WO2008044465A1

WO2008044465A1 - Method of utilizing thrombin as adjuvant

Info

Publication number: WO2008044465A1
Application number: PCT/JP2007/068544
Authority: WO
Inventors: Kazuyoshi Kaminaka; Junichi Matsuda; Chikateru Nozaki
Original assignee: Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
Priority date: 2006-10-12
Filing date: 2007-09-25
Publication date: 2008-04-17
Also published as: JPWO2008044465A1; JP5047181B2

Abstract

Disclosed are: a method of utilizing a thrombin as an adjuvant; and a vaccine comprising the thrombin as an adjuvant. Specifically disclosed are: a thrombin capable of enhancing the production of IgG and IgA against an antigen when administered together with the antigen by a non-injection technique; a method of utilizing the thrombin as an adjuvant for a vaccine; and a vaccine useful for local immunization, which comprises the thrombin as an adjuvant.

Description

Specification

Methods of using thrombin as an adjuvant

Technical field

[0001] The present invention relates to thrombin having an immune response inducing ability, and more specifically, a method of using thrombin as an immune response inducer of a vaccine (hereinafter sometimes referred to as "adjuvant"), a vaccine antigen, and the thrombin. It relates to a vaccine comprising.

Background art

[0002] Thrombin is a trypsin-like serine protease that has an indispensable effect on the maintenance of life such as the formation of hemostatic thrombus and wound healing. Thrombin includes mesothrombin, α-thrombin, 13 thrombin, and γ thrombin, but α-thrombin is physiologically important. Prothrombin, the precursor of thrombin, is biosynthesized in liver cells in a vitamin Κ-dependent manner, and the FXa- FVa complex on the plasma membrane phospholipid undergoes limited degradation of Arg320 I Le321 binding of prothrombin, resulting in the Gla domain and the kringle domain. Mesothrombin is formed. Next, Arg271-Thr272 binding is limited and α-thrombin is generated and released from the cell membrane. Various plasma proteins and thrombin receptors on various cell membranes are limited and show various physiological activities. α-thrombin is a double-stranded molecule consisting of a cocoon chain and a cocoon chain. However, when the B chain, which is important for enzyme activity, is further self-digested, it becomes / 3-thrombin and γ-thrombin and activates fibrinogen and platelets. Loses ability.

[0003] The generated thrombin binds to a fibrin thrombus and is involved in the formation of a larger thrombus locally. Thrombin is an enzyme that converts fibrinogen to fibrin, and also triggers FXIII to crosslink fibrin. Furthermore, it has a coagulation-promoting function that activates FVIII and FV, which are cofactors of FI X and FXa, respectively, and promotes coagulation. Thus, thrombin is an extremely useful protein as a hemostatic agent because it plays an important role in hemostasis.

[0004] On the other hand, when thrombin binds to thrombomodulin on vascular endothelial cells, its substrate specificity changes, and protein C is activated to promote anticoagulation. Using the enzyme activity of thrombin to produce activated protein c, an anticoagulant It is a useful protein that is also used as a process enzyme. In addition, thrombin acts on monocytes to promote tissue factor production, acts on vascular endothelial cells to produce and secrete prostacyclin and plasminogen activator inhibitors, and further contracts endothelial cells to produce cells. It is known to widen the gap, increase the substance permeability of the endothelium, and promote the proliferation of smooth muscle cells and fibroblasts in the subendothelial tissue.

[0005] Thrombin having such various functions has been widely used as a hemostatic agent, a process enzyme, and a research reagent. For example, for upper gastrointestinal bleeding, thrombin derived from blood is endoscopically sprayed on the affected area of the bleeding or administered orally, and a hemostatic effect has been observed. In addition, fibrin glue used as tissue adhesive contains blood-derived thrombin along with fibrinogen and Fxm factor.

[0006] As described above, thrombin is widely used because of its various physiological actions.

1S In recent years, it has the effect of suppressing the production of interleukin 12 (IL-12), a cytokine that acts on human monocytes and induces a Thl immune response in vitro, and shifts to a Th2 immune response. (For example, see Non-Patent Document 1). This suggests that thrombin may bridge the inflammatory and immune responses! However, whether the shift to a Th2-type immune response observed in vitro provokes the ability to produce antibodies against foreign substances in vivo, or in other words, has the effect of inducing an immune response It ’s not clear so far.

[0007] Currently, adjuvants are used to compensate for weaknesses of inactivated vaccines with weak antigenicity because of their ability to enhance antibody production against foreign substances. Of these, only aluminum hydroxide gel is permitted for use in humans, and when administered parenterally, it sufficiently raises antibodies (IgG) against vaccine antigens in the blood. Can do. However, sufficient immune response inducing ability has not been observed on the mucosal surface (nasal cavity, oral cavity, intestinal tract, genitourinary tract, periocular area, etc.) having the largest area in contact with the outside world, where many pathogens invade.

[0008] What plays a major role in eliminating pathogens on the mucosal surface is an antibody called IgA that binds invading pathogens and prevents them from entering the body through the mucosa. Have the work to do. Therefore, to effectively prevent infection from pathogens It is important to induce IgA antibody on the mucosal surface, which is not only the rise in blood IgG antibody expected by conventional vaccination. From this point of view, various substances including bacterial toxin components have been examined so far, and it is known that animal antibodies can induce both antibodies (see, for example, Non-Patent Document 2). However, there are no known substances that can be used clinically while having sufficient ability to induce an immune response in transmucosal administration.

[0009] Non-Patent Document 1: Naldini A. et al., British J. Pharmacol., 140, 980-986, 2003

Non-Patent Document 2: Holmgen J. et al., Immunology Letters, 97, 181-188, 2005

Disclosure of the invention

Problems to be solved by the invention

[0010] As described above, thrombin is known to act on human monocytes to induce cytodynamic force-in, but has so far been effective in inducing immune responses against antigens administered simultaneously in vivo. It has not been revealed. Therefore, the purpose of the present invention is to focus on this point, to clarify the ability to induce immune response by transmucosal administration of thrombin, which has already been approved for oral administration as a pharmaceutical, and to provide an adjuvant that can be used for vaccination by the administration route It is to provide.

Means for solving the problem

[0011] As a result of studying the in vitro action of thrombin, the present inventors surprisingly found that when thrombin was administered transmucosally with egg white albumin, blood IgG against the egg white albumin It was found that there is an adjuvant activity that raises antibodies. Furthermore, the present inventors have found that antigen-specific IgA antibodies on the mucosal surface are also induced to complete the present invention.

[0012] Therefore, the present invention provides a method of using thrombin shown below as an adjuvant, and a vaccine comprising the vaccine antigen and the thrombin having adjuvant activity.

1. Using thrombin as an adjuvant for vaccines.

2. The method of 1 above, wherein thrombin can induce IgG antibody and IgA antibody.

3. The method according to 1 or 2 above, wherein the thrombin is a recombinant thrombin obtained by a gene recombination technique. 4. The method according to any one of 1 to 3 above, wherein thrombin is derived from an animal.

5. The method of 4 above, wherein thrombin is derived from a primate.

6. The method according to 5 above, wherein thrombin is derived from human.

7. The method according to any one of 1 to 6 above, wherein the vaccine is a vaccine for local immunization.

8. The method according to 7 above, wherein the local immunity is transdermal or transmucosal.

9. The method according to 8 above, wherein the transmucosal membrane is any mucosa of the nasal cavity, oral cavity, intestinal tract, genitourinary tract, or periocular area.

10. A vaccine comprising a vaccine antigen and thrombin having adjuvant activity.

11. The vaccine according to 10 above, which can induce IgG antibody and IgA antibody.

12. The vaccine according to 10 or 11 above, wherein thrombin is a recombinant thrombin obtained by a gene recombination technique.

13. The vaccine according to any one of 10 to 12 above, wherein thrombin is derived from an animal.

14. The above 13 vaccines wherein thrombin is derived from primates.

15. The vaccine according to 14 above, wherein thrombin is derived from a human.

The invention's effect

[0013] According to the present invention, a method of using thrombin as an adjuvant and a vaccine containing the same as an adjuvant are provided. By administering thrombin transmucosally with an antigen according to the present invention, a specific IgG antibody against the antigen in the blood is induced early and in a large amount, and a specific IgA antibody against the antigen is induced in a large amount on the mucosal surface. The power S Therefore, according to the present invention, it was proved that thrombin is useful as an adjuvant for vaccine antigens administered mainly transmucosally.

[0014] Moreover, since thrombin has already been orally administered as a pharmaceutical, it can be safely administered transmucosally.

Brief Description of Drawings

[0015] [Fig. 1] Fig. 1 is a graph showing the time course of anti-ovalbumin IgG antibody titer in blood when mice were immunized with ovalbumin alone or a mixture of thrombin and ovalbumin. [0016] FIG. 2 is a graph showing anti-ovalbumin IgA antibody titer in nasal lavage fluid when mice were immunized with ovalbumin alone or a mixture of thrombin and ovalbumin.

[0017] FIG. 3 is a graph showing the anti-ovalbumin IgA antibody titer in the intestinal lavage fluid when mice were immunized with ovalbumin alone or a mixture of thrombin and ovalbumin.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] The present invention is characterized by thrombin having an immune response inducing ability and a vaccine containing the same as an adjuvant. The thrombin can be prepared from the blood of a primate or other animal by low-temperature ethanol fractionation, chromatography operation, or the like. Alternatively, it can be prepared using a genetic recombination technique as shown below. When a gene recombination technique is used, the gene encoding thrombin may be a gene obtained from either a primate or another animal. In the Examples of the present application, recombinant human thrombin was obtained starting from the human prothrombin gene (Preparation Example 1; see WO2007 / 040162 for details). The thrombin is derived from a force S that can use thrombin derived from mammals such as primates or other animals, preferably from humans, and a force S that can be appropriately selected according to the animal (or human) to be immunized.

[0019] For example, the human prothrombin gene is obtained by using a general gene recombination technique (Molecular Cloning, A Laboratory Manual Second) described by Sambrook et al., Starting from total RNA, mRNA, or genomic DNA extracted from human liver. edition. old spring Haroor Laboratory Press, NY, 1989). Today, various kits are available on the market. For example, for RNA extraction, reagents such as TRIzol reagent (Invitrogen), ISOGEN (Nitsubon Gene), StrataPr mark Total RNA Purificatio Kit (Toyobo), and for mRNA purification, mRNA Purification Kit (Amersham Bioscience) ), P ₀ l _y (A) Kits such as Quick mRNA Isolation Kit (Toyobo), mRNA Separator it (Clontech), and T-Primed First Strand Kit (Amanhamfano Remanno) Superscript plasmia system for cDNA synthesis and plasmid cloning (Invitrogen), cDNA Synthesis Kit (Takara Shuzo), SMART PCR cDN A Synthesis & Library Construction Kits (Clontech), Directional cDNA Library Construction Systems (Novagen), GeneAmp PCR Gold (Applied Biosystems), etc. are used.

[0020] The human prothrombin gene thus obtained is inserted into an appropriate expression vector, and a host, for example, an animal cell is transformed with the expression vector. Although there are no particular restrictions on the expression vector, an expression control region such as an appropriate promoter, a stop codon, a poly A addition signal arrangement IJ, a Kozak arrangement IJ, a secretion signal, etc. are added to the foreign gene. When using animal cells as the host, the promoter contained in the expression vector should be able to use early SV40, late SV40, cytomegalovirus promoter, chicken β-actin, etc. Anything can be used. When an animal cell is used as a host, a chicken β-actin promoter system expression plasmid pCAGG (Japanese Patent Laid-Open No. 3-168087) is preferably used. Marker genes for selection and gene amplification such as neo gene, dihydrofolate reductase (dhfr) gene, puromycin resistance enzyme gene, and glutamine synthetase (GS) gene can be used as marker genes for selection and gene amplification. .

[0021] As a host to be used in the present invention, cultured cells derived from various mammals can be widely used. Preferred examples include Chinese nomstar ovary cells (CHO cells), human-derived 293 cells, or bird-derived cells. Is done. For example, the calcium phosphate method, the DEAE dextran method, the method using lipofectin ribosomes, the protoplast polyethylene glycol fusion method, and the electopore position method can be used. a suitable method may be selected by the host cell (Molecular Clonin g (3rd Ed. ), Vol 3, Cold Spring Harbor Laboratory Press (2001)) 0 as culture land used for culturing, the agar medium from its shape, a liquid medium, The type force, YMM-01, DMEM, RPMI, a MEM, etc. may be selected as appropriate according to the cell, purpose of culture, or culture stage. In accordance with the protocol of each medium, those supplemented with serum, amino acids, vitamins, sugars, antibiotics, pH adjusting buffer, etc. are used. The pH of the medium is set at 6-8, and the culture temperature is set at 30 ° C-39 ° C. The amount of medium, additives, and culture time are appropriately adjusted according to the culture scale. [0022] Human prothrombin-producing cells can be detected in humans in a culture solution of drug-resistant cells cloned by a detection method using a specific reaction using an antithrombin antibody, such as a dot blot, Western blot, or ELISA. It can be obtained by detecting prothrombin. The human prothrombin-producing cells thus obtained are acclimated to a serum-free medium and then subjected to mass production at an industrial production level. As a method for mass culture, any method generally used, such as fedbatch culture or notch culture, may be used.

[0023] When purifying human prothrombin from such prothrombin-producing cells, purification methods generally used in protein chemistry, such as salting-out, ultrafiltration, isoelectric precipitation, Methods such as electrophoresis, ion exchange chromatography, gel filtration chromatography, affinity mouth-mat method, hydrophobic chromatography, and hydroxyapatite chromatography are used. Actually, since many kinds of cell-derived contaminants coexist, it may be fibrated by a combination of the above methods.

[0024] For conversion of human prothrombin to human thrombin, it is preferable to use recombinant ecarin. Recombinant ecarin can be prepared according to the method described in the patent publication (WO2003 / 004641). Briefly, a snake venom ecarin cDNA was prepared according to the method of Nishida et al. (S. Nishida et. Α1, Biochemistry, 34, 1771-1778, 1995), and this was used for the chicken β-actin used in the present invention. Incorporate into promoter system expression plasmid pCAGG. The obtained expression vector is introduced into animal cells such as CHO cells to obtain recombinant ecarin producing cells. Recombinant ecarin can be purified by cation exchange chromatography and gel filtration. The recombinant ecarin thus purified is added to the purified human prothrombin solution and allowed to react for a time sufficient for the conversion reaction. The reaction temperature is 35 to 38 ° C, preferably 37 ° C, and the reaction time is 1-4 hours, preferably 2 hours. Human thrombin converted by ecarin treatment is purified by chromatography using a benzamidine column and a cation exchange column. The purified recombinant ecarin is allowed to act on human prothrombin and converted to human thrombin. The reaction conditions are the same as those for normal enzyme reactions. For example, human prothrombin is converted to human thrombin by adding ecariline at a final concentration of 2-81¾ / ½ to human prothrombin 1000 _{8 /} ½ and reacting at 36-38 ° C for 1-4 hours. . [0025] Human thrombin is purified by subjecting the ecarin-treated solution to the protein purification method described above. Preferably, a purification method using a combination of a benzamidine column and cation exchange chromatography is used. As in the case of anion exchange, any buffer can be used as long as it retains the buffer capacity with the neutral region as the center. The pH range is 7-9, preferably 8, and the buffer concentration is 0.1M or less, preferably 0.01M. When human thrombin is adsorbed to the column, it contains 0.3-0.7M salt. Preferably, 0.5M NaCl is used. Human thrombin is washed with the same buffer, and then eluted with a buffer obtained by adding 0.1 M benzamidine hydrochloride to the buffer used for adsorption and washing.

[0026] In order to highly purify human thrombin, it is further subjected to cation exchange chromatography. As the cation exchanger, a force S including a sulfopropyl group type, a carboxymethyl group type and the like may be appropriately selected and used. In the present embodiment, SP Toyopearl 550C (Toso Soichi) is used. Adsorption, washing, and elution conditions are generally in the range used for cation exchange chromatography. For example, the pH is 6-8, preferably 6-7, and the buffer concentration is 5-200 mM, preferably 10 mM. More specifically, a 10 mM citrate buffer solution with 0.2 M NaCl added is used. Eluting human thrombin is done by increasing the salt concentration. Preferably 0.6M NaCl is used. By such a method, high-purity human thrombin can be obtained.

[0027] The thus obtained human thrombin is subjected to enzymatic chemical properties such as clotting activity, synthetic substrate (S-22338) cleavage activity, and sugar chain analysis. The clotting activity value is a relative value obtained by comparing the time of fibrinogen clotting with the standard curve of the standard. As such standard, the Japanese Pharmacopoeia Standard and WHO International A standard product (MBSC) thrombin is used. S-2338 cleavage activity is based on the reaction between thrombin and its specific substrate, and the amount of p-nitrotropilin released when thrombin cleaves the synthetic substrate S-2238. It is determined by measuring the change in absorbance of OD405 / 650.

[0028] For measurement of sialic acid, N-Acetylneuraminic acid and N-Glycolylneuraminic acid are used as standard substances, and the elution time from these ion exchange chromatography columns and the elution time of the test substance are determined. This is done by comparing. That is, reference material Each regression equation is created from the peak area at each concentration, and the measured value of the test substance is added to this to obtain the sialic acid concentration.

[0029] The usefulness of human thrombin thus obtained as an adjuvant is that a mixture of the human thrombin and an appropriate antigen can be used in various animals, such as mice, rats, guinea pigs, mice, musters, dogs, monkeys, etc. The amount of IgG in the serum obtained or the amount of IgA produced on the mucosal surface can be examined. As the antigen, vaccine antigens against various bacterial and viral infections that are not particularly limited can be used. The vaccine antigen may be in any form such as a peptide, a protein, a bacterial cell, or a virus particle. In the examples of the present application, ovalbumin, which is easy to obtain and analyze, was used. The administration method is not particularly limited as long as it is administered by a non-injection method. For example, it is administered by a commonly used inoculation route such as transdermal, oral, nasal drop or eye drop. When investigating the effects of local immunity, the use of a route that requires treatment to suppress the effects of proteolytic enzymes, transdermal, nasal, and eye drops is preferable, but treatment such as encapsulation in capsules is performed. Oral administration is also available. The solvent for mixing human thrombin and antigen is not particularly limited as long as it does not show toxicity to humans or animals, for example, physiological saline and phosphate buffer (PBS).

[0030] Specifically, for immunization, C57BL / 6 mice (SLC) were used, and 0-40 Hg human thrombin was mixed with PBS for 5; ^ ovalbumin per mouse. This is done by dripping the mixture into the nasal cavity once a week (5 times in total). Blood is collected from the tail vein 4 days before the first immunization and from the second immunization to the fifth immunization on the 4th to 5th day from the day of immunization.

[0031] On the other hand, for the mucosal surface cleaning solution, force-tailored from the incised trachea of the killed mouse, inserted into the nasal cavity opening from the throat to the nasal cavity, washed with PBS containing PMSF (Sigma), Collect the washing solution. Intestinal lavage fluid is collected by the following method. First, the pyloric part of the stomach is peeled off to the immediate front of the cecum without damaging the intestinal tract, and is collected in a dish containing EDTA (DOJINDO) solution containing Soybean Trypsin inhibitor (GIBCO BRL) in advance. Centrifuge after pipetting, mix with PMSF in the supernatant, and centrifuge. Protease inhibitor Cocktail set III, NaN3 (Katayama Chemical Co., Ltd.) is added to the supernatant, and the mixture is allowed to stand for 15 minutes after mixing. Combined and used as intestinal lavage fluid.

[0032] Specific antibodies in serum and mucosal lavage fluid are measured by ELISA. After blocking ovalbumin on a 96-well plate (Nunc, Maxisorp) with Block Ace (Dainippon Sumitomo Pharma), skim milk, etc., add diluted serum to react. After washing, react with HRP-labeled anti-mouse IgG goat antibody (American Qualax, A131PS). After washing, add chromogenic substrate solution TMB + (Dako) and react at room temperature in the dark. Color development is stopped with 1N sulfuric acid, and absorbance at 450 nm (OD450 value) is measured. When measuring IgA in the intestinal tract and nasal lavage fluid, the same procedure is performed using an HRP-labeled anti-mouse IgA goat antibody (Funakoshi, A90-103P). The anti-ovalbumin antibody titer is calculated from the standard curve of the standard solution.

[0033] As a result, it is clear that human thrombin has the effect of increasing the production of anti-ovalbumin IgG antibody in blood and anti-ovalbumin IgA antibody on the transmucosal surface when administered together with ovalbumin. became. Therefore, human thrombin can be used as an adjuvant for various vaccines. This is particularly useful when expecting an adjuvant effect on local immunity on the mucosal surface.

[0034] Thus, human thrombin has adjuvant activity, and is administered to animals or humans together with an appropriate vaccine antigen. In the formulation of a vaccine, the amount of human thrombin is set based on various conditions such as the properties of the vaccine antigen, the dose, the animal (or human) to be immunized, and the immunization schedule. It is effective to administer the amount of thrombin with the required antigen in the concentration range of 0.1 to 10 mg / mL. Preferably, 0.5 to 4 mg / mL thrombin is effective.

[0035] Additives such as stabilizers and protective agents may be prepared for formulation of vaccines containing human thrombin. Such additives include polysorbate 80, stabilizers such as sugars such as amino acids and ratatosucrose, preservatives such as formalin, thimerosal, 2-phenoxyethanol, benzyl alcohol, benzethonium chloride and benzalkonium chloride. Are listed. In addition, when a sugar such as ratatoose or sucrose having an effect as an excipient is added, it can be formulated as a lyophilized preparation. Furthermore, it can be used in combination with immunostimulants such as aluminum hydroxide, aluminum phosphate, mineral oil and non-mineral oil. [0036] Preparation Example 1: Preparation of human thrombin

An expression plasmid in which the human prothrombin gene is linked downstream of the chicken β-actin promoter was prepared by a modified calcium phosphate method (C. Chen et al., Mol. Cell. Biol., 7, p.2745-27 52, 1987). Introduced into Chinese nomstar ovary (CHO) cells. Prothrombin-producing CHO cells were screened by ELISA using an anti-human thrombin antibody. The obtained prothrombin-producing cells were acclimated to a serum-free medium and then cultured in a large amount with a fermenter to purify human thrombin.

[0037] Prothrombin production CHO cell culture supernatant 3200ml, 2 volumes of 20mM phosphate buffer

A sample obtained by adding (ρΗ7.4) and filtering through a 0.22 Hm filter was used. This sample was applied to a Q Sepharose first flow (Amersham) 80 ml column equilibrated with 10 mM phosphate buffer (ρΗ7 · 4) containing 0.1 M NaC 1 at a flow rate of 6.6 ml / min. After washing the column with 1600 ml of the above buffer, elution was performed with 450 ml of 10 mM phosphate buffer (pH 7.4) containing 0.35 M NaCl at a flow rate of 6.6 ml / min.

[0038] Purified recombinant ecarin was added to 360 ml of the anion exchange chromatography eluate to a final concentration of 4 U / ml, and reacted at 37 ° C for 2 hours to convert prothrombin to thrombin. This reaction solution was applied at 2.5 ml / min to a benzamidine fast flow (Amersham) 40 ml column equilibrated with 10 mM Tris_HCl (pH 8.0) buffer containing 0.5 N NaCl. After washing the column with 800 ml of the above buffer, elution was performed with 360 ml of 10 mM Tris-HCl + 0.5 M NaCl (pH 8.0) buffer containing lOOmM benzamidine hydrochloride.

[0039] Next, a sample obtained by adding 2.5 volumes of 10 mM citrate buffer (ρ 倍 7.0) to 160 ml of benzamidine column eluate was equilibrated with 10 mM citrate buffer (ρΗ7.0) containing 0.2Μ NaCl. SP Toyopearl 550C (Tosoichi) was applied to a 40 ml column at 6.6 ml / min. After washing the column with 800 ml of the above buffer, elution was carried out with 120 ml of 10 mM citrate buffer (pH 7.0) containing 0.6N NaCl to obtain highly pure recombinant human thrombin. The protein concentration of the prepared recombinant human thrombin is 7.57 mg / mL according to the Lowry method using BSA as standard.

Example 1

[0040] Ovalbumin exemption (1) Preparation of ovalbumin solution

Dissolve commercially available ovalbumin (Sigma, Grade IV, A2512-250MG) in 10 mL of PBS (GIBCO BRL), dilute 200 L 2.5 times with PBS (10 mg / mL), and add 30 L in 0.5 mL tubes. And stored at -20 ° C or lower until use.

[0041] (2) Mice treated

Male C57BL / 6 (7 weeks old, SPF) was purchased from SLC and bred for 10 days in an SPF environment.

[0042] (3) Immune group composition

As shown in Table 1, 22 mice were divided into 4 groups, which were divided into ovalbumin-only immunized group (group 1) and ovalbumin plus recombinant thrombin immunized group (groups 2, 3, 4).

[0043] [Table 1]

[0044] (4) Preparation of immune product

Group 1: 4 L of the previously prepared ovalbumin solution was diluted with PBS to 80. Group 2: Recombinant thrombin (4.57 mg / mL) 8.8 μL in 4 L of the above ovalbumin solution was calorie-free and diluted to 80 μm with PBS.

Group 3; 35 L of recombinant thrombin (4.57 mg / mL) was calorie-free and diluted with PBS to 4 μL in 4 L of the above ovalbumin solution.

Group 4: 70 L of recombinant thrombin (4.57 mg / mL) was calorie-free and diluted with PBS to 4 μL in 4 L of the above ovalbumin solution.

[0045] (5) Immunization method and schedule

Under ether anesthesia, 10 L of each immunized substance prepared in (4) above was administered from the nasal cavity using Pipetman Ρ-20 (Gilson) per mouse. Mice were immunized 6 times at weekly intervals.

[0046] (6) Blood collection and collection of nasal cavity and intestinal lavage fluid About 150 HL was collected from the tail vein 4 to 5 days after the first immunization and from the second immunization to the fifth immunization on the 4th to 5th day. On the 6th day from the 6th final immunization, all mice were anesthetized with pentobarbital sodium (Kyoritsu Pharmaceutical Co., Ltd., Somnopentil) and blood was collected from the abdominal vena cava and killed. The collected blood was transferred to Microtina (BECTON DIC INS ON) and coagulated sufficiently at room temperature, and then centrifuged at 5,000 rpm for 10 minutes. Separated sera were dispensed into two 0.5 mL tubes and stored at -80 ° C until measurement.

[0047] After blood collection, a catheter was inserted through the incised trachea, inserted into the nasal cavity leading from the throat to the nasal cavity, and washed with 0.5 mL of PBS containing ImM PMSF (Sigma). The washing solution was collected in a 1.5 mL tube, and 5 μL of Protease inhibitor Cocktail set III (Calbiochem, 539134) was collected and stored as a nasal washing solution at −80 ° C. until measurement.

[0048] On the other hand, the intestinal lavage fluid was collected and pretreated by the following method. First, the small intestine from the stomach pyloric region to the immediate front of the cecum was removed without damaging the intestinal tract. Pre-prepared lmL 0.2mg / mL Soybean Trypsin inhibitor (GIBCO BRL) containing 50mM EDTA (DOJINDO) in a 10cm diameter dish peeled intestinal lavage solution (washed with 2mL PBS) with intestinal contents Collected. Pipet this, transfer to a 15 mL tube, and make up to 3 mL with 50 mM EDTA containing 0.2 mg / mL Soybean Trypsin inhibitor 4. Centrifuge at 2,500 rpm for 10 minutes. 2 mL of the supernatant was transferred to a new 15 mL tube, 1/100 volume of lOOmM P MSF was added and mixed, and then centrifuged at 4190C for 14,190 rpm for 20 minutes. 1 mL of the separated supernatant was put into a 1.5 mL tube, 10 L of Protease inhibitor Cocktail set III, 10% 1% NaN (Katayama Chemical Co., Ltd.) was added, and the mixture was allowed to stand for 15 minutes after mixing. To this, 50 L of fetal calf serum (Hyclone) was added, mixed and stored as an intestinal lavage solution at -80 ° C until measurement.

Example 2

(1) Preparation of standard serum

In order to compare the increase in anti-ovalbumin antibody between the above groups, a standard serum of anti-egg albumin antibody was prepared by the method shown below. Ovalbumin (100 g / head) was administered intranasally to 5 mice (C57BL / 6, male) for 3 days. Four courses were administered at intervals. One week after the last course administration, booster immunization was carried out once by nasal administration, and sera obtained by collecting blood after 5 days were pooled and used as standard sera.

[0050] (2) Measurement of anti-ovalbumin IgG antibody

Dilute ovalbumin used for immunization to 10 g / mL with 0.1M Carbonate buffer, ρΗ9 · 6, and transfer to 100 L / well in a 96-well plate (Nunc, Maxisorp) and leave at 4 ° C overnight. To solidify. The next day, each well was washed 5 times with 350 L of PBS, and Block Ace (Dainippon Sumitomo Pharma Co., Ltd., hereinafter abbreviated as BA) diluted 4 times with PBS was added 350 L / well at room temperature for 2 hours. Left to stand.

[0051] After 2 hours, the 4-fold diluted BA was sufficiently removed, and the sample was diluted 10-fold with 0.05% Tween20-containing PBS (PBST) and added at 100 L / well. (Duplicate for each sample). After the reaction at 37 ° C for 2 hours, each diluted serum added was discarded and washed 5 times with 350 L / well PBST. After washing, thoroughly remove the wash solution in the well, add HRP-labeled anti-mouse IgG goat antibody (American Qualax, A131PS) diluted 2000-fold with the sample diluent at 100 L / well, and add 37 ° C, reacted for 1 hour. After the reaction, discard the diluted antibody diluted solution, wash 4 times with 350 L / well PBST and 2 times with the same amount of distilled water, add lOO L / well of chromogenic substrate solution TMB + (Dako), and protect from light. And reacted at room temperature for 30 minutes. Thereafter, 1 L sulfuric acid was added at 100 L / well to stop the color development, and the absorbance at 450 nm (OD450 value) was measured.

[0052] (3) Calculation of antibody titer of standard serum

Serum of 28 unimmunized mice (C57BL / 6) was diluted 50-fold with the sample diluent, and the OD450 value of each diluted sample was measured in duplicate. A value obtained by adding twice the standard deviation value to the average value of the measured OD450 values was set as the cutoff value. Next, the standard serum was diluted 2-fold from 200-fold to 204,800-fold, and its OD450 value was measured. The maximum dilution ratio exceeding the cutoff value was defined as the antibody titer of the standard serum. In this test system, the antibody titer of the standard serum was set at 102,400 units because the cut-off value was exceeded up to a dilution of 102,400.

[0053] (4) Anti-ovalbumin antibody titer

The antibody titer of the serum of each group of mice immunized with ovalbumin by the method shown in Example 1 was calculated as follows. First, the standard serum was diluted to 0.5, 1, 2, 4, 8, 16, 32 units with the sample diluent to prepare a standard for antibody titer measurement. Next, the immune group Usus serum was diluted with a specimen diluent so as to fall within the range of the prepared standard. The test sample prepared as described above was measured in the system shown in (2), and the anti-ovalbumin antibody titer of each test mouse serum was calculated from the standard unit obtained and the standard line of OD450 value.

Yes

[0054] Fig. 1 shows the change over time of the calculated average antibody titer of each immunization group. As shown in FIG. 1, it can be confirmed that anti-ovalbumin antibody appears earlier and has a higher antibody titer depending on the dose of thrombin compared to the ovalbumin-only immunized group (Group 1). It was. In particular, in the group (Groups 3 and 4) in which 20 Hg and 40 Hg thrombin were co-administered for one immunization per animal, the appearance time and antibody titer of the antibody were almost the same. It was considered that 20 Hg was sufficient for the amount of thrombin.

Example 3

[0055] m ^ ψ of! _G A pile ί this I measurement

(1) Measurement of specific IgA antibody

Dilute ovalbumin used for immunization to 10 g / mL with 0.1M Carbonate buffer, ρΗ9 · 6, and transfer to 100 L / well in a 96-well plate (Nunc, Maxisorp) and leave at 4 ° C overnight. To solidify. On the next day, each well was washed 5 times with 350 L of PBS, BA diluted 4 times with PBS was added in 350 L / well, and allowed to stand at room temperature for 1 hour.

[0056] After 1 hour, 4-fold dilution BA was sufficiently removed, and each individual nasal wash was diluted 5-fold with BA (specimen dilution solution) diluted 10-fold with PBS containing 0.05% Tween20 (PBST). (Replicated at 37 ° C for 1 hour in each sample, discard each diluted nasal wash and add 5 times with 350 L / well of PBST. After washing, wash in well) HRP-labeled anti-mouse IgA goat antibody (Funakoshi, A90-103 P) diluted 5,000-fold with a sample dilution solution was added at 100 L / well and reacted at 37 ° C for 1 hour. After the reaction, discard the labeled antibody dilution, wash 4 times with 350 L / well PBST and 2 times with the same amount of distilled water, and add 100 L / well of chromogenic substrate solution TMB + (Dak 0). Then, the reaction was allowed to proceed for 5 minutes at room temperature in the dark, after which color development was stopped by adding 100 N / well of 1N sulfuric acid, and the absorbance at 450 nm (OD450 value) was measured.

[0057] (2) Measurement of total IgA antibody

All IgA antibodies in the nasal lavage fluid are available from Funakoshi MOUSE IgA ELISA QUANTITATION Measurement was performed using KIT (E90-103) according to the attached protocol. At the time of measurement, the nasal wash was diluted 20 times.

[0058] (3) Calculation of anti-ovalbumin IgA antibody titer in nasal wash

In order to standardize the dispersion at the time of collecting the washing solution and compare the ovalbumin-specific IgA antibody titer, the OD450 value of each sample obtained in (1) and the measured value obtained in (2) (mg / mL The anti-ovalbumin IgA antibody titer against all IgA antibodies was calculated from the following formula (I).

(I) Anti-ovalbumin antibody titer = (specific IgA antibody OD450 value x 5) ÷ total IgA antibody measurement mg / mL)

[0059] Fig. 2 shows the calculated anti-ovalbumin IgA antibody titers and standard errors of each group. Compared to the ovalbumin-only immunized group (Group 1), Group 2 (Thrombin 5 g co-immunized group) is more prominent! /, But Groups 2 and 3 (Thrombin 20 and 40 ag co-immunized groups respectively) Then, the specific IgA antibody titer increased!

Example 4

[0060] IgA pile body price measurement of intestinal lavage fluid

(I) Measurement of specific IgA antibody

The measurement was performed in the same manner as in the specific IgA antibody measurement method in the nasal cavity washing solution shown in Example 3. However, at the time of measurement, the intestinal lavage fluid was diluted 20 times with the sample diluent.

[0061] (2) Measurement of total IgA antibody

As in Example 3, MOUSE IgA ELISA QUANTITATION KIT (E90_1 03) from Funakoshi was used. However, at the time of measurement, the intestinal lavage fluid was diluted 2,000 times.

[0062] (3) Calculation of anti-ovalbumin IgA antibody titer in intestinal lavage fluid

Like the nasal wash, the OD450 value of each sample obtained in (1) and the measurement obtained in (2) were used to compare the ovalbumin-specific IgA antibody titer by standardizing variations at the time of collecting the wash. From the value (converted to mg / mL), anti-ovalbumin IgA antibody titer against all IgA antibodies was calculated by the following formula (II).

(II) Anti-ovalbumin antibody titer = (specific IgA antibody OD450 value x 20) ÷ total IgA antibody measured value (mg / mL)

[0063] Fig. 3 shows the calculated anti-ovalbumin IgA antibody titers and their standard errors for each group. . Similar to the nasal lavage fluid, the specific IgA antibody titer increased according to the amount of co-administered thrombin (groups 2 and 3 versus group 1) compared to the ovalbumin immunized group (group 1). The increase in specific IgA antibody titers was similar at 20 and 40 g of thrombin.

Industrial applicability

In accordance with the present invention, thrombin can be utilized as an adjuvant when administering vaccine antigens primarily transmucosally.

Claims

The scope of the claims

[I] A method of using thrombin as an adjuvant for a vaccine.

[2] The method of claim 1, wherein thrombin can induce IgG antibody and IgA antibody.

[3] The method according to any one of claims 1 and 2, wherein the thrombin is a recombinant thrombin obtained by a gene recombination technique.

[4] The method according to any one of claims 1 to 3, wherein the thrombin is derived from an animal.

[5] The method according to claim 4, wherein the thrombin is derived from a primate.

6. The method according to claim 5, wherein the thrombin is derived from a human.

7. The method according to any one of claims 1 to 6, wherein the vaccine is a vaccine for performing local immunization.

8. The method according to claim 7, wherein the local immunity is transdermal or transmucosal.

[9] The method according to claim 8, wherein the transmucosal membrane is any mucosa of the nasal cavity, oral cavity, intestinal tract, genitourinary tract, or periocular area.

[10] A vaccine comprising a vaccine antigen and thrombin having an adjuvant activity.

[I I] The vaccine according to claim 10, which can induce IgG antibody and IgA antibody.

[12] The vaccine according to any one of claims 10 and 11, wherein the thrombin is a recombinant thrombin obtained by a gene recombination technique.

13. The vaccine according to any one of claims 10 to 12, wherein thrombin is derived from an animal.

14. The vaccine according to claim 13, wherein the thrombin is derived from a primate.

15. The vaccine according to claim 14, wherein the thrombin is derived from human.