CN106511996A - Multiple emulsion type adjuvant for foot-and-mouth disease vaccine and preparation method of multiple emulsion type adjuvant - Google Patents
Multiple emulsion type adjuvant for foot-and-mouth disease vaccine and preparation method of multiple emulsion type adjuvant Download PDFInfo
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- CN106511996A CN106511996A CN201610963355.XA CN201610963355A CN106511996A CN 106511996 A CN106511996 A CN 106511996A CN 201610963355 A CN201610963355 A CN 201610963355A CN 106511996 A CN106511996 A CN 106511996A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention provides a multiple emulsion type adjuvant for a foot-and-mouth disease vaccine and a preparation method of the multiple emulsion type adjuvant and relates to the field of medicine preparation. The multiple emulsion type adjuvant for the foot-and-mouth disease vaccine is composed of the following components including, by mass, 50-65 parts of mineral oil, 10-35 parts of vitamin E, 3-5 parts of castor oil ethyl oxide, 2-8 parts of tween 80, 2-6 parts of n-amyl alcohol, 1-4 parts of stearyl alcohol polyoxyethylene ether and 1-5 parts of deionized water. The invention further provides the foot-and-mouth disease vaccine containing the multiple emulsion type adjuvant and a preparation method of the foot-and-mouth disease vaccine. The foot-and-mouth disease vaccine containing the adjuvant can be completely absorbed by animals in short time on the premise of good stability and immunological enhancement effect, inflammation of the inoculation position is avoided, and therefore, side reaction is reduced remarkably.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, and in particular to the emulsion type adjuvant and its preparation side for aftosa vaccine
Method.
Background technology
The medium oil adjuvant of animal vaccine is most widely used, and immunoenhancement result is best.There are three kinds containing oil-adjuvant vaccine
Formulation：Water-In-Oil (W/O) type, W/O/W (W/O/W) type and oil-in-water (O/W) type.
At present, containing oil-adjuvant vaccine with W/O (oil emu) it is the most universal, to strengthen immune effect optimal, can not but exist
The mineral oil content of metabolism is high, big viscosity and the shortcomings of strong toxicity.The animals such as pig, dog, cat are particularly quick to the dose vaccine
Sense, which has limited the application in practice of W/O formulations.And O/W formulations overcome the shortcoming of W/O, but adjuvanticity is too weak, improves
Immune effect it is limited in one's ability, it is impossible to meet the demand of aquaculture.
The adjuvant commonly used in W/O/W (W/O/W) type vaccine is W/O/W (W/O/W) type emulsion.Prior art
In, W/O/W (W/O/W) type emulsion includes206 adjuvants of ISA (French SEPPIC companies) and contain
The adjuvant of a large amount of mineral oil, although the vaccine immunocompetence containing these adjuvants is high, after immunity, the long period can be by
Animal fully absorbs, the easy inflammation of inoculation position, therefore side reaction is larger.
The content of the invention
It is an object of the invention to provide for the emulsion type adjuvant of aftosa vaccine, the aftosa vaccine containing the adjuvant exists
Under the premise of stability, immunoenhancement result are preferable, in the short period just can completely passive thing absorb, it is to avoid inoculation position is sent out
Inflammation, therefore significantly reduce side reaction.
It is a further object of the present invention to provide the aftosa vaccine containing emulsion type adjuvant, the vaccine stability, immunity effect
Fruit preferably, within a short period of time just can completely passive thing absorb, it is to avoid inoculation position inflammation, therefore significantly reduce secondary anti-
Should.
Another object of the present invention is to provide the preparation method of above-mentioned vaccine, and the method is simple, efficiently, safety.
The purpose of the present invention adopts the following technical scheme that realization：
For the emulsion type adjuvant of aftosa vaccine, it is made up of the component of following mass parts：
Mineral oil 50-65 parts,
Vitamin E 10-35 parts,
Castor oil ethyl oxide 3-5 parts,
Tween 80 2-8 parts,
N-amyl alcohol 2-6 parts,
Brij-721 1-4 parts,
Deionized water 1-5 part.
In the present invention, the castor oil ethyl oxide be Emulsogen EL360, the Brij-721
For stearyl alcohol polyoxyethylene (21) ether.
The present invention also provides the preparation method of the emulsion type adjuvant for aftosa vaccine, each component is mixed, is stirred
Mix uniform, sterilizing.
The present invention also provides the aftosa vaccine containing the emulsion type adjuvant.
In the present invention, the vaccine is made up of the emulsion type adjuvant and foot-and-mouth disease antigen.
In preferred technical scheme, the mass ratio of the emulsion type adjuvant and foot-and-mouth disease antigen is 1:05～1.5.
The present invention also provides the preparation method of the aftosa vaccine, and emulsion type adjuvant and foot-and-mouth disease antigen are preheated to 15
～30 DEG C, emulsion type adjuvant is mixed with foot-and-mouth disease antigen, it is hand or stir, obtain aftosa vaccine.
Emulsion type adjuvant of the present invention for aftosa vaccine, replaces part mineral oil using vitamin E, from hypotoxicity
Surfactant, the aftosa vaccine containing the adjuvant under the premise of stability, immunoenhancement result are preferable, in the short period
Inside easily fully absorbed by animal, it is to avoid inoculation position inflammation, therefore significantly reduce the toxic and side effect to animal.The emulsion type is helped
Agent can be stirred by hand, magnetic agitation or machine with inactivation antigen, make stable W/O/W type vaccines.Preparation process without
Precision instrument is needed, easy to operate, preparation cost is low.Aftosa vaccine containing emulsion type adjuvant of the invention, injection are convenient, prepare
Method is simple, efficient, safety.
Specific embodiment
Embodiment 1 prepares emulsion type adjuvant
In the present embodiment, Emulsogen EL360 are purchased from Clariant Chemical Co., Ltd., production code member CNP1004847；Firmly
Lipidol polyoxyethylene (21) ether is purchased from Shanghai Mei Hao Fine Chemical Co., Ltd, production code member PCN/4500713528.
Emulsion type adjuvant 1 is filled a prescription：
Emulsion type adjuvant 2 is filled a prescription：
Emulsion type adjuvant 3 is filled a prescription：
Control adjuvant 1 is filled a prescription：
Control adjuvant 2 fill a prescription (be 104398478 A of Publication No. CN, title " the emulsion type carrier of veterinary drug and its
Using " in application for a patent for invention embodiment 1 oil phase 3)：
Emulsion type adjuvant 1-3 and control adjuvant 1,2 are prepared, concrete grammar is as follows：Each component is weighed according to formula, is mixed,
Stir, autoclaving is adopted 20 minutes under the conditions of 115 DEG C.Adjuvant keeps in dark place in room temperature.
The preparation of 2 aftosa vaccine of embodiment (W/O/W types)
Schweineseuche inactivation of viruses liquid (O-shaped, Mya98 strains) is provided by Jinyu Baoling Biology Drugs Co., Ltd, before inactivation
In per 0.2 milliliter of virus liquid, viral level is 107.17LD50。
By emulsion type adjuvant 1-3, control adjuvant 1 respectively with Schweineseuche inactivation of viruses liquid according to mass ratio 1:1 mixing, leads to
Spend hand 5-10 minutes or stir 30 minutes under the conditions of 250rpm, be well mixed each component, obtain aftosa vaccine, mouth hoof
The numbering of epidemic disease vaccine is shown in Table 1 with the corresponding relation of adjuvant.Adjuvant, virus liquid are preferably preheated to 15～30 DEG C before combination.According to
Embodiment in 104398478 A of Publication No. CN, title " the emulsion type carrier of veterinary drug and its application " application for a patent for invention
Control adjuvant 2 is made aftosa vaccine with Schweineseuche inactivation of viruses liquid (O-shaped, Mya98 strains) by 3 methods, and numbering is vaccine E.
| Adjuvant is numbered | Vaccine is numbered |
| Emulsion type adjuvant 1 | Aftosa vaccine A |
| Emulsion type adjuvant 2 | Aftosa vaccine B |
| Emulsion type adjuvant 3 | Aftosa vaccine C |
| Control adjuvant 1 | Aftosa vaccine D |
| Control adjuvant 2 | Aftosa vaccine E |
Aftosa vaccine A-E is W/O/W types, and their physical behavior is consistent, emulsion be in pale pink, wall during shake
On slightly light blue formulation.Adopt detection aftosa vaccine A-E with the following method：250mL beakers are taken, 100mL cold water is held, is taken
One cleaning suction pipe, draws a little vaccine and drips in cold water surface, and the drop of each vaccine is spread in cloud.Viscosity：Use 1mL suction pipes
(exit inside diameter 1.2mm) draws 25 DEG C or so of vaccine 1.0mL, makes its vertical natural flow out, record flow out needed for 0.4mL when
Between, as a result：The delivery time of each vaccine is within 8 seconds.Particle diameter：10 μ L of vaccine are taken accurately in test cup with liquid-transfering gun, plus
Enter 290 μ L PBSs fully to mix, it is ensured that emulsified particles fine dispersion, test cup is put in Malvern nano particle size instrument
Determine, temperature of the measurement is set as 25 DEG C, and as a result the average grain diameter of each vaccine is in the range of 130-190nm.Dewatering ability：Take epidemic disease
During seedling 10mL adds centrifuge tube, 15min is centrifuged in 3000rpm/min, the ttom of pipe of each vaccine is held without precipitation water phase.37 DEG C steady
It is qualitative：Vaccine is placed in 37 DEG C of incubators, daily timing is observed, each vaccine place 14 days after not breakdown of emulsion.Aftosa vaccine A-E
Place 12 months in 4 DEG C of refrigerators, it is stable.
The safety verification of embodiment 3 aftosa vaccine A, B, C
In inspection embodiment 2, aftosa vaccine A-E is to mouse and the security of pig.
(1) mouse safety test
16-22g cleaning grades mouse 60 is taken, 6 groups are randomly divided into, one of which is not inoculated with as control, in addition five components
Not immune aftosa vaccine A, B, C, D or E, (0.5ml, 1/2 part here is 1/2 part of every mouse neck hypodermic injection
Relative to pig) aftosa vaccine.1 week after vaccine inoculation, 2 weeks, 3 weeks, each group analyses mouse 3 at random, observes injection site
Absorbing state, the results are shown in Table 2.It can be seen from Table 2 that：Mice Inoculated injection site is analysed, 2 weeks after the injection of vaccine A, B, C group
3/3 fully absorbs；Only 1/3 fully absorb within 2 weeks after the injection of D groups, just 3/3 fully absorb within 3 weeks after injection；E groups injection after 2 weeks only
Have 1/3 to fully absorb, just 3/3 fully absorb within 3 weeks after injection.Mouse safety test result explanation：Using adjuvant 1-3 systems of the present invention
Standby aftosa vaccine, just fully absorbed after injection for two weeks by total Test animal, it is to avoid due to vaccine long-time it is not complete
The inflammation that hypersorption causes in inoculation position, therefore compared with contrast adjuvant 1,2, side reaction is significantly reduced, it is safe.
The safety verification result of 2 aftosa vaccine of table
(2) pig safety test
Take the negative 30-35 age in days sodium selenites of foot-and-mouth disease antibody each 30, be randomly divided into 6 groups, one of which is not inoculated with
Group is compared, five groups are inoculated with aftosa vaccine A, B, C, D or E, 4 part (4ml) aftosa epidemic diseases of every intramuscular injection respectively in addition
Seedling.
After inoculation observe 14 days, all test pig observation index include body temperature, spirit, appetite etc. local, general reaction with
And injection site absorbing state is analysed, the results are shown in Table 3.It can be seen from Table 3 that：All test pig spirit, body temperature, appetite etc. are
No abnormality seen.Vaccine inoculation piglet has no the abnormal response that vaccine inoculation causes.Cut open high dose (4ml) Pigs Inoculated injection site
Solution, after aftosa vaccine A, B, C are inoculated with 14 days, all pigs are fully absorbed, and aftosa vaccine D only has 2/5 pig after being inoculated with 14 days
Fully absorb, after aftosa vaccine E is inoculated with 14 days, only 1/5 pig is fully absorbed.Pig safety test result illustrates that the present invention is helped
Aftosa vaccine prepared by agent 1-3, can be fully absorbed after injection for two weeks by total Test animal, it is to avoid due to vaccine it is long
Time does not fully absorb the inflammation caused in inoculation position, therefore, compared with contrast adjuvant 1,2, side reaction is significantly reduced, pacified
Quan Xinggao.
3 aftosa vaccine safety verification result of table
Above-mentioned safety testing result explanation, the present invention replaces part mineral oil using vitamin E, from hypotoxicity surface
Activating agent so that aftosa vaccine is easily fully absorbed by animal in the short time, it is to avoid as vaccine long-time is not fully absorbed
In the inflammation that inoculation position causes, so as to be substantially reduced the toxic and side effect to animal.
The Study On Immunogenicity of embodiment 4 aftosa vaccine A, B, C
1st, pig Study On Immunogenicity
The immunogenicity of detection aftosa vaccine A, B, C.Healthy 2-3 month pig 20 is carried out into group experiment, is divided into 4
Group, 5 per group.One of which is not inoculated with as blank, and three groups are inoculated with aftosa vaccine A, B, C respectively in addition, and per connects
Plant 1ml.Blood sampling in 28 days after immunity separates serum, with Schweineseuche ELISA antibody assay kits (purchased from animal life before the section of Wuhan
Tetramune Co., Ltd) detection ELISA value for antibody, value for antibody >=6log2 is positive.
4 are the results are shown in Table, vaccine single immunization pig ELISA antibody whole turn sun after 4 weeks.The result shows multiple with the present invention
Aftosa vaccine prepared by newborn adjuvant has preferable immunogenicity, and after immunity, antibody horizontal is high, is good vaccine adjuvant.
The ELISA antibody (log2) that 4 different aftosa vaccine immune swines of table are produced
| Packet | Aftosa vaccine A | Aftosa vaccine B | Aftosa vaccine C | Blank |
| 1 | 6 | 7 | 7 | 2 |
| 2 | 7 | 7 | 7 | 3 |
| 3 | 7 | 7 | 7 | 2 |
| 4 | 7 | 8 | 7 | 2 |
| 5 | 7 | 7 | 7 | 3 |
| Mean value | 6.8 | 7.2 | 7.0 | 2.4 |
2nd, Mouse immunogenicity comparative test
Experiment vaccine：1. the aftosa vaccine A for preparing in embodiment 2；2.206 Adjuvanted vaccines：ISA206 adjuvants (are purchased from
SEPPIC companies) foot-and-mouth disease antigen in the same manner as in Example 2 is with mass ratio 1:Prepare after 1 mixing, emulsification；3. white oil is helped
Vaccinating agent：By foot-and-mouth disease antigen white oil in the same manner as in Example 2 with mass ratio 2:1 mixing, obtains after emulsification.4. in embodiment 2
The aftosa vaccine E of preparation.
20g or so cleaning grades mouse 25 is taken, aftosa vaccine A groups, 206 Adjuvanted vaccines groups, white-oil adjuvant is randomly divided into
Vaccine group, aftosa vaccine E groups and blank control group, have 5 mouse per group, in addition to blank control group is not immune, other each groups
The immunity vaccine corresponding with its group name, in addition to white-oil adjuvant vaccine group immunity 0.3ml/, other subcutaneous notes of immune group neck
0.2ml/ is penetrated only.28 days after immunity, (have purchased from animal biological product before the section of Wuhan with aftosa ELISA antibody assay kit
Limit responsible company) detection ELISA value for antibody.
Mouse immunogenicity trial the results are shown in Table 5,4 weeks ELISA antibody horizontals after visible mouse single immunization from the table,
Aftosa vaccine A groups antibody titer is higher than 206 Adjuvanted vaccines groups and aftosa vaccine E groups, shows emulsion adjuvant immunity of the present invention
Effect meets or exceeds ISA206 adjuvants and the existing emulsion type adjuvant containing a large amount of mineral oil；Aftosa vaccine A groups potency is omited
Less than white-oil adjuvant vaccine group, show that adjuvant immunity effect of the present invention is close to white-oil adjuvant, meet W/O/W type adjuvanted immunogenics
Infer less than W/O adjuvants.
28 days ELISA antibody titers (log2) after 5 mouse immune of table
Claims (7)
1. it is used for the emulsion type adjuvant of aftosa vaccine, is made up of the component of following mass parts：
Mineral oil 50-65 parts,
Vitamin E 10-35 parts,
Castor oil ethyl oxide 3-5 parts,
Tween 80 2-8 parts,
N-amyl alcohol 2-6 parts,
Brij-721 1-4 parts,
Deionized water 1-5 part.
2. the emulsion type adjuvant of aftosa vaccine according to claim 1, it is characterised in that the castor oil ethyl oxide
For Emulsogen EL360, the Brij-721 is stearyl alcohol polyoxyethylene（21）Ether.
3. it is used for the preparation method of the emulsion type adjuvant of aftosa vaccine described in claim 1 or 2, each component is mixed, stirs
Uniformly, sterilize.
4. the aftosa vaccine containing emulsion type adjuvant described in claim 1 or 2.
5. aftosa vaccine according to claim 4, it is characterised in that the vaccine is by one of claim 1-2 emulsion
Type adjuvant and foot-and-mouth disease antigen composition.
6. aftosa vaccine according to claim 5, it is characterised in that the quality of the emulsion type adjuvant and foot-and-mouth disease antigen
Than for 1:05～1.5.
7. the preparation method of aftosa vaccine described in claim 4, it is characterised in that will be emulsion type adjuvant and foot-and-mouth disease antigen pre-
Emulsion type adjuvant is mixed with foot-and-mouth disease antigen by heat to 15～30 DEG C, hand or stir, and obtains aftosa vaccine.
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| CN201610963355.XA CN106511996B (en) | 2016-11-04 | 2016-11-04 | Emulsion type adjuvant and preparation method thereof for aftosa vaccine |
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| CN201610963355.XA CN106511996B (en) | 2016-11-04 | 2016-11-04 | Emulsion type adjuvant and preparation method thereof for aftosa vaccine |
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Cited By (2)
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| CN111358944A (en) * | 2020-02-29 | 2020-07-03 | 浙江大学 | Composite vegetable oil vaccine adjuvant and application thereof |
| CN111789943A (en) * | 2019-04-08 | 2020-10-20 | 洛阳赛威生物科技有限公司 | Oil-in-water adjuvant composition and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111789943A (en) * | 2019-04-08 | 2020-10-20 | 洛阳赛威生物科技有限公司 | Oil-in-water adjuvant composition and preparation method and application thereof |
| CN111789943B (en) * | 2019-04-08 | 2024-01-19 | 洛阳赛威生物科技有限公司 | Oil-in-water adjuvant composition and preparation method and application thereof |
| CN111358944A (en) * | 2020-02-29 | 2020-07-03 | 浙江大学 | Composite vegetable oil vaccine adjuvant and application thereof |
| CN111358944B (en) * | 2020-02-29 | 2021-12-31 | 浙江大学 | Composite vegetable oil vaccine adjuvant and application thereof |
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