JP3045168B1 - Adjuvants and vaccines using them - Google Patents

Abstract

Abstract: PROBLEM TO BE SOLVED: To provide an adjuvant with high safety, high adjuvant activity and high stability for livestock, poultry, pet animals, and the like, and furthermore, to use this adjuvant for water-based or oil-based high-performance and stable Provide vaccines at low cost. SOLUTION: By using an ester, amide or ester-amide mixture of a high molecular weight polysaccharide and a fatty acid as an adjuvant or an emulsion stabilizer in an aqueous vaccine or an oil-based vaccine, the efficacy, safety and stability of a vaccine not found in conventional adjuvants are obtained. Nature can be secured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an adjuvant which can be suitably used as a vaccine for livestock, poultry or pets, and a vaccine using the same.

[0002]

2. Description of the Related Art Inactivated or component vaccines for humans or animals may be added with a composition called an adjuvant for the purpose of enhancing the immune effect. An adjuvant has been added. Representative examples of adjuvants include aluminum phosphate gel, aluminum hydroxide gel, alum, calcium hydroxide gel, quillajasaponin, and oil adjuvant, which have already been widely put into practical use. However, these existing adjuvants had the following problems.

[0003] In the case of aluminum gel and calcium gel, depending on the condition and properties of the antigen, pH, salt concentration, etc., the adsorption of the antigen to the gel matrix is poor, and sufficient effects cannot be obtained in many cases. Aluminum has also been linked to Alzheimer's disease. Quiraya saponin has a relatively excellent adjuvant effect, but its hemolytic activity and tissue toxicity are extremely high, and its application range is limited. The W / O type oil adjuvant is excellent in adjuvant effect and antibody persistence, but has a strong inoculation reaction, and may cause induration, abscess, necrosis, etc. at the site of vaccination.

[0004] As vaccine adjuvants in the livestock industry, W / O type oil adjuvants mainly composed of mineral oil have been developed and put to practical use in chickens, in addition to gel adjuvants. However, this type of adjuvant has a very strong inoculation local reaction, and is difficult to apply to mammals except for special uses such as sow immunization. In particular,
(1) Japanese Patent Publication No. 6-81731 discloses an oil-in-water type animal vaccine comprising anhydrous mannitol oleate and liquid paraffin. (2) Japanese Patent Application No. 5-255117 discloses: O comprising at least one or more hydrophobic synthetic lipopolysaccharide, surfactant and aqueous solute
/ W vaccine adjuvants have been disclosed (3)
Japanese Patent Publication No. 7-509733 discloses a liquid emulsion for water-in-oil vaccines containing a metabolizable oil. However, since all of them use a low molecular weight hydrophilic surfactant, there are problems such as strong cell damage and cell infiltration at the inoculation site, fever and edema-like disease at the injection site. Further, the W / O / W type adjuvant vaccine has a drawback that it cannot be stored for several months or more due to lack of emulsion stability.

As described above, it has been difficult for conventional adjuvants to satisfy all of safety, efficacy and stability.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an adjuvant having high safety and high adjuvant activity and stability for livestock, poultry, pets, and the like. An object of the present invention is to provide an inexpensive aqueous or oily high-performance and stable vaccine. In addition, the adjuvant activity refers to a function of stimulating an immune mechanism and enhancing an immune response to an antigen.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the problems in the conventional adjuvants and vaccines using the same as described above. The present inventors have found that the use of an ester-amide mixture as an adjuvant or an emulsion stabilizer in an aqueous vaccine or an oil-based vaccine can ensure the efficacy, safety and stability of a vaccine that has not been used in conventional adjuvants. Was.

That is, the adjuvant according to the present invention is an aqueous or oily adjuvant characterized in that it contains at least one kind of ester, amide or ester-amide mixture of a high-molecular polysaccharide and a fatty acid. In addition, the ester / amide of the high molecular polysaccharide and the fatty acid refers to a compound in which both the part where the fatty acid is ester-bonded and the part where the fatty acid is amide-bonded are mixed with the sugar chain of the high molecular polysaccharide. .

The high molecular weight polysaccharide has a sugar chain of 100 to 100.
Those composed of 5,000 monosaccharides are preferred.
In the case of a high-molecular-weight polysaccharide fatty acid ester, glucose, galactose, mannose and the like are preferable as the monosaccharide constituting the high-molecular-weight polysaccharide, and the high-molecular-weight polysaccharide fatty acid amide or the high-molecular weight polysaccharide fatty acid ester / amide is preferable. In this case, glucosamine, galactosamine, neuraminic acid and the like are preferable as the constituent monosaccharide of the sugar chain of the high molecular polysaccharide.

As the fatty acid, in the case of a high molecular weight polysaccharide fatty acid ester, a linear saturated fatty acid having 2 to 24 carbon atoms,
A branched saturated fatty acid having 4 to 24 carbon atoms or 6 to 22 carbon atoms
In the case of a high molecular weight polysaccharide fatty acid amide or a high molecular weight fatty acid ester / amide, a linear saturated fatty acid having 3 to 24 carbon atoms,
And unsaturated fatty acids having 6 to 22 carbon atoms are preferred. Further, it is preferable that the average introduction number of the fatty acid per one constituent monosaccharide of the high molecular polysaccharide is in the range of 0.001 to 2.0.

The high-molecular polysaccharide fatty acid esters include:
High molecular weight polysaccharide is cellulose, high molecular weight polysaccharide is mannan, high molecular weight polysaccharide is pullulan, and high molecular weight fatty acid ester / amide is high molecular weight polysaccharide is chitosan. Can be.

[0012] The vaccine according to the present invention is characterized by containing the adjuvant as described above. Also,
The vaccine of the present invention is an aqueous vaccine obtained by adding the above-mentioned ester of a polysaccharide and a fatty acid, an amide or an ester / amide mixture, the ester of the high-molecular polysaccharide and a fatty acid,
O / W type or W / O containing amide or ester-amide mixture as adjuvant or emulsion stabilizer
A / W type oil-based vaccine may be used.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The details of the present invention will be described below.
Monosaccharides constituting the sugar chains of the high molecular polysaccharide of the high molecular polysaccharide and fatty acid ester, amide or ester / amide mixture (hereinafter, these are collectively referred to as “high molecular polysaccharide fatty acid ester or amide”) used in the present invention. The number of 1
The range is from 00 to 5,000, preferably from 100 to 5,000.
4,000, more preferably 100 to 3,20
There are zero. By making these preferred ranges,
Aqueous and oleaginous vaccines are more stable and have higher adjuvant activity. The high-molecular-weight polysaccharide fatty acid ester or amide used in the present invention may be a mixture of compounds having different numbers of constituent monosaccharides in the sugar chain of the high-molecular-weight polysaccharide, but mainly contains those having the number of constituent monosaccharides in the above range. It is preferable that the number of constituent monosaccharides of the high molecular polysaccharide be within the above range is 50% by weight or more based on the total weight of the high molecular weight polysaccharide fatty acid ester or amide. It is preferred that all of the molecular polysaccharide fatty acid esters or amides have the number of monosaccharides constituting the sugar chain of the high molecular polysaccharide within the above range.

Among the high molecular weight polysaccharide fatty acid esters or amides used in the present invention, the high molecular weight polysaccharide fatty acid ester is a compound in which the high molecular weight polysaccharide is glucose, galactose or mannose alone or a compound based on two or more of these monosaccharides. preferable. As the high-molecular-weight polysaccharide fatty acid amide or the high-molecular-weight polysaccharide fatty acid ester / amide mixture, a high-molecular-weight polysaccharide is preferably a compound based on a single amino sugar such as glucosamine, galactosamine, neuraminic acid or the like, or a combination of two or more of these monosaccharides. In addition, the above-mentioned monosaccharide is basically used.
Specifically, it means that the monosaccharide is contained in 50 mol% or more, preferably 100 mol%, of all the monosaccharides constituting the sugar chain of the high molecular polysaccharide.

The structure of the basic sugar chain of the high-molecular polysaccharide in the high-molecular polysaccharide fatty acid ester or amide used in the present invention may be a straight-chain structure or a branched structure.

Specific examples of the high molecular polysaccharide include dextran, cellulose, pullulan in which the monosaccharide constituting the sugar chain is glucose, mannan in which the monosaccharide constituting the sugar chain is mannose, and glucosamine are the monosaccharides constituting the sugar chain. And the like.

Examples of the high molecular polysaccharide having mannose as a basic sugar chain include mannan such as ivory palm mannan, yeast mannan, seaweed mannan, and suppressor mannan.

Examples of the high molecular polysaccharide having glucosamine as a basic sugar chain include chitosan and soluble chitosan obtained by partially hydrolyzing it to make it water-soluble.

The high molecular weight polysaccharide having maltotriose, which is a glucose oligosaccharide, as a basic sugar chain includes pullulan derived from black yeast.

Further, examples of the high molecular polysaccharide based on a complex sugar chain containing a plurality of the above constituent monosaccharides include glucomannan, galactomannan and the like.

The fatty acid in the ester or amide of the high molecular weight polysaccharide fatty acid used in the present invention includes a linear fatty acid, a branched fatty acid and an unsaturated fatty acid. As the linear fatty acid, in the case of a high molecular weight polysaccharide fatty acid ester, a linear saturated fatty acid having 2 to 24 carbon atoms is preferable, and a high molecular weight polysaccharide fatty acid amide or a high molecular weight polysaccharide fatty acid ester.
In the case of an amide, a straight-chain saturated fatty acid having 3 to 24 carbon atoms is preferable. Examples of linear fatty acids include, for example, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid,
Examples include tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexanedecanoic acid, heptadecanoic acid, octadecanoic acid, arachidic acid, behenic acid, and lignoceric acid.

The branched fatty acid may have 4 to 2 carbon atoms.
The branched saturated fatty acids of 4 are preferable, and examples thereof include isobutyric acid, isovaleric acid, 2-ethylbutyric acid, isopentanoic acid, and mycolic acid.

Further, unsaturated fatty acids include those having 6 carbon atoms.
To 22 unsaturated fatty acids are preferred, for example, oleic acid, elaidic acid, linoleic acid, linolenic acid, palmitoleic acid, icosadienoic acid, icosatrienoic acid, arachidonic acid, icosapentaenoic acid, erucic acid, docosatetraenoic acid, docosapentaenoic acid And unsaturated fatty acids such as docosahexaenoic acid and tetracosenoic acid.

The fatty acids are preferably propionic acid, oleic acid, linoleic acid, mycolic acid and stearic acid, and more preferably oleic acid. By introducing one or more of these fatty acids into the polysaccharide, a polysaccharide fatty acid ester or amide having high adjuvant activity can be obtained.

The high molecular weight polysaccharide fatty acid ester used in the present invention is obtained by a substitution reaction between a high molecular weight polysaccharide and a fatty acid chloride, a transesterification reaction between a high molecular weight polysaccharide and a fatty acid methyl ester, a condensation reaction between a high molecular weight polysaccharide and a fatty acid, and the like. The polysaccharide fatty acid amide or ester-amide mixture can be easily synthesized, and the fatty acid chloride is added dropwise at a low temperature to a hydrated gel obtained by adding dimethylformamide to an aqueous solution of an aminopolysaccharide such as soluble chitosan. Thus, it can be easily synthesized.

As a commercially available product, a high-molecular-weight polysaccharide fatty acid ester in which the high-molecular-weight polysaccharide is cellulose is hydroxypropylmethylcellulose (for example, Shin-Etsu Chemical; product name "Metroze") or a small amount of hydroxypropylmethylcellulose. Hydrophobized hydroxypropyl methylcellulose into which a long-chain alkyl group has been introduced (for example, Sankyo Chemical; product name "Sangelose") can be used.

The average introduction number of the fatty acid per monosaccharide constituting the sugar chain of the high molecular polysaccharide in the high molecular polysaccharide fatty acid ester or amide is preferably in the range of 0.001 to 2.0, more preferably 0.005 to 2.0. To 1.0, more preferably 0.005 to 0.5. If the average number of introduced fatty acids is less than 0.001, the adjuvant activity may be insufficient, and if it exceeds 2.0, synthesis is difficult. The amount of the fatty acid to be introduced into the high-molecular polysaccharide can be easily adjusted by changing the molar ratio of the raw materials.

The average number of introduced fatty acids per constituent monosaccharide of these high-molecular-weight polysaccharide fatty acid esters or amides was determined by using 400 MHz ¹ H-NMR, measuring the proton ratio using chloroform as an internal standard substance, and measuring the infrared ray. It can be quantified by spectroscopic analysis (IR).

The weight average molecular weight of the high molecular weight polysaccharide fatty acid ester or amide is preferably 16,000.
~ 640,000, more preferably 16,000
~ 55,000. The weight average molecular weight can be measured by gel filtration chromatography.

The adjuvant of the present invention is an aqueous adjuvant or an oil-based adjuvant containing a high-molecular-weight polysaccharide fatty acid ester or amide as described above. Examples of the method for preparing the adjuvant include hand shaking, mechanical shaking, ultrasonic dispersion, dispersion using a homomixer, self-emulsification, film emulsification, D-phase emulsification, vacuum emulsification, and ultrahigh-pressure emulsification.

The content of the high molecular weight polysaccharide fatty acid ester or amide in the aqueous adjuvant is usually 0.01 to 2.0 w / v with respect to an aqueous medium such as water, physiological saline, or phosphate buffered physiological saline. %, Preferably 0.0%
5 to 1.0 w / v%, more preferably 0.1 to 0.5 w
/ V%. When used for an oily adjuvant, 0.05 to 5.0 w / v% in the outer layer water of O / W type or W / O / W type preparations such as liquid paraffin, squalene, squalane, synthetic alkane, edible vegetable oil, etc. Preferably 0.1 to 1.0 w / v%, more preferably 0.1 to 1.0 w / v%.
0.5 w / v%.

In the adjuvant of the present invention, additives such as oily components such as oleic acid, squalane, squalene, liquid paraffin, synthetic linear alkane oil, alcohol fatty acid ester oil and the like, and surfactants such as polyglycerin ester are used as additives. , Aluminum phosphate gel, aluminum hydroxide gel and the like. Its content is oleic acid, squalane, squalene, liquid paraffin,
10 to 50 w / v% for synthetic linear alkane oil and alcohol fatty acid ester oil, 5 to 20 v / v for aluminum phosphate gel and aluminum hydroxide gel
v%.

The vaccine of the present invention contains the adjuvant of the present invention and a desired antigen. This vaccine is obtained by adding and mixing a desired antigen solution to the adjuvant of the present invention as described above and emulsifying / dispersing as necessary, or by adding a high molecular polysaccharide to an aqueous vaccine or an oil-based vaccine containing a desired antigen. It can be prepared by adding and mixing a fatty acid ester or amide, and emulsifying / dispersing as necessary. The content of the high-molecular-weight polysaccharide fatty acid ester or amide in the vaccine varies depending on the dosage form.
~ 1.0w / v%, more preferably 0.1%
０．５0.5 w / v%. The content of the desired antigen in the vaccine is preferably 0.05 to 5.0 w / v%,
More preferably, it is 0.1 to 1.0 w / v%.

As the desired antigen, any antigen can be used.

[0035]

EXAMPLES The present invention will now be described specifically with reference to examples, but these examples do not limit the present invention.

(1) Production example of high-molecular-weight polysaccharide fatty acid ester or amide [Production Example 1] Synthesis of oleoyl chitosan (a mixture of chitosan oleate and amide) 10 g of chitosan was dissolved in 500 mL of 5 v / v% formic acid, and 90 ° C. For 40 minutes, 1 hour, 2 hours, 3 hours and 6 hours, respectively. One of these hydrolysates
Formic acid was neutralized by dropwise addition of N sodium hydroxide, and salts were removed by dialysis, followed by lyophilization to obtain five types of soluble chitosan. The average number of constituent monosaccharides of the five types of soluble chitosans calculated from the molecular weights measured by the gel filtration method was about 100, 290, 1000, 2000 and 2 respectively.
750 estimated.

Next, among the above five types, the average number of constituent monosaccharides was 2
2.5 g of 90 soluble chitosan was dissolved in 50 mL of purified water, and a 10-fold amount of dimethylformamide was added dropwise and mixed to form a low-viscosity gel. After adding 1 v / v% of pyridine to this gel, an amount of oleic acid chloride which reacts with 20 mol% of the amino group of the chitosan used is dissolved in dimethylformamide, and the mixture is added dropwise at 4 ° C. and uniformly mixed. After leaving for 60 minutes, the solvent was removed by evaporation. Next, this was redissolved in purified water, dialyzed against water, and freeze-dried to obtain 9.25 g of oleoyl chitosan (weight average molecular weight: 60,000, average of fatty acids (oleoyl groups) per constituent monosaccharide). 0.17) were obtained.

In this synthesis method, a part of oleic acid chloride reacts with the active hydroxyl group of chitosan to produce chitosan oleic acid ester. The length of the sugar chain of oleoyl chitosan and the viscosity of the solution can be easily adjusted by changing the hydrolysis time of chitosan.

[Production Example 2] Synthesis of mannan oleate 100 g of yeast paste was suspended in 50 mL of 0.002 M citrate buffer (pH 7.0) and heated in an autoclave at 120 to 125 ° C for 90 minutes. . After cooling, the mixture was centrifuged, and the supernatant was separated. 8
0 mL Fehling's reagent was added. The gummy precipitate of the mannan-copper complex was filtered and dissolved by adding 6 mL of 3N hydrochloric acid. This solution was mixed with methanol / acetic acid (1/1)
The solution was added dropwise to 100 mL with stirring. After leaving for 2 hours to precipitate mannan, the precipitated mannan was filtered, added to 100 mL of methanol, and washed to obtain 1.2 g of mannan (average constituent monosaccharide number: 340).

1 g of the above mannan was suspended in 50 mL of dimethylformamide, and 1 g of triethylamine was added.
While stirring this suspension, a container was put into an ultrasonic water bath, and 1.7 g of oleic acid chloride was added thereto in a state where mannan was finely suspended and dispersed by applying ultrasonic waves.
Reaction was performed at 24 ° C. for 24 hours. After the reaction, the residue was evaporated to dryness, redissolved in water and dialyzed to remove triethylamine hydrochloride, and concentrated to obtain 1.86 g of a crude product. This crude product was suspended and stirred in 200 mL of methanol and then separated by filtration to obtain 1.08 g of a purified mannan oleate. The weight average molecular weight is 58,000,
The average number of fatty acids (oleoyl groups) introduced per constituent monosaccharide was 0.032.

[Production Example 3] Synthesis of pullulan oleate Commercially available pullulan (manufactured by Wako Pure Chemical Industries, average constituent monosaccharide number 550)
1 g was suspended in 50 mL of dimethylformamide, and 1 g of triethylamine was added. While stirring this suspension, a container was put into an ultrasonic water bath, and 1.7 g of oleic acid chloride was added thereto in a state where pullulan was suspended and dispersed by ultrasonic waves (100 W), and reacted at 20 to 25 ° C. for 24 hours. did. After the reaction, the reaction product was evaporated to dryness, redissolved in water and dialyzed to remove triethylamine hydrochloride, and concentrated to obtain 1.36 g of a crude product. This crude product was suspended and stirred in 200 mL of methanol, followed by filtration and separation to obtain 1.08 g of purified pullulan oleate. The weight average molecular weight was 88,000, and the average number of introduced fatty acids (oleoyl groups) per constituent monosaccharide was 0.021.

(2) Adjuvant activity test [Example 1] Adjuvant effect on ovalbumin 10 μg of ovalbumin (OVA) was added to 0.2% w / v% of hydroxypropyl methylcellulose (HPMC, “Metroze 90SH30000F” manufactured by Shin-Etsu Chemical; average composition unit) Number of sugars 3,100, weight average molecular weight 600,000, average number of introduced fatty acids per constituent monosaccharide [methoxyl group 1.25 to 1.58, hydroxypropoxyl group 0.1
8-0.54]), 0.2% w / v hydrophobized hydroxypropylmethylcellulose (hydrophobized HPMC, “Sangelose 90L” manufactured by Sankyo Chemical; average number of monosaccharides 3,10)
0, weight-average molecular weight 650,000, average number of fatty acids introduced per constituent monosaccharide [methoxyl group 1.25 to 1.
58, hydroxypropoxyl group 0.18 to 0.54
Stearyloxyhydroxypropoxyl group 0.02-
0.16]) or 0.2% w / v oleoyl chitosan (Production Example 1; average number of constituent monosaccharides: 290, weight average molecular weight: 60,000, fatty acid (oleoyl group) per constituent monosaccharide) It was dissolved in 100 μL of a phosphate buffered saline solution containing an average number of transfection of 0.17) and injected into the hind limb muscle of a 5-week-old mouse. Blood was collected at 3 weeks and 4 weeks after injection, and antibodies were measured by indirect hemagglutination (PHA method). Aluminum phosphate gel (10 v / v%) was used as a control as a representative example of a conventional adjuvant. These results are shown in FIG. The measurement of the PHA antibody titer is as follows.

[Method for measuring anti-OVA antibody by PHA]
[Method of preparing OVA-fixed red blood cells] Phosphate buffered saline 10
In mL, OVA 50 mg and sheep live blood cells 0.4 mL
(Packed cell vol.), And 3 mL of 2.5 w / v% glutaraldehyde was added dropwise.
Stir for hours. After centrifugation, the blood cells are washed with phosphate buffered saline 1
After washing with 0 mL, 1 M glycine NaOH buffer (pH
7.2) Suspend in 1 mL and leave at 4 ° C overnight. The blood cells are again washed with 10 mL of a phosphate buffered saline, and a glycine veronal buffer solution is used so that the final blood cell concentration is 1 v / v%.
(GVB; pH 7.2), which was used as OVA-fixed red blood cells.

[Measurement of PHA value] Serum of the test mouse was subjected to GV
The mixture is serially diluted 2-fold with B and added to a 96-well U-bottom microtiter plate (manufactured by Sumitomo Bakelite Co., Ltd.) in an amount of 100 μL each. Next, 25 μL of OVA-fixed red blood cells are added to each well, stirred with a plate mixer, and allowed to stand at room temperature for 4 hours. The size and color tone of the sedimentation circle of the hemagglutination at the bottom of the plate were compared with those of the positive / negative control sera, and the minimum dilution that showed positive was defined as the PHA titer, that is, the anti-OVA antibody titer of the test serum. .

Referring to FIG. 1, no antibody response was observed in the group of mice to which no adjuvant was added (“Non Adj.” In the figure), whereas hydroxypropyl methylcellulose (HPMC) and hydrophobized hydroxy When propylmethylcellulose (hydrophobized HPMC) or oleoylchitosan was used as an adjuvant, high antibody production was observed. In addition, when these high-molecular-weight saccharide fatty acid esters or amides were used as adjuvants, the antibody titer was determined as an aluminum phosphate gel as a control (addition amount of 10%).
v / v%), and it is confirmed that the polysaccharide fatty acid ester or amide is useful as a non-oil type (aqueous suspension type) adjuvant. Was done.

Example 2 Adjuvant Effect on Feline Herpesvirus Feline Herpesvirus (FHV) -infected CRFK cells were solubilized with the detergent NonidetP-40, and the ultracentrifuged supernatant was used as an antigen (100 HA unit / mL). ,
To 0.5 mL of the antigen, the same hydroxypropylmethylcellulose, hydrophobized hydroxypropylmethylcellulose or oleoylchitosan as in Example 1 was added at 0.2 w / v.
%, The rat was immunized subcutaneously, and an antibody against FHV was added to p.
The measurement was performed by the laque reduction method. Aluminum phosphate gel (10 v / v%) was used as a control as a typical example of a conventional adjuvant. The result is shown in FIG. The method for measuring the neutralizing antibody titer is as follows.

[Method for measuring neutralizing antibody titer against feline herpes virus (plaque reduction method)]
The test serum is inactivated at 56 ° C. for 30 minutes and serially diluted 4-fold with a diluent. 0.1 mL of diluted serum and about 200 puf /
Feline herpesvirus 0.1mL adjusted to 0.1mL
And sensitize for 1 hour at 37 ° C. 0.1 mL of the above sensitizing solution is inoculated to cat renal-derived CRFK cells that have become confluent on a 60 mm-diameter dish, and adsorbed at 37 ° C. for 1 hour. The inoculum is removed and a culture solution containing 1 w / v% agar is overlaid to solidify the agar. 5% v / v at 37 ° C
The cells are cultured in a carbon dioxide incubator for 4 days, and the agar obtained by adding 0.01% w / v of neutral red to the composition of the above-described agar layer is further layered and solidified, and the culture is continued. The next day, plaques (white circles) were counted, and the serum dilution that suppressed 50% of the plaque was calculated by the Behrens-Kelper method, and the reciprocal of the dilution was used as the neutralizing antibody titer.

As shown in FIG. 2, the antibody response to FHV was higher in oleoyl chitosan and hydroxypropylmethylcellulose, and slightly lower in hydrophobized hydroxypropylmethylcellulose. The addition of amide as an adjuvant confirmed that the higher antibody response persisted longer than conventional aluminum gel vaccines. That is, the increase and the duration of the antibody titer were compared with the conventional aluminum phosphate gel adjuvant (addition amount: 10 v / v).
%), Aluminum gel adjuvant transiently increased the antibody, but the duration was extremely short, whereas the high antibody titer was 3 months when the ester or amide of high molecular polysaccharide fatty acid was used as the adjuvant. It was confirmed that it lasted.

Example 3 Pig Aujeszky's Disease W / O / W
Example of application to type oil vaccine Formalin-inactivated antigen of Aujeszky's disease virus Iwate strain and anhydrous mannitol oleate (3.1 w / v
%) The added squalane was mixed at a ratio of 3: 7, and emulsified with an ultra homomixer to obtain a W / O emulsion. Next, 10 mL of this emulsion was mixed and emulsified with a homogenizer while being dropped into a phosphate buffered saline solution of the following compounds (1) to (6) to prepare a W / O / W vaccine. (1) 0.2% w / v oleoyl chitosan (Production Example 1; average constituent monosaccharide number: 290, weight average molecular weight: 60,00)
0, average number of introduced fatty acids (oleoyl groups) per constituent monosaccharide: 0.17). (2) 0.2 w / v% hydroxypropyl methylcellulose ("Metroze 90SH30000" manufactured by Shin-Etsu Chemical Co., Ltd.)
F "; average constituent monosaccharide number 3,100, weight average molecular weight 60
0000, average number of fatty acids introduced per constituent monosaccharide
[Methoxyl group 1.25 to 1.58, hydroxypropoxyl group 0.18 to 0.54]). (3) 0.2 w / v% hydrophobized hydroxypropyl methylcellulose ("Sangelose 90L" manufactured by Sankyo Chemical;
Average constituent monosaccharide number 3,100, weight average molecular weight 650,0
00, average number of fatty acids introduced per constituent monosaccharide [methoxyl group 1.25 to 1.58, hydroxypropoxyl group 0.18 to 0.54, stearyloxyhydroxypropoxyl group 0.02 to 0.16 ]). (4) 0.2% w / v pullulan oleate (PLOE, Production Example 3; average number of constituent monosaccharides: 550, weight average molecular weight: 88,000, average of fatty acids (oleoyl groups) per constituent monosaccharide) 0.021). (5) 0.2% w / v mannan oleate (MNOE, Production Example 2) Average number of constituent monosaccharides 340, weight average molecular weight 58,000, average of fatty acids (oleoyl groups) per constituent monosaccharide 0.032). (6) A mixture of 0.1 w / v% of the pullulan oleate (4) and 0.1 w / v% of the mannan oleate (5) (MNOE + PLOE).

The above-mentioned W was introduced into the thigh muscle of a 3-week-old mouse.
/ O / W type vaccine 0.1mL injected, its after four weeks, was attacked by injecting 100 LD ₅₀ of Aujeszky's Disease Virus Iwate strain, as shown in Table 1, hydrophobic hydroxypropyl an antibody titer The methylcellulose-applied group had the highest results, but in the protective effect against infection, the group administered with a mixture of oleic acid esters of mannan and pullulan showed the best performance.

[Method of Measuring Mouse Anti-Aussky Disease Virus Antibody Titer by ELISA] 1) 2 × 10 ⁷ TCID _{50 of} inactivated Aujeszky's disease virus Iwate strain as an antigen was placed on a flat-bottomed 96-well ELISA plate as an antigen.
Add 0.1 mL / mL, sensitize at room temperature for 30 minutes, and discard the antigen solution. 2) Add 250 μL of 10% skim milk and let stand for 1 hour at room temperature. 3) Discard the skim milk and wash with PBS, then 400
Add 0.1 mL of test serum diluted 1-fold and let stand at 37 ° C for 1 hour. 4) After washing the plate with PBS, PO diluted with PBS
After adding a D-labeled anti-mouse IgG antibody, the mixture is left to stand for 30 minutes and then washed with PBS. 5) Add 0.1 mL of orthophenylenediamine reagent and add 2
After color development at 5 ° C. for 30 minutes, the reaction is stopped by adding 0.1 mL of 2N sulfuric acid, and the absorbance is measured with a plate reader. 6) The value obtained by subtracting the absorbance of the negative serum from the absorbance of the test serum is defined as the ELISA antibody titer.

[0052]

[Table 1]

[Example 4] Example of application of high molecular weight polysaccharide fatty acid ester or amide to Newcastle disease, infectious coryza aqueous vaccine, O / W and W / O / W type oil adjuvant vaccine (Newcastle disease, chicken infectivity) Application to Coryza A and C vaccines)

Example of Application to Aqueous Vaccine Newcastle disease virus was added to 10 ^9.5 EID50 / m
L, Haemophilus paragalinarum (H.pg) type A and type C thimerosal-inactivated cells were each 1.0 ×
Antigen solutions containing 10 ⁸ cells / mL at the following concentrations (a) to
An aqueous vaccine was prepared by mixing equal amounts of each of the high molecular weight polysaccharide fatty acid esters or amides of (i). (A) 0.4 w / v% hydroxypropyl methylcellulose (“Metroze 90SH30000” manufactured by Shin-Etsu Chemical Co., Ltd.)
F "; average constituent monosaccharide number 3,100, weight average molecular weight 60
0000, average number of fatty acids introduced per constituent monosaccharide
[Methoxyl group 1.25 to 1.58, hydroxypropoxyl group 0.18 to 0.54]). (B) 0.4 w / v% hydrophobized hydroxypropyl methylcellulose (“Sangelose 90L” manufactured by Sankyo Chemical;
Average constituent monosaccharide number 3,100, weight average molecular weight 650,0
00, average number of fatty acids introduced per constituent monosaccharide [methoxyl group 1.25 to 1.58, hydroxypropoxyl group 0.18 to 0.54, stearyloxyhydroxypropoxyl group 0.02 to 0.16 ]). (C) 0.4% w / v oleoyl chitosan (Production Example 1; average constituent monosaccharide number: 290, weight average molecular weight: 60,00)
0, average number of introduced fatty acids (oleoyl groups) per constituent monosaccharide: 0.17). (D) 0.4% w / v mannan oleate (Production Example 2; average number of constituent monosaccharides: 340, weight average molecular weight: 5)
8,000, average number of introduced fatty acids (oleoyl groups) per constituent monosaccharide 0.032). (E) 0.4 w / v% pullulan oleate (Production Example 3; average number of constituent monosaccharides: 550, weight average molecular weight: 8)
8,000, average number of introduced fatty acids (oleoyl groups) per constituent monosaccharide 0.021). (F) An equal mixture of 0.2 w / v% of the hydrophobized hydroxypropylmethylcellulose (b) and 0.2 w / v% of the oleoylchitosan (c). (G) Equivalent mixture of 0.2 w / v% of the hydrophobized hydroxypropylmethylcellulose (b) and 0.2 w / v% of the pullulan oleate (e). (H) An equal mixture of 0.2 w / v% of the hydrophobized hydroxypropylmethylcellulose (b) and 0.2 w / v% of the mannan oleate (d). (I) Equivalent mixture of 0.2 w / v% of the mannan oleate (d) and 0.2 w / v% of the pullulan oleate (e).

Preparation of O / W type oil adjuvant vaccine In a glass container, 50 parts of a phosphate buffered saline containing 0.2 w / v% of the same high molecular weight polysaccharide fatty acid ester or amide as in the above (a) to (i) was added. The mixture was added and emulsified with a high-speed homogenizer while dropping 50 parts of squalane with 5% anhydrous mannitol oleate to prepare an O / W adjuvant emulsion. Next, the adjuvant emulsion and the antigen solution [10 ^9.5 EID of Newcastle disease virus
50 / mL, Haemophilus paragalinarum (H. p.
g) Type A and Type C thimerosal-inactivated bacterial cells at a concentration of 1.0 × 10 ⁸ cells / mL] are mixed in equal amounts and mixed with a low-speed mixer to obtain an O / W type oil adjuvant vaccine. Prepared.

Preparation of W / O / W Type Oil Adjuvant Vaccine An oil adjuvant was prepared by mixing and dissolving 3 parts of anhydrous mannitol oleate in 97 parts of squalane. To 7 volumes of this oil adjuvant, add the above antigen solution [Newcastle disease virus to 10 ^9.5 EID50 / m
L, Haemophilus paragalinarum (H.pg) type A and type C thimerosal-inactivated cells were each 1.0 ×
10 ⁸ / at a concentration of mL] 3 volumes was added thereto to prepare a W / O type oil adjuvant vaccine by emulsifying using a high speed homogenizer. 5 parts of this W / O emulsion was mixed with (a)
W / O / W type by mixing and emulsifying with a high-speed homogenizer while gradually dropping into 5 parts of a phosphate buffered saline containing 0.2 w / v% of the same high molecular weight polysaccharide fatty acid ester or amide as in (i). An oil adjuvant vaccine was prepared.

Various aqueous vaccines, O / W type oil adjuvant vaccines and W / Os prepared as described above
0.5mL of / W oil adjuvant vaccine
Is injected into the lower leg muscles of a 60-day-old white leghorn (Supernic), and blood is collected over time to suppress hemagglutination.
(HI) The antibody titer was measured. As Comparative Examples 1 to 4, commercially available W / O type and O / W type oil adjuvants were used. Some of the results are shown in FIGS. The measurement of the antibody titer was performed using the HI antibody titer described below.

Immediately after the same injection as described above, swelling, induration and other general symptoms at the injection site were observed.
Blood was collected at 3 and 5 weeks after injection and HI antibody titer was measured. In addition, necropsy was performed 5 weeks later, and the injection site was observed. Table 2 shows some of the results. In addition, the observation of general findings was observed immediately after injection, and examples in which findings were observed until the necropsy were described. Among the side effects in Table 2, the larger the value of +, the more serious the side effect, and the sign of-, the side effect was not observed.

[Hamophilus paragalinarum (H. p.
g) Method for measuring HI antibody titer against type A bacteria] 0.025 mL of the test serum was serially diluted 2-fold with phosphate buffered saline. 4 units of H.O. pg-A type bacteria (2
21 strain) A phosphate buffered saline solution containing HA was added thereto, mixed, sensitized at room temperature for 10 minutes, 0.05 mL of a 0.5 v / v% chicken erythrocyte suspension was added, shaken, and then room temperature for 60 minutes. The sample was allowed to stand still, and the highest dilution of the test serum that completely suppressed red blood cell aggregation was defined as the HI antibody titer.

[HI for Newcastle Disease Virus]
Method for measuring antibody titer] Plastic hemagglutination plate (HAtr
ay) Dilute the test serum five-fold with phosphate buffered saline above, and prepare a two-fold serial dilution series for 0.2 mL based on this. To this, 0.2 mL of NDVHA antigen (4 units; hemagglutinin for diagnosis of Newcastle disease (NDV Ishii strain HA antigen; manufactured by Nissei Laboratories)) is added to 0.2 mL, and the mixture is left to stand for 10 minutes after shaking. 0.5 mL of a / v% chicken erythrocyte suspension was added, and the mixture was allowed to stand for 60 minutes after shaking, and the highest dilution factor of the test serum that completely suppressed red blood cell aggregation was defined as the HI antibody titer.

[0061]

[Table 2]

As a result, an aqueous gel vaccine containing a high-molecular-weight polysaccharide fatty acid ester or amide showed a higher antibody elevation than those not containing the polysaccharide fatty acid ester or amide. Clearly higher titers compared to vaccine. On the other hand, O / W using high molecular weight polysaccharide fatty acid ester or amide as an emulsifier / stabilizer
In both of the W / O / W preparations and the W / O / W preparations, an increase in the antibody was confirmed to be equal to or higher than that of the conventional W / O adjuvant. In terms of safety, as a result of the side effect tests shown in Table 2, aluminum phosphate gels (Comparative Example 2, addition amount 10 v / v%) and aluminum hydroxide gels (Comparative Example 3, With an addition amount of 10 v / v%),
An inoculation reaction was also observed at necropsy 5 weeks after the inoculation. On the other hand, in the case of an aqueous vaccine in which an ester, amide or a mixture of a high-molecular polysaccharide and a fatty acid was adjuvanted, the inoculation reaction was weak, and slight glue-like infiltration was observed at the injection site 1 to 2 hours after injection. Only swelling and induration were not observed at all. In the case of the W / O / W type vaccine using the same ester or amide, the formation of induration was observed slightly in the second to third weeks after injection. Although microparticles remained slightly, no inoculation reaction such as abscess formation and swelling was observed, and no trace was observed with the O / W vaccine. From these, it was confirmed that the ester or amide of the high-molecular polysaccharide and the fatty acid has high safety as an adjuvant.

Example 5 Monosaccharide 1 Constituting High-Polymer Polysaccharide Portion
With respect to the adjuvant effect of the high-molecular-weight polysaccharide fatty acid ester or amide when the number of introduced fatty acids per unit was fixed and the number of monosaccharides constituting the high-molecular-weight polysaccharide portion was changed, oleoyl obtained in the same manner as in Production Example 1 When the relationship between the sugar chain length and the adjuvant effect was examined using chitosan and various hydrophobized hydroxypropylmethylcellulose (manufactured by Sankyo Chemical Co., Ltd.) having different numbers of constituent monosaccharides, the adjuvant effect was 100 to 5000 constituent monosaccharides. Was confirmed in the range of
As shown in FIG. 12, it was confirmed that the strength was strongest in the range of 100 to 3200.

[0064]

As described above, the adjuvant containing the high-molecular-weight polysaccharide fatty acid ester or amide of the present invention has higher adjuvant activity than conventional aluminum gel adjuvant, and has side effects such as swelling and induration at the inoculation site. No safety was improved. In addition, the above compounds can be provided at a lower cost because of their ease of synthesis. This compound, in addition to aqueous vaccines,
It can also be used as an adjuvant or emulsifier (stabilizer) in vaccine emulsions of type W and W / O / W. With the above performance improvement, it can be expected to be applied as a safe, effective and stable adjuvant and further as a vaccine.

[Brief description of the drawings]

FIG. 1 is a graph showing the adjuvant effect on ovalbumin.

FIG. 2 is a graph showing an adjuvant effect on feline herpesvirus antigen.

FIG. 3 is a graph showing the adjuvant effect of the aqueous vaccine on Newcastle disease virus.

FIG. 4 is a graph showing an adjuvant effect on Coryza type A bacteria in an aqueous vaccine.

FIG. 5 is a graph showing the adjuvant effect on Coryza type C bacteria in an aqueous vaccine.

FIG. 6 is a graph showing the adjuvant effect of the O / W vaccine on Newcastle disease virus.

FIG. 7 is a graph showing the adjuvant effect of the W / O / W vaccine on Newcastle disease virus.

FIG. 8 is a graph showing the adjuvant effect of the O / W vaccine on Coryza type A bacteria.

FIG. 9 is a graph showing an adjuvant effect on Coryza type C bacteria in an O / W type vaccine.

FIG. 10. Coryza A in W / O / W vaccine
It is a graph which shows the adjuvant effect with respect to a mold.

FIG. 11. Coryza C in W / O / W vaccine
It is a graph which shows the adjuvant effect with respect to a mold.

FIG. 12 is a graph showing a change in the number of constituent monosaccharides of a high-molecular-weight polysaccharide and the adjuvant effect when the average number of introduced fatty acids per constituent monosaccharide of a high-molecular-weight polysaccharide fatty acid ester or amide is kept constant.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 31/12 A61P 31/12 (72) Inventor Yoshio Nagai 5-2-3 Higashiyawata, Hiratsuka-shi, Kanagawa Prefecture Sankyo Chemical Co., Ltd. Synthetic Chemistry (58) Investigated field (Int. Cl. ⁷ , DB name) A61K 39/39 A61K 39/00 A61K 39/102 A61K 39/245 A61P 31/04 A61P 31/12 CAPLUS (STN)

Claims (16)

(57) [Claims] 1. An aqueous or oily adjuvant comprising at least one ester, amide or mixture of esters and amides of a high molecular weight polysaccharide and a fatty acid. 2. The method according to claim 1, wherein the high molecular weight polysaccharide has a sugar chain of 100 to 5.0.
The adjuvant according to claim 1, which is composed of 00 monosaccharides. 3. The method according to claim 1, wherein the monosaccharide constituting the sugar chain of the high molecular polysaccharide contains a high molecular polysaccharide fatty acid ester which is at least one kind of monosaccharide selected from the group consisting of glucose, galactose and mannose. The adjuvant as described. 4. The fatty acid according to claim 3, wherein the fatty acid is at least one of a linear saturated fatty acid having 2 to 24 carbon atoms, a branched saturated fatty acid having 4 to 24 carbon atoms, and an unsaturated fatty acid having 6 to 22 carbon atoms. Adjuvant. 5. The high molecular weight polysaccharide fatty acid amide or high molecular weight polysaccharide fatty acid amide, wherein the monosaccharide constituting the sugar chain of the high molecular weight polysaccharide is at least one amino sugar selected from the group consisting of glucosamine, galactosamine and neuraminic acid. The adjuvant according to claim 1 or 2, comprising an ester. 6. The fatty acid according to claim 5, wherein the fatty acid is at least one of a linear saturated fatty acid having 3 to 24 carbon atoms, a branched saturated fatty acid having 4 to 24 carbon atoms, and an unsaturated fatty acid having 6 to 22 carbon atoms. Adjuvant. 7. The adjuvant according to claim 1, wherein the average number of introduced fatty acids per monosaccharide constituting the high-molecular-weight polysaccharide is 0.001 to 2.0. 8. The adjuvant according to claim 1, which comprises a cellulose fatty acid ester. 9. The method according to claim 1, further comprising a mannan fatty acid ester.
The adjuvant according to any one of to 4, 7 and 8. 10. The adjuvant according to claim 1, which contains a pullulan fatty acid ester. 11. The adjuvant according to claim 1, which comprises a chitosan fatty acid ester. 12. The adjuvant according to claim 1, comprising chitosan fatty acid amide. 13. The adjuvant according to claim 1, which comprises a chitosan fatty acid ester / amide mixture. A vaccine comprising the adjuvant according to any one of claims 1 to 13. 15. An aqueous vaccine comprising an ester, an amide or a mixture of an ester and an amide of a high molecular weight polysaccharide and a fatty acid. 16. An O / W-type or W / O / W-type oleaginous vaccine comprising an ester, amide or ester-amide mixture of a high molecular weight polysaccharide and a fatty acid as an adjuvant or an emulsion stabilizer.