US 20130266612A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0266612 A1 FUKASAKA et al. (43) Pub. Date: Oct. 10, 2013

(54) VACCINE COMPOSITION Publication Classification (71) Applicants: NITTO DENKO CORPORATION, (51) Int. Cl. Osaka (JP): OSAKA UNIVERSITY, A 6LX39/39 (2006.01) Osaka (JP) A639/45 (2006.01) (52) U.S. Cl. (72) Inventors: Masahiro FUKASAKA, Osaka (JP): CPC ...... A61K 39/39 (2013.01); A61 K39/145 Arimichi OKAZAKI, Osaka (JP): (2013.01) Daisuke ASARI, Osaka (JP); Mitsuhiko USPC ...... 424/210.1; 424/184.1; 424/234.1; HORI, Osaka (JP); Shizuo AKIRA, 424/257.1; 424/258.1 Osaka (JP): Osamu TAKEUCHI, Osaka (JP) (57) ABSTRACT (73) Assignees: OSAKA UNIVERSITY, Osaka (JP); NTTO DENKO CORPORATION An intraorally administrable vaccine composition useful to Osaka (JP) s be a preventive or therapeutic agent for infectious diseases, and effectively induces a systemic immune response or a (21) Appl. No.: 13/856,002 mucosal immune response is provided. A vaccine composi tion for administration to the oral cavity of a human or an (22) Filed: Apr. 3, 2013 animal, the vaccine composition containing at least one anti gen derived from an infectious disease, and at least one (30) Foreign Application Priority Data selected from the group consisting of a toll-like receptor 4 (TLR4)agonist, a toll-like receptor 2/6 (TLR2/6) agonist, and Apr. 4, 2012 (JP) ...... 2012-0858.39 cyclic dinucleotide, or a derivative or salt thereof. Patent Application Publication Oct. 10, 2013 Sheet 1 of 7 US 2013/0266612 A1

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VACCINE COMPOSITION the gastric juice by a large content of an antacid, or to protect an antigen using coating techniques such as microspheres, or TECHNICAL FIELD the like. 0008. However, the development was practically success 0001. The present invention relates to a sublingually ful only on vaccines intrinsically highly stable in the gastric administrable vaccine composition useful to be a preventive juice Such as live attenuated poliovirus vaccine and live or therapeutic agent for infectious diseases. The present attenuated rotavirus Vaccine. invention particularly relates to a vaccine composition 0009. Alternatively, an allergy vaccine is an example of capable of effectively inducing a systemic immune response the oral administration preparation to induce an immune and a mucosal immune response when at least one selected response via the oral cavity mucosa (particularly Sublingual from the group consisting of a toll-like receptor 4 (TLR4) mucosa) delivery without Swallowing. This vaccine is termed agonist, a toll-like receptor 2/6 (TLR2/6) agonist, and cyclic as Sublingual immunotherapy (SLIT) and works by continu dinucleotide, or a derivative or salt thereof is sublingually ously administering Sublingually a plant-derived extract con administered as an adjuvant together with an antigen derived taining a protein to be an allergy antigen (allergen) to boost from an infectious disease. the immunotolerance against the allergen and reduce the allergy reaction. In recent years, SLIT is now widely accepted BACKGROUND ART in Europe and many products are available in the market today. 0002 Currently, the commercial vaccine preparations are 0010. The therapy using such a preparation which induces mostly available in the injection form. The injection form the immune response via the oral cavity mucosa route, par vaccine induces the immune response (IgG antibody produc ticularly the Sublingual mucosa route, is the focus of attention tion) in blood (systemic) but does not induce the mucosal because it provides better patient’s QOL and has a lower risk immune response (IgA antibody production), and thus pre of anaphylactic shock, critical adverse effect, than the con vents the post-infection pathogen growth but has a problem in ventional therapy which required the Subcutaneous injection the protection against the pathogen infection by mucosal of an allergen (Subcutaneous immunotherapy). rOute. 0011. However, SLIT has been to use preparations only for 0003. Under the circumstances, the vaccination via boosting a specific immunotolerance, but has been not a mucosal route has been drawing attention in recent years. In therapy to activate the immunity. The oral cavity mucosa is particular, the development of a vaccine containing an influ generally not likely to develop immunity, and the activation of enza virus as an antigen for mucosal administration (transna immunity, even if the immunotolerance is developed, has sal administration) has been in a high profile. been considered to be difficult. 0004. The mucosal administration vaccine induces the 0012 Examples of the induction of the mucosal immunity systemic immunity (IgG antibody production) and also and systemic immunity via the oral cavity mucosa route, induces the mucosal immunity (IgA antibody production). particularly the Sublingual mucosa route, are reported includ The IgA antibody does not strictly discriminate the pathogen ing the following. type of a disease to be targeted, is adaptable to ever-changing 0013 OVA-specific systemic immune response (IgG pro pathogen epidemic every year and is thus considered to be duction) and OVA-specific mucosal immune response (IgA effective for the pandemic prevention. production) are proposed to have been confirmed when OVA 0005. One of the reasons for the transnasal administration used as an antigen and cholera toxin used as an adjuvant were vaccine to attract attention is that the antigen administration administered Sublingually (see, for example, Patent Litera to the gastrointestinal mucosa is susceptible to the influences ture 1). However, in the proposal, highly neurotoxic cholera of gastric juice and proteases which are hardly evitable, toxin was used as the adjuvant and the safety issue was left to whereas the antigen administration to the transnasal mucosa be cleared. is free of these influences. Additionally, there is an antigen 0014. Using OVA as an antigen and 3 de-O-acylated recognition tissue called NALT on the nasal cavity mucosa monophosphoryl lipid A, a TLR4 agonist, as an adjuvant and which is beneficial for immune response. This is another administering them Sublingually have been proposed to bring reason for the transnasal administration vaccine to gain inter about OVA-specific systemic immune response (IgG produc eStS. tion) and OVA-specific mucosal immune response (IgA pro duction) as well (see, for example, Patent Literature 2). In this 0006. However, the antigen administration to the nasal proposal, a TLR4 agonist was Sublingually administered as cavity mucosa, while being highly effective, have had draw an adjuvant, however, no example regarding an antigen backs in that it is most likely to cause critical adverse effects derived from an infectious disease was presented and the Such as acute encephalopathy, or the like; the transnasal versatility of the effect to the antigen type was not evident. administration perse is cumbersome and difficult to practice Additionally, the comparatively large doses of OVA being 80 on the elderly, infant, or the like; and the stable effect is not to 160 g and 3 de-O-acylated monophosphoryllipid Abeing assured due to physical factors such as nasal mucus, or the 20 to 40 ug are not practical when considering the safety. like. 0015. A proposal on a method for synthesizing glucopy 0007. On the other hand, there have also been many ranosyl lipid, a synthetic adjuvant, (see, for example, Patent attempts to induce the systemic immunity and mucosal Literature 3) also describes the mucosal immune response immunity via oral administration of an antigen through the induction by the mucosal administration of an antigen in gastrointestinal mucosa (intestines), and the like, after Swal combination with the adjuvant. The inductions of serum IgG lowing. The concern here is how the antigen breakdown and IgA in a nasal wash are also proposed by the administra caused by the gastric juice or proteases is prevented. To solve tion of MALP-2, a TLR2/6 ligand, together with B-galactosi the problem, techniques have been developed to neutralize dase used as an antigen to the nasal cavity of a mouse (see, for US 2013/0266612 A1 Oct. 10, 2013 example, Patent Literature 4). However, no example regard 0027. The toll-like receptor 4 (TLR4) agonist preferably ing an antigen derived from an infectious disease or admin comprises at least one selected from the group consisting of istration thereof to the oral cavity mucosa was presented, and lipopolysaccharide, or salt thereof, and monophosphoryl thus the versatility of the effect was not evident. Using c-di lipid, or salt thereof. GMP or c-di-AMP. cyclic dinucleotide, as an adjuvant 0028. The lipopolysaccharide, or salt thereof is preferably together with B-galactosidase as an antigen and intranasally derived from , Salmonella, Pantoea, Aceto administering them to a mouse have been also proposed to bacterium, Zymomonas, Xanthomonas or Enterobacter; and bring about the induction of serum IgG (see, for example, the monophosphoryllipid, or salt thereof is preferably mono Patent Literature 5), however, the proposal does not mention phosphoryl lipid derived from Salmonella or synthetic glu the IgA induction via transnasal administration and no copyranosyl lipid. example regarding an antigen derived from an infectious 0029. The toll-like receptor 2/6 (TLR2/6) agonist com disease or administration thereof to the oral cavity mucosa prises preferably diacylated lipopeptide, or a derivative or salt was presented, and thus the versatility of the effect was not thereof. evident. 0030 The diacylated lipopeptide preferably comprises Pam CSK, MALP-2, FSL-1, or a derivative or salt thereof. CITATION LIST 0031. The cyclic dinucleotide preferably comprises c-di GMP, c-di-AMP or a derivative or salt thereof. Patent Literature 0032. In the vaccine composition of the present invention, 0016 Patent Literature 1: U.S. Patent Application Serial the administration to the oral cavity is preferably administra No. 2008/O112974 tion to the Sublingual mucosa. 0017 Patent Literature 2: JP 2003-5 19669 T 0033. The vaccine composition of the present invention 0018 Patent Literature 3: U.S. Patent Application Serial preferably induces a mucosal immune response and a sys No. 2010/03106O2 temic immune response, and the mucosal immune response is 0019 Patent Literature 4: U.S. Patent Application Serial antigen-specific IgA antibody production, and the systemic No. 2005/0276813 immune response is antigen-specific IgG antibody produc 0020 Patent Literature 5: U.S. Patent Application Serial tion and antigen-specific cell-mediated immune production. No. 2008/028.6296 0034. Hereinafter, the present invention is described in detail. SUMMARY OF INVENTION 0035. The vaccine composition of the present invention contains at least one antigen derived from an infectious dis Technical Problem CaSC. 0036. The above antigen derived from an infectious dis 0021. Under the current circumstances described above, ease refers to any Substances targeted by the immune the present invention has an object to provide a Sublingually response developed in a test organism. The above antigen administerable vaccine composition useful to be a preventive derived from an infectious disease may also be the target of or therapeutic agent for infectious diseases, and effectively the immune response (e.g., aging of immunocompetent cells, induces a systemic immune response or a mucosal immune cytokine production, antibody production, etc.) at the contact response. with the immunocompetent cell. Solution to Problem 0037. The antigen derived from an infectious disease used in the present invention is not limited insofar as an antigen is 0022. The present inventors conducted extensive studies an infectious pathogen or derived from an infectious patho to solve the above problem and found that a systemic immune gen. response and a mucosal immune response are effectively 0038. The disease developed from the above infectious induced when at least one selected from the group consisting pathogen is not limited and examples include viral diseases of a toll-like receptor 4 (TLR4) agonist, a toll-like receptor caused by the infection of viruses such as adenovirus, herp 2/6 (TLR2/6) agonist, and cyclic dinucleotide, or a derivative esvirus (e.g., HSV-I, HSV-II, CMV or VZV), poxviruses or salt thereof is orally administered, particularly, sublin (e.g., variola or vaccinia, or orthopoxviruses Such as mollus gually administered, as an adjuvant together with an antigen cum contagiosum, or the like), picornaviruses (e.g., rhinovi derived from an infectious disease, whereby the present rus or enterovirus), orthomyxoviruses (e.g., influenza invention was accomplished. viruses), paramyxoviruses (e.g., parainfluenza virus, mumps 0023. More specifically, the present invention relates to a virus, measles virus, respiratory syncytial virus (RSV)), coro vaccine composition for administration to the oral cavity of a naviruses (e.g., SARS), papovaviruses (e.g., papilloma virus human or an animal, the vaccine composition comprising: at which causes genital warts, common warts or plantar warts), least one antigen derived from an infectious disease, and at hepadnaviruses (e.g., hepatitis B virus), flaviviruses (e.g., least one selected from the group consisting of a toll-like hepatitis C virus or dengue virus), or retroviruses (e.g., len receptor 4 (TLR4) agonist, a toll-like receptor 2/6 (TLR2/6) tiviruses such as HIV, or the like), and the like; bacterial agonist, and cyclic dinucleotide, or a derivative or salt thereof. diseases caused by the infection of such as Escheri 0024. In the vaccine composition of the present invention, chia, Enterobacter; Salmonella, Staphylococcus, Shigella, the antigen derived from an infectious disease is preferably an Listeria, Aerobacter; Helicobacter, Klebsiella, Proteus, influenza virus-derived antigen. Pseudomonas, Streptococcus, Chlamydiaceae, , 0025. The influenza virus-derived antigen is preferably Pneumococci, Neisseria, Clostridium, Bacillus, Corynebac hemagglutinin protein. terium, Mycobacterium, Campyrobacter, Vibrion, Serratia, 0026. The influenza virus-derived antigen is preferably an Providencia, Chromobacterium, Brucella, Yersinia, Haemo inactivated whole virus. philus, or Bordetella, and the like; fungus diseases such as US 2013/0266612 A1 Oct. 10, 2013

Chlamydia, Candidiasis, Aspergillosis, Histoplasmosis, toll-like receptor 4 (TLR4) agonist, a toll-like receptor 2/6 Cryptococcal Meningitis, to begin with, but not limited (TLR2/6) agonist, and cyclic dinucleotide, or a derivative or thereto; Malaria, Pneumocystis carinii pneumonia, Leishma salt thereof. niasis, Cryptosporidiosis, Toxoplasmosis, Trypanosomal 0046. These compounds serve as adjuvants in the vaccine infection, and the like. composition of the present invention. 0039. In the present invention, the antigen derived from an 0047 Preferable examples of the toll-like receptor 4 infectious disease is preferably an influenza virus-derived (TLR4) agonist include lipopolysaccharide, or salt thereof. antigen. Additionally, the term “lipopolysaccharide' is intended to 0040. The influenza virus used herein refers to a RNA include, in addition to lipopolysaccharide, derivatives thereof enveloped virus belonging to Orthomyxoviridae and having a as long as they maintain properties of lipopolysaccharide. particle diameter size of about 100 nm, and is classified into The term "salt” refer to any organic acid salt or inorganic acid Types A, B and C based on the antigenicity of internal protein salt and a pharmaceutically acceptable salt is preferred. thereof. The influenza virus is composed of a core of an 0048. The lipopolysaccharide may be an extract from the internal nucleocapsid Surrounded by a virus envelope having of gram-negative bacteria or a modified product a structure or of ribonucleic acid (RNA) associ thereof, or a synthetic product. ated with nucleoprotein, and an external glycoprotein. The 0049. Examples of the gram-negative bacteria include inner layer of the above virus envelope is consisted mainly of Escherichia sp., Shigella sp., Salmonella sp., Klebsiella sp., matrix proteins and the outer layer is consisted mostly of lipid Proteus sp., Yersinia sp., V. cholerae sp., Vparahaemolyticus materials derived from a host cell. The RNA of the influenza sp., Haemophilus sp., Pseudomonas sp., Legionella sp., Bor virus has a segmental structure. Additionally, influenza detella sp., Brucella sp., Francisella tularensis sp., Bacteroi worldwide pandemics are caused by Type A influenza virus. des sp., Neisseria sp., Chlamydia sp., Plesiomonas sp., Pro Type A influenza virus has 2 types of envelope glycoproteins, phyromonas sp., Pantoea sp., Agrobacterium sp., hemagglutinin (HA) and neuraminidase (NA), and, based on Stenortophomonas sp., Enterobacter sp., Acetobacter sp., the antigen type, HA is classified into 16 subtypes and NA Xanthomonas sp., Zymomonas sp., and the like. into 9 subtypes. 0050 Preferable gram-negative bacteria among them are those derived from Escherichia sp., Salmonella sp., Pantoea 0041. In the present invention, the antigens derived from sp., Acetobacter sp., Zymomonas sp., Xanthomonas or Types A and B influenza viruses are preferably used to be the Enterobacter sp. These bacteria have been used in many food above antigen derived from an infectious disease. The sub products and herbal medicine for many years and the safety in type of Type A and Type B influenza viruses described above the living body is guaranteed. The Pantoea bacteria, in par is not limited, and may be any of the subtypes isolated so far ticular, are currently used as health food products and assure or subtypes to be isolated in a future. more effectiveness. The extracts derived from these bacteria 0042. In the present invention, the antigen derived from or modified products thereofmay also be used without further influenza viruses is not limited insofar as it is at least part of treatment. the many components composing the above influenza 0051. When the lipopolysaccharide is used in the form of viruses. Examples include an inactivated whole virus in an extract from the cell wall of the above gram-negative which a purified virus particle is inactivated with an organic bacteria or a purified lipopolysaccharide, the safety in the Solvent/surfactant or other reagents, or a virus subunit pre living body should generally be considered and a modified pared by removing impurities from the inactivated whole product may also be used for the purpose of detoxifying them. virus and purifying HA and/or NA, and the like. The HA On the other hand, Acetobacter sp. (Acetobacter aceti, Aceto subunit or inactivated whole virus is preferable in light of the bacter xylinum, Acetobacter Orientalis, or the like), Zymomo immunogenicity. The inactivated whole virus is preferably nas sp. (Zymomonas mobilis, or the like), Xanthomonas sp. inactivated using formalin, or the like. The HA subunit (split), (Xanthomonas campestris, or the like), Enterobacter sp. (En having very few impurities and requiring an adjuvant, is par terobacter cloacae, or the like), and Pantoea sp. (Pantoea ticularly effective. agglomerans, or the like) have been used in many food prod 0043. The method for preparing the influenza virus anti ucts and herbal medicine for many years, and the safety in the gen is not limited and any known method is used with no living body is guaranteed. The extracts derived from these limitation. Examples include a method in which a virus strain bacteria or purified lipopolysaccharide may also be used isolated from an influenza-infected animal or an influenza without further treatment. patient is caused to infect a hen's egg, or the like, and incu 0.052 The toll-like receptor 4 (TLR4) agonist may be a bated in a routine manner, thereby preparing an antigen from derivative of the lipopolysaccharide and examples include the purified undiluted virus solution. Alternatively, an antigen lipid A wherein the polysaccharide moiety is removed from derived from a virus prepared in culture cells using a genetic the lipopolysaccharide or monophosphoryl lipid A, 3 de engineering technique may also be used. acylated MPL, and the like, and salts thereof. 0044) The vaccine composition of the present invention 0053. The lipid A wherein the polysaccharide moiety is may contain the antigen derived from an infectious disease in removed from the lipopolysaccharide may be an isolate an effective amount, but, preferably contains in an amount, derived from the above gram-negative bacteria, or a product for example, ranging from 0.001 to 1000 ug to the total synthesized so as to have the same structure as the isolate amount thereof per individual per administration. The derived from the above gram-negative bacteria. amount is more preferably 0.01 to 100 ug and still more 0054 Preferably used examples of the lipid A modified preferably 0.1 to 50LL. An amount below 0.001 g may result product are dephosphorylated monophosphoryl lipid (MPL) in the insufficient function to be a preventive or therapeutic or salt thereof. Additionally, the term “monophosphoryl lipid agent for infectious diseases, whereas an amount exceeding (MPL) is intended to include, in addition to monophospho 1000 ug may raise a safety issue. Additionally, the term “per ryl lipid (MPL), derivatives thereofas long as they maintain individual” refers to any mammals and a human is preferred. properties of lipopolysaccharide. 0045. The vaccine composition of the present invention 0055. In particular, 3 de-acylated monophosphoryl lipid contains at least one selected from the group consisting of a (3D-MPL) which has been already used as an adjuvant for the US 2013/0266612 A1 Oct. 10, 2013 medical purpose, or the non-deacylated synthetic glucopyra 0069. The solid preparation herein encompasses tablets, nosyl lipid proposed in the US Patent Application Ser. No. coated tablets, powders, granules, fine granules, orally disin 2010/0310602 specification are preferable in light of the tegrating tablet, oral patches, jellies and films, and is not safety in the living body. limited insofar as it is a Solid preparation to be administered to 0056 Salmonella bacteria, previously used and are safe, the oral cavity mucosa or Sublingual mucosa. are also preferably used as the monophosphoryl lipid. 0070 The vaccine composition of the present invention is 0057 The toll-like receptor 2/6 (TLR2/6) agonist prefer administered to the oral cavity of human or animals (mam ably contains, for example, diacylated lipopeptide, or a mals, avian, or the like), but the oral cavity administration is derivative or salt thereof. preferably Sublingual mucosa administration. As described 0058. The diacylated lipopeptide preferably contains at earlier, the oral cavity mucosa is generally not likely to least one selected from the group consisting of previously develop immunity, and the activation of immunity, even if the used and thus safe Pam CSK, MALP-2 and FSL-1, or a immunotolerance is developed, has been considered to be derivative or salt thereof. difficult. However, the vaccine composition of the present 0059. The toll-like receptor 2/6 (TLR2/6) agonist may be invention uses the specific adjuvants described above in com an extract from mycoplasma cell membrane or a modified bination with at least one antigen derived from an infectious product thereof, or a synthetic product. disease, and thus effectively induces the systemic immune 0060 Examples of the mycoplasma include Mycoplasma response and mucosal immune response even when adminis pneumoniae, , Mycoplasma hominis, tered to the oral cavity mucosa. Ureaplasma, Mycoplasma salivarium, Mycoplasma fermen 0071. The sublingual mucosa administration also enables tans, Mycoplasma gallisepticum, Mycoplasma hyopneumo the composition to be less susceptible to the influences of niae, Mycoplasma laboratorium, , gastric juice and proteases unlike the antigen administration Mycoplasma ovipneumoniae, and the like. to the gastrointestinal mucosa; and to be free of potential 0061. When the toll-like receptor 2/6 (TLR2/6) agonist is critical adverse effects such as acute encephalopathy, or the used in the form of an extract from the mycoplasma cell like: to be easy to practice on the elderly, infant, or the like: membrane or a purified diacylated lipopeptide, the safety in and to provide stable effects uninterrupted by physical factors the living body should generally be considered and a modified Such as nasal mucus, or the like, unlike the antigen adminis product may also be used for the purpose of detoxifying them. tration to the transnasal mucosa. 0072 The method for administering the vaccine compo 0062. The cyclic dinucleotide may be cyclic bis-dipurine sition of the present invention is as described earlier. The dose nucleotide, or a derivative or salt thereof and preferable is determined in accordance with animal species, age, sex, examples include, in light of the safety, c-di-GMP c-di-AMP. body weight to be administered, and the like, and, for or a derivative or salt thereof. example, 0.1 ug to 50 ug is typically administered once or 0063. The above TLR4 agonist, TLR2/6 agonist and c-di more than twice when HA is used as the antigen derived from GMP each sufficiently function to serve as a sublingual adju an infectious disease. The vaccine composition is preferably vant. The TLR4 agonist, particularly, is available at a low administered in several doses, and the composition in this price and has been used to human. For example, Pantoea LPS, case is preferably administered with 1 to 4 week intermittent. one of the above TLR4 agonists, has been commonly used in To use inactivated whole virus as the antigen derived from an health food products and offers a benefit of being easily infectious disease, the dose is determined in terms of HA. adaptable to human. Additionally, the HA weight is a value measured by SRD titer 0064. The vaccine composition of the present invention or the Lowry method. may preferably contain the above 3 adjuvants (TLR4, TLR2/ 6, cyclic dinucleotide) in an amount, for example, ranging Advantageous Effects of Invention from 0.1 ug to 100 mg to the total amount thereof. An amount below 0.1 g may result in the insufficient function to be a 0073. The vaccine composition of the present invention preventive or therapeutic agent for infectious diseases, uses the specific adjuvants described above in combination whereas an amount exceeding 100 mg may raise a safety with at least one antigen derived from an infectious disease, issue. More preferable lower limit content of the above adju and thus effectively induces the systemic immune response vants is 0.3 ug, and more preferable upper limit content is 50 and mucosal immune response when administered to the oral ng. cavity mucosa, particularly Sublingual mucosa. 0065. Additionally, the vaccine composition of the present invention may use other conventionally used known adju BRIEF DESCRIPTION OF DRAWINGS vants in combination with these adjuvants insofar as the com position contains at least one adjuvant selected from the 0074 FIG. 1 is a graph showing the results of mouse serum group consisting of the above three adjuvants. influenza HA-specific IgG titer in Examples 1 to 6 and Com 0066. The vaccine composition of the present invention parative Examples 1 to 9. may be prepared by adding other components (e.g., phos 0075 FIG. 2 is a graph showing the results of mouse nasal phate buffer, and the like) as necessary to the above antigen wash influenza HA-specific IgA titer in Examples 1 to 6 and derived from an infectious disease and the adjuvants and Comparative Examples 1 to 9. mixing with stirring by a known method. 0076 FIG.3 is a graph showing the results of mouse serum 0067. The vaccine composition of the present invention influenza HA-specific IgG titer in Examples 7 to 9 and Com may also be formulated into a solution, a solid preparation or parative Examples 10 to 12. a spray, and Suitably use, if desired, excipient, binder, per 0077 FIG. 4 is a graph showing the results of mouse nasal fume, flavor, Sweetener, colorant, preservative, antioxidant, wash influenza HA-specific IgA titer in Examples 7 to 9 and stabilizer, surfactant, and/or the like, in addition to the mate Comparative Examples 10 to 12. rials described above. 0078 FIG. 5 is a graph showing the survival rates of mice 0068. These additives are not limited and the convention infected with influenza virus in Example 10, Comparative ally used known materials may be used. Examples 13 and 14. US 2013/0266612 A1 Oct. 10, 2013

007.9 FIG. 6 is a graph showing the results of mouse serum Comparative Example 1 influenza HA-specific IgG titer in Examples 11, Comparative (0093. Five mice (7-week old, C57BL/6 female mice, Examples 1 and 14. Japan SLC, Inc.) prepared in advance were anaesthetized, 0080 FIG. 7 is a graph showing the results of mouse nasal 120 uL of phosphate buffer (Nacalai Tesque, Inc.) was pre wash influenza HA-specific IgA titer in Examples 11, Com pared and 20 LL thereof was Sublingually administered to parative Examples 1 and 14. each of the mice. The test was carried out by the same sub 0081 FIG. 8 is a graph showing the results of mouse serum sequent procedure as Example 1. influenza HA-specific IgG titer in Examples 12 and 13, Com parative Examples 15, 16 and 1. Comparative Example 2 0082 FIG.9 is a graph showing the results of mouse nasal 0094. In place of the phosphate buffer, phosphate buffer wash influenza HA-specific IgA titer in Examples 12 and 13, (Nacalai Tesque, Inc.) was added to 76.3 uL (236 ug/mL) of Comparative Examples 15, 16 and 1. an influenza HA antigen-containing solution (A/IvPRB/34 0083 FIG. 10 is a graph showing the results of mouse (H1N1), The Research Foundation for Microbial Diseases of serum influenza HA-specific IgG titer in Examples 12, 14 and Osaka University) to prepare 120 uL of a vaccine composi 15, Comparative Examples 16 and 1. tion. The test was carried out by the same Subsequent proce 0084 FIG. 11 is a graph showing the results of mouse dure as Example 1 in the dose shown in Table 1. nasal wash influenza HA-specific IgA titer in Examples 12, 14 and 15, Comparative Examples 16 and 1. Comparative Examples 3 to 9 0085 FIG. 12 is a graph showing the results of mouse 0.095 Vaccine compositions were prepared in the same serum influenza HA-specific IgG titer in Examples 16 and 17. al Comparative Examples 17 and 18. 0096 Comparative Example 2, except that, in addition to I0086 FIG. 13 is a graph showing the results of mouse the influenza HA antigen-containing solution (A/IvPR8/34 nasal wash influenza HA-specific IgA titer in Examples 16 (H1N1), The Research Foundation for Microbial Diseases of and 17, Comparative Examples 17 and 18. Osaka University), peptidoglycan (PGN derived from Salmo nella, InvivoGen) was used in Comparative Example 3, Zymosan (Nacalai Tesque, Inc.) was used in Comparative DESCRIPTION OF EMBODIMENTS Example 4, Pam CSK (InvivoGen) was used in Comparative 0087 Hereinafter, the present invention is described fur Example 5, Poly (I:C) (InvivoGen) was used in Comparative ther in detail with reference to the following examples, but is Example 6, flagellin (InvivoGen) was used in Comparative not limited thereto. Example 7, imiquimod (InvivoGen) was used in Comparative Example 8 and CpG (InvivoGen) was used in Comparative Example 1 Example 9. The test was carried out by the same procedure as Example 1 in the doses shown in Table 1. 0088 Phosphate buffer (Nacalai Tesque, Inc.) was added to 76.3 uL (236 g/mL) of an influenza HA antigen-contain TABLE 1 ing solution (A/IvPR8/34(H1N1), The Research Foundation for Microbial Diseases of Osaka University) and 30 uL (1 Antigen Adiuvant mg/mL) of a solution containing E. Coli lipopolysaccharide Dose Dose (Nacalai Tesque, Inc.) to prepare 120 uL of a vaccine com (Igbody Substance (ig/body position. No. time) Ligand l8le time) I0089. Five mice (7-week old, C57BL/6 female mice, Example 1 3 TLR4 E. coli LPS 5 Japan SLC, Inc.) prepared in advance were anaesthetized Example 2 3 TLR4 Patrioed LPS 5 (Somnopentyl, Kyoritsu Seiyaku Corporation), and 20 uL of Example 3 3 TLR4 GLA 5 the prepared vaccine composition was Sublingually adminis Example 4 3 TLR2?6 FSL-1 5 tered to each of the mice. Example 5 3 TLR2?6 Pam CSK 5 Example 6 3 unclear c-di-GMP 138 0090 1 week later from the administration, the mice were Comparative anaesthetized again and 20 Jull of the prepared vaccine com Example 1 Comparative 3 position was Sublingually administered to each of the mice. Example 2 0.091 Another week later from the second administration, Comparative 3 TLR2 PGN 100 the serum and nasal wash were collected from the mice, and Example 3 the serum influenza HA-specific IgG titer and nasal wash Comparative 3 TLR2/Dectin1 Zymosan 5 influenza HA-specific IgA titer were measured by ELISA. Example 4 Comparative 3 TLR2?1 Pam CSK 5 The measurement method is described later in detail. Example 5 Comparative 3 TLR3 poly I:C 5 Examples 2 to 6 Example 6 Comparative 3 TLRS flagelin 5 0092. Vaccine compositions were prepared in the same Example 7 Comparative 3 TLR7 imiquimod 5 manner as Example 1, except that, in place of the E. coli Example 8 lipopolysaccharide, Pantoea lipopolysaccharide (macrophi Comparative 3 TLR9 CpG 5 inc.) was used in Example 2, glucopyranosyl lipid (MPLAS, Example 9 InvivoGen) was used in Example 3, FSL-1 (InvivoGen) was used in Example 4, Pam CSK (InvivoGen) was used in Example 5 and c-di-GMP (Cyclic diguanosine monophos phate, Biolog Inc.) was used in Example 6. The test was Examples 7 to 9 carried out by the same procedure as Example 1 in the doses 0097. In addition to 19 ul. (236 ug/mL) of the influenza shown in Table 1. HA antigen-containing solution (A/IvPR8/34(H1N1), The US 2013/0266612 A1 Oct. 10, 2013

Research Foundation for Microbial Diseases of Osaka Uni 0101 Ten mice (7-week old, BALB/c female mice, Japan versity), phosphate buffer (Nacalai Tesque, Inc.) was added to SLC, Inc.) prepared in advance were anaesthetized and 20 ul, 30 uL. (1 mg/mL) of glucopyranosyl lipid (MPLAs, Invivo of the prepared vaccine composition was Sublingually admin Gen) to prepare 120 L of a vaccine composition in Example istered to each of the mice. 7. 20 uL thereof was sublingually administered to the mice 0102 1 week later from the administration, the mice were (7-week old BALE/c female mice, Japan SLC, Inc.) under anaesthetized again and 20 uL of the prepared vaccine com anaesthesia. In place of the glucopyranosyl lipid used in position was Sublingually administered to each of the mice. Example 7. FSL-1 (InvivoGen) was used in Example 8, and (0103) Another week later from the second administration, c-di-GMP (Cyclic diguanosine monophosphate, Biolog Inc.) the mice were infected with the lethal dose of influenza virus was used in Example 9 to prepare vaccine compositions. The (A/IvPRB/34(H1N1) and monitored for the following 2 doses are as shown in Table 2. The test was carried out by the weeks to measure the Survival rates. The measurement same procedure as Example 1 after the preparation, except method is described later in detail. that the mouse type was different. Comparative Examples 13 and 14 Comparative Example 10 0104. In Comparative Example 13, a solution containing only the influenza HA antigen-containing solution (A/IvPR8/ 0098 Phosphate buffer (Nacalai Tesque, Inc.) was added 34(H1N1), The Research Foundation for Microbial Diseases to 19LL (236 g/mL) of the influenza HA antigen-containing of Osaka University) was prepared, and in Comparative solution (A/IvPR8/34(H1N1), The Research Foundation for Example 14, a phosphate buffer was prepared. The test was Microbial Diseases of Osaka University) to prepare 120 uL of carried out by the same procedure as Example 10 in the doses a vaccine composition. The dose was as shown in Table 2. The shown in Table 3. test was carried out by the same procedure as Example 1 after the preparation, except that BALE/c mice were used. TABLE 3 Comparative Examples 11 and 12 Antigen Adiuvant Dose Dose 0099. In addition to 76.3 uL (236 g/mL) of the influenza (1g (1g HA antigen-containing solution (A/IvPR8/34(H1N1), The body Substance body/ Research Foundation for Microbial Diseases of Osaka Uni No. Type time) Ligand l8le time) versity), in Comparative Example 11, phosphate buffer (Na Example 10 AIvPR8/34(H1N1) 3 TLR4 Pantoea 5 calai Tesque, Inc.) was added to 30 LL (20 mg/mL) of pepti LPS doglycan (PGN derived from Salmonella, InvivoGen) to Comparative AIvPR8/34(H1N1) 3 prepare 120 uL of a vaccine composition. In place of the Example 13 peptidoglycan used in Comparative Example 11, Pam CSK Comparative — (InvivoGen) was used in Comparative Example 12 to prepare Example 14 a vaccine composition. The doses were as shown in Table 2. The test was carried out by the same procedure as Example 1 after the preparation, except that BALB/c mice were used. Example 11 0105. Phosphate buffer (Nacalai Tesque, Inc.) and 30 uI. TABLE 2 (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 10.1 uL (1776 ug/mL) of an Antigen Adiuvant influenza HA antigen-containing inactivated whole virus solution (A/California/7/2009(H1N1), The Research Foun Dose Dose dation for Microbial Diseases of Osaka University) to prepare body Substance body/ 120 uL of a vaccine composition. No. Type time) Ligand name time) 0106 The test was carried out by the same procedure as Example 7 AIvPR8/34(H1N1) O.75 TLR4 GLA 5 Example 1 in the dose shown in Table 4. Example 8 AIvPR8/34(H1N1) O.75 TLR2.6 FSL-1 5 Example 9 A/IvPR8/34(H1N1) 0.75 unclear c-di-GMP 138 Comparative Example 14 Comparative AIvPR8/34(H1N1) 0.75 - Example 10 0107. In Comparative Example 14, a solution containing Comparative AIvPR8/34(H1N1) 0.75 TLR2 PGN 1OO only the influenza HA antigen-containing inactivated whole Example 11 virus solution (A/California/7/2009(H1N1)) was prepared. Comparative AIvPR8/34(H1N1) 0.75 TLR2/1 Pam CSK 5 Example 12 The test was carried out by the same procedure as Example 1 in the dose shown in Table 4.

TABLE 4 Example 10 Antigen Adiuvant

0100 Phosphate buffer (Nacalai Tesque, Inc.) and 60 LL Dose Dose (1 mg/mL) of lipopolysaccharide derived from Pantoea (1g (1g (macrophi inc.) were added to 152.6 uL (236 g/mL) of the body Substance body/ influenza HA antigen-containing solution (A/IvPR8/34 No. Type time) Ligand l8le time) (H1N1), The Research Foundation for Microbial Diseases of Example 11 Inactivated whole 3 TLR4 Pantoea 5 Osaka University). The Research Foundation for Microbial virus (A/California LPS Diseases of Osaka University) to prepare 240 u, of a vaccine 7/2009(H1N1)) composition. US 2013/0266612 A1 Oct. 10, 2013

TABLE 4-continued TABLE 5-continued Antigen Adiuvant Antigen Adiuvant

Dose Dose Dose Dose (ig/ (1g body Substance body body/ Substance body/ No. Type time) Ligand l8le time) No. Type time) Ligand l8le time) Comparative Inactivated whole 3 Comparative — Example 14 virus (A/California Example 1 7/2009(H1N1)) Comparative — Example 1 Example 14 0108 Example 12 0114 Phosphate buffer (Nacalai Tesque, Inc.) and 30 uI. 0109 Phosphate buffer (Nacalai Tesque, Inc.) and 30 uI. (1 mg/mL) of lipopolysaccharide derived from Pantoea (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 33.7 uL (534 ug/mL) of an (macrophi inc.) were added to 36 uL (500 g/mL) of an influenza HA antigen-containing solution (A/Victria/361/ influenza HA antigen-containing solution (A/California/7/ 2009(H3N2), The Research Foundation for Microbial Dis 2009(H1N1), The Research Foundation for Microbial Dis eases of Osaka University) to prepare 120 uL of a vaccine eases of Osaka University) to prepare 120 uL of a vaccine composition. The test was carried out by the same procedure composition. as Example 1 in the dose shown in Table 6. 0110. The test was carried out by the same procedure as Example 15 Example 1 in the dose shown in Table 5. 0115 Phosphate buffer (Nacalai Tesque, Inc.) and 30 uI. Example 13 (1 mg/mL) of lipopolysaccharide derived from Pantoea (macrophi inc.) were added to 47.4 uL (380 ug/mL) of the 0111. In place of the lipopolysaccharide derived from influenza HA antigen-containing solution (B/Brisbane/60/ Pantoea used in Example 12, c-di-GMP (Cyclic diguanosine 2008, The Research Foundation for Microbial Diseases of monophosphate, Biolog Inc.) was used in Example 13 to Osaka University) to prepare 120 uL of a vaccine composi prepare a vaccine composition in the same manner as tion. The test was carried out by the same procedure as Example 12. The test was carried out by the same procedure Example 1 in the dose shown in Table 6. as Example 1 in the dose shown in Table 5. TABLE 6 Comparative Example 15 Antigen Adiuvant 0112. In place of the lipopolysaccharide derived from Dose Dose Pantoea used in Example 12, imiquimod (InvivoGen) was (ig/ (1g used in Comparative Example 15 and the test was carried out body/ Substance body/ by the same procedure as Example 1 in the dose shown in No. Type time) Ligand l8le time) Table 5. Example 12 A/California 3 TLR4 Pantoea 5 7/2009(H1N1) LPS Comparative Example 16 Example 14 AVictoria 3 TLR4 Pantoea 5 361/2009 (H3N2) LPS 0113. In place of the lipopolysaccharide derived from Example 15 B/Brisbane? 3 TLR4 Pantoea 5 Pantoea used in Example 12, a solution containing only the 60,2008 LPS influenza HA antigen-containing solution (A/IvPR8/34 Comparative A/California 3 (H1N1), The Research Foundation for Microbial Diseases of Example 16 7/2009(H1N1) Osaka University) was prepared without adding any other Comparative — components in Comparative Example 16. The test was carried Example 1 out by the same procedure as Example 1 in the dose shown in Table 5. Example 16 TABLE 5 0116 A vaccine composition was prepared in the same manner as Example 12. More specifically, phosphate buffer Antigen Adiuvant (Nacalai Tesque, Inc.) and 30 uL (1 mg/mL) of lipopolysac Dose Dose charide derived from Pantoea (macrophi inc.) were added to (ig/ (1g 36 uL (500 ug/mL) of an influenza HA antigen-containing body/ Substance body solution (A/California/7/2009(H1N1), The Research Foun No. Type time) Ligand l8le time) dation for Microbial Diseases of Osaka University) to prepare Example 12 A/California 3 TLR4 Panioea 5 120 uL., of a vaccine composition. 7/2009(H1N1) LPS 0117 The test was carried out by the same procedure as Example 13 A/California 3 unclear c-di-GMP 5 Example 1 in the dose shown in Table 7, except that BALB/c 7/2009(H1N1) mice were used. Comparative A/California 3 TLR7 imiquimod 5 Example 15 7/2009(H1N1) Example 17 Comparative A/California 3 Example 16 7/2009(H1N1) 0118 Phosphate buffer (Nacalai Tesque, Inc.) and 30 uI. (1 mg/mL) of lipopolysaccharide derived from Pantoea US 2013/0266612 A1 Oct. 10, 2013

(macrophi inc.) were added to 18 uL (1 mg/mL) of OVA DS Pharma Biomedical Co., Ltd.) was diluted to 4 g/100 mL (Ovalbumin, Sigma-Aldrich Co. LLC.) to prepare 120 uL of with purified water was added to each well, which was a vaccine composition. allowed to stand at room temperature for 2 hours. Subse 0119 The test was carried out by the same procedure as quently, the wells were washed 3 times with the wash Example 1 in the dose shown in Table 7, except that BALB/c (Tween20-containing PBS). mice were used. 0127. The serum collected from the mice in advance was centrifuged at 4° C. at 3000 G for 10 minutes, 300 uL of Comparative Example 17 phosphate buffer (Nacalai Tesque, Inc.) was added to 20 uL of 0120 In place of the lipopolysaccharide derived from the Supernatant to prepare a diluted serum Solution. Pantoea used in Example 16, a solution containing only the I0128. Using a solution wherein a blocking agent (Block influenza HA antigen-containing solution (A/IvPR8/34 Ace, DS Pharma Biomedical Co., Ltd.) wad diluted to 0.4 (H1N1), The Research Foundation for Microbial Diseases of g/100 mL with phosphate buffer (Nacalai Tesque, Inc.), the Osaka University) was prepared without adding any other above diluted serum solution was diluted two-fold in 16 steps, components in Comparative Example 17. The test was carried and 50 uL of the solution was added and allowed to stand at out by the same procedure as Example 1 in the dose shown in room temperature for 2 hours. Table 7, except that BALB/c mice were used. 0129. The wells were washed 3 times with a wash (Tween20-containing PBS), an HRP-labeled anti-mouse IgG Comparative Example 18 antibody (Goat-anti-mouse IgG Fc HRP, BETHYL Labora tories, Inc.) was diluted 10000-fold with the solution wherein 0121. In place of the lipopolysaccharide derived from a blocking agent (Block Ace, DS Pharma Biomedical Co., Pantoea used in Example 17, a solution containing only OVA Ltd.) was diluted to 0.4 g/100 mL with phosphate buffer (Ovalbumin, Sigma-Aldrich Co. LLC.) was prepared without (Nacalai Tesque, Inc.), and 100 uL each was added and adding any other components in Comparative Example 18. allowed to stand at room temperature for 1 hour. The test was carried out by the same procedure as Example 1 0.130. Subsequently, the wells were washed 3 times with in the dose shown in Table 7, except that BALB/c mice were the wash (Tween20-containing PBS) and 100 uL each of a used. TMB solution (ELISA PODTMB kit, Nacalai Tesque, Inc.) was added. 100 uL of a 1M sulfuric acid solution was added TABLE 7 thereto and the 96-well plate was measured using a micro plate reader (168-11135CAM, Bio-rad Laboratories, Inc.) for Antigen Adiuvant the 450 nm absorbance. The mouse serum IgG titers were Dose Dose determined in log 2 based on the absorbance at the time of (ig/ (1g step dilution. body/ Substance body 0131 Measurement Method of Mouse Nasal Wash Influ No. Type time) Ligand l8le time) enza HA-Specific IgA Titer (ELISA) Example 16 A/California 3 TLR4 Panioea 5 (0132) 100 uL of each influenza HA (e.g., A/IvPR8/34 7/2009(H1N1) LPS (H1N1) influenza HA for measuring an A/IvPR8/34(H1N1)- Example 17 Ovalbumin 3 TLR4 Panioea 5 specific IgA antibody titer) diluted with carbonate buffer or (OVA) LPS Comparative A/California 3 an OVA-containing Solution (5 ug/mL) was added to an Example 17 7/2009(H1N1) ELISA 96-well plate and allowed to stand overnight. Comparative Ovalbumin 3 0133. The wells were washed 3 times with a wash Example 18 (OVA) (Tween20-containing PBS) prepared in advance, and 200 ul, of a blocking solution wherein a blocking agent (Block Ace, DS Pharma Biomedical Co., Ltd.) was diluted to 4 g/100 mL Test Method with purified water was added to each well, which was allowed to stand at room temperature for 2 hours. 0122) The systemic immune response was evaluated by I0134. The wells were subsequently washed 3 times with a measuring the mouse serum influenza HA- or OVA-specific wash (Tween20-containing PBS). The nasal washes collected IgG titer. The mucosal immune response was evaluated by from the mice were diluted two-fold in 12 steps with the measuring the mouse nasal wash influenza HA- or OVA solution wherein a blocking agent (Block Ace, DS Pharma specific IgA titer. Each of the evaluation method is described Biomedical Co., Ltd.) wad diluted to 0.4 g/100 mL with below. Each of the evaluation results are shown in FIGS. 1 to phosphate buffer (Nacalai Tesque, Inc.), and 50LL each of the 13 (except FIG. 5). Solution was added and allowed to stand at room temperature 0123. A virus infection experiment was meanwhile car for 2 hours. ried out to reveal the vaccine efficacy by infecting the mice 0.135 The wells were washed 3 times with a wash with the influenza virus. The evaluation results are shown in (Tween20-containing PBS), an HRP-labeled anti-mouse IgA FIG.S. antibody (Goat-anti-mouse IgA Fc HRP, BETHYL Labora 012.4 Measurement Method of Mouse Serum Influenza tories, Inc.) was diluted 10000-fold with the solution wherein HA-Specific IgG Titer (ELISA) a blocking agent (Block Ace, DS Pharma Biomedical Co., 0125 100 uL of each influenza HA (e.g., A/IvPR8/34 Ltd.) was diluted to 0.4 g/100 mL with phosphate buffer (H1N1) influenza HA antigen solution for measuring an (Nacalai Tesque, Inc.), and 100 uL each thereof was added A/IvPR8/34(H1N1)-specific IgG antibody titer) diluted with and allowed to stand at room temperature for 1 hour. Subse carbonate buffer or an OVA-containing solution (5 ug/mL) quently, the wells were washed 3 times with the wash was added to an ELISA 96-well plate and allowed to stand (Tween20-containing PBS) and 100 uL each of a TMB solu overnight. tion (ELISA PODTMB kit, Nacalai Tesque, Inc.) was added. 0126 The wells were washed 3 times with a wash 100 uL of a 1M sulfuric acid solution was added thereto and (Tween20-containing PBS) prepared in advance, and 200 uL the 96-well plate was measured using a microplate reader of a blocking Solution wherein a blocking agent (Block Ace, (168-11135CAM, Bio-rad Laboratories, Inc.) for the 450 nm US 2013/0266612 A1 Oct. 10, 2013

absorbance. The mouse nasal wash IgA titers were deter with at least one antigen derived from an infectious disease, mined in log 2 based on the absorbance at the time of step and thus effectively induces the systemic immune response dilution. and mucosal immune response even when administered to the oral cavity mucosa. A/IvPR8/34 (H1N1) Influenza Virus Infection Experiment to 1. A vaccine composition for administration to the oral cavity of a human or an animal, the vaccine composition Mice (Survival Rate Measurement) comprising: 0.136) 15uLofdiluted A/IvPR8/34 (H1N1) influenza virus at least one antigen derived from an infectious disease, and was administered under anaesthesia to the vaccine-adminis at least one selected from the group consisting of: tered mice 1 week later from the final administration (10x a toll-like receptor 4 (TLR4) agonist, LD50: 50% Lethal Dose). Defining the virus administration a toll-like receptor 2/6 (TLR2/6) agonist, and day as Day 0, the mice were monitored up to Day 14 to cyclic dinucleotide, or a derivative or salt thereof. measure the Survival rates. 2. The vaccine composition according to claim 1, 0.137 As shown in FIGS. 1 and 2, the influenza HA-spe wherein the antigen derived from an infectious disease is cific IgG and IgA were produced in high yields in Examples an influenza virus-derived antigen. 1 to 6, whereas the influenza-specific IgA was produced in 3. The vaccine composition according to claim 2, low yields and the influenza HA-specific IgG was only some wherein the influenza virus-derived antigen is hemagglu what produced in Comparative Examples 1 to 9. These results tinin protein. revealed that the TLR4 agonist and TLR2/6 agonist, cyclic 4. The vaccine composition according to claim 2, dinucleotide are effective as adjuvants for inducing the wherein the influenza virus-derived antigen is an inacti mucosal immunity when Sublingually administered. vated whole virus. 0.138. As shown in FIGS. 3 and 4, the comparison between 5. The vaccine composition according to claim 1, Examples 7 to 9 and Comparative Examples 10 to 12 revealed wherein the toll-like receptor 4 (TLR4) agonist comprises that the Sufficient systemic immunity and mucosal immunity at least one selected from the group consisting of: are likely to be induced by the sublingual administration even lipopolysaccharide, or salt thereof, and in the system wherein the dose of influenza HA antigen was monophosphoryl lipid, or salt thereof. reduced to 0.75ug. 6. The vaccine composition according to claim 5. 0.139. As shown in FIG. 5, the comparison between wherein the lipopolysaccharide, or salt thereof is derived Example 10 and Comparative Examples 13 and 14 revealed from Escherichia coli, Salmonella, Pantoea, Acetobac that the Sublingual immunity provides the complete infection terium, Zymomonas, Xanthomonas or Enterobacter; and protection against the lethal dose of the influenza virus. the monophosphoryllipid, or salt thereof is monophospho 0140. As shown in FIGS. 6 and 7, the comparison between ryl lipid derived from Salmonella or synthetic glucopy Example 11 and Comparative Examples 14 and 1 revealed ranosyl lipid. that the Sufficient systemic immunity and mucosal immunity 7. The vaccine composition according to claim 1, are likely to be induced in the inactivated whole virus vaccine wherein the toll-like receptor 2/6 (TLR2/6) agonist com when the adjuvants are used. prises diacylated lipopeptide, or a derivative or salt 0141. As shown in FIGS. 8 and 9, the comparison between thereof. Examples 12 and 13 and Comparative Examples 15, 16 and 1 8. The vaccine composition according to claim 7. revealed that the systemic immunity and mucosal immunity wherein the diacylated lipopeptide comprises Pam CSK, are likely to be induced in A/California/7/2009(H1N1), a MALP-2, FSL-1, or a derivative or salt thereof. seasonal influenza infectious to human. 9. The vaccine composition according to claim 1, 0142. As shown in FIGS. 10 and 11, the comparison wherein the cyclic dinucleotide comprises c-di-GMP c-di between Examples 12, 14 and 15 and Comparative Examples AMP, or a derivative or salt thereof. 16 and 1 revealed that the sublingual immunity is likely to 10. The vaccine composition according to claim 6. induce the systemic immunity and mucosal immunity against wherein the lipopolysaccharide, or salt thereof is derived different types of influenza viruses. from Pantoea. 0143. As shown in FIGS. 12 and 13, the comparison 11. The vaccine composition according to claim 1, between Examples 16 and 17 and Comparative Examples 17 wherein the vaccine composition induces a mucosal and 18 revealed that the influenza HA-specific and OVA immune response and a systemic immune response, specific systemic immunity and mucosal immunity are highly the mucosal immune response is antigen-specific IgA anti induced. body production, and the systemic immune response is antigen-specific IgG anti INDUSTRIAL APPLICABILITY body production and antigen-specific cell-mediated 0144. The vaccine composition of the present invention immune production. uses the specific adjuvants described above in combination k k k k k