CN108187036A - A kind of zika virus combines inactivated vaccine with encephalitis B virus - Google Patents
A kind of zika virus combines inactivated vaccine with encephalitis B virus Download PDFInfo
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Abstract
The present invention provides a kind of zika virus and encephalitis B virus combined vaccine, belong to technical field of biological product preparation.Contain 0.5 10ug/ml encephalitis B virus and 0.5 10ug/ml zika virus in the combined vaccine of the present invention.The present invention also provides the preparation method of zika virus and encephalitis B virus combined vaccine, inoculation, purifying and inactivation step including encephalitis B virus and zika virus, wherein encephalitis B virus inoculation MOI are 0.001 0.1CCID50/ ml, zika virus inoculation MOI is 0.001 0.1CCID50/ml.Zika virus and encephalitis B virus can be immunized in the vaccine of the present invention simultaneously, reduce immune time, and there is good prevention and control zika virus and encephalitis B virus to cause the ability of disease for infection and allergic reaction caused by avoiding attenuated vaccine.
Description
Technical field
The invention belongs to technical field of biological product preparation, specifically, being related to a kind of containing zika virus and encephalitis disease
The joint inactivated vaccine of poison.
Background technology
Zika virus is found from 20th century mid-term, mainly in the torrid areas such as South America, Africa and Southeast Asia prevalence, there is no
Vaccine is protected.And 2015 to 2016, in Brazil's up to thousands of examples of newborn microcephaly disease as caused by zika virus, cause huge
It is big panic.With the raising of China's economic level, the people go on a tour it is aforementioned area commercial affairs and travelling chance it is more and more, sense
The probability of dye zika virus will increase.And there is no stockaded village's card vaccine listing at present, there is an urgent need for exploitation one kind to have protection to full age bracket
Stockaded village's card vaccine of effect, the health for our people provide safety guarantee.
Epidemic encephalitis B virus is one of Infectious Diseases of China's summer and autumn prevalence, in addition to Xinjiang, Tibet, Qinghai, entirely
There are a case in state various regions, year number of the infected 2.5 ten thousand, case fatality rate 10%, there are after different degrees of by about 15% patient
Lose disease.Encephalitis B virus belongs to Flavivirus with zika virus, and the two is all togavirus, and in China's encephalitis B virus vaccine
It is one of fundamental immunity vaccine, there is presently no the reports of the joint inactivated vaccine development of encephalitis B virus and zika virus.
Invention content
The object of the present invention is to provide a kind of bigeminy vaccines for safely, effectively preventing zika virus and infection and encephalitis B virus infection.
A kind of zika virus provided by the invention contains zika virus and encephalitis B virus, and two with encephalitis B virus combined vaccine
Person's virus inactivates.
Further, zika virus of the invention combines inactivated vaccine with encephalitis B virus and contains 0.5-10ug/ml encephalitis B virus
With 0.5-10ug/ml zika virus.
Preferably, zika virus of the invention combines inactivated vaccine with encephalitis B virus and contains 1-8ug/ml encephalitis B virus and 1-
8ug/ml zika virus.
It is highly preferred that the present invention zika virus combine with encephalitis B virus inactivated vaccine contain 4-6ug/ml encephalitis B virus and
4-6ug/ml zika virus.
The combined vaccine of the present invention is also sweet containing 0.25%-1% sodium glutamates, 0.5%-1% mannitol, 0.5%-1%
Propylhomoserin trimethylamine oxide and 0.2%-0.62% aluminium hydroxides, the % are mass volume ratio.Final pH is adjusted to 7.0-
8.0。
The present invention provides the preparation method of a kind of zika virus and encephalitis B virus combined vaccine, goes out including preparing encephalitis B virus
Living liquid, prepare zika virus inactivation liquid, two kinds of inactivation of virus liquid are diluted according to protein content after mix, addition addition
Agent makes to contain 0.5-10ug/ml encephalitis B virus and 0.5-10ug/ml zika virus in combined vaccine.
In the preparation method of the present invention, the method for preparing encephalitis B virus inactivation liquid includes the following steps：
(1) encephalitis B virus is inoculated with host cell MOI as 0.001-0.1CCID in the way of point kind or miscegenation50/ ml, training
34-36 DEG C of temperature is supported, supernatant of every 2 days harvests after thin inoculation；It harvests 4-6 times altogether, merges each batch supernatant and obtain second
Encephalovirus harvest liquid；
(2) concentrate is obtained after the clarification of encephalitis B virus harvest liquid, ultrafiltration, using molecular sieve and ion exchange two-step chromatography
Concentrate is purified, obtains encephalitis B virus refined solution；
(3) encephalitis B virus refined solution filters, and adjusts pH value 7.5-8.5, inactivates to obtain the final product.
During step (1), in addition to last time, every time after harvest, fresh virus-culturing fluid is all added, contributes to disease
The follow-up breeding of poison.
In step (2), encephalitis B virus purifying can remove the host more than 99% using two step of molecular sieve and ion exchange
Albumen and host's nucleotide.The rate of recovery that can ensure encephalitis B virus using the purification process of such combination is more than 50%.
Inactivation is the addition 0.01-0.1% β propiolactone into encephalitis B virus refined solution in step (3), stirs and goes out at 4 DEG C
It is hydrolyzed after 20-26 hours living；Or the encephalitis B virus refined solution addition 30-180ug/ml formaldehyde after filtering goes out in 22-28 sites
It is 96 hours living, formaldehyde is removed, obtains encephalitis B virus inactivation liquid.Using 0.22 μm of membrane filtration.
The zika virus of the present invention and the side in the preparation method of encephalitis B virus combined vaccine, preparing zika virus inactivation liquid
Method includes the following steps：
(1) zika virus inoculation host cell MOI is 0.001-0.1CCID50/ ml, 25-35 DEG C of cultivation temperature, treats cell
The supernatant of lesion 25%-35% harvests 80%, adds fresh virus-culturing fluid, continues to cultivate to cytopathy 45%-55%,
The supernatant of harvest 80%, adds fresh virus-culturing fluid, continues culture to cytopathy 70%-80%, harvest 80% it is upper
Clear liquid adds fresh virus-culturing fluid, continues culture to cytopathy 90%-100%, harvests whole supernatants；By aforementioned 4 batches
Supernatant merges the harvest liquid of as zika virus；Or
It treats cytopathy 45%-55%, harvests 80% supernatant, add fresh virus-culturing fluid, continue to cultivate to thin
Born of the same parents lesion 70%-80% harvests 80% supernatant, adds fresh virus-culturing fluid, continues culture to cytopathy 90%-
100%, harvest whole supernatants；Aforementioned 3 batches of supernatants are merged to the harvest liquid of as zika virus；
(2) harvest liquid clarification, the ultrafiltration of zika virus, obtains zika virus concentrate；It is purified using two-step chromatography dense
Contracting liquid obtains zika virus refined solution；
(3) pH is adjusted between 7.5-8.5, inactivated to obtain the final product through 0.22 μm of filtering by zika virus refined solution.
In step (2), zika virus purifying can be removed using molecular sieve and ion exchange two-step chromatography more than 99%
Host protein and host's nucleotide.
Inactivation is the β propiolactone of the zika virus refined solution addition 0.01-0.1% after filtering at 4 DEG C in step (3)
Inactivation hydrolyzes after 20-26 hours or the zika virus refined solution addition 30-180ug/ml formaldehyde after filtering goes out at 22-28 DEG C
It is 96 hours living, remove formaldehyde, as zika virus inactivation liquid.
The preparation method of the joint inactivated vaccine of the present invention, which is further included, carries out two kinds of inactivation of virus liquid according to protein content
It is mixed after dilution, adds the aluminum hydroxide adjuvant of final concentration of 0.2%-0.62%, 0.5-1% glutamic acid, 0.5-1% sweet dews
Alcohol, 0.5-1% Glycine Oxidation trimethylamines adjust the combined vaccine of pH to 7.0-8.0, final obtained stockaded village's card and encephalitis B virus.
The vaccine stability of the present invention is preferable, and stable in physicochemical property, two kinds of immunogenes can be immunized simultaneously without interfering with each other
Zika virus and encephalitis B virus, infection and allergic reaction caused by avoiding attenuated vaccine, being inoculated with the vaccine of the present invention can subtract
Few transfer needle is secondary, simplified immune programme, more succinct in operation, greatly improves the coverage rate of inoculation.It can effectively prevent people simultaneously
And animal, because of above-mentioned virogenetic disease, covering is more extensive, and immune effect is more preferable, have good prevention and control zika virus and
Encephalitis B virus causes the ability of disease.The method that the present invention prepares combined vaccine is easy, at low cost, extensive raw convenient for industrializing
Production.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of the invention
In the case of essence, to the modifications or substitutions that the method for the present invention, step or condition are made, all belong to the scope of the present invention.Such as
Lower adopted embodiment has used the technology of standard, unless otherwise detailed description, be those skilled in the art it is known that and
For routine techniques.Embodiment is illustrative, but is not intended to limit the present invention.
The present invention is not limited to the biomaterial used in following embodiment, such as Strain, those skilled in the art can select
Virus isolated strain the method according to the invention of the identical kind in this field carries out the preparation of combined vaccine, is not limited to the present embodiment institute
Strain.
The culture process of 1 encephalitis B virus vaccine of embodiment
Vero cells are cultivated on microcarrier to individual layer, discard cell culture fluid, cell surface is rinsed with PBS, connects
Kind encephalitis B virus, using P3 plants of second generations work seeds culture of viruses passed on Vero cells of encephalitis mouse brain strain, inoculation MOI is
0.01CCID50/ ml, virus-culturing fluid pH are 7.6, are cultivated at 35 DEG C.It is inoculated with after encephalitis B virus after 2 days in harvest for the first time
Clearly, harvest yield is the 80% of whole liquid volumes.Later, it harvests supernatant according to preceding description within every two days, harvests 5 times altogether.It will receive
Each batch of supernatant obtained merges, as encephalitis B virus harvest liquid.
The purifying of 2 encephalitis B virus of embodiment
By encephalitis B virus harvest liquid after deep layer membrane stack filters removal cell fragment and large crumb, harvest on cell
Clearly.With the ultrafiltration concentration of 300,000 molecular weight of retention, concentrate is obtained.Afterwards by gel permeation chromatography and ion-exchange chromatography.
Wherein the available filler of gel permeation chromatography is GE Sepharose 4Fast Flow, Sepharose 4Fast
The separating ranges such as Flow, Sephacryl S-300HR, Sephacryl S-400HR and Sephacryl S-500HR are suitable, easy
In the high carrying capacity filler of high throughput of amplification.The available filler of ion-exchange chromatography is the fillers such as GE DEAE, CM and ANX.Tool
Gymnastics is made as follows.
(1) first by encephalitis B virus vaccine concentrate by chromatographic column filtration chromatography, ultraviolet detection wavelength is 280nm, is flowed
Speed is 6cm/h, and mobile phase is the 10mmol/LPBS (NaCl0.2-0.6mol/L) of pH7.4, collects first absorption peak, i.e. second
Pure liquid at the beginning of encephalopathy toxalbumin.
(2) it is balanced with 10mmol/LPBS (NaCl0.2-0.6mol/L) balance anion chromatographic columns to ultraviolet, conductance, stream
Fast 3cm/h.
(3) liquid pure at the beginning of encephalitis B virus is loaded by 2 times of bed volumes in ion-exchange chromatography media, host nucleic acids are secured
Absorption is on anion chromatography medium, and encephalitis B virus albumen is taken in the binding site of Ion Exchange Medium by the ion in buffer solution
In generation, is directed through, and ultraviolet detection wavelength is 280nm, collects absorption peak.
(4) after sample all loads, continue to wash ion with 10mmol/LPBS (NaCl0.2-0.6mol/L) buffer solution
2 times of bed volumes of displacement chromatography column, until ultraviolet absorption value drops to baseline, collection spreads peak for encephalitis refined solution.
After two-step chromatography, albumen removal rate is averagely more than 99.9%DNA removal rates and is averagely more than 99.9%.Chromatography effect
Fruit is as shown in table 1.
1 impurity protein of table and host DNA removal rate
The inactivation technology of 3 encephalitis B virus of embodiment
Encephalitis B virus refined solution made from embodiment 2 is inactivated, is inactivated according to two kinds of inactivation modes, respectively final concentration
0.025% 25 ± 3 DEG C of inactivations of the inactivation of 4 DEG C of β propiolactone and final concentration of 30-180ug/ml formaldehyde, respectively at 0,6,12,24,
36th, it samples within 48,72,96 hours, detects virus titer, data analysis is as a result carried out using SPSS17.0 softwares, with (x ± s) table
Show, examined as needed using variance analysis and t, it is statistically significant for difference with P ＜ 0.05.The results are shown in Table 2, formaldehyde
After inactivation 6 hours, virus titer is begun to decline, and most of viruses have been inactivated after 24 hours, and disease is not detected after 48 hours
Malicious titre.Beta-propiolactone can't check virus after inactivating 12 hours.
β propiolactone is taken to inactivate 24 hours, the formalin-inactivated sample of 96 hours is inoculated with Vero cells, blind passage three generations, verification disease
Malicious inactivating efficacy.As a result Vero cytopathic effects will not be caused by showing the virus of two kinds of ablation method inactivations.
The encephalitis B virus (containing 4 μ g albumen) that formaldehyde and β propiolactone are inactivated, while it is pure to set up the encephalitis B virus not inactivated
Change liquid (containing 4 μ g albumen) and PBS negative controls, immune according to 0,14 day, the program immunity mouse of blood sampling in 28 days passes through detection
ELISA serum antibody titers come determine inactivation encephalitis B virus induce mouse generate antibody level.The encephalitis B virus of inactivation is exempted from
Epidemic focus testing result is as shown in table 3, formaldehyde, β propiolactone and the antibody drop of encephalitis B virus refined solution initiation mouse not inactivated
Degree is suitable, no significant difference.3 times reproducible results is similar.The present embodiment result illustrates that formaldehyde may be used in encephalitis B virus refined solution
Inactivation can also use β propiolactone to inactivate, and inactivating efficacy is suitable.
2 formaldehyde of table and β propiolactone inactivation different time encephalitis B virus titre (lgCCID50/ml, x ± s, n=3)
Note:"-" expression does not detect infection titer
3 formaldehyde of table and the inactivation of β propiolactone induce mice antibody titer and compare
| Ablation method | Antibody titer |
| Formaldehyde | 20066.17±6506.69 |
| β propiolactone | 21342.08±9036.78 |
| Ultrafiltrate | 21462.17±10376.89 |
The screening of 4 zika virus of embodiment
By zika virus seed Z-3 on Vero cells Adaptable growth to the 15th generation, select the 1st, 4,7,10,13,16 generations
Virus titer measure is carried out respectively, measures mouse neutralizing antibody titers with cell method and ELISA method respectively.As a result SPSS is used
17.0 softwares carry out data analysis, and measurement data is represented with (x ± s), are examined as needed using variance analysis and t, P ＜
0.05, there is statistical significance.The results are shown in Table 4, according to as a result, the original species of the present invention the 10th generation zika virus of selection
Son, and main viral seed and working virus seed are established, main virus seed generation was 13 generations, and working virus seed generation is 15
Generation.
Each generation virus titer (lgCCID of 4 zika virus Z-3 of table50/ ml, x ± s, n=3) and neutralizing antibody titers
| Generation | Virus titer (lgCCID50/ml) | Neutralizing antibody titers (GMT) |
| 1 | 6.18±0.13 | 16 |
| 4 | 6.77±0.17 | 64 |
| 7 | 7.02±0.23 | 64 |
| 10 | 7.70±0.22 | 128 |
| 13 | 7.68±0.19 | 96 |
| 16 | 7.82±0.09 | 128 |
The culture process of 5 zika virus vaccine of embodiment
1st, it is 0.001-0.1CCID according to MOI on Cytodex1 after Cultivation of Vero individual layer50/ ml inoculation stockaded village's card diseases
Poison work seed, cultivates after there is 30% lesion to cell, harvests supernatant, be placed in 2-8 DEG C.Fresh virus-culturing fluid is added, after
Continuous culture 24-60 hours, after 50% lesion occurs in cell, harvests supernatant, is placed in 2-8 DEG C again.Add fresh Virus culture
Liquid after continuing culture to cell 75% lesion of appearance, harvests supernatant, is placed in 2-8 DEG C.Fresh virus-culturing fluid is added, until cell
There is lesion and reach 85-100%, harvest supernatant.Merge the supernatant of above-mentioned Multiple harvests, as zika virus harvest liquid, put
It is placed in 2-8 DEG C to keep in, as 001 batch.
2nd, on Cytodex1 after Cultivation of Vero individual layer, stockaded village's card is inoculated with for 0.001-0.1CCID50/ml according to MOI
Working viral seed is cultivated to cell and 30-40% lesions occurs, harvests 80% supernatant, is placed in 2-8 DEG C.Add fresh virus training
Nutrient solution.Continue culture to cell and lesion occur to reach 60-75% lesions, harvest 80% supernatant, be placed in 2-8 DEG C.Add fresh training
Nutrient solution continues culture to cell and 85-100% lesions occurs, harvests whole supernatants.Merge supernatant, as zika virus harvests
Liquid is positioned over 2-8 DEG C and keeps in, as 002 batch.
3rd, on Cytodex1 after Cultivation of Vero individual layer, stockaded village's card is inoculated with for 0.001-0.1CCID50/ml according to MOI
Working viral seed is cultivated to cell and 50% lesion occurs, harvests 80% supernatant, is placed in 2-8 DEG C.Add fresh Virus culture
Liquid, continues culture to cell and lesion occurs to reach 85-100%, harvests supernatant, merges harvest liquid.As zika virus harvest liquid,
It is positioned over 2-8 DEG C to keep in, is denoted as 003 batch.
4th, it is 0.001-0.1CCID according to MOI on Cytodex1 after Cultivation of Vero individual layer50/ ml inoculation stockaded village's card diseases
Poison work seed, cultivates to cell and 85-100% lesions occurs, harvests supernatant, as zika virus harvest harvest liquid, is positioned over
2-8 DEG C temporary, is denoted as 004 batch.
The virus titer (in triplicate) for the virus harvest liquid that more above-mentioned four kinds of methods obtain determines best culture side
Method.It is as shown in the table, the titre highest of the harvest liquid of 001 batch and 002 batch, while the volume of harvest liquid is also maximum, therefore select four
Secondary harvest or the method harvested three times can reach preferable result.
The zika virus titre of the different harvesting approaches of table 5 compares (LgCCID50/ml)
| Batch | Virus titer (lgCCID50/ml) | Harvest liquid volume (fermenter volume) |
| 001 | 8.1±0.09 | ≈4 |
| 002 | 8.25±0.12 | ≈3 |
| 003 | 7.8±0.17 | ≈2 |
| 004 | 7.7±0.19 | 1 |
The present invention selects the zika virus culture process of the 002nd batch, is harvested altogether 4 times according to different cytopathy degree,
Harvest volume is improved to the full extent, and ensure that the content of harvest liquid unit volume inner virus titre.
The purifying process of 6 zika virus vaccine of embodiment
Zika virus culture solution is clarified and ultrafiltration after obtain zika virus liquid be concentrated by ultrafiltration, chromatograph to obtain stockaded village by two steps
Card viral purification liquid.Gel permeation chromatography of the first step chromatography for removal impurity protein, second step chromatography are removal contaminant nucleic acid
Ion-exchange chromatography.The available filler of gel permeation chromatography is GE Sepharose 4Fast Flow, Sepharose
The separating ranges such as 4Fast Flow, Sephacryl S-300HR, Sephacryl S-400HR and Sephacryl S-500HR are closed
Fit, be easy to the high carrying capacity filler of high throughput of amplification.The available filler of ion-exchange chromatography is filled out for GE DEAE, CM and ANX etc.
Material.Concrete operations are as follows.
(1) first by zika virus vaccine concentrate by chromatographic column filtration chromatography, ultraviolet detection wavelength is 280nm, is flowed
Speed is 6cm/h, and mobile phase is the 10mmol/L PBS (NaCl 0.2-0.6mol/L) of pH7.4, collects first absorption peak, i.e.,
Pure liquid at the beginning of zika virus albumen.
(2) it is balanced with 10mmol/L PBS (NaCl 0.2-0.6mol/L) balance anion chromatographic columns to ultraviolet, conductance,
Flow velocity 3cm/h.
(3) liquid pure at the beginning of zika virus is loaded by 2 times of bed volumes in ion-exchange chromatography media, host nucleic acids are secured
Absorption is on anion chromatography medium, and zika virus albumen is taken in the binding site of Ion Exchange Medium by the ion in buffer solution
In generation, is directed through, and ultraviolet detection wavelength is 280nm, collects absorption peak.
(4) after sample all loads, continue to wash ion with 10mmol/LPBS (NaCl0.2-0.6mol/L) buffer solution
2 times of bed volumes of displacement chromatography column, until ultraviolet absorption value drops to baseline, collection spreads peak for final refined solution.
After two-step chromatography, albumen removal rate is averagely more than 99.9%DNA removal rates and is averagely more than 99.9%.Chromatography knot
Fruit is as shown in the table.
6 impurity protein of table and host DNA removal rate
The inactivation technology of 7 zika virus vaccine of embodiment
Zika virus is inactivated using two kinds of ablation methods, and liquid, a kind of β propiolactone 4 for final concentration 0.025% is concentrated by ultrafiltration
DEG C inactivation, another kind is the inactivation of 25 ± 3 DEG C of 30-180ug/ml formaldehyde, is taken respectively at 0,6,12,24,36,48,72,96 hour
Sample detects virus titer, as a result carries out data analysis using SPSS17.0 softwares, is represented with (x ± s), use side as needed
Difference is analysed and t is examined, statistically significant for difference with P ＜ 0.05.The results are shown in Table 7, and formalin-inactivated is after 6 hours, virus
Titre is begun to decline, and most of viruses have been inactivated after 24 hours, and virus titer is not detected after 48 hours.Beta-propiolactone goes out
Virus is can't check after 12 hours living.
β propiolactone is taken to inactivate 24 hours, the formalin-inactivated sample of 96 hours is inoculated with Vero cells, blind passage three generations, verification disease
Malicious inactivating efficacy.As a result Vero cytopathic effects will not be caused by showing the virus of two kinds of ablation method inactivations.
The zika virus (containing 5 μ g albumen) that formaldehyde and β propiolactone are inactivated, while set up the zika virus not inactivated and surpass
Filter concentration liquid (containing 5 μ g albumen) and PBS negative controls, immune according to 0,14 day, the program immunity mouse of blood sampling in 28 days passes through
ELISA serum antibody titers are detected to determine that inactivation zika virus induces the level that mouse generates antibody.The zika virus of inactivation
Immunogenicity testing result it is as shown in the table, formaldehyde, β propiolactone and inactivation before ultrafiltrate cause mouse antibody titer phase
When no significant difference.3 times reproducible results is similar.
7 formaldehyde of table and β propiolactone inactivation different time zika virus titre (lgCCID50/ml, x ± s, n=3)
Note:"-" expression does not detect infection titer
8 formaldehyde of table and the inactivation of β propiolactone induce mice antibody titer and compare
| Ablation method | Antibody titer |
| Formaldehyde | 18066.67±6609.86 |
| β propiolactone | 21500.00±13063.87 |
| Ultrafiltrate | 22533.33±14368.89 |
8 combined vaccine proportioning process of embodiment
By the encephalitis B virus stoste and zika virus stoste after inactivation made from above-described embodiment, carried out according to protein content
After dilution, after mixing, addition stabilizer so that zika virus protein content is 4ug/ml, and viral encephalitis protein content is 4ug/
Ml adds 0.2% (aluminum hydroxide adjuvant and 0.5%-1% glutamic acid, 0.5%-1% mannitol, the 0.5%-1% of (w/v)
Glycine Oxidation trimethylamine adjusts pH to 7.5.More than % is mass volume ratio.
Stockaded village's card and encephalitis B combined vaccine totally three batches made from being formulated with this, are denoted as 001,002 and 003 batch respectively.With not
It with formula, i.e., by stockaded village's card and encephalitis stoste, dilutes according to a certain percentage, and adds 0.2% (the aluminum hydroxide adjuvant system of (w/v)
The vaccine obtained is denoted as 004 batch of progress stability comparison.The amount of antigen that above-mentioned vaccine every batch of contains is 400 antigen units.
Above-mentioned vaccine is deposited in 25 DEG C respectively, detects the antigen of 7 days, 14 days, 28 days, 60 days, 90 days and 6 months respectively
Content the results are shown in Table 9.
Table 9
| Batch | 7 days | 14 days | 21 days | 28 days | 60 days | 90 days | 6 months |
| 001 | 425 | 418 | 407 | 405 | 390 | 380 | 340 |
| 002 | 407 | 412 | 407 | 400 | 387 | 375 | 330 |
| 003 | 418 | 400 | 395 | 390 | 400 | 385 | 310 |
| 004 | 420 | 392 | 360 | 289 | 106 | —— | —— |
According to 25 DEG C of stability studies the results show that 001,002 and 003 batch is stored 90 days, antigenic content does not occur bright
It is aobvious to decline, and 004 batch at 21 days, antigenic content is substantially reduced.Therefore the vaccine containing this formula can greatly improve
The stability of vaccine.
The immunogenicity of 9 stockaded village's card of embodiment and encephalitis B combined vaccine
The combined vaccine of stockaded village's card and encephalitis made from Example 8, stockaded village's card inactivated vaccine, Japanese encephalitis inactivated vaccine are respectively compared
Immunogenicity.
Immune according to 0,14 day, the program immunity mouse of blood sampling in 28 days is inoculated with a dosage, passes through cytopathy every time
Method detects neutralizing antibody, calculates neutralizing antibody titers (GMT).As a result as shown in table 10,11.
10 combined vaccine of table is to the neutralizing antibody titers (GMT) of zika virus
| Batch | Neutralizing antibody titers (GMT) |
| The combined vaccine of stockaded village's card and encephalitis | 1024 |
| Stockaded village's card inactivated vaccine | 768 |
| Negative control | ＜ 8 |
11 combined vaccine of table is to the dilution factor of encephalitis B virus
It is above-mentioned the experimental results showed that, stockaded village's card made from the embodiment of the present invention 8 and encephalitis B combined vaccine are in Mice Body to stockaded village
The immunogenicity of card is high compared to stockaded village's card inactivated vaccine is used alone.To the immunogenicity of encephalitis B virus, compared to exclusive use encephalitis
Inactivated vaccine is 1 times high.Therefore, it is higher to compare single seedling immunogenicity with encephalitis B combined vaccine for stockaded village's card made from the embodiment of the present invention 8.
10 combined vaccine of embodiment attacks malicious protection
To evaluate the validity of combined vaccine, by stockaded village's card made from embodiment 8 and encephalitis B combined vaccine immunoprophylaxis defect
Mouse, immune programme 0,14 are immune, after 28 days and with 106TCID50ZIKV/Homo sapiens/PRI/PRVABC59/
The survival condition of mouse is observed in NaK plants of abdominal cavity attacks of 2015 plants of abdominal cavity attacks and encephalitis B virus.This experiment is set as negative right
According to 2 groups of group, 1 group of test sample and test sample, negative control group uses aluminum hydroxide solution 3mg/ml, and 1 group of test sample contains respectively
Stockaded village's card of 2ug/m and encephalitis B virus combined vaccine, 2 groups of test sample containing respectively having stockaded village's card of 1ug/ml and encephalitis B virus combined vaccine,
Every group of 10 immunodeficient mouses.The experimental results are shown inthe following table：
The malicious Protection of attacking of 12 stockaded village's card of table and encephalitis B virus combined vaccine designs
After attacking poison, the changes of weight and survival condition of 12 days mouse, all survivals of 1 group and 2 groups of test sample, control group are observed
8th day dead 6, the 9th day is all dead.This is it is demonstrated experimentally that stockaded village's card and encephalitis B virus combined vaccine of the invention can be with
It protects the mouse of immune deficiency from the attack of stockaded village's card and encephalitis B virus, there is good protectiveness.
Claims (10)
1. a kind of zika virus and encephalitis B virus combined vaccine, which is characterized in that containing zika virus and encephalitis B virus, and two kinds
Virus inactivates.
2. zika virus according to claim 1 and encephalitis B virus combined vaccine, which is characterized in that contain 0.5-10ug/
Ml encephalitis B virus and 0.5-10ug/ml zika virus.
3. zika virus according to claim 1 and encephalitis B virus combined vaccine, which is characterized in that contain 2-8ug/ml second
Encephalovirus and 2-8ug/ml zika virus.
4. inactivated vaccine is combined with encephalitis B virus according to any zika virus of claim 1-3, which is characterized in that contain
0.25%-1% sodium glutamates, 0.5%-1% mannitol, 0.5%-1% Glycine Oxidations trimethylamine and 0.2%-0.62% hydrogen
Aluminium oxide, the % are mass volume ratio.
5. the preparation method of a kind of zika virus and encephalitis B virus combined vaccine, which is characterized in that go out including preparing encephalitis B virus
Work liquid prepares zika virus inactivation liquid, will be mixed after two kinds of inactivation of virus liquid dilutions, adds additive, makes to contain in combined vaccine
There are 0.5-10ug/ml encephalitis B virus and 0.5-10ug/ml zika virus.
6. preparation method according to claim 5, it is characterised in that：The method for preparing encephalitis B virus inactivation liquid includes following
Step：
(1) encephalitis B virus is inoculated with host cell MOI as 0.001-0.1CCID in the way of point kind or miscegenation50/ ml, culture temperature
34-36 DEG C of degree, supernatant of every 2 days harvests after thin inoculation；It harvests 4-6 times altogether, merges each batch supernatant and obtain encephalitis disease
Malicious harvest liquid；
(2) concentrate is obtained after the clarification of encephalitis B virus harvest liquid, ultrafiltration, is purified using molecular sieve and ion exchange two-step chromatography
Concentrate obtains encephalitis B virus refined solution；
(3) encephalitis B virus refined solution filters, and adjusts pH value 7.5-8.5, inactivates to obtain the final product.
7. preparation method according to claim 6, which is characterized in that inactivation is to encephalitis B virus refined solution in step (3)
Middle addition 0.01-0.1% β propiolactone, hydrolyzes after inactivation being stirred at 4 DEG C 20-26 hours；Or the encephalitis B virus after filtering is pure
Change liquid addition 30-180ug/ml formaldehyde to inactivate 96 hours in 22-28 sites, remove formaldehyde, obtain encephalitis B virus inactivation liquid.
8. preparation method according to claim 5, it is characterised in that：The method for preparing zika virus inactivation liquid includes following
Step：
(1) zika virus inoculation host cell MOI is 0.001-0.1CCID50/ ml, 25-35 DEG C of cultivation temperature, treats cytopathy
The supernatant of 25-35% harvests 80%, adds fresh virus-culturing fluid, continues culture to cytopathy 45-55%, harvest 80%
Supernatant, add fresh virus-culturing fluid, continue culture to cytopathy 70-80%, harvest 80% supernatant, add new
Fresh virus-culturing fluid continues culture to cytopathy 90-100%, harvests whole supernatants；It is by the merging of aforementioned 4 batches of supernatants
Harvest liquid for zika virus；Or
It treats cytopathy 45-55%, harvests 80% supernatant, add fresh virus-culturing fluid, continue culture to cytopathy
70-80% harvests 80% supernatant, adds fresh virus-culturing fluid, continues culture to cytopathy 90-100%, harvest is complete
Portion's supernatant；Aforementioned 3 batches of supernatants are merged to the harvest liquid of as zika virus；
(2) harvest liquid clarification, the ultrafiltration of zika virus, obtains zika virus concentrate；Concentrate is purified using two-step chromatography,
Obtain zika virus refined solution；
(3) zika virus refined solution is filtered, pH is adjusted between 7.5-8.5, is inactivated to obtain the final product.
9. preparation method according to claim 8, which is characterized in that inactivation is stockaded village's card disease after filtering in step (3)
The β propiolactone of malicious refined solution addition 0.01-0.1% is hydrolyzed after being inactivated 20-26 hours at 4 DEG C or stockaded village's card disease after filtering
Malicious refined solution addition 30-180ug/ml formaldehyde inactivates 96 hours at 22-28 DEG C, removes formaldehyde, as zika virus inactivation liquid.
10. according to any preparation methods of claim 5-9, it is characterised in that：It is mixed after two kinds of inactivation of virus liquid are diluted
Close, add the aluminum hydroxide adjuvant of final concentration of 0.2%-0.62%, 0.5%-1% glutamic acid, 0.5%-1% mannitol,
0.5%-1% Glycine Oxidation trimethylamines adjust pH to 7.0-8.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020226831A1 (en) * | 2019-05-08 | 2020-11-12 | Takeda Vaccines, Inc. | Inactivated virus compositions and zika vaccine formulations |
| AU2022200351B2 (en) * | 2017-11-30 | 2022-06-23 | Takeda Vaccines, Inc. | Method for inactivating ZIKA Virus and related methods |
| US11478541B2 (en) | 2017-11-03 | 2022-10-25 | Takeda Vaccines, Inc. | Method for inactivating Zika virus and for determining the completeness of inactivation |
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| CN105749268A (en) * | 2016-04-11 | 2016-07-13 | 北京科兴中维生物技术有限公司 | Inactivated Zika virus vaccine |
| WO2017009873A1 (en) * | 2015-07-16 | 2017-01-19 | Bharat Biotech International Limited | Vaccine compositions |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017009873A1 (en) * | 2015-07-16 | 2017-01-19 | Bharat Biotech International Limited | Vaccine compositions |
| CN105749268A (en) * | 2016-04-11 | 2016-07-13 | 北京科兴中维生物技术有限公司 | Inactivated Zika virus vaccine |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11478541B2 (en) | 2017-11-03 | 2022-10-25 | Takeda Vaccines, Inc. | Method for inactivating Zika virus and for determining the completeness of inactivation |
| US11648304B2 (en) | 2017-11-03 | 2023-05-16 | Takeda Vaccines, Inc. | Zika vaccines and immunogenic compositions, and methods of using the same |
| US11730802B2 (en) | 2017-11-03 | 2023-08-22 | Takeda Vaccines, Inc. | Zika vaccines and immunogenic compositions, and methods of using the same |
| US11964008B2 (en) | 2017-11-03 | 2024-04-23 | Takeda Vaccines, Inc. | Method for inactivating zika virus and for determining the completeness of inactivation |
| AU2022200351B2 (en) * | 2017-11-30 | 2022-06-23 | Takeda Vaccines, Inc. | Method for inactivating ZIKA Virus and related methods |
| US11975062B2 (en) | 2017-11-30 | 2024-05-07 | Takeda Vaccines, Inc. | Zika vaccines and immunogenic compositions, and methods of using the same |
| WO2020226831A1 (en) * | 2019-05-08 | 2020-11-12 | Takeda Vaccines, Inc. | Inactivated virus compositions and zika vaccine formulations |
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