JP2006206539A - Adjuvant and vaccine using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe and stably suppliable adjuvant having high immunopotentiating actions and to provide a vaccine using the adjuvant. <P>SOLUTION: The adjuvant is obtained by mixing aluminum gel with 1-20% of a higher fatty acid, especially an unsaturated fatty acid. No expression of toxicity or adverse effect is recognized in the adjuvant and a raw material in a sufficient amount can inexpensively be procured. Immune responses to an antigen to be the object can safely and effectively be enhanced by administering the vaccine composed of the adjuvant and an antigen to an animal. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to the field of immunology, and particularly to a technique for preparing an immunopotentiating substance contained in a vaccine that is a biologic.

Substances added for the purpose of enhancing the immunity of a vaccine are collectively called an adjuvant. The terms and concepts of adjuvants have already been proposed in the 1920s (see Non-Patent Document 1). Examples of adjuvants include aluminum gel (see Non-Patent Document 2), nonionic block copolymer (see Non-Patent Document 3), and saponin (for example, Non-Patent Document 4, Patent Document 1, Patent Document 2 and the like). There are reports on various substances such as muramyl dipeptide (see Non-Patent Document 5) and carbohydrate polymer (see Non-Patent Document 6). In addition, it is not limited to such a single substance, and a formulation using fine particles formed by combining liquid paraffin, lipid, or polymer substance and a plurality of surfactants or adjuvant substances as a carrier has also been developed as an adjuvant. Including Freund's complete adjuvant (see Non-patent Document 7), Immunizing Complex (abbreviated as ISCOM) (see Non-Patent Document 8), or liposome (Non-Patent Document 9). Etc.) are known. As described above, various types of adjuvants have been devised, but the adjuvants that have been put into practical use at present are roughly classified into two types: aluminum gel adjuvant and oil adjuvant.

Aluminum gel is one of the indispensable materials for understanding vaccine adjuvants, with many years of clinical experience and accumulated data from human vaccines to animal vaccines (see Non-Patent Document 10). .) In particular, human vaccines have a track record in inactivated vaccines including influenza vaccines and tetanus toxoid vaccines. Aluminum gel is highly valued not only for its performance, but also for its safety, stable supply of raw materials, and its relatively low price. It is a vaccine not only for humans but also for domestic animals and pets. Has also been widely used to contribute to the prevention of human infectious diseases.

The oil adjuvant is a kind of emulsion type adjuvant prepared by mixing liquid paraffin or squalane with an oily base and various surfactants. In a broad sense, it is classified as a particulate adjuvant (see, for example, Non-Patent Document 11). Oil adjuvants are roughly divided into two types: water-in-oil type (water-in-oil type, hereinafter referred to as W / O type) and oil-in-water type (oil-in-water type, hereinafter referred to as O / W type). Is done. The W / O type oil adjuvant is particularly excellent in a depot (meaning “retention”) effect and can maintain an immune response for a long period of time. O / W type oil adjuvants are particularly known to have a strong effect of stimulating the presentation of antigen-presenting cells (meaning “antigen presentation”) and enhance a series of immune responses.

Means for combining these adjuvants have also been developed. For example, an animal vaccine preparation comprising an aluminum gel adjuvant and an oil adjuvant is known (see Patent Document 3). This is a mixture of two vaccine preparations with different adjuvants rather than a mixed adjuvant. In addition, as an adjuvant in which other substances are mixed in an aluminum gel, an adjuvant in which an aluminum salt is added to an adjuvant composed of squalene, glycerin or phospholipid and a surfactant (see Patent Documents 4 and 5), a trivalent metal. An oil adjuvant is added to an adjuvant composed of a salt of an ion and an organic anion (such as aluminum salicylate) (see Patent Document 6), as a biocompatible optimized adjuvant (SBA), and further to solid lipid-based particles. A composition containing an aluminum gel (see Patent Document 7), a composition containing a fatty phase and an organometallic gel (complex compound of an anionic polymer and a polyvalent metal cation) (see Patent Document 8), and the like. is there. However, none of the adjuvants used in these inventions uses the higher fatty acids themselves contained in the vaccine adjuvant of the present invention.

JP 2000-219638 A JP 11-322632 A JP-A-8-27028 JP-A-8-315162 JP-A-8-315163 JP 2000-507610 A Special table 2003-500365 gazette JP-T-2004-527615 Levis and Loomis, J Exp Med 40, 503, 1924 Gupta, Adv Drug Deliv Rev 32, 155-72, 1998 (1998) Hunter et al., Vaccine 9, 250-6, 1991 Oda et al., Biol Chem 381, 67-74, 2000 Lefrancier et al., Int J Pep Prot Res 11, 289-96, 1978. China et al., Vaccine 10, 551-7, 1992. Stuart-Tall, The Theory and Practical Application of Adjuvants, 1-19, 1995 Moline et al., Nature 308, 457-60, 1984 Alvink, Biochem Biophys Acta Rev Biomember 1113, 307-22, 1992 Ikehoff and Meyer, Vaccine 20 Suppl, S1-S4, 2002 Cox and Coulter, Vaccine 15, 248-56, 1997 Katayama et al., Vaccine 17, 2733-9, 1999 Oda et al., Vaccine 22, 2812-21, 2004 Aucor Turrier, Vaccine 19, 2666-72, 2001 Tizard, Veterinary immunology. An introduction. 2000

Although aluminum gel has been widely used as a vaccine adjuvant, it has been pointed out that the adjuvant effect cannot be satisfactorily exhibited in diseases that require the activation of Th1-type immunity due to its weak ability to activate Th1-type immunity. (See Non-Patent Document 12). In addition, it has been pointed out that the antibody production inducing ability of aluminum gel is slightly inferior to that of oil adjuvant (see Non-Patent Document 13). Thus, the aluminum gel alone cannot always cope with all antigens and diseases, and there remains room for improvement.

In addition, the oil adjuvant is added at a high content ratio of about 50 to 70% in the vaccine composition in the W / O type and about 15 to 25% in the O / W type (Non-patent Document 14). It has been pointed out that this causes injection marks and persistence at the vaccine injection site. The problem of injection marks and residues due to oil adjuvants leads to a decrease in the edible part, ie the value of meat, especially in livestock. In pet animals or humans, it is not preferable because it causes hair loss, swelling, lump, or pain at the site (see Non-Patent Document 15).

In addition, basic research has been progressing on other adjuvant materials other than aluminum gel and oil adjuvant, but toxicity, mass production, stable supply, cost, and various regulations due to chemical substances are barriers. As a result, there were many things that could not be put to practical use.

Although it is necessary to continue to search for new compounds having adjuvant activity, the screening often requires enormous time and labor. In addition, even when a new compound is obtained, the fact that the strength of side effects is unknown due to the fact that it is new and there is a possibility of having a strong toxicity can also be an uneasy material in early commercialization. The vaccine adjuvants that will be required in the future are not only immunostimulatory, but as mentioned above, it is obvious that it is desirable for the product to be safe, stable supply, and low cost. It is also important to reduce the cost of the process leading to the process and shorten the development period.

In addition, with the globalization of society, transportation of goods and transportation has been dramatically improved, and it has been realized that people can enjoy a comfortable social life. However, when serious infections broke out, it spread through these networks at a rate that could not have been anticipated in the past, and it was an era that could adversely affect social life. In particular, early development and production of vaccines are essential for the prevention of serious or emerging infectious diseases. In order to respond quickly to such social needs, it is an important concern that early practical use is possible as a condition required for adjuvants in the future.

Considering the above background, we thought that applying a method that takes advantage of the advantages of the prior art would be useful as a means to satisfy these conditions. Therefore, as a result of repeated studies focusing on the above-mentioned two types of typical and proven adjuvants, the inventors have devised the vaccine adjuvant of the present invention.

An object of the present invention is to further enhance the adjuvant activity while taking advantage of the advantages of the conventional aluminum gel, that is, having a relatively high adjuvant activity, safe, stable supply, and low cost. The present invention in which a higher fatty acid, which is a biological component, is added as a partner of an aluminum gel that has a clinical track record as a vaccine adjuvant is an invention that satisfies the conditions for solving the various problems described above.

A method itself for improving the performance of an aluminum gel by adding any substance has been already proposed, including a report by Gupta et al. (See Non-Patent Document 2), and is well known. However, the invention of a specific combination of an aluminum gel and a higher fatty acid is novel in the present invention, and there have been no reports on concentration studies or examples using various antigens.

In the present invention, aluminum phosphate or aluminum hydroxide is used as the aluminum gel. As these aluminum gels, those already used as vaccine adjuvants can be used. In addition, aluminum phosphate and aluminum hydroxide are mixed inadvertently by the reaction with phosphate ions or hydroxide ions that coexist in the antigen stock solution, or for the purpose of adjusting the adsorption effect of the antigen, etc. Aluminum gel may be prepared. Such phosphoric acid / aluminum hydroxide gel mixtures can also be used in the present invention. These aluminum gels have an aluminum equivalent concentration of 0.2 to 1.2 w / v%, preferably 0.3 to 0.5 w /%, as described in Examples (adjuvant preparation method) described later. Used in the range of v%.

The higher fatty acid used in the present invention needs to be uniformly dispersed from room temperature to body temperature when it finally becomes a vaccine. The higher fatty acid satisfying such conditions is selected from fatty acids having 12 to 30 carbon atoms and 1 to 6 unsaturated degrees. For example, unsaturated higher fatty acids such as oleic acid, linoleic acid, and linolenic acid are relatively easily available higher fatty acids that satisfy this condition. In addition, in recent years, docosahexaenoic acid, eicosapentaenoic acid, and the like, whose physiological activities have been attracting attention, are a kind of higher fatty acids that can be used. These higher fatty acids can be used in a single type, but can be used even when a plurality of higher fatty acids are mixed. It is indispensable to use a plurality of higher fatty acids in a mixed state depending on the purpose, for example, when adjusting the viscosity of the vaccine liquid when used as a vaccine. Moreover, the higher fatty acid obtained as a mixture from the beginning as a higher fatty acid material may be used. In addition, a saturated higher fatty acid having a relatively high melting point, such as stearic acid, palmitic acid, myristic acid, lauric acid or the like, may be used in combination with the aforementioned unsaturated higher fatty acid. In the present invention, the concentration of the higher fatty acid in the adjuvant solution was examined as an example in the range of 1.0 to 20.0 w / v%, but the adjuvant activity can be exhibited even at a concentration higher than that. . In other words, the concentration of higher fatty acid may be determined within a range as desired by repeatedly examining the antigen factor (antigen properties and amount), cost, or the strength of adjuvant activity. For example, recombinant protein antigens and peptide antigens having a relatively small molecular weight may have a lower antigenicity than general protein antigens. When preparing a vaccine with such an antigen, there are 1) a method of increasing the concentration of the antigen itself, or 2) a method of increasing the concentration of the adjuvant. The latter method can be selected particularly when it is difficult to secure the amount of antigen to be added.

In addition, a uniform and stable dispersion can be easily obtained by adding an appropriate amount of an appropriate substance, for example, a surfactant as a dispersant, to the higher fatty acid. Examples of the dispersant include nonionic surfactants such as polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, mannitol fatty acid ester, oligosaccharide fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene sorbite fatty acid ester. Polyoxyethylene castor oil / hardened castor oil, polyoxyethylene sterol, hydrogenated sterol, or the like can be used. These surfactants are available as commercial products and are handled, for example, by Nikko Chemicals Co., Ltd. under advanced quality control. The above surfactants may be used in combination of a plurality of surfactants in consideration of the case where a single type is added and in consideration of composition, addition amount, purpose of use, vaccine preparation procedure, or cost. In addition, the method itself using combining surfactant is a well-known means. Also, vegetable oils such as soybean oil, olive oil, grape seed oil, safflower oil, almond oil, corn oil, sunflower oil, sesame oil, hazelnut oil, apricot oil, kukui nut oil, jojoba oil, macadamia nut oil, Meadowfoam oil, rosehip oil, or the like can be added as an auxiliary. In addition, in order to achieve uniform dispersion, if a method of applying strong stirring energy to the adjuvant material of the present invention is used with a special dispersing device, for example, a high-speed stirring device described in JP 2000-15073 A, It is possible to suppress the amount of the additive such as the surfactant described above.

The higher fatty acid used in the present invention is one of the biological components indispensable for life support of animals and plants, and it is known that fatty acid metabolic pathways including β-oxidation exist. Therefore, if these fatty acids are used, it is predicted that they will have a high affinity for the living body, that is, they will be metabolized at a high rate after administration, and may not only be an adjuvant enhancing activity but also a residual adjuvant. Is expensive. It was confirmed that the characteristics and effects shown in the next section can be obtained by using the vaccine adjuvant of the present invention obtained by adding the higher fatty acid to the aluminum gel obtained by the method described above.

(1) Adjuvant activity : A basic test using mice confirmed that the addition of higher fatty acids elicited higher antibody-producing ability than the conventional aluminum gel adjuvant alone. Furthermore, in a practical test using pigs, it was confirmed by an attack test that not only the effect of enhancing the antibody production ability but also the effect of reducing the lung lesion area rate of porcine mycoplasma pneumonia was enhanced.

(2) Safety : Observing the change in body weight after injecting the immunogen into the abdominal cavity of the mouse, the change in the body weight of the mice to which the higher fatty acid of the present invention was added was the same as that of the conventional aluminum gel adjuvant group. There was no significant difference from the fluctuation, confirming low toxicity. In the swine test, a transient fever was observed after the injection of the prototype vaccine, but the fever temperature and fever duration were comparable to the conventional aluminum gel adjuvant simple group. There were no clinical signs of loss of energy, loss of appetite, vomiting, diarrhea, or sitting after injection. In addition, no injection mark was observed at the injection site. From the above test results, it was found that the adjuvant obtained by adding a higher fatty acid to the aluminum gel of the present invention is a highly safe adjuvant equivalent to the conventional aluminum gel adjuvant alone.

(3) Stable supply : Aluminum, which is a raw material for aluminum gel, is one of the elements present in large quantities on the earth as one of the mineral resources, and is obtained as an underground reserve resource. Although there are some market fluctuations depending on social conditions, there is currently no depletion of the raw material itself, and stable supply as an adjuvant is possible. Higher fatty acids are usually obtained from animals or plants. It is also synthesized from petroleum. Higher fatty acids are not only used, but are indispensable as raw materials for various ester compounds. Since ester compounds are in demand in all industrial fields such as pharmaceuticals, quasi-drugs, foods, and industrial products, the supply route of higher fatty acids as raw materials is sufficiently secured at present.

(4) BSE measures : In recent years, the problem of BSE so-called mad cow disease has begun, and it has been demanded that raw materials derived from bovine organs should not be used as much as possible as raw materials for foods and pharmaceuticals. Even if it is unavoidably used, 1) use cattle from the country of origin that has been proven to be a country without BSE, and 2) contain as little prion as the causative substance of BSE, or contain no prion. It is required to use an organ as a raw material, and 3) to perform appropriate heating, purification, and processing for decomposing and eliminating prions. In the present invention, in consideration of use as an adjuvant for livestock vaccines in particular, higher fatty acids are premised on the use of raw materials not derived from bovine organs, such as synthetic products derived from petroleum or plant-derived products. Higher fatty acids derived from bovine organs are basically not used. Therefore, BSE does not occur in the administration subject or in humans or animals that eat livestock when the administration subject is livestock, due to the adjuvant of the present invention. The adjuvant of the present invention is an adjuvant that not only has high adjuvant activity, but also consists of a material that is highly safe, can be stably supplied, and satisfies BSE countermeasures in addition to low cost. Examples of the above verification and confirmation will be shown below.

(1) Basic test using mice: Examples of the performance of the adjuvant of the present invention using mice are shown below. The practical use of an adjuvant is advanced on the assumption that it is used in vaccines for humans, livestock, and pets (see, for example, Non-Patent Document 7). It is known that the immune response and toxicity differ depending on the animal species (see, for example, Non-Patent Document 2). However, in basic research of adjuvants, it is fundamental to use small animals such as mice first, as with other pharmaceuticals. Has been.

1.1 Materials and Methods 1.1.1 Preparation of Adjuvant : Aluminum gel is 0.4 w / v% in terms of aluminum and higher fatty acids are 1.0, 2.0, 5.0, 10.0, or 20. 0 w / v% and polysorbate 80 as a higher fatty acid dispersion aid in an amount equivalent to higher fatty acid, added to sterilized purified water, and using a disperser TK Robotics (Special Machine Industries Co., Ltd.) at room temperature 3 An adjuvant solution was obtained by uniformly dispersing at 1,000 rpm for 5 minutes. The aluminum gel was studied based on aluminum phosphate and compared with aluminum hydroxide. The higher fatty acid was studied based on oleic acid, and in the examination of the type of higher fatty acid, palmitooleic acid, linoleic acid, and linolenic acid were used in addition to oleic acid.

1.1.2 Preparation of antigen : Ovalbumin (hereinafter referred to as OVA) (manufactured by Sigma-Aldrich) was dissolved in physiological saline, and then centrifuged at 4 ° C., 100,000 × g for 90 minutes to obtain insoluble and aggregated components. Was removed. This centrifugal supernatant was used as a soluble OVA antigen.

1.1.3 Preparation of immunogen : 30 mL of the above-described adjuvant solution and an OVA antigen solution were mixed and prepared so that the OVA amount was 100 μg / mL in the immunogen, and 100 mL of the immunogen was prepared. Each 0.1 mL of this immunogen was injected once into the leg muscles of each group of 5 6-week-old ddY mouse females (Japan SLC), and the anti-OVA mouse antibody titer in the serum 4 weeks later Was measured.

1.1.4 Measurement of antibody titer in serum : An antibody titer measurement method carried out with reference to a report by Oda et al. (For example, Non-patent Document 13, Patent Documents 1 and 2) will be described. Enzyme linked immunosorbent assay (hereinafter referred to as ELISA) was performed using a polycarbonate 96-well round bottom plate (manufactured by Greiner BioOne). A 0.1% (w / v) OVA solution prepared with a carbonate buffer (pH 9.6) was dispensed at 150 μL per well and subjected to solid phase treatment at 4 ° C. overnight. The plate was washed 3 times with 0.05% Tween 20 phosphate buffered saline (hereinafter referred to as Tween PBS), and 100 μL of mouse test serum diluted 100-fold with Tween PBS was dispensed at 30 ° C. Sensitized for 1 hour. After washing three times, 100 μL each of anti-mouse total IgG-POD labeled antibody (Biosource) diluted with Tween PBS was dispensed and sensitized for 1 hour at 30 ° C. After washing 3 times again, 100 μL of 10 mg / mL orthophenylenediamine solution (0.003% hydrogen peroxide solution added) prepared with phosphate citrate buffer (pH 5.6) was dispensed. After sensitization at 30 ° C. for 1 hour, 50 μL of 1 mol / L sulfuric acid aqueous solution was dispensed to stop the color reaction. The absorbance at a wavelength of 492 nm was used as the anti-OVA mouse ELISA antibody titer in serum.

1.1.5 Mouse Acute Toxicity Test : The toxicity of sensitized materials was examined in accordance with the abnormal toxicity denial test method stipulated in the Animal Biologics Standards published by the Ministry of Agriculture, Forestry and Fisheries (p650, 2002). . A group of 10 5-week-old ddY mice (weight approximately 24 g) (Japan SLC) was injected intraperitoneally with 0.5 mL of sensitizing material, and the weight of each individual was measured once a day for 7 days. The changes in weight recovery were compared between the groups.

1.2 Results 1.2.1 Amount of higher fatty acid added : The serum antibody ELISA antibody titer was higher in the adjuvant group to which higher fatty acid was added than in the aluminum gel plain immunogen injection group. . Further, among the groups to which higher fatty acids were added, the group with the larger amount of addition showed a higher value tendency (FIG. 1 above). Regarding mouse acute toxicity, none of the mice experienced any extreme weight loss after injection and recovered on the fourth day after injection. Strong toxicity was not recognized within the range of the higher fatty acid content examined, and there was no significant difference from the immunogen injection group of aluminum gel alone (FIG. 2). From the above results, it was confirmed that the addition of a higher fatty acid to the aluminum gel can further enhance the adjuvant activity of the aluminum gel while maintaining the same safety as that of the conventional aluminum gel adjuvant.

1.2.2 Types of higher fatty acids and aluminum : The above-described examination was a test in which one oleic acid as a higher fatty acid and one aluminum phosphate as an aluminum gel were combined. Then, next, the case where five types of higher fatty acids (oleic acid, palmitooleic acid, linoleic acid, and linolenic acid) and aluminum hydroxide in addition to aluminum phosphate was used as the aluminum gel was examined. As a result, even if any higher fatty acid was added, the same adjuvant activity enhancement effect as that of oleic acid was shown (FIG. 3). It was also confirmed that the same effect of enhancing the adjuvant activity as that of aluminum phosphate was obtained with aluminum hydroxide to which higher fatty acid was added (FIG. 4).

1.2.3 Mixing example of higher fatty acids : As an application example of the above-mentioned adjuvant, a composition in which higher fatty acids to be added to aluminum gel are composed of a plurality of types and vegetable oil is added is described as an example. An adjuvant could not be prepared with stearic acid alone, but an adjuvant could be prepared by adding oleic acid and soybean oil (Japanese Pharmacopoeia) as a vegetable oil. Based on this, an immunogen was prepared and injected into mice. As a result, an increase in serum antibody titer could be confirmed. The above results are shown in the following table. This result shows that the adjuvant of the present invention can be prepared by using an appropriate amount of a plurality of higher fatty acids and dispersion aids, even if the composition uses higher fatty acids that were originally difficult to prepare due to a high melting point. It was confirmed that there was.

複数の高級脂肪酸を混合した場合に得られる効果（アジュバント物性の改良）。

Effect obtained by mixing a plurality of higher fatty acids (improvement of physical properties of adjuvant).

(2) Application test in pigs : Although the sensitivity may vary depending on the animal species, the effect of enhancing the adjuvant activity of aluminum gel obtained by adding higher fatty acids is similar to the above results even if the animal species changes. It was predicted. Therefore, in conjunction with the confirmation of the effects in animal species other than mice, the following are the results of studies on animal vaccines, particularly swine vaccines, as one of the best vaccine candidates for practical use.

Chronic respiratory disease is one of the most important issues in pig farming management. Among swine respiratory diseases, both mycoplasma pneumonia and atrophic rhinitis caused by Pasteurella multocida are prevalent before and after weaning, and serious respiratory symptoms are caused by mixed infection with other pathogens. Moreover, swine influenza becomes a hotbed for infection of various pathogens involved in respiratory infections including the above-mentioned pathogens, and also seriously causes respiratory symptoms. Therefore, for the purpose of reducing respiratory complex infection before and after the weaning period, a mixed vaccine for fattening pigs with the addition of Mycoplasma hyopneumoniae (hereinafter referred to as Mhp), Pasteurella multocida (hereinafter referred to as Pm), and swine influenza antigens And the usefulness thereof was examined to evaluate the performance of the adjuvant of the present invention.

2.1 Materials and Methods 2.1.1 Preparation of prototype vaccine: Preparation of the prototype vaccine was in accordance with the above-described immunogen preparation method. Aluminum phosphate (0.4 w / v% in the adjuvant solution in terms of aluminum) was used as the aluminum gel, and oleic acid (10 w / v% in the adjuvant solution) was used as the higher fatty acid. As antigens, Mycoplasma hyopneumoniae (Mhp) inactivated whole cells, Pasteurella multocida (Pm) inactivated skin necrosis toxin (toxoid), and inactivated influenza virus (hereinafter referred to as SIV) were used. Specific methods for preparing each antigen are shown below.

2.1.2 Preparation of Mhp inactivated whole cell antigen stock solution : Mhp was cultured by aeration and agitation in Friis liquid medium (Ross et al., Am J Vet Res 45, 1899-1905, 1984) at 210C for 210 hours and collected. Formalin was added to the cultured bacterial solution at a rate of 0.1 vol%, and the mixture was allowed to stand at 37 ° C. for 24 hours or more to be inactivated. After inactivation, the bacterial solution was collected by centrifugation. A suspension of the centrifugal sediment obtained by converting the number of viable bacteria before inactivation using a physiological saline solution to 1.0 × 10 ¹⁰ CCU or more / mL was used as an Mhp stock solution.

2.1.3 Preparation of Pm toxoid stock solution : Pm was cultured with agitation at 37 ° C. for 60 hours in a peptone liquid medium made from soybean, and the centrifuged supernatant was concentrated 30 times by ultrafiltration (fractionated molecular weight 300,000). did. Formalin was added to the supernatant obtained by centrifugation at 10,000 × g for 30 minutes at a rate of 0.5 vol%, sensitized at 37 ° C. for 3 days, and inactivated. A diluted Pm toxoid solution was obtained by diluting this antigen solution to 10,000 U / mL in terms of skin necrosis toxin value before inactivation.

2.1.4 Preparation of inactivated SIV stock solution : SIV Kyoto strain (H1N1) and Wadayama strain (H3N2) (strain used as an antigen of “Kyoto Microken” swine influenza vaccine) Inoculated into the cavity, cultured at 37 ° C. for 3 days, and then allowed to stand at 4 ° C. overnight to collect the infected allantoic fluid. After concentration by ultrafiltration (fractional molecular weight 300,000), formalin was added at a ratio of 0.04 vol%, and sensitized for 48 hours at 22 ° C. to obtain an inactivated stock solution. Further, the antigen fraction was collected by zonal centrifugation at 100,000 × g, and concentrated and prepared as an influenza virus stock solution.

2.1.5 Antigen Amount in Prototype Vaccine: The final antigen amount in 10 mL of the prototype vaccine is the number of viable bacteria before Mhp inactivation 1.0 × 10 ¹⁰ CCU, skin necrotic toxin activity before Pm inactivation 10,000 U Inactivated SIV Kyoto strain (H1N1) stock solution 1,280 HA and Wadayama strain (H3N2) stock solution 1,280 HA were added. As a prototype vaccine, one using an adjuvant obtained by adding oleic acid to aluminum phosphate gel and one containing aluminum phosphate gel alone were prepared.

2.1.6 Injection into pigs and clinical observation : Allocate 5 pigs per group, 2 mL of the above-mentioned prototype vaccine, 23-day-old weaning pigs (specific pathogen-free pigs, steers) Were injected twice at 4-week intervals. In the control group, the same amount of antigen was used, and a prototype vaccine using an aluminum phosphate gel alone as an adjuvant was injected under the same vaccine program. A clinical observation was performed after each injection, and the antibody titers in the serum against each antigen at the time of the first injection and 4 weeks after the second injection were measured. At the time of dissection, the presence or absence of an injection mark of the vaccine at the injection site was observed.

2.1.7 Mhp challenge test : In the Mhp challenge test, the group was assigned according to the above, and after 4 weeks after the second injection, live Mhp was inoculated via the nasal cavity, and mycoplasma pneumonia of the lung 4 weeks later The lesion area rate was calculated. The attack test was in accordance with the technique described in the report by Shim et al. (Vaccine 21, 532-7, 2003). In the Mhp challenge test, a negative control group not injected with the prototype vaccine was also set.

2.1.8 Measurement of antibody titer in serum (a) Measurement of anti-Mhp antibody titer and anti-Pm toxoid antibody titer : With reference to the method for measuring ELISA antibody titer in mouse serum described above, the method for measuring porcine serum was Reorganized and implemented. Using a polycarbonate 96-well round bottom plate (Greiner BioOne), Mhp-inactivated whole cell disruption ELISA antigen solution (Mhp antibody titer) or Pm skin prepared with carbonate buffer (pH 9.6) Necrotic toxin (Pm antibody titer measurement) was dispensed at 150 μL per well and subjected to solid phase treatment at 4 ° C. overnight. The plate was washed 3 times with Tween PBS, and 100 μL of porcine test serum diluted 2-fold with Tween PBS was dispensed and sensitized at 30 ° C. for 1 hour. After washing three times, 100 μL each of anti-porcine total IgG-POD labeled antibody (Cappel) diluted with Tween PBS was dispensed and sensitized at 30 ° C. for 1 hour. After washing 3 times again, 100 μL of 10 mg / mL orthophenylenediamine solution (0.003% hydrogen peroxide solution added) prepared with phosphate citrate buffer (pH 5.6) was dispensed. After sensitization at 30 ° C. for 1 hour, 50 μL of 1 mol / L sulfuric acid aqueous solution was dispensed to stop the color reaction. Absorbance at a wavelength of 492 nm is measured. The highest dilution factor of sera showing an absorbance of 0.5 or higher was defined as anti-Mhp porcine ELISA antibody titer or anti-Pm porcine ELISA antibody titer.

(B) Measurement of influenza HI antibody titer : It was measured by a hemagglutination inhibition (hereinafter referred to as HI) test. Test serum is treated with RDE and chicken erythrocytes. This is diluted 2-fold with PBS, and an equal amount of 8 units of hemagglutinating antigen in 0.2 mL is added to 0.2 mL of each diluted solution, followed by treatment at room temperature for 60 minutes. To this, 0.1 mL of 2 vol% chicken erythrocyte suspension is added and allowed to stand at room temperature for 60 minutes to determine the presence or absence of hemagglutination. The highest dilution factor of serum that suppresses hemagglutination is defined as the HI antibody titer.

2.2 Results 2.2.1 Clinical observation after injection : At the time of the first injection, individuals with fever were observed in the higher fatty acid added experimental vaccine injection group after 1 hour. The fever recovered as a peak at 9 hours after injection and recovered to normal temperature at 18 hours. In the group of aluminum phosphate gel alone, the peak of fever was from 3 to 6 hours, which was earlier than in the group injected with the higher fatty acid added prototype vaccine. However, there was no significant difference in the overall body temperature fluctuation between the two groups. At the time of the second injection, the peak of fever in both groups was 6 hours after the injection, and then recovered to the original body temperature at 18 hours as in the first injection (FIG. 5). In addition, none of the individuals showed signs of loss of energy, loss of appetite, diarrhea, vomiting, or sitting after injection. Furthermore, during the subsequent rearing period, no abnormal clinical symptoms were observed with the prototype vaccine. The results are shown in the table.

本発明のワクチン注射後の豚の臨床症状観察記録。

The clinical symptom observation record of the pig after the vaccine injection of this invention.

2.2.2 Antibody response : Each antibody titer against Mhp, Pm, and SIV was higher in the higher fatty acid-added prototype vaccine injection group than in the aluminum gel simple group. Regarding Pm, the minimum effective antibody titer (anti-Pm porcine ELISA antibody titer 2 or higher) necessary for prevention of onset described in the previous report ("Kyoto Microken" Pigwin AR-BP2 technical data collection, 2003), and for SIV, onset prevention The minimum effective antibody titer (HI antibody titer of 80 or higher) required for the above was satisfied (FIG. 6 above).

2.2.3 Mhp challenge test : The lesion area rate of mycoplasma pneumonia exhibited by individuals in the high-fatty acid added experimental vaccine injection group was lower than that of the aluminum gel simple group, and was found to exhibit a stronger preventive effect. (FIG. 7).

2.2.4 Vaccine residue at the injection site : The individual in the challenge test was observed for the presence or absence of cervical muscle injection marks at the time of dissection. At the time of dissection, the second month after the injection (= second injection site) and the third month after the injection (= first injection site) were observed, but there was no marked change in the injection site muscle in any individual. There were no abscesses, muscle discoloration, or fibrotic tissue. From this result, it has been found that the adjuvant of the present invention has already disappeared macroscopically at the most 2 months after the injection. In the adjuvant of the present invention, it was confirmed that the injection mark disappeared in the same period as in the case of the aluminum gel alone. The results are shown in the table.

本発明のワクチン注射部位における解剖時の肉眼所見（注射痕の有無）。

Macroscopic findings at the time of dissection at the site of injection of the vaccine of the present invention (presence or absence of injection marks).

From the above results, the adjuvant effect of the present invention was confirmed to be enhanced in the animal species other than mice, for example, in pigs in the examples. It was also found that the side effects were as low as the conventional aluminum gel, and the usefulness of the present invention was proved. In this study, the effect of the prototype vaccine on a total of three different antigens including inactivated bacterial cells, toxoid, and virus, and thus the adjuvant effect of the adjuvant of the present invention on a wide range of antigen types was confirmed. . At the same time, it was confirmed that the basic test results in mice could be applied to heterogeneous animals (pigs in the examples), so it was not limited to pigs, but domestic animals such as cattle and chickens, dogs and cats, etc. It can be applied to vaccines for humans, including pet animals, and to farmed fish and fish vaccines for which preventive hygiene measures have been strengthened in recent years.

Relationship between higher fatty acid concentration in adjuvant and anti-OVA antibody titer in mouse serum. Aluminum phosphate was used for the aluminum gel, and oleic acid was used for the higher fatty acid. Relationship between the number of days elapsed after the intraperitoneal injection of the adjuvant of the present invention and the average mouse body weight. Using aluminum phosphate as the aluminum gel and oleic acid as the higher fatty acid, the relationship between the amount of higher fatty acid added and weight fluctuation was compared. Anti-OVA antibody titer in mouse serum when various higher fatty acids are used. The higher fatty acid was added at 10 w / v% in the adjuvant solution. Anti-OVA antibody titer in mouse serum when two types of aluminum gel are used. The aluminum gel amount was 0.4 w / v% in the adjuvant solution in terms of aluminum. Changes in body temperature of pigs after injection of the vaccine of the present invention. Results of antibody titer in pigs 4 weeks after the second injection of the prototype vaccine of the present invention. Lesions of mycoplasma pneumonia (Mhp challenge test)

Claims (11)

An adjuvant consisting of aluminum gel and higher fatty acids. The adjuvant according to claim 1, wherein the aluminum gel contains 0.2 to 1.2 w / v% in terms of aluminum and 1.0 to 20.0 w / v% higher fatty acid. The adjuvant according to claim 1, wherein the aluminum gel is aluminum phosphate. The adjuvant according to claim 1, wherein the aluminum gel is aluminum hydroxide. The adjuvant according to claim 1, wherein the higher fatty acid is an unsaturated higher fatty acid. The adjuvant according to claim 1, wherein the unsaturated higher fatty acid has 12 to 30 carbon atoms and 1 to 6 degrees of unsaturation. The adjuvant according to claims 1 to 6, wherein the unsaturated higher fatty acid is selected from at least one selected from oleic acid, palmitooleic acid, linoleic acid and linolenic acid. Furthermore, the adjuvant of Claims 1-7 containing a vegetable oil. The adjuvant according to claim 8, wherein the vegetable oil is soybean oil. A vaccine comprising the adjuvant according to claim 1 and an antigen. The vaccine according to claim 10, comprising at least one bacterial component and / or virus-derived component as an antigen.

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