CN1109782A - Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster - Google Patents

Abstract

A process for preparing epidemic hemorrhagic fever vaccine I and II, and bivalent vaccine I and II by kidney cells of golden hamster and a concentration and purification process for these vaccines are disclosed.

Description

Production of inactivated vaccine for epidemic hemorrhagic fever using primary cells of kidney of golden yellow hamster

The present invention relates to epidemic hemorrhagic fever vaccine, relate to particularly that to use golden hamster kidney cells be cellular matrix and be seed culture of viruses, produce the method for epizootic hemorrhagic heat-inactivated vaccine with the epidemic haemorrhagic fever virus strain that is adapted to golden hamster kidney cells.

Epidemic hemorrhagic fever has another name called renal syndrome-hemorrhagic fever (being called for short EHF or HFRS), is with the disease of natural focus of Mus as communication media, is a kind of viral acute infectious disease of serious harm people ' s health.In recent years, there are 150,000 cases in the whole world every year, and mortality rate reaches 3%～10%.Popular epidemic-stricken area is in continuous expansion, and serious symptom type epidemic-stricken area mainly occurs in the east in Asia and Europe, and light disease type is all over the world.In China, existing 26 provinces have the generation of primary disease and in various degree popular.110,000 cases once took place in 1986, and this all causes the people's health and the national economic development and has a strong impact on.For this reason, to the epidemic-stricken area high-risk group and will enter epidemic-stricken area work or the people of tourism carry out the specific immunity inoculation, be to reduce sickness rate, effectively control the fundamental way of the outbreak of epidemic of primary disease.

The eighties initial stage, Korea S Lee pick Wang reports that at first being separated to the epidemic hemorrhagic fever cause of disease from nature confirms virus (Lee HW.eTal.Jinfect Dis 146,638,1982), after this, domestic and international many researcheres all set about carrying out the epidemic hemorrhagic fever Inactivated Vaccine.Korea S LeeHW at first develop suckling mouse brain purification vaccine (Lee HW.eTal.Arch virol[suppl, I], 35,1990), Korea gold Lip river Ji etc. is developed white mouse and suslik suckling mouse brain purification vaccine (Jin Luoji etc., China's experiment and clinical virology magazine 5,487,1991).In China, the Mus brain purification vaccine of the also developing (Sun Zhuchen etc. of institute are ground in the Lanzhou life, Chinese media biology and control magazine 3＜7 phases of special issue 〉, 203,1992), institute is ground in the Shanghai life and Zhejiang Province's epidemic prevention station is developed long melon gerbil jird nephrocyte vaccine (Zhu Zhiyong etc., Chinese media biology and control magazine 3＜7 phases of special issue 〉, 279,1992), stream grinds the chick embryo cell vaccine of the having developed (Yan Yuchen etc. of institute, Chinese media biology and control magazine 3:166,1992), the calibrating Vero cell vaccine of having developed (Chinese biological goods such as Nie Zilin magazine 4:17,1991).

Main purpose of the present invention provides a kind of with the cellular matrix of golden hamster kidney cells as infective virus, prepares the method for epizootic hemorrhagic heat-inactivated vaccine.Its main points are to select known JR strain or L99 virus strain infection golden hamster kidney cells for use, with preparation I type (hantaan type) or II type (soul type) or two valency type epizootic hemorrhagic heat-inactivated vaccine.

Another main purpose of the present invention provides a kind of preparation method of golden hamster kidney cells of popularity hemorrhagic fever virus strain sensitivity.This method is included in and gets 10～14 age in days Golden Hamster kidneys in the sterilizing room, with making cell suspension after the trypsinization, cultivates 2-3 day at the Tissue Culture Flask internal rotation, and obtaining can be for the cell monolayer of infective virus.

A further object of the present invention provides a kind of popularity hemorrhagic fever vaccine semi-finished product and concentrates the method for purifying.This method is semi-finished product vaccine liquid to be carried out ultrafiltration dialysis concentrate after high speed centrifugation and the filtration of G2 filter bulb, purifies through Cellufinl Sulfate gel chromatographic column again.

The preparation of I, epidemic hemorrhagic fever vaccine seed culture of viruses

With separating the nephridial tissue specimen of prolonging the geographic morbidity in limit rabbit from Jilin Province [is the JR strain, draw from " Chinese public health journal " 9(2): 74,1990] or the lung tissue sample that separates from the Jiangxi Province Rattus losea [be the L99 strain, draw from " Jiangxi Medical College's journal " (3): 1,1984], prior to 2-4 age in days neonatal rat encephalocoele inoculation adaptation of virus, when viral infection neonatal rat encephalocoele LD50 reaches 8.0Log, transferred species is in hamster kidney cell, and passes more than 10 generations continuously with last diluted passage method eventually.Proceed to when going down to posterity for the last time, after treating the hamster kidney cell 7-9 of virus strain infection days, check intracellular virus antigen with immunofluorescence (IFA), IFA reaches when infection cell ++ +～++ ++ the time gather in the crops viral liquid, after adding the packing of 10-20% Ox blood serum, put-60 ℃ of preservations.Seed culture of viruses carries out infectious titration at hamster kidney cell, titre 〉=8.5Log TCLD50/ml, and mycoplasma inspection and sterility test are all negative.Using monoclonal antibody typing reagent is determined the specificity and the interior no exogenous factor of proof seed culture of viruses of the antigenicity and the serotype of seed culture of viruses simultaneously.With the seed culture of viruses of this seed culture of viruses as the production epidemic hemorrhagic fever vaccine.

II, production of vaccine

1, preparation hamster kidney cell

Select the Golden Hamster of 10-14 age in days health for use, take out nephridial tissue with aseptic formality, shred the back and add 0.125% pancreatin, put 4-8 ℃ 18-20 hour, discard trypsin solution, use the nutritional solution cell dispersion that contains 0.2% lactalbumin hydrolysate Earle's liquid and be added with 8% calf serum, make cell suspension, inject in the 10 liter culture bottles, every bottle is injected cell suspension 1500ml.Tissue Culture Flask is put 37 ℃ of thermostatic chambers, rotating and culturing 2-3 day, can form even, fine and close cell monolayer.

2, virus inoculation and vaccine semi-finished product results

Select the well-grown Tissue Culture Flask of cell monolayer for infective virus.If production I type vaccine need infect the JR seed culture of viruses; If production II vaccine then need infect the L99 seed culture of viruses.Infective virus dosage is 3-4Log TCID50/1.0ml.Infecting with liquid is 2% Ox blood serum, 0.2% lactalbumin hydrolysate Earle's liquid.Place 32-35 ℃ of thermostatic chamber internal rotation to cultivate 1-3 days the Tissue Culture Flask of infective virus, with Earle's liquid flushing cell, and then add the immersion of 0.1% human albumin, 0.07 ‰ cysteine Earle's liquid after about 20 hours, reuse with the method flushing once, and then change to 0.2% human albumin, 199 comprehensive culture fluid, put 32-35 ℃ of thermostatic chamber and continue to cultivate.7-9 days results behind the infective virus are collected earlier culture fluid, cell are hit down again, mix with culture fluid after putting-60 ℃ of freeze thawing again, by the silk filtration, sample and do sterility test and titration of virus.Culture fluid and Cell sap mixed liquor after filtering are added 1/4000 formalin (inactivator), put 33-37 ℃ earlier after 24 hours, transposition 2-8 ℃ deactivation, this is the semi-finished product of inactivated vaccine.Get the I type and II vaccine semi-finished product mixed in equal amounts can be made into the epidemic hemorrhagic fever bivalent vaccine.

The semi-finished product vaccine liquid that so makes, by the requirement of new biological product vertification regulation, carry out stock solution virus titer mensuration, antigen quantitative determination, remaining Ox blood serum content, deactivation safety test and potency test etc. are after the assay was approved a series of, can packing carry out finished product inspection.Finished product calibrating comprises sterility test, pH value to should be 7.0-8.0, aluminium hydroxide content is that 0.4-0.7mg/ml, free formaldehyde content inaccurately surpass 0.025%, thimerosal content is inaccurate and surpass 0.01% that 4 Mus of mouse toxicity test should be good for and be deposited.

3, the purification of vaccine concentrates

Select qualified semi-finished product vaccine liquid, remove most of cell debris through high speed centrifugation (7000rpm40 minute), reuse G2 filter bulb filters once, afterwards through ultrafiltration dialyse concentrated (dialysis solution is PH7.2 0.2MPB), with PH7.2 0.2MPB balance Cellufine Sulfate gel chromatographic column, the vaccine sample upper prop that dialysis equilibrium is good, wash post with same buffer afterwards and do not flow out (A280nm monitoring) to there being albumen, change PH8.5 0.2MPB eluting virus protein, collect the eluting peak part, the back adding preservative agent (1:20000 thimerosal) that keeps sample is put 4 ℃ of preservations.The purification sample detection comprises: remaining Ox blood serum assay (＜50ng/ml) effectiveness is examined and determine (〉=1:20, sun rate of rotation 〉=3/4) after above-mentioned detection is all qualified, the purification vaccine carries out aseptic filtration, add AL(OH), to 0.5mg/ml, then, do sterility test and pyrogen testing, carry out the packing lyophilizing after qualified, be finished product.The finished product calibrating comprises sterility test, toxicity test, antiseptic test, formaldehyde determination, AL(OH) measures, and PH measures, hydrargyrum is measured, the water content mensuration of freeze dried vaccine etc., and quality index is as the criterion with the inactivated vaccine index.

III, crowd's clinic trial

Ministry of Public Health approval II type vaccine carried out clinical observation first in 1989, and the human vaccination does not have obvious adverse reaction, and serum neutralizing antibody all sun changes.Nineteen ninety Ministry of Public Health approval bivalent vaccine carries out human clinical's observation, and we are to volunteer's intramuscular injection 2 pins (0-28) of 206 preimmune serum antibody response feminine genders, intramuscular injection 3 pin (0-28-42; 0-7-28), 56 days inspection serum I FAT antibody male rotary rates are 95.9% after the first pin vaccination; Check that with the ElisA method antibody male rotary rate is 93.5%.Check that with enzyme plaque reduction neutralization test (EFRNT) to II type virucidin sun rate of rotation be 100%; To I type virus is 88.2%.Three batches of Ministry of Public Health approval II type unit price Seedlings carried out pilot scale 1500 people's II phase clinical observations in 1992.Injecting 2 pins (0-28) MCPENT method detection neutralizing antibody sun rate of rotation is 96%, and injecting the positive rate of rotation of 3 pins (0-28-42) is 99%.I type vaccine carries out the clinical observation of II phase in approval in 1993, observes 500 people for every batch, and injecting 2 pins (0-28) neutralizing antibody sun rate of rotation is 92%, and injecting 3 pins is 93%.People more than 4000 is inoculated in I type and three batches of clinical observations of II type vaccine pilot scale altogether, inject first pin occur in strong responder be 8 people, inject second pin and the 3rd pin is 22 people.In strong response rate＜1%.Proof epidemic hemorrhagic fever I type, II type and two valency type vaccine are all safe and effective.

Embodiment:

Time: 8 ,～July 19,94 on April

Get the Golden Hamster of 12-14 age in days, put to death the back and sterilize with 0.3% bromo geramine.The aseptic kidney of getting shreds, and cleans blood cell, adds 0.125% the trypsin solution of PH8.0, puts 8 ℃ and carries out cold digestion, takes out after 18-19 hour.Discard trypsin solution, wash with Earle's liquid, add a small amount of bead jog 5～6 times, collect each time suspension, through 2 layers of filtered through gauze, make in the outstanding dried growth-promoting media of cell (growth-promoting media is 8% Ox blood serum lactalbumin liquid), shake up the back branch and install in the 15 liter bottles, 1500 milliliters every bottle, put 37 ℃ of rotating and culturing, after three days, cell grows up to fine and close monolayer.

Choose the Tissue Culture Flask that grows up to fine and close monolayer, discard the growth-promoting media in the former bottle, seed culture of viruses is diluted in the lactalbumin liquid of 2% Ox blood serum, be sub-packed in the culture bottle 1500 milliliters every bottle after the jolting evenly.Put 33-35 ℃ of rotating and culturing 2 days, and discarded viral liquid,, add soak then, 1500 milliliters every bottle with Earle's liquid hydro-peening.Soak is 0.07 ‰ cysteine Earle's liquid, contains 0.1% human albumin.Soak after 20 hours, discard soak, with the same method again hydro-peening once change to 1500 milliliters of cell maintenance mediums then.Keeping liquid is: 199 comprehensive culture fluid contain 0.2% human albumin.The cell bottle is put 33-35 ℃ of rotating and culturing.Gather in the crops after 8 days, receive earlier 1200 milliliters of each flask culture liquid, retains 300 milliliters, the cell bottle is put-60 ℃ freeze taking-up after 5-6 hour, jolt cell and make its fragmentation, then with culture fluid and Cell sap hybrid filtering.Add formalin to 1:4000 by liquid measure, thimerosal 1:20000, shake well rearmounted 37 ℃ 24 hours, 4 ℃ on transposition is 14 days again, in the meantime, jolting every day vaccine 2 times.The qualified back end hydrogenation aluminum adjuvant of safety test is to 0.5mg/ml.

The above-mentioned vaccine of gathering in the crops is semi-finished product, through sterility test, after the assay approvals such as titration of virus, antigen quantitative determination, remaining Ox blood serum content, inactivation of virus safety test, potency test, carries out packing, 1.2 milliliters every.Carry out the finished product calibrating after the packing, the finished product calibrating comprises sterility test, and visual examination, pH value, aluminium hydroxide content, free formaldehyde content, thimerosal content, toxicity test etc. are the epizootic hemorrhagic heat-inactivated vaccine behind the finished product assay approval.

Claims (3)

1, a kind of method for preparing the epizootic hemorrhagic heat-inactivated vaccine.This method comprises the cellular matrix of the golden hamster kidney cells of application popularity hemorrhagic fever virus sensitivity as infective virus, infects JR strain, L respectively ₉₉Strain prepares I type, II type and two valency type epizootic hemorrhagic heat-inactivated vaccine, and vaccine is concentrated purification.

2, according to the process of claim 1 wherein said golden hamster kidney cells be meant get 10-40 age in days Golden Hamster kidney with trypsinization after, make cell suspension, in Tissue Culture Flask, execute and change to cultivate, the cell monolayer of preparation.

3, saidly vaccine is concentrated purification be meant with semi-finished product vaccine liquid through high speed centrifugation G according to the process of claim 1 wherein ₂Carry out ultrafiltration dialysis after filter bulb filters and concentrate, purify through Cellufine Sulfate gel chromatographic column again.