CN101020053B - Preparation process of inactivated rotavirus vaccine - Google Patents
Preparation process of inactivated rotavirus vaccine Download PDFInfo
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- CN101020053B CN101020053B CN2007100657085A CN200710065708A CN101020053B CN 101020053 B CN101020053 B CN 101020053B CN 2007100657085 A CN2007100657085 A CN 2007100657085A CN 200710065708 A CN200710065708 A CN 200710065708A CN 101020053 B CN101020053 B CN 101020053B
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Abstract
The present invention discloses preparation process of inactivated rotavirus vaccine. Rotavirus strains G1, G2, G3 and G4 are made to adapt human diploid cell line and propagate on human diploid cell line to obtain the producing strain of inactivated rotavirus vaccine, with the human diploid cell line being KMB17. The human diploid cell line used as the cell matrix for producing inactivated rotavirus vaccine is safer compared with available cell matrixes. Therefore, by means of the established technological platform, it is possible to develop inactivated rotavirus vaccine with other serotypesof rotavirus as the target.
Description
Technical field
The present invention relates to a kind of preparation method of inactivated rotavirus vaccine, more particularly, the present invention relates to utilize human diploid cell system to cultivate rotavirus, and then prepare the method for unit price or multivalent inactivated vaccine.
Background technology
Human reoviruslike agent (human rotavirus, infection HRV) is the main cause that causes the acute colliquative diarrhea of infant, HRV worldwide propagates.No matter at present in developed country or developing country, rotavirus remains infant disease and the main causes of death that cause.The infectiousness of rotavirus is very strong, can form propagation and popular in the short period of time.Developing vaccine is the effective way of control and prevention rotavirus infection.
The development of Rotavirus Vaccine is in earlier stage based on oral attenuated live vaccine, and for multiple HRV serotype is produced the excellent protection effect, research worker has developed multivalence reassortant vaccine.Comprise at present the various countries research institution of China, the U.S., Belgium, Australia, India and company respectively 6 kinds of rotavirus live vaccine (single serotype or reassortant) of development all entered preclinical study or put on market.Though attenuated live vaccine taking convenience; can breed in vivo; stimulate and produce intestinal protection antibody; but aspect safety, but exist defective, for example; the people monkey reprovision vaccine Rotashield of drugs approved by FDA in 1998 has withdrawn from American market owing to existing the hidden danger that causes intussusception); especially HRV belongs to RNA viruses, has the characteristics of high variability, and vaccine strain in human body and the circulation in the environment also exist the virulence back mutation and produce the risk of new variant.Aspect the safety of vaccine, inactivated vaccine has absolute advantage.But the inactivated vaccine of rotavirus still is in conceptual phase at present, does not also enter clinical.
Because inactivated vaccine is to inoculate in the mode of injection, the selection permission, it is very important that the cellular matrix of safety increases to virus.Human diploid cell system is the cellular matrix of present production of vaccine first-selection.From present development, preparing vaccine with diploid cell will have the trend that replaces other primary cell and passage cell.As inactivated vaccine,, may cause immune allergy, so the cellular matrix of inactivated vaccine requires higher through repeatedly immunity of intramuscular injection.Rotavirus is adapted to human diploid cell, adopt the cellular matrix of human diploid cell system, will have low allergy, the DEVELOPMENT PROSPECT of high value is arranged as large-scale culture.
The propagation of rotavirus on cell exists following characteristics: promptly the rotavirus of animal origin is more easily bred, the multiplication capacity of people's rotavirus when In vitro culture a little less than, titre is also lower.Rotavirus is at its sensitive cells such as MA104, and propagation is very fast on the cells such as CV-1, but then can not breed on human diploid cell, or propagation is very weak.In the prior art, still there is not relevant bibliographical information about the high efficiently multiplying of rotavirus strain on human diploid cell.
United States Patent (USP) 5,610,049, Human rotavirus HCR3A and method of growing saidrotavirus have been described a kind of rotavirus strain HCR3A paramount titre (10 that increases on human diploid cell WI38
6-10
9PFU/mL) ability.This patent adopts unique this virus of the separation of method from feces, and has adopted the method that progressively adapts to that it is adapted on the human diploid cell.This patent has covered utilizes this Strain to prepare vaccine, or becomes the application power of vaccine with the method for preparing reassortant of other serotype rotavirus and exploitation.Mention in the patent, the new rotavirus that HCR3A is separated to from clinical samples as a strain, belong to the G3 type, SA11 has similar antigenicity to the monkey rotavirus, but the nucleic acid electrophoresis banding pattern is different fully, and the reason that this strain is adapted to the human diploid cell cultivation is not done deep analysis and elaboration, and is that specific rotavirus strain HCR3A cultivates at human diploid cell WI38, have only a kind of serotype, and unresolved rotavirus is cultivated the difficult problem of amplification on human diploid cell.
U.S. Pat 2005/0277194A1, mention among the A Packaging Cell Line, utilize adenovirus 35 type E1 districts to transform human diploid cell system, make it express Ad35, E1B sequence, this cell line can effectively breed except that adenovirus as human influenza virus, herpesvirus, rotavirus and Measles virus.But do not see further relevant report.
Chinese invention patent application CN 1686540A has reported a kind of preparation method of tetravalent wheel shaped virus inactivated vaccine, utilizes little bovine kidney cells or vero cell amplification rotavirus G1, G2, G3 and G4 type.Rotavirus after the amplification is after physical mixed, and is concentrated and purified, and with formalin or the deactivation of β propiolactone, as vaccinogen liquid, but the cultured cell that uses system still is not a human diploid cell, may have higher allergy after aseptic calibrating.
Therefore, how to prepare safe, have good immunogenic inactivated rotavirus vaccine, be the anxious technical barrier to be solved in this area.
Summary of the invention
The objective of the invention is at deficiency of the prior art, it is the preparation method of the inactivated rotavirus vaccine of culture matrix that a kind of diploid cell with the people is provided.
Purpose of the present invention is achieved by following technical proposals.
*Except as otherwise noted, the percent that is adopted among the present invention is percetage by weight.
The invention provides a kind of preparation method of inactivated rotavirus vaccine, it is characterized in that, this method makes rotavirus strain G1, G2, G3, G4 adapts to the pure man diploid cell line, and fastens efficient breeding at human diploid cell, with the rotavirus that the makes production strain as inactivated rotavirus vaccine.
Wherein, described human diploid cell is KMB17,
Simultaneously, the invention provides the deactivation and the purification process of rotavirus, be applicable to the development of different serotypes inactivated rotavirus vaccine; Behind inactivation of viruses behind the purification and the adjuvant mixed immunity experimental guinea pig, gather the serum of animal, with the rotavirus behind the purification or its coat protein as antigen coated ELISA Plate, detect the special neutralizing antibody of rotavirus with the ELISA method, NAT is up to 1: 1024, minimum is 1: 128, and geometric mean titer is 415.8.
Compared with prior art, the present invention has following beneficial effect:
1. the present invention adapts to rotavirus on the human diploid cell KMB17 for the first time, is the breeding of MA104 from rotavirus at its permissive cell, filter out and to fasten the strain of efficient breeding at human diploid cell, simultaneously human diploid cell system is screened, find that KMB17 cell line is suitable for the cultivation of these rotavirus strains most, and found responsive clone.The rotavirus that amplification obtains can be used as the Rotavirus Vaccine seed culture of viruses, after concentrated and purified deactivation, makes rotavirus unit price or multivalent inactivated vaccine, is used to prevent the rotavirus infection disease of infant.
2. the cellular matrix of selecting for use human diploid cell system to produce as the deactivation Rotavirus Vaccine is compared with existing other cellular matrix (for example vero cell), and is safer.Therefore, utilize the technology platform of setting up, other serotype that can adopt rotavirus is as object of study, and exploitation becomes inactivated rotavirus vaccine.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is done further clearly explanation, but they are not the qualification to protection domain of the present invention.
Embodiment 1
---unit price adopts following steps to the preparation method of pentavalent deactivation rotavirus:
(1) cultivation of cell: human diploid cell 2BS, SL-7, WI38, KMB17, preferred KMB17 cultivates with the MEM culture medium, and the composition of culture medium is: the hyclone of 5%-10% (volume ratio), the L glutamine of 300-592mg/L, each 100～200U/mL of penicillin and streptomycin, the sodium bicarbonate of 0.66g-1.32g/L; In 37 ℃, after 5% carbon dioxide is cultured to and grows up to monolayer, be used to inoculate rotavirus.
(2) Bing Du inoculation: the titre of viral seed culture of viruses is more than or equal to 10
7.5TCID
50/ mL, use the pancreatin of final concentration as 15-50 μ g/mL, the pancreatin of preferred 25 μ g/mL activates 20-45 minute (preferred 45 minutes) in 37 ℃, with the infection multiplicity is m.o.i=0.1～3 inoculating cells, in 37 ℃ of absorption 40-90 minute, viral liquid is abandoned in suction, adds to contain the serum-free MEM culture medium that final concentration is a 0.5-1 μ g/mL pancreatin, is cultured in 37 ℃ and produces tangible cytopathy.
(3) Bing Du results: when 75% cell generation pathological changes, results virus.With culture in 37 ℃ and-20 ℃ of multigelations 2-4 time, with 8000-10, the centrifugal 30-60 of 000rpm/min minute, abandon cell precipitation, collect supernatant, in-80 ℃ of preservations.
(4) concentrating of virus: is the ultrafilter membrane ultrafiltration and concentration of 100kD-300kD with the supernatant of results with the molecular weight that dams.Use the QuickStand ultrafiltration apparatus, virus results liquid is concentrated, by calculating the volume ratio before and after concentrating, rotavirus granule content in the outer liquid of ultrafiltration detects, adding up that spissated ratio and yield should be equal to or greater than is 1: 35 and 95%, also can concentrate with PEG 8000, concentrated purpose reaches and gets final product.
(5) Bing Du purification: utilize gel filtration, sample-loading buffer is the PBS of 0.5-1M, and elution buffer is the PBS of 20-30mM, and pH value is 7.0-7.4.Virion will mainly be present in the eluting peak, and nucleic acid and the less foreign protein of other molecular weight will appear in the follow-up peak.After the process gel filtration is slightly pure, use anion-exchange chromatography further consummate.Also available sucrose density gradient centrifugation comes purification, and purity reaches 95% and gets final product.
(6) Bing Du deactivation: the beta-propiolactone that adopts 1: 4000 was in 4 ℃ of deactivation 24-72 hours.After the filter membrane aseptic filtration of 0.2 μ m or 0.45 μ m, be monovalent virus stock solution used.Also can come deactivation with formalin, after the deactivation deactivation complete, deactivation reagent residual quantity touches the mark and gets final product.
(7) preparation of finished product: monovalent virus stock solution used is pressed equal-volume than mixing.Mixed vaccinogen liquid is finished product.
Embodiment 2
The preparation method of tetravalent wheel shaped virus inactivated vaccine, adopt following processing step:
(1) preparation of cell: the KMB17 cell is disperseed with trypsinization, add the MEM complete medium, be cultured in 37 ℃ and grow up to the shape monolayer that confluxes, culture medium was replaced by the MEM that does not contain serum in preceding 12 hours in kind of poison and keeps liquid.
(2) Bing Du inoculation and cultivation: with rotavirus strain G1, G2 strain, G3 strain, G4 strain final concentration is the pancreatin of 20 μ g/mL, in 37 ℃ of activation 45 minutes, inhale to abandon and keep liquid, with the same liquid of keeping the KMB17 cell is cleaned once again, with the infection multiplicity is the m.o.i=3 inoculating cell, in 37 ℃ of absorption 90 minutes, inhale and abandon viral liquid, add and contain the serum-free MEM culture medium that final concentration is 0.5 μ g/mL pancreatin, being cultured to the tangible cytopathy of generation in 37 ℃ (was generally 3-5 days, m.o.i=3).
(3) collection virus: when 75% cell generation pathological changes is arranged approximately, results virus.In 37 ℃ and-20 ℃ of multigelations 3 times, with 10, centrifugal 30 minutes of 000rpm/min abandons cell precipitation, collects supernatant, in-80 ℃ of preservations with culture.
(4) concentrating of virus: is the ultrafilter membrane ultrafiltration and concentration of 100kD with the supernatant of results with the molecular weight that dams.Use the QuickStand ultrafiltration apparatus, virus results liquid is concentrated, by calculating the volume ratio before and after concentrating, the rotavirus granule content in the outer liquid of ultrafiltration detects, and adding up spissated ratio and yield is 1: 35 and 95%.
(5) Bing Du purification: utilize gel filtration, sample-loading buffer is the PBS of 1M, and elution buffer is the PBS of 20mM, and pH value is 7.2.Virion will mainly be present in the eluting peak, and nucleic acid and the less foreign protein of other molecular weight will appear in the follow-up peak.After the process gel filtration is slightly pure, use anion-exchange chromatography to carry out further consummate.
(6) Bing Du deactivation: the beta-propiolactone that adopts 1: 4000 was in 4 ℃ of deactivations 24 hours.After the filter membrane aseptic filtration of 0.2 μ m, be monovalent virus stock solution used.
(7) monovalent viral vaccine stock solution should be through the deactivation potency test, i.e. blind passage 3 times on the rotavirus sensitive cells, and the result should be negative.With each monovalent inactivation of viruses stock solution filtration sterilization, be deactivation Rotavirus Vaccine stock solution, inactivation of viruses stock solution add final concentration be the glycine of 3mg/mL as stabilizing agent, final concentration is that the 2-phenoxyethanol of 5.0mg/mL is as antiseptic.Selecting aluminium hydroxide as adjuvant, is that the ammonia spirit of 6-8% slowly is added in the aluminum chloride that concentration is 4% (g/V) with concentration, and the speed of changeing with per minute 15-20 under 60 ℃ stirs, and makes aluminum hydroxide adjuvant to pH6.9.The final concentration of aluminium hydroxide is 1.0mg/mL in the deactivation sample of immunity, mixes in 30 minutes in stirring at room with inactivated vaccine.When joining the different price vaccine, the final concentration of adjuvant all is the same with mixed method.
Embodiment 3
---carry out the stock solution calibrating with reference to Chinese biological goods rules,
(1) determining the protein quantity
Utilize the Lowry method to measure, protein content should not be higher than 20 μ g/mL.Measurement result is all less than 20 μ g/mL.
(2) antigen amount
With buying or making by oneself, detect at two kinds of commercialization monoclonal antibody coated elisa plates that the rotavirus type is special.Adopt the double-antibody sandwich euzymelinked immunosorbent assay (ELISA), the antigen amount should be not less than 3200EU/mL.
(3) sterility test
Check that in accordance with the law sample is aseptic, up to specification.
(4) bovine serum albumin residual quantity
Detect with euzymelinked immunosorbent assay (ELISA), the bovine serum albumin residual quantity should not be higher than 50ng/mL.The meansigma methods of testing result is 20ng/mL.
(5) inactivation of virus demonstration test
Sampling in 37 ℃ of cultivations 5 days, with the continuous blind passage three generations of method, detects wheel virus antigen with the ELISA method after the deactivation on the KMB17 cell, should be negative.It is qualified to detect.
(6) pH value
Should be 6.0～7.0.It is qualified to detect.
(7) bacterial endotoxin inspection
Should be less than 10EU/mL (gel limit the quantity of test method(s)).It is qualified to detect.
(8) adjuvant content detection
The content of aluminium hydroxide should be 0.80～1.20mg/mL.It is qualified to detect.
(9) adjuvant assay
The content of 2-phenoxyethanol should be 4.0～6.0mg/mL.
(10) efficacy determinations
Virus stock solution used through assay approval is the inactivated virus vaccine finished product.
Embodiment 4～8
Change the rotavirus strain of inoculation into G1 respectively, the G3 strain, the G2 strain, G4, a kind of in the G9 strain, other process repeats embodiment 2.
Embodiment 9～18
Change the rotavirus strain of inoculation into G1 and G3 strain respectively, G1 and G2 strain, G1 and G4 strain, G3 and G2 strain, G3 and G4 strain, G2 and G4 strain, G1 and G9 strain, G2 and G9 strain, G3 and G9 strain, a kind of combination in G4 and the G9 strain, other process repeats embodiment 2.
Embodiment 19
The preparation method of pentavalent inactivated rotavirus vaccine---change the rotavirus strain of inoculation into G1, the G3 strain, the G2 strain, G4 strain and G9 strain, other process is with embodiment 4.
Embodiment 20～27
Change the rotavirus strain of inoculation into the G1G2G3 strain, the G2G3G4 strain, the G1G3G4 strain, the GlG2G9 strain, the G2G3G9 strain, the G1G3G9 strain, the G3G4G9 strain, a kind of combination in the G2G4G9 strain, other processing step is with embodiment 2.
Embodiment 28
The preparation method of tetravalent wheel shaped virus inactivated vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell 2BS, other process is identical with embodiment 2.
Embodiment 27～31
The preparation method of unit price inactivated rotavirus vaccine---
The rotavirus strain of inoculation changes G1 into, the G3 strain, and the G2 strain, G4 strain and G9 strain, other processing step is with Application Example 4～8.
Embodiment 32～41
The preparation method of bivalence inactivated rotavirus vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell 2BS, other processing step is with Application Example 9～18.
Embodiment 42
The preparation method of pentavalent inactivated rotavirus vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell 2BS, other processing step is with Application Example 17.
Embodiment 43～50
The preparation method of trivalent inactivated rotavirus vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell 2BS, other processing step is with Application Example 20～27.
Embodiment 51
The preparation method of tetravalent wheel shaped virus inactivated vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell SL-7, other processing step is with Application Example 2.
Embodiment 51～56:
The preparation method of unit price inactivated rotavirus vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell SL-7, other processing step is with Application Example 4～8.
Embodiment 57～66
The preparation method of bivalence inactivated rotavirus vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell SL-7, other processing step is with Application Example 9～18.
Embodiment 67
The preparation method of pentavalent inactivated rotavirus vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell SL-7, other processing step is with Application Example 19.
Embodiment 68～75
The preparation method of trivalent inactivated rotavirus vaccine---
Change the cellular matrix of cultivating rotavirus into human diploid cell SL-7, other processing step is with Application Example 20～27.
Claims (3)
1. the preparation method of an inactivated rotavirus vaccine, it is characterized in that, it is G1 that this method makes serotype, G2, G3, the rotavirus strain of G4 adapts to the pure man diploid cell line, and fastens efficient breeding at human diploid cell, with the rotavirus that the makes production strain as inactivated rotavirus vaccine.
2. preparation method according to claim 1 is characterized in that described human diploid cell is KMB17.
3. preparation method according to claim 1 and 2, it is characterized in that, behind inactivation of viruses behind the purification and adjuvant mixed immunity experimental guinea pig, gather the serum of animal, as antigen coated ELISA Plate, detect rotavirus special neutralizing antibody with the ELISA method with the rotavirus behind the purification or its coat protein, NAT is up to 1: 1024, minimum is 1: 128, and geometric mean titer is 415.8.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2698392C (en) | 2007-09-04 | 2019-12-03 | Baoming Jiang | Thermal inactivation of rotavirus |
| CN101899422B (en) * | 2010-05-21 | 2011-09-28 | 中国医学科学院医学生物学研究所 | Adaptive culture method of rotavirus P(2) G3 strain and P(8) G1 strain on KMB17 cells and immunogenicity |
| CN102343087A (en) * | 2011-10-21 | 2012-02-08 | 长春祈健生物制品有限公司 | Preparation of diploid cell attenuated live vaccine from measles long-47 strain and preparation process thereof |
| CN104524562A (en) * | 2014-12-08 | 2015-04-22 | 武汉生物制品研究所有限责任公司 | Oral hexavalent reassorted rotavirus live vaccine |
| CN105749268B (en) * | 2016-04-11 | 2020-09-11 | 北京科兴中维生物技术有限公司 | Inactivated Zika virus vaccine |
| CN106769928A (en) * | 2016-12-14 | 2017-05-31 | 中国医学科学院医学生物学研究所 | Wheel virus antigen ELISA quantitative detecting methods |
| CN115216454A (en) * | 2022-09-21 | 2022-10-21 | 中国医学科学院医学生物学研究所 | Method for serum-free large-scale culture of rotavirus by using bioreactor |
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| CN1686540A (en) * | 2005-03-19 | 2005-10-26 | 兰州生物制品研究所 | Preparation of tetravalent wheel shaped virus inactivated vaccine and application |
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