CN112516325B - Stable foot-and-mouth disease vaccine composition and application thereof - Google Patents
Stable foot-and-mouth disease vaccine composition and application thereof Download PDFInfo
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- 229960005486 vaccine Drugs 0.000 title claims abstract description 70
- 241000710198 Foot-and-mouth disease virus Species 0.000 title claims abstract description 46
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 title claims abstract description 42
- 239000000203 mixture Substances 0.000 title claims abstract description 42
- 239000000427 antigen Substances 0.000 claims abstract description 54
- 102000036639 antigens Human genes 0.000 claims abstract description 53
- 108091007433 antigens Proteins 0.000 claims abstract description 53
- 239000003381 stabilizer Substances 0.000 claims abstract description 34
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
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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
- A61K39/125—Picornaviridae, e.g. calicivirus
- A61K39/135—Foot- and mouth-disease virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- 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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- 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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- 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
- 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 stabilizer for stabilizing foot-and-mouth disease antigens, wherein the stabilizer comprises: 0.5-2 w/V% DEAE dextran, 0.5-2 w/V% hydroxypropyl-beta-cyclodextrin, based on the final concentration in the vaccine composition. The stabilizer can stabilize the inactivated foot-and-mouth disease antigen in the vaccine composition for a long time, ensure the content of 146S to be stable, and ensure the stability and the consistent appearance of the vaccine composition. The invention also provides a vaccine composition containing the stabilizer.
Description
Technical Field
The invention belongs to the field of medical preparations, and particularly relates to a stable foot-and-mouth disease vaccine composition.
Background
Instability of inactivated foot-and-mouth disease virus antigens is one of the great challenges faced in vaccine production. The foot-and-mouth disease inactivated virus antigen is mainly complete virus with a sedimentation coefficient of 146S, has a size of about 30nm and has strong immunogenicity. However, 146S is susceptible to disruption of particle structure during production and storage, is cleaved into smaller sized particles 12S, is less than 10nm in size, and releases viral nucleic acid, whereas cleaved 12S is hardly immunogenic. Thus, the yield of active antigen complete virus 146S is critical for foot and mouth disease vaccine production.
CN108504638A discloses a method for purifying or storing foot-and-mouth disease inactivated virus antigen, which increases the yield of active antigen by adding polyhydroxy compound during purification or storage. As is apparent from the results of the fifth page table 1 of the patent specification, among the many substances which are generally used as ordinary protein protectants, compounds having a polyhydric hydroxyl group such as saccharides, sugar alcohols, glycerol and the like have an inhibitory effect on cleavage of 146S, while amino acids, surfactants, albumin and the like have no protective effect, and even have an effect of promoting cleavage.
"research on preservation technology of foot-and-mouth disease vaccine" (Li Zhengfeng, etc.) discloses a stabilizer for foot-and-mouth disease, which comprises trehalose, sorbitol and PVP in a proportion of 4:5:1, and discloses that adding a stabilizer into a foot-and-mouth disease vaccine influences the stability of the vaccine, and trehalose, sorbitol and PVP all have an influence on the stability of the vaccine, wherein PVP has a larger influence.
Accordingly, there is a need in the art for a foot-and-mouth disease vaccine composition that does not affect the stability of the vaccine, but also enhances the stability of the foot-and-mouth disease antigen.
Disclosure of Invention
To solve the above technical problems, the present invention provides a stabilizer for stabilizing foot-and-mouth disease antigen, wherein the stabilizer comprises: 0.5-2 w/V% DEAE dextran, 0.5-2 w/V% hydroxypropyl-beta-cyclodextrin, based on the final concentration in the vaccine composition.
By using DEAE dextran and hydroxypropyl-beta-cyclodextrin as stabilizers, a synergistic effect is generated, and the combination has remarkable effect on the stability of foot-and-mouth disease antigens and can reduce the use concentration of the DEAE dextran and the hydroxypropyl-beta-cyclodextrin.
The final concentration of DEAE-dextran in the vaccine composition may be selected from 0.5w/V, 0.6w/V, 0.7w/V, 0.8w/V, 0.9w/V, 1.0w/V, 1.1w/V, 1.2w/V, 1.3w/V, 1.4w/V, 1.5w/V, 1.6w/V, 1.7w/V, 1.8w/V, 1.9w/V, 2w/V%.
The final concentration of hydroxypropyl-beta-cyclodextrin in the vaccine composition may be selected from 0.5w/V%, 0.6w/V%, 0.7w/V%, 0.8w/V%, 0.9w/V%, 1.0w/V%, 1.1w/V%, 1.2w/V%, 1.3w/V%, 1.4w/V%, 1.5w/V%, 1.6w/V%, 1.7w/V%, 1.8w/V%, 1.9w/V%, 2w/V%. In one embodiment of the invention, the stabilizing agent for stabilizing foot-and-mouth disease antigen further comprises 0.1-0.4% w/V EDTA2Na.
The stability is slightly improved by adding EDTA2Na.
The final EDTA2Na concentration in the vaccine composition may be selected from 0.1% w/V, 0.2% w/V, 0.3% w/V, 0.4w/V.
In one embodiment of the present invention, the stabilizer for stabilizing an antigen of foot-and-mouth disease according to the present invention comprises: 0.5w/V% DEAE dextran, 0.5w/V% hydroxypropyl-beta-cyclodextrin, based on the final concentration in the vaccine composition; or the stabilizer comprises: 0.5w/V% DEAE dextran, 0.5w/V% hydroxypropyl-beta-cyclodextrin, 0.1w/VEDTA2Na, at the final concentration in the vaccine composition; or the stabilizer comprises: 1w/V% DEAE dextran, 1w/V% hydroxypropyl-beta-cyclodextrin, 0.2w/VEDTA2Na, at the final concentration in the vaccine composition; or the stabilizer comprises: 2w/V% DEAE dextran, 2w/V% hydroxypropyl-beta-cyclodextrin, 0.1w/VEDTA2Na, at the final concentration in the vaccine composition; or the stabilizer comprises: 2w/V% DEAE-dextran, 2w/V% hydroxypropyl-beta-cyclodextrin, 0.4w/VEDTA2Na at the final concentration in the vaccine composition.
In one embodiment of the present invention, the stabilizing agent for stabilizing a foot-and-mouth disease antigen according to the present invention is an inactivated foot-and-mouth disease antigen.
In one embodiment of the present invention, the stabilizing agent for stabilizing foot-and-mouth disease antigen according to the present invention is an inactivated foot-and-mouth disease 146S antigen.
In a preferred embodiment of the present invention, the stabilizing agent for stabilizing a foot-and-mouth disease antigen according to the present invention is an inactivated O-type foot-and-mouth disease antigen.
In a more preferred embodiment of the present invention, the stabilizing agent for stabilizing foot-and-mouth disease antigen according to the present invention is an inactivated MYA 98/O146S antigen.
The invention also provides a vaccine composition, wherein the vaccine composition comprises the stabilizer, an immune quantity of foot-and-mouth disease inactivated antigen and tris buffer solution.
The addition of the stabilizer to the vaccine composition of the present invention has no effect on the stability of the vaccine, and the stabilization of the antigen can be achieved in the vaccine for a long period of time.
In one embodiment of the present invention, in the vaccine composition of the present invention, the inactivated antigen for foot-and-mouth disease is 146S.
As a preferred embodiment of the invention, in the vaccine composition of the invention, the foot-and-mouth disease antigen is an inactivated MYA 98/O146S antigen.
As one embodiment of the present invention, in the vaccine composition of the present invention, the tris buffer is 0.12% w/v tris buffer and the pH is 7.5.
The invention also provides application of the stabilizer in stabilizing the stability of the inactivated foot-and-mouth disease antigen in the vaccine composition.
The beneficial effects of the invention are that
The vaccine composition of the invention has good stability and thermal stability (and vaccine appearance stability, and can ensure that the vaccine can realize antigen stabilization for a long time).
Detailed Description
The advantages and features of the present invention will become more apparent from the following description of the embodiments. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
The chemical reagents used in the examples of the invention are all analytically pure and purchased from the national drug group. The experimental methods provided by the invention are conventional methods unless specified; the biological material, unless otherwise specified, is commercially available.
EXAMPLE 1 Effect of additives on foot and mouth disease Virus antigen stability
Taking virus stock solution containing O type (MYA 98/O) foot-and-mouth disease virus antigen with the concentration of 27 mug/mL, respectively adding different substances and additives with different concentrations in table 1, storing at 45 ℃ for 30min, measuring the residual antigen content, calculating the percentage of the residual antigen content accounting for 0min according to a sucrose density centrifugation method by a measuring method, and comparing the percentage with the residual antigen content (blank control) without adding any substances.
TABLE 1 residual antigen content after storage of foot and mouth disease Virus antigen with different additives and different concentrations of additives at 45℃for 30min
| Additive agent | Additive concentration (w/V) | Residual antigen content |
| Blank control | 23％ | |
| Sucrose | 5 | 76％ |
| Sucrose | 15 | 82％ |
| DEAE dextran | 1 | 22％ |
| DEAE dextran | 5 | 26％ |
| Hydroxypropyl-beta-cyclodextrin | 1 | 23％ |
| Hydroxypropyl-beta-cyclodextrin | 5 | 47％ |
| Hydroxypropyl-beta-cyclodextrin | 10 | 63％ |
The test results in Table 1 show that the addition of DEAE-dextran at low concentration has little effect on the stability improvement of foot-and-mouth disease antigen, and that hydroxypropyl-beta-cyclodextrin and sucrose both need higher concentrations to function as stabilizers.
Example 2 Effect of foot and mouth disease antigen solution addition of stabilizers on vaccine stability
Taking foot-and-mouth disease antigen solutions added with different additives and different concentrations in table 2 as water phase, adding adjuvant (206 adjuvant) and emulsifying at 31 ℃ for 5 minutes according to the weight ratio of 1:1, performing stability test at 25 ℃ after emulsification, observing the appearance stability of the vaccine, and the test results are shown in table 2.
TABLE 2 appearance stability of foot and mouth disease vaccine at 25℃
| Additive agent | Additive and concentration (w/V) in antigen | Stability at 25 DEG C |
| Blank control | —— | No obvious change in 14 days |
| Sucrose | 5 | Obvious delamination after 5 days |
| Sucrose | 15 | Obvious delamination after 2 days |
| DEAE dextran | 1 | No obvious change in 14 days |
| DEAE dextran | 5 | No obvious change in 14 days |
| Hydroxypropyl-beta-cyclodextrin | 1 | No obvious change in 14 days |
| Hydroxypropyl-beta-cyclodextrin | 5 | No obvious change in 14 days |
| Hydroxypropyl-beta-cyclodextrin | 10 | Obvious delamination in 7 days |
As can be seen from the test results in Table 2, sucrose has a significant effect on the stability (appearance stability) of the foot-and-mouth disease vaccine, while DEAE dextran and low concentration of hydroxypropyl-beta-cyclodextrin have little effect on the stability (appearance stability) of the vaccine.
EXAMPLE 3 preparation of foot-and-mouth disease vaccine and stability test
3.1 preparation of aqueous phase
The foot-and-mouth disease antigen aqueous phase was prepared with 27. Mu.g/mL of foot-and-mouth disease O-antigen as antigen stock solution according to the additives and contents of Table 3, the balance being diluent, the diluent being 0.12% (w/v) tris alkali solution, and the final pH being 7.5. The remaining antigen content was determined after 30min storage of the aqueous phase at 45℃by sucrose density centrifugation, and the percentage of antigen content at 0min was calculated for the remaining antigen content.
TABLE 3 formulation and stability of antigen aqueous phase
The test results in Table 3 show that DEAE dextran and hydroxypropyl-beta-cyclodextrin are used as stabilizers to produce synergistic effect, and the combination has remarkable effect on the stability of foot-and-mouth disease antigen and can reduce the use concentration of the DEAE dextran and hydroxypropyl-beta-cyclodextrin. And the stability is slightly improved after EDTA2Na is added.
In the vaccine composition of the invention, any combination of DEAE-dextran final concentration of 0.5-2% w/V, hydroxypropyl-beta-cyclodextrin final concentration of 0.5-2% w/V and EDTA2Na final concentration of 0.1-0.4% w/V can ensure good stability of the vaccine.
3.2 preparation of vaccine
Taking the antigen solution added with additives with different concentrations in 3.1 as a water phase, adding an adjuvant (206 adjuvant) according to the weight ratio of 1:1, and emulsifying for 5 minutes at 31 ℃ to prepare the vaccine.
3.3 vaccine stability test
3.2, respectively carrying out stability appearance stability test at 25 ℃, demulsifying a stability test sample at 4 ℃ for 9 months according to the CN 102380232 method, measuring 146S content, calculating the ratio of 146S measured on day 0, and obtaining the result shown in Table 4.
TABLE 4 appearance stability of foot and mouth disease vaccine at 25℃
| Group of | Appearance stability at 25 DEG C | Stability at 4℃at 9 months |
| Test example 1 preparation of vaccine | No obvious change in 14 days | 88％ |
| Test example 2 preparation of vaccine | No obvious change in 14 days | 90％ |
| Test example 3 preparation of vaccine | No obvious change in 14 days | 89％ |
| Test example 4 preparation of vaccine | No obvious change in 14 days | 93％ |
| Test example 5 preparation of vaccine | No obvious change in 14 days | 92％ |
| Comparative example 1 formulation of vaccine | No light is seen in 14 daysDisplay changes | 81％ |
From the results of Table 4, it is understood that the addition of the stabilizer to the vaccine composition of the present invention has no effect on the stability of the vaccine, and that the stabilization of the antigen can be achieved in the vaccine for a long period of time.
The present invention is not limited to the above-mentioned embodiments, but is capable of modification and variation in all embodiments without departing from the spirit and scope of the present invention.
Claims (6)
1. A vaccine composition, wherein the vaccine composition comprises a stabilizer and an immunizing amount of foot-and-mouth disease inactivated 146S antigen, and tris buffer;
the stabilizer consists of the following components: 0.5-2 w/V% DEAE dextran, 0.5-2 w/V% hydroxypropyl-beta-cyclodextrin, based on the final concentration in the vaccine composition.
2. The vaccine composition of claim 1, wherein the stabilizer further comprises 0.1-0.4 w/v% EDTA2Na.
3. Vaccine composition according to claim 1 or 2, wherein the stabilizer consists of: 0.5w/V% DEAE dextran, 0.5w/V% hydroxypropyl-beta-cyclodextrin, based on the final concentration in the vaccine composition; or (b)
The stabilizer consists of the following components: 0.5w/V% DEAE dextran, 0.5w/V% hydroxypropyl-beta-cyclodextrin, 0.1w/V% EDTA2Na, based on the final concentration in the vaccine composition; or (b)
The stabilizer consists of the following components: 1w/V% DEAE dextran, 1w/V% hydroxypropyl- β -cyclodextrin, 0.2w/V% EDTA2Na, based on the final concentration in the vaccine composition; or (b)
The stabilizer consists of the following components: 2w/V% DEAE dextran, 2w/V% hydroxypropyl- β -cyclodextrin, 0.1w/V% EDTA2Na, based on the final concentration in the vaccine composition; or (b)
The stabilizer consists of the following components: 2w/V% DEAE dextran, 2w/V% hydroxypropyl-beta-cyclodextrin, 0.4w/V% EDTA2Na, based on the final concentration in the vaccine composition.
4. The vaccine composition of claim 1, wherein the foot-and-mouth disease inactivated 146S antigen is an inactivated O-type foot-and-mouth disease 146S antigen.
5. The vaccine composition of claim 4, wherein the foot-and-mouth disease inactivated 146S antigen is an inactivated MYA 98/O146S antigen.
6. The vaccine composition of claim 1, wherein the tris buffer is 0.12% w/v tris buffer, pH 7.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910883507.9A CN112516325B (en) | 2019-09-18 | 2019-09-18 | Stable foot-and-mouth disease vaccine composition and application thereof |
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