CN103251941A - Animal viral vaccine pulsatile release system, and preparation method and application thereof - Google Patents
Animal viral vaccine pulsatile release system, and preparation method and application thereof Download PDFInfo
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- CN103251941A CN103251941A CN2012100404364A CN201210040436A CN103251941A CN 103251941 A CN103251941 A CN 103251941A CN 2012100404364 A CN2012100404364 A CN 2012100404364A CN 201210040436 A CN201210040436 A CN 201210040436A CN 103251941 A CN103251941 A CN 103251941A
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Abstract
The invention relates to the field of biomedical engineering, and specifically relates to an animal viral vaccine pulsatile release system, a preparation method thereof, and an application thereof in animal disease prevention. According to the invention, a vaccine pulsatile release system composed of polymer microspheres comprising vaccine antigens and with different release characteristics and compositions of the microspheres; injection medication is adopted; and full immunization is realized with single dose injection. Therefore, immunization process is simplified, immunization effect is improved, labor intensity is reduced, and immunization missing rate is reduced.
Description
Technical field
The present invention relates to biological medicine engineering field, be specifically related to a kind of animal vaccine pulse releasing system, its preparation method and the application in the animal epidemic control.
Background technology
Vaccine virus immunization is prevention, control and the most effective and the most practical method of eliminating animal infectious disease.But traditional inactivated vaccine and the genetic engineering subunit vaccine, synthetic peptide vaccine, dna vaccination etc. that represent future thrust all exist not enough, and namely immunogenicity is low, needs repeatedly repeated inoculation could produce effective immanoprotection action.The approach that addresses this problem has two, and the one, develop new and effective immunological adjuvant; The 2nd, develop the controlled release system of injectable vaccine antigen, in constant, that continue, predetermined mode with antigen delivery to particular organization.
The particulate antigen of similar pathogen is effectively engulfed by antigen-presenting cell (APC) than dissolubility antigen is easier on the form, thereby causes more strong cellular immunization and humoral immunization.Studying both at home and abroad and using polyester, gelatin, polyacryl starch, chitosan etc. as adjuvant, the parcel vaccine is made the Biodegradable polymer microballoon, makes antigen particlesization, thereby more effectively promotes cellular immunization and the humoral immunization of body.The biological degradation polyalcohol microsphere is the research focus of vaccine adjuvant in recent years, also is the maximum also the most successful antigen vectors system of research.
The principal character of microsphere is the release that they can control antigen.Microsphere can be made up of two or more different polymer, the antigen that carries of wrapping can be different speed discharge.The long-term release of antigen is equivalent to repeatedly vaccination, thereby produces high-level antibody, and this will make research and development inoculation single vaccinating agent once become possibility.
Modern animal produces the pattern that adopts intensive culture.The animal epidemic particularly control of viral blight is one of key issue of facing of modern farming.But explore the pulse releasing system of the vaccine antigen of single needle injection, can reduce immune cost and labor intensity effectively, reduce animal stress, improve immune effect.This all has important value in scientific research and production practices.
Summary of the invention
The invention discloses a kind of zoosis toxicity vaccine pulse releasing system, by adopting polymer manufacture to contain the vaccine microsphere with pulsatile release characteristics or slow release characteristic of vaccine antigen, the vaccine microsphere appropriate combination with the different release characteristics of tool namely constitutes the vaccine pulse releasing system again.This kind vaccine pulse releasing system can be used for the single needle immunity of zoosis toxicity eqpidemic disease, replaces the spininess immunity of common vaccine, realizes the immune protection of zoosis toxicity eqpidemic disease.It uses more convenient than vaccine regular dosage form, can reduce labor intensity greatly, reduce animal stress, have adjuvant effect simultaneously, make immune effect better.
The preparation method of zoosis toxicity vaccine pulse releasing system of the present invention is as follows:
1. adopt the emulsion intra-liquid desiccation method to prepare the vaccine microsphere, its basic step comprises: get colostrum and make emulsion, be transferred in the beaker, volatilize solvent under the magnetic agitation, room temperature, centrifugal collection vaccine microsphere, with the sterilized water for injection washing, vacuum lyophilization namely gets the vaccine microsphere under the aseptic condition.
2. adopt spraying low-temperature extraction legal system to be equipped with the vaccine microsphere, its basic step comprises: vaccine antigen and polymer solution are mixed and made into suspension, be sprayed onto in the freezing alcoholic solution with vaporific through shower nozzle, refilter and remove ethanol, with the sterilized water for injection washing, vacuum lyophilization namely gets the vaccine microsphere under the aseptic condition.
3. adopt spray drying method for preparation vaccine microsphere, its basic step comprises: vaccine antigen and polymer solution are mixed and made into suspension, and spray-dried device spray drying namely gets the vaccine microsphere.
4. the vaccine microsphere is pressed release characteristics and antigenic content appropriate combination the vaccine pulse releasing system.
The specific embodiment
Embodiment 1
The preparation of newcastle killed vaccine antigen
Get Newcastle disease attenuated La Sota strain, inoculate the susceptible Embryo Gallus domesticus according to a conventional method, through cultivating, results Embryo Gallus domesticus liquid is through the formalin deactivation.Get the Avian pneumo-encephalitis virus Embryo Gallus domesticus liquid after the deactivation, in 4 ℃ of centrifuging and taking supernatant, supernatant is that 30,000 ultrafilter membrane carries out ultrafiltration and concentration under 4 ℃ of aseptic conditions through molecular cut off again under aseptic condition.Supernatant after ultrafiltration purification concentrates is the antigen liquid of preparation vaccine microsphere.
Embodiment 2
The preparation I of newcastle inactivated vaccine microsphere
The preparation of microsphere: adopt the emulsion intra-liquid desiccation method to prepare newcastle inactivated vaccine PLGA microsphere.With the newcastle antigen liquid as water, with PLGA (50: 50) be dissolved in make debita spissitudo in the dichloromethane solution as oil phase.Water and oil phase are mixed in 1: 10 (V/V) ratio, and ultrasonic emulsification got colostrum in 10 seconds under the condition of ice bath.Colostrum is added in the 2%PVA solution, through emulsator high-speed stirred 30 seconds, get emulsion under the 10000rpm rotating speed.The gained emulsion is transferred in the beaker, stirred 5 hours at 400rpm rotating speed lower magnetic force, volatilize dichloromethane under the room temperature condition, under the 6000rpm rotating speed centrifugal 10 minutes again, collect microsphere, with aquesterilisa washing 3 times, vacuum lyophilization namely gets newcastle inactivated vaccine PLGA microsphere I.
The quality evaluation of microsphere: microscopic method is observed the microsphere form and is measured particle diameter; Measure antigenic content in the microsphere with ultraviolet spectrophotometry or Bradford method, by formula computational envelope rate and drug loading, in vitro method is measured the release profiles of vaccine microsphere.
After measured, the release in vitro of newcastle inactivated vaccine microsphere I is two facies models, first prominent the releasing in later half day of injection, and second prominent the releasing in injection back 35 days is slow release phase between biphase.
Embodiment 3
The preparation II of newcastle inactivated vaccine microsphere
The preparation of microsphere: adopt the emulsion intra-liquid desiccation method to prepare newcastle inactivated vaccine PLGA microsphere.With the newcastle antigen liquid as water, with PLGA (75: 25) be dissolved in make debita spissitudo in the dichloromethane solution as oil phase.Water and oil phase are mixed in 1: 10 (V/V) ratio, and ultrasonic emulsification got colostrum in 10 seconds under the condition of ice bath.Colostrum is added in the 2%PVA solution, through emulsator high-speed stirred 30 seconds, get emulsion under the 4000rpm rotating speed.The gained emulsion is transferred in the beaker, stirred 5 hours at 200rpm rotating speed lower magnetic force, volatilize dichloromethane under the room temperature condition, under the 5000rpm rotating speed centrifugal 10 minutes again, collect microsphere, with aquesterilisa washing 3 times, vacuum lyophilization namely gets newcastle inactivated vaccine PLGA microsphere II.
The quality evaluation of microsphere: microscopic method is observed the microsphere form and is measured particle diameter; Measure antigenic content in the microsphere with ultraviolet spectrophotometry or Bradford method, by formula computational envelope rate and drug loading, in vitro method is measured the release profiles of vaccine microsphere.
After measured, the release in vitro of newcastle inactivated vaccine microsphere II is two facies models, first prominent the releasing in later half day of injection, and second prominent the releasing in injection back 96 days is slow release phase between biphase.
Embodiment 4
The preparation III of newcastle inactivated vaccine microsphere
The preparation of microsphere: adopt the emulsion intra-liquid desiccation method to prepare newcastle inactivated vaccine PLGA microsphere.With the newcastle antigen liquid as water, with PLGA (85: 15) be dissolved in make debita spissitudo in the dichloromethane solution as oil phase.Water and oil phase are mixed in 1: 10 (V/V) ratio, and ultrasonic emulsification got colostrum in 10 seconds under the condition of ice bath.Colostrum is added in the 2%PVA solution, through emulsator high-speed stirred 30 seconds, get emulsion under the 4000rpm rotating speed.The gained emulsion is transferred in the beaker, stirred 5 hours at 200rpm rotating speed lower magnetic force, volatilize dichloromethane under the room temperature condition, under the 5000rpm rotating speed centrifugal 10 minutes again, collect microsphere, with aquesterilisa washing 3 times, vacuum lyophilization namely gets newcastle inactivated vaccine PLGA microsphere III.
The quality evaluation of microsphere: microscopic method is observed the microsphere form and is measured particle diameter; Measure antigenic content in the microsphere with ultraviolet spectrophotometry or Bradford method, by formula computational envelope rate and drug loading, in vitro method is measured the release profiles of vaccine microsphere.
After measured, the release in vitro of newcastle inactivated vaccine microsphere I is two facies models, first prominent the releasing in later half day of injection, and second prominent the releasing in injection back 182 days is slow release phase between biphase.
Embodiment 5
The preparation of newcastle inactivated vaccine pulse releasing system and chicken immune test
Get newcastle inactivated vaccine microsphere I, microsphere II and microsphere III, the three is mixed in 1: 1: 1 ratio (in antigenic content), total amount is equivalent to 1/10 of common newcastle inactivated vaccine single injection antigen amount, be packaged under the aseptic condition in the sterilization cillin bottle, 4 ℃ of storages are standby, face the suspension that is made into 0.1mL with sterile saline with preceding.
Get 2 all SPF chickens in age (not having newcastle epidemic disease antibody after testing), random packet, every group 10, be divided into negative control group (sterile saline), positive controls (common newcastle inactivated vaccine) and test group (newcastle inactivated vaccine pulse releasing system), inject 0.1mL under every Corium Gallus domesticus, every chicken blood sampling separation of serum weekly after the immunity, the HI method detects in the serum newcastle epidemic disease antibody and tires routinely.
After testing, the geometrical mean of negative control group HI antibody titer all＜2log
2, the geometrical mean 〉=4log of positive controls 2-6 HI antibody titer in week after immunity
2, drop to gradually afterwards≤2log
2, test group the geometrical mean of whole test phase (6 months) HI antibody titer all 〉=7.5log
2, wherein in the 2nd, 6,13,27 weeks, serum HI tires and peak value occurs.
Claims (6)
1. zoosis toxicity vaccine pulse releasing system is characterized by: 1. this delivery system is made up of the polymer microballoon that one or more comprise the different release characteristics of tool of corresponding antigens; 2. comprise or do not comprise all acceptable accessories simultaneously; 3. comprise or do not comprise acceptable immunological adjuvant on all vaccinologies simultaneously; 4. its administering mode comprises intradermal injection, subcutaneous injection, intramuscular injection for injection; 5. its delivery mode is pulse release, presents pulse release more than 2 times or 2 times in 1-12 month scope.
2. the described zoosis toxicity of claim 1 vaccine comprises: the inactivated vaccine, genetic engineering subunit vaccine, the synthetic peptide vaccine that are used for animal virus sexually transmitted disease immune protection.
3. the described polymer microballoon of claim 1, wherein polymer refers to all pharmaceutically acceptable polymeric materials, wherein preferably have biocompatibility, biodegradable polymer, comprise chitosan, collagen, gelatin, albumin, polyamino acid, polyester (polyester), poly-anhydride (polyanhydride), polylactic acid-polyglycol copolymer (PELA) etc.
4. the described polymer microballoon of claim 1, its size have pulsatile release characteristics or slow release characteristics between 10nm-1000 μ m.
5. the described polymer microballoon of claim 1, its preparation method are emulsion intra-liquid desiccation method, spraying low-temperature extraction method or spray drying method.
6. the described zoosis toxicity of claim 1 vaccine pulse releasing system is used for the single needle immunity of zoosis toxicity eqpidemic disease, replaces the spininess immunity of common vaccine, reaches the effect of control animal virus sexually transmitted disease.
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Cited By (3)
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| WO2015095230A1 (en) * | 2013-12-16 | 2015-06-25 | Massachusetts Institute Of Technology | Micromolded or 3-d printed pulsatile release vaccine formulations |
| CN112516070A (en) * | 2020-12-16 | 2021-03-19 | 南开大学 | Single injection vaccine of protein antigen and preparation method thereof |
| US11541017B2 (en) | 2013-12-16 | 2023-01-03 | Massachusetts Institute Of Technology | Fortified micronutrient salt formulations |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015095230A1 (en) * | 2013-12-16 | 2015-06-25 | Massachusetts Institute Of Technology | Micromolded or 3-d printed pulsatile release vaccine formulations |
| AU2014364930B2 (en) * | 2013-12-16 | 2017-06-15 | Massachusetts Institute Of Technology | Micromolded or 3-D printed pulsatile release vaccine formulations |
| US10300136B2 (en) | 2013-12-16 | 2019-05-28 | Massachusetts Institute Of Technology | Micromolded or 3-D printed pulsatile release vaccine formulations |
| EP3082852B1 (en) * | 2013-12-16 | 2020-06-17 | Massachusetts Institute of Technology | Micromolded or 3-d printed pulsatile release vaccine formulations |
| US10960073B2 (en) | 2013-12-16 | 2021-03-30 | Tokitae Llc | Micromolded or 3-D printed pulsatile release vaccine formulations |
| US11541017B2 (en) | 2013-12-16 | 2023-01-03 | Massachusetts Institute Of Technology | Fortified micronutrient salt formulations |
| US11975069B2 (en) | 2013-12-16 | 2024-05-07 | Massachusetts Institute Of Technology | Micromolded or 3-D printed pulsatile release vaccine formulations |
| CN112516070A (en) * | 2020-12-16 | 2021-03-19 | 南开大学 | Single injection vaccine of protein antigen and preparation method thereof |
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Application publication date: 20130821 |