Nasal mucosal vaccine composition

Abstract

The present invention provides a nasal mucosal vaccine composition which is safe, useful as a preventive or therapeutic agent for infectious diseases or cancers, and capable of inducing systemic immune responses and mucosal immune responses effectively. The present invention provides a nasal mucosal vaccine composition to be administered to a human or animal nasal mucous membrane, the nasal mucosal vaccine composition containing at least one antigen excluding antigens derived from influenza viruses; 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 nasal mucosal vaccine composition to a human or animal nasal mucous membrane, the nasal mucosal vaccine composition comprising: at least one antigen derived from a pathogen excluding antigens derived from influenza viruses; 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.

2. The method according to claim 1, wherein a mass ratio between a total mass of the adjuvant and a total mass of the antigen is 0.002 to 500.

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

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

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

6. The method according to claim 1, wherein the mucosal vaccine composition is a spray, semi-solid medicine, or solid preparation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the results of measuring the Streptococcus pneumoniae-specific IgA titers in mouse nasal lavage fluid in Examples 1 to 5 and Comparative Examples 1 to 4.

(2) FIG. 2 is a graph showing the results of measuring the Streptococcus pneumoniae-specific IgG titers in mouse serum in Examples 1 to 5 and Comparative Examples 1 to 4.

(3) FIG. 3 is a graph showing the results of measuring the HPV16 recombinant protein-specific IgA titers in mouse nasal lavage fluid in Examples 6 to 10 and Comparative Examples 5 to 7.

(4) FIG. 4 is a graph showing the results of measuring the HPV16 recombinant protein-specific IgG titers in mouse serum in Examples 6 to 10 and Comparative Examples 5 to 7.

DESCRIPTION OF EMBODIMENTS

(5) The present invention will be described in detail below referring to, but not limited to, the following examples.

Examples 1 to 5 and Comparative Examples 1to 4

(6) The vaccine composition was prepared on the assumption that each administration group consists of 10 mice.

(7) A solution containing a Streptococcus pneumoniae capsule polysaccharide (PNEUMOVAX NP, MSD K.K.) (1150 ?g/mL) and a solution of a Pantoea agglomerans-derived lipopolysaccharide (MACROPHI Inc.) (50 mg/mL) were prepared in doses for each administration group shown in Table 1. A phosphate buffer (NACALAI TESQUE, INC.) was added to the solutions to provide 100 ?L of a vaccine composition. In Example 1, for example, 8.7 ?L of the solution containing a Streptococcus pneumoniae capsule polysaccharide and 20 ?L of the solution of a Pantoea agglomerans-derived lipopolysaccharide were mixed with each other, and then a phosphate buffer was added to the mixture to make the whole volume 100 ?L. In the other examples and comparative examples, the solutions were appropriately diluted such that the amounts of the ingredients corresponded to the doses. In Comparative Example 4, neither vaccine antigen nor adjuvant was added and only a phosphate buffer (NACALAI TESQUE, INC.) was administered to mice.

(8) Six mice (eight-week-old female BALB/C mice, Japan SLC, Inc.) were anesthetized, and then each mouse received 10 ?L of the vaccine composition by nasal administration. One week after the administration, the mice were again anesthetized, and then each mouse received 10 ?L of the vaccine composition by nasal administration. One week after the second administration, the serums and the nasal lavage fluids were collected from the respective mice. The Streptococcus pneumoniae-specific IgG titer in the serum and the Streptococcus pneumoniae-specific IgA titer in the nasal lavage fluid were measured by ELISA.

(9) In the administration group with 1000 ?g of the adjuvant (Comparative Example 1), the mice showed a bad coat of hair and weight loss 24 hours after the first administration, so that they were euthanized. Thus, the antibody titers were not measured. The adjuvant is a substance which activates the immunity and it clearly gives better immunity as the amount of the adjuvant increases. Still, administration of an excessive amount of the adjuvant causes safety issues, so that 1000-?g administration to the mice was never performed after Comparative Example 1.

(10) The measuring method will be described in detail below.

(11) TABLE-US-00001 TABLE 1 Adjuvant (LPS derived from Vaccine antigen Pantoea agglomerans) Ratio Amount Amount (adjuvant/ Administration No. Species [?g/mouse/dose] [?g/mouse/dose] antigen) route Comparative Streptococcus 1 1000 1000 Intranasal Example 1 pneumoniae capsule polysaccharide Pneumovax NP Example 1 Streptococcus 1 100 100 Intranasal pneumoniae capsule polysaccharide Pneumovax NP Example 2 Streptococcus 1 10 10 Intranasal pneumoniae capsule polysaccharide Pneumovax NP Example 3 Streptococcus 1 1 1 Intranasal pneumoniae capsule polysaccharide Pneumovax NP Example 4 Streptococcus 1 0.1 0.1 Intranasal pneumoniae capsule polysaccharide Pneumovax NP Example 5 Streptococcus 1 0.01 0.01 Intranasal pneumoniae capsule polysaccharide Pneumovax NP Comparative Streptococcus 1 0.001 0.001 Intranasal Example 2 pneumoniae capsule polysaccharide Pneumovax NP Comparative Streptococcus 1 0 0 Intranasal Example 3 pneumoniae capsule polysaccharide Pneumovax NP Comparative Intranasal Example 4

Examples 6 to 10, Comparative Examples 5 to 7

(12) Vaccine compositions shown in Table 2 were prepared in manners similar to those in Examples 1 to 5 and Comparative Examples 1 to 4 except that a solution containing a HPV16 recombinant protein (HPV16, PROSPEC) (820 ?g/mL) was used instead of the solution containing a Streptococcus pneumoniae capsule polysaccharide. In Example 6, for example, a solution containing a HPV16 recombinant protein (12.2 ?L) and a solution of a Pantoea agglomerans-derived lipopolysaccharide (20 ?L) were mixed with each other, and then a phosphate buffer was added to the mixture to make the whole volume 100 ?L.

(13) Six mice (eight-week-old female BALB/C mice, Japan SLC, Inc.) were anesthetized, and then each mouse received 10 ?L of the vaccine composition by nasal administration. One week after the administration, the mice were again anesthetized, and then each mouse received 10 ?L of the vaccine composition by nasal administration. One week after the second administration, the serum and the nasal lavage fluid were collected from the respective mice. The HPV16 recombinant protein-specific IgG titer in the serum and the HPV16 recombinant protein-specific IgA titer in the nasal lavage fluid were measured by ELISA. The measuring method will be described in detail below.

(14) TABLE-US-00002 TABLE 2 Adjuvant (LPS derived from Vaccine antigen Pantoea agglomerans) Ratio Amount Amount (adjuvant/ Administration No. Species [?g/mouse/dose] [?g/mouse/dose] antigen) route Example 6 HPV16 1 100 100 Intranasal recombinant protein Example 7 HPV16 1 10 10 Intranasal recombinant protein Example 8 HPV16 1 1 1 Intranasal recombinant protein Example 9 HPV16 1 0.1 0.1 Intranasal recombinant protein Example 10 HPV16 1 0.01 0.01 Intranasal recombinant protein Comparative HPV16 1 0.001 0.001 Intranasal Example 5 recombinant protein Comparative HPV16 1 0 0 Intranasal Example 6 recombinant protein Comparative Intranasal Example 7

Examples 11 to 13, Comparative Example 8

(15) A solution containing live attenuated rotaviruses (ROTATEQ oral liquid, MSD K.K.) (50 ?L) was mixed with a solution of a Pantoea agglomerans-derived lipopolysaccharide (2 mg/mL) (NACALAI TESQUE, INC.) (50 ?L in Example 11; 5 ?L in Example 12; or 0.5 ?L in Example 13) or a solution of a glucopyranosyl lipid (2 mg/mL) (MPLAs, InvivoGen) (5 ?L) in Comparative Example 8. A phosphate buffer (NACALAI TESQUE, INC.) was then added to the mixture to provide 100 ?L of a vaccine composition. Six mice (eight-week-old female BALE/C mice, Japan SLC, Inc.) were anesthetized, and then each mouse received 10 ?L of the vaccine composition by nasal administration. One week after the administration, the mice were again anesthetized, and then each mouse received 10 ?L of the vaccine composition by nasal administration. One week after the second administration, the serum and the nasal lavage fluid were collected from the respective mice. The antigen-specific IgG titer in the serum and the antigen-specific IgA titer in the nasal lavage fluid were measured by ELISA.

Examples 14 to 52, Comparative Examples 9 to 21

(16) Examples 14 to 16 and Comparative Example 9 used an inactivated poliovirus-containing solution (IMOVAX POLIO subcutaneous, Sanofi K.K.). Examples 17 to 19 and Comparative Example 10 used an inactivated hepatitis A virus-containing solution (Aimmugen, Kaketsuken (The Chemo-Sero-Therapeutic Research Institute)). Examples 20 to 22 and Comparative Example 11 used an inactivated Japanese encephalitis virus-containing solution (ENCEVAC for hypodermic injection, Kaketsuken (The Chemo-Sero-Therapeutic Research Institute)). Examples 23 to 25 and Comparative Example 12 used a live attenuated mumps virus-containing solution (live mumps vaccine, Kitasato Daiichi Sankyo Vaccine Co., Ltd.). Examples 26 to 28 and Comparative Example 13 used a live attenuated measles virus-containing solution (live measles vaccine, Kitasato Daiichi Sankyo Vaccine Co., Ltd.). Examples 29 to 31 and Comparative Example 14 used a live attenuated rubella virus-containing solution (dried live attenuated rubella vaccine, Kitasato Daiichi Sankyo Vaccine Co., Ltd.). Examples 32 to 34 and Comparative Example 15 used a tetanus toxoid-conjugated Haemophilus influenzae type b polysaccharide-containing solution (ActHIB, Sanofi K.K.). Examples 35 to 37 and Comparative Example 16 used a recombinant HBs antigen protein-containing solution (Bimmugen, Kaketsuken (The Chemo-Sero-Therapeutic Research Institute)). Examples 38 to 40 and Comparative Example 17 used a live attenuated yellow fever virus-containing solution (yellow fever vaccine, Sanofi K.K.). Examples 41 to 43 and Comparative Example 18 used a tetanus toxoid-containing solution (tetanus toxoid, DENKA SEIKEN CO., LTD.). Examples 44 to 46 and Comparative Example 19 used a live attenuated varicella-zoster virus-containing solution (dried live attenuated varicella vaccine, The Research Foundation for Microbial Diseases of Osaka University). Examples 47 to 49 and Comparative Example 20 used a live BCG-containing solution (dried BCG vaccine, Japan BCG Laboratory). Examples 50 to 52 and Comparative Example 21 used an inactivated rabies virus-containing solution (Inactivated Tissue Culture Rabies Vaccine, Kaketsuken (The Chemo-Sero-Therapeutic Research Institute)). The vaccine compositions as shown in Table 3 were prepared in a manner similar to that in Examples 11 to 13 and Comparative Example 8. The immunity tests were performed in a manner similar to that in Examples 11 to 13 and Comparative Example 8.

(17) TABLE-US-00003 TABLE 3 Amount Adminis- Vaccine antigens Amount Adjuvant [mg/mouse/ tration No. Species [/mouse/dose] Substance name Ligand dose] route Note Example 11 Live attenuated rotavirus (RIX4414 Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid strain) Pantoea agglomerans Example 12 Live attenuated rotavirus (RIX4414 Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid strain) Pantoea agglomerans Example 13 Live attenuated rotavirus (RIX4414 Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid strain) Pantoea agglomerans Example 14 Inactivated poliovirus (type 1, type 2, Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid type 3) Pantoea agglomerans Example 15 Inactivated poliovirus (type 1, type 2, Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid type 3) Pantoea agglomerans Example 16 Inactivated poliovirus (type 1, type 2, Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid type 3) Pantoea agglomerans Example 17 Inactivated hepatitis A virus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 18 Inactivated hepatitis A virus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 19 Inactivated hepatitis A virus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 20 Inactivated Japanese encephalitis virus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 21 Inactivated Japanese encephalitis virus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 22 Inactivated Japanese encephalitis virus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 23 Live attenuated mumps virus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 24 Live attenuated mumps virus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 25 Live attenuated mumps virus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 26 Live attenuated measles virus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 27 Live attenuated measles virus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 28 Live attenuated measles virus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 29 Live attenuated rubella virus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 30 Live attenuated rubella virus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 31 Live attenuated rubella virus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 32 Tetanus toxoid-conjugated Haemophilus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid influenzae type b polysaccharide Pantoea agglomerans Example 33 Tetanus toxoid-conjugated Haemophilus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid influenzae type b polysaccharide Pantoea agglomerans Example 34 Tetanus toxoid-conjugated Haemophilus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid influenzae type b polysaccharide Pantoea agglomerans Example 35 Recombinant HBs antigen protein Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 36 Recombinant HBs antigen protein Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 37 Recombinant HBs antigen protein Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 38 Live attenuated yellow fever virus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 39 Live attenuated yellow fever virus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 40 Live attenuated yellow fever virus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 41 Tetanus toxoid Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 42 Tetanus toxoid Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 43 Tetanus toxoid Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 44 Live attenuated varicella-zoster virus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 45 Live attenuated varicella-zoster virus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 46 Live attenuated varicella-zoster virus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 47 Live BCG Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 48 Live BCG Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 49 Live BCG Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Example 50 Inactivated rabies virus Vaccine 5 ?L equivalent LPS derived from TLR4 10 Intranasal Liquid Pantoea agglomerans Example 51 Inactivated rabies virus Vaccine 5 ?L equivalent LPS derived from TLR4 1 Intranasal Liquid Pantoea agglomerans Example 52 Inactivated rabies virus Vaccine 5 ?L equivalent LPS derived from TLR4 0.1 Intranasal Liquid Pantoea agglomerans Comparative Live attenuated rotavirus (RIX4414 Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 8 strain) Comparative Inactivated poliovirus (type 1, type 2, Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 9 type 3) Comparative Inactivated hepatitis A virus Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 10 Comparative Inactivated Japanese encephalitis virus Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 11 Comparative Live attenuated mumps virus Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 12 Comparative Live attenuated measles virus Vaccine 5 ?L equivalent Glucopyrancsyl lipid TLR4 1 Intranasal Liquid Example 13 Comparative Live attenuated rubella virus Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 14 Comparative Tetanus toxoid-conjugated Haemophilus Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 15 influenzae type b polysaccharide Comparative Recombinant HBs antigen protein Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 16 Comparative Live attenuated yellow fever virus Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 17 Comparative Tetanus toxoid Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 18 Comparative Live attenuated varicella-zoster virus Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 19 Comparative Live BCG Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 20 Comparative Inactivated rabies virus Vaccine 5 ?L equivalent Glucopyranosyl lipid TLR4 1 Intranasal Liquid Example 21
(Mouse Immunity Test)

(18) The vaccine composition was administered to an eight-week-old female BALB/c mouse twice at a one-week interval. One week after the final administration, the blood and the nasal lavage fluid was collected from the mouse. The blood was centrifuged at 3000 G at 4? C. for 10 minutes. The serum (20 ?L) was mixed with a phosphate buffer (NACALAI TESQUE, INC.) (300 ?L) to provide a serum sample. The nasal lavage fluid was collected as follows. Specifically, a slit was formed at the lower portion of the respiratory tract of the BALB/c mouse, and 200 ?L of a phosphate buffer (NACALAI TESQUE, INC.) was poured into the respiratory tract through the slit to allow the buffer to flown out of the nasal cavity. This flown-out sample was collected as a nasal lavage fluid sample.

(19) The Streptococcus pneumoniae- or HPV16 recombinant protein-specific IgG titer in the mouse serum was measured, thereby evaluating the systemic immune response. The Streptococcus pneumoniae- or HPV16 recombinant protein-specific IgA titer in the nasal lavage fluid of mouse was measured, thereby evaluating the mucosal immune response. The methods of evaluating the titers will be described below.

(20) FIGS. 1 to 4 show the respective evaluation results.

(21) (Method of Measuring Antigen-specific IgG Titer in Mouse Serum (ELISA))

(22) Each antigen was diluted with a carbonate buffer (for example, a solution of a Streptococcus pneumoniae capsule polysaccharide antigen was prepared for the measurement of a Streptococcus pneumoniae capsule polysaccharide-specific IgG antibody titer), and 100 ?L portions of the diluted antigen (2.5 ?g/mL) were put into the wells of a 96-well plate for ELISA. They were left to stand overnight.

(23) The wells were then washed three times with a polysorbate (TWEEN 20)-containing PBS (hereinafter, referred to as a washing liquid) prepared in advance. A blocking agent (BLOCK ACE, DS Pharma Biomedical Co., Ltd.) was diluted to 4 g/400 mL with purified water to provide a blocking solution. Then, 200 ?L portions of the blocking solution were added to the respective wells and left to stand for two hours at room temperature. Thereafter, the wells were washed three times with the washing liquid.

(24) A blocking agent (BLOCK ACE, DS Pharma Biomedical Co., Ltd.) was diluted to 0.4 g/100 mL with a phosphate buffer (NACALAI TESQUE, INC.) to provide a solution (hereinafter, referred to as a diluted reagent). The aforementioned serum sample was serially diluted 2-fold 15 times using the diluted reagent. Then, 50 ?L portions of the resulting solution were added to the wells, and left to stand for two hours at room temperature.

(25) Next, the wells were washed three times using the washing liquid, and 100 ?L portions of a TMB solution (ELISA POD TMB kit, NACALAI TESQUE, INC.) were added to the wells. Then, 100 ?L portions of a 1 M sulfuric acid solution were added thereto, and the absorbance at 450 nm of the 96-well plate was measured using a microplate reader (168-11135CAM, Bio-Rad Laboratories, Inc.). Based on the absorbance values in the serial dilutions, the maximum dilution factor among the dilution factors with an absorbance of not breaking 0.1 was defined as the IgG titer in a mouse serum. The values were represented in terms of Log2.

(26) (Method of Measuring Antigen-Specific IgA Titer in Nasal Lavage Fluid of Mouse (ELISA))

(27) This method is fundamentally similar to the method of measuring an antigen-specific IgG titer. The measurement sample is a nasal lavage fluid, and a HRP-labeled anti-mouse IgA antibody (Goat-anti-mouse IgA a HRP, Bethyl Laboratories, Inc.) was used instead of the HRP-labeled anti-mouse IgG antibody. The other operations were performed in a similar manner.

(28) FIGS. 1 to 4 show that the Streptococcus pneumoniae-or HPV16 recombinant protein-specific IgG and IgA were produced at high levels in the examples. In contrast, the Streptococcus pneumoniae- or HPV16recombinant protein-specific IgG and IgA were produced at low levels in the comparative examples.

(29) These results prove that combination use of an antigen and a specific gram-negative bacterium-derived lipopolysaccharide or a salt thereof as an adjuvant is effective in inducing mucosal immunity at the nasal mucosa.

INDUSTRIAL APPLICABILITY

(30) Since the nasal mucosal vaccine composition of the present invention contains at least one antigen together with the aforementioned specific adjuvant, it can safely and effectively induce systemic immune responses and mucosal immune responses.