BISPHOSPHONATE-CONTAINING VACCINE PHARMACEUTICAL COMPOSITION FOR HUMORAL IMMUNITY

Abstract

The present invention aims to provide a vaccine pharmaceutical composition universally usable for induction of humoral immunity against various antigens and exerting a high antibody production inducing effect. The present invention relates to a vaccine pharmaceutical composition for inducing humoral immunity, including: an antigen; and an immunity induction promoter that is a bisphosphonate.

Claims

1. A vaccine pharmaceutical composition for inducing humoral immunity, comprising: an antigen; and an immunity induction promoter that is a bisphosphonate.

2. The vaccine pharmaceutical composition according to claim 1, structured and arranged to be administered to a body surface.

3. The vaccine pharmaceutical composition according to claim 1, wherein the immunity induction promoter that is a bisphosphonate is at least one selected from the group consisting of etidronate, clodronate, tiludronate, pamidronate, neridronate, alendronate, ibandronate, zoledronate, risedronate, and minodronate.

4. A method for inducing humoral immunity, comprising administering a vaccine pharmaceutical composition comprising: an antigen; and an immunity induction promoter that is a bisphosphonate, wherein the administration induces a humoral immune response.

5. The method for inducing humoral immunity according to claim 4, comprising administering the vaccine pharmaceutical composition to a body surface.

6. The method for inducing humoral immunity according to claim 4, wherein the immunity induction promoter that is a bisphosphonate is at least one selected from the group consisting of etidronate, clodronate, tiludronate, pamidronate, neridronate, alendronate, ibandronate, zoledronate, risedronate, and minodronate.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0117] FIG. 1 is a graph showing antigen (OVA)-specific IgG titers in mouse serums after transnasal administration of solutions for transmucosal administration obtained in examples and comparative examples.

[0118] FIG. 2 is a graph showing antigen (OVA)-specific IgG titers in mouse serums after sublingual administration of solutions for transmucosal administration obtained in examples and comparative examples.

[0119] FIG. 3 is a graph showing antigen (OVA)-specific IgG titers in mouse serums after sublingual administration of solid formulations for sublingual administration obtained in examples and comparative examples.

[0120] FIG. 4 is a graph showing antigen (OVA)-specific IgG titers in mouse serums after transdermal administration of creams for transdermal administration obtained in examples and comparative examples.

DESCRIPTION OF EMBODIMENTS

[0121] The present invention is described specifically in detail in the following with reference to, but not limited to, examples.

Examples 1 to 20, Comparative Examples 1 and 2

(Preparation of Solution for Transmucosal Administration)

[0122] Solutions for transmucosal administration (transnasal administration or sublingual administration) having a composition shown in Table 1 and 2 were prepared. Specifically, an antigen (ovalbumin (OVA), Sigma-Aldrich Co. LLC) and an immunity induction promoter that is a bisphosphonate each in an amount as shown in Table 1 or 2 were blended, followed by addition of saline thereto. The mixture was mixed to give a solution for transmucosal administration (transnasal administration or sublingual administration).

[0123] As the immunity induction promoter that is a bisphosphonate, etidronate (LKT Laboratories, Inc.), clodronate (LKT Laboratories, Inc.), tiludronate (Sigma-Aldrich Co. LLC), pamidronate (Sigma-Aldrich Co. LLC), neridronate (Sigma-Aldrich Co. LLC), alendronate (medichem), ibandronate (URQUIMA S.A.), zoledronate (Konan Chemical Industry co., ltd.), risedronate (Propharma S.A.), or minodronate (Ava Chem Scientific) was used.

TABLE-US-00001 TABLE 1 Composition [μg] Comparative Example Example Component 1 2 3 4 5 6 7 8 9 10 1 Antigen OVA 1 1 1 1 1 1 1 1 1 1 1 Immunity Bisphosphonate Etidronate 10 — — — — — — — — — — induction Clodronate — 10 — — — — — — — — — promoter Tiludronate — — 10 — — — — — — — — Pamidronate — — — 10 — — — — — — — Neridronate — — — — 10 — — — — — — Alendronate — — — — — 10 — — — — — Ibandronate — — — — — — 10 — — — — Zoledronate — — — — — — — 10 — — — Risedronate — — — — — — — — 10 — — Minodronate — — — — — — — — — 10 — Saline [μL] 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Dosage form Solution Administration route Transnasal administration

TABLE-US-00002 TABLE 2 Composition [μg] Comparative Example Example Component 11 12 13 14 15 16 17 18 19 20 2 Antigen OVA 1 1 1 1 1 1 1 1 1 1 1 Immunity Bisphosphonate Etidronate 100 — — — — — — — — — — induction Clodronate — 100 — — — — — — — — — promoter Tiludronate — — 100 — — — — — — — — Pamidronate — — — 100 — — — — — — — Neridronate — — — — 100 — — — — — — Alendronate — — — — — 100 — — — — — Ibandronate — — — — — — 100 — — — — Zoledronate — — — — — — — 50 — — — Risedronate — — — — — — — — 50 — — Minodronate — — — — — — — — — 50 — Saline [μL] 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 Dosage form Solution Administration route Sublingual administration

Examples 21 to 32, Comparative Examples 3 and 4

(Preparation of Solid Formulation for Sublingual Administration)

[0124] Solid formulations (freeze dry formulations or films) for sublingual administration having a composition as shown in Table 3 were prepared. Specifically, an antigen (ovalbumin (OVA), Sigma-Aldrich Co. LLC), an immunity induction promoter that is a bisphosphonate, and hydroxypropyl cellulose (HPC-SSL, Nippon Soda Co., Ltd.) as a base each in an amount as shown in Table 3 were blended, followed by addition of saline thereto. The mixture was mixed to give a drug solution. The drug solution was divided into 25-mg portions, and then freeze-dried to give a freeze dry formulation or dried under reduced pressure to give a film. The immunity induction promoter that is a bisphosphonate used was the same as that used for preparation of a solution for transmucosal administration.

TABLE-US-00003 TABLE 3 Composition [parts by weight] Comparative Example Example Component 21 22 23 24 25 26 27 28 29 30 31 32 3 4 Antigen OVA 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Immunity Bisphosphonate Etidronate 10 — — — — — 10 — — — — — — — induction Pamidronate — 10 — — — — — 10 — — — — — — promoter Alendronate — — 10 — — — — — 10 — — — — — Zoledronate — — — 5 — — — — — 5 — — — — Risedronate — — — — 5 — — — — — 5 — — — Minodronate — — — — — 5 — — — — — 5 — — Base HPO-SSL 250 250 250 250 250 250 250 250 250 250 250 250 250 250 Saline 739.9 739.9 739.9 744.9 744.9 744.9 739.9 739.9 739.9 744.9 744.9 744.9 749.9 749.9 Dispensing amount [mg/mouse] 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 Dosage form Solid (freeze dry) Film Solid Film (freeze dry) Administration route Sublingual administration

<Evaluation 1>

[0125] The solutions for transmucosal administration or solid formulations for sublingual administration obtained in the examples and comparative examples were evaluated by the following methods.

(Evaluation of the Humoral Immunity Inducing Effect)

[0126] In accordance with the following procedure, a mouse immunity test using an animal model for immunological evaluation was performed with the solution for transmucosal administration or the solid formulation for sublingual administration. Then, the antigen (OVA)-specific IgG antibody in the mouse serum was analyzed for evaluation of the systemic immune response. FIGS. 1 to 3 show the evaluation results.

(1) Mouse Immunity Test of Solution for Transmucosal Administration or Solid Formulation for Sublingual Administration

[0127] A mouse (BALB/c mice, female, 7 weeks old) prepared was anesthetized. The solution for transmucosal administration was administered to the mouse by transnasal administration (10 μL, Examples 1 to 10, Comparative Example 1 (Table 1)) or sublingual administration (30 μL, Examples 11 to 20, Comparative Example 2 (Table 2)). Similarly, the solid formulation for sublingual administration (Examples 21 to 32, Comparative Example 3 and 4 (Table 3)) was administered. One week after the administration, the mouse was again anesthetized, and the administration was performed again in the same manner. One week after the second administration, the mouse serum was taken.

(2) ELISA

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

[0128] To each well of a 96-well plate for ELISA was added 100 μL of an OVA-containing solution (100 μg/mL) diluted with carbonate buffer, followed by standing overnight.

[0129] The wells were washed three times with preliminarily prepared wash (Tween 20-containing PBS), and to each well was added 200 μL of a blocking solution prepared by diluting a blocking agent (Block Ace, Sumitomo Dainippon Pharma Co., Ltd.) in purified water to 4 g/100 mL. This was followed by standing for 2 hours at room temperature. The wells were then washed three times with wash.

[0130] The serum taken from the mouse was centrifuged at 4° C. and 3,000 g for 10 minutes, and the supernatant was recovered. The supernatant was diluted in two-fold increments using a solution prepared by diluting a blocking agent in a phosphate buffer (Nacalai Tesque, Inc.) to 0.4 g/100 mL. The diluted solutions were added to wells (50 μL for each well), followed by standing for 2 hours at room temperature.

[0131] The wells were then washed three times with wash. An HRP-labeled anti-mouse IgG antibody (Goat-anti mouse IgG Fc HRP, BETHYL) was diluted 10,000-fold using a solution prepared by diluting a blocking agent in a phosphate buffer (Nacalai Tesque, Inc.) to 0.4 g/100 mL. To each well was added 100 μL of the resulting solution, followed by standing for 1 hour at room temperature.

[0132] The wells were then washed three times with wash, and 100 μL of a TMB solution (ELISA POD TMB kit, Nacalai Tesque, Inc.) was added to each well, followed by standing for 30 minutes at dark place.

[0133] Thereafter, 100 μL of a 1M sulfuric acid solution was added to each well, and the 96-well plate was subjected to measurement of absorbance at 450 nm with a microplate reader (Spectra Max M2®, Molecular Devices). The IgG titer in the mouse serum was determined as Log 2 titer based on the absorbance at the incremental dilution.

Examples 33 to 42, Comparative Example 5

(Preparation of Cream for Transdermal Administration)

[0134] Creams for transdermal administration having a composition shown in Table 4 were prepared. Specifically, an antigen (ovalbumin (OVA), Sigma-Aldrich Co. LLC) and an immunity induction promoter that is a bisphosphonate each in an amount as shown in Table 4 were blended, followed by addition of a base (base cream) thereto in such an amount that the entire amount became 100 parts by weight. The mixture was mixed to give a cream for transdermal administration. The base cream used was prepared by mixing materials in amounts as shown in Table 5.

[0135] The immunity induction promoter that is a bisphosphonate used was the same as that used for the preparation of the solution for transnasal or sublingual administration. White Vaseline, sorbitan monostearate, isostearic acid, benzyl alcohol, stearyl alcohol, polysorbate 60, concentrated glycerin, and dimethyl sulfoxide were purchased from Wako Pure Chemical Industries, Ltd. Cetanol was purchased from Tokyo Chemical Industry Co., Ltd.

[0136] A PET film/PET nonwoven fabric laminate (area: 0.7 cm.sup.2) was attached to an adhesive tape for fixation at a central portion in such a manner that the PET film was in contact with the tape, thereby preparing a complex base. To the nonwoven fabric part of the obtained complex base, 4 mg of each cream for transdermal administration was applied. The resulting product was used as an administration sample in a mouse immunity test.

TABLE-US-00004 TABLE 4 Composition [μg] Comparative Example Example Component 33 34 35 36 37 38 39 40 41 42 5 Antigen OVA 5 5 5 5 5 5 5 5 5 5 5 Immunity Bisphosphonate Etidronate 2 — — — — — — — — — — induction Clodronate — 2 — — — — — — — — — promoter Tiludronate — — 2 — — — — — — — — Pamidronate — — — 2 — — — — — — — Neridronate — — — — 1 — — — — — — Alendronate — — — — — 1 — — — — — Ibandronate — — — — — — 1 — — — — Zoledronate — — — — — — — 0.5 — — — Risedronate — — — — — — — — 0.5 — — Minodronate — — — — — — — — — 0.5 — Ointment cream (base cream) 93.0 93.0 93.0 93.0 94.0 94.0 94.0 94.5 94.5 94.5 95.0 Amount [mg/mouse] 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 Dosage form Cream Administration route Transdermal administration

TABLE-US-00005 TABLE 5 Amount Additive [parts by weight] Base White Vaseline 60.7 cream Sorbitan monostearate 0.7 Isostearic acid 12 Benzyl alcohol 2.4 Cetanol 2.4 Stearyl alcohol 3.5 Polysorbate 60 3.5 Concentrated glycerin 2.4 Purified water 12.4 Total 100

<Evaluation 2>

[0137] The creams for transdermal administration obtained in the examples and comparative examples were evaluated by the following methods.

(Evaluation of the Humoral Immunity Inducing Effect)

[0138] In accordance with the following procedure, a mouse immunity test using an animal model for immunological evaluation was performed with the cream for transdermal administration. Then, the antigen (OVA)-specific IgG antibody in the mouse serum was analyzed for evaluation of the systemic immune response. FIG. 4 shows the evaluation results.

(1) Mouse Immunity Test of Cream for Transdermal Administration

[0139] The right back of a mouse (C57BL6 NCr mouse, female, 7 weeks old) was shaved in advance. After a rearing period for recovery from the skin damage caused by the shaving, 4 mg of the cream for transdermal administration was administered to the skin of the right back, and the left back was shaved at the same time. Twenty-four hours later, the cream for transdermal administration on the right back was removed. One week after the administration, the cream for transdermal administration was similarly administered to the skin of the left back of the mouse and removed 24 hours later. One week after the second administration, the mouse serum was taken.

(2) ELISA

[0140] The antigen (OVA)-specific IgG antibody in the mouse serum was analyzed by ELISA by the same procedure as in <Evaluation 1>, the evaluation of the solution for transmucosal administration or the solid formulation for sublingual administration.

Examples 43 to 162, Comparative Examples 6 to 45

[0141] Solutions for transmucosal administration (transnasal administration or sublingual administration) having a composition as shown in Tables 6 to 10 were prepared. Specifically, an antigen and an immunity induction promoter that is a bisphosphonate were blended in amounts specified in Tables 6 to 10. For transnasal administration, saline was added thereto such that the amount of the resulting mixture was 10 μL. For sublingual administration, saline was added such that the amount of the resulting mixture was 30 μL. This was followed by mixing to provide a solution for transmucosal administration (transnasal administration or sublingual administration).

[0142] Influenza vaccine antigens used were an influenza vaccine antigen-containing solution H1N1 (A/California/07/2009, The Research Foundation for Microbial Diseases of Osaka University), H3N2 (A/Victoria361/2011, The Research Foundation for Microbial Diseases of Osaka University), Influenza B virus (B/Wisconsin/1/2010, The Research Foundation for Microbial Diseases of Osaka University), and Influenza B virus (B/Brisbane/60/2008, The Research Foundation for Microbial Diseases of Osaka University). Also used were a pneumococcal capsular polysaccharide-containing solution (Pneumovax NP, MSD), a HPV16 recombinant protein-containing solution (HPV16, PROSPEC), a live attenuated rotavirus-containing solution (RotaTeq Oral Solution, MSD), an inactivated poliovirus-containing solution (IMOVAX POLIO for subcutaneous injection, Sanofi), an inactivated hepatitis A virus-containing solution (Aimmugen, The Chemo-Sero-Therapeutic Research Institute), an inactivated Japanese encephalitis virus-containing solution (Encevac for subcutaneous injection, The Chemo-Sero-Therapeutic Research Institute), a live attenuated mumps virus-containing solution (live mumps vaccine, Kitasato Daiichi Sankyo Vaccine Co., Ltd), a live attenuated measles virus-containing solution (live measles vaccine, Kitasato Daiichi Sankyo Vaccine Co., Ltd), a live attenuated rubella virus-containing solution (dried live attenuated rubella vaccine, Kitasato Daiichi Sankyo Vaccine Co., Ltd), a solution containing haemophilus influenzae type b polysaccharide-tetanus toxoid conjugate (ActHIB, Sanofi), a recombinant HBs antigen protein-containing solution (Bimmugen, The Chemo-Sero-Therapeutic Research Institute), a live attenuated yellow fever virus-containing solution (yellow fever vaccine, Sanofi), a tetanus toxoid-containing solution (tetanus toxoid, Denka Seiken Co., Ltd.), a live attenuated varicella virus-containing solution (dried live attenuated varicella vaccine, The Research Foundation for Microbial Diseases of Osaka University), a live BCG-containing solution (dried BCG vaccine, Japan BCG Laboratory), and an inactivated rabies virus-containing solution (tissue-cultured inactivated rabies vaccine, The Chemo-Sero-Therapeutic Research Institute).

[0143] As the immunity induction promoter that is a bisphosphonate, etidronate (LKT Laboratories, Inc.), alendronate (medichem), and zoledronate (Konan Chemical Industry co., ltd.) were used.

Examples 163 to 172, Comparative Example 46

[0144] Creams for transdermal administration having a composition shown in Table 11 were prepared in the same manner as in the case of the creams for transdermal administration shown in Table 4. A mouse (C57BL6 NCr mouse, female, 7 weeks old) was prepared and its right back was shaved. Corneum exfoliation treatment was performed thereon five times using an OPP tape (EZ Dunplon No. 3301EZ) produced by Nitto Denko Corporation. The cream (4 mg) was administered to the treated skin (minimally invasive administration), and the left back of the mouse was shaved at the same time. Twenty-four hours later, the cream for transdermal administration on the right back was removed. One week after the administration, corneum exfoliation treatment was similarly performed on the skin of the left back of the mouse. The cream for transdermal administration was administered and removed 24 hours later. One week after the second administration, the mouse serum was taken. The antigen (OVA)-specific IgG antibody in the mouse serum was analyzed by ELISA.

<Evaluation 3>

[0145] The solutions for transmucosal administration obtained in the examples and comparative examples were evaluated by the following methods.

(Evaluation of the Humoral Immunity Inducing Effect)

[0146] A mouse immunity test using an animal model for immunological evaluation was performed with the solution for transmucosal administration by the following procedure. Then, the systemic immune response was evaluated by analyzing the antigen-specific IgG antibody in the mouse serum.

(1) Mouse Immunity Test of Solution for Transmucosal Administration

[0147] Mouse serum was taken by the same procedure as in <Evaluation 1>, evaluation of the solutions for transmucosal administration or solid formulations for sublingual administration.

(2) ELISA

[0148] The antigen-specific IgG antibody in the mouse serum was analyzed by ELISA by the same procedure as in <Evaluation 1>, evaluation of the solutions for transmucosal administration or solid formulations for sublingual administration.

[0149] The evaluation of the humoral immunity inducing effect shows that the transmucosal administration (transnasal administration or sublingual administration) of a solution for transmucosal administration containing an immunity induction promoter that is a bisphosphonate (Examples 43 to 162) provides a higher antigen-specific IgG titer than the administration of a solution for transmucosal administration free from an immunity induction promoter that is a bisphosphonate (Comparative Examples 6 to 45).

[0150] Accordingly, also when antigens such as those shown in Tables 6 to 10 below are used, the use of an immunity induction promoter that is a bisphosphonate leads to a high antigen-specific IgG titer.

[0151] Also in the case of an immunization method by minimally invasive administration as shown in Table 11, the humoral immunity specific to the administered antigen can be induced.

TABLE-US-00006 TABLE 6 Immunity Antigen induction promoter Amount Amount Dosage Administration Amount Name [μg] Bisphosphonate [μg] form route [μL] Comparative Example 6 A/California/07/2009 [H1N1] 1.0 — — Solution Transnasal 10 Example 43 A/California/07/2009 [H1N1] 1.0 Etidronate 10 Solution Transnasal 10 Example 44 A/California/07/2009 [H1N1] 1.0 Alendronate 10 Solution Transnasal 10 Example 45 A/California/07/2009 [H1N1] 1.0 Zoledronate 10 Solution Transnasal 10 Comparative Example 7 A/California/07/2009 [H1N1] 1.0 — — Solution Sublingual 30 Example 46 A/California/07/2009 [H1N1] 1.0 Etidronate 100  Solution Sublingual 30 Example 47 A/California/07/2009 [H1N1] 1.0 Alendronate 50 Solution Sublingual 30 Example 48 A/California/07/2009 [H1N1] 1.0 Zoledronate 50 Solution Sublingual 30 Comparative Example 8 A/Victoria361/2011 [H3N2] 1.0 — — Solution Transnasal 10 Example 49 A/Victoria361/2011 [H3N2] 1.0 Etidronate 10 Solution Transnasal 10 Example 50 A/Victoria361/2011 [H3N2] 1.0 Alendronate 10 Solution Transnasal 10 Example 51 A/Victoria361/2011 [H3N2] 1.0 Zoledronate 10 Solution Transnasal 10 Comparative Example 9 A/Victoria361/2011 [H3N2] 1.0 — — Solution Sublingual 30 Example 52 A/Victoria361/2011 [H3N2] 1.0 Etidronate 100  Solution Sublingual 30 Example 53 A/Victoria361/2011 [H3N2] 1.0 Alendronate 50 Solution Sublingual 30 Example 54 A/Victoria361/2011 [H3N2] 1.0 Zoledronate 50 Solution Sublingual 30 Comparative Example 10 B/Wisconsin/1/2010 1.0 — — Solution Transnasal 10 Example 55 B/Wisconsin/1/2010 1.0 Etidronate 10 Solution Transnasal 10 Example 56 B/Wisconsin/1/2010 1.0 Alendronate 10 Solution Transnasal 10 Example 57 B/Wisconsin/1/2010 1.0 Zoledronate 10 Solution Transnasal 10 Comparative Example 11 B/Wisconsin/1/2010 1.0 — — Solution Sublingual 30 Example 58 B/Wisconsin/1/2010 1.0 Etidronate 100  Solution Sublingual 30 Example 59 B/Wisconsin/1/2010 1.0 Alendronate 50 Solution Sublingual 30 Example 60 B/Wisconsin/1/2010 1.0 Zoledronate 50 Solution Sublingual 30 Comparative Example 12 B/Brisbane/60/2008 1.0 — — Solution Transnasal 10 Example 61 B/Brisbane/60/2008 1.0 Etidronate 10 Solution Transnasal 10 Example 62 B/Brisbane/60/2008 1.0 Alendronate 10 Solution Transnasal 10 Example 63 B/Brisbane/60/2008 1.0 Zoledronate 10 Solution Transnasal 10 Comparative Example 13 B/Brisbane/60/2008 1.0 — — Solution Sublingual 30 Example 64 B/Brisbane/60/2008 1.0 Etidronate 100  Solution Sublingual 30 Example 65 B/Brisbane/60/2008 1.0 Alendronate 50 Solution Sublingual 30 Example 66 B/Brisbane/60/2008 1.0 Zoledronate 50 Solution Sublingual 30

TABLE-US-00007 TABLE 7 Immunity Antigen induction promoter Amount Amount Dosage Administration Amount Name [μg] Bisphosphonate [μg] form route [μL] Comparative Pneumococcal capsular polysaccharide 20 — — Solution Transnasal 10 Example 14 Pneumovax NP Example 67 Pneumococcal capsular polysaccharide 20 Etidronate 10 Solution Transnasal 10 Pneumovax NP Example 68 Pneumococcal capsular polysaccharide 20 Alendronate 10 Solution Transnasal 10 Pneumovax NP Example 69 Pneumococcal capsular polysaccharide 20 Zoledronate 10 Solution Transnasal 10 Pneumovax NP Comparative Pneumococcal capsular polysaccharide 20 — — Solution Sublingual 30 Example 15 Pneumovax NP Example 70 Pneumococcal capsular polysaccharide 20 Etidronate 100  Solution Sublingual 30 Pneumovax NP Example 71 Pneumococcal capsular polysaccharide 20 Alendronate 50 Solution Sublingual 30 Pneumovax NP Example 72 Pneumococcal capsular polysaccharide 20 Zoledronate 50 Solution Sublingual 30 Pneumovax NP Comparative HPV16 recombinant protein 10 — — Solution Transnasal 10 Example 16 Example 73 HPV16 recombinant protein 10 Etidronate 10 Solution Transnasal 10 Example 74 HPV16 recombinant protein 10 Alendronate 10 Solution Transnasal 10 Example 75 HPV16 recombinant protein 10 Zoledronate 10 Solution Transnasal 10 Comparative HPV16 recombinant protein 10 — — Solution Sublingual 30 Example 17 Example 76 HPV16 recombinant protein 10 Etidronate 100  Solution Sublingual 30 Example 77 HPV16 recombinant protein 10 Alendronate 50 Solution Sublingual 30 Example 78 HPV16 recombinant protein 10 Zoledronate 50 Solution Sublingual 30 Comparative Live attenuated rotavirus (RIX4414 strain) 10 — — Solution Transnasal 10 Example 18 Example 79 Live attenuated rotavirus (RIX4414 strain) 10 Etidronate 10 Solution Transnasal 10 Example 80 Live attenuated rotavirus (RIX4414 strain) 10 Alendronate 10 Solution Transnasal 10 Example 81 Live attenuated rotavirus (RIX4414 strain) 10 Zoledronate 10 Solution Transnasal 10 Comparative Live attenuated rotavirus (RIX4414 strain) 10 — — Solution Sublingual 30 Example 19 Example 82 Live attenuated rotavirus (RIX4414 strain) 10 Etidronate 100  Solution Sublingual 30 Example 83 Live attenuated rotavirus (RIX4414 strain) 10 Alendronate 50 Solution Sublingual 30 Example 84 Live attenuated rotavirus (RIX4414 strain) 10 Zoledronate 50 Solution Sublingual 30 Comparative Inactivated poliovirus (type 1, type 2, type 3) Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 20 Example 85 Inactivated poliovirus (type 1, type 2, type 3) Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 86 Inactivated poliovirus (type 1, type 2, type 3) Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 87 Inactivated poliovirus (type 1, type 2, type 3) Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Inactivated poliovirus (type 1, type 2, type 3) Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 21 Example 88 Inactivated poliovirus (type 1, type 2, type 3) Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 89 Inactivated poliovirus (type 1, type 2, type 3) Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 90 Inactivated poliovirus (type 1, type 2, type 3) Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30

TABLE-US-00008 TABLE 8 Immunity Antigen induction promoter Amount Amount Dosage Administration Name [μg] Bisphosphonate [μg] form route Amount [μL] Comparative Inactivated hepatitis A virus Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 22 Example 91 Inactivated hepatitis A virus Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 92 Inactivated hepatitis A virus Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 93 Inactivated hepatitis A virus Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Inactivated hepatitis A virus Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 23 Example 94 Inactivated hepatitis A virus Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 95 Inactivated hepatitis A virus Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 96 Inactivated hepatitis A virus Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30 Comparative Inactivated Japanese encephalitis virus Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 24 Example 97 Inactivated Japanese encephalitis virus Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 98 Inactivated Japanese encephalitis virus Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 99 Inactivated Japanese encephalitis virus Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Inactivated Japanese encephalitis virus Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 25 Example 100 Inactivated Japanese encephalitis virus Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 101 Inactivated Japanese encephalitis virus Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 102 Inactivated Japanese encephalitis virus Vaccine 100 μL equivalent Zoiedronate 50 Solution Sublingual 30 Comparative Live attenuated mumps virus Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 26 Example 103 Live attenuated mumps virus Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 104 Live attenuated mumps virus Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 105 Live attenuated mumps virus Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Live attenuated mumps virus Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 27 Example 106 Live attenuated mumps virus Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 107 Live attenuated mumps virus Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 108 Live attenuated mumps virus Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30 Comparative Live attenuated measles virus Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 28 Example 109 Live attenuated measles virus Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 110 Live attenuated measles virus Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 111 Live attenuated measles virus Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Live attenuated measles virus Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 29 Example 112 Live attenuated measles virus Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 113 Live attenuated measles virus Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 114 Live attenuated measles virus Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30

TABLE-US-00009 TABLE 9 Immunity Antigen induction promoter Amount Bis- Amount Dosage Administration Amount Name [μg] phosphonate [μg] form route [μL] Comparative Live attenuated rubella virus Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 30 Example 115 Live attenuated rubella virus Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 116 Live attenuated rubella virus Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 117 Live attenuated rubella virus Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Live attenuated rubella virus Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 31 Example 118 Live attenuated rubella virus Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 119 Live attenuated rubella virus Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 120 Live attenuated rubella virus Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30 Comparative Haemophilus influenzae type b Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 32 polysaccharide-tetanus toxoid conjugate Example 121 Haemophilus influenzae type b Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 polysaccharide-tetanus toxoid conjugate Example 122 Haemophilus influenzae type b Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 polysaccharide-tetanus toxoid conjugate Example 123 Haemophilus influenzae type b Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 polysaccharide-tetanus toxoid conjugate Comparative Haemophilus influenzae type b Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 33 polysaccharide-tetanus toxoid conjugate Example 124 Haemophilus influenzae type b Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 polysaccharide-tetanus toxoid conjugate Example 125 Haemophilus influenzae type b Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 polysaccharide-tetanus toxoid conjugate Example 126 Haemophilus influenzae type b Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30 polysaccharide-tetanus toxoid conjugate Comparative Recombinant HBs antigen protein Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 34 Example 127 Recombinant HBs antigen protein Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 128 Recombinant HBs antigen protein Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 129 Recombinant HBs antigen protein Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Recombinant HBs antigen protein Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 35 Example 130 Recombinant HBs antigen protein Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 131 Recombinant HBs antigen protein Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 132 Recombinant HBs antigen protein Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30 Comparative Live attenuated yellow fever virus Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 36 Example 133 Live attenuated yellow fever virus Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 134 Live attenuated yellow fever virus Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 135 Live attenuated yellow fever virus Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Live attenuated yellow fever virus Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 37 Example 136 Live attenuated yellow fever virus Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 137 Live attenuated yellow fever virus Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 138 Live attenuated yellow fever virus Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30

TABLE-US-00010 TABLE 10 Immunity Antigen induction promoter Amount Amount Dosage Administration Amount Name [μg] Bisphosphonate [μg] form route [μL] Comparative Tetanus toxoid Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 38 Example 139 Tetanus toxoid Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 140 Tetanus toxoid Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 141 Tetanus toxoid Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Tetanus toxoid Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 39 Example 142 Tetanus toxoid Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 143 Tetanus toxoid Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 144 Tetanus toxoid Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30 Comparative Live attenuated varicella virus Vaccine 100 μL equivalent — — Solution Transnasal 10 Example 40 Example 145 Live attenuated varicella virus Vaccine 100 μL equivalent Etidronate 10 Solution Transnasal 10 Example 146 Live attenuated varicella virus Vaccine 100 μL equivalent Alendronate 10 Solution Transnasal 10 Example 147 Live attenuated varicella virus Vaccine 100 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Live attenuated varicella virus Vaccine 100 μL equivalent — — Solution Sublingual 30 Example 41 Example 148 Live attenuated varicella virus Vaccine 100 μL equivalent Etidronate 100  Solution Sublingual 30 Example 149 Live attenuated varicella virus Vaccine 100 μL equivalent Alendronate 50 Solution Sublingual 30 Example 150 Live attenuated varicella virus Vaccine 100 μL equivalent Zoledronate 50 Solution Sublingual 30 Comparative Live BCG Vaccine 30 μL equivalent — — Solution Transnasal 10 Example 42 Example 151 Live BCG Vaccine 30 μL equivalent Etidronate 10 Solution Transnasal 10 Example 152 Live BCG Vaccine 30 μL equivalent Alendronate 10 Solution Transnasal 10 Example 153 Live BCG Vaccine 30 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Live BCG Vaccine 30 μL equivalent — — Solution Sublingual 30 Example 43 Example 154 Live BCG Vaccine 30 μL equivalent Etidronate 100  Solution Sublingual 30 Example 155 Live BCG Vaccine 30 μL equivalent Alendronate 50 Solution Sublingual 30 Example 156 Live BCG Vaccine 30 μL equivalent Zoledronate 50 Solution Sublingual 30 Comparative Inactivated rabies virus Vaccine 200 μL equivalent — — Solution Transnasal 10 Example 44 Example 157 Inactivated rabies virus Vaccine 200 μL equivalent Etidronate 10 Solution Transnasal 10 Example 158 Inactivated rabies virus Vaccine 200 μL equivalent Alendronate 10 Solution Transnasal 10 Example 159 Inactivated rabies virus Vaccine 200 μL equivalent Zoledronate 10 Solution Transnasal 10 Comparative Inactivated rabies virus Vaccine 200 μL equivalent — — Solution Sublingual 30 Example 45 Example 160 Inactivated rabies virus Vaccine 200 μL equivalent Etidronate 100  Solution Sublingual 30 Example 161 Inactivated rabies virus Vaccine 200 μL equivalent Alendronate 50 Solution Sublingual 30 Example 162 Inactivated rabies virus Vaccine 200 μL equivalent Zoledronate 50 Solution Sublingual 30

TABLE-US-00011 TABLE 11 Composition [parts by weight] Comparative Example Example Component 163 164 165 166 167 168 169 170 171 172 46 Antigen OVA 5 5 5 5 5 5 5 5 5 5 5 Immunostimulant Bisphosphonate Etidronate 2 — — — — — — — — — — Clodronate — 2 — — — — — — — — — Tiludronate — — 2 — — — — — — — — Pamidronate — — — 2 — — — — — — — Neridronate — — — — 1 — — — — — — Alendronate — — — — — 1 — — — — — Ibandronate — — — — — — 1 — — — — Zoledronate — — — — — — — 0.5 — — — Risedronate — — — — — — — — 0.5 — — Minodronate — — — — — — — — — 0.5 — Ointment cream (base cream) 93.0 93.0 93.0 93.0 94.0 94.0 94.0 94.5 94.5 94.5 95.0 Amount [mg/mouse] 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 Dosage form Cream Administration route Transdermal administration (minimally invasive)

INDUSTRIAL APPLICABILITY

[0152] The vaccine pharmaceutical composition of the present invention is universally usable for induction of humoral immunity against various antigens, exerts a high antibody production inducing effect, and favorably used for transdermal administration or transmucosal administration.