CN105396131A - Adjuvant for reovirus inactivated vaccine and preparing method thereof - Google Patents
Adjuvant for reovirus inactivated vaccine and preparing method thereof Download PDFInfo
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- CN105396131A CN105396131A CN201511022856.XA CN201511022856A CN105396131A CN 105396131 A CN105396131 A CN 105396131A CN 201511022856 A CN201511022856 A CN 201511022856A CN 105396131 A CN105396131 A CN 105396131A
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- adjuvant
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- inactivated 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
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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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
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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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
- A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12211—Orthoreovirus, e.g. mammalian orthoreovirus
Abstract
The invention provides an adjuvant for reovirus inactivated vaccine and a preparing method thereof. The ISCOM matrix component is adopted innovatively, so that an organism is stimulated to generate high cellular immunity capacity and humoral immunity capacity, immunologic enhancement is better achieved, and the immune protection period of the vaccine is prolonged. When the adjuvant is used for preparing the reovirus inactivated vaccine, the dosage form is water-in-oil (W/O) form, the reovirus inactivated vaccine prepared with the adjuvant has high stability due to reasonable design of constituents, and stable property is kept after the vaccine is stored at 2-8 DEG C for 18 months. Furthermore, practical use shows that after the reovirus inactivated vaccine prepared with the adjuvant is inoculated onto chicken, local or general adverse reaction can not be caused, safety performance is reliable, and a better immune effect is realized compared with reovirus inactivated vaccine prepared with the existing adjuvant. A prominent technical effect is realized through innovative technical improvement, cost is low, implementation is easy, and therefore popularization prospects are broad.
Description
Technical field
The present invention relates to live vaccine technical field, being specifically related to a kind of for exhaling adjuvant of the lonely inactivated vaccine of intestinal and preparation method thereof.
Background technology
Reovirus tool has the genomic viroid of segmented double-stranded RNA, this viral distribu-tion is extremely wide, easily eruption and prevalence between birds, fatality rate is higher, host's barrier can also be broken through simultaneously, infect the mammal comprising the mankind, therefore this disease has serious harm to human health and economic development.Exhale the lonely host of intestinal extensive, therefore prevention and control have larger difficulty, and except ensureing suitable sanitary condition and quarantine measures, vaccination is the key measure controlling to exhale intestinal lonely popular.In prior art, although exhale the lonely vaccine of intestinal to form product already, but still also exist such as Vaccine effectiveness not enough, need repeatedly immunity could onset, cost comparatively high-technology defect, thus be difficult to meet user demand.
In order to promote the immune effect of vaccine, the researcher of prior art has carried out effort from many aspects, such as, develop the better antigenic substance of immune effect, at vaccinated immunopotentiating agent etc. of feeding simultaneously.Adjuvant (adjuvant) be to be injected in after mixing with antigen in organism, can non-specific change or enhancing body to the material of the specific immune response of this antigen, therefore develop suitable vaccine adjuvant will contribute to promoting vaccine quality, strengthen immune effect.
In prior art, immunological adjuvant multiselect white-oil adjuvant, aluminum hydroxide adjuvant, lipoid adjuvant and propolis adjuvant etc., wherein white-oil adjuvant is believed to be helpful in strengthening vaccine immune efficacy, and application is comparatively extensive at present.White-oil adjuvant common formulations comprises oil-in-water (O/W), W/O/W (W/O/W), Water-In-Oil (W/O) three kinds, wherein the application of Water-In-Oil (W/O) type is the most general, but also there is certain technological deficiency, such as its viscosity be higher, injections difficult; Simultaneously because white oil content in vaccine is relatively high, usually cause serious Side effects, form the granuloma around aseptic suppuration infringement and inoculation position, outside visible edema, swelling, scleroma and necrosis can be produced; In addition have research think this dosage form adjuvant inoculation after may be formed residual at inoculation position, thus bring potential safety hazard.
Summary of the invention
The present invention is intended to the technological deficiency for prior art, provides a kind of for exhaling adjuvant of the lonely inactivated vaccine of intestinal and preparation method thereof, to solve the adjuvant of prior art to exhaling the technical problem that intestinal lonely inactivated vaccine immune efficacy facilitation is lower.
Another technical problem that the present invention will solve has side effects after the water-in-oil type adjuvant of prior art easily makes vaccine injection.
The technical problem again that the present invention will solve is that the adjuvant of prior art is unstable, the easily character of passing generation in time change.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of for exhaling the adjuvant of the lonely inactivated vaccine of intestinal, this adjuvant is divided into oil phase and ISCOM substrate two parts, wherein said oil phase comprises following composition: injection stage white oil 80 ~ 85% (w/w), span806 ~ 8% (w/w), Tween 80 6 ~ 8% (w/w); Described ISCOM substrate comprises following composition: QuilA0.2 ~ 5mg/ml, cholesterol 0.2 ~ 5mg/ml, levamisole 3 ~ 8mg/ml.
Preferably, the content of described QuilA is 1 ~ 5mg/ml.
Preferably, the content of described cholesterol is 1 ~ 5mg/ml.
The present invention additionally provides the preparation method of above-mentioned adjuvant simultaneously, and the method comprises the following steps:
1) get the QuilA of formula ratio, levamisole, cholesterol be dissolved in PBS buffer and obtain ISCOM substrate;
2) get the injection stage white oil of formula ratio, add span80 and the Tween 80 of formula ratio after sterilizing wherein, after emulsifying, namely obtain oil phase.
Preferred on the method basis, step 2) be: the injection stage white oil getting formula ratio, the span80 of formula ratio is added wherein after sterilizing, 4 ~ 6min is stirred with the rotating speed of 60 ~ 100r/min, then under stirring, add Tween 80 wherein, stir 4 ~ 6min with the rotating speed of 60 ~ 100r/min, namely obtain oil phase; More excellent, it is above-mentioned that twice speed of agitator is 80r/min, mixing time is 5min.
Adjuvant of the present invention can use by the following method in the preparation process of vaccine: mix as aqueous phase using vaccine antigen solution with the ISCOM substrate of adjuvant of the present invention, this aqueous phase is mixed in (1:1) ~ (1:3) (v/v) ratio with the oil phase of adjuvant of the present invention, namely obtains vaccine.
In above technical scheme, ISCOM (immunostimulatingcomplex) substrate is a kind of immunostimulating complex containing antigenic component, be called iscom matrix, by QuilA (saponin active component), cholesterol etc. prepare, its feature is effectively activate auxiliary type T cell and cytotoxic T cell, promote cellullar immunologic response, especially Th1 type cellullar immunologic response, secondly, the usual people of ISCOM substrate apply it in live vaccine, the present invention creatively applies it in inactivated vaccine, experiment confirms, can more effectively activate immunity, significantly improve the antigen presentation of totivirus and the persistence of vaccine.Levamisole is a kind of broad-spectrum de-worming medicine, and research simultaneously finds that it has obvious immunoregulation effect.
The present invention introduces ISCOM matrix components for what exhale the adjuvant novelty of the lonely inactivated vaccine of intestinal, improves immunological enhancement effect, body can be stimulated to produce stronger cellular immunization and humoral immunization, have better protective effect, extend the vaccine immunity protection period.When adjuvant of the present invention is used for exhaling the lonely inactivated vaccine of intestinal to prepare, dosage form is Water-In-Oil (W/O) type, rational Design on Plane due to component prescription makes the lonely inactivated vaccine of intestinal of exhaling utilizing this adjuvant to prepare have good stability, preserves and within 18 months, still keep character to stablize under 2-8 DEG C of condition.In addition, practical application finds, for preparing with this adjuvant exhales without local or Systemic reaction after intestinal lonely inactivated vaccine inoculation chicken, and safety is reliable, and has better immune effect compared with the lonely inactivated vaccine of intestinal of exhaling prepared by prior art adjuvant.The present invention achieves outstanding technique effect with the technological improvement of novelty, and cost is lower simultaneously, is easy to realize, therefore has outstanding promotion prospect.
Detailed description of the invention
Below will be described in detail the specific embodiment of the present invention.In order to avoid too much unnecessary details, in the examples below to belonging to known structure or function will not be described in detail.Apart from outside definition, technology used in following examples and scientific terminology have the identical meanings generally understood with those skilled in the art of the invention.
Embodiment 1
(1) preparation of adjuvant oil phase
Oil phase adds aluminium stearate in the mixed liquor formed according to percentage ratio (white oil: span80=17:1), and its addition is that every 100ml mixed liquor adds 2g aluminium stearate.120 DEG C, 20min high-temp steam sterilizing, room temperature preservation is for subsequent use.
(2) preparation of adjuvant ISCOM substrate
Take QuilA100mg, cholesterol 100mg, levamisole 500mg respectively, dissolve with 10mlPBS and mix to obtain the ISCOM substrate of 10 times of concentration.
(3) preparation of the lonely inactivation of viruses liquid of intestinal is exhaled
According to " veterinary biologics quality standard compilation " (2012), related request prepares the reovirus antigen aqueous solution of deactivation, every 0.2ml virus liquid viral level Ying≤10 before deactivation
7.0lD
50.
(4) preparation of the lonely vaccine of intestinal is exhaled
In sterile working's situation, the ISCOM3ml of preparation in above-mentioned (2) and (3) middle intestinal lonely inactivation of viruses liquid 15g that exhales prepared are put into the beaker of 50ml, mixing; The oil phase separately getting preparation in (1) is heated to 30 DEG C, get 13g and also put into this beaker, add magnetic rotor to put on magnetic stirring apparatus (containing heating function) and carry out stirring and emulsifying simultaneously, rotating speed is 2000 turns/min, temperature is 30 DEG C, stir 30min, called after exhales the lonely vaccine A of intestinal.
Do not add ISCOM substrate with method and make white oil control vaccine, called after control vaccine B
Substitute compound oil adjuvant of the present invention with the method ISA206 adjuvant that French SEPPIC company produces, prepare control vaccine, name control vaccine M.
(5) vaccine physical behavior and immunocompetently to compare in table 1.
The comparison of the lonely vaccine A of intestinal and control vaccine B, M exhaled by table 1 vaccine
Can be found out by table 1, adopt vaccine prepared by composite adjuvant of the present invention, stability is 18 months, and stablize than control vaccine M, meanwhile, its immune effect is better than control vaccine M, and side reaction is fewer than control vaccine M, has extraordinary actual use value.
Table 2: broiler chicks inoculation is different exhale the lonely vaccine of intestinal after exhale the lonely hemagglutination inhibition antibody titer (log2 meansigma methods) of intestinal
According to the grouping situation of table 2, often group repetition 3 batches is obtained exhales the lonely composite adjuvant inactivated vaccine of intestinal, after broiler chicks injects vaccine of the present invention and control vaccine, the hemagglutination inhibition antibody titer rate of climb of chicken group is faster than matched group, along with the increase of injection natural law, exhale the lonely vaccine antibody persistent levels of intestinal better, white oil matched group and control vaccine M group antibody horizontal persistence are all low than exhaling the lonely vaccine group of intestinal.
Embodiment 2
A kind of for exhaling the adjuvant of the lonely inactivated vaccine of intestinal, this adjuvant is divided into oil phase and ISCOM substrate two parts, wherein said oil phase consists of the following composition: injection stage white oil 85% (w/w), span807% (w/w), Tween 80 8% (w/w); Described ISCOM substrate comprises following composition: QuilA0.2mg/ml, cholesterol 0.2mg/ml, levamisole 3mg/ml.
Described adjuvant is prepared by the following method:
1) get the QuilA of formula ratio, levamisole, cholesterol be dissolved in PBS buffer and obtain ISCOM substrate;
2) get the injection stage white oil of formula ratio, after sterilizing, add the span80 of formula ratio wherein, stir 4min with the rotating speed of 60r/min, then under stirring, add Tween 80 wherein, stir 4min with the rotating speed of 60r/min, namely obtain oil phase.
Embodiment 3
A kind of for exhaling the adjuvant of the lonely inactivated vaccine of intestinal, this adjuvant is divided into oil phase and ISCOM substrate two parts, wherein said oil phase consists of the following composition: injection stage white oil 84% (w/w), span808% (w/w), Tween 80 8% (w/w); Described ISCOM substrate comprises following composition: QuilA5mg/ml, cholesterol 5mg/ml, levamisole 8mg/ml.
Described adjuvant is prepared by the following method:
1) get the QuilA of formula ratio, levamisole, cholesterol be dissolved in PBS buffer and obtain ISCOM substrate;
2) get the injection stage white oil of formula ratio, after sterilizing, add the span80 of formula ratio wherein, stir 6min with the rotating speed of 100r/min, then under stirring, add Tween 80 wherein, stir 6min with the rotating speed of 100r/min, namely obtain oil phase.
Embodiment 4
A kind of for exhaling the adjuvant of the lonely inactivated vaccine of intestinal, this adjuvant is divided into oil phase and ISCOM substrate two parts, wherein said oil phase consists of the following composition: injection stage white oil 84.5% (w/w), span808% (w/w), Tween 80 7.5% (w/w); Described ISCOM substrate comprises following composition: QuilA2.2mg/ml, cholesterol 1mg/ml, levamisole 6mg/ml.
Described adjuvant is prepared by the following method:
1) get the QuilA of formula ratio, levamisole, cholesterol be dissolved in PBS buffer and obtain ISCOM substrate;
2) get the injection stage white oil of formula ratio, add span80 and the Tween 80 of formula ratio after sterilizing wherein, after emulsifying, namely obtain oil phase.
Embodiment 5
A kind of for exhaling the adjuvant of the lonely inactivated vaccine of intestinal, this adjuvant is divided into oil phase and ISCOM substrate two parts, wherein said oil phase consists of the following composition: injection stage white oil 85% (w/w), span808% (w/w), Tween 80 7% (w/w); Described ISCOM substrate comprises following composition: QuilA1mg/ml, cholesterol 2.3mg/ml, levamisole 5mg/ml.
Described adjuvant is prepared by the following method:
1) get the QuilA of formula ratio, levamisole, cholesterol be dissolved in PBS buffer and obtain ISCOM substrate;
2) get the injection stage white oil of formula ratio, after sterilizing, add the span80 of formula ratio wherein, stir 5min with the rotating speed of 80r/min, then under stirring, add Tween 80 wherein, stir 5min with the rotating speed of 80r/min, namely obtain oil phase.
Above embodiments of the invention have been described in detail, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All make in application range of the present invention any amendment, equivalent to replace and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. one kind for exhaling the adjuvant of the lonely inactivated vaccine of intestinal, it is characterized in that this adjuvant is divided into oil phase and ISCOM substrate two parts, wherein said oil phase comprises following composition: injection stage white oil 80 ~ 85% (w/w), span806 ~ 8% (w/w), Tween 80 6 ~ 8% (w/w); Described ISCOM substrate comprises following composition: QuilA0.2 ~ 5mg/ml, cholesterol 0.2 ~ 5mg/ml, levamisole 3 ~ 8mg/ml.
2. adjuvant according to claim 1, is characterized in that the content of described QuilA is 1 ~ 5mg/ml.
3. adjuvant according to claim 1, is characterized in that the content of described cholesterol is 1 ~ 5mg/ml.
4. a preparation method for adjuvant described in claim 1, is characterized in that comprising the following steps:
1) get the QuilA of formula ratio, levamisole, cholesterol be dissolved in PBS buffer and obtain ISCOM substrate;
2) get the injection stage white oil of formula ratio, add span80 and the Tween 80 of formula ratio after sterilizing wherein, after emulsifying, namely obtain oil phase.
5. preparation method according to claim 4, it is characterized in that step 2) be: the injection stage white oil getting formula ratio, the span80 of formula ratio is added wherein after sterilizing, 4 ~ 6min is stirred with the rotating speed of 60 ~ 100r/min, then under stirring, add Tween 80 wherein, stir 4 ~ 6min with the rotating speed of 60 ~ 100r/min, namely obtain oil phase.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201511022856.XA CN105396131A (en) | 2015-12-29 | 2015-12-29 | Adjuvant for reovirus inactivated vaccine and preparing method thereof |
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| CN201511022856.XA CN105396131A (en) | 2015-12-29 | 2015-12-29 | Adjuvant for reovirus inactivated vaccine and preparing method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105770887A (en) * | 2016-03-04 | 2016-07-20 | 邓招红 | Adjuvant for HBV (hepatitis B virus) vaccine and preparation method of adjuvant |
| CN112641939A (en) * | 2020-12-28 | 2021-04-13 | 吉林冠界生物技术有限公司 | Water-in-oil type composite adjuvant, preparation method and application thereof, and preparation method of vaccine |
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2015
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105770887A (en) * | 2016-03-04 | 2016-07-20 | 邓招红 | Adjuvant for HBV (hepatitis B virus) vaccine and preparation method of adjuvant |
| CN112641939A (en) * | 2020-12-28 | 2021-04-13 | 吉林冠界生物技术有限公司 | Water-in-oil type composite adjuvant, preparation method and application thereof, and preparation method of vaccine |
| CN112641939B (en) * | 2020-12-28 | 2024-04-02 | 吉林冠界生物技术有限公司 | Water-in-oil type composite adjuvant, preparation method and application thereof, and preparation method of vaccine |
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Application publication date: 20160316 |