the 96-well plate was measured using a microplate reader (168-11135CAM, Bio-rad Laboratories, Inc.) for the 450 nm absorbance. The mouse serum IgG titers were determined in log 2 based on the absorbance at the time of step dilution.

Measurement Method of Mouse Nasal Wash Influenza HA-Specific IgA Titer (ELISA)

[0129] 100 L of each influenza HA (e.g., A/IvPR8/34(H1N1) influenza HA for measuring an A/IvPR8/34 (H1N1)-specific IgA antibody titer) diluted with carbonate buffer or an OVA-containing solution (5 g/mL) was added to an ELISA 96-well plate and allowed to stand overnight.

[0130] The wells were washed 3 times with a wash (Tween20-containing PBS) prepared in advance, and 200 L of a blocking solution wherein a blocking agent (Block Ace, DS Pharma Biomedical Co., Ltd.) was diluted to 4 g/100 mL with purified water was added to each well, which was allowed to stand at room temperature for 2 hours.

[0131] The wells were subsequently washed 3 times with a wash (Tween20-containing PBS). The nasal washes collected from the mice were diluted two-fold in 12 steps with the solution wherein a blocking agent (Block Ace, DS Pharma Biomedical Co., Ltd.) wad diluted to 0.4 g/100 mL with phosphate buffer (Nacalai Tesque, Inc.), and 50 L each of the solution was added and allowed to stand at room temperature for 2 hours.

[0132] The wells were washed 3 times with a wash (Tween20-containing PBS), an HRP-labeled anti-mouse IgA antibody (Goat-anti-mouse IgA Fc HRP, BETHYL Laboratories, Inc.) was diluted 10000-fold with the solution wherein a blocking agent (Block Ace, DS Pharma Biomedical Co., Ltd.) was diluted to 0.4 g/100 mL with phosphate buffer (Nacalai Tesque, Inc.), and 100 L each thereof was added and allowed to stand at room temperature for 1 hour. Subsequently, the wells were washed 3 times with the wash (Tween20-containing PBS) and 100 L each of a TMB solution (ELISA POD TMB kit, Nacalai Tesque, Inc.) was added. 100 L of a 1M sulfuric acid solution was added thereto and the 96-well plate was measured using a microplate reader (168-11135CAM, Bio-rad Laboratories, Inc.) for the 450 nm absorbance. The mouse nasal wash IgA titers were determined in log 2 based on the absorbance at the time of step dilution.

A/IvPR8/34 (H1N1) Influenza Virus Infection Experiment to Mice (Survival Rate Measurement)

[0133] 15 L of diluted A/IvPR8/34 (H1N1) influenza virus was administered under anaesthesia to the vaccine-administered mice 1 week later from the final administration (10LD50: 50% Lethal Dose). Defining the virus administration day as Day 0, the mice were monitored up to Day 14 to measure the survival rates.

[0134] As shown in FIGS. 1 and 2, the influenza HA-specific IgG and IgA were produced in high yields in Examples 1 to 6, whereas the influenza-specific IgA was produced in low yields and the influenza HA-specific IgG was only somewhat produced in Comparative Examples 1 to 9. These results revealed that the TLR4 agonist and TLR2/6 agonist, cyclic dinucleotide are effective as adjuvants for inducing the mucosal immunity when sublingually administered.

[0135] As shown in FIGS. 3 and 4, the comparison between Examples 7 to 9 and Comparative Examples 10 to 12 revealed that the sufficient systemic immunity and mucosal immunity are likely to be induced by the sublingual administration even in the system wherein the dose of influenza HA antigen was reduced to 0.75 g.

[0136] As shown in FIG. 5, the comparison between Example 10 and Comparative Examples 13 and 14 revealed that the sublingual immunity provides the complete infection protection against the lethal dose of the influenza virus.

[0137] As shown in FIGS. 6 and 7, the comparison between Example 11 and Comparative Examples 14 and 1 revealed that the sufficient systemic immunity and mucosal immunity are likely to be induced in the inactivated whole virus vaccine when the adjuvants are used.

[0138] As shown in FIGS. 8 and 9, the comparison between Examples 12 and 13 and Comparative Examples 15, 16 and 1 revealed that the systemic immunity and mucosal immunity are likely to be induced in A/California/7/2009(H1N1), a seasonal influenza infectious to human.

[0139] As shown in FIGS. 10 and 11, the comparison between Examples 12, 14 and 15 and Comparative Examples 16 and 1 revealed that the sublingual immunity is likely to induce the systemic immunity and mucosal immunity against different types of influenza viruses.

[0140] As shown in FIGS. 12 and 13, the comparison between Examples 16 and 17 and Comparative Examples 17 and 18 revealed that the influenza HA-specific and OVA-specific systemic immunity and mucosal immunity are highly induced.

INDUSTRIAL APPLICABILITY

[0141] The vaccine composition of the present invention uses the specific adjuvants described above in combination with at least one antigen derived from an infectious disease, and thus effectively induces the systemic immune response and mucosal immune response even when administered to the oral cavity mucosa.