Mucosal vaccine composition

Abstract

The present invention aims at providing a mucosal vaccine composition that can be administered to an intraoral mucous membrane, ocular mucous membrane, ear mucous membrane, genital mucous membrane, pharyngeal mucous membrane, respiratory tract mucous membrane, bronchial mucous membrane, pulmonary mucous membrane, gastric mucous membrane, enteric mucous membrane, or rectal mucous membrane, that is useful as a prophylactic or therapeutic agent for infectious diseases or cancers, and is capable of safely and effectively inducing the systemic immune response and mucosal immune response. The present invention provides a mucosal vaccine composition to be administered to at least one mucous membrane selected from the group consisting of a human or animal intraoral mucous membrane, ocular mucous membrane, ear mucous membrane, genital mucous membrane, pharyngeal mucous membrane, respiratory tract mucous membrane, bronchial mucous membrane, pulmonary mucous membrane, gastric mucous membrane, enteric mucous membrane, and rectal mucous membrane, containing: at least one antigen; and as an adjuvant, a lipopolysaccharide derived from at least one gram-negative bacterium selected from the group consisting of Serratia, Leclercia, Rahnella, Acidicaldus, Acidiphilium, Acidisphaera, Acidocella, Acidomonas, Asaia, Belnapia, Craurococcus, Gluconacetobacter, Gluconobacter, Kozakia, Leahibacter, Muricoccus, Neoasaia, Oleomonas, Paracraurococcus, Rhodopila, Roseococcus, Rubritepida, Saccharibacter, Stella, Swaminathania, Teichococcus, Zavarzinia, Pseudomonas, Achromobacter, Bacillus, Methanoculleus, Methanosarcina, Clostridium, Micrococcus, Flavobacterium, Pantoea, Acetobacter, Zymomonas, Xanthomonas, and Enterobacter, or a salt thereof.

Claims

1. A method comprising: administering a mucosal vaccine composition to a mucous membrane selected from the group consisting of a human or animal intraoral mucous membrane, ocular mucous membrane, ear mucous membrane, genital mucous membrane, pharyngeal mucous membrane, respiratory tract mucous membrane, bronchial mucous membrane, pulmonary mucous membrane, gastric mucous membrane, enteric mucous membrane, and rectal mucous membrane, the mucosal vaccine composition comprising: at least one antigen derived from a pathogen; and as an adjuvant, a lipopolysaccharide derived from at least one gram-negative bacterium selected from the group consisting of Pantoea, Acetobacter, Zymomonas, and Xanthomonas, or a salt of the lipopolysaccharide; wherein the adjuvant does not comprise a proteosome.

2. The method according to claim 1, wherein: the mucosal vaccine composition is a liquid preparation, a nebular, a semisolid preparation, or a solid preparation, and the semi-solid preparation and the solid preparation dissolve by a body fluid and/or body temperature.

3. The method according to claim 2, wherein the mucosal vaccine composition is a solid preparation that dissolves by a body fluid and/or body temperature.

4. The method according to claim 1, wherein the method induces humoral immunity.

5. The method according to claim 2, wherein the method induces humoral immunity.

6. The method according to claim 3, wherein the method induces humoral immunity.

7. The method according to claim 1, wherein the lipopolysaccharide is derived from Pantoea agglomerans.

8. The method according to claim 1, wherein the solid preparation is a film preparation, a disintegrating tablet, or a soluble tablet.

9. The method according to claim 1, wherein the solid preparation is a soluble tablet.

10. The method according to claim 9, wherein the soluble tablet is a freeze-dried tablet.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a graph showing results of influenza HA (type B)-specific IgA titers in a mouse nasal cavity washing liquid in Example 1, and Comparative Examples 1 to 5.

(2) FIG. 2 is a graph showing results of influenza HA (type B)-specific IgG titers in a mouse serum in Example 1, and Comparative Examples 1 to 5.

(3) FIG. 3 is a graph showing results of influenza HA (H1N1)-specific IgA titers in a mouse nasal cavity washing liquid in Example 2, and Comparative Examples 6 to 10.

(4) FIG. 4 is a graph showing results of influenza HA (H1N1)-specific IgG titers in a mouse serum in Example 2, and Comparative Examples 6 to 10.

(5) FIG. 5 is a graph showing mouse survival rates in Reference Example 1, and Reference Comparative Examples 1 and 2.

(6) FIG. 6 is a graph showing results of influenza HA (H1N1)-specific IgA titers in a mouse nasal cavity washing liquid in Examples 2, 3, and 4, and Comparative Example 10.

(7) FIG. 7 is a graph showing results of influenza HA (H1N1)-specific IgG titers in a mouse serum in Examples 2, 3, and 4, and Comparative Example 10.

(8) FIG. 8 is a graph showing results of pneumococcal capsular polysaccharide-specific IgA titers in a mouse nasal cavity washing liquid in Example 5, and Comparative Examples 11 and 12.

(9) FIG. 9 is a graph showing results of pneumococcal capsular polysaccharide-specific IgG titers in a mouse serum in Example 5, and Comparative Examples 11 and 12.

(10) FIG. 10 is a graph showing results of HPV16-specific IgA titers in a mouse nasal cavity washing liquid in Example 6, and Comparative Examples 13 and 14.

(11) FIG. 11 is a graph showing results of HPV16-specific IgG titers in a mouse serum in Example 6, and Comparative Examples 13 and 14.

(12) FIG. 12 is a graph showing results of pneumococcal capsular polysaccharide-specific IgA titers in a mouse nasal cavity washing liquid in Examples 5, 7, and 8, and Comparative Example 12.

(13) FIG. 13 is a graph showing results of pneumococcal capsular polysaccharide-specific IgG titers in a mouse serum in Examples 5, 7, and 8, and Comparative Example 12.

(14) FIG. 14 is a graph showing results of HPV16-specific IgA titers in a mouse nasal cavity washing liquid in Examples 6, 9, and 10, and Comparative Example 14.

(15) FIG. 15 is a graph showing results of HPV16-specific IgG titers in a mouse serum in Examples 6, 9, and 10, and Comparative Example 14.

(16) FIG. 16 is a graph showing results of OVA-specific IgA titers in a mouse nasal cavity washing liquid in Example 25 and Comparative Example 15.

(17) FIG. 17 is a graph showing results of OVA-specific IgA titers in a mouse saliva in Example 25 and Comparative Example 15.

(18) FIG. 18 is a graph showing results of OVA-specific IgA titers in a mouse alveolus washing liquid in Example 25 and Comparative Example 15.

(19) FIG. 19 is a graph showing results of OVA-specific IgA titers in a mouse vaginal washing liquid in Example 25 and Comparative Example 15.

(20) FIG. 20 is a graph showing results of OVA-specific IgA titers in a mouse fecal extract in Example 25 and Comparative Example 15.

(21) FIG. 21 is a graph showing results of OVA-specific IgG titers in a mouse serum in Example 25 and Comparative Example 15.

(22) FIG. 22 is a graph showing results of OVA-specific IgA titers in a mouse nasal cavity washing liquid in Example 26 and Comparative Example 16.

(23) FIG. 23 is a graph showing results of OVA-specific IgA titers in a mouse saliva in Example 26 and Comparative Example 16.

(24) FIG. 24 is a graph showing results of OVA-specific IgA titers in a mouse alveolus washing liquid in Example 26 and Comparative Example 16.

(25) FIG. 25 is a graph showing results of OVA-specific IgA titers in a mouse vaginal washing liquid in Example 26 and Comparative Example 16.

(26) FIG. 26 is a graph showing results of OVA-specific IgA titers in a mouse fecal extract in Example 26 and Comparative Example 16.

(27) FIG. 27 is a graph showing results of OVA-specific IgG titers in a mouse serum in Example 26 and Comparative Example 16.

(28) FIG. 28 is a graph showing results of OVA-specific IgA titers in a mouse vaginal washing liquid in Example 27 and Comparative Example 17.

(29) FIG. 29 is a graph showing results of OVA-specific IgA titers in a mouse fecal extract in Example 27 and Comparative Example 17.

(30) FIG. 30 is a graph showing results of OVA-specific IgG titers in a mouse serum in Example 27 and Comparative Example 17.

(31) FIG. 31 is a graph showing results of OVA-specific IgA titers in a mouse vaginal washing liquid in Example 28 and Comparative Example 18.

(32) FIG. 32 is a graph showing results of OVA-specific IgA titers in a mouse fecal extract in Example 28 and Comparative Example 18.

(33) FIG. 33 is a graph showing results of OVA-specific IgG titers in a mouse serum in Example 28 and Comparative Example 18.

DESCRIPTION OF EMBODIMENTS

(34) The present invention will be described in more detail with reference to the following examples, but is not limited to these examples.

Example 1

(35) To 2.25 L (445 g/mL) of an influenza vaccine antigen-containing solution (B/Wisconsin/1/2010, available from The Research Foundation for Microbial Diseases of Osaka University), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans(available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 300 L of a mucosal vaccine composition.

(36) Six mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 30 L of the prepared vaccine composition was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and 30 L of the prepared vaccine composition was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and an influenza HA (type B)-specific IgG titer in a serum and an influenza HA (type B)-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later.

Comparative Examples 1 to 5

(37) A mucosal vaccine composition was prepared in the same manner as in Example 1 except that, in place of a lipopolysaccharide derived from Pantoea agglomerans, a lipopolysaccharide derived from Escherichia coli (available from WAKO) was used in Comparative Example 1, a lipopolysaccharide derived from Salmonella typhimurium (available from WAKO) was used in Comparative Example 2, glucopyranosyl lipid (MPLAs, available from InvivoGen) was used in Comparative Example 3, and Imiquimod (available from InvivoGen) was used in Comparative Example 4, and the test was conducted in the same manner as in Example 1 with the administration amount shown in Table 1. In Comparative Example 5, only a phosphate buffer (available from Nacalai Tesque) was administered to mice while a vaccine antigen and an adjuvant were not added.

(38) TABLE-US-00001 TABLE 1 Vaccine antigen Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Example 1 B/Wisconsin/1/2010 0.1 LPS derived from TLR4 1 Sublingual Pantoea agglomerans Comparative B/Wisconsin/1/2010 0.1 LPS derived from TLR4 1 Sublingual Example 1 Escherichia coli Comparative B/Wisconsin/1/2010 0.1 LPS derived from TLR4 1 Sublingual Example 2 Salmonella typhimurium Comparative B/Wisconsin/1/2010 0.1 Glucopyranosyl lipid TLR4 1 Sublingual Example 3 Comparative B/Wisconsin/1/2010 0.1 Imiquimod TLR7 1 Sublingual Example 4 Comparative Sublingual Example 5

Example 2

(39) To 1.25 L (801 g/mL) of an influenza vaccine antigen-containing solution (A/California/07/2009 (H1N1), available from The Research Foundation for Microbial Diseases of Osaka University), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 300 L of a mucosal vaccine composition.

(40) Six mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 30 L of the prepared mucosal vaccine composition was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and 30 L of the prepared mucosal vaccine composition was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and an influenza HA (H1N1)-specific IgG titer in a serum and an influenza HA (H1N1)-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later.

Comparative Examples 6 to 10

(41) A mucosal vaccine composition was prepared in the same manner as in Example 2 except that, in place of a lipopolysaccharide derived from Pantoea agglomerans, a lipopolysaccharide derived from Escherichia coli (available from WAKO) was used in Comparative Example 6, a lipopolysaccharide derived from Salmonella typhimurium (available from WAKO) was used in Comparative Example 7, glucopyranosyl lipid (MPLAs, available from InvivoGen) was used in Comparative Example 8, and Imiquimod (available from InvivoGen) was used in Comparative Example 9, and the test was conducted in the same manner as in Example 2 with the administration amount shown in Table 2. In Comparative Example 10, only a phosphate buffer (available from Nacalai Tesque) was administered to mice while a vaccine antigen and an adjuvant were not added.

(42) TABLE-US-00002 TABLE 2 Vaccine antigen Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Example 2 A/California/07/2009(H1N1) 0.1 LPS derived from TLR4 1 Sublingual Pantoea agglomerans Comparative A/California/07/2009(H1N1) 0.1 LPS derived from TLR4 1 Sublingual Example 6 Escherichia coli Comparative A/California/07/2009(H1N1) 0.1 LPS derived from TLR4 1 Sublingual Example 7 Salmonella typhimurium Comparative A/California/07/2009(H1N1) 0.1 Glucopyranosyl lipid TLR4 1 Sublingual Example 8 Comparative A/California/07/2009(H1N1) 0.1 Imiquimod TLR7 1 Sublingual Example 9 Comparative Sublingual Example 10

Reference Example 1

(43) A sample containing the same antigen and adjuvant as those in the sample administered in Example 1, and having the same (antigen/adjuvant) as Example 1 was prepared, and the safety of the sample was evaluated. To be more specific, to 225 L (445 g/mL) of an influenza vaccine antigen-containing solution (B/Wisconsin/1/2010, available from The Research Foundation for Microbial Diseases of Osaka University), and 500 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 1000 L of a vaccine composition.

(44) Eight mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 100 L of the prepared vaccine composition was subcutaneously administered to each mouse. The mice were followed up to 72 hours from the administration, and the survival rate was observed.

Reference Comparative Examples 1 and 2

(45) A vaccine composition was prepared in the same manner as in Reference Example 1 except that, in place of a lipopolysaccharide derived from Pantoea agglomerans, a lipopolysaccharide derived from Escherichia coli (available from WAKO) was used in Reference Comparative Example 1, and a lipopolysaccharide derived from Salmonella typhimurium (available from WAKO) was used in Reference Comparative Example 2, and the test was conducted in the same manner as in Reference Example 1 with the administration amount shown in Table 3.

(46) TABLE-US-00003 TABLE 3 Vaccine antige Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Reference B/Wisconsin/1/2010 10 LPS derived from TLR4 100 Subcutaneous Example 1 Pantoea agglomerans Reference B/Wisconsin/1/2010 10 LPS derived from TLR4 100 Subcutaneous Comparative Escherichia coli Example 1 Reference B/Wisconsin/1/2010 10 LPS derived from TLR4 100 Subcutaneous Comparative Salmonella typhimurium Example 2

Examples 3 and 4

(47) To 2.5 L (801 g/mL) of an influenza vaccine antigen-containing solution (A/California/07/2009 (H1N1), available from The Research Foundation for Microbial Diseases of Osaka University), and 10 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), 45 mg of hydroxypropylcellulose (HPC-SSL, available from Nippon Soda Co., Ltd.) as a base material was added, and a phosphate buffer (available from Nacalai Tesque) was added and mixed uniformly to give 500 mg of a mixture. Then, the mixture was dispensed by 25 mg, and freeze-dried to prepare rapid soluble tablets in Example 3, and was dried under reduced pressure to prepare film preparations in Example 4.

(48) Six mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and the prepared rapid soluble tablet or film preparation was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and the prepared rapid soluble tablet or film preparation was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and an influenza HA (H1N1)-specific IgG titer in a serum and an influenza HA (H1N1)-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later. In Table 4, results of Example 2 and Comparative Example 10 are also shown.

(49) TABLE-US-00004 TABLE 4 Vaccine antigen Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Note Example 3 A/California/07/2009(H1N1) 0.1 LPS derived from TLR4 1 Sublingual Dry Pantoea agglomerans preparation Example 4 A/California/07/2009(H1N1) 0.1 LPS derived from TLR4 1 Sublingual Film Pantoea agglomerans Example 2 A/California/07/2009(H1N1) 0.1 LPS derived from TLR4 1 Sublingual Liquid Pantoea agglomerans Comparative Sublingual Example 10

Example 5

(50) To 87 L (1150 g/mL) of a pneumococcal capsular polysaccharide-containing solution (PNEUMOVAX NP, MSD K.K.), and 2.5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 100 L of a mucosal vaccine composition.

(51) Four mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 20 L of the prepared mucosal vaccine composition was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and 20 L of the prepared mucosal vaccine composition was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and a pneumococcal capsular polysaccharide-specific IgG titer in a serum and a pneumococcal capsular polysaccharide-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later.

Comparative Examples 11 and 12

(52) A mucosal vaccine composition was prepared in the same manner as in Example 5 except that, in place of a lipopolysaccharide derived from Pantoea agglomerans, glucopyranosyl lipid (MPLAs, available from InvivoGen) was used in Comparative Example 11, and the test was conducted in the same manner as in Example 5 with the administration amount shown in Table 5. In Comparative Example 12, only a phosphate buffer (available from Nacalai Tesque) was administered to mice while a vaccine antigen and an adjuvant were not added.

(53) TABLE-US-00005 TABLE 5 Vaccine antigen Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Example 5 Pneumococcal capsular 20 LPS derived from TLR4 1 Sublingual polysaccharide Pneumovax NP Pantoea agglomerans Comparative Pneumococcal capsular 20 Glucopyranosyl lipid TLR4 1 Sublingual Example 11 polysaccharide Pneumovax NP Comparative Sublingual Example 12

Example 6

(54) To 61 L (820 g/mL) of an HPV16 recombinant protein-containing solution (HPV16, available from PROSPEC), and 2.5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 100 L of a mucosal vaccine composition.

(55) Four mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 20 L of the prepared mucosal vaccine composition was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and 20 L of the prepared mucosal vaccine composition was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and an HPV16-specific IgG titer in a serum and an HPV16-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later.

Comparative Examples 13 and 14

(56) A mucosal vaccine composition was prepared in the same manner as in Example 6 except that, in place of a lipopolysaccharide derived from Pantoea agglomerans, glucopyranosyl lipid (MPLAs, available from InvivoGen) was used in Comparative Example 13, and the test was conducted in the same manner as in Example 6 with the administration amount shown in Table 6. In Comparative Example 14, only a phosphate buffer (available from Nacalai Tesque) was administered to mice while a vaccine antigen and an adjuvant were not added.

(57) TABLE-US-00006 TABLE 6 Vaccine antige Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Example 6 HPV16 recombinant 10 LPS derived from TLR4 1 Sublingual protein Pantoea agglomerans Comparative HPV16 recombinant 10 Glucopyranosyl lipid TLR4 1 Sublingual Example 13 protein Comparative Sublingual Example 14

Examples 7 and 8

(58) To 174 L (1150 g/mL) of a pneumococcal capsular polysaccharide-containing solution (PNEUMOVAX NP, available from MSD K.K.), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), 22.5 mg of hydroxypropylcellulose (HPC-SSL, available from Nippon Soda Co., Ltd.) was added as a base material, and a phosphate buffer (available from Nacalai Tesque) was added and mixed uniformly to give 250 mg of a mixture. Then, the mixture was dispensed by 25 mg, and freeze-dried to prepare rapid soluble tablets in Example 7, and was dried under reduced pressure to prepare film preparations in Example 8.

(59) Four mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and the prepared rapid soluble tablet or film preparation was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and the prepared rapid soluble tablet or film preparation was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and a PNEUMOVAX NP-specific IgG titer in a serum and a PNEUMOVAX NP-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later. In Table 7, results of Example 5 and Comparative Example 12 are also shown.

(60) TABLE-US-00007 TABLE 7 Vaccine antigen Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Note Example 7 Pneumococcal capsular 20 LPS derived from TLR4 1 Sublingual Dry polysaccharide Pneumovax NP Pantoea agglomerans preparation Example 8 Pneumococcal capsular 20 LPS derlved from TLR4 1 Sublingual Flm polysaccharide Pneumovax NP Pantoea agglomerans Example 5 Pneumococcal capsular 20 LPS derived from TLR4 1 Sublingual Liquid polysaccharide Pneumovax NP Pantoea agglomerans Comparative Sublingual Example 12

Examples 9 and 10

(61) To 122 L (820 g/mL) of an HPV16 recombinant protein-containing solution (HPV16, available from PROSPEC), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), 22.5 mg of hydroxypropylcellulose (HPC-SSL, available from Nippon Soda Co., Ltd.) was added as a base material, and a phosphate buffer (available from Nacalai Tesque) was added and mixed uniformly to give 250 mg of a mixture. Then, the mixture was dispensed by 25 mg, and freeze-dried to prepare rapid soluble tablets in Example 9, and was dried under reduced pressure to prepare film preparations in Example 10.

(62) Four mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and the prepared rapid soluble tablet or film preparation was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and the prepared rapid soluble tablet or film preparation was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and an HPV16-specific IgG titer in a serum and an HPV16-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later. In Table 8, results of Example 6 and Comparative Example 14 are also shown.

(63) TABLE-US-00008 TABLE 8 Vaccine antigen Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Note Example 9 HPV16 recombinant 10 LPS derived from TLR4 1 Sublingual Dry protein Pantoea agglomerans preparation Example 10 HPV16 recombinant 10 LPS derived from TLR4 1 Sublingual Film protein Pantoea agglomerans Example 6 HPV16 recombinant 10 LPS derived from TLR4 1 Sublingual Liquid protein Pantoea agglomerans Comparative Sublingual Example 14

Example 11

(64) To 1000 L of an attenuated live rotavirus-containing solution (ROTATEQ mixture for internal use, available from MSD K.K.), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), 22.5 mg of hydroxypropylcellulose (HPC-SSL, available from Nippon Soda Co., Ltd.) was added as a base material to give 1005 L of a mixture. Then, the mixture was dispensed by 100 L, and freeze-dried to prepare rapid soluble tablets.

(65) Four mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and the prepared rapid soluble tablet was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and the prepared rapid soluble tablet was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and an attenuated live rotavirus-specific IgG titer in a serum and an attenuated live rotavirus-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method.

Examples 12 to 22

(66) A rapid soluble tablet was prepared in the same manner as in Example 11 by using an inactivated poliovirus-containing solution (IMOVAX POLIO subcutaneous, available from Sanofi K.K.) in Example 12, an inactivated hepatitis A virus-containing solution (Aimmugen, available from KAKETSUKEN) in Example 13, an inactivated Japanese encephalitis virus-containing solution (ENCEVAC for subcutaneous injection, available from KAKETSUKEN) in Example 14, an attenuated live mumps virus-containing solution (mumps live vaccine, available from KITASATO DAIICHISANKYO VACCINE CO., LTD.) in Example 15, an attenuated live measles virus-containing solution (measles live vaccine, available from KITASATO DAIICHISANKYO VACCINE CO., LTD.) in Example 16, an attenuated live rubella virus-containing solution (dry attenuated live rubella vaccine, available from KITASATO DAIICHISANKYO VACCINE CO., LTD.) in Example 17, a tetanus toxoid conjugate Haemophilus influenzae type b polysaccharide-containing solution (ActHIB, available from Sanofi K.K.) in Example 18, a recombinant HBs antigen protein-containing solution (Bimmugen, available from KAKETSUKEN) in Example 19, an attenuated live yellow fever virus-containing solution (yellow fever vaccine, available from Sanofi K.K.) in Example 20, a tetanus toxoid-containing solution (tetanus toxoid, available from DENKA SEIKEN CO., LTD.) in Example 21, and an attenuated live chickenpox virus-containing solution (dry attenuated live chickenpox vaccine, available from The Research Foundation for Microbial Diseases of Osaka University) in Example 22. Also immunological experiments are conducted as described in Example 12.

Example 23

(67) To 300 L of a live BCG-containing solution (dry BCG vaccine, available from Japan BCG Laboratory), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), 22.5 mg of hydroxypropylcellulose (HPC-SSL, available from Nippon Soda Co., Ltd.) was added as a base material to give 305 L of a mixture. Then, the mixture was dispensed by 30 L, and freeze-dried to prepare rapid soluble tablets.

(68) Four mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and the prepared rapid soluble tablet was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and the prepared rapid soluble tablet was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and a live BCG-specific IgG titer in a serum and an attenuated live BCG-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method.

Example 24

(69) To 2000 L of an inactivated rabies virus-containing solution (tissue-cultured inactivated rabies vaccine, available from KAKETSUKEN), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), 22.5 mg of hydroxypropylcellulose (HPC-SSL, available from Nippon Soda Co., Ltd.) was added as a base material to give 2005 L of a mixture. Then, the mixture was dispensed by 200 L, and freeze-dried to prepare rapid soluble tablets.

(70) Four mice (female BALE/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and the prepared rapid soluble tablet was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and the prepared rapid soluble tablet was sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse were collected, and an inactivated rabies virus-specific IgG titer in a serum and an inactivated rabies virus-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method.

(71) TABLE-US-00009 TABLE 9 Vaccine antigen Adjuvant Amount Amount [g/ Administration No. Species [/mouse/dose] Substance name Ligand mouse/dose] route Note Example 11 Live attenuated rotavirus Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry (RIX4414 strain) Pantoea agglomerans preparation Example 12 Inactivated poliovirus Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry (type 1, type 2, type 3) Pantoea agglomerans preparation Example 13 Inactivated hepatitis Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry A virus antigen Pantoea agglomerans preparation Example 14 Inactivated Japanese Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry encephalitis virus Pantoea agglomerans preparation Example 15 Live attenuated Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry mumps virus Pantoea agglomerans preparation Example 16 Live attenuated Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry measles virus Pantoea agglomerans preparation Example 17 Live attenuated Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry rubella virus Pantoea agglomerans preparation Example 18 Tetanus toxoid- Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry conjugated Haemophilus Pantoea agglomerans preparation influenzae type b polysaccharide Example 19 Recombinant HBs Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry antigen protein Pantoea agglomerans preparation Example 20 Live attenuated Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry yellow fever virus Pantoea agglomerans preparation Example 21 Tetanus toxoid Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry Pantoea agglomerans preparation Example 22 Live attenuated Vaccine 100 uL equivalent LPS derived from TLR4 1 Sublingual Dry varicella-zoster virus Pantoea agglomerans preparation Example 23 Live BCG Vaccine 30 uL equivalent LPS derived from TLR4 1 Sublingual Dry Pantoea agglomerans preparation Example 24 Inactivated rabies virus Vaccine 200 uL equivalent LPS derived from TLR4 1 Sublingual Dry Pantoea agglomerans preparation

Example 25

(72) To 100 L (1000 g/mL) of ovalbumin (OVA) (Sigma-Aldrich Japan), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 200 L of a mucosal vaccine composition.

(73) Six mice (female BALE/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 20 L of the prepared vaccine composition was sublingually administered to each mouse. After one week from the administration, the mice were anesthetized again, and sublingual administration was conducted to each mouse in the same manner. After one week from the second administration, a serum and mucosal samples of each mouse were collected, and an ovalbumin-specific IgG titer in a serum, and ovalbumin-specific IgA titers in a nasal cavity washing liquid, saliva, an alveolus washing liquid, a vaginal washing liquid, and a fecal extract were determined by the ELISA method. Specific determination methods will be described later.

Comparative Example 15

(74) To 100 L (1000 g/mL) of ovalbumin (OVA) (Sigma-Aldrich Japan), a phosphate buffer (available from Nacalai Tesque) was added to prepare 300 L of a mucosal vaccine composition. The subsequent operation and evaluation are as described in Example 25.

(75) TABLE-US-00010 TABLE 10 Vaccine antigen Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Example 25 Ovalbumin 10 LPS derived from TLR4 1 Sublingual Pantoea agglomerans Comparative Ovalbumin 10 Sublingual Example 15

Example 26

(76) To 100 L (1000 g/mL) of ovalbumin (OVA) (Sigma-Aldrich Japan), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 500 L of a mucosal vaccine composition.

(77) Six mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 50 L of the prepared vaccine composition was spray-administered to the bronchial tube of each mouse using a liquid sprayer (available from Penn-Century, Inc.). After one week from the administration, the mice were anesthetized again, and pulmonary administration was conducted to each mouse in the same manner. After one week from the second administration, a serum and mucosal samples of each mouse were collected, and an ovalbumin-specific IgG titer in a serum, and ovalbumin-specific IgA titers in a nasal cavity washing liquid, saliva, an alveolus washing liquid, a vaginal washing liquid, and a fecal extract were determined by the ELISA method. Specific determination methods will be described later.

Comparative Example 16

(78) To 100 L (1000 g/mL) of ovalbumin (OVA) (Sigma-Aldrich Japan), a phosphate buffer (available from Nacalai Tesque) was added to prepare 500 L of a mucosal vaccine composition. The subsequent operation and evaluation are as described in Example 26.

(79) TABLE-US-00011 TABLE 11 Vaccine antigen Adjuvant Amount [g/ Amount [g/ Administration No. Species mouse/dose] Substance name Ligand mouse/dose] route Example 26 Ovalbumin 10 LPS derived from TLR4 1 Pulmonary Pantoea agglomerans Comparative Ovalbumin 10 Pulmonary Example 16

Example 27

(80) To 100 L (1000 g/mL) of ovalbumin (OVA) (Sigma-Aldrich Japan), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 200 L of a mucosal vaccine composition.

(81) Six mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 20 L of the prepared vaccine composition was administered to the vagina of each mouse with the use of a pipette. After one week from the administration, the mice were anesthetized again, and vaginal administration was conducted to each mouse in the same manner. After one week from the second administration, a serum and mucosal samples of each mouse were collected, and an ovalbumin-specific IgG titer in a serum, and ovalbumin-specific IgA titers in a vaginal washing liquid and a fecal extract were determined by the ELISA method. Specific determination methods will be described later.

Comparative Example 17

(82) To 100 L (1000 g/mL) of ovalbumin (OVA) (Sigma-Aldrich Japan), a phosphate buffer (available from Nacalai Tesque) was added to prepare 200 L of a mucosal vaccine composition. The subsequent operation and evaluation are as described in Example 27.

(83) TABLE-US-00012 TABLE 12 Vaccine antigen Adjuvant Amount Amount [g/ Administration No. Species [g/mouse/dose] Substance name Ligand mouse/dose] route Example 27 Ovalbumin 10 LPS derived from TLR4 1 Transvaginal Pantoea agglomerans Comparative Ovalbumin 10 Transvaginal Example 17

Example 28

(84) To 100 L (1000 g/mL) of ovalbumin (OVA) (Sigma-Aldrich Japan), and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), a phosphate buffer (available from Nacalai Tesque) was added to prepare 500 L of a mucosal vaccine composition.

(85) Six mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) were anesthetized, and 50 L of the prepared vaccine composition was administered to the rectum of each mouse with the use of a 1 mL syringe and a sonde for mouse (Fuchigami Kikai). After one week from the administration, the mice were anesthetized again, and rectal administration was conducted to each mouse in the same manner. After one week from the second administration, a serum and mucosal samples of each mouse were collected, and an ovalbumin-specific IgG titer in a serum, and ovalbumin-specific IgA titers in a vaginal washing liquid and a fecal extract were determined by the ELISA method. Specific determination methods will be described later.

Comparative Example 18

(86) To 100 L (1000 g/mL) of ovalbumin (OVA) (Sigma-Aldrich Japan), a phosphate buffer (available from Nacalai Tesque) was added to prepare 500 L of a mucosal vaccine composition. The subsequent operation and evaluation are as described in Example 28.

(87) TABLE-US-00013 TABLE 13 Vaccine antigen Adjuvant Amount Amount [g/mouse/ [g/mouse/ Administration No. Species dose] Substance name Ligand dose] route Example 28 Ovalbumin 10 LPS derived from TLR4 1 Rectal Pantoea agglomerans Comparative Ovalbumin 10 Rectal Example 18
(Mouse Immunological Experiments)

(88) For female BALB/c mice aged 8 weeks, administration was conducted twice at an interval of one week. After one week from the last administration, blood and a nasal cavity washing liquid of each mouse were collected. The blood was centrifuged at 3000 G for 10 minutes at 4 C., and 300 L of a phosphate buffer (available from Nacalai Tesque) was added to 20 L of the supernatant to prepare a serum sample. Mucous membrane samples were collected in the following manner. Regarding a nasal cavity washing liquid, a cut was made in a lower part of the respiratory tract of a BALB/c mouse, 200 L of a phosphate buffer (available from Nacalai Tesque) was poured into the respiratory tract, and a sample came into the nasal cavity was collected as a nasal cavity washing liquid sample. Regarding saliva, 500 L of 12 g/mL carbamylcholine chloride was administered to the abdominal cavity of a mouse to promote production of saliva, and then 20 L of saliva was collected. Regarding an alveolus washing liquid, a cut was made in a lower part of the respiratory tract of a BALB/c mouse, 500 L of a phosphate buffer (available from Nacalai Tesque) was poured into the lung, and the phosphate buffer came into the lung was collected as an alveolus washing liquid sample. Regarding a vaginal washing liquid, 150 L of a phosphate buffer (available from Nacalai Tesque) was poured into the vagina of a BALB/c mouse, and a sample after pipetting 10 times was collected as a vaginal washing liquid sample. Regarding a fecal extract, 100 L of a phosphate buffer (available from Nacalai Tesque) per 10 mg of collected faces was added, and the mixture was vortexed for 10 minutes. Thereafter, centrifugation at 3000 G was conducted for 10 minutes at 4 C., and the supernatant was collected as a fecal extract sample. By measuring an immunogen-specific IgG titer in a mouse serum, the systemic immune response was evaluated. Also, by measuring an immunogen-specific IgA titer in a mouse mucous membrane sample, the mucosal immune response was evaluated. The respective evaluation methods will be described below.

(89) The respective evaluation results are shown in FIGS. 1 to 4 and 6 to 33.

(90) (Method for Measuring Antigen-Specific IgG Titer in Mouse Serum (ELISA Method))

(91) In a 96-well plate for ELISA, each 100 L of each antigen (for example, a B/Wisconsin/1/2010(B) influenza HA antigen solution in measurement of a B/Wisconsin/1/2010(B)-specific IgG antibody titer) diluted with a carbonate buffer (2.5 g/mL) was added, and the plate was left still overnight.

(92) Wells were washed with a preliminarily prepared TWEEN 20-containing PBS (hereinafter, referred to as a washing liquid) three times, and after adding each 200 L of a blocking solution prepared by diluting a blocking agent (BLOCK ACE, available from DS Pharma Biomedical Co., Ltd.) in purified water into 4 g/400 mL, the plate was left still for 2 hours at room temperature. Then, wells were washed with the washing liquid three times.

(93) Using a solution prepared by diluting a blocking agent (BLOCK ACE, available from DS Pharma Biomedical Co., Ltd.) with a phosphate buffer (available from Nacalai Tesque) into 0.4 g/100 mL (hereinafter, referred to as a reagent diluent), the aforementioned serum sample was diluted 15 times by doubling serial dilution, each 50 L of the solutions were added, and the plate was left still for 2 hours at room temperature.

(94) Then, the wells were washed three times with a washing liquid, and each 100 L of an HRP-labeled anti-mouse IgG antibody (Goat-anti-mouse IgG Fc HRP, available from BETHYL) diluted 10000 times with the reagent diluent was added, and the plate was left still for 1 hour at room temperature.

(95) Then, the wells were washed three times with a washing liquid, and each 100 L of a TMB solution (ELISA POD TMB kit, available from Nacalai Tesque) was added. Then, each 100 L of a 1 M sulfuric acid solution was added, and absorbance at 450 nm of the 96-well plate was measured by a micro plate reader (168-11135CAM, available from Bio-Rad). Based on the absorbance in the serial dilution, the maximum dilution fold at which the absorbance was not less than 0.1 was determined as an IgG titer in a mouse serum, and the value was determined as a value of Log 2.

(96) (Method for Measuring Antigen-Specific IgA Titer in Mouse Mucous Membrane Sample Washing Liquid (ELISA Method))

(97) The method is basically the same as the method for measuring antigen-specific IgG titer, and the same operations were conducted except that the measurement sample was various mucous membrane samples, and an HRP-labeled anti-mouse IgA antibody (Goat-anti-mouse IgA a HRP, available from BETHYL) was used in place of the HRP-labeled anti-mouse IgG antibody.

(98) (Examination Regarding Safety of LPS)

(99) Each 100 L of samples of vaccine compositions according to Reference Example 1 and Reference Comparative Examples 1 and 2 containing a type B vaccine and various LPSs was subcutaneously administered to BALB/c mice be injection. As the follow-up, the conditions of the mice were checked every 24 hours, and life or death thereof was observed. The observation was continued to 72 hours after the administration, and the survival rate was calculated. The result is shown in FIG. 5. The evaluation results are adopted as the result of safety of an LPS in mucosal vaccine compositions.

(100) As shown in FIGS. 1 to 4 and 6 to 15, according to the examples and comparative examples, antigen-specific IgG and IgA were produced at high levels by the use of a lipopolysaccharide. On the other hand, in other comparative examples, the production amount was low with respect to the antigen-specific IgA although antigen-specific IgG was produced in some comparative examples. These results reveal that a lipopolysaccharide or a salt thereof as an adjuvant is effective for the sublingual mucosal immune induction. Also, as can be seen in FIGS. 16 to 33, it was confirmed that by administering an antigen and a lipopolysaccharide to a mucous membrane, immunity is induced not only on the mucosal surface but also on a remote mucosal surface (for example, when an antigen and a lipopolysaccharide were administered sublingually, production of antigen-specific IgA was observed on the intestinal tract and the vaginal surface). That is, it was found that a lipopolysaccharide or a salt thereof functions as an adjuvant that is effective on any mucosal surface, and is capable of sufficiently inducing antigen-specific IgA all over the body.

(101) Also, as shown in FIG. 5, a vaccine composition containing a lipopolysaccharide derived from Pantoea agglomerans, a vaccine composition containing a lipopolysaccharide derived from Escherichia coli, and a vaccine composition containing a lipopolysaccharide derived from Salmonella typhimurium were compared by injection immunization, and it was confirmed that the safety of the vaccine composition containing a lipopolysaccharide derived from Pantoea agglomerans was high.

(102) Therefore, considering both the immune inducing effect of mucosal administration and the safety of the administered composition, excellence of the vaccine composition containing a lipopolysaccharide derived from Pantoea agglomerans was confirmed.

INDUSTRIAL APPLICABILITY

(103) Since the mucosal vaccine composition of the present invention contains the aforementioned specific adjuvant together with at least one antigen, it can induce the systemic immune response and mucosal immune response safely and effectively even when it is administered to an intraoral mucous membrane, ocular mucous membrane, ear mucous membrane, genital mucous membrane, pharyngeal mucous membrane, respiratory tract mucous membrane, bronchial mucous membrane, pulmonary mucous membrane, gastric mucous membrane, enteric mucous membrane, or rectal mucous membrane.