CN106075423A - A kind of combined vaccine preventing hand-foot-mouth disease - Google Patents
A kind of combined vaccine preventing hand-foot-mouth disease Download PDFInfo
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Abstract
The invention provides a kind of combined vaccine preventing hand-foot-mouth disease, it contains the EV71 antigen of inactivation, CA16 antigen, CA6 antigen.The present invention by by causing Major Epidemic Strain EV71 of hand-foot-mouth disease, CA16, CA6 type to carry out cultivating, be concentrated by ultrafiltration, sucrose density gradient centrifugation, ultrafiltration desaccharide and aseptic filtration, obtain the virus liquid of purification, further this virus liquid is prepared as the hand-foot-mouth disease combined vaccine that immunogenicity is good, safety and stability is high, the vaccine of the present invention can prevent multiple enterovirus to infect body simultaneously, and there is not the phenomenon interfered between these antigens, the immunogenicity that corresponding immunogenicity excites compared with antigen alone will not reduce；And use vaccine of the present invention can substantially simplify vaccination program, improve inoculation efficiency and reduce cost.
Description
Technical field
The present invention relates to technical field of biological product preparation, in particular it relates to a kind of prevention hands containing enterovirus antigen
The combined vaccine of foot stomatosis.
Background technology
Hand-foot-mouth disease (hand-foot-mouth disease, HFMD) is classified as Class C in China's Law on the Prevention and Control of Infectious Diseases
Infectious disease, is a kind of acute infectious disease caused by multiple enterovirus, relatively conventional with infant morbidity.Patient shows as mouth
Bitterly, there is exanthema vesiculosum or aphtha, most infant spontaneous recoverys in about 1 week, minority infant in the position such as anorexia, low grade fever, hands, foot, oral cavity
The complication such as myocarditis, pulmonary edema, AME can be caused.Indivedual children with serious disease PD are fast, cause death.Draw
Play the virus of hand-foot-mouth disease except EV71 (Enterovirus 71, EV71) and CA16 type (Coxasckievirus A16, CA16)
Outside relatively conventional, also include 2,4,5,6,10 types etc. of CA group (Coxasckievirus A, CA), B group
1,2,3,4,5 types etc. of (Coxasckievirus B, CB), Enterovirus 68 type (Enterovirus 68) and echovirus
(Echovirus, Echo) etc..
Thinking, EV71 and CA16 is the main pathogens causing a lot of HFMD of Asia-Pacific nations to break out in the past, but in recent years by
The report that CA6 causes HFMD to break out gradually increases, and before 2012, CA6 is mainly popular in distributing in China region of Southeast,
After 2012, CA6 China many substitute EV71 or CA16 and become the main pathogens causing HFMD to break out.Hand-foot-mouth disease becomes
One of public health problem for serious threat children's health and social stability.At present, there is no clinically and effectively treat brothers
The antiviral drugs of stomatosis, although the most granted existing EV71 inactivated vaccine, but due to the change of HFMD enterovirus cause of disease spectrum
And the leading strain type that causes HFMD occurred frequently is various, and between Strain, there is not Cross immunogenicity effect, it is impossible to real
Now viral to infecting other type hand-foot-mouth disease body protective, proposes new challenge to prevention and control HFMD.Inoculation EV71 vaccine is difficult
To control the outburst of hand-foot-mouth disease that CA16 and CA6 cause and popular.Therefore, it is badly in need of preparing corresponding multivalence, multiple vaccines, with
Reply HFMD break out with seriously disease popular.There is presently no the relevant report that EV71, CA16 and CA6 combined vaccine is developed
Road, first, the hand-foot-mouth disease that EV71, CA16 and CA6 virus cause just there occurs large-scale outburst within the recent short period,
Secondly, there is certain regionality distribution in the hand-foot-mouth disease that virus causes, and for the developed country that incidence rate is relatively low, puts into
The mechanism of research and development is less, and again, the immune interference effect existed between different virus antigen is unknown, and this interference effect may be straight
Connect the immunogenicity reducing vaccine one-component, affect immune effect.Therefore, in order to protect body from hand-foot-mouth disease more broadly
The infringement of serial viral, in the HFMD prevention and control system of China's Erecting and improving, expands protection by preparation connection Seedling imperative.
Summary of the invention
For solving the problems referred to above, it is an object of the invention to provide that a kind of immunogenicity is good, safety and stability is high
Comprise the hand-foot-mouth disease combined vaccine of enterovirus antigen.
The combined vaccine of the prevention hand-foot-mouth disease of the present invention, contains EV71 antigen, the CA16 antigen of inactivation inactivated and goes out
The CA6 antigen lived.
Further, in the combined vaccine of the present invention, the antigenic content of EV71, CA16, CA6 is respectively 100-1000U/ people
Part, 100-1000U/ person-portion, 200-1200U/ person-portion.
Preferably, the antigenic content of EV71, CA16, CA6 is respectively 200-800U/ person-portion, 200-800U/ person-portion, 400-
1000U/ person-portion.
It is highly preferred that the antigenic content of EV71, CA16, CA6 is respectively 400U/ person-portion, 400U/ person-portion, 600U/ person-portion.
The combined vaccine of the present invention, its viral purification liquid is prepared by following steps:
(1) respectively EV71, CA16, CA6 virus liquid is inactivated, be concentrated by ultrafiltration；
(2) respectively above-mentioned three kinds of viral concentration liquid are carried out sucrose density gradient centrifugation；
(3) merging centrifugal liquid is collected.
In step (1), the preparation method of EV71 virus liquid is: EV71 virus inoculation human diploid cell, and inoculation MOI is:
0.0001-0.001, cultivation serum-concentration: 2% Ox blood serum, cultivation temperature: 36 ± 1 DEG C, harvest time is 7-9d；
The preparation method of CA16 virus liquid is: CA16 virus inoculation human diploid cell, and inoculation MOI is: 0.001-0.01,
Cultivation serum-concentration: 1% Ox blood serum, cultivation temperature: 35 ± 1 DEG C, harvest time is 8-10d；
The preparation method of CA6 virus liquid is: CA6 virus inoculation human diploid cell, and inoculation MOI is: 0.0001-0.001,
Cultivation serum-concentration: 1% Ox blood serum, cultivation temperature: 33 ± 1 DEG C, harvest time is 8-10d.
Preferably, the present invention cultivates the cell of EV71, CA16, CA6 virus is human diploid cell SLF-1.This cell is
Disclosed in Chinese patent CN103255102A.
Further, in step (1), ablation method is formalin-inactivated, final concentration of 67～200 μ g/ml of formalin-inactivated.
Wherein, the formalin-inactivated time is: under the conditions of 37 ± 1 DEG C, inactivate 2.8～5.5d.Preferably inactivate 4d.
Wherein, the virus liquid of step (1) is concentrated by ultrafiltration and is realized by the following method:
Inactivation of virus liquid concentrates through slipstream membrane filtration or hollow fibre filtering, and cycles of concentration is 50-200 times, uses and cuts
Staying molecular weight is 1,000,000-500 ten thousand.
Preferably, the ultrafilter membrane bag that use aperture be 300kD is concentrated by ultrafiltration.
Wherein, the sucrose density gradient centrifugation method of step (2) is:
Surpassing from buffer from lateral opening pump into successively with 50-100rpm of centrifuge successively, virus is concentrated by ultrafiltration liquid, low concentration sugarcane
Sugar juice, finally pumps into high concentration sucrose solution with 50-150rpm until mesopore trickle centrifugal rotational speed is as 28000-
35000rpm, centrifugation time is: 6-20 hour, and centrifuging temperature is 2-8 DEG C.
Surpassing volume ratio liquid being concentrated by ultrafiltration from buffer and virus is 1:5-1:10；Virus is concentrated by ultrafiltration liquid and low concentration sugarcane
The volume ratio of sugar juice is 1:1-2:1；Can stop adding for centrifuge hollow trickle as long as high concentration sucrose adds standard
Enter high concentration sucrose.
Wherein, described low concentration sucrose mass percent is 25%-45%, and high concentration sucrose mass percent is 55%-
60%.
Wherein, surpass from buffer selected from 0.01-0.5M PB, PBS and PBST in one.
Wherein, in density gradient centrifugation method, during purification EV71, CA16, CA6 virus, collect and merge antigenic content with
Protein content is than the centrifugal liquid more than 5U/ μ g.Using 300KD film bag ultrafiltration desaccharide, method is: EV71 virus liquid equal-volume surpasses
Filter 8 times, 3 times of volume ultrafiltration of CA16 virus liquid 8 times, CA6 virus liquid equal-volume ultrafiltration 8 times.
Further, the combined vaccine of the present invention is possibly together with aluminium adjuvant.
Described aluminium adjuvant is aluminium hydroxide, its final concentration of 0.36-1.73mg/ml in combined vaccine.
Preferably, aluminium hydroxide, its final concentration of 1.44mg/ml in combined vaccine.
Further, the dosage form of described combined vaccine is injection.
The human diploid cell that the present invention has independent intellectual property right by using applicant cultivates virus, compares inhuman source
Cellular matrix vaccine, has had lifting on cellular matrix, eliminates inhuman source protein, inhuman source DNA and potential oncogenicity etc. no
Safety factors；With time vaccines purge process using completely physical method carry out, eliminate the residual risk of PEG, DNA enzymatic etc.,
Guarantee is provided for vaccine safety.
Based on technique scheme, the present invention utilizes the prevention hand-foot-mouth disease connection that current cause hand-foot-mouth disease epidemic strain prepares
Close vaccine at least to have the following advantages and beneficial effect:
(1) vaccine uses human diploid cell substrate to cultivate virus, provides guarantee for vaccine safety；Use simultaneously
Human diploid cell is the SLF-1 strain of applicant's independent intellectual property right, inscience property right barrier.
(2) seedling Seedling process consistency is good, process stabilizing；Through great many of experiments, screen optimal inactivation technology ginseng
Number, can improve the safety of vaccine, it is achieved complete inactivation, can control, in Min., to save into by the usage amount of formaldehyde again
This.
(3) combined vaccine of the present invention comprises hand-foot-mouth disease principal causative cause of disease, it is possible to well prevention is by EV71, CA16
Generation with the hand-foot-mouth disease that CA6 virus causes.Present invention research shows, above-mentioned three kinds of antigens immunity by after kind person mutual the most not
Interference antigenicity and immune effect, have good immunogenicity and safety.
(4) combined vaccine that the present invention provides can prevent between infecting of multiple pathogens, and these antigens simultaneously
There is not the phenomenon interfered, the immunogenicity that corresponding immunogenicity excites compared with antigen alone will not reduce.Combined vaccine
Use can substantially simplify vaccination program, improve inoculation efficiency and also reduce cost, be the main trend of future technical advances,
Also it is demand place, future market.
(5) vaccine stability of the present invention is good, can preserve for a long time.
Accompanying drawing explanation
Fig. 1 is different formalin-inactivated concentration EV71 virus titer change curves under different time.
Fig. 2 A is EV71 virus harvest liquid electromicroscopic photograph (100000 ×).
Fig. 2 B is EV71 vaccinogen liquid electromicroscopic photograph (100000 ×).
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.Without departing substantially from present invention spirit
In the case of essence, the amendment that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.
If not specializing, experiment material, reagent and instrument etc. used in the embodiment of the present invention are all commercially available, if
Do not particularly point out, the conventional means that technological means used in embodiment is well known to the skilled person.
EV71 Strain preserving number of the present invention is CGMCC No.3544, disclosed in Chinese patent CN103386126A；
CA16 Strain preserving number is CGMCC No.5371, disclosed in Chinese patent CN103386126A；CA6 Strain in
Be preserved on May 11st, 2016 China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address:
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is
CGMCC No.12294, entitled CA group 6 type of classifying.The present invention cultivates the cell of EV71, CA16, CA6 virus and behaves
Diploid cell SLF-1, this cell is disclosed in Chinese patent CN103255102A.
The preparation of embodiment 1 virus liquid
1, cell is cultivated
Recovery SLF-1 cell, uses containing the MEM solution of 20% Ox blood serum, puts 37 ± 1 DEG C of cultivation, adherent after change liquid, use
MEM solution containing 10% Ox blood serum.Washing, 0.25% trypsinization after monolayer, divided in 1:4 ratio and plant to culture bottle or thin
In born of the same parents factory, reach 40 layer cell factory, long to monolayer, prepare virus inoculation.
2, Virus culture
EV71 virus, with MOI 0.0001 inoculating cell, uses the culture fluid containing 2% Ox blood serum, puts 36 ± 1 DEG C of cultivations, 8d
Results virus.
CA16 virus, with MOI 0.001 inoculating cell, uses the culture fluid containing 1% Ox blood serum, puts 35 ± 1 DEG C of cultivations, 10d
Results virus.
CA6 virus, with MOI 0.001 inoculating cell, uses the culture fluid containing 1% Ox blood serum, puts 33 ± 1 DEG C of cultivations, and 9d receives
Obtain virus.
The inactivation of embodiment 2 virus
(1) clarification
It is 3-0.8 μm, the filter element filtering of 0.65-0.22 μm by virus liquid through continuous 2 grades of apertures or centrifugal (includes flowing continuously
Centrifugal) rotating speed is 3000rpm, centrifugation time is 0.5 hour, obtains virus clear liquor；
(2) inactivation
EV71, CA16, CA6 virus clear liquor is respectively classified into 9 groups, and often group adds the 1:200 formalin of different volumes
Solution so that it is theoretical final concentration of 200,100,67 μ g/ml, 3 groups of parallel sample of each concentration, inactivates under the conditions of putting 37 ± 1 DEG C,
Grouping information is shown in Table 1.Being sampled for 0,2,4,6,8,10,12,14,16,18,20,22,24 hours of inactivation, sampling terminates
After immediately with in sodium sulfite solution and formaldehyde, carry out virus titer mensuration.The virus titer often organizing inactivator concentration is taken
Meansigma methods (the results are shown in Table 2).EV71, CA16 and CA6 inactivation of virus kinetic curve (different first are drawn respectively according to virus titer
Under aldehyde inactivation concentration, EV71 virus titer change curve is as shown in Figure 1).According to Inactivation Dynamics curve calculating virus complete inactivation
Required time (the results are shown in Table 3).
Table 1 grouping information
Table 2 variable concentrations formalin-inactivated restrovirus titre (lg CCID50/ml)
Note: when drawing Inactivation Dynamics curve, if titre value is less than just calculating by this value during certain value, such as < 1.50lg
CCID50/ ml presses 1.50lg CCID50/ ml draws curve
The reckoning of the different concentration of formaldehyde inactivation time of table 3
All EV71 can be gone out completely in 31h by the visible three kinds of variable concentrations formalins of table 3 (200,100,67 μ g/ml)
Live, all can be by CA16 complete inactivation in 32h, all can be by CA6 complete inactivation in 33h, it is contemplated that inactivated vaccine scale
Produce the security risks of inactivation of virus, prudent for the sake of select 1:2000 as the final concentration of formalin-inactivated, i.e. 200 μ g/ml first
Aldehyde final concentration is as inactivation dosage, and 37 ± 1 DEG C inactivate 4 days.
The purification of embodiment 3 virus
(1) ultrafiltration concentration of virus liquid
Selective retention molecular weight is the ultrafilter membrane bag in tri-kinds of apertures of 100kD, 300kD and 500kD, respectively by EV71 (or
CA16 or CA6) it is concentrated by ultrafiltration with a collection of inactivation liquid, fixing ultrafiltration pressure, concentrate identical multiple (100 times), fixing filter wash
Number of times (filter wash 8 times), before and after comparing the antigen response rate of ultrafiltration time and three kinds of film bags, foreign protein clearance and ultrafiltration concentration
The specific activity (antigenic content/protein content) of product, and detect the antigenic content of each filter liquor.Determine super according to testing result
Filter film bag aperture.The results are shown in Table 4.
The different film bag aperture ultrafiltration effectiveness comparison result of table 4
The result of study of table 4 shows, filtered solution EV71 (or CA16 or CA6) antigenic content testing result is respectively less than under detection
Limit, shows three kinds of film Bao Junneng effectively catching purpose viruses.Along with the increase in film bag aperture, the ultrafiltration used time is gradually shortened, although
The antigen response rate declines, but protein removal rate and specific activity improve.The synthetic antigen response rate, protein removal rate and to mesh
Virus rejection effect result of study, determine that ultrafiltration concentration process film bag aperture is 300kD.
(2) sucrose density gradient centrifugation
Low, the high concentration of sucrose solution are respectively 40%, and 55%.Density gradient centrifugation buffer is that 0.5mol/L PBS delays
Rushing liquid, its pH value is: 6.5.Use Hitachi CP70MX/ME supercentrifuge, successively from lateral opening with 100rpm pump into 100ml surpass from
The step (1) of heart buffer, about 700ml is concentrated by ultrafiltration EV71 (or CA16 or CA6) the virus ultrafiltrate obtained, low concentration sucrose
Solution 500ml, finally pumps into high concentration sucrose solution with 100rpm until mesopore trickle, and 4 DEG C are centrifuged 12 hours, first collect
800ml waste liquid, after be in charge of collection 500ml virus liquid, after antigenic content and protein content detect, merge antigenic content and egg
White matter content ratio is more than 5U/ μ g place section.
300KD film bag ultrafiltration desaccharide, equal-volume or triploid amass filter wash 8 times, molten with the 0.01M PBST (pH7.2) of 50ml
Liquid top is washed 1 time, washes film 2 times with 0.01M PBST (pH7.2) solution of 50ml.Obtain each virus desaccharide liquid.Detection BSA content,
Filter liquor antigenic content, compares the antigen response rate and protein removal rate (the results are shown in Table 5).Determine ultrafiltration desaccharide filter wash volume.
Table 5 ultrafiltration desaccharide difference filter wash volume effect compares
Table 5 result shows, filtered solution EV71 (or CA16 or CA6) antigenic content testing result is respectively less than Monitoring lower-cut, table
Bright three kinds of film Bao Junneng effectively catching purposes virus.BSA is the major impurity in each virus liquid, it is contemplated that the quality of vaccine, knot
Close the antigen response rate and albumen clearance, determine that EV71 virus ultrafiltration desaccharide technique is equal-volume ultrafiltration 8 times, CA16 virus ultrafiltration
Desaccharide technique is 3 times of volume ultrafiltration 8 times, and CA6 virus ultrafiltration desaccharide technique is equal-volume ultrafiltration 8 times.
(3) aseptic filtration each virus desaccharide liquid is through 0.22 μm aperture filter membrane aseptic filtration, i.e. obtains each viral vaccine stock solution.
The determination of embodiment 4 animal model
(1) determination of immunogenic animal model
With 0.85% normal saline, aluminium adjuvant is diluted to 1.0mg/ml, with 0.01M PBS by EV71 (or CA16 or CA6)
Aluminium adjuvant after dilution, to 1600U/ml, is added dropwise to EV71 (or CA16 or the CA6) antigen after dilution by antigen diluent under room temperature
In, make the two equal-volume adsorb, stirring while adding, after continue mixed at room temperature 30 minutes, it is thus achieved that EV71 (or CA16 or
CA6) aluminum adsorbed vaccine.
EV71 (or CA16 or CA6) vaccine with same dose, respectively a pin (0d exempts from 28d blood sampling), (0d, 14d exempt from two pins
28d takes a blood sample) peritoneal immunity mice and intramuscular immunisation rat.Measure serum NAT, the results are shown in Table 6.
Table 6EV71 (or CA16 or CA6) vaccine immunogenicity result
Being shown by table 6 result of study, EV71 vaccine (or CA6 vaccine) immune rat, mice all can produce and quite drip
The antibody of degree, for convenience operation later, it is contemplated that cost, selecting mice is EV71 vaccine (or CA6 vaccine) animal immune mould
Type.CA16 vaccine immunity rat can produce the antibody of suitable titre, but mice antibody titer is low, and selecting rat is CA16 vaccine
Animal immune model.
(2) counteracting toxic substances protection animal model
Establishing the counteracting toxic substances protection model of EV71 vaccine (or CA16 Seedling or CA6 vaccine), concrete grammar is as follows:
Vaccine immunity and counteracting toxic substances program: vaccine is diluted to respectively 200U, 50U, 12.5U, 3.12U, 0.78U/0.5ml,
Use the female Mus of peritoneal immunity parent, 10 female Mus of each dose immunization, 0.5ml/ only, often organize female Mus all with hero Mus with cage 1～2 days
Rear point of cage, just exempts from the first pin, immune second pin after 14 days, about 21 days (two exempt from after about 1 week) galactopoiesis Mus, treats that neonatal rat length is to 5-7
During age in days, respectively every nest neonatal rat being carried out abdominal cavity counteracting toxic substances with Mus adaptability counteracting toxic substances strain, only, counteracting toxic substances amount is 10LD to 0.1ml/50/ only.
Observe 14 days after neonatal rat counteracting toxic substances, record neonatal rat morbidity, dead quantity, add up neonatal rat morbidity and Death prevention rate under each dosage
Experimental control: as above method two pin immunity parent dams 0.01M PBS, 0.5ml/, with Mus adaptability counteracting toxic substances strain
Respectively every nest neonatal rat being carried out counteracting toxic substances, only, counteracting toxic substances amount is 10LD to abdominal cavity 0.1ml/50/ only.
Test establishment condition: matched group neonatal rat should be all dead.
Process of the test controls: indicate experimental quantities, group, immunity date on every cage.
Clinical observation project and frequency: after counteracting toxic substances, every day observes animal with or without limb paralysis, erythra symptom and death condition.
Within whole experimental period, all itemized record such as the health condition of all animals, Behavioral change, result is shown in embodiment 8.
Embodiment 5 immunizing dose and the determination of immune programme for children
(1) determination of immunizing dose
For controlling the protein content (each component protein matter content≤4 μ g/ person-portion) of every vaccinating agent, according to many batches of vaccinogens
Liquid purification result is added up, and in every dose of EV71/CA16/CA6 combined vaccine, EV71 antigenic content should not be greater than 1000U/ person-portion, CA16
Antigenic content should not be greater than 1000U/ person-portion, and CA6 antigenic content should not be greater than 1200U/ person-portion.
Connection Seedling dosage choice is designed comparing with reference to following principle, and determines, it may be assumed that A. is according to having listed EV71 epidemic disease now
The dosage (400U/ person-portion) of Seedling, in every dose of connection Seedling, EV71 vaccine dose selects design dosage 25U, 100U, 400U, 1000U；B.
Determine that experiment, plan clinical immunization dosage are 400U/ person-portion according to CA16 mono-Seedling immunizing dose, CA16 vaccine dose in every dose of connection Seedling
Amount selects design dosage 25U, 100U, 400U, 1000U；C. experiment, plan clinical immunization are determined according to CA6 mono-Seedling immunizing dose
Dosage is 600U/ person-portion, and in every dose of connection Seedling, CA6 vaccine dose selects design dosage 50U, 200U, 600U, 1200U；D. connection Seedling is each
Component proportion principle, by the design dosage of above-mentioned each component, first proportioning is for selecting each component clinic dosage to join
Ratio, i.e. EV71, CA16, CA6 antigenic content is respectively 400U, 400U, 600U, and second proportioning is in view of possible between each component
Mutual inhibitory action select maximum dose level carry out proportioning, i.e. EV71, CA16, CA6 antigenic content be respectively 1000U, 1000U,
1200U, another proportioning mode is to select low dosage to carry out proportioning, i.e. in view of mutual potentiation possible between each component
EV71, CA16, CA6 antigenic content is respectively (100U, 100U, 120U) or (25U, 25U, 50U).
Big with above-mentioned other two pins of four kinds of proportioning dosage component (0d, 14d exempt from 28d blood sampling) peritoneal immunity mice and intramuscular immunisation
Mus, sets up single Seedling comparison and negative control simultaneously.In triplicate.NAT the results are shown in Table 7.Each virus in connection and vaccine
Component immunogenicity carries out statistical analysis with corresponding unit price Seedling respectively, and statistic analysis result is shown in Table 7.
Table 7 unit price Seedling and combined vaccine each virus ingredient immunity neutralizing antibody result
Note: in table, data are the meansigma methods of three experimental results
Connection Seedling 1:EV71, CA16, CA6 antigenic content is respectively 25U, 25U, 50U
Connection Seedling 2:EV71, CA16, CA6 antigenic content is respectively 100U, 100U, 120U
Connection Seedling 3:EV71, CA16, CA6 antigenic content is respectively 400U, 400U, 600U
Connection Seedling 4:EV71, CA16, CA6 antigenic content is respectively 1000U, 1000U, 1200U
Shown by table 7 result of study, the immune neutralizing antibodies of 4 kinds of connection Seedlings and single Seedling than there are no significant difference (P >
0.05), show to join between each component of Seedling without immune interference.These four kinds connection Seedling each component immunogenicities are compared two-by-two simultaneously
Understanding, the connection each component immunity neutralizing antibody water product of Seedling 2,3,4 compare two-by-two, no significant difference (P > 0.05).Connection Seedling 2,
3,4 each component immunity neutralizing antibody levels compare with connection Seedling 1, and difference has statistical significance (P < 0.05).According to result above,
The plan clinical dosage of EV71, CA16, CA6 in evaluating in conjunction with single Seedling, determines EV71, CA16, CA6 antigen component in every dose of connection Seedling
Optimum combination is 400U, 400U, 600U.
(2) determination of immune programme for children
According to the immunogenic animal model determined in embodiment 4 (1), EV71/CA16/CA6 combined vaccine is with identical dose
Amount 400U/400U/600U/ person-portion, respectively a pin (0d exempts from 28d blood sampling), two pins (0d, 14d exempt from 28d blood sampling) peritoneal immunity mice
With intramuscular immunisation rat.Measure serum NAT, the results are shown in Table 8.
Table 8 Different immunizing schedule connection Seedling immunity neutralizing antibody result
Being shown by table 8 result of study, EV71/CA16/CA6 combined vaccine is with same dose 400U/400U/600U/ people
Part, a pin (0d exempts from 28d blood sampling), two pins (0d, 14d exempt from 28d blood sampling) peritoneal immunity mice and intramuscular immunisation rat, record respectively
The immune animal anti-neutralizing antibody level of each component all has significant difference (P < 0.05), EV71/CA16/CA6 combined vaccine 2
Pin immunity NAT is higher than 1 pin immunity NAT.
According to EV71/CA16/CA6 combined vaccine various dose, Different immunizing schedule immunogenicity result of study, the most really
Determining EV71/CA16/CA6 combined vaccine immunizing dose is 400U/400U/600U/ person-portion, and immune programme for children is two pin immunity.
Embodiment 6 preparation process
(1) preparation prescription
The preparation of aluminum adsorbed vaccine: with 0.85% normal saline by aluminium adjuvant (Brenntag Biosector company of Denmark
The aluminium hydroxide produced) it is diluted to 1.0mg/ml, with 0.01M PBS by EV71 (or CA16 or CA6) antigen diluent to 1600U/
Ml, is added dropwise to the aluminium adjuvant after dilution under room temperature in EV71 (or CA16 or the CA6) antigen after dilution, makes the two equal-volume enter
Row absorption, stirring while adding, after continue mixed at room temperature 30 minutes, it is thus achieved that EV71 (or CA16 or CA6) aluminum adsorbed vaccine.
The preparation of EV71 (or CA16 or CA6) aluminum-free adsorbed vaccine: EV71 (or CA16 or CA6) is resisted with 0.01M PBS
Former it is diluted to 800U/ml.
EV71 (or CA16 or CA6) vaccine prepared by two kinds of different preparation prescriptions is with same dose, and two pin abdominal cavities are exempted from respectively
Epidemic disease mice and intramuscular immunisation rat.Measure serum NAT, the results are shown in Table 9.
Table 9 aluminum adsorbed vaccine and aluminum-free Seedling immunogenicity result
Table 9 result shows, under the identical immunizing dose of EV71 vaccine and immune programme for children, containing aluminium adjuvant vaccine and without aluminium adjuvant
Vaccine is significantly higher than aluminum-free vaccine (P < 0.05) in rats with antibody horizontal；In mice level, two groups of neutralizing antibodies are without significantly
Difference (P > 0.05), analyzing reason may may reach immunologic tolerance with this dosage through two pin immunity relative to mice
Dosage range is relevant；Under the identical immunizing dose of CA16 vaccine and immune programme for children, containing aluminium adjuvant vaccine no matter in rat level or
Aluminum-free vaccine (P < 0.05) it is all remarkably higher than in mice level；Under the identical immunizing dose of CA6 vaccine and immune programme for children, containing aluminium adjuvant
No matter vaccine is all remarkably higher than aluminum-free vaccine (P < 0.05) in rat level or mice level.
The lasting level considering rat, mice neutralizing antibody result, and the slow release of antigen and antibody needs, and determines
EV71 (or CA16 or CA6) bacterin preparation prescription is: vaccinogen liquid adds aluminium adjuvant after dilution.
(2) aluminium adjuvant absorption carrying capacity to EV71 (or CA16 or CA6) antigen
With 0.85% normal saline, aluminium adjuvant being diluted to aluminium hydroxide content is 2.89mg/ml, with 0.01M PBS respectively
By EV71, CA16 and CA6 antigen diluent to 12000U, 8000U, 4000U, under room temperature, the aluminium adjuvant after dilution is added dropwise to dilution
After each virus antigen in, make the two equal-volume adsorb, stirring while adding, after continue mixed at room temperature 30 minutes.Inspection
Antigenic content after surveying SNAg content and dissociating, calculates adsorption rate and dissociation yield, sees that aluminium adjuvant is to EV71 (or CA16 or CA6)
The absorption carrying capacity of antigen.The results are shown in Table 10.
Adsorption rate=(1-SNAg content ÷ theory adsorption antigen amount) × 100%
Dissociation yield=dissociate antigenic content ÷ theory adsorption antigen amount × 100%
Table 10 aluminium adjuvant absorption carrying capacity to EV71 (or CA16 or CA6) antigen
Being shown by table 10 result of study, every milliliter of aluminium adjuvant is 8000U/ml to the absorption carrying capacity of EV71 antigen, and aluminum is helped
Agent is 12000U/ml to the absorption carrying capacity of CA16 antigen, and aluminium adjuvant is 8000U/ml to the absorption carrying capacity of CA6 antigen.
(3) aluminium adjuvant absorption carrying capacity to EV71/CA16/CA6 hybrid antigen
With 0.85% normal saline, aluminium adjuvant being diluted to aluminium hydroxide content is 2.89mg/ml, with 0.01M PBS respectively
By EV71, CA16 and CA6 antigen diluent mix, make EV71/CA16/CA6 hybrid antigen be respectively 8000U/8000U/8000U,
4000U/4000U/4000U, 1000U/1000U/1000U, be added dropwise to mixed each disease by the aluminium adjuvant after dilution under room temperature
In poison antigen, make the two equal-volume carry out adsorbing (after adsorbing with adjuvant equal-volume, antigenic content be in table correspondence 4000,
2000,1000), stirring while adding, after continue mixed at room temperature 30 minutes.After detecting SNAg content and dissociating, antigen contains
Amount, calculates adsorption rate and dissociation yield, and (absorption carrying capacity is the most originally to the absorption carrying capacity of EV71/CA16/CA6 hybrid antigen to see aluminium adjuvant
The protein content corresponding to maximum antigenic content that secondary experiment can be adsorbed).The results are shown in Table 11.
Table 11 aluminium adjuvant absorption carrying capacity to EV71/CA16/CA6 hybrid antigen
Being shown by table 11 result, EV71, CA16, CA6 antigen presses antigenic content 8000U/8000U/8000U, 4000U/
After 4000U/4000U, 1000U/1000U/1000U mixing, all can inhale with the aluminium adjuvant that aluminium hydroxide content is 2.89mg/ml
Attached.
(4) the aluminum absorbing process of vaccine
According to the antigenic content of vaccinogen liquid, suitably dilute with 0.01M PBS (pH 7.2) solution and aluminum hydroxide solution,
Making the final concentration of 1.44mg/ml of aluminium hydroxide content in semi-finished product, EV71, CA16, CA6 antigenic content of semi-finished product is respectively
800U/ml, 800U/ml, 1200U/ml, be stirred at room temperature absorption 30 ± 5 minutes, is vaccine semi-finished product.
(5) fill carries out syringe fill to the vaccine semi-finished product obtained, and vaccine finished product dosage is 0.5ml/ person-portion.Associating
Vaccine each virus component protein content≤4 μ g/ person-portion.Each component protein concentration (μ g/ml)=[each component total protein content (μ
G/ml) × target antigen content (U/ml)] ÷ each virus stock solution used antigenic content (U/ml).
7 three batches of vaccine consistency checies of embodiment
(1) prepared by continuous three batches of EV71, CA16, CA6 vaccinogen liquids
The technique determined according to embodiment 1～3, carries out the production of continuous three batches of extensive EV71 vaccinogen liquids, to each epidemic disease
Seedling stock solution carries out antigenic content, protein content, BSA content, purity (HPLC method) detection, and to each batch inactivation of virus liquid and
Vaccinogen liquid carries out inactivating efficacy checking, calculates total antigen response rate and gross protein clearance.The results are shown in Table 12.And to EV71
Virus harvest liquid and stock solution carry out electron microscopic observation, and result is shown in Fig. 2 A and Fig. 2 B.
The continuous three batches of EV71 vaccine purification effects of table 12
The technique determined according to embodiment 1～3, carries out the production of continuous three batches of extensive CA16 vaccinogen liquids, to each epidemic disease
Seedling stock solution carries out antigenic content, protein content, BSA content, purity (HPLC method) detection, and to each batch inactivation of virus liquid and
Vaccinogen liquid carries out inactivating efficacy checking, calculates total antigen response rate and gross protein clearance.The results are shown in Table 13.And to CA16
Virus harvest liquid and stock solution carry out electron microscopic observation.
The continuous three batches of CA16 vaccine purification effects of table 13
The technique determined according to embodiment 1～3, carries out the production of continuous three batches of extensive CA6 vaccinogen liquids, to each vaccine
Stock solution carries out antigenic content, protein content, BSA content, purity (HPLC method) detection, and to each batch inactivation of virus liquid and epidemic disease
Seedling stock solution carries out inactivating efficacy checking, calculates total antigen response rate and gross protein clearance.The results are shown in Table 14.And it is sick to CA6
Poison harvest liquid and stock solution carry out electron microscopic observation.
The continuous three batches of CA6 vaccine purification effects of table 14
Being shown by table 12-14 result of study, display inactivation technology is safe and reliable, purifying process is stablized controlled, can be linear
Amplify for large-scale production.
(2) preparation of continuous three batches of combined vaccines
Three batches of stock solutions of preparation in embodiment 7 (1) are utilized to carry out continuous three batches of associatings by the preparation process that embodiment 6 determines
Preparation (EV71 aim parameter 400U/ person-portion, CA16 aim parameter 400U/ person-portion, the CA6 aim parameter 600U/ person-portion of vaccine；0.5ml/
Person-portion).Detection EV71/CA16/CA6 connection Seedling finished product in aluminium hydroxide content, SNAg content, dissociate after antigenic content,
Calculate adsorption rate and dissociation yield, the results are shown in Table 15.
Table 15 Seedling finished product detection result
(3) immunogenicity uses three batches of connection animal models of determining by embodiment 4 (1) of Seedlings, respectively two pin immune rats with
Mice, the NAT in detection serum.Set up the comparison of unit price Seedling and negative control simultaneously.In triplicate.Thin with trace
Born of the same parents' pathological changes method detection NAT.Calibrating cell is RD cell, neutralizes experiment and cultivates 7 days at 37 DEG C, and observation of cell is sick
Become.NAT and Conversion rate the results are shown in Table 16.Connection and vaccine in each virus ingredient immunogenicity respectively with corresponding list
Valency Seedling carries out statistical analysis, the results are shown in Table 17.According to statistical analysis, the neutralizing antibody GMT value of connection Seedling and single Seedling is all without notable
Difference (P value is all higher than 0.05).
Table 16 unit price Seedling and combined vaccine each virus ingredient immunity NAT
Note: in table, data are the meansigma methods of three experimental results.
Table 17 combined vaccine each virus ingredient immunity NAT compares (P value) with unit price Seedling
| Batch | EV71 | CA16 | CA6 |
| 1 | >0.05 | >0.05 | >0.05 |
| 2 | >0.05 | >0.05 | >0.05 |
| 3 | >0.05 | >0.05 | >0.05 |
(4) counteracting toxic substances protection uses three batches of connection Seedlings to carry out counteracting toxic substances by the counteracting toxic substances guard method of embodiment 4 (2), calculates half dead
Die and protect dosage and half morbidity protection dosage, the results are shown in Table 18.
Table 18 Seedling counteracting toxic substances protection result
Embodiment 8 combined vaccine safety evaluatio
(1) in the assessment immunogenic whole experiment periods of each combined vaccine, all healthy survival of all animals, clinical manifestation
Without exception.
(2) to three batches of EV71/CA16/CA6 combined vaccines of embodiment 7 and carried out animal safety evaluation, including different
Often toxicity test, acute toxicity test in mice and Cavia porcellus actively hypersensitive test.
Abnormal toxicity test includes mouse test and Cavia porcellus test.In mouse test, EV71/CA16/CA6 combined vaccine is noted
Penetrating 18～22g mice 5,0.5ml/ only, observes 7d.In observation period, mice is all strong deposits, and reaction without exception, little when expiring
Mus body weight increases.In Cavia porcellus test, EV71/CA16/CA6 combined vaccine injection 250～350g Cavia porcellus 2,5ml/ only, observes
7d.In observation period, Cavia porcellus is all strong deposits, and reaction without exception, and when expiring, Cavia porcellus body weight increases.
In acute toxicity test in mice, EV71/CA16/CA6 combined vaccine uses maximum dosage-feeding method intramuscular injection to give
Kunming mice, dosage is 8000U/kg body weight (being equivalent to about 120 times of 0.5 years old child's Clinical practice dosage of body weight), is administered dynamic
There is not abnormal clinical symptom and death in thing.
In Cavia porcellus actively hypersensitive test, Hartley Cavia porcellus gives combined vaccine 3 times through intramuscular injection sensitization, priming dose
0.5ml//time, it is administered every other day.After last sensitization is administered, the 14th day intravenous administration excites administration, booster dose 1.0ml/
Only/time, it is administered once.Experimental animal is Continuous Observation anaphylaxis after exciting administration, result of the test display EV71/CA16/CA6
Combined vaccine anaphylaxis in Cavia porcellus whole body actively hypersensitive test is negative.
Embodiment 9 combined vaccine estimation of stability
Connection Seedling finished product 1, finished product 2 and finished product 3 that embodiment 6 prepares are stored under 2-8 DEG C of environment, temporally sampling inspection
Survey antigenic content, outward appearance, loading amount, pH value, osmotic pressure molar density, bacterial endotoxin, content of formaldehyde etc..Record data are to 12
Month, and undue toxicity and the immunogenicity of vaccine is detected at December.Connection Seedling finished product 2-8 DEG C is placed 12 months, every Testing index
It is showed no and is decreased obviously.The results are shown in Table 19 and table 20.
Table 19 2～8 DEG C of connection Seedling finished product antigenic content testing result (percentage ratio of labelled amount)
Table 20 2～8 DEG C of connection Seedling finished product detection results
Note: "-" represents that not carrying out this detects.
Claims (10)
1. the combined vaccine preventing hand-foot-mouth disease, it is characterised in that it contains the EV71 antigen of inactivation, the CA16 of inactivation resists
CA6 antigen that is former and that inactivate.
Combined vaccine the most according to claim 1, it is characterised in that the antigen of described combined vaccine EV71, CA16, CA6
Content is respectively 100-1000U/ person-portion, 100-1000U/ person-portion, 200-1200U/ person-portion.
3. combined vaccine as claimed in claim 2, it is characterised in that the antigenic content of EV71, CA16, CA6 is respectively 200-
800U/ person-portion, 200-800U/ person-portion, 400-1000U/ person-portion.
4. the arbitrary described combined vaccine of claim 1-3, it is characterised in that its viral purification liquid is prepared into by following steps
Arrive:
(1) respectively EV71, CA16, CA6 virus liquid is inactivated, be concentrated by ultrafiltration；
(2) respectively above-mentioned three kinds of viral concentration liquid are carried out sucrose density gradient centrifugation；
(3) merging centrifugal liquid is collected.
5. combined vaccine as claimed in claim 4, it is characterised in that in step (1),
The preparation method of EV71 virus liquid is: EV71 virus inoculation human diploid cell, and inoculation MOI is: 0.0001-0.001, training
Foster serum-concentration: 2% Ox blood serum, cultivation temperature: 36 ± 1 DEG C, harvest time is 7-9d；
The preparation method of CA16 virus liquid is: CA16 virus inoculation human diploid cell, and inoculation MOI is: 0.001-0.01, cultivates
With serum-concentration: 1% Ox blood serum, cultivation temperature: 35 ± 1 DEG C, harvest time is 8-10d；
The preparation method of CA6 virus liquid is: CA6 virus inoculation human diploid cell, and inoculation MOI is: 0.0001-0.001, cultivates
With serum-concentration: 1% Ox blood serum, cultivation temperature: 33 ± 1 DEG C, harvest time is 8-10d.
6. combined vaccine as claimed in claim 4, it is characterised in that in step (1), ablation method is formalin-inactivated, formaldehyde
Final concentration of 67～200 μ g/m l of inactivation.
7. combined vaccine as claimed in claim 6, it is characterised in that the formalin-inactivated time is: under the conditions of 37 ± 1 DEG C, inactivation
2.8～5.5d.
8. the combined vaccine as described in claim 5-7 is arbitrary, it is characterised in that step (1) is concentrated by ultrafiltration employing aperture and is
The ultrafilter membrane bag of 300kD, EV71 inactivation of virus liquid equal-volume ultrafiltration 8 times, 3 times of volume ultrafiltration of CA16 inactivation of virus liquid 8 times, CA6
Inactivation of virus liquid equal-volume ultrafiltration 8 times.
9. the arbitrary described combined vaccine of claim 1-3, it is characterised in that possibly together with aluminium adjuvant.
10. the combined vaccine described in claim 9, it is characterised in that described aluminium adjuvant is aluminium hydroxide, and it is in combined vaccine
The final concentration of 0.36-1.73mg/ml of aluminium hydroxide content.
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Cited By (3)
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| CN108853490A (en) * | 2018-06-25 | 2018-11-23 | 中国医学科学院医学生物学研究所 | A kind of combined vaccine and preparation method thereof preventing hand-foot-and-mouth disease and hepatitis A |
| CN112717128A (en) * | 2020-12-30 | 2021-04-30 | 北京科兴生物制品有限公司 | Combined vaccine for preventing hand-foot-and-mouth disease and preparation method and application thereof |
| WO2022141266A1 (en) * | 2020-12-30 | 2022-07-07 | 北京科兴生物制品有限公司 | Combined vaccine for preventing hand, foot and mouth disease, preparation method therefor and use thereof |
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| CN103386126A (en) * | 2013-06-25 | 2013-11-13 | 北京科兴生物制品有限公司 | Multivalent immunogenic composition containing enterovirus antigens |
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| CN103386126A (en) * | 2013-06-25 | 2013-11-13 | 北京科兴生物制品有限公司 | Multivalent immunogenic composition containing enterovirus antigens |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108853490A (en) * | 2018-06-25 | 2018-11-23 | 中国医学科学院医学生物学研究所 | A kind of combined vaccine and preparation method thereof preventing hand-foot-and-mouth disease and hepatitis A |
| CN112717128A (en) * | 2020-12-30 | 2021-04-30 | 北京科兴生物制品有限公司 | Combined vaccine for preventing hand-foot-and-mouth disease and preparation method and application thereof |
| WO2022141266A1 (en) * | 2020-12-30 | 2022-07-07 | 北京科兴生物制品有限公司 | Combined vaccine for preventing hand, foot and mouth disease, preparation method therefor and use thereof |
| CN112717128B (en) * | 2020-12-30 | 2023-07-21 | 北京科兴生物制品有限公司 | Combined vaccine for preventing hand-foot-mouth disease and preparation method and application thereof |
| EP4272755A4 (en) * | 2020-12-30 | 2024-03-13 | Sinovac Biotech Co., Ltd. | Combined vaccine for preventing hand, foot and mouth disease, preparation method therefor and use thereof |
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