Mucosal vaccine composition

Abstract

The present disclosure relates to a method for inducing humoral immunity in a human or animal, that includes administering to the human or animal 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 includes 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 mass ratio between a total mass of the adjuvant and a total mass of the antigen is 0.002 to 500. The mucosal vaccine composition is administered in an amount effective to induce humoral immunity in the human or animal.

Claims

1. A method for inducing humoral immunity in a human or animal, comprising: administering to the human or animal 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: a mass ratio between a total mass of the adjuvant and a total mass of the antigen is 0.002 to 500; and the mucosal vaccine composition is administered in an amount effective to induce humoral immunity in the human or animal.

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, wherein 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 lipopolysaccharide is derived from Pantoea agglomerans.

5. The method according to claim 1, wherein the mass ratio between the total mass of the adjuvant and the total mass of the antigen is 0.01 to 100.

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 Examples 1 to 10, 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 Examples 1 to 10, 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 Examples 11 to 20, and Comparative Examples 6 to 9.

(4) FIG. 4 is a graph showing results of influenza HA (H1N1)-specific IgG titers in a mouse serum in Examples 11 to 20, and Comparative Examples 6 to 9.

(5) FIG. 5 is a graph showing results of pneumococcal-specific IgA titers in a mouse nasal cavity washing liquid in Examples 21 to 24, and Comparative Examples 10 to 12.

(6) FIG. 6 is a graph showing results of pneumococcal-specific IgG titers in a mouse serum in Examples 21 to 24, and Comparative Examples 10 to 12.

(7) FIG. 7 is a graph showing results of HPV16-specific IgA titers in a mouse nasal cavity washing liquid in Examples 25 to 28, and Comparative Examples 13 to 15.

(8) FIG. 8 is a graph showing results of HPV16-specific IgG titers in a mouse serum in Examples 25 to 28, and Comparative Examples 13 to 15.

(9) FIG. 9 is a graph showing results of OVA-specific IgA titers in a mouse nasal cavity washing liquid in Examples 71 to 73, and Comparative Example 30.

(10) FIG. 10 is a graph showing results of OVA-specific IgA titers in mouse saliva in Examples 71 to 73, and Comparative Example 30.

(11) FIG. 11 is a graph showing results of OVA-specific IgA titers in a mouse alveolus washing liquid in Examples 71 to 73, and Comparative Example 30.

(12) FIG. 12 is a graph showing results of an OVA-specific IgA titer in a mouse vaginal washing liquid in Examples 71 to 73, and Comparative Example 30.

(13) FIG. 13 is a graph showing results of OVA-specific IgA titers in a mouse fecal extract in Examples 71 to 73, and Comparative Example 30.

(14) FIG. 14 is a graph showing results of OVA-specific IgG titers in a mouse serum in Examples 71 to 73, and Comparative Example 30.

(15) FIG. 15 is a graph showing results of OVA-specific IgA titers in a mouse nasal cavity washing liquid in Examples 74 to 76, and Comparative Example 31.

(16) FIG. 16 is a graph showing results of OVA-specific IgA titers in mouse saliva in Examples 74 to 76, and Comparative Example 31.

(17) FIG. 17 is a graph showing results of OVA-specific IgA titers in a mouse alveolus washing liquid in Examples 74 to 76, and Comparative Example 31.

(18) FIG. 18 is a graph showing results of OVA-specific IgA titers in a mouse vaginal washing liquid in Examples 74 to 76, and Comparative Example 31.

(19) FIG. 19 is a graph showing results of OVA-specific IgA titers in a mouse fecal extract in Examples 74 to 76, and Comparative Example 31.

(20) FIG. 20 is a graph showing results of OVA-specific IgG titers in a mouse serum in Examples 74 to 76, and Comparative Example 31.

(21) FIG. 21 is a graph showing results of OVA-specific IgA titers in a mouse vaginal washing liquid in Examples 77 to 79, and Comparative Example 32.

(22) FIG. 22 is a graph showing results of OVA-specific IgA titers in a mouse fecal extract in Examples 77 to 79, and Comparative Example 32.

(23) FIG. 23 is a graph showing results of OVA-specific IgG titers in a mouse serum in Examples 77 to 79, and Comparative Example 32.

(24) FIG. 24 is a graph showing results of OVA-specific IgA titers in a mouse vaginal washing liquid in Examples 80 to 82, and Comparative Example 33.

(25) FIG. 25 is a graph showing results of an OVA-specific IgA titer in a mouse fecal extract in Examples 80 to 82, and Comparative Example 33.

(26) FIG. 26 is a graph showing results of OVA-specific IgG titers in a mouse serum in Examples 80 to 82, and Comparative Example 33.

DESCRIPTION OF EMBODIMENTS

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

(28) Each of the following administration groups was prepared for ten animals.

(29) As an appropriate dose of a vaccine antigen to mice, two patterns of 1.0 g and 0.1 g were examined in the case of an influenza vaccine. If the dose is more than 1.0 g, an antibody may be produced even in the absence of an adjuvant. On the other hand, if the dose is less than 0.1 g, an antibody may not be produced even in the presence of an adjuvant because the amount of the vaccine antigen is too small.

Examples 1 to 10, Comparative Examples 1 to 5

(30) An influenza vaccine antigen-containing solution (B/Wisconsin/1/2010, produced by The Research Foundation for Microbial Diseases of Osaka University) (445 g/mL), and a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Institute of applied technology for innate immunity) (50 mg/mL) were prepared to give doses in each group of table 1, and then a phosphate buffer (available from Nacalai Tesque) was added to prepare 300 L of a vaccine composition. For example, in Example 1, after adding 22.5 L of the influenza vaccine antigen-containing solution, and 20 L of the solution of a lipopolysaccharide derived from Pantoea agglomerans, a phosphate buffer was added to make the total amount 300 L. For other examples and comparative examples, vaccine compositions were prepared to have contents corresponding to the doses by appropriate dilution, and in Comparative Example 5, only a phosphate buffer (available from Nacalai Tesque) was administered to mice without adding a vaccine antigen or an adjuvant.

(31) 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.

(32) In the group in which 1000 g of the adjuvant was administered (Comparative Example 1), impairment in the lie of hair, and weight loss of mice were observed after 24 hours from the first administration, and the mice were euthanized. Therefore, the subsequent measurement of the antibody titer was not conducted. An adjuvant is a substance that activates immunity, and it is apparent that the immunity can be obtained more easily as the amount added increases. However, administering an excessive amount is problematic in terms of safety, and administration of 1000 g in mice was not conducted after Comparative Example 1.

(33) Specific determination methods will be described later.

(34) TABLE-US-00001 TABLE 1 Adjuvant (LPS derived Vaccine antigen from Pantoea agglomerans) Amount Amount Ratio Administration No. Species [g/mouse/dose] [g/mouse/dose] (adjuvant/antigen) route Comaprative B/Wisconsin/1/2010 1 1000 1000 Sublingual Example 1 Example 1 B/Wisconsin/1/2010 1 100 100 Sublingual Example 2 B/Wisconsin/1/2010 1 10 10 Sublingual Example 3 B/Wisconsin/1/2010 1 1 1 Sublingual Example 4 B/Wisconsin/1/2010 1 0.1 0.1 Sublingual Example 5 B/Wisconsin/1/2010 1 0.01 0.01 Sublingual Comaprative B/Wisconsin/1/2010 1 0.001 0.001 Sublingual Example 2 Comaprative B/Wisconsin/1/2010 1 0 0 Sublingual Example 3 Example 6 B/Wisconsin/1/2010 0.1 10 100 Sublineual Example 7 B/Wisconsin/1/2010 0.1 1 10 Sublingual Example 8 B/Wisconsin/1/2010 0.1 0.1 1 Sublingual Example 9 B/Wisconsin/1/2010 0.1 0.01 0.1 Sublingual Example 10 B/Wisconsin/1/2010 0.1 0.001 0.01 Sublingual Comparative B/Wisconsin/1/2010 0.1 0 0 Sublingual Example 4 Comparative Sublingual Example 5

Examples 11 to 20, Comparative Examples 6 to 9

(35) Vaccine compositions corresponding to Table 2 were prepared in the procedure based on that in Examples 1 to 10 and Comparative Examples 1 to 5 except that the influenza vaccine antigen-containing solution was changed from B/Wisconsin/1/2010 to A/California/07/2009 (H1N1, produced by The Research Foundation for Microbial Diseases of Osaka University) (801 g/mL). For example, in Example 11, after adding 12.5 L of an influenza vaccine antigen-containing solution and 20 L of a solution of a lipopolysaccharide derived from Pantoea agglomerans, a phosphate buffer was added to make the total amount 300 L.

(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 (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.

(37) TABLE-US-00002 TABLE 2 Adjuvant (LPS derived Vaccine antigen from Pantoea agglomerans) Amount Amount Ratio Administration No. Species [g/mouse/dose] [g/mouse/dose] (adjuvant/antigen) route Example 11 A/California/07/2009(H1N1) 1 100 100 Sublingual Example 12 A/California/07/2009(H1N1) 1 10 10 Sublingual Example 13 A/California/07/2009(H1N1) 1 1 1 Sublingual Example 14 A/California/07/2009(H1N1) 1 0.1 0.1 Sublingual Example 15 A/California/07/2009(H1N1) 1 0.01 0.01 Sublingual Comparative A/California/07/2009(H1N1) 1 0.001 0.001 Sublingual Example 6 Comparative A/California/07/2009(H1N1) 1 0 0 Sublingual Example 7 Example 16 A/California/07/2009(H1N1) 0.1 10 100 Sublingual Example 17 A/California/07/2009(H1N1) 0.1 1 10 Sublingual Example 18 A/California/07/2009(H1N1) 0.1 0.1 1 Sublingual Example 19 A/California/07/2009(H1N1) 0.1 0.01 0.1 Sublingual Example 20 A/California/07/2009(H1N1) 0.1 0.001 0.01 Sublingual Comparative A/California/07/2009(H1N1) 0.1 0 0 Sublingual Example 8 Comparative Sublingual Example 9

Examples 21 to 24, Comparative Examples 10 to 12

(38) Vaccine compositions corresponding to Table 3 were prepared in the procedure based on that in Examples 1 to 10 and Comparative Examples 1 to 5 except that the vaccine antigen was changed from influenza to a pneumococcal capsular polysaccharide-containing solution (PNEUMOVAX NP, available from MSD K.K.) (1150 g/mL). For example, in Example 21, after adding 8.7 L of a pneumococcal capsular polysaccharide-containing solution and 2 L of a solution of a lipopolysaccharide derived from Pantoea agglomerans, a phosphate buffer was added to make the total amount 300 L.

(39) 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 a pneumococcal-specific IgG titer in a serum and a pneumococcal-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later.

(40) TABLE-US-00003 TABLE 3 Adjuvant (LPS derived Vaccine antigen from Pantoea agglomerans) Amount Amount Ratio Administration No. Species [g/mouse/dose] [g/mouse/dose] (adjuvant/antigen) route Example 21 Pneumococcal capsular 1 10 10 Sublingual polysaccharide Pneumovax NP Example 22 Pneumococcal capsular 1 1 1 Sublingual polysaccharide Pneumovax NP Example 23 Pneumococcal capsular 1 0.1 0.1 Sublingual polysaccharide Pneumovax NP Example 24 Pneumococcal capsular 1 0.01 0.01 Sublingual polysaccharide Pneumovax NP Comparative Pneumococcal capsular 1 0.001 0.001 Sublingual Example 10 polysaccharide Pneumovax NP Comparative Pneumococcal capsular 1 0 0 Sublingual Example 11 polysaccharide Pneumovax NP Comparative Sublingual Example 12

Examples 25 to 28, Comparative Examples 13 to 15

(41) Vaccine compositions corresponding to Table 4 were prepared in the procedure based on that in Examples 1 to 10 and Comparative Examples 1 to 5 except that the vaccine antigen was changed from influenza to an HPV16 recombinant protein-containing solution (HPV16, available from PROSPEC) (820 g/mL). For example, in Example 25, after adding 12.2 L of an HPV16 recombinant protein-containing solution and 2 L of a solution of a lipopolysaccharide derived from Pantoea agglomerans, a phosphate buffer was added to make the total amount 300 L.

(42) 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 HPV16 recombinant protein-specific IgG titer in a serum and an HPV16 recombinant protein-specific IgA titer in a nasal cavity washing liquid were determined by the ELISA method. Specific determination methods will be described later.

(43) TABLE-US-00004 TABLE 4 Adjuvant (LPS derived Vaccine antigen from Pantoea agglomerans) Amount Amount Ratio Administration No. Species [g/mouse/dose] [g/mouse/dose] (adjuvant/antigen) route Example 25 HPV16 recombinant protein 1 10 10 Sublingual Example 26 HPV16 recombinant protein 1 1 1 Sublingual Example 27 HPV16 recombinant protein 1 0.1 0.1 Sublingual Example 28 HPV16 recombinant protein 1 0.01 0.01 Sublingual Comparative HPV16 recombinant protein 1 0.001 0.001 Sublingual Exampl 13 Comparative HPV16 recombinant protein 1 0 0 Sublingual Exampl 14 Comparative Sublingual Exampl 15

Examples 29 to 31, Comparative Example 16

(44) To 200 L of an attenuated live rotavirus-containing solution (ROTATEQ mixture for internal use, available from MSD K.K.), 50 L (2 mg/mL) in Example 29, 5 L in Example 30, or 0.5 L in Example 31 of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), or 5 L of a glucopyranosyl lipid (MPLAs, available from InvivoGen) solution (2 mg/mL) in Comparative Example 16 was added, and a phosphate buffer (available from Nacalai Tesque) was added to prepare 300 L of a vaccine composition.

(45) Six mice (female BALB/C mice aged 8 weeks, Japan SLC, Inc.) are anesthetized, and 30 L of the prepared vaccine composition is sublingually administered to each mouse. After one week from the administration, the mice are anesthetized again, and 30 L of the prepared vaccine composition is sublingually administered to each mouse. After one week from the second administration, a serum and a nasal cavity washing liquid of each mouse are collected, and an antigen-specific IgG titer in a serum and an antigen-specific IgA titer in a nasal cavity washing liquid are determined by the ELISA method.

Examples 32 to 70, Comparative Examples 17 to 29

(46) In Examples 32 to 34, and Comparative Example 17, an inactivated poliovirus-containing solution (IMOVAX POLIO subcutaneous, available from Sanofi K.K.) was used, in Examples 35 to 37, and Comparative Example 18, an inactivated hepatitis A virus-containing solution (AIMMUGEN, available from KAKETSUKEN) was used, in Examples 38 to 40, and Comparative Example 19, an inactivated Japanese encephalitis virus-containing solution (ENCEVAC for subcutaneous injection, available from KAKETSUKEN) was used, in Examples 41 to 43, and Comparative Example 20, an attenuated live mumps virus-containing solution (mumps live vaccine, available from KITASATO DAIICHISANKYO VACCINE CO., LTD.) was used, in Examples 44 to 46, and Comparative Example 21, an attenuated live measles virus-containing solution (measles live vaccine, available from KITASATO DAIICHISANKYO VACCINE CO., LTD.) was used, in Examples 47 to 49, and Comparative Example 22, an attenuated live rubella virus-containing solution (dry attenuated live rubella vaccine, available from KITASATO DAIICHISANKYO VACCINE CO., LTD.) was used, in Examples 50 to 52, and Comparative Example 23, a tetanus toxoid conjugate Haemophilus influenzae type b polysaccharide-containing solution (ACTHIB, available from Sanofi K.K.) was used, in Examples 53 to 55, and Comparative Example 24, a recombinant HBs antigen protein-containing solution (BIMMUGEN, available from KAKETSUKEN) was used, in Examples 56 to 58, and Comparative Example 25, an attenuated live yellow fever virus-containing solution (yellow fever vaccine, available from Sanofi K.K.) was used, in Examples 59 to 61, and Comparative Example 26, a tetanus toxoid-containing solution (tetanus toxoid, available from DENKA SEIKEN CO., LTD.) was used, in Examples 62 to 64, and Comparative Example 27, an attenuated live chickenpox virus-containing solution (dry attenuated live chickenpox vaccine, available from The Research Foundation for Microbial Diseases of Osaka University) was used, in Examples 65 to 67, and Comparative Example 28, a live BCG-containing solution (dry BCG vaccine, available from Japan BCG Laboratory) was used, and in Examples 68 to 70, and Comparative Example 29, an inactivated rabies virus-containing solution (tissue-cultured inactivated rabies vaccine, available from KAKETSUKEN) was used.

(47) A vaccine composition was prepared in the same manner as in Table 5 and Examples 29 to 31, and Comparative Example 16. Also immunological experiments are conducted in the same manner as in Examples 29 to 31, and Comparative Example 16.

(48) TABLE-US-00005 TABLE 5 Vaccine antigen Adjuvant Amount Amount Administration No. Species [/mouse/dose] Substance name Ligand [g/mouse/dose] route Note Example 29 Live attenuated rotavirus Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid (RIX4414 strain) Pantoea agglomerans Example 30 Live attenuated rotavirus Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid (RIX4414 strain) Pantoea agglomerans Example 31 Live attenuated rotavirus Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid (RIX4414 strain) Pantoea agglomerans Example 32 Inactivated poliovirus Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid (type 1, type 2, type 3) Pantoea agglomerans Example 33 Inactivated poliovirus Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid (type 1, type 2, type 3) Pantoea agglomerans Example 34 Inactivated poliovirus Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid (type 1, type 2, type 3) Pantoea agglomerans Example 35 Inactivated hepatitis Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid A virus Pantoea agglomerans Example 36 Inactivated hepatitis Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid A virus Pantoea agglomerans Example 37 Inactivated hepatitis Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid A virus Pantoea agglomerans Example 38 Inactivated Japanese Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid encephalitis virus Pantoea agglomerans Example 39 Inactivated Japanese Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid encephalitis virus Pantoea agglomerans Example 40 Inactivated Japanese Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid encephalitis virus Pantoea agglomerans Example 41 Live attenuated mumps Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid virus Pantoea agglomerans Example 42 Live attenuated mumps Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid virus Pantoea agglomerans Example 43 Live attenuated mumps Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid virus Pantoea agglomerans Example 44 Live attenuated Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid measles virus Pantoea agglomerans Example 45 Live attenuated Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid measles virus Pantoea agglomerans Example 46 Live attenuated Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid measles virus Pantoea agglomerans Example 47 Live attenuated Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid rubella virus Pantoea agglomerans Example 48 Live attenuated Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid rubella virus Pantoea agglomerans Example 49 Live attenuated Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid rubella virus Pantoea agglomerans Example 50 Tetanus toxoid-conjugated Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid Haemophilus influenzae type Pantoea agglomerans b polysaccharide Example 51 Tetanus toxoid-conjugated Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid Haemophilus influenzae type Pantoea agglomerans b polysaccharide Example 52 Tetanus toxoid-conjugated Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid Haemophilus influenzae type Pantoea agglomerans b polysaccharide Example 53 Recombinant HBs antigen Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid protein Pantoea agglomerans Example 54 Recombinant HBs antigen Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid protein Pantoea agglomerans Example 55 Recombinant HBs antigen Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid protein Pantoea agglomerans Example 56 Live attenuated yellow Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid fever virus Pantoea agglomerans Example 57 Live attenuated yellow Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid fever virus Pantoea agglomerans Example 58 Live attenuated yellow Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid fever virus Pantoea agglomerans Example 59 Tetanus toxoid Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid Pantoea agglomerans Example 60 Tetanus toxoid Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid Pantoea agglomerans Example 61 Tetanus toxoid Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid Pantoea agglomerans Example 62 Live attenuated varicella- Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid zoster virus Pantoea agglomerans Example 63 Live attenuated varicella- Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid zoster virus Pantoea agglomerans Example 64 Live attenuated varicella- Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid zoster virus Pantoea agglomerans Example 65 Live BCG Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid Pantoea agglomerans Example 66 Live BCG Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid Pantoea agglomerans Example 67 Live BCG Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid Pantoea agglomerans Example 68 Inactivated rabies virus Vaccine 20 L equivalent LPS derived from TLR4 10 Sublingual Liquid Pantoea agglomerans Example 69 Inactivated rabies virus Vaccine 20 L equivalent LPS derived from TLR4 1 Sublingual Liquid Pantoea agglomerans Example 70 Inactivated rabies virus Vaccine 20 L equivalent LPS derived from TLR4 0.1 Sublingual Liquid Pantoea agglomerans Comparative Live attenuated rotavirus Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 16 (RIX44I4 strain) Comparative Inactivated poliovirus Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 17 (type 1, type 2, type 3) Comparative Inactivated hepatitis Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 18 A virus Comparative Inactivated Japanese Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 19 encephalitis virus Comparative Live attenuated mumps Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 20 virus Comparative Live attenuated measles Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 21 virus Comparative Liva attenuated rubella Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 22 virus Comparative Tetanus toxoid-conjugated Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 23 Haemophilus influenzae type b polysaccharide Comparative Recombinant HBs antigen Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 24 protein Comparative Live attenuated yellow Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 25 fever virus Comparative Tetanus toxoid Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 26 Comparative Live attenuated varicella- Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 27 zoster virus Comparative Live BCG Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 28 Comparative Inactivated rabies virus Vaccine 20 L equivalent Glucopyranosyl lipid TLR4 1 Sublingual Liquid Example 29

Examples 71 to 73, Comparative Example 30

(49) Vaccine compositions corresponding to Table 6 were prepared in the procedure based on that in Examples 1 to 10 and Comparative Examples 1 to 5 except that the vaccine antigen was changed from influenza to ovalbumin (OVA) (Sigma-Aldrich Japan). For example, in Example 71, after adding 100 L (1000 g/mL) of ovalbumin (OVA) 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.

(50) 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 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.

(51) TABLE-US-00006 TABLE 6 Vaccine antigen Adjuvant Amount Amount Administration No. Species [g/mouse/dose] Substance name Ligand [g/mouse/dose] route Example 71 Ovalbumin 10 LPS derived from Pantoea agglomerans TLR4 1 Sublingual Example 72 Ovalbumin 1 LPS derived from Pantoea agglomerans TLR4 1 Sublingual Example 73 Ovalbumin 0.1 LPS derived from Pantoea agglomerans TLR4 1 Sublingual Comparative Ovalbumin 0.001 LPS derived from Pantoea agglomerans TLR4 1 Sublingual Example 30

Examples 74 to 76, Comparative Example 31

(52) Vaccine compositions corresponding to Table 7 were prepared in the procedure based on that in Examples 1 to 10 and Comparative Examples 1 to 5 except that the vaccine antigen was changed from influenza to ovalbumin (OVA) (Sigma-Aldrich Japan). For example, in Example 74, a phosphate buffer (available from Nacalai Tesque) was added to 100 L (1000 g/mL) of ovalbumin (OVA) and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), to prepare 500 L of a mucosal vaccine composition.

(53) 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.

(54) TABLE-US-00007 TABLE 7 Vaccine antigen Adjuvant Amount Amount Administration No. Species [g/mouse/dose] Substance name Ligand [g/mouse/dose] route Example 74 Ovalbumin 10 LPS derived from Pantoea agglomerans TLR4 1 Pulmonary Example 75 Ovalbumin 1 LPS derived from Pantoea agglomerans TLR4 1 Pulmonary Example 76 Ovalbumin 0.1 LPS derived from Pantoea agglomerans TLR4 1 Pulmonary Comparative Ovalbumin 0.001 LPS derived from Pantoea agglomerans TLR4 1 Pulmonary Example 31

Examples 77 to 79, Comparative Example 32

(55) Vaccine compositions corresponding to Table 8 were prepared in the procedure based on that in Examples 1 to 10 and Comparative Examples 1 to 5 except that the vaccine antigen was changed from influenza to ovalbumin (OVA) (Sigma-Aldrich Japan). For example, in Example 77, a phosphate buffer (available from Nacalai Tesque) was added to 100 L (1000 g/mL) of ovalbumin (OVA) and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), to prepare 200 L of a mucosal vaccine composition.

(56) 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.

(57) TABLE-US-00008 TABLE 8 Vaccine antigen Adjuvant Amount Amount Administration No. Species [g/mouse/dose] Substance name Ligand [g/mouse/dose] route Example 77 Ovalbumin 10 LPS derived from Pantoea agglomerans TLR4 1 Transvaginal Example 78 Ovalbumin 1 LPS derived from Pantoea agglomerans TLR4 1 Transvaginal Example 79 Ovalbumin 0.1 LPS derived from Pantoea agglomerans TLR4 1 Transvaginal Comparative Ovalbumin 0.001 LPS derived from Pantoea agglomerans TLR4 1 Transvaginal Example 32

Examples 80 to 82, Comparative Example 33

(58) Vaccine compositions corresponding to Table 9 were prepared in the procedure based on that in Examples 1 to 10 and Comparative Examples 1 to 5 except that the vaccine antigen was changed from influenza to ovalbumin (OVA) (Sigma-Aldrich Japan). For example, in Example 80, a phosphate buffer (available from Nacalai Tesque) was added to 100 L (1000 g/mL) of ovalbumin (OVA) and 5 L (2 mg/mL) of a solution of a lipopolysaccharide derived from Pantoea agglomerans (available from Nacalai Tesque), to prepare 500 L of a mucosal vaccine composition.

(59) 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.

(60) TABLE-US-00009 TABLE 9 Vaccine antigen Adjuvant Amount Amount Administration No. Species [g/mouse/dose] Substance name Ligand [g/mouse/dose] route Example 80 Ovalbumin 10 LPS derived from Pantoea agglomerans TLR4 1 Rectal Example 81 Ovalbumin 1 LPS derived from Pantoea agglomerans TLR4 1 Rectal Example 82 Ovalbumin 0.1 LPS derived from Pantoea agglomerans TLR4 1 Rectal Comparative Ovalbumin 0.001 LPS derived from Pantoea agglomerans TLR4 1 Rectal Example 33
(Mouse Immunological Experiments)

(61) 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.

(62) 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.

(63) The respective evaluation results are shown in FIGS. 1 to 26.

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

(65) 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.

(66) 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 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.

(67) 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.

(68) 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.

(69) 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.

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

(71) 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 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.

(72) As shown in FIGS. 1 to 26, according to examples, antigen-specific IgG and IgA were produced at high levels. On the other hand, in comparative examples, the production amounts of both antigen-specific IgG and IgA were small. Also, as can be seen in FIGS. 9 to 26, 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.

(73) These results revealed that a lipopolysaccharide derived from a specific gram-negative bacterium or a salt thereof as an adjuvant contained within a predetermined range with respect to the antigen is effective for mucosal immune induction on the surface of a mucous membrane.

INDUSTRIAL APPLICABILITY

(74) Since the mucosal vaccine composition of the present invention contains the aforementioned specific adjuvant together with at least one antigen in contents within predetermined ranges, 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.