U.S. patent application number 13/126357 was filed with the patent office on 2011-08-25 for mucosal vaccine using cationic nanogel.
Invention is credited to Kazunari Akiyoshi, Hiroshi Kiyono, Tomonori Nochi, Yoshikazu Yuki.
Application Number | 20110206729 13/126357 |
Document ID | / |
Family ID | 42128937 |
Filed Date | 2011-08-25 |
United States Patent
Application |
20110206729 |
Kind Code |
A1 |
Akiyoshi; Kazunari ; et
al. |
August 25, 2011 |
MUCOSAL VACCINE USING CATIONIC NANOGEL
Abstract
A mucosal vaccine for the prevention or treatment of microbial
infections is described that is capable of inducing vaccine
antigen-specific immune responses in an organism without the
addition of a mucosal adjuvant. The mucosal vaccine comprises a
composite of a nanogel comprising a hydrophilic polysaccharide
having a cationic functional group and a hydrophobic cholesterol
added thereto as a side chain and a vaccine antigen. The vaccine is
administered via a mucosal route.
Inventors: |
Akiyoshi; Kazunari; (Tokyo,
JP) ; Kiyono; Hiroshi; (Tokyo, JP) ; Yuki;
Yoshikazu; (Tokyo, JP) ; Nochi; Tomonori;
(Chapel Hill, NC) |
Family ID: |
42128937 |
Appl. No.: |
13/126357 |
Filed: |
October 30, 2009 |
PCT Filed: |
October 30, 2009 |
PCT NO: |
PCT/JP2009/068647 |
371 Date: |
April 27, 2011 |
Current U.S.
Class: |
424/204.1 ;
424/184.1; 424/234.1; 424/236.1; 424/265.1; 424/274.1 |
Current CPC
Class: |
A61P 33/00 20180101;
A61P 31/00 20180101; A61K 47/36 20130101; A61K 2039/55583 20130101;
A61K 47/61 20170801; A61K 9/0043 20130101; A61P 31/12 20180101;
A61K 9/06 20130101; A61K 39/39 20130101; A61P 31/18 20180101; A61K
2039/543 20130101; A61P 33/02 20180101; A61K 39/08 20130101; A61P
37/04 20180101; A61K 2039/541 20130101; A61K 9/006 20130101; A61P
37/00 20180101; A61K 47/6903 20170801; A61K 9/5161 20130101; A61K
47/554 20170801; A61P 31/10 20180101; A61K 2039/6087 20130101; A61P
31/04 20180101 |
Class at
Publication: |
424/204.1 ;
424/184.1; 424/234.1; 424/265.1; 424/274.1; 424/236.1 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 39/00 20060101 A61K039/00; A61K 39/02 20060101
A61K039/02; A61K 39/002 20060101 A61K039/002; A61P 37/00 20060101
A61P037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 2008 |
JP |
2008-281065 |
Claims
1. A mucosal vaccine preparation used for prevention or treatment
of a microbial infection, comprising a composite of a nanogel, the
nanogel comprising a hydrophilic polysaccharide having a cationic
functional group with a hydrophobic cholesterol added thereto as a
side chain and a vaccine antigen, wherein the vaccine preparation
is administered via the mucosal route.
2. The mucosal vaccine preparation according to claim 1, wherein
the cationic functional group is an amino group.
3. The mucosal vaccine preparation according to claim 1, wherein
the nanogel is cholesterol-bearing pullulan.
4. The mucosal vaccine preparation according to claim 1, wherein
the vaccine antigen is derived from a microorganism.
5. The mucosal vaccine preparation according to claim 4, wherein
the microorganism is selected from the group consisting of a virus,
a bacterium, a protozoan, and a fungus.
6. The mucosal vaccine preparation according to claim 5, wherein
the vaccine antigen is selected from the group consisting of a
C-terminal avirulent region of the heavy chain of botulinus toxin,
tetanus toxoid, and the AIDS virus membrane antigen molecule (gag
p24).
7. The mucosal vaccine preparation according to claim 1, wherein
the vaccine antigen is combined with the nanogel at a molar ratio
of 1:1 to 1:10.
8. The mucosal vaccine preparation according to claim 1, wherein
the vaccine preparation is a nasal preparation.
9. The mucosal vaccine preparation according to claim 1, wherein
the vaccine preparation is an oral preparation.
10. A method for producing the mucosal vaccine preparation of claim
1 comprising mixing a nanogel comprising a hydrophilic
polysaccharide having a cationic functional group with hydrophobic
cholesterol added thereto as a side chain and a vaccine antigen at
about 4.degree. C. to about 37.degree. C. for about 2 to about 48
hours.
11. The method for producing a mucosal vaccine preparation
according to claim 10, wherein the cationic functional group is an
amino group.
12. The method for producing a mucosal vaccine preparation
according to claim 10, wherein the nanogel is cholesterol-bearing
pullulan.
13. The method for producing a mucosal vaccine preparation
according to claim 10, wherein the vaccine antigen is derived from
a microorganism.
14. The method for producing a mucosal vaccine preparation
according to claim 13, wherein the microorganism is selected from
the group consisting of a virus, a bacterium, a protozoan, and a
fungus.
Description
TECHNICAL FIELD
[0001] The present invention relates to a mucosal vaccine
comprising a composite of a vaccine antigen and a cationic nanogel
that is transnasally or orally administered.
BACKGROUND ART
[0002] Non-injection mucosal vaccines are safe and convenient to
use, and thus they have drawn attention as the next-generation
vaccines. It was necessary to administer a mucosal vaccine
simultaneously with a mucosal adjuvant in order to induce effective
antigen-specific immune responses with the use of a mucosal
vaccine. As mucosal adjuvants, toxin-related proteins, such as
cholera toxins (CT) or detoxicated cholera toxins (mCT), are known.
Addition of such mucosal adjuvants to mucosal vaccines enables
transnasal vaccines to induce mucosal IgA in addition to
antigen-specific systemic IgG. However, such mucosal adjuvant may
disadvantageously migrate to the brain, and the safety thereof on
organisms has remained problematic.
[0003] The present inventors developed nanogels comprising
molecules such as cholesterol-bearing pulullan (CHP), which is
composed of hydrophilic polysaccharides and hydrophobic cholesterol
added thereto as a side chain, as DDS substrates (see WO 00/12564,
JP Patent Publication (kokai) No. 2005-298644 A, WO 2006/049032, JP
Patent Publication (kokai) No. 2006-143808 A, WO 2007/083643, JP
Patent Publication (kokai) No. 2007-252304 A, and Hasegawa et al.,
Saibou Kougaku (Cell Technology), Vol. 26, No. 6, 2007, pp.
679-685). Specifically, CHP is capable of self-assembly in an
aqueous environment, and it is converted into colloids (nanogels)
with diameters of 20 to 30 nm capable of enclosing various
substances therein. An excellent feature of CHP is the "molecular
chaperone effects." That is, upon enclosure of a molecule, such as
a protein molecule, inside CHP nanogels, followed by release
thereof, refolding takes place at the time of release, a
physiological 3-D structure is formed, and normal activity is
exerted.
[0004] While the use of such nanogels for vaccine preparations has
been reported (see JP Patent No. 4033497), such nanogels become
usable upon activation of cytotoxic T cells (CTL) for anti-cancer,
anti-virus, or autoimmune disease treatment applications. That is,
it could not be said that nanogels can always exert the effects of
mucosal vaccines.
[0005] Also, use of a liposome having a lipid membrane comprising
glycolipids and phospholipids for the delivery of oral vaccines had
been reported (see JP Patent Publication (kokai) No. H05-339169 A
(1993)).
DISCLOSURE OF THE INVENTION
Object to be Attained by the Invention
[0006] The present invention provides a mucosal vaccine for
transnasal or oral administration, which is capable of inducing
vaccine antigen-specific immune responses in organisms without the
addition of a mucosal adjuvant such as a toxin-related protein
(e.g., cholera toxin (CT) or a detoxicated cholera toxin
(mCT)).
Means for Attaining the Object
[0007] The present inventors previously developed a nanogel
comprising a hydrophilic polysaccharide with hydrophobic
cholesterol added to the hydrophilic polysaccharide as a side
chain, which can be used for the delivery of a substance such as a
physiologically active protein.
[0008] The present inventors conducted concentrated studies in
order to examine the applicability of such nanogel to the
production of mucosal vaccines. As a result, they discovered that
administration of a composite of nanogels comprising cationic
functional groups such as amino groups and vaccine antigens (i.e.,
viral or bacterial proteins) through the mucous membrane of the
nasal cavity or the mucous membrane of the intestinal canal would
induce systemic immune responses and mucosal immune responses more
effectively than would be possible with the use of a liposome, and
such administration would be useful for prevention or treatment of
viral or bacterial infections. This has led to the completion of
the present invention.
[0009] Specifically, the present invention is as follows.
[0010] [1] A mucosal vaccine preparation used for prevention or
treatment of a microbial infection, which comprises a composite of
a nanogel comprising a hydrophilic polysaccharide having a cationic
functional group with hydrophobic cholesterol added thereto as a
side chain and a vaccine antigen and is administered via the
mucosal route.
[0011] [2] The mucosal vaccine preparation according to [1],
wherein the cationic functional group is an amino group.
[0012] [3] The mucosal vaccine preparation according to [1] or [2],
wherein the nanogel is cholesterol-bearing pullulan.
[0013] [4] The mucosal vaccine preparation according to any of [1]
to [3], wherein the vaccine antigen is derived from a
microorganism.
[0014] [5] The mucosal vaccine preparation according to [4],
wherein the microorganism is selected from the group consisting of
a virus, a bacterium, a protozoan, and a fungus.
[0015] [6] The mucosal vaccine preparation according to [5],
wherein the vaccine antigen is selected from the group consisting
of a C-terminal avirulent region of the heavy chain of botulinus
toxin, tetanus toxoid, and the AIDS virus membrane antigen molecule
(gag p24).
[0016] [7] The mucosal vaccine preparation according to any of [1]
to [6], wherein the vaccine antigen is combined with the nanogel at
a molar ratio of 1:1 to 1:10.
[0017] [8] The mucosal vaccine preparation according to any of [1]
to [7], which is a nasal preparation.
[0018] [9] The mucosal vaccine preparation according to any of [1]
to [7], which is an oral preparation.
[0019] [10] A method for producing a mucosal vaccine preparation
comprising a composite of the vaccine antigen and the nanogel,
[0020] the method comprising mixing a nanogel comprising a
hydrophilic polysaccharide having a cationic functional group with
hydrophobic cholesterol added thereto as a side chain and a vaccine
antigen at 4.degree. C. to 37.degree. C. for 2 to 48 hours.
[0021] [11] The method for producing a mucosal vaccine preparation
according to [10], wherein the cationic functional group is an
amino group.
[0022] [12] The method for producing a mucosal vaccine preparation
according to [10] or [11], wherein the nanogel is
cholesterol-bearing pullulan.
[0023] [13] The method for producing a mucosal vaccine preparation
according to any of [10] to [12], wherein the vaccine antigen is
derived from a microorganism.
[0024] [14] The method for producing a mucosal vaccine preparation
according to [13], wherein the microorganism is selected from the
group consisting of a virus, a bacterium, a protozoan, and a
fungus.
[0025] This description includes the contents as disclosed in the
description and/or drawings of Japanese Patent Application No.
2008-281065, which is a priority document of the present
application.
EFFECTS OF THE INVENTION
[0026] The mucosal vaccine preparation of the present invention
prepared by combining a vaccine antigen and a cationic nanogel
effectively induces systemic and mucosal immune responses in an
animal via transmucosal administration, such as transnasal or oral
administration. The mucosal vaccine of the present invention
involves the use of cationic nanogels. Accordingly, vaccine
antigens can be efficiently delivered to the immune system, and
immune responses are induced more effectively than a case in which
non-cationic nanogels or cationic liposomes are used. The mucosal
vaccine of the present invention can be effectively used for
prevention or treatment of viral or bacterial infections of an
animal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows the total IgG antibody titer to Hc in the serum
of a transnasally immunized mouse.
[0028] FIG. 2 shows the IgG1, IgG2a, IgG2b, and IgG3 antibody
titers to Hc in the serum of a transnasally immunized mouse. The
four aligned bar graphs each show IgG1, IgG2a, IgG2b, and IgG3 from
the left.
[0029] FIG. 3 shows the total IgG antibody titer to TT in the serum
of a transnasally immunized mouse.
[0030] FIG. 4 shows the IgG1, IgG2a, IgG2b, and IgG3 antibody
titers to TT in the serum of a transnasally immunized mouse. The
four aligned bar graphs each show IgG1, IgG2a, IgG2b, and IgG3 from
the left.
[0031] FIG. 5 shows the total IgG antibody titer to gag p24 in the
serum of a transnasally immunized mouse.
[0032] FIG. 6 shows the IgA antibody titer to He in the nasal wash
solution used for a transnasally immunized mouse.
[0033] FIG. 7 shows the IgA antibody titer to TT in the nasal wash
solution used for a transnasally immunized mouse.
[0034] FIG. 8 shows the number of He antigen-specific IgA-producing
cells in the nasal wash solution used for a transnasally immunized
mouse.
[0035] FIG. 9 shows the viability of a mouse transnasally immunized
with He after intraperitoneal administration of botulinum toxins
with the elapse of time.
[0036] FIG. 10 shows the viability of a mouse transnasally
immunized with He after transnasal administration of botulinum
progenitor toxins with the elapse of time.
[0037] FIG. 11 shows the total IgG antibody titer to botulinus
toxin in the serum of a mouse transnasally immunized with cationic
nanogels or cationic liposomes.
[0038] FIG. 12 shows the IgA antibody titer to gag p24 in the nasal
wash solution used for a mouse transnasally immunized with cationic
nanogels or cationic liposomes.
[0039] FIG. 13 shows the effects of cationic nanogel-based vaccines
for retaining an antigen in nasal cavity tissue and the transition
thereof to the cerebral nervous system.
PREFERRED EMBODIMENTS OF THE INVENTION
[0040] Hereafter, the present invention is described in detail.
[0041] In the present invention, the term "nanogel" refers to a
hydrophobized polymer gel nanoparticle comprising a hydrophilic
polysaccharide with hydrophobic cholesterol added thereto as a side
chain. Nanogels can be produced by the method described in, for
example, WO 00/12564 (the title of the invention: High-purity
polysaccharide containing hydrophobic groups and process for
producing the same).
[0042] At the outset, a hydroxyl-group-containing hydrocarbon or
sterol having 12 to 50 carbon atoms is allowed to react with a
diisocyanate compound represented by the formula 0CN--R1 NCO,
wherein R1 represents a hydrocarbon group having 1 to 50 carbon
atoms, to produce an isocyanate-group-containing hydrophobic
compound that had reacted with a molecule of a
hydroxyl-group-containing hydrocarbon or sterol having 12 to 50
carbon atoms. Subsequently, the resulting
isocyanate-group-containing hydrophobic compound is subjected to a
further reaction with a polysaccharide to produce a polysaccharide
containing a hydrophobic group comprising a hydrocarbon or steryl
group having 12 to 50 carbon atoms as a hydrophobic group. The
reaction product may be purified using a ketone-based solvent to
produce a high-purity hydrophobic-group-containing polysaccharide.
Examples of polysaccharides include pullulan, amylopectin, amylose,
dextran, hydroxyethyl dextran, mannan, levan, inulin, chitin,
chitosan, xyloglucan, and water-soluble cellulose.
[0043] Examples of nanogels that are preferably used in the present
invention include cholesterol-bearing pullulan (hereafter referred
to as "CHP") and a CHP derivative. In CHP, 1 to 10 and preferably 1
to several cholesterol molecules are substituted with pullulan
having a molecular weight of 30,000 to 200,000 (e.g., 100,000) per
100 monosaccharide units. CHP properties can be modified in terms
of the amount of cholesterol substitution depending on protein size
or degree of hydrophobicity. In order to control the hydrophobic
properties of CHP, an alkyl group having 10 to 30 and preferably
about 12 to 20 carbon atoms may be introduced. The nanogels used in
the present invention have a particle diameter of 10 to 40 nm and
preferably 20 to 30 nm. Nanogels are extensively commercialized,
and such commercialized nanogels can be used in the present
invention.
[0044] In the present invention, the mucosal vaccine involves the
use of nanogels into which positively charged functional groups,
such as amino groups, have been introduced. The number of amino
groups introduced into nanogels is 1 to 50 and preferably 5 to 30
per 100 glucose monosaccharides of CHP. A preferable example of a
method for introducing amino groups into nanogels is a method
involving the use of amino-group-added cholesterol pullulan
(CHPNH.sub.2) described below.
[0045] CHP dried under reduced pressure (0.15 g) is dissolved in 15
ml of a dimethyl sulfoxide (DMSO) solvent, 75 mg of
1-1'-carbonyldiimidazole is added thereto under a nitrogen stream,
and the reaction is allowed to proceed at room temperature for 4
hours. Ethylenediamine (300 mg) is slowly added to the reaction
solution, and the resultant is agitated for 24 hours. The reaction
solution is dialyzed against distilled water for 6 days. The
resultant is lyophilized to obtain an opalescent solid. The degree
of substitution of ethylenediamine is determined via elemental
analysis or H-NMR analysis. The number of substituents to be
introduced can be changed as necessary. By changing the number of
substituents introduced, the magnitude of the positive charge can
be regulated, and the efficiency for vaccine antigen delivery from
the vaccine antigen/cationic nanogel composite can be
regulated.
[0046] The mucosal vaccine preparation of the present invention can
efficiently induce vaccine antigen-specific systemic and mucosal
immune responses in animals without the addition of another mucosal
adjuvant.
[0047] Examples of vaccine antigens used for the mucosal vaccine of
the present invention include antigens of microorganisms, such as
bacteria, viruses, fungi, and protozoans, that cause infections in
animals. Such antigens induce antigen-specific immune responses in
animals, they can be used for vaccines, and they are thus referred
to as "vaccine antigens."
[0048] Specific examples of microbial antigens include protein
antigens of the following microorganisms: pathogenic viruses, such
as influenza virus A, influenza virus B, hepatitis C virus,
hepatitis A virus, hepatitis B virus, rotavirus, cytomegalovirus,
respiratory syncytial (RS) virus, adenovirus, HIV, varicella-zoster
virus, herpes simplex virus type 1 and type 2, ATL (adult T-cell
leukemia) virus, coxsackie virus, enterovirus, exanthema subitum
virus (HHV-6), measles virus, rubella virus, mumps (epidemic
parotiditis) virus, poliovirus, Japanese encephalitis virus, rabies
virus, hepatitis C virus, Norwalk virus (norovirus), rabies virus,
respiratory syncytial (RS) virus, cytomegalovirus, foot and mouth
disease virus, transmissible gastroenteritis virus, rubella virus,
ATL virus, adenovirus, ECHO virus, herpes virus, smallpox virus,
dengue fever virus, yellow fever virus, West Nile virus, SARS
(coronavirus), ebola hemorrhagic fever virus (phyllovirus), Marburg
virus (phyllovirus), Lassa fever virus, hantavirus, and Nipah
virus; pathogenic bacteria, such as enteropathogenic Escherichia
coli (e.g., enterohemorrhagic E. coli), Staphylococcus (e.g.,
Staphylococcus aureus), meningococci, Pseudomonas aeruginosa,
Streptococcus mutans, Vibrio cholera, Bacillus typhosus, chlamydia,
Shigella, pneumococcus, Bordetella pertussis, Corynebacterium
diphtheriae, Clostridium tetani, Haemophilus influenzae, Yersinia
pestis, Clostridium botulinum, Bacillus anthracis, Francisella
tularensis, Salmonella, VRE (Enterococcus), Mycobacterium
tuberculosis, Shigella, Salmonella typhi, Salmonella paratyphi,
Chlamydia, amebic dysentery, Legionella, Lyme Disease Borrelia, and
Brucellosis (undulant fever); Rickettsia, such as Q fever
rickettsia and chlamydia; protozoans, such as causal agents of
malaria and Cryptosporidium; and fungi, such as Cryptococcosis and
Aspergillus. Examples of proteins derived from pathogenic
microorganisms include proteins or peptides constituting pathogenic
microorganisms (e.g., surface proteins, capsid proteins, and
ciliary proteins), proteins or peptides produced by pathogenic
microorganisms (e.g., toxins, enzymes, hormones, immunomodulating
substances, receptors, and ligands thereof), and fragments or
domains thereof. Protein antigens capable of inducing the
production of antibodies that can attack and neutralize the
aforementioned microorganisms may be used. A protein antigen to be
used is not limited to only one type, and the mucosal vaccine of
the present invention may contain a plurality of types of vaccine
antigens derived from homologous or heterologous microorganisms. In
the case of the influenza virus, for example, either or both the
hemagglutinin (HA) receptor and the neuraminidase (NA) receptor may
be combined with a cationic nanogel to produce a mucosal vaccine.
Vaccine antigens can be obtained from microorganisms via
processing, purification, or other means. Also, vaccine antigens
can be chemically synthesized or can be obtained in the form of
recombinant proteins via genetic engineering. The molecular weight
of vaccine antigens contained in the mucosal vaccine preparation of
the present invention is not limited. For example, it is
approximately 500 to 1,000,000, and preferably approximately 1,000
to 200,000.
[0049] The vaccine antigen/cationic nanogel composite can be
prepared by causing interactions between the cationic nanogels and
the vaccine antigens so as to incorporate the vaccine antigens into
the cationic nanogels. Preparation of a composite is referred to as
"composite formation." The mixing ratio of vaccine antigens to
cationic nanogels can be adequately determined in accordance with
types of vaccine antigens and cationic nanogels used. For example,
CHPNH.sub.2 can be mixed with vaccine antigens at a molar ratio of
1:1 to 1:100, and preferably 1:1 to 1:10.
[0050] A vaccine antigen/cationic nanogel composite can be prepared
by, for example, mixing vaccine antigens with cationic nanogels in
a buffer and allowing the mixture to stand at 4.degree. C. to
37.degree. C. for 2 to 48 hours, and preferably 20 to 30 hours. A
buffer used for preparation of a vaccine antigen/cationic nanogel
composite can be adequately prepared in accordance with protein and
nanogel types. An example is Tris-HCl buffer (50 mM, pH 7.6). The
resulting vaccine antigen/nanogel composite can be analyzed in
accordance with a conventional technique, such as gel permeation
chromatography (GPC), atomic force microscopy (AFM), fluorescence
microscopy, or confocal fluorescence microscopy.
[0051] The mucosal vaccine preparation of the present invention is
administered via a mucosal route. Transmucosal administration is
preferably carried out through the mucous membrane of the nasal
cavity or the mucous membrane of the intestinal canal. In the
former case, the vaccine preparation is administered transnasally.
In the latter case, the vaccine preparation is administered orally.
Nasal vaccine preparations induce immune responses in the nasal
cavity via transnasal administration. Specifically, such vaccine
preparations are capable of inducing local immune responses on the
mucosal membrane of the respiratory tract (the upper respiratory
tract, in particular), which is the route of microbial infection
that causes viral or other infections. Nasal vaccine preparations
may be administered into the nasal cavity via, for example,
spraying, coating, or dropping. Oral vaccine preparations induce
immune responses in the intestinal tract via oral administration.
Mucosal vaccine preparations remain in the mucosal membrane,
nasal-associated lymphoid tissue (NALT), or gut-associated
lymphatic tissue (GALT) and releases vaccine antigens. Both nasal
vaccine preparations and oral vaccine preparations induce systemic
immune responses, produce virus-specific IgG or the like in
organisms, induce mucosal immune responses, produce IgA antibodies
in the mucosal membrane, and block infections via systemic and
mucosal immune mechansisms. Thus, infections can be treated.
[0052] The mucosal vaccine preparation may contain known
pharmaceutically acceptable stabilizers, antiseptics, antioxidants,
and the like. Examples of stabilizers include gelatin, dextran, and
sorbitol. Examples of antiseptics include thimerosal and
.beta.-propiolactone. An example of an antioxidant is
.alpha.-tocopherol.
[0053] The mucosal vaccine preparation of the present invention can
be administered to, for example, mammalians, such as humans,
monkeys, mice, rats, rabbits, cats, cattles, dogs, horses, and
goats, and birds, such as chickens.
[0054] A dose of the mucosal vaccine preparation can be adequately
determined based on immunogen type, age or body weight of a
subject, and other conditions. The mucosal vaccine preparation
contains pharmaceutically effective amounts of vaccine antigens.
The term "pharmaceutically effective amount(s)" refers to an amount
of an antigen that is necessary for inducing immune responses to a
vaccine antigen. For example, a dose of several .mu.g to several
ten mg of a vaccine antigen may be administered once to several
times per day, and administration may take place several times at
intervals of 1 to several weeks (e.g., administration may take
place 1 to 5 times).
EXAMPLES
[0055] The present invention is described in detail with reference
to the following examples, although the technical scope of the
present invention is not limited to these examples.
Example 1
Preparation of Mucosal Vaccine
[0056] Cationic nanogels (cationic CHP) in which the degree of
cholesterol substitution was 1.4 and the degree of ethylenediamine
substitution was 18 per 100 monosaccharides were used (CHPNH.sub.2
nanogels). A CHP derivative or cationic Pullulan was dissolved in a
1 mg/ml phosphate buffer solution (PBS). The CHPNH2 nanogels were
subjected to sonication for 15 minutes and then filtered through a
0.22-mm filter.
[0057] The C-terminal avirulent region of a heavy chain of
botulinus toxin (Hc; molecular weight: 45,000), tetanus toxoid (TT;
molecular weight: 150,000), or the AIDS virus membrane antigen
molecule (gag p24; molecular weight: 24,000) expressed in E. coli
and purified was mixed with the equimolar amount of cationic
nanogels prepared in the manner described above, and the resulting
mixture was subjected to reaction at 45.degree. C. for 5 hours to
prepare a composite. The obtained antigen/cationic nanogel
composite was used as a mucosal vaccine using cationic nanogels.
The gene of the purified C-terminal avirulent region of the heavy
chain of botulinus toxin was inserted into a GST fusion protein
expression vector (pGEX-6P3, GE Healthcare), transformed into E.
coli Rossetta 2 (Novagen), and induced to express with the addition
of 0.1 mM IPTG. Hc was centrifuged after ultrasonic disintegration
of cells suspended in PBS, the resulting supernatant was purified
via anion exchange chromatography (DAEA Sepharose; GE Healthcare),
affinity chromatography (Glutathione Sepharose; GE Healthcare), or
gel permeation chromatography (Sephacryl S-100; GE healthcare). GST
fused to the N terminus of Hc was subjected to affinity
chromatography and then removed via ablation with the addition of
PreScission Protease (GE Herthcare) to the column. Tetanus toxoid
was obtained from the Research Foundation for Microbial Diseases of
Osaka University and gag p24 was obtained from Kyoko Yokota of the
Department of Immunology at the National Institute of Infectious
Diseases.
Example 2
Transnasal Immunization
[0058] The mucosal vaccine using cationic nanogels prepared in
Example 1 or the antigen alone was administered to 6- to 8-week-old
Balb/c mice (female) through the nasal cavity in an amount of 10
.mu.g of Hc (88.9 .mu.g of nanogel), 30 .mu.g of TT (80.0 .mu.g of
nanogel), or 10 .mu.g of gag p24 (166.7 .mu.g of nanogel) per mouse
once a week (3 times in total) to immunize mice transnasally. The
amount of antigens administered (i.e., the amount of the solution)
was adjusted to 15 .mu.l in every experimental group, and 7.5 .mu.l
of the solution was administered to each nostril. PBS was
administered as a control.
[0059] The blood was sampled before immunization and a week after
immunization, and IgG antibody titers to botulinus toxin, TT, or
gag p24 in the blood serum were measured to evaluate the systemic
immune responses. The nasal cavity was washed with 200 .mu.l of PBS
a week after the final immunization, and the IgA antibody titer in
the nasal wash solution was measured to evaluate immune responses
in the mucosal system. The antibody titer was evaluated via ELISA.
Regarding serum IgG, antibody titers of IgG1, IgG2a, IgG2b, and
IgG3 subclasses were measured, and antibody production pattern at
the subclass level was evaluated in order to predict the Th1/Th2
immune balance after immunization. Further, the number of
antigen-specific IgA-producing cells (blood plasma cells) in the
nasal tissue a week after the final immunization was evaluated via
ELISPOT.
[0060] FIG. 1 shows the total IgG antibody titer to botulinus toxin
in the serum. FIG. 2 shows the IgG1, IgG2a, IgG2b, and IgG3
antibody titers to botulinus toxin in the serum sampled after 3
immunization procedures. Further, FIG. 3 shows the total IgG
antibody titer to TT in the serum, FIG. 4 shows the IgG1, IgG2a,
IgG2b, and IgG3 antibody titers to TT in the serum sampled after 3
immunization procedures, and FIG. 5 shows the gag p24-specific IgG
antibody titer after 3 immunization procedures.
[0061] FIG. 6 shows the IgA antibody titer to botulinus toxin in
the nasal wash solution after 3 immunization procedures, and FIG. 7
shows the IgA antibody titer to TT in the nasal wash solution after
3 immunization procedures.
[0062] As shown in FIGS. 1, 3, and 5, the total IgG antibody titers
to botulinus toxin, TT, or gag p24 were significantly higher when
the composite of Hc, TT, or gag p24 and cationic nanogels was
administered, compared with the case when Hc, TT, or gag p24 was
administered alone. This indicates that more potent systemic immune
responses would be induced when the composite of Hc, TT, or gag p24
and cationic nanogels was administered, compared with the case when
Hc, TT, or gag p24 was administered alone. As shown in FIGS. 2 and
4, also, a majority of antigen-specific IgG antibodies were of the
IgG1 subclass, and the IgG2a level was significantly low. Thus, it
was deduced that transnasal administration of a vaccine
antigen/cationic nanogel composite would effectively induce
Th2-type humoral immunity responses.
[0063] As shown in FIGS. 6 and 7, substantially no IgA antibody
titer was recognized when Hc or TT was administered alone. When a
composite of Hc or TT and cationic nanogels was administered,
however, a high IgA antibody titer to botulinus toxin or IT was
observed. This indicates that mucosal immune responses would be
induced in the nasal mucous membrane only via transnasal
administration of the mucosal vaccine of the present invention in
the form of an antigen/cationic nanogel composite.
[0064] FIG. 8 shows a comparison of the number of botulinus toxin
antigen-specific IgA-producing cells in the mucous membrane of the
nasal cavity. As shown in FIG. 8, no IgA-producing cells were
produced when Hc was administered alone; however, IgA-producing
cells were produced when a Hc/cationic nanogel composite was
administered.
Example 3
Neutralization Effects after Transnasal Immunization using Mucosal
Vaccine using Nanogels
[0065] The vaccine using cationic nanogels using a C-terminal
avirulent region of the heavy chain of botulinus toxin (Hc;
molecular weight: 45,000) as the antigen prepared in Example 1 or
Hc alone was administered transnasally to 5 mice for immunization
in the same manner as in Example 2. PBS was administered as a
negative control. After the mice were subjected to immunization 3
times, botulinus toxin (obtained from Professor Shunji Kozaki,
Division of Veterinary Science, School of Life and Environmental
Sciences, Osaka Prefecture University) was administered
intraperitoneally in an amount 25,000 times greater than the lethal
dose thereof via intraperitoneal administration (i.g., 500 ng) to
analyze the survival effects. For the purpose of analyzing the
neutralization effects of Hc-specific IgA induced in the nasal
tissue, 10 .mu.g of botulinum progenitor toxins (obtained from Wako
Pure Chemical Industries, Ltd.) was administered transnasally and
the later survival effects were also analyzed.
[0066] FIG. 9 shows the viability of a mouse after intraperitoneal
administration of botulinum toxin with the elapse of time. As shown
in FIG. 9, all mice that had been immunized with Hc alone died
within a day; however, all mice that had been immunized with an
Hc/cationic nanogel composite remained alive 1 week later. This
indicates that potent systemic neutralization and immunization
would be induced via transnasal administration of the Hc/cationic
nanogel composite.
[0067] FIG. 10 shows the viability after transnasal administration
of botulinum progenitor toxin with the elapse of time. As shown in
FIG. 10, all mice that had been immunized with Hc alone died within
a day; however, all mice that had been immunized with an
Hc/cationic nanogel composite remained alive 1 week later. This
indicates that botulinus toxin-specific mucosal IgA induced via
transnasal administration of an Hc/cationic nanogel composite would
effectively block mucosal infection by botulinum.
Example 4
Effects of Vaccine using Cationic Nanogel for Immunity Induction in
Comparison with Vaccine using Cationic Liposome
[0068] The vaccine using cationic nanogels using a C-terminal
avirulent region of the heavy chain of botulinus toxin (Hc;
molecular weight: 45,000) as the antigen prepared in Example 1 or
the cationic liposome comprising the same amounts of the antigens
of the same type (Project) was administered transnasally to 5 mice
for immunization in the same manner as in Example 2. Project was
obtained from PIERCE.
[0069] FIG. 11 shows the total IgG antibody titer to botulinus
toxin after 3 immunization procedures.
[0070] As shown in FIG. 11, the total IgG antibody titer to
botulinus toxin was significantly higher when the Hc/cationic
nanogel composite was administered, compared with the case when Hc
was administered in the form of an Hc/cationic liposome.
[0071] FIG. 12 shows the total IgA antibody titer to botulinus
toxin after 3 immunization procedures.
[0072] As shown in FIG. 12, the total IgA antibody titer to
botulinus toxin was significantly higher when the Hc/cationic
nanogel composite was administered, compared with the case when Hc
was administered in the form of a Hc/cationic liposome.
Example 5
Effects of Vaccine using Cationic Nanogels for Retaining Antigen in
Nasal Tissue and Migration to Cerebral Nervous System
[0073] The C-terminal avirulent region of the heavy chain of
botulinus toxin (Hc; molecular weight: 45,000) was labeled with
111In (indium) in accordance with a known technique with the use of
DTPA anhydride. Labelling efficiency was 728.3233.+-.115.3543
CPM/ng. Thereafter, the labelled Hc was combined with nanogels. The
mucosal vaccine using nanogels combined with the labelled Hc
(1,000,000 CPM) or the labeled Hc alone was administered
transnasally to mice. The disposition thereafter in the brain, the
olfactory bulb, the nasal cavity, the nasal-associated lymphoid
tissue (NALT), the cervical lymph node, and the spleen was
subjected to follow-up evaluation using a gamma counter.
Specifically, the brain, the olfactory bulb, the nasal cavity, the
nasal-associated lymphoid tissue (NALT), the cervical lymph node,
and the spleen were extracted from mice 0.17, 1, 6, 12, 24, and 48
hours after transnasal administration, the samples were weighed,
and gamma rays emitted by the samples were measured using a gamma
counter.
[0074] FIG. 13 shows the results of gamma ray measurements in the
brain (A), the olfactory bulb (B), the nasal tissue (C), the
nasal-associated lymphoid tissue (NALT) (D), the cervical lymph
node (E), and the spleen (F).
[0075] As shown in FIG. 13, mucosal vaccines using nanogels
remained, particularly in the nasal tissue (C), for a long period
of time, although migration to the brain or the olfactory bulb was
not observed. The results demonstrate that transnasal
administration of the mucosal vaccines comprising cationic nanogels
of the present invention yields the higher effects of antigen
retention in the nasal cavity, compared with a case in which a
mucosal vaccine is administered alone. In addition, the results
demonstrate that such mucosal vaccines can be used as preparations
for intranasal administration with excellent safety and
effectiveness, and they do not migrate to the central nervous
system as some adjuvants would.
[0076] All publications, patents, and patent applications cited
herein are incorporated herein by reference in their entirety.
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