JP5047181B2 - Methods of using thrombin as an adjuvant - Google Patents

Description

  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. Regarding vaccines.

  Thrombin is a trypsin-like serine protease that has an indispensable action in the maintenance of life such as hemostatic thrombus formation and wound healing. Thrombin includes mesothrombin, α-thrombin, β-thrombin, and γ-thrombin, but α-thrombin is physiologically important. Prothrombin, a precursor of thrombin, is biosynthesized in hepatocytes in a vitamin K-dependent manner, and the Arg320-ILe321 bond of prothrombin undergoes limited degradation by the FXa-FVa complex on the plasma membrane phospholipid, resulting in the Gla domain and the kringle domain. Mesothrombin is formed. Subsequently, Arg271-Thr272 binding is limited and α-thrombin is produced and released from the cell membrane, and various plasma proteins and thrombin receptors of various cell membranes are limited and show various physiological activities. α-Thrombin is a double-stranded molecule consisting of A and B chains. However, when the B chain important for enzyme activity is further self-digested, it becomes β-thrombin and γ-thrombin and loses the ability to activate fibrinogen and platelets. .

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

  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. It is a useful protein that is also used as a process enzyme for producing activated protein C, an anticoagulant, by utilizing the enzyme activity of thrombin. 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 widen cell gaps, endothelium It is known to increase the permeability of the substance and promote the proliferation of smooth muscle cells and fibroblasts in the subendothelial tissue.

  Thrombin having such various functions has been widely used so far as a hemostatic agent, a process enzyme, and a research reagent. For example, for upper gastrointestinal bleeding, blood-derived thrombin is endoscopically sprayed on the affected part of the bleeding or orally administered, and a hemostatic effect is recognized. Furthermore, fibrin glue used as a tissue adhesive contains thrombin derived from blood together with fibrinogen and FXIII factor.

  As described above, thrombin is widely used due to its various physiological functions. Recently, interleukin-12 (IL-12), a cytokine that acts on human monocytes and induces a Th1-type immune response in vitro. 12) was found to have the effect of suppressing the production of (2) and shifting to a Th2-type immune response (see, for example, Non-Patent Document 1). This suggests that thrombin may bridge inflammatory and immune responses. However, it is not clear so far whether the shift to the Th2-type immune response observed in vitro induces the ability to produce antibodies against foreign substances in vivo, in other words, has the effect of inducing an immune response.

  Currently, adjuvants are used to compensate for the weaknesses of inactivated vaccines with weak antigenicity due to the effect of enhancing antibody-producing ability against foreign substances. Among them, only the aluminum hydroxide gel is permitted to be used for humans. When administered parenterally, the antibody (IgG) against the vaccine antigen in the blood is sufficiently increased. Can do. However, sufficient ability to induce an immune response 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 enter the gate.

  An antibody called IgA plays a major role in eliminating pathogens on the mucosal surface and binds the invading pathogens to prevent them from entering the living body through the mucosa. Therefore, in order to more effectively prevent infection from pathogens, it is important to induce IgA antibodies on the mucosal surface as well as the rise of blood IgG antibodies expected by conventional vaccination. From this point of view, various substances including bacterial-derived toxin components and the like have been investigated so far, and those that can induce both antibodies in an animal system are known (for example, see Non-Patent Document 2). However, a substance that can be used clinically while having a sufficient ability to induce an immune response in transmucosal administration has not been known so far.

Naldini A. et al., British J. Pharmacol., 140, 980-986, 2003 Holmgen J. et al., Immunology Letters, 97, 181-188, 2005

  As described above, thrombin is known to act on human monocytes to induce cytokines, but the action to induce an immune response against an antigen administered simultaneously in vivo has not been clarified so far. Therefore, the purpose of the present invention is to focus on this point, to clarify the immune response inducing ability by transmucosal administration of thrombin, which is already 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.

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

Therefore, the present invention provides a method using the thrombin shown below as an adjuvant, and a vaccine comprising the vaccine antigen and the thrombin having adjuvant activity.
1. A method of using thrombin as an adjuvant for a vaccine.
2. The method of 1 above, wherein thrombin can induce IgG antibody and IgA antibody.
3. The method according to any one of 1 and 2 above, wherein the thrombin is a recombinant thrombin obtained by a gene recombination technique.
4). 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 the thrombin is derived from a primate.
6). 6. The method according to 5 above, wherein the thrombin is derived from a human.
7). 7. The method according to any one of 1 to 6 above, wherein the vaccine is a vaccine for performing local immunization.
8). The method according to 7 above, wherein the local immunity is transdermal or transmucosal.
9. 9. The method according to 8 above, wherein the transmucosal membrane is a mucosa of any of the nasal cavity, oral cavity, intestinal tract, urogenital tract, and peripheries.
10. A vaccine comprising thrombin having vaccine antigen and adjuvant activity.
11. 10. The vaccine according to the above 10, which can induce IgG antibody and IgA antibody.
12 The vaccine according to any of 10 or 11 above, wherein thrombin is a recombinant thrombin obtained by a gene recombination technique.
13. The vaccine according to any one of the above 10 to 12, wherein thrombin is derived from an animal.
14 13. The vaccine according to 13 above, wherein thrombin is derived from a primate.
15. 14. The vaccine according to 14 above, wherein thrombin is derived from a human.

  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. can do. Therefore, according to the present invention, it was found that thrombin is useful as an adjuvant for vaccine antigens administered mainly transmucosally.

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

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.

FIG. 2 is a graph showing the anti-ovalbumin IgA antibody titer in nasal lavage fluid when mice were immunized with ovalbumin alone or a mixture of thrombin and ovalbumin.

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 mixed solution of thrombin and ovalbumin.

  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 primates or other animals, such as by low-temperature ethanol fractionation or chromatography. Alternatively, it can be prepared using a gene 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 Examples of the present application, recombinant human thrombin was obtained starting from a human prothrombin gene (Preparation Example 1; see WO2007 / 040162 for details). The thrombin can be derived from thrombin derived from mammals such as primates or other animals, preferably from humans, and can be appropriately selected depending on the animal (or human) to be immunized.

  For example, the human prothrombin gene is obtained by using a general genetic recombination technique (Molecular Cloning, A Laboratory Manual Second Edition. Cold Spring) described by Sambrook et al., Starting from total RNA, mRNA or genomic DNA extracted from human liver. Harbor Laboratory Press, NY, 1989). Today, various kits are commercially available and can be used. For example, for RNA extraction, reagents such as TRIzol reagent (Invitrogen), ISOGEN (Nippon Gene), StrataPrep Total RNA Purification Kit (Toyobo), for mRNA purification, mRNA Purification Kit (Amersham Bioscience), Poly (A) Kits such as Quick mRNA Isolation Kit (Toyobo), mRNA Separator Kit (Clontech), T-Primed First Strand Kit (Amersham Pharmacia), SuperScript plasmid system for cDNA synthesis and plasmid cloning (Invitrogen), cDNA Synthesis Kit (Takara Shuzo), SMART PCR cDNA Synthesis & Library Construction Kits (Clontech), Directional cDNA Library Construction systems (Novagen), GeneAmp PCR Gold (Applied Biosystems), etc. are used .

  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 sequence, a Kozak sequence, a secretion signal, etc. are added to the foreign gene. The promoter contained in the expression vector can be SV40 early, SV40 late, cytomegalovirus promoter, chicken β-actin, etc. when animal cells are used as the host. Anything can be used. When animal cells are used as a host, the chicken β-actin promoter system expression plasmid pCAGG (Japanese Patent Laid-Open No. 3-168087) is preferably used. As marker genes for selection and gene amplification, generally known 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 the host used in the present invention, cultured cells derived from various mammals can be widely used, and preferable examples include Chinese hamster ovary cells (CHO cells), human-derived 293 cells, and bird-derived cells. There are no particular restrictions on the method of gene transfer into animal cells. For example, the calcium phosphate method, the DEAE dextran method, the method using lipofectin-based liposomes, the protoplast polyethylene glycol fusion method, the electroporation method, and the like can be used. An appropriate method may be selected depending on the cell (Molecular Cloning (3rd Ed.), Vol 3, Cold Spring Harbor Laboratory Press (2001)). As the medium used for the culture, agar medium, liquid medium, and YMM-01, DMEM, RPMI, αMEM, etc. are used depending on the shape, but it can be appropriately selected depending on the cell, the purpose of culture, or the culture stage. good. In accordance with the protocol of each medium, those supplemented with serum, amino acids, vitamins, sugars, antibiotics, pH adjusting buffers and the like are used. The pH of the medium is set to 6 to 8, and the culture temperature is set to a range of 30 ° C to 39 ° C. The amount of medium, additives, and culture time are appropriately adjusted according to the culture scale.

  Human prothrombin-producing cells can be detected in the culture solution of cloned drug-resistant cells using a detection method that utilizes a specific reaction using an antithrombin antibody, such as dot blot, western blot, or ELISA. Can be obtained. 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 mass culture method, fed-batch culture, batch culture, or the like is generally used, but any method may be used.

  When purifying human prothrombin from such prothrombin-producing cells, purification methods generally used in protein chemistry, such as salting-out method, ultrafiltration method, isoelectric point precipitation method, electrophoresis method, ion Methods such as exchange chromatography, gel filtration chromatography, affinity chromatography, hydrophobic chromatography, and hydroxyapatite chromatography are used. Actually, since many types of impurities derived from cells coexist, it may be performed by a combination of the above methods.

  For the 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. Al., Biochemistry, 34, 1771-1778, 1995), and this was used to express the chicken β-actin promoter system used in the present invention. Integrate into 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 period of time during which the conversion reaction is sufficiently performed. 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 thus purified recombinant ecarin is allowed to act on human prothrombin and converted to human thrombin. As the reaction conditions, the same conditions as those for normal enzyme reactions are used. For example, the conversion of human prothrombin to human thrombin is completed by adding ecarin having a final concentration of 2-8 Unit / mL to 1000 μg / mL of human prothrombin and reacting at 36-38 ° C. for 1-4 hours.

  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 solution can be used as long as it retains a buffering ability mainly in the neutral region. The pH range is 7-9, preferably 8, and the concentration of the buffer 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.

  In order to highly purify human thrombin, it is further subjected to cation exchange chromatography. Examples of the cation exchanger include a sulfopropyl group type and a carboxymethyl group type, and may be appropriately selected and used. In this embodiment, SP Toyopearl 550C (Tosoh) is used. The conditions for adsorption, washing, and elution 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. When eluting human thrombin, it is carried out by increasing the salt concentration. Preferably 0.6M NaCl is used. By such a method, high-purity human thrombin can be obtained.

  The thus obtained human thrombin is subjected to enzyme chemical properties such as clotting activity, synthetic substrate (S-22338) cleavage activity, and sugar chain analysis. The clotting activity value represents a relative value obtained by comparing the time for clotting of fibrinogen with a standard calibration curve. As such a standard product, the Japanese Pharmacopoeia standard product and the WHO international standard product (NIBSC) Thrombin) is used. The S-2338 cleavage activity utilizes the reaction between thrombin and its specific substrate. The amount of p-nitroaniline released by cleavage of the synthetic substrate S-2238 by thrombin is determined by OD405 / 650. This is performed by measuring the change in the absorbance.

  For the measurement of sialic acid, N-Acetylneuraminic acid and N-Glycolylneuraminic acid are used as standard substances. By comparing the elution time from these ion exchange chromatography columns with the elution time of the test substance, Done. That is, each regression equation is created from the peak area at each concentration of the standard substance, and the measured value of the test substance is extrapolated to this to obtain the sialic acid concentration.

  The usefulness of the human thrombin thus obtained as an adjuvant is that serum obtained by immunizing various animals, for example, mice, rats, guinea pigs, hamsters, dogs, monkeys, etc., with a mixture of the human thrombin and an appropriate antigen It can be shown by examining the amount of IgG in it or the amount of IgA produced on the mucosal surface. The antigen is not particularly limited, and vaccine antigens against various bacterial and viral infections 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. When investigating the effect of local immunity, the inoculation route by transcutaneous, nasal drop and eye drop which does not require treatment to suppress the influence of proteolytic enzymes is preferable, but oral administration is performed by performing treatment such as encapsulation in capsules Is also available. As a solvent for mixing human thrombin and antigen, for example, physiological saline and phosphate buffer (PBS) are used, but are not particularly limited as long as they are not toxic to humans or animals.

  Specifically, for immunization, C57BL / 6 mice (SLC) were used, and a mixed solution of 0-40 μg human thrombin in PBS to 5 μg ovalbumin per mouse was measured once a week (total). 5 times), by dropping into the nasal cavity. 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, and the serum is collected.

  On the other hand, the mucosal surface cleaning solution is inserted into the nasal cavity opening from the trachea incised for the killed mouse, inserted into the nasal cavity opening from the throat to the nasal cavity, washed with PBS containing PMSF (Sigma), and the cleaning solution is collected. Intestinal lavage fluid is collected by the following method. First, the small intestine from the stomach pyloric region to the immediate front of the cecum is peeled without damaging the intestinal tract, and then collected in a dish containing a soybean trypsin inhibitor (GIBCO BRL) -containing EDTA solution (DOJINDO). This is pipetted and then centrifuged, and PMSF is mixed with the supernatant and then centrifuged. Protease inhibitor Cocktail set III and NaN3 (Katayama Chemical Co., Ltd.) are added to the supernatant. After mixing, the mixture is allowed to stand for 15 minutes. Fetal calf serum (Hyclone) is added to the supernatant, mixed, and used as an intestinal lavage solution.

  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., diluted serum is added and allowed to react. After washing, an HRP-labeled anti-mouse IgG goat antibody (American Qualax, A131PS) is reacted, and after washing, a chromogenic substrate solution TMB + (Dako) is added and reacted at room temperature in the dark. Color development is stopped with 1N sulfuric acid, and the absorbance at 450 nm (OD450 value) is measured. When measuring IgA in the intestinal tract and nasal lavage fluid, it is similarly 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.

  As a result, it was revealed that human thrombin has an 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. Therefore, human thrombin can be used as an adjuvant for various vaccines. It is particularly useful when an adjuvant effect on local immunity on the mucosal surface is expected.

  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 nature 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 a concentration range of 0.1 to 10 mg / mL. Preferably, it is effective to use 0.5 to 4 mg / mL thrombin.

  In addition, additives such as stabilizers and protective agents may be added to the formulation of vaccines containing human thrombin. Such additives include polysorbate 80, amino acids and stabilizers such as sugars such as lactose and sucrose, preservatives such as formalin, thimerosal, 2-phenoxyethanol, benzyl alcohol, benzethonium chloride and benzalkonium chloride. Further, when a sugar such as lactose or sucrose having an effect as an excipient is added, it can be formulated as a lyophilized preparation. Furthermore, it can also be used in combination with immunostimulants such as aluminum hydroxide, aluminum phosphate, mineral oil and non-mineral oil.

Preparation Example 1: Preparation of human thrombin An expression plasmid in which a human prothrombin gene was ligated downstream of a chicken β-actin promoter was transformed into a modified calcium phosphate method (C. Chen et al., Mol. Cell. Biol., 7, p. 2745). -2752, 1987) was introduced into Chinese hamster ovary (CHO) cells. Prothrombin-producing CHO cells were screened by ELISA using an anti-human thrombin antibody. The obtained prothrombin-producing cells were adapted to a serum-free medium, and then cultured in a large amount with a fermenter to purify human thrombin.

  A sample obtained by adding 2 volumes of 20 mM phosphate buffer (pH 7.4) to 3200 ml of the culture supernatant of prothrombin-producing CHO cells and filtering through a 0.22 μm filter was used as a sample. This sample was applied to a Q Sepharose Fast Flow (Amersham) 80 ml column equilibrated with 10 mM phosphate buffer (pH 7.4) containing 0.1 M NaCl 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.

  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. The 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 100 mM benzamidine hydrochloride.

  Next, a sample obtained by adding 2.5 volumes of 10 mM citrate buffer (pH 7.0) to 160 ml of benzamidine column eluate and equilibrated with 10 mM citrate buffer (pH 7.0) containing 0.2 M NaCl. Toyopearl 550C (Tosoh) 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 performed with 120 ml of 10 mM citrate buffer (pH 7.0) containing 0.6 N NaCl to obtain highly pure recombinant human thrombin. The protein concentration of the prepared recombinant human thrombin was 4.57 mg / mL by the Lowry method using BSA as a standard.

Immunization of ovalbumin (1) Preparation of ovalbumin solution Commercially available ovalbumin (Sigma, Grade IV, A2512-250MG) was dissolved in 10 mL of PBS (GIBCO BRL), and 200 μL was diluted 2.5 times with PBS (10 mg). / mL) and dispensed into 0.5mL tubes in 30μL aliquots and stored at -20 ° C or lower until use.

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

(3) Immunization group composition As shown in Table 1, 22 mice were divided into 4 groups, ovalbumin single immunization group (group 1) and ovalbumin and recombinant thrombin immunization group (groups 2, 3, 4) did.

(4) Preparation of immunized substances Group 1; 4 μL of the previously prepared ovalbumin solution was diluted with PBS to 80 μL.
Group 2: 8.8 μL of recombinant thrombin (4.57 mg / mL) was added to 4 μL of the above ovalbumin solution, and diluted with PBS to 80 μL.
Group 3: 35 μL of recombinant thrombin (4.57 mg / mL) was added to 4 μL of the above ovalbumin solution and diluted with PBS to 80 μL.
Group 4: 70 μL of recombinant thrombin (4.57 mg / mL) was added to 4 μL of the above ovalbumin solution and diluted with PBS to 80 μL.

(5) Immunization method and schedule Under ether anesthesia, 10 μL of each immunized substance prepared in (4) above was administered from the nasal cavity using Pipetman P-20 (Gilson) per mouse. The mice were immunized 6 times at weekly intervals.

(6) Blood collection and collection of nasal cavity and intestinal lavage fluid About 150 μL of blood was collected from the tail vein on the 4th to 5th day from the day of immunization 4 days before the first immunization and from the second immunization to the fifth immunization. On the 6th day after the 6th final immunization, all mice were anesthetized with pentobarbital sodium (Kyoritsu Seiyaku, Somnopentyl) and blood was collected from the abdominal vena cava and killed. The collected blood was transferred to Microtina (BECTON DICKINSON), sufficiently coagulated 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.

  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 1 mM PMSF (Sigma). The washing solution was collected in a 1.5 mL tube, 5 μL of protease inhibitor cocktail set III (Calbiochem, 539134) was added, and stored as a nasal washing solution at −80 ° C. until measurement.

On the other hand, the intestinal lavage fluid was prepared and collected by the following method and pretreated. First, the small intestine from the stomach under the pylorus to the immediate front of the cecum was removed without damaging the intestinal tract. An intestinal lavage solution (washed with 2 mL of PBS) exfoliated into a 10 cm diameter dish containing 1 mL of 0.2 mg / mL Soybean Trypsin inhibitor (GIBCO BRL) containing 50 mM EDTA (DOJINDO) was collected together with the intestinal contents. This was pipetted, transferred to a 15 mL tube, diluted to 3 mL with 0.2 mg / mL Soybean Trypsin inhibitor-containing 50 mM EDTA, and centrifuged at 2,500 rpm for 10 minutes at 4 ° C. 2 mL of the supernatant was transferred to a new 15 mL tube, added 1/100 volume of 100 mM PMSF, 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 μL of protease inhibitor cocktail set III and 10 μL of 1% NaN ₃ (Katayama Chemical Co., Ltd.) were added, and the mixture was allowed to stand for 15 minutes after mixing. 50 μL of fetal calf serum (Hyclone) was added thereto, mixed and stored as an intestinal rinsing solution at −80 ° C. until measurement.

Measurement of antibody titer in blood (1) Preparation of standard serum In order to compare the increase in anti-ovalbumin antibody among the above groups, standard serum of anti-ovalbumin antibody was prepared by the method shown below. Ovalbumin (100 μg / head) was administered intranasally to 5 mice (C57BL / 6, male) for 3 consecutive days, and this was treated as 4 courses at 1 week intervals. One week after the last course of administration, one nasal administration was given for booster immunization. Five days later, sera obtained by blood collection were pooled and used as standard sera.

(2) Measurement of anti-ovalbumin IgG antibody The ovalbumin used for immunization was diluted to 10 μg / mL with 0.1 M Carbonate buffer, pH 9.6, and added to a 96-well plate (Nunc, Maxisorp) at 100 μL / well. The solution was allowed to stand overnight at 0 ° C. to be solid-phased. The next day, each well was washed 5 times with 350 μL of PBS, and Block Ace (Dainippon Sumitomo Pharma Co., Ltd., hereinafter abbreviated as BA) diluted 4 times with PBS was added 350 μL / well and allowed to stand at room temperature for 2 hours. did.

  Two hours later, the 4-fold diluted BA was sufficiently removed, and the sample was diluted with BA (sample dilution solution) diluted 10-fold with 0.05% Tween20-containing PBS (PBST) and added at 100 μL / well (each sample was duplicated). ). After the reaction at 37 ° C. for 2 hours, each diluted serum added was discarded and washed 5 times with 350 μL / 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 dilution solution at 100 μL / well, 37 ° C, 1 hour Reacted. After the reaction, the labeled antibody dilution is discarded, washed 4 times with 350 μL / well of PBST and twice with the same amount of distilled water, and 100 μL / well of chromogenic substrate solution TMB + (Dako) is added. Reacted for 30 minutes. Thereafter, 1N sulfuric acid was added at 100 μL / well to stop color development, and the absorbance at 450 nm (OD450 value) was measured.

(3) Calculation of antibody titer of standard serum
Sera of 28 non-immunized mice (C57BL / 6) were diluted 50-fold with a sample diluent, and the OD450 values of each diluted sample were measured with duplicate. A value obtained by adding twice the standard deviation value to the average value of the measured OD450 values was set as a 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 cut-off value was defined as the antibody titer of the standard serum. In this test system, the antibody titer of the standard serum was set to 102,400 units because the cut-off value was exceeded up to a dilution of 102,400.

(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 with a specimen diluent to 0.5, 1, 2, 4, 8, 16, 32 units, and a standard for antibody titer measurement was prepared. Next, the mouse serum of each immunization group was diluted with a specimen diluent so as to be within the range of the prepared standard. The test sample prepared as described above was measured by the system shown in (2), and the anti-ovalbumin antibody titer of each test mouse serum was calculated from the standard units obtained and the standard line of OD450 values.

  FIG. 1 shows the change over time of the calculated average antibody titer of each immunization group. As shown in FIG. 1, it was confirmed that the anti-ovalbumin antibody appeared earlier and had a high antibody titer according to the dose of thrombin compared to the ovalbumin-only immunized group (Group 1). . In particular, in the group (Groups 3 and 4) in which 20 μg and 40 μg of thrombin were co-administered for one immunization per animal, the time of antibody appearance and antibody titer were almost the same, so the amount of thrombin administered simultaneously was 20 μg Was considered sufficient.

Measurement of IgA antibody titer in nasal wash (1) Measurement of specific IgA antibody Dilute ovalbumin used for immunization to 10μg / mL with 0.1M Carbonate buffer, pH 9.6, 96-well plate (Nunc, Maxisorp) 100 μL / well, and left at 4 ° C. overnight to solidify. The next day, each well was washed 5 times with 350 μL of PBS, BA diluted 4-fold with PBS was added 350 μL / well, and allowed to stand at room temperature for 1 hour.

  After 1 hour, remove the 4-fold diluted BA sufficiently, dilute the nasal wash of each individual 5 times with BA (specimen dilution) diluted with PBS containing 0.05% Tween20 (PBST) and add at 100 μL / well. (Each specimen was duplicated). After reaction at 37 ° C. for 1 hour, each diluted nasal wash was added and washed 5 times with 350 μL / well PBST. After washing, thoroughly remove the wash solution in the well, add HRP-labeled anti-mouse IgA goat antibody (Funakoshi, A90-103P) diluted 5,000 times with the sample diluent at 100 μL / well, 37 ° C, 1 Reacted for hours. After the reaction, the labeled antibody dilution is discarded, washed 4 times with 350 μL / well of PBST and twice with the same amount of distilled water, and 100 μL / well of chromogenic substrate solution TMB + (Dako) is added. Reacted for 5 minutes. Thereafter, 1N sulfuric acid was added at 100 μL / well to stop color development, and the absorbance at 450 nm (OD450 value) was measured.

(2) Measurement of total IgA antibody Total IgA antibody in the nasal cavity washing solution was measured using MOUSE IgA ELISA QUANTITATION KIT (E90-103) manufactured by Funakoshi according to the attached protocol. At the time of measurement, the nasal wash was diluted 20 times.

(3) Calculation of anti-ovalbumin IgA antibody titer in nasal lavage fluid In order to standardize the variation in the collection of the lavage fluid and compare the ovalbumin-specific IgA antibody titer, the OD450 value of each specimen obtained in (1) And the anti-ovalbumin IgA antibody titer with respect to all the IgA antibodies was computed by following Formula (I) from the measured value (converted into mg / mL) obtained by (2).
(I) Anti-ovalbumin antibody titer = (specific IgA antibody OD450 value × 5) ÷ total IgA antibody measured value (mg / mL)

  FIG. 2 shows the calculated anti-ovalbumin IgA antibody titers and standard errors of each group. Compared to the ovalbumin-only immunization group (Group 1), it is not significant in Group 2 (Thrombin 5 μg co-immunization group), but in Groups 2 and 3 (Thrombin 20 and 40 μg co-immunization groups respectively), specific IgA It was confirmed that the antibody titer increased.

Measurement of IgA antibody titer in intestinal lavage fluid (1) Measurement of specific IgA antibody The measurement was carried out in the same manner as the measurement method of specific IgA antibody in the nasal lavage fluid shown in Example 3. However, at the time of measurement, the intestinal lavage fluid was diluted 20 times with the sample diluent.

(2) Measurement of total IgA antibody As in Example 3, MOUSE IgA ELISA QUANTITATION KIT (E90-103) from Funakoshi was used. However, the intestinal lavage fluid was diluted 2,000 times during the measurement.

(3) Calculation of anti-ovalbumin IgA antibody titer in intestinal lavage fluid Similar to nasal lavage fluid, it was obtained in (1) in order to standardize the variation at the time of collecting the lavage fluid and compare the ovalbumin-specific IgA antibody titer. The anti-ovalbumin IgA antibody titer against all IgA antibodies was calculated from the OD450 value of each specimen and the measured value (converted to mg / mL) obtained in (2) by the following formula (II).
(II) Anti-ovalbumin antibody titer = (specific IgA antibody OD450 value × 20) ÷ total IgA antibody measured value (mg / mL)

  FIG. 3 shows the calculated anti-ovalbumin IgA antibody titers and standard errors of each group. Similar to the nasal lavage fluid, specific IgA antibody titers increased according to the amount of co-administered thrombin compared to the ovalbumin immunized group (Group 1) (Groups 2 and 3 versus Group 1). The increase in specific IgA antibody titer was similar at 20 and 40 μg of thrombin.

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

Claims (13)

An adjuvant for immunization containing thrombin as an active ingredient. It can induce IgG antibodies and IgA antibody according to claim 1, wherein the immune auxiliary adjuvant. The adjuvant for immune support according to claim 1 or 2 , wherein thrombin is derived from an animal. The adjuvant for immune support according to claim 3 , wherein the animal is a primate. The adjuvant for immune support according to claim 4 , wherein the primate is a human. Thrombin is a recombinant thrombin obtained by genetic recombination technology, according to claim 1 or immune auxiliary adjuvant 2 wherein. The adjuvant for immune support according to claim 6 , wherein the gene of thrombin is derived from an animal. The adjuvant for immune support according to claim 7 , wherein the animal is a primate. The adjuvant for immune support according to claim 8 , wherein the primate is a human. The adjuvant for immune assistance according to any one of claims 1 to 9 , which can induce local immunity. Local immunity is due to transdermal or transmucosal, 10. immunological auxiliary adjuvants according. Transmucosal nasal cavity, oral cavity, intestinal tract, or genitourinary tract is any mucosa surrounding the eye, according to claim 11, wherein the immune auxiliary adjuvant. A vaccine comprising a vaccine antigen and the adjuvant for immune support according to any one of claims 1 to 12 .

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