US20150306214A1 - Adjuvant for mucosal vaccine - Google Patents

Adjuvant for mucosal vaccine Download PDF

Info

Publication number
US20150306214A1
US20150306214A1 US14/649,279 US201314649279A US2015306214A1 US 20150306214 A1 US20150306214 A1 US 20150306214A1 US 201314649279 A US201314649279 A US 201314649279A US 2015306214 A1 US2015306214 A1 US 2015306214A1
Authority
US
United States
Prior art keywords
viruses
adjuvant
amino acid
antigens
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/649,279
Inventor
Yukako Fujinaga
Takuhiro Matsumura
Masahiro Yutani
Nao JONAI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Osaka University NUC
Daiichi Sankyo Co Ltd
Original Assignee
Osaka University NUC
Daiichi Sankyo Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Osaka University NUC, Daiichi Sankyo Co Ltd filed Critical Osaka University NUC
Assigned to OSAKA UNIVERSITY, DAIICHI SANKYO COMPANY, LIMITED reassignment OSAKA UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FUJINAGA, YUKAKO, JONAI, NAO, MATSUMURA, Takuhiro, YUTANI, Masahiro
Publication of US20150306214A1 publication Critical patent/US20150306214A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • A61K39/145Orthomyxoviridae, e.g. influenza virus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76Albumins
    • C07K14/77Ovalbumin
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525Virus
    • A61K2039/5252Virus inactivated (killed)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/54Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541Mucosal route
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/54Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541Mucosal route
    • A61K2039/543Mucosal route intranasal
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55516Proteins; Peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55544Bacterial toxins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/57Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/40Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/16011Orthomyxoviridae
    • C12N2760/16111Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an effective and safe adjuvant for a mucosal vaccine and a mucosal vaccine preparation containing such adjuvant and vaccine antigens.
  • mucosal immunity In recent years, the mechanisms of mucosal immunity on the respiratory apparatus, the digestive apparatus, the reproductive organs, and other organs have been gradually elucidated as the immune system to prevent infectious diseases such as influenza or acquired immunodeficiency syndrome (AIDS).
  • infectious diseases such as influenza or acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • mucosal IgA antibody mucosal IgA antibody
  • serum IgG antibody to neutralize the viruses
  • cytotoxic T cells that lyse infected cells to interrupt virus transmission.
  • mucosal immune mechanisms are functional at the initial phase of infection, and play a key role in biophylaxis at the time of infection or during the initial phase of infection.
  • mucosal vaccines inducing immune protection response against infection on the mucosa which is the first barrier at portals of entry for pathogens, are considered as effective vaccine for various infectious diseases through mucosae.
  • mucosal vaccines induce secretory IgA antibody in mucosal tissue upon mucosal administration (e.g., intranasal administration), and also induce IgG antibody in the serum.
  • mucosal vaccines are capable of inducing immune responses in both the mucosal and systemic systems against pathogens.
  • mucosal vaccines are superior to conventional vaccination with needles and syringe in terms of operability, safety, and economic efficiency. Accordingly, mucosal vaccines are expected as novel vaccines, and have been developed.
  • mucosal adjuvants with toxins such as CT or LT might be difficult in terms of safety.
  • MPL resulting from attenuation of activity of endotoxin LPS, bacterial flagellin proteins (Patent Document 1), double-stranded RNA (poly(I:C)) (Patent Document 2), and other substances have been studied as mucosal adjuvants, which are not derived from toxins.
  • those candidates induce excessive inflammatory responses, they are not satisfactory for mucosal adjuvants in terms of safety. That is, no effective and safe adjuvants for mucosal vaccines are being put to practical use at present.
  • hemagglutinin (HA) and the nontoxic-nonhemagglutinin (NTNH) component bind to the botulinum neurotoxin (NIX) produced by botulinum bacilli causing food poisoning, and those components form three types of neurotoxin complex (progenitor toxin (PTX)) whose molecular weight are 300,000, 500,000, or 900,000.
  • PTX neurotoxin complex
  • Botulinum toxin blocks neuron transmission, and leads to death in human. Taking advantage of the activity thereof, botulinum toxin is used as an effective neurotransmission inhibitor for medical purposes.
  • a botulinum toxin type A (BOTOX) complex is known to be used for treatment of blepharospasm, hemifacial spasm, spasmodic torticollis, heterotropia, and the reduction of wrinkles.
  • non-toxic HA is known to have functions of disrupting the epithelial barrier and transporting botulinum neurotoxins and macromolecules.
  • NTX and albumin antigens are subcutaneously administered to mice in combination with HA, production of serum antibody specific for antigens is enhanced through IL-6 production (Non-Patent Document 3).
  • Patent Documents 3 and 4 describe the adjuvant activity of an HA subcomponent (HA1 or HA3) and the use as a carrier of nucleic acids into cells, no protein complex composed of HA subcomponents (HA1, HA2, and HA3) has been discussed.
  • the present inventors previously reported that HA acts on M cells in the epithelial cell layer of the Peyer's patch (i.e., M cells on the Peyer's patch), and that HA assists migration of neurotoxin complex from apical side of to basolateral side of M cells via transcytosis (Non-Patent Document 4).
  • an object of the present invention is to provide an adjuvant for mucosal vaccines with high both efficacy and safety.
  • the present inventors focused on hemagglutinin (HA), a non-toxic component of botulinum toxin, and the mice were intranasally immunized with a protein complex composed of HA subcomponents (HA1, HA2, and HA3) intranasal in combination with ovalbumin antigens or influenza HA antigens.
  • HA hemagglutinin
  • HA subcomponents HA1, HA2, and HA3
  • the present invention includes the following.
  • An adjuvant for a mucosal vaccine comprising a protein complex composed of hemagglutinin (HA) subcomponents HA1, HA2, and HA3 of botulinum toxin.
  • HA hemagglutinin
  • the adjuvant according to (1) or (2) which is used simultaneously with vaccine antigens or before or after vaccine antigens are administered.
  • the adjuvant according to (3), wherein the vaccine antigens are subunit antigens or inactivated antigens.
  • the adjuvant according to (3) or (4), wherein the vaccine antigens are derived from pathogens causing mucosal infections.
  • the adjuvant according to (5), wherein the pathogens causing mucosal infections are viruses or bacteria.
  • influenza viruses human immunodeficiency viruses (HIV), chickenpox viruses, measles viruses, rubella viruses, mumps viruses, polioviruses, rotaviruses, adenoviruses, herpes viruses, RS viruses, dengue viruses, Japanese encephalitis viruses, severe acute respiratory syndrome (SARS) viruses, or hepatitis viruses (type A, type B, or type C).
  • influenza viruses human immunodeficiency viruses (HIV), chickenpox viruses, measles viruses, rubella viruses, mumps viruses, polioviruses, rotaviruses, adenoviruses, herpes viruses, RS viruses, dengue viruses, Japanese encephalitis viruses, severe acute respiratory syndrome (SARS) viruses, or hepatitis viruses (type A, type B, or type C).
  • HAV human immunodeficiency viruses
  • chickenpox viruses measles viruses
  • rubella viruses rubella viruses
  • mumps viruses polioviruses
  • Neisseria meningitidis type B influenza, pneumococcus, tuberculosis bacteria, tetanus bacilli, or cholera bacilli.
  • the adjuvant according to any of (1) to (8) which is administered with any mucosal routes.
  • the adjuvant according to (9), wherein the administration with mucosal routes is intranasal administration.
  • a mucosal vaccine preparation comprising vaccine antigens and the adjuvant according to any of (1) to (10).
  • the adjuvant of the present invention When the adjuvant of the present invention is administered to mucosa such as the intranasal mucosa in combination with vaccine antigens derived from pathogens causing mucosal infections, such as influenza viruses, production of serum IgG antibody and that of secretory IgA antibody on the mucosa are accelerated, and antigen-specific systemic and mucosal immune responses are enhanced. Accordingly, the adjuvant of the present invention is useful as an adjuvant for a mucosal vaccine against diseases of the respiratory apparatus or the digestive apparatus.
  • vaccine antigens derived from pathogens causing mucosal infections such as influenza viruses
  • production of serum IgG antibody and that of secretory IgA antibody on the mucosa are accelerated, and antigen-specific systemic and mucosal immune responses are enhanced.
  • the adjuvant of the present invention is useful as an adjuvant for a mucosal vaccine against diseases of the respiratory apparatus or the digestive apparatus.
  • the adjuvant of the present invention uses hemagglutinin (HA) subcomponent, which is a non-toxic botulinum toxin component, the adjuvant does not activate innate immunity, and the adjuvant is less likely to cause inflammations on mucosa after administration. Therefore, the adjuvant of the present invention is very safe for mucosal vaccines to use.
  • HA hemagglutinin
  • FIG. 1 shows the amino acid sequence of recombinant botulinus HA1-3 used to prepare the botulinus HA (BHA) complex in Example 1.
  • the underlined regions indicate vector-derived amino acid sequences (FLAG tag sequence: SEQ ID NO: 7; Strep tag sequence: SEQ ID NO: 8).
  • FIG. 2 shows purification of the BHA complex via gel filtration chromatography.
  • FIG. 3 shows interaction between M cells and each subcomponcnt HA1, HA2, or HA3 of botulinus (a microscope photograph showing localization of subcomponents on the follicle-associated epithelium (FAE)).
  • FIG. 4 shows interaction between M cell and the HA2+3 complex or the HA1+2+3 complex of botulinus (a microscope photograph showing localization of complexes on the follicle-associated epithelium (FAE)).
  • FIG. 5 shows the results of ELISA that measured the concentration of ovalbumin-specific IgG in sera and that of ovalbumin-specific IgA in the nasal cavity lavage or in the bronchoalveolar lavage (OVA: the group to which ovalbumin alone is administered; OVA+CTB: the group to which ovalbumin with the cholera toxin B subunit are administered; OVA+BHA: the group to which ovalbumin with the BHA complex are administered; Reciprocal log 2 titer: the antibody titer represented by the logarithm of the reciprocal of the maximal dilution factor exhibiting absorbance that is higher than the sample before immunization by 0.1).
  • OVA the group to which ovalbumin alone is administered
  • OVA+CTB the group to which ovalbumin with the cholera toxin B subunit are administered
  • OVA+BHA the group to which ovalbumin with the BHA complex are administered
  • Reciprocal log 2 titer the antibody titer represented by the
  • FIG. 6 shows activation of innate immunity by the BHA complex (the amount of IL-6 produced).
  • FIG. 7 shows the results of ELISA that measured the concentration of influenza-antigen-specific IgG in the sera (SV: the group to which influenza split vaccine alone is administered; SV+BHA: the group to which influenza split vaccine with BHA complex are administered; SV+CTB: the group to which influenza split vaccine with cholera toxin B subunit are administered; NC: the group to which no antigens with adjuvants is administered (*** p ⁇ 0.0001 ** p ⁇ 0.001 * p ⁇ 0.01)).
  • FIG. 8 shows the results of ELISA that measured the concentration of influenza-antigen-specific IgA in the nasal cavity lavage and in the bronchoalveolar lavage (SV: the group to which influenza split vaccine alone is administered; SV+BHA: the group to which influenza split vaccine with BHA complex are administered; SV+CTB: the group to which influenza split vaccine with cholera toxin B subunit are administered: NC: the group to which no antigens with adjuvants is administered (*** p ⁇ 0.0001 ** p ⁇ 0.001 * p ⁇ 0.01)).
  • FIG. 9 shows the results of ELISA that measured the concentration of influenza-antigen-specific IgG in the sera
  • SV the group to which influenza split vaccine alone is administered
  • SV+BHA the group to which influenza split vaccine with BHA complex are administered
  • SV+BHA1-3 the group to which influenza split vaccine with BHA1, BHA2, or BHA3 are administered
  • SV+CTB the group to which influenza split vaccine with cholera toxin B subunit are administered: NC: the group to which no antigens with adjuvants is administered (*** p ⁇ 0.0001 ** p ⁇ 0.001 * p ⁇ 0.01)).
  • FIG. 10 shows the results of ELISA that measured the concentration of influenza-antigen-specific IgA in the nasal cavity lavage and in the bronchoalveolar lavage
  • SV the group to which influenza split vaccine alone is administered
  • SV+BHA the group to which influenza split vaccine with BHA complex are administered
  • SV+BHA1-3 the group to which influenza split vaccine with BHA1, BHA2, or BHA3 are administered
  • SV+CTB the group to which influenza split vaccine with cholera toxin B subunit are administered: NC: the group to which no antigens with adjuvants is administered (*** p ⁇ 0.0001 ** p ⁇ 0.001 * p ⁇ 0.01)).
  • the adjuvant for a mucosal vaccine of the present invention (hereafter it is merely referred to as an “adjuvant”) is a protein complex composed of HA1, HA2, and HA3, which are hemagglutinin (HA) subcomponents of botulinum toxin.
  • HA hemagglutinin
  • the term “adjuvant” used herein refers to a substance that is administered so as to enhance the immunogenicity of a vaccine antigen.
  • Botulinum toxins are classified as type A to type G in accordance with the different antigenicities of toxins produced by botulinum bacilli ( Clostridium botulinum ).
  • the botulinum toxin complex for the adjuvant of the present invention is preferably of type A or type B.
  • the first component of the protein complex contained in the adjuvant of the present invention is the botulinum toxin complex HA1
  • the second component is the botulinum toxin complex HA2
  • the third component is the botulinum toxin complex HA3.
  • HA1, HA2, and HA3 are a protein consisting of the amino acid sequence as shown in SEQ ID NO: 1, a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, and a protein consisting of the amino acid sequence as shown in SEQ ID NO: 3, respectively.
  • the adjuvant of the present invention is preferably a protein complex composed of the first component, the second component, and the third component.
  • the three proteins composing the protein complex may be mutant proteins of the protein consisting of the amino acid sequence as shown in SEQ ID NO: 1, the protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, and the protein consisting of the amino acid sequence as shown in SEQ ID NO: 3, respectively, provided that such mutant proteins have activities equivalent to those of the relevant original proteins.
  • mutant proteins “have activities equivalent to” those of the original proteins
  • the protein complex composed of such mutant proteins has mucosal adjuvant activity equivalent to that of the protein complex composed of the protein consisting of the amino acid sequence as shown in SEQ ID NO: 1, the protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, and the protein consisting of the amino acid sequence as shown in SEQ ID NO: 3.
  • mutant protein refers to activity that enhances production of antigen-specific antibody when the adjuvant is administered transmucosally in combination with vaccine antigens in both the mucosal and systemic immune response.
  • the influence of such activity on innate immunity is insignificant, and production of antigen-specific antibody is enhanced in both the mucosal and systemic immunity.
  • innate immunity is not influenced and production of antigen-specific antibody is enhanced in both the mucosal and systemic immunity.
  • An example of such mutant protein is a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 1, 2, or 3 by deletion, substitution, insertion, or addition of one to several amino acids.
  • a mutant protein may consist of an amino acid sequence having 90% or higher identity to the amino acid sequence as shown in SEQ ID NO: 1, 2, or 3.
  • the term “90% or higher identity” used herein refers to sequence identity of preferably 95% or higher, more preferably 97% or higher, and most preferably 98% or higher. Amino acid sequence identity can be determined by FASTA or BLAST search. While the term “mutation” used herein primarily refers to a mutation that is artificially introduced in accordance with a known method of producing a mutant protein, an equivalent mutation existing in nature may be employed.
  • a method for producing the adjuvant of the present invention is not particularly limited.
  • the protein complex may be derived from nature.
  • proteins composing such protein complex may be produced via a genetic recombination technique, and the protein complex may be formulated using such proteins.
  • the protein complex may be produced in accordance with a conventional genetic recombination technique using genes encoding the proteins of interest.
  • HA1, HA2, and HA3 can be produced by constructing expression vectors containing genes encoding the amino acid sequences as shown in SEQ ID NOs: 1, 2, and 3 (the nucleotide sequences are shown in SEQ ID NOs: 4, 5, and 6, respectively), introducing the expression vectors into adequate host cells, and culturing the host cells. Mutant proteins of HA1.
  • HA2, and HA3 can be also produced by a well-known recombinant DNA technique by, for example, subjecting genes encoding the amino acid sequences as shown in SEQ ID NOs: 1, 2, and 3 to site-directed mutagenesis, obtaining genes encoding the mutant proteins, and using such genes.
  • the protein productions can be easily carried out with reference to, for example. Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press, 1989.
  • HA1, HA2, and HA3 can be chemically synthesized on the basis of the amino acid sequences thereof.
  • the resulting HA1. HA2, and HA3 proteins may be incubated in a solvent such as a phosphate buffer for 2 to 8 hours, preferably 3 to 5 hours, and more preferably 3 hours at 25° C. to 40° C. and preferably 37° C. and the protein complex may be thus composed.
  • a fusion protein may be prepared from the HA1, HA2, and HA3 proteins.
  • the adjuvant of the present invention may be administered to organisms simultaneously with vaccine antigens.
  • the adjuvant may be administered before the administration of vaccine antigens or after the administration of antigens.
  • the adjuvant may be administered substantially simultaneously with the vaccines.
  • the adjuvant and vaccine antigens may be administered to the target at exactly the same time, or they may be continuously administered within a given period of time (preferably within several minutes).
  • the vaccine antigens are preferably inactivated antigens or subunit antigens.
  • the term “inactivated antigens” refers to antigens of pathogens (e.g., viruses or bacteria) deprived of infectivity. Examples thereof include complete virus particles (virions), incomplete virus particles, virion-constituting particles, virus nonstructural proteins, the antigens to prevent infections, and neutralizing epitopes.
  • Antigens may be inactivated by physical treatments (e.g., x-rays, heat, or ultrasound), chemical treatments (e.g., formalin, mercury, alcohol, or chlorine), or via other means.
  • subunit vaccines refers to vaccines selectively containing particular antigens (i.e., the antigens to prevent infections) that are effective vaccine components among various types of antigens contained in inactivated vaccines.
  • An example of a subunit vaccine against the influenza virus is a vaccine selectively containing hemagglutinin (HA) and neuraminidase (NA) that are surface antigens purified.
  • HA hemagglutinin
  • NA neuraminidase
  • the vaccine antigens are not particularly limited, provided that the vaccine antigens are capable of inducing a mucosal immune response together with the adjuvant of the present invention.
  • Typical antigens are derived from pathogens causing mucosal infections. Pathogens causing mucosal infections may be viruses or bacteria.
  • viruses include, but are not limited to, influenza viruses, human immunodeficiency viruses (HIV), chickenpox viruses, measles viruses, rubella viruses, mumps viruses, polioviruses, rotaviruses, adenoviruses, herpes viruses, RS viruses, dengue viruses, Japanese encephalitis viruses, severe acute respiratory syndrome (SARS) viruses, and hepatitis viruses (type A, type B, and type C).
  • bacteria include, but are not limited to, Bordetella pertussis, Neisseria meningitidis , type B influenza, pneumococcus, tuberculosis bacteria, tetanus bacilli, and cholera bacilli.
  • antigens derived from pathogens may be derived from nature or artificially prepared via gene recombination or other techniques.
  • the vaccine antigens include allergens used for hyposensitization therapy. Accordingly, the adjuvant of the present invention can be used as an adjuvant for allergen vaccines. Allergen vaccines are used to block IgE causing allergies by producing IgG antibody against allergens or to increase allergen-specific type I helper T cells (Th1 cells) in vivo by administering allergens to organisms, thereby decreasing type II helper T cells (Th2 cells) associated with allergy symptoms. Allergen vaccines are capable of suppressing allergy symptoms via hyposensitization.
  • allergens are not particularly limited, and examples of allergens include food allergens (e.g., casein, lactalbumin, lactoglobulin, ovomucoid, ovalbumin, and conalbumin), house dust allergens (e.g., mite allergens), pollen allergens (e.g., cedar pollen allergens, ragweed allergens, and cocksfoot grass allergens), and allergens of animal body hair.
  • food allergens e.g., casein, lactalbumin, lactoglobulin, ovomucoid, ovalbumin, and conalbumin
  • house dust allergens e.g., mite allergens
  • pollen allergens e.g., cedar pollen allergens, ragweed allergens, and cocksfoot grass allergens
  • allergens of animal body hair e.g., casein, lactalbumin, lactoglobulin, ovomucoid
  • the adjuvant of the present invention is administered transmucosally in combination with the mucosal vaccine antigens.
  • an agent is “administered transmucosally,” it is administered through the mucosa.
  • mucosae include inner walls of hollow organs that lead to the exterior, such as the digestive apparatus, the respiratory apparatus, and the urogenital apparatus, and specific examples include the nasal cavity, oral cavity, pharynx, alveolus, air tube, intestinal tract, and vagina, with the nasal cavity being preferable.
  • examples of forms of transmucosal administration include intranasal, intraoral, intra-alveolar, intratracheal, intravaginal, and intrarectal administration with the intranasal administration being preferable.
  • Adjuvants and mucosal vaccines can be administered transmucosally in an adequate manner in accordance with the site of administration.
  • the agents can be sprayed, added dropwise, or applied to the nasal cavity or oral cavity.
  • Intra-alveolar administration can be carried out by a method involving the use of an inhaler or a sprayer or a method of administering a preparation comprising an aerosol preparation.
  • the amount of the adjuvant of the present invention to be administered varies in accordance with the age of the subject, body weight, disease type, route of administration, form of administration, and other conditions.
  • oral administration for example, 10 ⁇ g to 100 mg, and preferably 1 ⁇ g to 10 mg of the adjuvant of the present invention can be administered simultaneously with vaccine antigens per instance per adult human.
  • nasal administration 0.1 ⁇ g to 100 mg, and preferably 1 ⁇ g to 10 mg of the adjuvant can be administered, for example.
  • Subjects of administration can be adequately determined in accordance with the types of vaccine antigens used in combination with the adjuvant. Examples thereof include, in addition to humans, non-human mammalians, birds, and crustaceans.
  • a person skilled in the art can easily determine the frequency of administration of the adjuvant of the present invention in combination with vaccine antigens to the subjects by taking, for example, age, body weight, medical history, clinical course of the subject, disease type, and other factors into consideration.
  • administration may be carried out at an adequate time before the onset of the disease at the frequency of, in general, one to several instances per day for a day, or administration may be carried out several times at intervals of one to several weeks.
  • Administration is preferably carried out while observing progress, and booster immunization is preferably carried out at intervals of at least a week. Intervals of booster immunization are preferably at least about two weeks. By providing booster immunization, more effective infection-protective effects can be expected.
  • the adjuvant may be mixed with vaccine antigens together with pharmaceutically acceptable carriers suitable for the dosage form, and vaccine preparations may be produced by various known techniques.
  • the amount of the adjuvant to be incorporated into vaccine preparations can be adequately determined in accordance with the types of vaccine antigens to be mixed.
  • the content of the adjuvant in the preparations is not particularly limited, provided that sufficient antigen immune responses are induced via transmucosal administration.
  • Such amount is generally 0.1% to 90% by weight, preferably 0.5% to 80% by weight, and more preferably 1% to 50% by weight relative to the entire preparation amount.
  • Dosage forms of the mucosal vaccine preparations of the present invention are not particularly limited, provided that the mucosal vaccine preparations can be administered transmucosally. Examples thereof include liquid preparations, suspensions, sprays, and powders. According to need, various additives that are generally used for vaccine preparations, such as solubilizers, anticoagulants, viscosity modifiers, pH adjusters, isotonizing agents, emulsifiers, antioxidants, fillers, surfactants, diluents, preservatives, stabilizers, desiccating agents, or moisturizing agents, can be added to the mucosal vaccine preparations of the present invention.
  • solubilizers such as solubilizers, anticoagulants, viscosity modifiers, pH adjusters, isotonizing agents, emulsifiers, antioxidants, fillers, surfactants, diluents, preservatives, stabilizers, desiccating agents, or moisturizing agents.
  • the vaccine preparations of the present invention can be in a liquid state or a dried state, and such vaccine preparations can be introduced into hermetically sealed vial bottles, syringes, atomizers, or scaled ampules.
  • the botulinus HA (BHA) complex was prepared in the manner described below.
  • BHA1, BHA2, and BHA3 The genes encoding the proteins of the botulinus HA subcomponents (BHA1, BHA2, and BHA3) (BHA1: a protein consisting of amino acids 7 to 294 of the amino acid sequence as shown in SEQ ID NO: 1; BHA2: a protein consisting of amino acids 2 to 146 of the amino acid sequence as shown in SEQ ID NO: 2; and BHA3: a protein consisting of amino acids 19 to 626 of the amino acid sequence as shown in SEQ ID NO: 3) were amplified by PCR from genomic DNA of the Clostridium botulinum B-Okra strain as a template using the primers described below.
  • BHA1 forward primer cactataagcttatccaaaattcattaaatg
  • BHA1 reverse primer gttgataggtaccttatgggttactcatag
  • BHA forward primer (SEQ ID NO: 11) tgaataagctttcagctgaaagaacttttc
  • BHA2 reverse primer (SEQ ID NO: 12) cactttggtaccttatattttttcaagtttga
  • BHA3 forward primer (SEQ ID NO: 13) gaaaaagggtaccaatatagtgatactattg
  • BHA3 reverse primer (SEQ ID NO: 14) cgtgtcgacttaattagtaatatctatatgc
  • the amplified DNA fragments of BHA1 and BHA2 were each inserted into the HindIII-SalI site of pT7-FLAG-1 (Sigma), and the amplified DNA fragment of BHA3 was inserted into the KpnI-SalI site of pET52b(+) (Novagen) (pET-BHA3).
  • E. coli Rosetta2 (DE3) strains (Novagen). Protein expression was induced using the Overnight Express Autoinduction System 1 (Novagen). BHA1 and BHA3 were induced to express proteins at 30° C. for 36 hours, and BHA2 was induced to express a protein at 18° C. for 40 hours. E. coli strains were collected by centrifugation and stored at ⁇ 80° C.
  • BHA1 and BHA2 were purified using Anti-FLAG M2 agarose (Sigma).
  • BHA3 was purified using StrepTrap HP (GE Healthcare).
  • the amino acid sequences of the purified recombinant proteins. FLAG-BHA1, FLAG-BHA2, and Strep-BHA3, are shown in FIG. 1 .
  • the purified recombinant proteins were mixed at a ratio of BHA1:BHA2:BHA3 of 4:4:1 by mole, and the resultant was incubated at 37° C. for 3 hours, followed by purification with the use of StrepTrap HP.
  • BHA BHA complex
  • the BHA complex (BHA) prepared in Example 1 was separated using Superdex 200 10/300 GL (GE Healthcare). In this test, C-terminal FLAG tag HA1, N-terminal His tag HA2, and N-terminal Strep tag HA3 were used for HA1, HA2, and HA3 composing the BHA complex (BHA). The results are shown in FIG. 2 .
  • HA1, HA2, and HA3 of botulinus type A were labeled with Alexa 568 and injected into ligated intestinal loop of the mouse. Two hours later, HA subcomponent localization was observed under a confocal microscope. M cells were stained with FITC-labeled UEA-1. Neither M-cell binding nor transcytosis was substantially observed as a result when HA1, HA2, or HA3 alone was used ( FIG. 3 ).
  • the HA213 complex and the HA1+2+3 complex of botulinus type A were labeled with Alexa 568 and injected into ligated intestinal loop of the mouse. Two hours later, localization of complexes was observed under a confocal microscope. M cells were stained with FITC-labeled UEA-1. Neither M-cell binding nor transcytosis was substantially observed as a result when the HA2+3 complex was used. As with the case of native 16S toxin. M-cell binding and transcytosis were observed when the HA1+2+3 complex was used ( FIG. 4 ). Thus, formation of a complex of HA1, HA2, and HA3 was found to be necessary for interaction between M cell and HA.
  • the amount of IL-6 cytokine production resulting from treatment with the BHA complex adjuvant was measured using mouse splenocytes, and the ability of the BHA complex adjuvant to activate innate immunity was evaluated.
  • Splenocytes were sampled from native mice raised under SPF conditions (C57BL/6, 6-week-old, female, purchased from CLEA Japan. Inc.) and seeded onto a 96-well plate at a cell density of 1 ⁇ 10 6 cells/well. Thereafter, the BHA complex (BHA) was serially diluted from 20 ⁇ g/ml (20 ⁇ g/ml, 2 ⁇ g/ml. 0.2 ⁇ g/ml), and the splenocytes were stimulated. The splenocytes were further stimulated with the BHA complex adjuvant in combination with a TLR ligand of CpG oligo DNA (K3 or D35, 20 ⁇ g/ml) or LPS (1 ⁇ g/ml).
  • BHA BHA complex
  • the culture supernatant was recovered 24 hours after the initiation of stimulation and the amount of cytokine (IL-6) in the culture supernatant was measured (R&D systems).
  • the results are shown in FIG. 6 .
  • the induction of IL-6 by the BHA complex adjuvant alone was below the detection limit.
  • the amounts of TNF- ⁇ , IL-1 ⁇ , and IL-12 were also below the detection limit. Since the BHA complex adjuvant would not influence IL-6 production mediated by CpG or LPS stimulation, it was considered that the BHA complex adjuvant would not enhance or suppress signals to activate any other innate immunity. Thus, the BHA complex adjuvant was considered to be a non-inflammatory adjuvant that would not influence signals to activate innate immunity.
  • Influenza split vaccines were used as antigens to evaluate adjuvant effects of the BHA complex.
  • mice and C57BL/6 mice (6-week-old, female) were purchased from CLEA Japan, Inc. Mice were raised under SPF conditions.
  • split vaccines The mouse-adapted A/Puerto Rico/8/34 (H1N1) split vaccines (hereafter referred to as “split vaccines”) received from Kitasato Daiichi Sankyo Vaccine Co., Ltd. were used as vaccine antigens. During the experiment, antigens were refrigerated at 4° C. in the dark.
  • the BHA complex (BHA) prepared in Example 1 was used as the adjuvant. Endotoxin content was determined by designating the standard for purification at 0.5 EU/ml or lower.
  • the BHA adjuvant was cryopreserved at ⁇ 80° C. thawed immediately before use, and then used for immunization.
  • the cholera toxin adjuvant (CTB) was prepared by mixing 1 ⁇ g of cholera toxin B subunit (Catalog No. 033-20611, Wako Pure Chemical Industries. Ltd.) and 1 ⁇ g of cholera toxin (Catalog No. 033-20621. Wako Pure Chemical Industries, Ltd.) for each mouse.
  • the cholera toxin adjuvant was cryopreserved at ⁇ 80° C., thawed immediately before use, and then used for immunization.
  • PBS( ⁇ ) was added to the mixture of 1 ⁇ g of split vaccine antigens with 20 ⁇ g of the BHA complex (BHA) adjuvant or with 2 ⁇ g of the cholera toxin adjuvant to adjust the amount of each vaccine preparation to 12 ⁇ l used for each mouse.
  • the vaccine preparations were administered to 6-week-old mice through both nasal cavities in amounts of 6 ⁇ l each. Administration was carried out four times in total at intervals of 2 weeks (day 0, day 14, day 28, and day 42).
  • mice were anesthetized using Ketalar (Daiichi Sankyo Company, Limited)/Selactar (Bayer), and blood samples were obtained from the orbital venous plexus.
  • the sampled blood was allowed to stand at 4° C., overnight, and serum separation was carried out using a refrigerated benchtop centrifuge (9,100 g, 10 minutes, 4° C.).
  • the obtained serum specimens were cryopreserved at ⁇ 20° C.
  • IgG levels total IgG, IgG1, IG2a, and IG2c levels
  • mice were anesthetized using Ketalar/Selactar 56 days after the initiation of immunization, exsanguinated via cardiopuncture, and euthanized. Immediately thereafter, nasal cavity lavages and bronchoalveolar lavages were sampled. Thereafter, the nasal cavity lavages and the bronchoalveolar lavages were stored on ice or refrigerated until ELISA assays were initiated.
  • ELISA assays were carried out in the manner described below.
  • the split vaccine antigens were applied to a plate at a concentration of 1 ⁇ g/ml (4° C. overnight), and blocking was carried out with 1% BSA/PBST (Tween 20: 0.5%) by allowing the plate to stand at room temperature for 2 hours.
  • the serum sample was serially diluted using 1% BSA/PBST (Tween 20: 0.5%).
  • HRP-labeled antibody in accordance with subclasses was used.
  • OD was measured using a plate reader after coloring, and the amounts of influenza-antigen-specific antibody were calculated.
  • FIG. 7 shows the results of measurement of the level of influenza-antigen-specific IgG in the sera (56 days after the initiation of immunization).
  • the level of the antigen-specific antibody reactions in the sera induced in the group subjected to immunization with the BHA complex (BHA) adjuvant in combination with the influenza antigens was significantly higher than that induced in the group subjected to immunization with influenza antigens alone. Such phenomenon was observed in all the evaluated IgG subclasses.
  • BHA BHA complex
  • FIG. 8 shows the results of measurement of the amount of secretory IgA produced in the nasal cavity lavages and in the bronchoalveolar lavages.
  • the amount of antigen-specific IgA production was high in the group subjected to immunization with the BHA complex (BHA) adjuvant in combination with the influenza antigens.
  • BHA BHA complex
  • secretory IgA production was not substantially observed in the group of mice subjected to immunization with influenza antigens alone.
  • mice (6-week-old, female) were purchased from CLEA Japan. Inc. Mice were raised under SPF conditions.
  • split vaccines The mouse-adapted A/Puerto Rico/8/34 (H1N1) split vaccines (hereafter referred to as “split vaccines”) received from Kitasato Daiichi Sankyo Vaccine Co., Ltd. were used as immunogens. During the experiment, antigens were refrigerated at 4° C. in the dark.
  • the BHA complex (BHA) prepared in Example 1 or BHA1, BHA2, and BHA3 that are composing elements of the BHA complex were used as the adjuvant. Endotoxin content was determined by designating the standard for purification at 0.5 EU/ml or lower.
  • the BHA adjuvant was cryopreserved at ⁇ 80° C., thawed immediately before use, and then used for immunization.
  • the cholera toxin adjuvant (CTB) was prepared by mixing 1 ⁇ g of cholera toxin B subunit (Catalog No. 033-20611, Wako Pure Chemical Industries, Ltd.) and 1 ⁇ g of cholera toxin (Catalog No. 033-20621, Wako Pure Chemical Industries, Ltd.) for each mouse.
  • the cholera toxin adjuvant was cryopreserved at ⁇ 80° C., thawed immediately before use, and then used for immunization.
  • PBS( ⁇ ) was added to the mixture of 1 ⁇ g of split vaccine antigens and 20 ⁇ g each of the BHA complex (BHA) adjuvant, the BHA1 adjuvant, the BHA2 adjuvant, or the BHA3 adjuvant or 2 ⁇ g of the CTB adjuvant to adjust the amount of each vaccine preparation to 12 ⁇ l used for each mouse.
  • the vaccine preparations were administered to 6-week-old mice through both nasal cavities in amounts of 6 ⁇ l each. Administration was carried out four times in total at intervals of 2 weeks (day 0, day 14, day 28, and day 42).
  • mice were anesthetized using Ketalar (Daiichi Sankyo Company, Limited)/Selactar (Bayer), and blood samples were obtained from the orbital venous plexus.
  • the sampled blood was allowed to stand at 4° C. overnight, and serum separation was carried out using a refrigerated benchtop centrifuge (9.100 g, 10 minutes. 4° C.).
  • the obtained serum specimens were cryopreserved at ⁇ 20° C.
  • IgG levels total IgG, IgG1, and IG2a levels
  • mice were anesthetized using Ketalar/Selactar 56 days after the initiation of immunization, exsanguinated via cardiopuncture, and euthanized. Immediately thereafter, nasal cavity lavages and bronchoalveolar lavages were sampled. Thereafter, the nasal cavity lavages and the bronchoalveolar lavages were stored on ice or refrigerated until ELISA assays were initiated.
  • ELISA assays were carried out in the manner described below.
  • the split vaccine antigens were applied to a plate at concentration of 1 ⁇ g/ml (4° C., overnight), and blocking was carried out with 1% BSA/PBST (Tween 20: 0.5%) by allowing the plate to stand at room temperature for 2 hours.
  • the serum sample was serially diluted using 1% BSA/PBST (Tween 20: 0.5%).
  • HRP-labeled antibody in accordance with subclasses was used. After color had developed, OD was measured using a plate reader, and the amounts of influenza-antigen-specific antibody produced were measured.
  • FIG. 9 shows the results of measurement of the level of influenza-antigen-specific IgG in the sera (56 days after the initiation of immunization).
  • the level of the antigen-specific antibody reactions in the sera induced in the group subjected to immunization with the BHA complex (BHA) adjuvant in combination with the influenza antigens was significantly higher than that induced in the group subjected to immunization with influenza antigens alone.
  • BHA BHA complex
  • Such phenomenon was observed in all the evaluated IgG subclasses.
  • antibody reactions in the sera were not significantly potentiated, compared with the group subjected to immunization with the influenza antigens alone.
  • FIG. 10 shows the results of measurement of the amount of secretory IgA in the nasal cavity lavages and in the bronchoalveolar lavages.
  • the amount of antigen-specific IgA was significantly higher in the group subjected to immunization with the BHA complex (BHA) adjuvant in combination with the influenza antigens than in the group subjected to immunization with the influenza antigens alone.
  • BHA BHA complex
  • the amount of secretory IgA was not significantly increased in the group subjected to immunization with the BHA1, BHA2, or BHA3 adjuvants that are composing elements of the BHA complex in combination with the influenza antigens, compared with the group subjected to immunization with the influenza antigens alone.
  • BHA BHA complex
  • the present invention is applicable in the field of production of a mucosal adjuvant and a mucosal vaccine preparation comprising such adjuvant.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pulmonology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

An object of the present invention is to provide an adjuvant for a mucosal vaccine with high safety that induces a sufficient immune response on the mucosa. According to the present invention, an adjuvant for a mucosal vaccine comprising a protein complex composed of hemagglutinin (HA) subcomponents HA1, HA2, and HA3 of botulinum toxin is provided.

Description

    TECHNICAL FIELD
  • The present invention relates to an effective and safe adjuvant for a mucosal vaccine and a mucosal vaccine preparation containing such adjuvant and vaccine antigens.
  • BACKGROUND ART
  • In recent years, the mechanisms of mucosal immunity on the respiratory apparatus, the digestive apparatus, the reproductive organs, and other organs have been gradually elucidated as the immune system to prevent infectious diseases such as influenza or acquired immunodeficiency syndrome (AIDS). For example, immune response to prevent influenza virus infection is associated with mucosal IgA antibody, serum IgG antibody to neutralize the viruses, and cytotoxic T cells that lyse infected cells to interrupt virus transmission. Such mucosal immune mechanisms are functional at the initial phase of infection, and play a key role in biophylaxis at the time of infection or during the initial phase of infection. Accordingly, mucosal vaccines inducing immune protection response against infection on the mucosa, which is the first barrier at portals of entry for pathogens, are considered as effective vaccine for various infectious diseases through mucosae.
  • While mucosal vaccines induce secretory IgA antibody in mucosal tissue upon mucosal administration (e.g., intranasal administration), and also induce IgG antibody in the serum. Thus, mucosal vaccines are capable of inducing immune responses in both the mucosal and systemic systems against pathogens. In addition, mucosal vaccines are superior to conventional vaccination with needles and syringe in terms of operability, safety, and economic efficiency. Accordingly, mucosal vaccines are expected as novel vaccines, and have been developed.
  • However, because mucosal vaccines with antigens alone are not capable of inducing sufficient immune responses, mucosal adjuvants for mucosal vaccines is necessary in order to induce effective immune responses on the mucosal surface. Up to the present, many mucosal adjuvants have been reported. For example, bacterial endotoxins such as cholera toxin (CT) and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli, have been known as representative mucosal adjuvants (Non-Patent Documents 1 and 2). However, previous reports showed that clinical trials with LT intranasal administration caused facial nerve palsy (Bell's palsy). Accordingly, development of mucosal adjuvants with toxins such as CT or LT might be difficult in terms of safety. MPL resulting from attenuation of activity of endotoxin LPS, bacterial flagellin proteins (Patent Document 1), double-stranded RNA (poly(I:C)) (Patent Document 2), and other substances have been studied as mucosal adjuvants, which are not derived from toxins. However, since those candidates induce excessive inflammatory responses, they are not satisfactory for mucosal adjuvants in terms of safety. That is, no effective and safe adjuvants for mucosal vaccines are being put to practical use at present.
  • The hemagglutinin (HA) and the nontoxic-nonhemagglutinin (NTNH) component bind to the botulinum neurotoxin (NIX) produced by botulinum bacilli causing food poisoning, and those components form three types of neurotoxin complex (progenitor toxin (PTX)) whose molecular weight are 300,000, 500,000, or 900,000. Botulinum toxin blocks neuron transmission, and leads to death in human. Taking advantage of the activity thereof, botulinum toxin is used as an effective neurotransmission inhibitor for medical purposes. For example, a botulinum toxin type A (BOTOX) complex is known to be used for treatment of blepharospasm, hemifacial spasm, spasmodic torticollis, heterotropia, and the reduction of wrinkles. In the neurotoxin complex as described above, non-toxic HA is known to have functions of disrupting the epithelial barrier and transporting botulinum neurotoxins and macromolecules. When NTX and albumin antigens are subcutaneously administered to mice in combination with HA, production of serum antibody specific for antigens is enhanced through IL-6 production (Non-Patent Document 3). While Patent Documents 3 and 4 describe the adjuvant activity of an HA subcomponent (HA1 or HA3) and the use as a carrier of nucleic acids into cells, no protein complex composed of HA subcomponents (HA1, HA2, and HA3) has been discussed. The present inventors previously reported that HA acts on M cells in the epithelial cell layer of the Peyer's patch (i.e., M cells on the Peyer's patch), and that HA assists migration of neurotoxin complex from apical side of to basolateral side of M cells via transcytosis (Non-Patent Document 4). While the functions of the neurotoxin complex (HA to which the toxin component has been bound) to breach the intestinal epithelial barrier have been investigated in the study described above, interaction of toxin-free HA with M cells or adjuvant effects for delivering vaccine antigens for mucosal vaccines to infectious diseases have not yet been examined.
  • PRIOR ART DOCUMENTS Patent Documents
    • Patent Document 1: WO 2005/070455
    • Patent Document 2: JP 2005-97267 A
    • Patent Document 3: JP 2009-132686 A
    • Patent Document 4: JP 2009-81997 A
    Non-Patent Documents
    • Non-Patent Document 1: J. Xu-Amano et al., J. Exp. Med., 178, 1309, 1993
    • Non-Patent Document 2: I. Takahashi et al., J. Infect. Dis. 173, 627, 1996
    • Non-Patent Document 3: J. Lee et al., Microbiology, 151, 3739, 2005
    • Non-Patent Document 4: Takuhiro Matsumura et al., Japanese Journal of Bacteriology 64 (1) 79, 2009
    SUMMARY OF THE INVENTION
  • Accordingly, an object of the present invention is to provide an adjuvant for mucosal vaccines with high both efficacy and safety.
  • The present inventors focused on hemagglutinin (HA), a non-toxic component of botulinum toxin, and the mice were intranasally immunized with a protein complex composed of HA subcomponents (HA1, HA2, and HA3) intranasal in combination with ovalbumin antigens or influenza HA antigens. As a result, they confirmed that production of serum IgG antibody and that of secretory IgA antibody on the mucosa would be accelerated by vaccine antigens with HA subcomponent, suggesting that HA augments systemic immunity and mucosal immunity to vaccine antigens. In addition, innate immunity (e.g., production of IL-6) caused by CpG or LPS would not be affected by additional HA. Thus, they discovered that the HA complex would be effective as an adjuvant for a mucosal vaccine without induction of inflammation.
  • The present invention includes the following.
  • (1) An adjuvant for a mucosal vaccine comprising a protein complex composed of hemagglutinin (HA) subcomponents HA1, HA2, and HA3 of botulinum toxin.
    (2) The adjuvant according to (1), wherein the protein complex is composed of the first component, the second component, and the third component described below:
    the first component:
  • (a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 1, or
  • (b) a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 1 by deletion, substitution, or addition of one to several amino acids and having functions equivalent to those of the protein (a);
  • the second component:
  • (c) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, or
  • (d) a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by deletion, substitution, or addition of one to several amino acids and having functions equivalent to those of the protein (c): and the third component:
  • (e) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 3, or
  • (f) a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 3 by deletion, substitution, or addition of one to several amino acids and having functions equivalent to those of the protein (e).
  • (3) The adjuvant according to (1) or (2), which is used simultaneously with vaccine antigens or before or after vaccine antigens are administered.
    (4) The adjuvant according to (3), wherein the vaccine antigens are subunit antigens or inactivated antigens.
    (5) The adjuvant according to (3) or (4), wherein the vaccine antigens are derived from pathogens causing mucosal infections.
    (6) The adjuvant according to (5), wherein the pathogens causing mucosal infections are viruses or bacteria.
    (7) The adjuvant according to (6), wherein the viruses are influenza viruses, human immunodeficiency viruses (HIV), chickenpox viruses, measles viruses, rubella viruses, mumps viruses, polioviruses, rotaviruses, adenoviruses, herpes viruses, RS viruses, dengue viruses, Japanese encephalitis viruses, severe acute respiratory syndrome (SARS) viruses, or hepatitis viruses (type A, type B, or type C).
    (8) The adjuvant according to (6), wherein the bacteria are Bordetella pertussis. Neisseria meningitidis, type B influenza, pneumococcus, tuberculosis bacteria, tetanus bacilli, or cholera bacilli.
    (9) The adjuvant according to any of (1) to (8), which is administered with any mucosal routes.
    (10) The adjuvant according to (9), wherein the administration with mucosal routes is intranasal administration.
    (11) A mucosal vaccine preparation comprising vaccine antigens and the adjuvant according to any of (1) to (10).
  • When the adjuvant of the present invention is administered to mucosa such as the intranasal mucosa in combination with vaccine antigens derived from pathogens causing mucosal infections, such as influenza viruses, production of serum IgG antibody and that of secretory IgA antibody on the mucosa are accelerated, and antigen-specific systemic and mucosal immune responses are enhanced. Accordingly, the adjuvant of the present invention is useful as an adjuvant for a mucosal vaccine against diseases of the respiratory apparatus or the digestive apparatus. In addition, the adjuvant of the present invention uses hemagglutinin (HA) subcomponent, which is a non-toxic botulinum toxin component, the adjuvant does not activate innate immunity, and the adjuvant is less likely to cause inflammations on mucosa after administration. Therefore, the adjuvant of the present invention is very safe for mucosal vaccines to use.
  • This patent application claims priority from Japanese Patent Application No. 2012-265532 filed on Dec. 4, 2012, and it includes part or all of the contents as disclosed in the description thereof.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the amino acid sequence of recombinant botulinus HA1-3 used to prepare the botulinus HA (BHA) complex in Example 1. The underlined regions indicate vector-derived amino acid sequences (FLAG tag sequence: SEQ ID NO: 7; Strep tag sequence: SEQ ID NO: 8).
  • FIG. 2 shows purification of the BHA complex via gel filtration chromatography.
  • FIG. 3 shows interaction between M cells and each subcomponcnt HA1, HA2, or HA3 of botulinus (a microscope photograph showing localization of subcomponents on the follicle-associated epithelium (FAE)).
  • FIG. 4 shows interaction between M cell and the HA2+3 complex or the HA1+2+3 complex of botulinus (a microscope photograph showing localization of complexes on the follicle-associated epithelium (FAE)).
  • FIG. 5 shows the results of ELISA that measured the concentration of ovalbumin-specific IgG in sera and that of ovalbumin-specific IgA in the nasal cavity lavage or in the bronchoalveolar lavage (OVA: the group to which ovalbumin alone is administered; OVA+CTB: the group to which ovalbumin with the cholera toxin B subunit are administered; OVA+BHA: the group to which ovalbumin with the BHA complex are administered; Reciprocal log 2 titer: the antibody titer represented by the logarithm of the reciprocal of the maximal dilution factor exhibiting absorbance that is higher than the sample before immunization by 0.1).
  • FIG. 6 shows activation of innate immunity by the BHA complex (the amount of IL-6 produced).
  • FIG. 7 shows the results of ELISA that measured the concentration of influenza-antigen-specific IgG in the sera (SV: the group to which influenza split vaccine alone is administered; SV+BHA: the group to which influenza split vaccine with BHA complex are administered; SV+CTB: the group to which influenza split vaccine with cholera toxin B subunit are administered; NC: the group to which no antigens with adjuvants is administered (*** p<0.0001 ** p<0.001 * p<0.01)).
  • FIG. 8 shows the results of ELISA that measured the concentration of influenza-antigen-specific IgA in the nasal cavity lavage and in the bronchoalveolar lavage (SV: the group to which influenza split vaccine alone is administered; SV+BHA: the group to which influenza split vaccine with BHA complex are administered; SV+CTB: the group to which influenza split vaccine with cholera toxin B subunit are administered: NC: the group to which no antigens with adjuvants is administered (*** p<0.0001 ** p<0.001 * p<0.01)).
  • FIG. 9 shows the results of ELISA that measured the concentration of influenza-antigen-specific IgG in the sera (SV: the group to which influenza split vaccine alone is administered: SV+BHA: the group to which influenza split vaccine with BHA complex are administered: SV+BHA1-3: the group to which influenza split vaccine with BHA1, BHA2, or BHA3 are administered: SV+CTB: the group to which influenza split vaccine with cholera toxin B subunit are administered: NC: the group to which no antigens with adjuvants is administered (*** p<0.0001 ** p<0.001 * p<0.01)).
  • FIG. 10 shows the results of ELISA that measured the concentration of influenza-antigen-specific IgA in the nasal cavity lavage and in the bronchoalveolar lavage (SV: the group to which influenza split vaccine alone is administered: SV+BHA: the group to which influenza split vaccine with BHA complex are administered: SV+BHA1-3: the group to which influenza split vaccine with BHA1, BHA2, or BHA3 are administered; SV+CTB: the group to which influenza split vaccine with cholera toxin B subunit are administered: NC: the group to which no antigens with adjuvants is administered (*** p<0.0001 ** p<0.001 * p<0.01)).
  • EMBODIMENTS FOR CARRYING OUT THE INVENTION
  • The adjuvant for a mucosal vaccine of the present invention (hereafter it is merely referred to as an “adjuvant”) is a protein complex composed of HA1, HA2, and HA3, which are hemagglutinin (HA) subcomponents of botulinum toxin. The term “adjuvant” used herein refers to a substance that is administered so as to enhance the immunogenicity of a vaccine antigen.
  • Botulinum toxins are classified as type A to type G in accordance with the different antigenicities of toxins produced by botulinum bacilli (Clostridium botulinum). The botulinum toxin complex for the adjuvant of the present invention is preferably of type A or type B.
  • The first component of the protein complex contained in the adjuvant of the present invention is the botulinum toxin complex HA1 the second component is the botulinum toxin complex HA2, and the third component is the botulinum toxin complex HA3. Specifically, HA1, HA2, and HA3 are a protein consisting of the amino acid sequence as shown in SEQ ID NO: 1, a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, and a protein consisting of the amino acid sequence as shown in SEQ ID NO: 3, respectively. The adjuvant of the present invention is preferably a protein complex composed of the first component, the second component, and the third component.
  • The three proteins composing the protein complex may be mutant proteins of the protein consisting of the amino acid sequence as shown in SEQ ID NO: 1, the protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, and the protein consisting of the amino acid sequence as shown in SEQ ID NO: 3, respectively, provided that such mutant proteins have activities equivalent to those of the relevant original proteins. When mutant proteins “have activities equivalent to” those of the original proteins, the protein complex composed of such mutant proteins has mucosal adjuvant activity equivalent to that of the protein complex composed of the protein consisting of the amino acid sequence as shown in SEQ ID NO: 1, the protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, and the protein consisting of the amino acid sequence as shown in SEQ ID NO: 3. The term “mucosal adjuvant activity” refers to activity that enhances production of antigen-specific antibody when the adjuvant is administered transmucosally in combination with vaccine antigens in both the mucosal and systemic immune response. Preferably, the influence of such activity on innate immunity is insignificant, and production of antigen-specific antibody is enhanced in both the mucosal and systemic immunity. More preferably, innate immunity is not influenced and production of antigen-specific antibody is enhanced in both the mucosal and systemic immunity. An example of such mutant protein is a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 1, 2, or 3 by deletion, substitution, insertion, or addition of one to several amino acids. The term “one to several” used herein indicates the number of amino acids that can be deleted, substituted, or added by a known method for producing a mutant protein, such as site-directed mutagenesis. As long as the activity described above is retained, such number is not limited. For example, such number is 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5. A mutant protein may consist of an amino acid sequence having 90% or higher identity to the amino acid sequence as shown in SEQ ID NO: 1, 2, or 3. The term “90% or higher identity” used herein refers to sequence identity of preferably 95% or higher, more preferably 97% or higher, and most preferably 98% or higher. Amino acid sequence identity can be determined by FASTA or BLAST search. While the term “mutation” used herein primarily refers to a mutation that is artificially introduced in accordance with a known method of producing a mutant protein, an equivalent mutation existing in nature may be employed.
  • A method for producing the adjuvant of the present invention is not particularly limited. The protein complex may be derived from nature. Alternatively, proteins composing such protein complex may be produced via a genetic recombination technique, and the protein complex may be formulated using such proteins. The protein complex may be produced in accordance with a conventional genetic recombination technique using genes encoding the proteins of interest. Specifically, HA1, HA2, and HA3 can be produced by constructing expression vectors containing genes encoding the amino acid sequences as shown in SEQ ID NOs: 1, 2, and 3 (the nucleotide sequences are shown in SEQ ID NOs: 4, 5, and 6, respectively), introducing the expression vectors into adequate host cells, and culturing the host cells. Mutant proteins of HA1. HA2, and HA3 can be also produced by a well-known recombinant DNA technique by, for example, subjecting genes encoding the amino acid sequences as shown in SEQ ID NOs: 1, 2, and 3 to site-directed mutagenesis, obtaining genes encoding the mutant proteins, and using such genes. The protein productions can be easily carried out with reference to, for example. Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press, 1989. Alternatively, HA1, HA2, and HA3 can be chemically synthesized on the basis of the amino acid sequences thereof.
  • The resulting HA1. HA2, and HA3 proteins may be incubated in a solvent such as a phosphate buffer for 2 to 8 hours, preferably 3 to 5 hours, and more preferably 3 hours at 25° C. to 40° C. and preferably 37° C. and the protein complex may be thus composed. Alternatively, a fusion protein may be prepared from the HA1, HA2, and HA3 proteins. When production of a fusion protein is intended, a known method in which DNA fragments encoding the HA1, HA2, and HA3 proteins are bound to be in-frame with each other, the resultant is introduced into an adequate expression vector, and the resultant is transcribed and translated with the aid of an adequate host so as to express the protein may be employed.
  • In general, the adjuvant of the present invention may be administered to organisms simultaneously with vaccine antigens. Alternatively, the adjuvant may be administered before the administration of vaccine antigens or after the administration of antigens. When the adjuvant is administered simultaneously with vaccine antigens, the adjuvant may be administered substantially simultaneously with the vaccines. For example, the adjuvant and vaccine antigens may be administered to the target at exactly the same time, or they may be continuously administered within a given period of time (preferably within several minutes).
  • The vaccine antigens are preferably inactivated antigens or subunit antigens. The term “inactivated antigens” refers to antigens of pathogens (e.g., viruses or bacteria) deprived of infectivity. Examples thereof include complete virus particles (virions), incomplete virus particles, virion-constituting particles, virus nonstructural proteins, the antigens to prevent infections, and neutralizing epitopes. Antigens may be inactivated by physical treatments (e.g., x-rays, heat, or ultrasound), chemical treatments (e.g., formalin, mercury, alcohol, or chlorine), or via other means. The term “subunit vaccines” refers to vaccines selectively containing particular antigens (i.e., the antigens to prevent infections) that are effective vaccine components among various types of antigens contained in inactivated vaccines. An example of a subunit vaccine against the influenza virus is a vaccine selectively containing hemagglutinin (HA) and neuraminidase (NA) that are surface antigens purified.
  • The vaccine antigens are not particularly limited, provided that the vaccine antigens are capable of inducing a mucosal immune response together with the adjuvant of the present invention. Typical antigens are derived from pathogens causing mucosal infections. Pathogens causing mucosal infections may be viruses or bacteria. Examples of viruses include, but are not limited to, influenza viruses, human immunodeficiency viruses (HIV), chickenpox viruses, measles viruses, rubella viruses, mumps viruses, polioviruses, rotaviruses, adenoviruses, herpes viruses, RS viruses, dengue viruses, Japanese encephalitis viruses, severe acute respiratory syndrome (SARS) viruses, and hepatitis viruses (type A, type B, and type C). Examples of bacteria include, but are not limited to, Bordetella pertussis, Neisseria meningitidis, type B influenza, pneumococcus, tuberculosis bacteria, tetanus bacilli, and cholera bacilli. Such antigens derived from pathogens may be derived from nature or artificially prepared via gene recombination or other techniques.
  • The vaccine antigens include allergens used for hyposensitization therapy. Accordingly, the adjuvant of the present invention can be used as an adjuvant for allergen vaccines. Allergen vaccines are used to block IgE causing allergies by producing IgG antibody against allergens or to increase allergen-specific type I helper T cells (Th1 cells) in vivo by administering allergens to organisms, thereby decreasing type II helper T cells (Th2 cells) associated with allergy symptoms. Allergen vaccines are capable of suppressing allergy symptoms via hyposensitization. Allergens are not particularly limited, and examples of allergens include food allergens (e.g., casein, lactalbumin, lactoglobulin, ovomucoid, ovalbumin, and conalbumin), house dust allergens (e.g., mite allergens), pollen allergens (e.g., cedar pollen allergens, ragweed allergens, and cocksfoot grass allergens), and allergens of animal body hair.
  • The adjuvant of the present invention is administered transmucosally in combination with the mucosal vaccine antigens. When an agent is “administered transmucosally,” it is administered through the mucosa. Examples of mucosae include inner walls of hollow organs that lead to the exterior, such as the digestive apparatus, the respiratory apparatus, and the urogenital apparatus, and specific examples include the nasal cavity, oral cavity, pharynx, alveolus, air tube, intestinal tract, and vagina, with the nasal cavity being preferable. Accordingly, examples of forms of transmucosal administration include intranasal, intraoral, intra-alveolar, intratracheal, intravaginal, and intrarectal administration with the intranasal administration being preferable. Adjuvants and mucosal vaccines can be administered transmucosally in an adequate manner in accordance with the site of administration. In the case of nasal or oral administration, for example, the agents can be sprayed, added dropwise, or applied to the nasal cavity or oral cavity. Intra-alveolar administration can be carried out by a method involving the use of an inhaler or a sprayer or a method of administering a preparation comprising an aerosol preparation.
  • The amount of the adjuvant of the present invention to be administered varies in accordance with the age of the subject, body weight, disease type, route of administration, form of administration, and other conditions. In the case of oral administration, for example, 10 μg to 100 mg, and preferably 1 μg to 10 mg of the adjuvant of the present invention can be administered simultaneously with vaccine antigens per instance per adult human. In the case of nasal administration, 0.1 μg to 100 mg, and preferably 1 μg to 10 mg of the adjuvant can be administered, for example. Subjects of administration can be adequately determined in accordance with the types of vaccine antigens used in combination with the adjuvant. Examples thereof include, in addition to humans, non-human mammalians, birds, and crustaceans.
  • A person skilled in the art can easily determine the frequency of administration of the adjuvant of the present invention in combination with vaccine antigens to the subjects by taking, for example, age, body weight, medical history, clinical course of the subject, disease type, and other factors into consideration. As in the case of general vaccine preparations, administration may be carried out at an adequate time before the onset of the disease at the frequency of, in general, one to several instances per day for a day, or administration may be carried out several times at intervals of one to several weeks. Administration is preferably carried out while observing progress, and booster immunization is preferably carried out at intervals of at least a week. Intervals of booster immunization are preferably at least about two weeks. By providing booster immunization, more effective infection-protective effects can be expected.
  • In order to administer the adjuvant of the present invention simultaneously with vaccine antigens, the adjuvant may be mixed with vaccine antigens together with pharmaceutically acceptable carriers suitable for the dosage form, and vaccine preparations may be produced by various known techniques.
  • The amount of the adjuvant to be incorporated into vaccine preparations can be adequately determined in accordance with the types of vaccine antigens to be mixed. The content of the adjuvant in the preparations is not particularly limited, provided that sufficient antigen immune responses are induced via transmucosal administration. Such amount is generally 0.1% to 90% by weight, preferably 0.5% to 80% by weight, and more preferably 1% to 50% by weight relative to the entire preparation amount.
  • Dosage forms of the mucosal vaccine preparations of the present invention are not particularly limited, provided that the mucosal vaccine preparations can be administered transmucosally. Examples thereof include liquid preparations, suspensions, sprays, and powders. According to need, various additives that are generally used for vaccine preparations, such as solubilizers, anticoagulants, viscosity modifiers, pH adjusters, isotonizing agents, emulsifiers, antioxidants, fillers, surfactants, diluents, preservatives, stabilizers, desiccating agents, or moisturizing agents, can be added to the mucosal vaccine preparations of the present invention.
  • The vaccine preparations of the present invention can be in a liquid state or a dried state, and such vaccine preparations can be introduced into hermetically sealed vial bottles, syringes, atomizers, or scaled ampules.
  • Hereafter, the present invention is described in greater detail with reference to the examples, although the technical scope of the present invention is not limited thereto. The data obtained in the examples were statistically processed by the Student's t-test.
  • Example 1 Preparation of Botulinus HA (BHA) Complex
  • The botulinus HA (BHA) complex was prepared in the manner described below.
  • (1) Preparation of Plasmids
  • The genes encoding the proteins of the botulinus HA subcomponents (BHA1, BHA2, and BHA3) (BHA1: a protein consisting of amino acids 7 to 294 of the amino acid sequence as shown in SEQ ID NO: 1; BHA2: a protein consisting of amino acids 2 to 146 of the amino acid sequence as shown in SEQ ID NO: 2; and BHA3: a protein consisting of amino acids 19 to 626 of the amino acid sequence as shown in SEQ ID NO: 3) were amplified by PCR from genomic DNA of the Clostridium botulinum B-Okra strain as a template using the primers described below.
  • (Primers for BHA1 Amplification)
  • (SEQ ID NO: 9)
    BHA1 forward primer:
    cactataagcttatccaaaattcattaaatg
    (SEQ ID NO: 10)
    BHA1 reverse primer:
    gttgataggtaccttatgggttactcatag
  • (Primers for BHA2 Amplification)
  • BHA forward primer:
    (SEQ ID NO: 11)
    tgaataagctttcagctgaaagaacttttc
    BHA2 reverse primer:
    (SEQ ID NO: 12)
    cactttggtaccttatattttttcaagtttga
  • (Primers for BHA3 Amplification)
  • BHA3 forward primer:
    (SEQ ID NO: 13)
    gaaaaagggtaccaatatagtgatactattg
    BHA3 reverse primer:
    (SEQ ID NO: 14)
    cgtgtcgacttaattagtaatatctatatgc
  • The amplified DNA fragments of BHA1 and BHA2 were each inserted into the HindIII-SalI site of pT7-FLAG-1 (Sigma), and the amplified DNA fragment of BHA3 was inserted into the KpnI-SalI site of pET52b(+) (Novagen) (pET-BHA3).
  • (2) Protein Expression
  • The resulting plasmids were separately transformed into E. coli Rosetta2 (DE3) strains (Novagen). Protein expression was induced using the Overnight Express Autoinduction System 1 (Novagen). BHA1 and BHA3 were induced to express proteins at 30° C. for 36 hours, and BHA2 was induced to express a protein at 18° C. for 40 hours. E. coli strains were collected by centrifugation and stored at −80° C.
  • (3) Protein Purification and Complex Preparation
  • BHA1 and BHA2 were purified using Anti-FLAG M2 agarose (Sigma). BHA3 was purified using StrepTrap HP (GE Healthcare). The amino acid sequences of the purified recombinant proteins. FLAG-BHA1, FLAG-BHA2, and Strep-BHA3, are shown in FIG. 1.
  • The purified recombinant proteins were mixed at a ratio of BHA1:BHA2:BHA3 of 4:4:1 by mole, and the resultant was incubated at 37° C. for 3 hours, followed by purification with the use of StrepTrap HP. Thus, the BHA complex (BHA) was obtained.
  • (4) Gel Filtration Chromatography of Botulinus HA (BHA) Complex
  • The BHA complex (BHA) prepared in Example 1 was separated using Superdex 200 10/300 GL (GE Healthcare). In this test, C-terminal FLAG tag HA1, N-terminal His tag HA2, and N-terminal Strep tag HA3 were used for HA1, HA2, and HA3 composing the BHA complex (BHA). The results are shown in FIG. 2.
  • Example 2 Interaction Between M Cell and Botulinus HA Subcomponent Alone or Complex of Botulinus HA Subcomponents
  • HA1, HA2, and HA3 of botulinus type A (600 nM each) were labeled with Alexa 568 and injected into ligated intestinal loop of the mouse. Two hours later, HA subcomponent localization was observed under a confocal microscope. M cells were stained with FITC-labeled UEA-1. Neither M-cell binding nor transcytosis was substantially observed as a result when HA1, HA2, or HA3 alone was used (FIG. 3).
  • Separately, the HA213 complex and the HA1+2+3 complex of botulinus type A (600 nM each) were labeled with Alexa 568 and injected into ligated intestinal loop of the mouse. Two hours later, localization of complexes was observed under a confocal microscope. M cells were stained with FITC-labeled UEA-1. Neither M-cell binding nor transcytosis was substantially observed as a result when the HA2+3 complex was used. As with the case of native 16S toxin. M-cell binding and transcytosis were observed when the HA1+2+3 complex was used (FIG. 4). Thus, formation of a complex of HA1, HA2, and HA3 was found to be necessary for interaction between M cell and HA.
  • Example 3 Nasal Adjuvant Effects of BHA Complex Using Ovalbumin (OVA)
  • With the use of model antigens (ovalbumin, OVA), the efficacy of botulinus HA (BHA) as a mucosal vaccine adjuvant was inspected in the mouse with intranasal administration system. The BHA complex (BHA) prepared in Example 1 was used as BHA. OVA (5 μg only), OVA (5 μg)+BHA (15 μg), and OVA (5 μg)+cholera toxin B subunit (2 μg) (as the positive control) were intranasally administered to BALB/c mice (6-week-old: a group of 3 individuals) at intervals of one week (at day 0, day 7, day 14, day 21, and day 28), and five times of administration was totally carried out. Production of OVA-specific IgG in the sera, that of OVA-specific IgA in the nasal cavity lavage, and that of OVA-specific IgA in bronchoalveolar lavage were assayed by ELISA on day 34.
  • The results are shown in FIG. 5. Production of IgA was not observed in any nasal cavity lavage or bronchoalveolar lavage in the group to which OVA alone had been administered, although a slight increase was observed in the sera IgG level on day 34. In the group to which OVA and BHA had been administered and the group to which OVA and the cholera toxin B subunit had been administered, the IgA levels in the nasal cavity lavage and the bronchoalveolar lavagc and the amount of IgG in the sera significantly increased.
  • Example 4 Evaluation of Ability of BHA Complex Adjuvant to Activate Innate Immunity (Activity to IL-6 Production)
  • The amount of IL-6 cytokine production resulting from treatment with the BHA complex adjuvant was measured using mouse splenocytes, and the ability of the BHA complex adjuvant to activate innate immunity was evaluated.
  • Splenocytes were sampled from native mice raised under SPF conditions (C57BL/6, 6-week-old, female, purchased from CLEA Japan. Inc.) and seeded onto a 96-well plate at a cell density of 1×106 cells/well. Thereafter, the BHA complex (BHA) was serially diluted from 20 μg/ml (20 μg/ml, 2 μg/ml. 0.2 μg/ml), and the splenocytes were stimulated. The splenocytes were further stimulated with the BHA complex adjuvant in combination with a TLR ligand of CpG oligo DNA (K3 or D35, 20 μg/ml) or LPS (1 μg/ml). The culture supernatant was recovered 24 hours after the initiation of stimulation and the amount of cytokine (IL-6) in the culture supernatant was measured (R&D systems). The results are shown in FIG. 6. As shown in FIG. 6, the induction of IL-6 by the BHA complex adjuvant alone was below the detection limit. The amounts of TNF-α, IL-1β, and IL-12 were also below the detection limit. Since the BHA complex adjuvant would not influence IL-6 production mediated by CpG or LPS stimulation, it was considered that the BHA complex adjuvant would not enhance or suppress signals to activate any other innate immunity. Thus, the BHA complex adjuvant was considered to be a non-inflammatory adjuvant that would not influence signals to activate innate immunity.
  • Example 5 Effects of Intranasal Adjuvant of BHA Complex Using Influenza HA Antigens
  • Influenza split vaccines were used as antigens to evaluate adjuvant effects of the BHA complex.
  • (1) Experimental Animals and Materials
  • BALB/c mice and C57BL/6 mice (6-week-old, female) were purchased from CLEA Japan, Inc. Mice were raised under SPF conditions.
  • The mouse-adapted A/Puerto Rico/8/34 (H1N1) split vaccines (hereafter referred to as “split vaccines”) received from Kitasato Daiichi Sankyo Vaccine Co., Ltd. were used as vaccine antigens. During the experiment, antigens were refrigerated at 4° C. in the dark.
  • The BHA complex (BHA) prepared in Example 1 was used as the adjuvant. Endotoxin content was determined by designating the standard for purification at 0.5 EU/ml or lower. The BHA adjuvant was cryopreserved at −80° C. thawed immediately before use, and then used for immunization. The cholera toxin adjuvant (CTB) was prepared by mixing 1 μg of cholera toxin B subunit (Catalog No. 033-20611, Wako Pure Chemical Industries. Ltd.) and 1 μg of cholera toxin (Catalog No. 033-20621. Wako Pure Chemical Industries, Ltd.) for each mouse. The cholera toxin adjuvant was cryopreserved at −80° C., thawed immediately before use, and then used for immunization.
  • (2) Test Method
  • PBS(−) was added to the mixture of 1 μg of split vaccine antigens with 20 μg of the BHA complex (BHA) adjuvant or with 2 μg of the cholera toxin adjuvant to adjust the amount of each vaccine preparation to 12 μl used for each mouse. The vaccine preparations were administered to 6-week-old mice through both nasal cavities in amounts of 6 μl each. Administration was carried out four times in total at intervals of 2 weeks (day 0, day 14, day 28, and day 42). Immediately before booster immunizations were provided on day 14, day 28, and day 42, mice were anesthetized using Ketalar (Daiichi Sankyo Company, Limited)/Selactar (Bayer), and blood samples were obtained from the orbital venous plexus. The sampled blood was allowed to stand at 4° C., overnight, and serum separation was carried out using a refrigerated benchtop centrifuge (9,100 g, 10 minutes, 4° C.). The obtained serum specimens were cryopreserved at −20° C. In order to evaluate adjuvant effects of the BHA complex, IgG levels (total IgG, IgG1, IG2a, and IG2c levels) in the serum specimens were measured.
  • Mice were anesthetized using Ketalar/Selactar 56 days after the initiation of immunization, exsanguinated via cardiopuncture, and euthanized. Immediately thereafter, nasal cavity lavages and bronchoalveolar lavages were sampled. Thereafter, the nasal cavity lavages and the bronchoalveolar lavages were stored on ice or refrigerated until ELISA assays were initiated.
  • ELISA assays were carried out in the manner described below. The split vaccine antigens were applied to a plate at a concentration of 1 μg/ml (4° C. overnight), and blocking was carried out with 1% BSA/PBST (Tween 20: 0.5%) by allowing the plate to stand at room temperature for 2 hours. The serum sample was serially diluted using 1% BSA/PBST (Tween 20: 0.5%). As secondary antibody, HRP-labeled antibody in accordance with subclasses was used. OD was measured using a plate reader after coloring, and the amounts of influenza-antigen-specific antibody were calculated. The nasal cavity lavages and the bronchoalveolar lavages were serially diluted using 1% BSA/PBST (Tween 20: 0.5%). In order to evaluate adjuvant effects of the BHA complex to potentiate the antigen-specific mucosal immunity, the amount of influenza-antigen-specific mucosal IgA produced was measured.
  • (3) Test Results
  • FIG. 7 shows the results of measurement of the level of influenza-antigen-specific IgG in the sera (56 days after the initiation of immunization).
  • As shown in FIG. 7, the level of the antigen-specific antibody reactions in the sera induced in the group subjected to immunization with the BHA complex (BHA) adjuvant in combination with the influenza antigens was significantly higher than that induced in the group subjected to immunization with influenza antigens alone. Such phenomenon was observed in all the evaluated IgG subclasses.
  • FIG. 8 shows the results of measurement of the amount of secretory IgA produced in the nasal cavity lavages and in the bronchoalveolar lavages. As shown in FIG. 8, the amount of antigen-specific IgA production was high in the group subjected to immunization with the BHA complex (BHA) adjuvant in combination with the influenza antigens. In contrast, secretory IgA production was not substantially observed in the group of mice subjected to immunization with influenza antigens alone.
  • Example 6 Comparison of Effects of Intranasal Adjuvants of BHA Complex with BHA1, BHA2, or BHA3 Respectively
  • With the use of the influenza split vaccines as antigens, adjuvant effects of the BHA complex were compared with adjuvant effects of BHA1, BHA2, and BHA3 that are composing elements of the BHA complex.
  • (1) Experimental Animals and Materials
  • BALB/c mice (6-week-old, female) were purchased from CLEA Japan. Inc. Mice were raised under SPF conditions.
  • The mouse-adapted A/Puerto Rico/8/34 (H1N1) split vaccines (hereafter referred to as “split vaccines”) received from Kitasato Daiichi Sankyo Vaccine Co., Ltd. were used as immunogens. During the experiment, antigens were refrigerated at 4° C. in the dark.
  • The BHA complex (BHA) prepared in Example 1 or BHA1, BHA2, and BHA3 that are composing elements of the BHA complex were used as the adjuvant. Endotoxin content was determined by designating the standard for purification at 0.5 EU/ml or lower. The BHA adjuvant was cryopreserved at −80° C., thawed immediately before use, and then used for immunization. The cholera toxin adjuvant (CTB) was prepared by mixing 1 μg of cholera toxin B subunit (Catalog No. 033-20611, Wako Pure Chemical Industries, Ltd.) and 1 μg of cholera toxin (Catalog No. 033-20621, Wako Pure Chemical Industries, Ltd.) for each mouse. The cholera toxin adjuvant was cryopreserved at −80° C., thawed immediately before use, and then used for immunization.
  • (2) Test Method
  • PBS(−) was added to the mixture of 1 μg of split vaccine antigens and 20 μg each of the BHA complex (BHA) adjuvant, the BHA1 adjuvant, the BHA2 adjuvant, or the BHA3 adjuvant or 2 μg of the CTB adjuvant to adjust the amount of each vaccine preparation to 12 μl used for each mouse. The vaccine preparations were administered to 6-week-old mice through both nasal cavities in amounts of 6 μl each. Administration was carried out four times in total at intervals of 2 weeks (day 0, day 14, day 28, and day 42). Immediately before booster immunizations were provided on day 14, day 28, and day 42, mice were anesthetized using Ketalar (Daiichi Sankyo Company, Limited)/Selactar (Bayer), and blood samples were obtained from the orbital venous plexus. The sampled blood was allowed to stand at 4° C. overnight, and serum separation was carried out using a refrigerated benchtop centrifuge (9.100 g, 10 minutes. 4° C.). The obtained serum specimens were cryopreserved at −20° C. In order to evaluate adjuvant effects of the BHA complex. IgG levels (total IgG, IgG1, and IG2a levels) in the serum specimens were measured.
  • Mice were anesthetized using Ketalar/Selactar 56 days after the initiation of immunization, exsanguinated via cardiopuncture, and euthanized. Immediately thereafter, nasal cavity lavages and bronchoalveolar lavages were sampled. Thereafter, the nasal cavity lavages and the bronchoalveolar lavages were stored on ice or refrigerated until ELISA assays were initiated.
  • ELISA assays were carried out in the manner described below. The split vaccine antigens were applied to a plate at concentration of 1 μg/ml (4° C., overnight), and blocking was carried out with 1% BSA/PBST (Tween 20: 0.5%) by allowing the plate to stand at room temperature for 2 hours. The serum sample was serially diluted using 1% BSA/PBST (Tween 20: 0.5%). As secondary antibody. HRP-labeled antibody in accordance with subclasses was used. After color had developed, OD was measured using a plate reader, and the amounts of influenza-antigen-specific antibody produced were measured. The nasal cavity lavages and the bronchoalveolar lavages were serially diluted using 1% BSA/PBST (Tween 20: 0.5%). In order to evaluate adjuvant effects of the BHA complex to potentiate the antigen-specific mucosal immunity, the amount of influenza-antigen-specific mucosal IgA produced was measured.
  • (3) Test Results
  • FIG. 9 shows the results of measurement of the level of influenza-antigen-specific IgG in the sera (56 days after the initiation of immunization).
  • As shown in FIG. 9, the level of the antigen-specific antibody reactions in the sera induced in the group subjected to immunization with the BHA complex (BHA) adjuvant in combination with the influenza antigens was significantly higher than that induced in the group subjected to immunization with influenza antigens alone. Such phenomenon was observed in all the evaluated IgG subclasses. In the group subjected to immunization with the BHA1, BHA2, or BHA3 adjuvants that are composing elements of the complex in combination with the influenza antigens, in contrast, antibody reactions in the sera were not significantly potentiated, compared with the group subjected to immunization with the influenza antigens alone. When intradermal administration via injection was employed instead of intranasal administration, antibody reactions in the sera were not significant in any of the groups subjected to immunization with the BHA complex (BHA) adjuvant, the BHA1 adjuvant, the BHA2 adjuvant, or the BHA3 adjuvant in combination with the influenza antigens.
  • FIG. 10 shows the results of measurement of the amount of secretory IgA in the nasal cavity lavages and in the bronchoalveolar lavages. As shown in FIG. 10, the amount of antigen-specific IgA was significantly higher in the group subjected to immunization with the BHA complex (BHA) adjuvant in combination with the influenza antigens than in the group subjected to immunization with the influenza antigens alone. In contrast, the amount of secretory IgA was not significantly increased in the group subjected to immunization with the BHA1, BHA2, or BHA3 adjuvants that are composing elements of the BHA complex in combination with the influenza antigens, compared with the group subjected to immunization with the influenza antigens alone. When intradermal administration via injection was employed instead of intranasal administration, the amount of secretory IgA production was below the detection limit in all the groups subjected to immunization with the BHA complex (BHA) adjuvant, the BHA1 adjuvant, the BHA2 adjuvant, or the BHA3 adjuvant in combination with the influenza antigens.
  • INDUSTRIAL APPLICABILITY
  • The present invention is applicable in the field of production of a mucosal adjuvant and a mucosal vaccine preparation comprising such adjuvant.
  • All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (11)

1. An adjuvant for a mucosal vaccine comprising a protein complex composed of hemagglutinin (HA) subcomponents HA1, HA2, and HA3 of botulinum toxin.
2. The adjuvant according to claim 1, wherein the protein complex is composed of the first component, the second component, and the third component described below:
the first component:
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 1, or
(b) a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 1 by deletion, substitution, or addition of one to several amino acids and having functions equivalent to those of the protein (a);
the second component:
(c) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, or
(d) a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by deletion, substitution, or addition of one to several amino acids and having functions equivalent to those of the protein (c); and
the third component:
(e) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 3, or
(f) a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 3 by deletion, substitution, or addition of one to several amino acids and having functions equivalent to those of the protein (e).
3. The adjuvant according to claim 1, which is used simultaneously with vaccine antigens or before or after vaccine antigens are administered.
4. The adjuvant according to claim 3, wherein the vaccine antigens are subunit antigens or inactivated antigens.
5. The adjuvant according to claim 3, wherein the vaccine antigens are derived from pathogens causing mucosal infections.
6. The adjuvant according to claim 5, wherein the pathogens causing mucosal infections are viruses or bacteria.
7. The adjuvant according to claim 6, wherein the viruses are influenza viruses, human immunodeficiency viruses (HIV), chickenpox viruses, measles viruses, rubella viruses, mumps viruses, polioviruses, rotaviruses, adenoviruses, herpes viruses, RS viruses, dengue viruses, Japanese encephalitis viruses, severe acute respiratory syndrome (SARS) viruses, or hepatitis viruses.
8. The adjuvant according to claim 6, wherein the bacteria are Bordetella pertussis, Neisseria meningitidis, type B influenza, pneumococcus, tuberculosis bacteria, tetanus bacilli, or cholera bacilli.
9. The adjuvant according to claim 1, which is administered with any mucosal routes.
10. The adjuvant according to claim 9, wherein the administration with mucosal routes is intranasal administration.
11. A mucosal vaccine preparation comprising vaccine antigens and the adjuvant according to claim 1.
US14/649,279 2012-12-04 2013-11-15 Adjuvant for mucosal vaccine Abandoned US20150306214A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2012-265532 2012-12-04
JP2012265532 2012-12-04
PCT/JP2013/081459 WO2014087849A1 (en) 2012-12-04 2013-11-15 Adjuvant for mucous membrane vaccine

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/081459 A-371-Of-International WO2014087849A1 (en) 2012-12-04 2013-11-15 Adjuvant for mucous membrane vaccine

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/223,566 Continuation US9662386B2 (en) 2012-12-04 2016-07-29 Adjuvant for mucosal vaccine

Publications (1)

Publication Number Publication Date
US20150306214A1 true US20150306214A1 (en) 2015-10-29

Family

ID=50883272

Family Applications (2)

Application Number Title Priority Date Filing Date
US14/649,279 Abandoned US20150306214A1 (en) 2012-12-04 2013-11-15 Adjuvant for mucosal vaccine
US15/223,566 Active US9662386B2 (en) 2012-12-04 2016-07-29 Adjuvant for mucosal vaccine

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/223,566 Active US9662386B2 (en) 2012-12-04 2016-07-29 Adjuvant for mucosal vaccine

Country Status (5)

Country Link
US (2) US20150306214A1 (en)
EP (1) EP2929893B1 (en)
JP (1) JP6230028B2 (en)
TW (1) TW201427687A (en)
WO (1) WO2014087849A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9662386B2 (en) * 2012-12-04 2017-05-30 Osaka University Adjuvant for mucosal vaccine
US20170196970A1 (en) * 2014-06-04 2017-07-13 Osaka University Adjuvant for mucosal vaccine
US11230701B2 (en) 2014-06-27 2022-01-25 Osaka University Mutant hemagglutinin complex protein, and method for culturing pluripotent stem cells using same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI634899B (en) * 2016-11-22 2018-09-11 國立臺灣大學 Vaccine composition comprising hepatitisb virus-like particle as adjuvant

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005538954A (en) * 2002-05-31 2005-12-22 トーマス・ジェファーソン・ユニバーシティ Compositions and methods for transepithelial transport of molecules
EP1666059A4 (en) 2003-08-11 2008-08-27 Univ Osaka Res Found Novel vaccine containing adjuvant capable of inducing mucosal immunity
KR20050073865A (en) 2004-01-12 2005-07-18 대한민국(전남대학교총장) Vaccine adjuvants containing constituents of the flagellum of vibrio vulnificus as an active component
JP2009081997A (en) 2007-09-27 2009-04-23 Chemo Sero Therapeut Res Inst Method for utilizing botulinus toxin component ha as carrier for intracellular introduction of nucleic acid
JP2009132686A (en) 2007-10-26 2009-06-18 Okayama Univ Polypeptide derived from botulinum toxin and method for detecting botulinum toxin
TW201427687A (en) * 2012-12-04 2014-07-16 Daiichi Sankyo Co Ltd Adjuvant for mucosal vaccines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Amatsu et al (Journal of Biological Chemistry Vol. 288, No. 49, pp 35617-35625, 2013) *
Nakajima et al (Microbiol. Immunol. Vol. 42 (9), pp 599-605, 1998) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9662386B2 (en) * 2012-12-04 2017-05-30 Osaka University Adjuvant for mucosal vaccine
US20170196970A1 (en) * 2014-06-04 2017-07-13 Osaka University Adjuvant for mucosal vaccine
US10226529B2 (en) * 2014-06-04 2019-03-12 Osaka University Adjuvant for mucosal vaccine
US11230701B2 (en) 2014-06-27 2022-01-25 Osaka University Mutant hemagglutinin complex protein, and method for culturing pluripotent stem cells using same

Also Published As

Publication number Publication date
EP2929893B1 (en) 2020-10-14
EP2929893A4 (en) 2016-07-06
JP6230028B2 (en) 2017-11-15
US9662386B2 (en) 2017-05-30
TW201427687A (en) 2014-07-16
US20160324960A1 (en) 2016-11-10
JPWO2014087849A1 (en) 2017-01-05
WO2014087849A1 (en) 2014-06-12
EP2929893A1 (en) 2015-10-14

Similar Documents

Publication Publication Date Title
Rhee et al. Mucosal vaccine adjuvants update
US9662386B2 (en) Adjuvant for mucosal vaccine
Negri et al. Persistence of mucosal and systemic immune responses following sublingual immunization
US9795664B2 (en) Vaccine against streptococcal infections based on recombinant proteins
Wei et al. Immunogenicity and protective efficacy of orally or intranasally administered recombinant Lactobacillus casei expressing ETEC K99
Rivera-Hernandez et al. Self-adjuvanting modular virus-like particles for mucosal vaccination against group A streptococcus (GAS)
Staats et al. Mucosal targeting of a BoNT/A subunit vaccine adjuvanted with a mast cell activator enhances induction of BoNT/A neutralizing antibodies in rabbits
Keijzer et al. Inactivated influenza vaccine adjuvanted with bacterium-like particles induce systemic and mucosal influenza A virus specific T-cell and B-cell responses after nasal administration in a TLR2 dependent fashion
Tejera-Alhambra et al. New biological insights in the immunomodulatory effects of mucosal polybacterial vaccines in clinical practice
US10226529B2 (en) Adjuvant for mucosal vaccine
Garg et al. The respiratory syncytial virus fusion protein formulated with a novel combination adjuvant induces balanced immune responses in lambs with maternal antibodies
Del Campo et al. Mucosal immunization using proteoliposome and cochleate structures from Neisseria meningitidis serogroup B induce mucosal and systemic responses
Shi et al. The expression of membrane protein augments the specific responses induced by SARS-CoV nucleocapsid DNA immunization
US9119803B2 (en) Carious tooth vaccine and preparation method
Zhu et al. Protective efficacy of a Mycoplasma pneumoniae P1C DNA vaccine fused with the B subunit of Escherichia coli heat-labile enterotoxin
Gartner et al. Mucosal prime-boost vaccination for tuberculosis based on TLR triggering OprI lipoprotein from Pseudomonas aeruginosa fused to mycolyl-transferase Ag85A
Olivera et al. Immunization with the recombinant Cholera toxin B fused to Fimbria 2 protein protects against Bordetella pertussis infection
Jun et al. Sublingual immunization with adenovirus F protein-based vaccines stimulates protective immunity against botulinum neurotoxin A intoxication
KR20210141623A (en) Methods for modulating mucosal immunogenicity
Mann et al. CD71 targeting boosts immunogenicity of sublingually delivered influenza haemagglutinin antigen and protects against viral challenge in mice
Sloat et al. Evaluation of the immune response induced by a nasal anthrax vaccine based on the protective antigen protein in anaesthetized and non-anaesthetized mice
Silvestre et al. Display of FliC131 on the Surface of Lactococcus lactis as a Strategy to Increase its Adjuvanticity for Mucosal Immunization
KR20240031857A (en) Tetanus vaccine platform for embedding COVID-19 vaccines
Tsai et al. Mucosal vaccine delivery
WO2024050452A2 (en) Compositions and methods for raising immune responses to gram-negative bacteria

Legal Events

Date Code Title Description
AS Assignment

Owner name: OSAKA UNIVERSITY, JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FUJINAGA, YUKAKO;MATSUMURA, TAKUHIRO;YUTANI, MASAHIRO;AND OTHERS;REEL/FRAME:035774/0546

Effective date: 20150512

Owner name: DAIICHI SANKYO COMPANY, LIMITED, JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FUJINAGA, YUKAKO;MATSUMURA, TAKUHIRO;YUTANI, MASAHIRO;AND OTHERS;REEL/FRAME:035774/0546

Effective date: 20150512

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION