CN101780278B

CN101780278B - Bivalent hand-foot-and-mouth disease inactivated vaccine

Info

Publication number: CN101780278B
Application number: CN2009101774263A
Authority: CN
Inventors: 贾宝山; 石慧颖
Original assignee: FORWELL BIOPHARMACEUTICALS Co Ltd
Current assignee: Bohui Biopharmaceutical Co.,Ltd.
Priority date: 2009-09-29
Filing date: 2009-09-29
Publication date: 2012-02-01
Anticipated expiration: 2029-09-29
Also published as: CN101780278A

Abstract

The invention belongs to the technical field of novel viral inactivated vaccines. The inactivated vaccine is used for preventing the hand-foot-and-mouth disease and is technically characterized in that virus seeds, i.e. an EV71 virus and a CA16 virus, are both separated from cotton applicators of hand-foot-and-mouth disease patients, and cells for separation and production are from the American Type Culture Collection. A preparation method of the inactivated vaccine comprises the following steps in American Type Culture Collection (ATCC) of: culturing Vero cells by a reactor, a spinner bottle or a cell factory; infecting the EV71 virus or the CA16 virus after flakiness; culturing at 35 DEG C for 48-96 hours, and then obtaining the viruses; respectively preparing univalent inactivated stock solutions through multistage filter of a 0.65-0.22 micrometer filter membrane, 100 MW ultrafiltration concentration, Sepharose 6 Fast Flow chromatography, DEAE SepharoseFF chromatography and by inactivating 1:2000 formaldehyde at 37 DEG C for 12 days; and mixing the two inactivated stock solutions by the volume ratio of 1:1, and adding a human leukocyte protective agent to obtain the bigeminal hand-foot-and-mouth disease inactivated vaccine.

Description

The bigeminy hand-foot-and-mouth disease inactivated vaccine

Technical field:

The invention belongs to a kind of novel prevention and use viral inactivated vaccine.

Background technology:

Hand-foot-mouth disease (hand-foot-and-mouth disease; HFMD) be a kind of viral dermatosis that mainly betides the child, pilosity is born in infant, the also accidental generation of being grown up; Have another name called dermexanthesis property blister property stomatitis; Have very strong infectiousness, normal eruption and prevalence, the report of existing dozens of countries and regions takes place and is popular.Asian-Pacific area HFMD is popular in higher mortality rate.There is report HFMD to infect the part infant cardiac damage is in various degree arranged.1998～2000 years twice outburst HFMD is popular in Taiwan, dead 78 examples.It is main clinic symptoms that this disease becomes ulcer with hands, foot and oral mucosa herpes or ulceration, and severe complications can take place, like concurrent aseptic meningitis, encephalitis and paralysis.At present the extremely strong infectiousness of HFMD is more and more paid attention to by the clinicist, and HFMD betides summer, is prone to the secondary skin infection, so active treatment prevents secondary infection, the cut-out route of transmission, especially prevents eruption and prevalence to seem particularly important.

The infectious disease (like hepatitis B, measles) that is only caused by a kind of pathogen with major part is different; Hand-foot-mouth disease is caused by multiple viral infection; But the overwhelming majority all be by coxsackie virus A organize 16 types (Coxsackievirus A 16, CA16) and enterovirns type 71 (Enteroviruse EV71) cause.

The object of the invention is exactly a kind of hand-foot-mouth disease that can prevent CA16 and EV71 virus to cause simultaneously of development.

Summary of the invention:

Separate hand-foot-mouth disease pathogen EV71 virus and CA16 virus,, confirmed the two is united the method for processing inactivated vaccine, be used to prevent hand-foot-mouth disease popular through purification, deactivation, antigenicity and immunogenicity determining.

With reaction vessel, rolling bottle or cell factory Cultivation of Vero; Postoperative infection EV71 or CA16 are viral in flakes; Gather in the crops virus after 48-96 hour in 35 ℃ of cultivations; In 37 ℃ of deactivations 12 days, process unit price deactivation stock solution through 0.65-0.22 micron filter membrane multistage filtering, 100MW ultrafiltration and concentration, Sepharose 6 Fast Flow chromatographies, DEAE Sepharose FF chromatography, 1: 2000 formaldehyde respectively; Two kinds of deactivation stock solutions are mixed with 1: 1 volume ratio, be the bigeminy hand-foot-and-mouth disease inactivated vaccine after the white protective agent of adding people.

The specific embodiment:

1.EV71 all separate from the hand-foot-mouth disease patient with CA16 virus and to swallow examination son.

2. virus separation and vaccine production use cell as African green monkey kidney passage cell (Vero), available from U.S. ATCC.

3. the viral seed lot system for preparing EV71 and CA16 respectively: with the amplification of on the Vero cell, going down to posterity of isolating original seed culture of viruses, preparation EV71 and the main seed lot of CA16 virus and the seed lot of working.

4. viral seed lot is identified: " produce vaccine for man in two ones of the Chinese pharmacopoeia and seed culture of viruses is identified comprehensively that confirm no foreign aid's factor pollution, aseptic, no mycoplasma contamination carries out Biology identification with monoclonal antibody to two kinds of viruses with the requirement of seed culture of viruses according to 2005 editions.Virus titer, immunogenicity are checked.

5. set up cell bank: ATCC is purchased next germinal cell through the amplification of going down to posterity, respectively frozen master cell bank and working cell storehouse.And according to 2005 editions " two middle passage cells of Chinese pharmacopoeia are examined as the cytostromatic prescription of vaccine for man entirely.

6. recovery work storehouse cell and go down to posterity and be expanded to reaction vessel scale or cell factory scale or 3L Tissue Culture Flask.

7. work seed lot EV71 or CA16 virus is infected in the in blocks back (80% is in blocks) of cell, in 35-37 ℃ cultivate 48-96 hour after the harvesting culture supernatant.

8. carry out multistage filtering with 0.65-0.22 micron filter membrane, remove cell debris.

9. the ultrafilter membrane with the 100MW molecular cut off carries out ultrafiltration and concentration 50-100 doubly.

10. carry out sieve chromatography with Sepharose 6 Fast Flow, collect first peak.

11. carry out anion-exchange chromatography with DEAE-Separose FF, collect stream and wear the peak.

12. add 1 in the purified virus liquid: 2000-1: 4000 formalin shakes up, and puts 33-37 ℃ of effect 6-12 days.

13. the adding stabilizing agent is unit price deactivation stock solution.The unit price stock solution of EV71 and CA16 should prepare respectively, can not on a collection of cell, prepare simultaneously.

14. measure the virus titer that virus is gathered in the crops liquid with microtitrimetry, virus titer should be not less than 7.0 CCID50/ml.

15. use EV71 and CA16 monoclonal antibody wrapper sheet respectively, measure the antigenic content of each processing step with double antibody sandwich method.

Produce level 16. measure the immunogenicity and the protection antibody of inactivation antigen with microneutralization test.

17. according to antigen measuring result and immunogenicity determining result, two kinds of unit price deactivation stock solutions are mixed according to a certain percentage, process semi-finished product.

18. be distributed into 0.5ml/ extremely, be the bigeminy hand-foot-and-mouth disease inactivated vaccine.

Claims

1. one kind comprises the method for preparing that enterovirns type 71 (EV71) and coxsackie virus A organize the bigeminy hand-foot-and-mouth disease inactivated vaccine of 16 types (CA16) simultaneously, may further comprise the steps:

A) with reaction vessel or cell factory or glass rolling bottle Cultivation of Vero: recovery work storehouse cell also goes down to posterity and is expanded to reaction vessel scale or cell factory scale or 3L Tissue Culture Flask.

B) cell postoperative infection EV71 in blocks or CA16 virus was cultivated 48-96 hour in 33-35 ℃.

C) results culture supernatant.

D) remove cell debris through multistage filtering.

E) ultrafiltration and concentration viral suspension: the ultrafilter membrane with the 100MW molecular cut off carries out ultrafiltration and concentration 50-100 doubly.

F) sieve chromatography purified virus suspension.

G) ion-exchange chromatography viral suspension.

H) obtain EV71 or CA16 unit price deactivation stock solution through the inactivator inactivation of viruses.

I) mix EV71 and CA16 unit price deactivation stock solution preparation semi-finished product.

J) be distributed into 0.5ml/ and prop up, process bivalent inactivated vaccine.

2. the process of claim 1 wherein that EV71 seed culture of viruses and CA16 seed culture of viruses are isolating any homotype strain within Chinese territory.

3. the method for claim 1, the used microcarrier of reaction vessel cultured cell is cytodexl.

4. the method for claim 1, the sieve chromatography used medium is Sepharose 6 Fast Flow.

5. the method for claim 1, the used balance liquid of sieve chromatography is the PBS of pH 6.8-7.6.

6. the method for claim 1, the ion-exchange chromatography used medium is DEAE Sepharose Fast Flow.

7. the method for claim 1, the used balance liquid of ion-exchange chromatography is the PBS of pH 6.8-7.6, and eluent is for containing 0.1-0.5M sodium chloride, and pH is the buffer salt solution of 6.8-7.6.

8. the process of claim 1 wherein that inactivator is a formalin solution, reaction density is 1: 1000 to 1: 4000.

9. the process of claim 1 wherein that the inactivator operative temperature is 35 ℃～37 ℃.

10. the process of claim 1 wherein that inactivator action time is 6～12 days.

11. the process of claim 1 wherein that the mixed proportion of EV71 and CA16 unit price deactivation stock solution comprises any volume ratio.