Oral vaccine

专利摘要:

公开号:ES2563242T9
申请号:ES08751817.1T
申请日:2008-03-19
公开日:2016-05-12
发明作者:Toshiro Shirakawa；Masato Kawabata；Tetsuo Takata；Michiko Taniguchi；Asako Okamoto；Masanori Asada；Masaaki Nakatsuji
申请人:Morishita Jintan Co Ltd；
IPC主号:

专利说明:



Oral vaccine
Technical field
The present invention relates to an oral vaccine useful for preventing and treating a bacterial infectious disease, and a method of producing it.
Prior art
10 Typhoid fever is one of the infectious diseases caused by Salmonella enterica var. Typhi, which is a type of salmonella bacteria; infection caused by ingestion of contaminated drinking water, food, or the like. Typhoid fever is common throughout the world, particularly in the areas of Asia, the Middle East, Eastern Europe, Africa and Central and South America. Each year, 16 million people are affected by typhoid fever, and 0.6 million people die of this disease. Most of the dead are children in countries
15 in development. Currently, an attenuated Salmonella (Ty2la) or similar bacterium is administered orally in the form of a typhoid fever vaccine caused by Salmonella bacteria, but cannot be given to infants 5 years old or younger due to its effects side effects, such as diarrhea or vomiting. Once a person is affected by typhoid fever, an antibody against typhoid fever develops within the body, and immunity is acquired, but this effect does not last long.
20 Cholera is one of the infectious diseases caused by Vibrio cbolerae O1 or O139. Cholera is prevalent throughout the world, particularly in Asia, the Middle East and Africa. Classic cholera epidemics have occurred repeatedly and several million people have died of this disease due to its strong pathogenicity (20% mortality rate). Currently, people are inoculated against this disease, but the effect of such inoculation is
25 relatively low and is said to be approximately 50%.
Bacterial dysentery (shigellosis) is a bacterial infectious disease widely distributed throughout the world, and it is especially noticeable in countries with poor hygiene. Bacterial dysentery is caused by intestinal bacteria that belong to the genus Shigella, which includes four groups consisting of Shigella dysenteriae, S.
30 flexneri, S. boydii and S. sonnei, in order of pathogenicity.
As described above, there are various bacterial infectious diseases, and it is clear that effective vaccines against bacterial infectious diseases are necessary. In particular, vaccines for the prevention of infectious diseases of transmission among humans are necessary. Currently, for example,
35 some vaccines against several species of salmonella are commercially available. These vaccines are sometimes effective, but they have serious disadvantages. These vaccines normally induce antibodies caused by infection with the wild bacteria, and an excessive load is placed on the subjects.
In order to solve this problem, a study focused on the scourge of bacteria has also led to
40 out. A scourge is a long structure that protrudes from the cell surface of bacteria, and plays an important role when cells move and invade a host cell. The flagellum is made up of a protein known as flagelin. This flagelin protein has been known to induce a high level of antibodies. The flagelin of Salmonella typhimurium antigenic protein is described by M. McClelland M. et al. in Nature, vol. 413, p. 852 (2001). The antigenic protein flagelin of Vibrio cholerae is described by Heiderberg et al. in
45 Nature, vol. 406, p. 477 (2000). In addition, Shigella dysenteriae antigenic protein flagellin is described by Tominaga A. et al. in Genes Genet. Syst., Vol. 76, p. 111 (2001). However, an effective vaccine for the use of this type of antibody against scourge has not yet been provided.
Description of the invention
It is an object of the present invention to provide means for using, as a vaccine, a flagelin protein derived from a bacterium that causes an infectious disease, because the infectious disease is not caused by the flagelin protein alone.
The present invention provides an oral vaccine against a bacterial infectious disease, in the form of a capsule formulation, comprising:
a capsule membrane anda transformed microorganism that expresses a flagelin antigen protein,
60 where the capsule membrane is resistant to acids, and the transformed microorganism is encapsulated with the capsule membrane.
The present invention also provides a first method for producing an oral vaccine against a bacterial infectious disease, comprising the steps of:

the preparation of a transformed microorganism that expresses a lagelin antigen protein; and the envelope of the microorganism transformed into an acid-resistant capsule membrane, thereby producing an acid-resistant capsule formulation.
The present invention further provides a second method for producing an oral vaccine against a bacterial infectious disease, comprising the steps of:
the preparation of a transformed microorganism that expresses a flagelin antigen protein; the envelope of the microorganism transformed into a capsule membrane, thereby producing a capsule formulation; and the proportion of the capsule membrane of the formulation in capsules produced with acid resistance.
In one embodiment, the flagelin antigen protein that is expressed in the cell of the microorganism.
In another embodiment, the flagelin antigen protein is secreted from the microorganism cell.
In one embodiment, the microorganism is at least one selected from microorganisms belonging to the group consisting of the genus Bilidobacterium, the genus Lactobacillus, the genus Lactococcus, the genus Pediococcus, the genus Streptococcus, the genus Enterococcus, the genus Leuconostoc, the genus Tetragenococcus, the genus Oenococcus, and the genus Weissella.
In one embodiment, the oral vaccine is a vaccine against typhoid fever, cholera, or dysentery.
In one embodiment, the capsule formulation is a transparent capsule formulation, a soft capsule formulation, or a hard capsule formulation.
In accordance with the present invention, a transformed microorganism that expresses a flagellin antigenic protein is contained in an acid resistant capsule formulation. Therefore, the transformed microorganism is protected from gastric acid in order to allow it to be effectively delivered to the intestine alive. The formulation disintegrates in the intestine to release the transformed microorganism, which produces the flagellin antigenic protein. Flagelin itself is not infectious, however, an antibody is produced in the body. In particular, the transformed microorganism can be prepared from intestinal bacteria, commonly known as good bacteria, such as bifidobacteria or lactic acid bacteria, which is viable in the intestine. Consequently, the flagelin protein is produced in the intestine, and the flagelin protein produced is then considered as an antigen to induce the production of antibodies in the body. Therefore, infectious disease can be prevented.
Accordingly, the present invention can provide a method for the prevention and treatment of bacterial infectious diseases with a small antibody load.
Brief description of the drawings
FIG. 1 is a schematic view showing the structure of plasmid pBLES 100.FIG. 2 is a schematic view showing the structure of pBLES-FliC prepared as a vector offlagelin expressionFIG. 3 is a schematic cross-sectional view showing the configuration of a formulationof a transparent three-layer capsule containing a transformed flagelin microorganism.
The best way to carry out the invention
An oral vaccine against a bacterial infectious disease according to the present invention in the form of a capsule formulation. In this document, a capsule containing its contents is known as a "capsule formulation." The capsule formulation according to the present invention includes a capsule membrane and a transformed microorganism that expresses a flagellin antigen protein, in which the capsule membrane is acid resistant. The capsule formulation includes an acid resistant capsule membrane and a transformed microorganism that expresses a flagellin antigen protein that can have any configuration and any shape, as long as this capsule formulation has a capsule membrane resistant to acidic and contains a transformed microorganism that expresses a flagelin antigen protein as the capsule content, without excluding the formulation that also includes an additional constituent element. Accordingly, the transformed microorganism that expresses the flagelin antigen protein is encapsulated in or envelops the membrane of the acid-resistant capsule (i.e., contained within the capsule formed by the acid-resistant membrane). In this document, this capsule formulation is also known as an "acid resistant capsule formulation".
Hereinafter, the acquisition of a gene for flagellin (flagellin gene), the preparation of a vector for

expressing flagelin (flagelin expression vector), the preparation of a transformed microorganism that expresses flagelin, and the production of an acid-resistant capsule formulation containing the transformed microorganism for the preparation of an oral vaccine, and an oral vaccine against a bacterial infectious disease will be described sequentially in the following sections.
1. Acquisition of Flagelin Gene
A gene that encodes flagelin is available based on known gene sequences. A gene that encodes flagelin can be acquired, for example, by performing amplification through a polymerase chain reaction (PCR) using genomic DNA or cDNA prepared from infectious pathogenic bacteria (e.g., bacteria that cause salmonella, cholera or dysentery) as a template with a pair of primers prepared based on the sequence information of the bacterial flagellin gene of the bacterium.
A gene that encodes the flagelin of typhoid fever is available based on the sequence of the flagelin structural gene of S. typhimurium described by M. McClelland et al., In Nature, vol. 413, p. 852 (2001). For example, the gene can be acquired by performing amplification through a polymerase chain reaction (PCR) using S. typhimurium chromosomal DNA or cDNA as a template with the sequences of SEQ ID NOs: 1 and 2 Like a couple of primers.
A gene encoding cholera flagellin is available based on the structural flagelin gene of Vibrio cholerae described by Heiderberg et al., In Nature, vol. 406, p. 477 (2,000). For example, the gene can be acquired by performing amplification through PCR using the DNA or cDNA chromosome of V. cholerae as a template with the sequences of SEQ ID NOs: 3 and 4 as a pair of primers.
A gene that encodes dysentery flagelin is available on the basis of the structural flagellin gene of Shigella dysenteriae described by Tominaga A. et al., In Genes Genet. Syst., Vol. 76, p. 111 (2001). For example, the gene can be acquired by performing amplification through PCR, using the DNA dynasty or DNA dynasteriae as a template, with the sequences of SEQ ID NOs: 5 and 6 in the sequence list Like a couple of primers.
2. Preparation of flagelin expression vector
The flagelin gene prepared as in section 1 above is incorporated into a plasmid to prepare an expression vector. There is no particular limitation on the plasmid used to prepare an expression vector, while a plasmid can effect expression in intestinal bacteria. A plasmid derived from a microorganism belonging to the genus Bifidobacterium (for example, pTB4, pTB6, pTB10, pBL67 or pBL78), a plasmid derived from a microorganism belonging to the genus Streptococcus (for example, plasmid pC194), and the like are used. In addition, these plasmids can be complexed with an Escherichia coli plasmid (see Open Japanese Patent Publication No. 5-130876, for example).
In view of the stable expression and ease of DNA preparation for the preparation of a transformed strain, a complex plasmid of a Bifidobacterium longum plasmid (B. longum) with an Escherichia coli plasmid is preferable among the plasmids described above.
In view of selection for a transformed strain, the expression vector preferably has a selectable marker such as antibiotic resistance, auxotrophy, or the like.
The expression vector preferably has a control sequence to express or advantageously express flagelin. Examples of the control sequence include promoter sequences, leader sequences, propeptide sequences, enhancer sequences, signal sequences, termination sequences, and the like. There is no particular limitation on the origin of the control sequence, as long as the expression is made in intestinal bacteria. There is no particular limitation in the promoter sequence, as long as it effects expression in intestinal bacteria.
There is no particular limitation in a promoter sequence, as long as it effects the expression of the intestinal bacteria. In view of efficient expression, a promoter sequence of a histone-like protein (HU) (hereinafter, may be referred to as a "HUquot;) promoter of B. longum is preferably used. For example, a HU promoter gene can be obtained by amplifying and recovering the sequence of nucleotide positions 1 to 192 in the HU genes of SEQ ID Nos: 9 and 10 (Biosci. Biotechnol. Biochem. 66 (3), 598-603 (2002)), using the B. longum DNA or cDNA chromosome as a template with the sequences of SEQ ID 7 and 8 N0S in the sequence list as a pair of primers. To facilitate incorporation into a plasmid, an appropriate restriction enzyme site may be included in a primer sequence (HindIII for SEQ ID No. 7, NcoI for SEQ ID No. 8).

In addition, in view of improving expression efficiency, a terminator sequence is preferably included. As the terminator sequence, the terminator sequence of the HU gene is preferably used, which corresponds to a base sequence at positions 475 to 600 of SEQ ID NO: 9.
In addition to the above, a leader sequence, a propeptide sequence, an enhancer sequence, a signal sequence, and the like may be arranged as necessary. For example, it is preferable to contain a leader sequence and a signal sequence for secretion so that flagellin can be secreted outside the cell of the microorganism.
Thus, control sequences, such as a promoter sequence and terminator sequence, and a selectable marker gene are incorporated into the plasmid as necessary, to prepare a cloning vector. For example, a linker having a multi-cloning site is preferably disposed downstream of the cloning vector promoter. When using such a linker, a gene (DNA) encoding the flagellin is incorporated downstream of the promoter so that the flagellin can be expressed in frame.
Examples of the plasmid for a cloning vector include pBLES100, pBLEM100, and the like. FIG. 1 shows a schematic view of the structure of pBLES 100. Plasmid pBLES100 includes the Escherichia coli vector derived from pBR322, PstI-EcoRI fragment and PstI-HindIII fragment (in total 4.4 kbp; part of the line in FIG 1), PstI-PstI fragment of B. longum vector derived from pTB6 (3.6 kbp: black band portion in FIG. 1), and a region encoding Enterococcus faecalis (SpR) spectinomycin adenyltransferase (SpR) ( 1.1 kbp: arrow drawn in FIG. 1).
For example, a plasmid pBLES100 is prepared as follows. pTB6, which is a plasmid derived from B. longum, was cleaved with PstI, and inserted into the PstI site of Escherichia coli of cloning vector pBR322 (manufactured by Takara Bio Inc.). In addition, a region of HindIII-EcoRI fragment encoding SpR of Enterococcus faecalis is inserted into the EcoRI-HindIII site of pBR322.
The fragments acquired for promoter sequence and the HTJ flagellin gene (hereinafter, may be referred to as an "F1iCquot gene;) are incorporated into the frame in this plasmid pBLES 100 to prepare a vector that expresses flagelin. More specifically, the flagelin gene fragment that is prepared by PCR amplification is performed using S. typhimurium chromosomal DNA as a template with the sequence of SEQ ID NO: 1, which has the NcoI cleavage site and the sequence of SEQ ID NO: 2 having the BamHI cleavage site as a pair of primers, and the amplified fragment is cleaved with NcoI and BamIII. The HU promoter fragment is prepared because PCR amplification is performed using B. longum chromosomal DNA as a template with a primer of SEQ ID NO: 7 having the HindIII site and a primer of SEQ ID NO: 8 which It has the NcoI site as a pair of primers, and the amplified fragment is cleaved with HindIII and NcoI. These fragments were ligated to pBLES100 cleaved with HindIII and BamHI. Therefore, a pBLES-F1iC flagelin expression vector is obtained in which the salmonella flagelin gene ("flagelinaquot; in FIG. 2) is incorporated downstream of the HU promoter gene" (hupPquot; FIG. 2). FIG. 2 shows this expression vector pBLES-F1iC. The flagelin expression vector thus obtained is used for the transformation of intestinal bacteria.
For secretory expression outside the cell of the microorganism, a vector can be used that is made by incorporating fragments for the secretion gene of the signal peptide and for the flagellin gene (F1iC gene) under the plasmid pBLES100. More specifically, the flagelin gene fragment that is prepared by PCR amplification is performed using S. typhimurium chromosomal DNA as a template with the sequence of SEQ ID NO: 1 having the NcoI cleavage site and the sequence of SEQ ID NO: 2 having the BamHI cleavage site as a pair of primers, and the amplified fragment is cleaved with NcoI and BamHI. The gene fragment of the secretion signal peptide is prepared because PCR amplification is performed using B. bifidum chromosomal DNA as a template with a SEQ ED 15 NO: 11 primer having the HindIII site and a SEQ ID primer NO: 12 which has the NcoI site as a pair of primers, and the amplified fragment is cleaved with HindIII and NcoI. These fragments are combined with pBLES 100 cleaved with BamHI and HindIII. Therefore, a pBLESSP-FliC flagelin secretory expression vector is obtained in which the salmonella flagelin gene is incorporated downstream of the secretion signal peptide gene fragment. The flagelin expression vector thus obtained is used for the transformation of intestinal bacteria.
3. Preparation of transformed flagellin expression microorganism
There is no particular limitation on the host microorganism in which flagelin is expressed, as long as the bacteria is viable in the large intestine and small intestine of humans or animals (intestinal bacteria). When the host bacterium grows in the intestine, flagelin is expressed. The expressed flagelin exerts antigenicity, by which an antibody is induced. Any viable bacteria in the intestine (i.e. intestinal bacteria), commonly called good bacteria, such as bifidobacteria or lactic acid bacteria can be used favorably.
Preferable examples of the microorganism include microorganisms belonging to the genus Bifidobacterium, the genus Lactobacillus, the genus Lactococcus, the genus Pediococcus, the genus Streptococcus, the genus

Enterococcus, the genus Leuconostoc, the genus Tetragenococcus, the genus Oenococcus, and the genus Weisseilla (also collectively referred to as `` lactic acid bacteria '').
Examples of microorganisms that belong to the genus Bifidobacterium (also collectively referred to as `` bifidobacteriaquot;) include Bifidobacterium adolescentis, B. angulatum, B. animalis subsp. animalis, B. animalis subsp. lactis, B. asteroides, B. bifidum, B. boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B. cuniculi
B. denticolens, B. dentium, B. gallicum, B. gallinarum, B. balloonsum, B. indicum, B. infantis, B. inopinatum, B. lactis,
B. longum, B. magnum, B. merycicum, B. minimum, B. parvulorum, B. pseudocatenulatum, B. pseudolongum subsp. Globesum, B. pseudolongum subsp. pseudolongum, B. puilorum, B. ruminale, B. ruminantium, B. saeculare, B. scardovii B. subtile, B. suis, B. thermacidophilum, and B. thermophilum.
Of these, Bifidobacterium adolescentis, B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. lactis, B. longum, and B. pseudolongum. Pseudolongum subsp are preferably used.
Examples of microorganisms that belong to the genus Lactobacillus include Lactobacillus acidophilus, L. amylovorus, L. animalis, L. brevis, L. brevis subsp. gravesensis, L. L. buchneri, L. bulgaricus, L. casei, L. casei subsp. casei, L. casei subsp. plantarum, L. casei subsp. tolerans, L. cellobiosus, L. curvatus, L. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. divergens, L. fermentum, L. fructosus, L. gasseri L. hilgardii L. kefir, L. leichmannii L. paracasei, L. paracasei subsp. paracasei, L. pentosus, L. plantarum, L. reuteri, L. rhamnosus, L., L. sakei, L. sakei subsp. sakei, L. sanfrancisco, L. vaccinostrcus, Lactobacillus sp.
Examples of microorganisms belonging to the genus Lactococcus include Lactococcus garvieae, L. lactis, L. lactis subsp. hordniae, L. lactis subsp. lactis, L. piantarum, and L. raffinolactis.
Examples of microorganisms belonging to the genus Pediococcus include Pediococcus pentosaceus and P. acidilactici.
Examples of microorganisms belonging to the genus Streptococcus include Streptococcus bovis, S. cremoris, S. faecalis, S. lactis, S. pyogenes and S. thermophilus.
Examples of microorganisms belonging to the genus Enterococcus include Enterococcus casseliflavus and E. faecalis.
Examples of microorganisms belonging to the genus Leuconostoc include Leuconostoc citreum, Leuconostoc mesenteroides, L. mesenteroides subsp. mesenteroides and L. mesenteroides subsp. dextranicum.
Examples of microorganisms belonging to the genus Tetragenococcus include Tetragenococcus halophilus and T muriaticus.
Examples of microorganisms belonging to the genus Oenococcus include Oenococcus oeni.
Examples of microorganisms belonging to the Weissella genus include Weissella viridescens.
There is no particular limitation on the method for introducing a flagelin expression vector into intestinal bacteria, and methods commonly used by those skilled in the art can be used. Examples thereof include electroporation methods; Calcium phosphate; lipofection; the use of calcium ions; protoplast; and the like Electroporation is preferably used. Electroporation can be performed in 0.5 to 20 kV / cm and 0.5 µsec at 10 msec, more preferably 2 to 10 kV / cm and 50 µsec at 5 msec.
A transformed strain is selected with a selectable marker contained in the flagellin expression vector. A medium for culturing the transformed strain can be any medium suitable for the host microorganism. Examples of the medium include blood liver agar medium (BL), Man-Rogosa-Sharpe agar medium (MRS), Gifu anaerobic medium agar medium (GAM), GAM enhancement agar medium (TGAM), a Briggs agar medium and a yeast glucose peptone agar (YGP) medium. For selection pressure, antibiotics can be added to the medium, or amino acids can be suppressed or added to the medium, depending on the selectable marker.
Flagelin expression in a transformed microorganism can be confirmed, for example, using Western blotting. The expression of flagelin can be confirmed by: First, the transformed microorganism was lysed, for example, using a non-ionic surfactant, including polyoxyethylene sorbitan ester (Tween (registered trademark) 20, 40, 60, 65, 80, 85), and sorbitan ester (Span (registered trademark) 20, 40, 60, 65, 80, 85), and the like; then diluted with phosphate buffer, citrate buffer, borate buffer, tris (hydroxymethyl) aminomethane (Ts) -hydrochloride buffer, or the like; then it was electrophoresed with sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), tris-glycine-polyacrylamide gel, or the like; it was then transferred to the nitrocellulose membrane, polyvinylidene fluoride (PVF) of the membrane, or the like; and then I know

reacts with an antibody (immunoglobulin G (IgG)) against flagelin, and further reacts with a secondary antibody with a fluorescent label. For the secretory expression of flagelin by a transformed microorganism, which can be confirmed after selection for the transformed strain, a supernatant is obtained through centrifugal separation and subjected to Western Blot as described above.
The transformed microorganism in which flagelin expression has been confirmed can be cultured, recovered, and used directly for the production of a formulation, using any method commonly used by those skilled in the art. Alternatively, the transformed microorganism can be used in a dry form. The transformed microorganism can be dried by the treatment in which a low temperature treatment is performed, such as lyophilization or low temperature drying, so that the microorganism can grow when exposed to growth conditions such as those of an intestinal environment or a medium.
4. The production of an acid-resistant capsule formulation that contains the transformed microorganism.
In order to allow the transformed microorganism that expresses a flagellin protein to act as an oral vaccine, the transformed microorganism has to pass through the stomach, reach the intestine, and grow in it. However, intestinal bacteria ingested mostly by mouth, such as lactic acid bacteria, die due to significantly lower stomach pH, the pH from 1 to 3. Usually, the ratio of intestinal bacteria they reach is said to reach The intestine while maintaining its ability to proliferate is 10,000 or less of the amount of bacteria administered. Consequently, in order to use the transformed microorganism according to the present invention, it is necessary to prevent the transformed microorganism from being affected by gastric acid so that the transformed microorganism can reach the living human intestine and grow in the intestine to express flagelin .
Therefore, the present invention provides a capsule formulation that the transformed microorganism is encapsulated with or wrapped in an acid-resistant capsule membrane, in the other word, the pharmacist is in the form of a capsule formulation in which The transformed microorganism is contained within a capsule that has an acid resistant membrane. There is no particular limitation on the configuration, shape, or the like of the capsule formulation, as long as the membrane is resistant to gastric acid. Specifically, the configuration is desirable that prevents gastric acid from entering the capsule and in contact with the transformed microorganism. The capsule membrane may be an insoluble membrane at a pH of 4 or less, preferably at a pH of 1 to 3. There is no particular limitation on the method for encapsulation.
Seamless capsule formulation
The capsule to provide with resistance to gastric acid may preferably be in the form of a seamless capsule. In this document, the "capsule without seams"; It refers to a type of soft capsule in which the contents are wrapped in a seamless membrane. The seamless capsule may have a multilayer structure consisting of two or more layers, and preferably has a multilayer structure consisting of three or more layers. Typically, a more internal layer may contain the contents (the microorganism being transformed in the case of the present invention), and an external layer (or the outermost layer) may act as a membrane. Specifically, the transformed microorganism is encapsulated with the membrane.
Hereinafter, the preparation of a three-layer transparent capsule formulation will be described. FIG. 3 is a schematic view in a cross-sectional view of a three-layer transparent capsule formulation. This three layer structure consists of a more internal layer, an intermediate layer that covers the internal layer, and an external layer that covers the intermediate layer.
The innermost layer includes the transformed microorganism and a non-aqueous solvent or solid component to suspend or mix the transformed microorganism (hereafter referred to as a quot; more internal layer substance "). There is no particular limitation on the innermost layer substance. Examples thereof include various fats and oils, fatty acids, sugar fatty acid esters, aliphatic hydrocarbons, aromatic hydrocarbons, linear ethers, higher fatty acid esters, higher alcohols, and terpenes. Specific examples thereof include, but are not limited to, soybean oil, sesame oil, palm oil, palm kernel oil, corn oil, cottonseed oil, coconut oil, rapeseed oil, butter of cocoa, beef tallow, lard, oil horses, whale oil, fat and oils of these natural fats and oils have a melting point of 40 ° C or less, margarine, hydrogenated butter, esters of fatty acid glycerin, sucrose fatty acid esters, camphor oils, peppermint oil, a-pinene, D-limonene, and the like. These more inner layer substances can be used alone or in a combination of two or more.
A material used for the intermediate layer is, among the innermost layer substances listed above, a material that has a melting point of 20 ° C to 50 ° C and different from the layer substance plus

internal, more preferably a material that is in a solid state at room temperature. As, in the examples set forth below, hydrogenated palm kernel oil having a melting point of 34 ° C and hydrogenated palm kernel oil having a melting point of 43 ° C are used as the most layered substance. internal and the inner layer material, respectively, the same species of grease and oils can be used as the innermost layer substance and the inner layer material, which was subjected to hydrogenation in order to have different melting points. This intermediate layer can act as the prevention of water and oxygen permeation and the prevention of contact with gastric acid. The material to be selected can be determined in consideration of the storage duration of the capsule and the like.
A material used for the outer layer (the outermost layer in the case of a structure having three or more layers) can be a mixture of a protein and a water-soluble polyhydric alcohol; a mixture of a protein, a water-soluble polyvalent alcohol, and a polysaccharide; a mixture of a polysaccharide and a water-soluble polyvalent alcohol; or similar. Examples of the protein include gelatin and collagen. Examples of the water-soluble polyvalent alcohol include sorbitol, mannitol, glycerin, propylene glycol, and polyethylene glycol. Examples of polysaccharide include agar, gellan gum, xanthan gum, locust bean gum, pectin, alginate, carrageenan, gum arabic, dextrin, modified dextrin, starch, modified starch, pululane, pectin, and the carboxymethyl cellulose salt. In the case where pectin, alginate, gellan gum, or carrageenan is used, an alkali metal salt or an alkaline earth metal salt may be added as appropriate.
The transparent three-layer capsule formulation is prepared using any technique known to those skilled in the art, such as the drip method using a triple nozzle described in Japanese Patent No. 1398836. In this drip method, the layer substance plus internally combined with the transformed microorganism (for example, the cells of the microorganism were freeze dried), which is preferably a suspension of the transformed microorganism (preferably, lyophilized cells of the microorganism) into a hydrophobic solvent material that is non-fluid at 20 to 50 ° C, from the innermost nozzle of the concentric triple nozzle, an intermediate layer forming material (for example, a liquid obtained by melting a material in the form of a solid at room temperature) of the intermediate nozzle and a solution of a material that forms the outer layer (membrane) of the outermost nozzle is ejected simul tartar, and dropped into a carrier liquid (eg, corn oil, rapeseed oil, or the like), which flows under cooling, thus forming a capsule "without seams"; of three layers in the transformed microorganism is contained in the innermost layer. Consequently, the transformed microorganism is encapsulated with or wrapped in the membrane without seams.
The capsule formed in this way is then dried. For example, drying is done by ventilation at room temperature. Typically, the capsule is dried, for example, in the air at 5 ° C to 30 ° C. The drying time is preferably from 2 to 12 hours. As described in Japanese Open Patent Publication No. 07-069867, a capsule that has been dried ordinarily as described above may further be preferably subjected to vacuum or vacuum freeze-drying. The degree of vacuum can be maintained at 0.5 to 0.02 torr. The capsule can be frozen and dried at -20 ° C or lower in the case of vacuum freeze drying. There is no particular limitation on the time of vacuum drying or vacuum freeze drying, but it is typically 5 to 60 hours, preferably 24 to 48 hours. If the time is shorter than 5 hours, drying is insufficient and the water present in the capsule can adversely affect the content.
In the case of a capsule obtained using the method described in Japanese Open Patent Publication No. 07069867, the water is sufficiently removed from the capsule by vacuum freeze drying, and therefore the Aw value can be 0.20 or less, and the heat conductivity can be 0.16 kcallmh ° C or less. By vacuum drying or vacuum freeze drying, the amount of water is naturally reduced, while the capsule is sufficiently dry and becomes porous. Therefore, the thermal conductivity is significantly lower than in the case where it is performed simply by ordinary drying.
The Aw value does not refer to an absolute water content present in the sample, but to a value determined by the state in which the water is present, that is, the degrees of freedom for water in the sample. The Aw value is an indicator that indicates water that can directly affect the chemical reaction or the growth of microorganisms, and is measured using a method of measuring water resistance activity (for example, Aw WA-360 counter, Shibaura Electronics Co., Ltd.). Thermal conductivity is measured using the Fitch method or similar. The Aw value is preferably 0.20 or less, and the thermal conductivity is preferably 0.02 to 0.08 kcal / mh.
In order to provide the capsule membrane of the acid-resistant transparent capsule formulation, an acid-resistant outer layer is formed or the membrane (the outermost layer) of the transparent capsule prepared in a manner is treated. It is resistant to acids.
Examples of the method for the formation of an acid resistant outer layer include the addition of pectin, alginate, gum arabic, or the like in an amount of 0.01 to 20% by weight, preferably 0.1 to 10% by weight to gelatin, agar, carrageenan, or the like, which has a gelling capacity. Examples of the method for providing the membrane (the outermost layer) of the seamless capsule prepared with acid resistance include crosslinking the outer layer (the outermost layer) of the capsule

transparent and coating the surface of the capsule without seam, which can be done alone or in combination.
For the cross-linking of the outer layer containing a protein, the transparent capsule is prepared first, and then sufficiently washed with water and then the transparent water-washed capsule is added to an aqueous solution containing a cross-linking agent. Therefore, the surface of the outer layer is subjected to a crosslinking treatment. As a crosslinking agent, conventionally known crosslinking agents can be used. Examples of crosslinking agents include formaldehyde, acetaldehyde, propionaldehyde, glyoxal, glutaraldehyde, cinnamaldehyde, vanylyl aldehyde, acetone, methyl ethyl ketone, ethylene oxide, propylene oxide, potassium alum, and ammonium alum. Typically, the outer layer is treated by adding 1 part by weight of the seamless capsule to 50 to 100 parts by weight of aqueous solution containing 0.1 to 2 w / v%, preferably 0.5 to 2 w / v%, of a crosslinking agent, and stirring the mixture for 10 to 300 seconds. Here, the amount of crosslinking agent used and the period of time for action will vary depending on the type of crosslinking agent. After the surface of the outer membrane undergoes the crosslinking treatment, the outer membrane is sufficiently washed with water to remove the aqueous solution containing the crosslinking agent, and the water in the outer layer is dried.
For crosslinking of the outer layer containing proteins, crosslinking can be performed through enzymatic treatment with transglutaminase. In this case, the outer layer is treated by adding 1 part by weight of transparent capsule produced at 50 to 100 parts by weight of aqueous solution containing 0.1 to 10 w / v%, preferably 0.5 to 2 w / v%, of enzyme, and the mixture is stirred for 1 to 300 minutes. The result is washed with water and dried as described above.
For the coating, after the wet transparent capsule produced has dried, the transparent capsule is conventionally coated with shellac, ethyl cellulose, ropylmethyl cellulose hydroxy, hydroxypropyl cellulose, polyvinyl pyrrolidone, cellulose TC-5, vinyl vinyl acetate pyrrolidone copolymer, zein, wax of ethylene, or the like as the base material, and castor oil, rapeseed oil, dibutyl phthalate, polyethylene glycol, glycerin, stearic acid, fatty acid ester, sorbitan pairnitate, polyoxyethylene stearate, acetylated monoglyceride, or the like the plasticizer
The capsule membrane may also be provided with entericity. In this way, the capsule is protected from an acid solution and the like (such as gastric acid) in the stomach, and disintegrates in the intestine so that the transformed microorganism is released from inside the capsule to sufficiently produce of antigen in the intestine. The capsule membrane may be provided with entericity by producing an enteric capsule as commonly practiced by those skilled in the art. A mixture of gelatin and pectin can be used as the material of the outer layer of the seamless capsule to convert the membrane into enteric. The acid-resistant outer layer is also provided with entericity by the preparation by adding pectin, alginate, gum arabic, or the like in an amount of 0.01 to 20% by weight, preferably 0.1 to 10% in Gelatin weight, agar, carrageenan, or the like, which has a gelling capacity.
The transparent capsule formulation may be in the form of a sphere due to the production method. The average particle size of the seamless capsule is 0.3 to 10 mm, preferably 1.5 to 8.0 mm.
The transparent capsule formulation obtained in this way can be stored for six months or more while maintaining the activity of the transformed microorganism at room temperature. If the formulation is stored at 10 ° C or less, extended storage for a year or more is possible.
Soft capsule formulation
As in the case of seamless capsule formulation, a soft capsule formulation may be the encapsulation of a suspension of the microorganism transformed into a non-aqueous solvent (such as the contents of the capsule) with a membrane sheet. The membrane sheet material is as mentioned for the outer layer of the capsule without seams.
A soft capsule formulation can be prepared using any known method, for example, as described in Japanese Patent No. 2999535. For example, using a rotating matrix, while the contents are injected, and filled, the membrane sheet is heats through the matrix, in order to wrap and encapsulate the contents. For the action of releasing the transformed microorganism in the intestine, an oil, which is a releasing agent, is removed from the resulting soft capsule by washing with a polar solvent (for example, methanol, ethanol, propanol or isopropanol). Subsequently, the capsule can be made resistant to acids by performing a cross-linking treatment and coating treatment in combination, or performing any one of the treatments, as in the case of the seamless capsule.
The acid resistant membrane sheet can also be prepared on the basis of any of the known methods such as by adding pectin, alginate, gum arabic, or the like in an amount of 0.01 to 20% by weight, preferably of 0.1 to 10% by weight to gelatin, agar, carrageenan, or the like, which has a

gelling capacity Alternatively, the membrane sheet can be made resistant to acids, by performing cross-linking treatment and coating treatment in combination, or performing any one of the treatments. The acid-resistant membrane sheet thus obtained can be used to produce a soft capsule formulation in which the transformed microorganism is encapsulated with the acid-resistant membrane. For example, from the sheet obtained from an acid-resistant membrane, the shape of a capsule is obtained, the contents are introduced into the capsule, and then a seam of the capsule is melted and joined in order to wrap the contents , using known techniques.
The soft capsule formulation may be in the form of a sphere, an ellipse, or a rectangle. The soft capsule preferably has a major axis of 3 to 16 mm and a minor axis of 2 to 10 mm, and more preferably has a major axis of 5 to 7 mm and an axis of 2 to 3 mm.
The soft capsule formulation obtained in this way can be stored for six months or more while maintaining the activity of the transformed microorganism at room temperature. If the formulation is stored at 10 ° C or less, prolonged storage for a year or more is possible.
Hard capsule formulation
A hard capsule formulation can be produced by molding a capsule membrane in a body and a cap beforehand, filling the body of the capsule with the contents, and combining the resultant with the capsule lid.
Examples of the membrane material of the hard capsule formulation include gelatin, cellulose, pululane, carrageenan, and cellulose derivatives such as hydroxypropyl methylcellulose. The hard capsule can be molded using any of the methods commonly used by those skilled in the art. The molded capsule may consist of commercially available capsules. The content can be encompassed and wrapped in the membrane.
The contents may be a mixture obtained by sufficient mixing of the microorganism transformed with a vehicle (for example, silicic anhydride, synthetic aluminum silicate, lactose, corn starch, or crystalline cellulose), or powders containing dry powders of the transformed microorganism.
After the contents are stored in the capsule, the membrane of the capsule may be coated. For this coating, the materials and methods that have been mentioned for the outer layer of the capsule without seams can be applied to provide the membrane with acid resistance and preferably disintegration in the intestine (entericity). This coating also allows the capsule membrane to be sealed in order to encapsulate the contents.
The acid-resistant membrane sheet can also be prepared based on any of the known methods, such as through the addition of pectin, alginate, gum arabic, or the like in an amount of 0.01 to 20% by weight, preferably from 0.1 to 10% by weight to gelatin, agar, carrageenan, or the like, which has a gelation capacity. Alternatively, the membrane sheet can be made resistant to acids, by performing cross-linking treatment and coating treatment in combination, or performing any one of the treatments. The acid-resistant membrane sheet thus obtained can be used to produce a hard capsule formulation in which the transformed microorganism is encapsulated by the acid-resistant membrane. For example, from the sheet obtained from an acid-resistant membrane, the shape of a hard capsule is obtained, the contents are introduced into the hard capsule formed, and then a seam of the capsule melts and joins in order to wrap the contents, using a known technique.
The hard capsule formulation thus obtained can be stored for six months or more, while maintaining the activity of the transformed microorganism at room temperature. If the formulation is stored at 10 ° C or less, extended storage for a year or more is possible.
5. Oral vaccine against a bacterial infectious disease
After oral administration, the acid-resistant capsule formulation (the seamless capsule formulation, the soft capsule formulation, and the hard capsule formulation) obtained as explained in Section 4 described above passes through of the stomach, having a pH of 1 to 3, reaches the intestine and then disintegrates in the intestine. The transformed microorganism is released through the disintegration of the formulation, grows, produces, and preferably secretes flagelin out of the microorganism cell in an intestinal environment. Flagelin is then recognized as an antigen to produce an antibody. Accordingly, the acid-resistant capsule formulation can be an effective oral vaccine against a microorganism that has flagelin.
Examples

Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.
Example 1: Preparation of the formulation of the acid-resistant capsule containing typhoid fever antigen that produces bifidobacteria
A. The amplification of the S. Typhimurium flagellin gene through PCR.
S. typhimurium ATCC 14028 was grown in LB medium (manufactured by Invitrogen) at 37 ° C for 12 hours. After the culture was finished, genomic DNA was conventionally extracted from S. typhimurium. The extracted genomic DNA was amplified using a kit for the PCR reaction (manufactured by Applied Biosystems) with Ampli Taq DNA polymerase (0.5 units) according to the instruction. As a pair of primers, the following were used: SEQ ID NO: 1 (forward): 5'-CATGCCATGGATGGCACAGTCATTAATACA-3 '(CCATGG at positions 5 to 10 is the Ncol cleavage site), and SEQ ID No: 2 ( reverse): 5'-CGCGGATCCTTAACGCAGTAAAGAGAGGAC-3 '(GATCCT at positions 5 to 10 is the BamHI cleavage site). The PCR was performed using 40 µL of reaction liquid containing 125 ng of DNA template, 0.5 µmol of each primer, 2.5 units of Pfu of DNA polymerase, 4 µL of x10 buffer solution for polymerase of Pfu DNA, and 200 µmol of each dNTP, under 30 cycles at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 1 minute, and then at 72 ° C for 10 minutes. After completion of the PCR, the resulting product was cleaved with NcoI and BamHI. Therefore, the flagellin gene fragment was prepared.
B. Amplification of the HU promoter through PCR
B. The ATCC 15703 longum strain was grown in MRS medium (manufactured by Nippon Becton Dickinson Company, Ltd.) at 37 ° C for 12 hours. After the culture was finished, genomic DNA was conventionally extracted from B. longum. The PCR was performed as described in Section A above. As a pair of primers, the following were used: SEQ ID No: 7 (forward): 5'-CGCCAAGCTTTGGGCGCGGCGGCCATGAAG-3 '(AAGCTT at positions 5 to 10 is the HindIII cleavage site), and SEQ ID NO: 8 ( reverse): 5'-CGCGCCATGGAAAGCATCCTTCTTGGGTCA-3 '(CCATGG at positions 5 to 10 is the NcoI cleavage site). After completion of the PCR, the resulting product was cleaved with HindIII and NcoI. Thus, the fragment for the HU promoter gene was prepared.
C. Preparation of the expression vector
Plasmid pBLES100 was cleaved with BamHI and Hindili, and combined with and ligated to the salmonella flagelin gene fragment prepared in Section A as described above and the HU promoter gene fragment prepared in Section B described above. Thus, an expression vector pBLES-F1iC was obtained.
D. Introduction of the expression vector in B. Animalis
B. animalis ATCC 27536 was inoculated in the MRS medium, and cultured until the semi-logarithmic growth phase standing at 37 ° C for 12 hours under the nitrogen atmosphere containing 10% carbon dioxide. The resulting culture was centrifuged, and the microorganism cells were collected and washed three times with PBS (obtained by diluting 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0, 24 g of potassium dihydrogen phosphate with 1 L of distilled water, and adjusting the pH to 7.4). Then, PBS was added at 5x108 cells / mL in order to obtain a suspension of B. animalis. Then, 5 µL (1 µg of DNA / 5 µL) of pBLES-F1iC prepared in Section C described above was added to 50 µL of this suspension, and the resulting one is placed in a 0.2 cm wide electroporation cuvette and it was treated under the conditions of 5 µs,
1,000 V for the transformation.
The culture is carried out in a spectinomycin (50 µg / ml) containing BL agar medium (manufactured by Nissui Pharmaceutical Co., Ltd) at 37 ° C under the atmosphere of nitrogen containing 10% carbon dioxide. Thus, B. animalis transformed was obtained.
E. Western Blotting
Whether or not transformed B. animalis is expressed a flagelin protein was confirmed as follows. B Animalis was diluted with a phosphate buffer (pH 6.8) containing 1 w / v% of Tween (registered trademark) 80 and a solution of buffer A (126 mM Tris hydrochloride, 20 w / v% glycerin, 4 w / v% sodium dodecyl sulfate, 1.0 w / v% 2-mercaptoethanol, 0.05 w / v% bromophenol blue, pH 6.8). Then, 5 µg of the resulting was subjected to electrophoresis (polyacrylamide-tris glycine gel), and then subjected to electrotransfer to transfer resolved proteins to a nitrocellulose membrane, and then subjected to ELISA with specific IgG1 (manufactured by ViroStat) for flagelin common to Salmonella species and secondary horseradish peroxidase antibody (HRP) (1: 500). Therefore, flagelin expression was confirmed.
F. Preparation of lyophilized microorganism powder of the transformed microorganism

First, 2 platinum loops of the B. animalis transformed were inoculated into 1 L of MRS medium (manufactured by Nippon Becto Dickinson Company, Ltd.) containing 50 µM of spectinomycin, and grown at 37 ° C for 18 hours with the injection of nitrogen gas containing 10% carbon dioxide. The pH was adjusted to 5.5 with 10M of aqueous sodium hydroxide solution by an automatic pH adjuster to avoid lowering the pH during cultivation. After cultivation, for 15 hours, the cells were properly diluted with an anaerobic diluent, applied to the BL agar medium containing 50 µm of spectinomycin and counted for the number of viable cells in the colonies. Here, the anaerobic diluent was obtained by dissolving 6.0 g of disodium hydrogen phosphate, 4.5 g of potassium dihydrogen phosphate, 0.5 g of L-cysteine monohydroquioride, 0.5 g of Tween (registered trademark ) 80, and 1.0 g of agar in 1 L of distilled water and steam sterilizing the resultant at 121 ° C for 15 minutes.
After cultivation, the cells were collected by centrifugal separation (15,000 xg, 20 minutes), and to the cells, 120 g of distilled water, 12 g of sodium citrate and 8 g of sodium malate were added to obtain a suspension of the cells. Then, 8 g of Avicel FD-101 (manufactured by Asahi Kasei Corporation) was added to this suspension, and the resultant was sufficiently stirred, frozen, and then dried in a vacuum. Subsequently, dextrin was added in one. amount twice as much as the powders obtained. Therefore, lyophilized microorganism cell powders were obtained.
G. Preparation of acid-resistant capsule formulation
As described below, the acid-resistant seamless capsule formulation containing transformed microorganism cells was prepared using a capsule production apparatus provided with a triple coaxial nozzle.
First, 400 g of hardened oil (hydrogenated palm kernel oil having a melting point of 34 ° C) and 100 g of lyophilized microorganism cell powders obtained in section F described above were melted then it dispersed in it. As described below, the acid-resistant seamless capsule formulation containing the transformed microorganism cells was prepared using a capsule production apparatus provided with a triple coaxial nozzle. First, 400 g of hardened oil (hydrogenated palm kernel oil having a melting point of 34 ° C) and 100 g of the lyophilized microorganism cell powders obtained in section F were melted, described above. It disperses in it. This dispersion of the innermost nozzle of the concentric triple nozzle, a molten hardened oil (palm hydrogenated almond oil having a melting point of 43 ° C) of the intermediate nozzle located on the outer side of the innermost nozzle , and a solution of gelatin (obtained by dissolving 600 g of gelatin, 300 g of glycerin and 100 g of pectin in 4 kg of purified water) from the outermost nozzle expelled simultaneously, and dripped in rapeseed oil, which flows under cooling to 15 ° C, thereby forming a formulation in which the transformed microorganism cells are encapsulated in a transparent three-layer capsule with a diameter of 2.5 mm. This capsule formulation was dried by ventilation at 20 ° C for 10 hours, and then dried under vacuum at room temperature. Therefore, the value of the Aw water activity was reduced to 0.20 or less and the heat conductivity was reduced to 0.16 kmal / mh ° C or less in the capsule.
H. Preparation of acid-resistant soft capsule formulation
First, 50 g of lyophilized microorganism cell powders obtained in Section F described above were suspended in 300 g of rapeseed oil to prepare a fluid content of a soft capsule. Then, 400 g of gelatin and 100 g of glycerin were added to 200 g of distilled water, stirred at 60 ° C, and dissolved, and the resultant is formed into a sheet, thereby obtaining a jelly membrane, which It was used as the soft capsule membrane. The gelatin membranes are sent to a space between a pair of rotary cylindrical dies, and the fluid content was expelled to a space between the gelatin membranes by a pump that moves relative to the dies, thereby forming the encapsulation .
Next, 400 g of the encapsulates were placed in a rolling granulator, and a solution obtained by dissolving 10 g of shellac and 1 g of castor oil in 400 g of mixed methanol-ethyl acetate mixed liquor (1: 1, v / v) it was sprayed over the entire surface of the soft capsules at a thickness of the coating membrane of 0.3 mm. Thus, it was obtained that 400 g of soft capsule formulations having an axis greater than 4 mm and an axis less than 3 mm, encapsulating the transformed microorganism cells, and having an acid resistant coating.
I. Preparation of acid-resistant hard capsule formulation
Lyophilized microorganism cell powders obtained in Section F described above are used as the content of a hard capsule. For the hard capsule membrane, a commercially available No. 5 capsule as defined in the Japanese Pharmacopoeia was used. The contents were filled with the capsule body, and combined with the capsule lid, thus forming the encapsulation.

Next, 100 g of the encapsulates were placed in a rolling granulator, and a solution obtained by dissolving 10 g of shellac and 1 g of castor oil in 400 g of mixed methanol-acetate mixed liquor acetate (1, v / v) was sprayed over the entire surface of the hard capsules to a coating of membrane thickness of 0.3 mm. Therefore, 100 g of hard capsule formulations that encapsulate the microorganisms of transformed cells and have an acid resistant coating.
Example 2: Preparation of the formulation of the acid-resistant capsule containing lactic acid cholera antigen producing bacteria
V. cholerae ATCC 11628, which produces a cholera antigen, was grown in a LB medium at 37 ° C for 12 hours. After completion of the culture, genomic DNA was conventionally extracted from V cholerae. The PCR was performed as in Example 1, using the genomic DNA extracted as a template with the sequences of SEQ ID NO: 3 (forward) and SEQ. ID NO: 4 (inverse) as a pair of primers. The resulting amplified fragment was recovered and cleaved with NcoI and BamHI. Therefore, the cholera flagelin gene fragment was prepared. The pBLES-FliC prepared in Example 1 was digested with NcoI and BamHI, and a large fragment was recovered. This fragment and the cholera flagellin gene fragment were linked together. Thus, cholera was obtained that expresses the vector expression antigen pBLES-Vc.
The cholera flagellin expression vector pBLESVc obtained was used to transform Lb. ATCC BAA793 plantarum to prepare a Lb. plantarum that produces the cholera antigen. Cholera antigen expression was confirmed by ELISA using the antigen antibody response as explained in Section E of Example 1.
The Lb. Plantarum in which the expression of the cholera flagelin protein was used was confirmed to prepare a lyophilized microorganism cell powder as in section F of Example 1, and a seamless capsule formulation and a soft capsule formulation and a formulation hard capsule, each of which contains the cell powders of lyophilized microorganisms, were prepared, respectively, as in sections G, H, I and of Example 1. The membranes of the resulting transparent capsule formulation, the formulation of Soft capsule and hard capsule formulation were acid resistant.
Example 3: Preparation of the formulation of the acid-resistant capsule containing dysentery antigen that produces bifidobacteria.
S. dysenteriae ATCC 29026, which produces a dysentery antigen, was grown in LB medium at 37 ° C for 12 hours. After the culture was finished, genomic DNA was conventionally extracted from S. dysenteriae. The PCR was performed as in Example 1, using the genomic DNA extracted as a template with the sequences of SEQ ID No: 5 (forward) and SEQ ID NO: 6 (reverse) as a pair of primers. The resulting amplified fragment was recovered and cleaved with NcoI and BamHI. Therefore, the dysentery flagellin gene fragment was prepared. The pBLES-F1iC prepared in Example 1 was digested with NcoI and BamHI, and a large fragment was recovered. This fragment and the dysentery flagellin gene fragment were linked together. Thus, a pBLES-Sd dysentery antigen expression vector was obtained.
The pBLES-Sd flagelin dysentery expression vector obtained was used to transform B. longum ATCC 15697 to prepare a dysentery antigen that produces B. longum. Dysentery antigen expression was confirmed by ELISA, using the antigen antibody response as explained in Section E of Example 1.
B. longum in which the expression of the dysentery flagelin protein was confirmed to be used to prepare the cell powders of lyophilized microorganisms as in section F of Example 1, and a seamless capsule formulation, a capsule formulation soft, and a hard capsule formulation, each of which contains the cell powders of lyophilized microorganisms, were prepared, respectively, as in sections G, H, I and of Example 1. The membranes of the capsule formulation without Obtained seams, soft capsule formulation, and hard capsule formulation were acid resistant.
Comparative Example 1
A seamless capsule formulation was prepared as in Example 1, except that the gelatin solution for the membrane of section G of Example 1 was changed to a material obtained by dissolving 600 g of gelatin, 300 g of glycerin and 100 g of sorbitol in 4 kg of purified water. The membrane of the pharmaceutical obtained was not resistant to acids.
Comparative Example 2
A soft capsule formulation was prepared as in Example 1, except that the coating of section H of Example 1 was not carried out in the preparation of the soft capsule. The membrane of the pharmaceutical obtained was not resistant to acids.


Comparative Example 3
A hard capsule formulation was prepared as in Example 1, except that the coating of Section I of Example 1 was not made in the preparation of the hard capsule. The membrane of the pharmaceutical obtained was not resistant to acids.
Comparative Examples 4 to 6
In Comparative Examples 4 to 6, a seamless capsule formulation, a soft capsule formulation, and
A hard capsule formulation was prepared, respectively, as explained in Comparative Examples 1 to 3, except that the microorganism was changed to the transformed flagelin cholera expression microorganism as explained in Example 2. The membranes of the formulation The obtained seamless capsule, soft capsule formulation, and hard capsule formulation were not acid resistant.
15 Comparative Examples 7 to 9
In Comparative Examples 7 to 9, a seamless capsule formulation, a soft capsule formulation, and a hard capsule formulation were prepared, respectively, as explained in Comparative Examples 1 to 3, except that the microorganism was changed to flagelin dysentery bacillus expressing microorganism
Transformed prepared as explained in Example 3. The membranes of the obtained seamless capsule formulation, soft capsule formulation, and hard capsule formulation were not acid resistant.
Example 4: The test for the induction of antibodies, resulting from the administration of transformed microorganism of flagelin protein typhoid fever (Recombinant B. animalis)
First, female BALB / c mice 8 to 12 weeks old (provided by Charles River Laboratories Japan, Inc.) were purchased for a week on a standard diet. The mice were divided into nine groups (from 5 to 7 mice per group). For three groups, the seamless capsule formulation, the soft capsule formulation, and the hard capsule formulation, which were prepared in Example 1, were administered orally separately, each of which contained the transformed microorganism of typhoid flagelin fever expression. For three other groups, the non-acid-resistant seamless capsule formulation, the soft capsule formulation, and the hard capsule formulation, which were prepared in Comparative Examples 1 to 3, respectively, are administered orally separately. , each of which contained the
35 transformed microorganism expressing flagelin typhoid fever. For another three groups, the transformed microorganism of flagelin expression (recombinant B. animalis) living cells, living cells of host B. animalis, and a phosphate buffer separately, as controls. These capsule formulations, living cells, and the like are ingested once a day for three weeks.
40 After three weeks, the amounts of serum IgA and feces were measured as follows. PBS containing flagelin antigen was added to a 96-well plate (Nunc Immunoplate Maxisorb F96, manufactured by Nalge Nunc International K.K.), and kept at 4 ° C for 16 hours for coating the plate surface. Subsequently, the PBS containing 1 w / v% bovine serum albumin was used for blocking at room temperature for 2 hours. After washing three times with PBS, a secondary antibody was added
45 (IgA, IgG, goat-derived anti-mouse IgM (manufactured by Santa Cruz Biotechnology, inc) and incubated at room temperature for three hours. After washing with PBS three times, a tertiary antibody (fluorescein isothiocyanate ( FITC) labeled with rabbit-derived anti-goat IgG (manufactured by QED Bioscience, Inc.), and incubated at room temperature for three hours. Fluorescence was measured using FluoroscanII (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) Table 1 shows the resulting fluorescence values.
50 Table 1
Administration Sample BALB / c numberDaily dose 107 cfu / dayIgA in stool (OD + Std. Error)Serum IgA (OD + Std. Error)
Example 1: seamless capsule 72.50.16 + 0.0120.40 + 0.145
Example 1: soft capsule 73.20.15 + 0.0130.38 + 0.151
Example 1: hard capsule 730.14 + 0.0140.37 + 0.120

Comparative example 1: seamless capsule 52.50.05 + 0.0110.12 + 0.038
Comparative example 2: soft capsule 53.20.06 + 0.0100.14 + 0.041
Comparative Example 3: Hard Capsule 530.06 + 0.0100.13 + 0.028
Live cells of transformed microorganism 52.50.04 + 0.0120.11 + 0.041
Live host microorganism cells 5120.02 + 0.0080.10 + 0.038
Phosphate buffer 5-0.02 + 0.0060.14 + 0.032
It was observed that, in the cases of the acid-resistant seamless capsule, soft capsule formulations and
25 of hard capsules prepared in Example 1, regardless of the form of the acid-resistant capsule formulation, the amounts of IgA were larger in both feces and blood and the effect of inducing an antibody was greater, compared with the cases of non-acid resistant capsule formulations of Comparative Examples 1 to 3 or of living cells.
Example 5: Test for induction of antibodies resulting from transformed microorganisms of flagelin cholera administration
The seamless capsule formulation, the soft capsule formulation, and the hard capsule formulation, which were prepared in Example 2, and the capsule formulations of Comparative Examples 4 to 6 were examined.
35 for the induction of antibodies as in Example 4, each of which contained the transformed flagelin cholera microorganism expressing cells (recombinant Lb. plantarum).
In addition, the living cells of the transformed microorganism that expresses flagelin cholera, harbor live Lb cells. plantarum, and a phosphate buffer that was used for control administrations. Table 2 shows the results.
Table 2
Administration Sample BALB / c numberDaily dose 107 cfu / dayIgA in stool (OD + Std. Error)Serum IgA (OD + Std. Error)
Example 2: seamless capsule 72.80.15 + 0.0120.42 + 0.133
Example 2: soft capsule 73.30.13 + 0.0130.41 + 0.142
Example 2: hard capsule 73.20.13 + 0.0140.39 + 0.134
Comparative example 4: seamless capsule 52.80.05 + 0.0110.13 + 0.038
Comparative example 5: soft capsule 53.30.06 + 0.0100.12 + 0.052
Comparative Example 6: Hard Capsule 53.20.06 + 0.0100.11 + 0.028

Live cells of transformed microorganism 52.80.03 + 0.0120.12 + 0.032
Live host microorganism cells 58.30.02 + 0.0080.10 + 0.022
Phosphate buffer 5-0.02 + 0.0060.15 + 0.033
It was observed that, in the case of the acid-resistant seamless capsule, soft capsule and hard capsule formulations prepared in Example 2, regardless of the form of the acid resistant capsule formulation, the amounts of IgA they were larger in both feces and blood and the effect of inducing an antibody was greater, compared to the cases of non-acid resistant capsule formulations of Comparative Examples 4 to 6 or of living cells.
Example 6: Examination for the induction of antibodies resulting from the administration of Flagelin dysentery-Expressing transformed microorganism
The capsule seamless formulation, the soft capsule formulation, and the hard capsule formulation, which is
25 prepared in Example 3, and the capsule formulations of Comparative Examples 7 to 9 were examined for the induction of antibodies as in Example 4, each containing transformed microorganism cells expressing the flagelin dysentery bacillus (recombinant B. longum).
In addition, living cells of transformed microorganisms expressing flagellin dysentery, living B. host cells, and a phosphate buffer was used for control administrations. Table 3 shows the results.
Table 3
Administration Sample BALB / c numberDaily dose 107 cfu / dayIgA in stool (OD + Std. Error)Serum IgA (OD + Std. Error)
Example 3: seamless capsule 73.20.13 + 0.0120.38 + 0.142
Example 3: soft capsule 73.90.12 + 0.0130.37 + 0.153
Example 3: hard capsule 740.13 + 0.0140.39 + 0.131
Comparative example 7: seamless capsule 53.20.06 + 0.0110.11 + 0.038
Comparative example 8: soft capsule 53.90.05 + 0.0100.12 + 0.051
Comparative Example 9: Hard Capsule 540.06 + 0.0100.11 + 0.028
Live cells of transformed microorganism 53.20.02 + 0.0120.11 + 0.038
Live host microorganism cells 510.10.03 + 0.0080.13 + 0.036

Phosphate buffer 5-0.03 + 0.0060.14 + 0.031
It was observed that, in the case of the seamless acid resistant capsule, soft capsule and hard capsule formulations prepared in Example 3, regardless of the form of the acid resistant capsule formulation, the amounts of IgA were larger in both feces and blood and the effect of
10 inducing an antibody was higher, compared to the cases of non-acid resistant capsule formulations of Comparative Examples 7 to 9 or of living cells.
Example 7: Preparation of the formulation of the acid-resistant capsule containing bifidobacteria that secrete typhoid fever antigen outside the microorganism cell
A. Amplification of the Flagelin S. Typhimurium gene by PCR
The S. typhimurium flagelin gene fragment was prepared as in Section A of Example 1.
20 B. DNA amplification of the secretion signal peptide through PCR
B. bifidum ATCC 29521 was grown in MRS medium (manufactured by Nippon Becton Dickinson Company, Ltd.) at 37 ° C for 12 hours. After the end of the culture, the genomic DNA (Access # AJ224435) of B. bifidum is extracted conventionally. The PCR was performed as in Section A of Example 1, using the genomic DNA of B. bifidum
25 as a template with pair of primers of SEQ ID NO: 11 (forward): 5'CGGCAAGCTTTATGGGGGATACAGGATTGGCGAT-3 '(AAGCTT in positions 5 to 10 is the site of HindIII cleavage) and SEQ ID NO: 12 (back) : 5'-GCGCCCATGGAAATCGGGTGGCGTCCTCGACCG-3 '(CCATGG in positions 5 to 10 is the NcoI cleavage site). After completion of the PCR, the resulting product was cleaved with NcoI and HindIII. Therefore, the gene fragment of the secretion signal peptide was prepared.
C. Preparation of the secretion type expression vector
Plasmid pBLES100 was cleaved with BamHI and HindIII and combined with and ligated to the flagellin gene fragment obtained in section A described above and the secretion signal peptide gene fragment.
35 obtained in Section B described above. Thus, a pBLES-SP-FliC expression vector of the secretion type was obtained.
D. Introduction of secretion type expression vector in B. Brief
The transformation was performed as in Section D of Example 1, except that the pBLES-SP-F1iC obtained in Section C described above was used as an expression vector, and B. Brief ATCC 15700 was used as the microorganism to be transformed. . Therefore, B. brief transformed was obtained.
E. Secretion confirmation
The transformed brief B. obtained in section D described above was grown in MRS broth medium containing marble at 37 ° C for 12 hours, and subsequently, centrifuged at 4 ° C and 12,000 rpm, and the supernatant was obtained. The supernatant was subjected to Western Blotting as in Section E of Example 1. It was confirmed that a flagellin protein was secreted out of the transformed B brief cells.
F. Preparation of a lyophilized powder microorganism of the transformed microorganism
The brief B. confirmed for flagelin secretion of typhoid fever was confirmed to be used to prepare cell powders of lyophilized microorganisms as in section F of Example 1.
G. Preparation of acid-resistant seamless capsule formulation, soft capsule formulation and hard capsule formulation.
With the freeze-dried microorganism powders obtained as explained in Section F described above,
A seamless capsule formulation, a soft capsule formulation, and a hard capsule formulation were prepared as in sections G, H and I of Example 1, respectively. The membranes of the obtained seamless capsule formulation, soft capsule formulation, and acid resistant hard capsule formulation.
65 Comparative Examples 10 to 12


In Comparative Examples 10 to 12, a seamless capsule formulation, a soft capsule formulation, and a hard capsule formulation were prepared, respectively, as in Comparative Examples 1 to 3, except that the microorganism was changed to the transformed microorganism of typhoid fever expression of flagelin secretion prepared as in Example 7. Membranes of the capsule formulation without seams
5 obtained, the soft capsule formulation and hard capsule formulation were not acid resistant.
Example 8
The seamless capsule formulation, the soft capsule formulation and the hard capsule formulation, which
10 were obtained in Example 7, and the capsule formulations of Comparative Examples 10 through 12 were examined for the induction of antibodies as in Example 4, each of which contained the transformed microorganism cells of flagelin-secreting typhoid fever (recombinant B. Brief).
On the other hand, living cells of transformed microorganism expression of typhoid fever secretion of
15 flagellin (recombinant B. Brief), live B. Brief cells from host, and a phosphate buffer was used for administration control. Table 4 shows the results.
Table 4
Administration Sample BALB / c numberDaily dose 107 cfu / dayIgA in stool (OD + Std. Error)Serum IgA (OD + Std. Error)
Example 7: seamless capsule 73.50.18 + 0.0150.44 + 0.155
Example 7: soft capsule 74.10.16 + 0.0130.42 + 0.148
Example 7: hard capsule 73.60.20 + 0.0140.38 + 0.134
Comparative example 10: seamless capsule 53.50.05 + 0.0120.12 + 0.033
Comparative example 11: soft capsule 54.10.04 + 0.0110.11 + 0.046
Comparative Example 12: Hard Capsule 53.60.06 + 0.0130.14 + 0.034
Live cells of transformed microorganism 53.40.05 + 0.0110.13 + 0.036
Live host microorganism cells 510.50.03 + 0.0090.12 + 0.039
Phosphate buffer 5-0.02 + 0.0070.13 + 0.040
It was observed that, in the cases of the acid-resistant seamless capsule, soft capsule and hard capsule formulations containing the transformed microorganism expressing typhoid fever secretion of
60 flagelin prepared in Example 7, regardless of the form of acid-resistant capsule formulation, the amounts of IgA were larger in both feces and blood and the effect of inducing an antibody was greater, compared to cases of the non-acid resistant capsule formulations of Comparative Examples 10 to 12 or of living cells.
65 Industrial Applicability


A formulation in which a transformed microorganism that expresses flagelin is contained in an acid-resistant capsule increases the amount of anti-flagelin antibody produced, and is therefore effective as an oral vaccine against a bacterial infectious disease, such as typhoid fever. , cholera or dysentery. Therefore, a method to prevent and treat bacterial infectious diseases can be provided. In
5 Consideration of the latest infectious pathogenic bacteria resistant to prevalent medications, the administration of an oral vaccine to people living in an endemic area or people visiting a business or vacation region is an ideal strategy for prevention and treatment.
LIST OF SEQUENCES 10
<110gt; Morishita Jintan Co., Ltd.
<120gt; Oral Vaccine 15 <130gt; P39591EP
<150gt; JP 2007-70626
<151gt; 2007-03-19 20
<160gt; 12
<170gt; Patent version 3.1 25 <210gt; one
<211 gt; 30
<212gt; DNA 30
<213gt; Artificial
<220gt; 35 <223gt; Direct Salmonella Typhimurium Primer
<400gt; one
catgccatgg atggcacagt cattaataca 30 40
<210gt; 2
<211gt; 30 45 <212gt; DNA
<213gt; Artificial
<220gt; fifty
lt; 223gt; Salmonella typhimurium reverse primer
<400gt; 2 55 cgcggatcct taacgcagta aegagaggac 30
<210gt; 3
<211gt; 31 60
<212gt; DNA
<213gt; Artificial 65 <220gt;
<223gt; direct primer vibrio cholerae
<400gt; 3
5 catgccatgg atggcaatta atgtaaacac g
<210gt; 4
10 <211gt; 31 <212gt; DNA
<213gt; Artificial
fifteen <220gt;
<223gt; reverse primer vibrio cholerae
twenty <400gt; 4 cgcggatcct ttatcccaat aagctcagag c
<210gt; 5
25 <211gt; 30
<212gt; DNA
30 <213gt; Artificial <220gt;
<223gt; direct primer shigella dysenteriae
35 <400gt; 5
catgccatgg atggcacaag tcattaatac
40 <210gt; 6 <211gt; 30
<212gt; DNA
Four. Five <213gt; Artificial
<220gt;
fifty <223gt; reverse primer shiqella dysenteriae <400gt; 6
cgcggatcct ttaaccctgc tgcagagaca
55 <210gt; 7
<211gt; 30
60 <212gt; DNA <213gt; Artificial
<220gt;
65 <223gt; hup direct primer


<400gt; 7
cgccaagctt tgggcgcggc ggccatgaag 30
<210gt; 8
<211gt; 30
<212gt; DNA
<213gt; Artificial
<220gt;
<223gt; reverse hup primer
<400gt; 8
cgcgccatgg aaagcatcct tcttgggtca 30
<210gt; 9
<211gt; 600
<212gt; DNA
<213gt; Bifidobacterium longum
<220gt;
<221gt; promoter
<222gt; (1) .. (192)
<223gt;
<220gt;
<221gt; CDS
<222gt; (193) .. (474)
<223gt;
<220gt;
<221gt; terminator
<222gt; (475) .. (600)
<223gt;
<400gt; 9 5 <210gt; 10
<211gt; 93
<212gt; PRT
<213gt; Bifidobacterium longum
<400gt; 10
<210gt; eleven
<211gt; 3. 4
<212gt; DNA
<213gt; Artificial
<220gt;
<223gt; Bifidobacterium bifidum direct primer
<400gt; 11 cggcaagctt tatgggggat acaggattgg cgat 34
<210gt; 12
<211gt; 33
<212gt; DNA
<213gt; Artificial
<220gt;
<223gt; Bifidobacterium bifidum reverse primer
<400gt; 12 gcgcccatgg aaatcgggtg gcgtcctcga ccg 33

权利要求:
Claims (18)
[1]
one. An oral vaccine against a bacterial infectious disease, in the form of a capsule formulation, comprising:
a capsule membrane anda transformed microorganism that expresses a flagelin antigen protein,in which the capsule membrane is resistant to acids, and the transformed microorganism isencapsulates with the capsule membrane.
[2]
2. The oral vaccine of claim 1, wherein the flagellin antigen protein is expressed in the cell of the microorganism.
[3]
3. The oral vaccine of claim 1, wherein the flagellin antigen protein is secreted out of the microorganism cell.
[4]
Four. The oral vaccine of any one of claims 1 to 3, wherein the microorganism is at least one selected from microorganisms belonging to the group consisting of the genus Bifidobacterium, the genus Lactobacillus, the genus Lactococcus, the genus Pediococcus, the genus Streptococcus, the genus Enterococcus, the genus Leuconostoc, the genus Tetragenococcus, the genus Oenococcus, and the genus Weissella.
[5]
5. The oral vaccine according to any one of claims 1 to 4, wherein the oral vaccine is a vaccine against typhoid fever, cholera, or dysentery.
[6]
6. The oral vaccine of any one of claims 1 to 5, wherein the capsule formulation is a seamless capsule formulation, a soft capsule formulation, or a hard capsule formulation.
[7]
7. A method for producing an oral vaccine against a bacterial infectious disease, comprising the steps of the preparation of a transformed microorganism that expresses a flagellin antigen protein; and that wraps the transformed microorganism into an acid resistant capsule membrane, thereby producing an acid resistant capsule formulation
[8]
8. The method of claim 7, wherein the transformed microorganism secretes the flagelin antigen protein in the cell of the microorganism.
[9]
9. The method of claim 7, wherein the transformed microorganism secretes the flagelin antigen protein outside the cell of the microorganism.
[10]
10. The method of any one of claims 7 to 9, wherein the microorganism is at least one selected from microorganisms belonging to the group consisting of the genus Bifidobacterium, the genus Lactobacillus, the genus Lactococcus, the genus Pediococcus, the genus Streptococcus , the Enterococcus genus, the Leuconostoc genus, the Tetragenococcus genus, the Oenococcus genus, and the Weissella genus.
[11]
eleven. The method of any one of claims 7 to 10, wherein the oral vaccine is a vaccine against typhoid fever, cholera, or dysentery.
[12]
12. The method of any one of claims 7 to 11, wherein the capsule formulation is a seamless capsule formulation, a soft capsule formulation, or a hard capsule formulation.
[13]
13. A method for producing an oral vaccine against a bacterial infectious disease, which comprises the
steps of: preparing a transformed microorganism that expresses a flagelin antigen protein; wrap the transformed microorganism in a capsule membrane, thereby producing a capsule formulation; and providing the capsule membrane of the capsule formulation produced with acid resistance.
[14]
14. The method of claim 13, wherein the transformed microorganism expresses the flagelin antigen protein in the cell of the microorganism.
[15]
fifteen. The method of claim 13, wherein the transformed microorganism secretes the flagelin antigen protein outside the cell of the microorganism.
[16]
16. The method of any one of claims 13 to 15, wherein the microorganism is at least one selected from microorganisms belonging to the group consisting of the genus Bifidobacterlum, the genus Lactobacillus, the genus Lactococcus, the genus Pediococcus, the genus Streptococcus , the Enterococcus genus, the Leuconostoc genus, the Tetragenococcus genus, the Oenococcus genus, and the Weissella genus.

[17]
17. The method of any one of claims 13 to 16, wherein the oral vaccine is a vaccine against typhoid fever, cholera, or dysentery.
[18]
18. The method of any one of claims 13 to 17, wherein the capsule formulation is a seamless capsule formulation, a soft capsule formulation, or a hard capsule formulation.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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JP2007070626|2007-03-19|

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JP2007070626|2007-03-19|

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PCT/JP2008/055815|WO2008114889A1|2007-03-19|2008-03-19|Oral vaccine|

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