CN109464661A - A kind of vaccine antigen composition and preparation method thereof - Google Patents
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Abstract
The present invention provides a kind of vaccine antigen composition, the composition includes vaccine antigen and choline type ionic liquid.Vaccine antigen composition of the invention has the choline type ionic liquid of good biocompatibility as additive by addition, thermal stability, multigelation stability, accelerated stability and the extended storage stability for significantly improving vaccine antigen significantly improve stability of the antigen in solution and adjuvant environment and do not influence antigen active.
Description
Technical field
The invention belongs to vaccine research field, it is related to a kind of vaccine antigen composition and preparation method thereof more particularly to one
Kind includes the vaccine antigen composition and preparation method thereof of choline type ionic liquid.
Background technique
Antigen is the main constituent of vaccine, and the antigen based on virion is at present in vaccine for man and epidemic disease for animals
The antigenic type that seedling field is most widely used.This kind of antigen mainly includes virus type vaccine antigen and recombinant virus sample particle epidemic disease
Seedling antigen.This kind of antigen usually has complicated composition, the big feature of particle size in structure.Natural virus particle structure phase
To stabilization, but the virion used as antigen by attenuation, inactivate or re-assembly process after, natural structure by
It destroys, stability decline is extremely easy to happen cracking or aggregation and inactivates.It is mentioned with to vaccine quality and security requirement
Height needs to carry out raw material the antigen of purifying preparation high-purity, and the process and higher purity that purify then further reduce
The stability of antigen.On the other hand, antigen usually also needs to be used cooperatively with adjuvant, and the ingredient in adjuvant would generally also be fought
Former stability damages.
For example, aftosa (Foot and Mouth Disease, FMD) is a kind of very strong disease of infectiousness, the disease
It is fast with infection velocity, the features such as spread scope is wide, therefore infect the disease and can cause huge loss to animal husbandry.Inactivate mouth
Aphtovirus vaccine antigen is the prevention most effective vaccine of FMD.Complete foot and mouth disease virus particle sedimentation coefficient be 146S (i.e.
FMDV 146S), structure composition is complicated, and particle size is big (about 30nm), and stability is excessively poor, in heating and pH < 7.0
Time is extremely easy to happen cracking inactivation.Harmsen M M etc. is reported in Vaccine (2015,33 (21): 2477-2484)
Oil emulsion adjuvant ISA206 can further speed up the cracking of foot-and-mouth disease virus antigen.This transport of vaccine and saves to need strictly
Cold chain, increase research and development, production, transport, storage and the difficulty and cost used of vaccine.Therefore, it is steady to improve vaccine antigen
Qualitative is always one of the priority research areas of aftosa vaccine.
China is hepatitis B big country, and the vaccine for preventing hepatitis B at present is the hepatitis B surface antigen virus-like particle of genetic recombination
(HBsAg-VLP), it is made of a subunits up to a hundred, size 25nm or so.HBsAg-VLP is during isolating and purifying and storing, very
It is easy to happen cracking or aggregation.Valenzuela P etc. reports cracking in Nature (1982,298 (5872): 347-350)
Or the HBsAg-VLP of aggregation, activity only have the one thousandth of normal VLP antigen.
Human papillomavirus virus-like particles (HPV-VLP) play in the invasion of prevention HPV as the antigen of vaccine for cervical cancer
Important function.HPV-VLP is prepared by the method for genetic recombination, remain structure similar with natural HPV viruse and
Epitope.Shi Jingjie etc. reported in viral journal (2018,34 (02): 252-258) HPV-VLP particle integrality and
Thermal stability is the key that HPV vaccine reaches ideal immune effect.The subunit of depolymerization or aggregation does not only result in active reduction,
And the problem of becoming new impurity, may cause product quality.
The structural stability for how improving virus or virus sample particle vaccines antigen, avoids subunit depolymerization or aggregation, is true
Protect the key of its activity and safety.There are two main classes for the method for raising virion antigen stability at present, and first is to pass through
To the transformation of vaccine antigen grain structure such as amino is mutated or passed through to the key amino acid site for causing particle unstable
Acid mutation come enhance composition particle subunit between interaction force, prevent particle from cracking to improve the stability of particle.But
Such methods are effective only for the antigen of particular types specific subtype, poor universality, uncontrollable to the activity influence of antigen particles,
And such methods technology is complicated, higher cost, there are certain difficulty in large-scale application.Second is by being added in antigen
Particular kind of additive, such as inorganic salts, polyalcohol, sucrose and albumen.This method is usually used in improving protein drug
Stability, but for virion antigen, the more general protein drug of structure is increasingly complex, more to its factors affecting stability
It is more, although be added inorganic salts can certain antigens of raising to a certain extent stability, inorganic salts are steady to this antigen
Qualitative castering action is limited, and the inorganic salts of high concentration are larger to the activity influence of antigen；And the addition confrontation of albumen and carbohydrate
Former active castering action is usually limited, and higher cost, and addition while is also introduced into unnecessary big molecular impurity, influences epidemic disease
The safety of seedling, there are still larger difficulty in practical application.
Therefore it provides a kind of safety, biocompatibility are high, excellent in stability and the simple vaccine antigen of preparation method combine
Object is of great significance.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provides a kind of vaccine antigen composition and its preparation side
Method significantly improves the stability of vaccine antigen by adding choline type ionic liquid, reduces transport and storage cost.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provide composition described in a kind of vaccine antigen composition include vaccine antigen and choline type from
Sub- liquid.
Ionic liquid is the salt being in a liquid state under a kind of room temperature, has good dissolubility, physicochemical property designability, property
The advantages that stablizing, has been widely used in fields such as catalysis, synthesis, extraction and separation.Ionic liquid can be used for the extraction point of albumen
From, crystallization, stable and enzymatic, ionic liquid can be used for improving the stability of protein, but steady for ionic liquid at present
The research for determining albumen focuses primarily upon the simple model protein of structure such as BSA, insulin and nuclease etc..
The present invention is directed to virus type vaccine antigen or recombinant virus sample particle vaccines antigen, the antigenic source in animal or
People, is virus or recombinant virus sample particle by being attenuated, inactivating, mostly spherical viruses particle, compared to natural live virus,
Stability is worse, it is contemplated that the sample characteristics of for example of vaccine, the stabilization that the reagent of stabilization of vaccines not only needs to consider it to antigen,
With greater need for its safety of consideration.It is different from plant virus vaccine, the reagent for stablizing animal or source of people vaccine antigen needs
Preferable biocompatibility.The present invention is improved using the choline type ionic liquid with good biocompatibility as additive
Vaccine antigen stability, the choline type ionic liquid is by choline cation [cho]+It is formed with organic or inorganic anion,
Property is different from conventional inorganic salts or organic substance property, more preferable can obtain and stablize egg in conjunction with customary inorganic salts or organic substance
White advantage, at the same it is also with unique properties.
The present invention surprisingly has found by research, using choline type ionic liquid as additive, can significantly improve virus
It with the stability of recombinant virus sample particulate antigen in the solution, prevents it from assembling or cracking, and does not influence the activity of antigen itself,
Such ionic liquid have good biocompatibility, easily prepared, dissolubility is good and is easy to add.Therefore, choline type ion
Liquid adds simplicity as the stabilizer of vaccine antigen, and stablizing effect is good, has biggish practical value.
Preferably, the vaccine antigen includes virus type vaccine antigen and recombinant virus sample particle vaccines antigen.
Preferably, the virus type vaccine antigen packet foot-and-mouth disease virus antigen, Polio virus antigens, encephalitis B
Viral antigen, pig circular ring virus antigen or rabies virus antigen.
Preferably, the recombinant virus sample particle vaccines antigen includes recombination hepatitis B surface antigen virus-like particle, human milk
Head tumor virus sample particle or Hepatitis E virus sample particle.
In the present invention, the vaccine antigen can be prepared by the live virus obtained through cell culture by attenuation, inactivation, or
Method by genetic engineering prepares virus-like particle using acquisition prepared by conventional purification procedures.It is typical but unlimited
In the inactivation foot and mouth disease virus (FMDV) for being used as aftosa vaccine antigen, the hepatitis B surface antigen virus as hepatitis B vaccine antigen
Sample particle (HBsAg-VLP), human papillomavirus virus-like particles (HPV-VLP) as vaccine of cervical cancer antigen etc..
Preferably, the cation of the choline type ionic liquid is cholinium ion [cho]+。
Preferably, the anion of the choline type ionic liquid includes trimethylamine ion (tma), lactic acid ion
(lactate), propionate (propionate), caproic acid ion (hexanoate), hydrogen phosphate (HPO4 2-), dihydrogen phosphate
(H2PO4 2-), chloride ion (Cl-), bromide ion (Br-), iodide ion (I-), acetate ion (CH3COO-), sulfate ion
(SO4 2-), trifluoro methylsulfonimide ion (NTf2) any one of more library ester acid ions (AOT) or at least two group
It closes, the preferably combination of chloride ion and sulfate ion.
In the present invention, inventor has found that the anion can cooperate with cholinium ion, improves vaccine antigen stability,
The effect of middle chloride ion and sulfate ion is optimal, is remarkably improved the thermal stability of vaccine antigen.
Preferably, the molar concentration rate of the chloride ion and sulfate ion is 10:1-1:10, such as can be 1:1,1:
2,1:5,1:6,1:9,2:1,3:1,5:1,7:1,8:1 or 10:1.
Preferably, the pH value of the choline type ionic liquid be 7.0-8.0, such as can be 7.0,7.2,7.5,7.8 or
8.0, preferably 7.5.
Vaccine antigen particle is compared to structure relatively simple albumen such as monoclonal antibody, BSA etc., to the pH of solution environmental
Variation is more steady sensitive, and too high or too low pH easily leads to the cracking or aggregation of antigen particles.Therefore, in the present invention, first by gallbladder
The pH value of alkaline ionic liquid is adjusted in the range, is then added in vaccine antigen.
The choline type ionic liquid is added to the final concentration of 0.001-4mol/L in vaccine antigen solution, such as can be with
It is 0.001mol/L, 0.003mol/L, 0.005mol/L, 0.007mol/L, 0.009mol/L, 0.01mol/L, 0.015mol/
L、0.02mol/L、0.03mol/L、0.05mol/L、0.06mol/L、0.08mol/L、0.1mol/L、0.2mol/L、0.3mol/
L、0.5mol/L、0.8mol/L、0.85mol/L、0.9mol/L、1mol/L、1.1mol/L、1.2mol/L、1.5mol/L、
1.8mol/L、2mol/L、2.2mol/L、2.5mol/L、2.7mol/L、2.9mol/L、3mol/L、3.2mol/L、3.5mol/L、
3.7mol/L, 3.8mol/L or 4mol/L, preferably 0.005-1mol/L.
In the present invention, the addition final concentration of choline type ionic liquid can effectively improve vaccine antigen in the range
Stability and antigen active is not influenced, in preferred scope, choline type ionic liquid promotes effect the most to vaccine antigen stability
Significantly.
Preferably, the method also includes adjuvants.
Auxiliary agent of the adjuvant as antigen vaccine usually also has certain destructiveness, gallbladder used in the present invention to the stability of antigen
Alkaline ionic liquid can significantly improve the stability of vaccine antigen, and the vaccine antigen for being added to choline type ionic liquid can be straight
Preservation and use are connect, can also be further with use be saved after adjuvant mixing, choline type ionic liquid will not influence antigen or assistant
The effect of agent.
Preferably, the adjuvant includes the group of any one of oil emulsion adjuvant, aluminium adjuvant or particulate adjuvants or at least two
It closes.
Preferably, the particulate adjuvants include particles of polylactic acid or chitosan particle.
In the present invention, adjuvant refers to mixed with antigen after can increase the immunocompetent substance of antigen, adjuvant may include ISA206
Emulsion adjuvant, aluminium adjuvant, particulate adjuvants etc..
Second aspect, the present invention provides a kind of method of vaccine antigen composition prepared as described in relation to the first aspect, described
Method includes the following steps: choline type ion liquid dissolving in buffer, adjusts the pH value of mixed solution, and it is anti-that vaccine is added
It is uniformly mixed in raw water solution, obtains vaccine antigen composition.
Preferably, the buffer includes phosphate buffer.
Preferably, the temperature of the vaccine antigen aqueous solution is 4-25 DEG C, such as can be 4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C
Or 25 DEG C.
Preferably, the pH value be 7.0-8.0, preferably 7.5.
Preferably, final concentration of 0.001-4mol/L of the choline type ionic liquid in vaccine antigen composition.
Preferably, the method specifically comprises the following steps: by choline type ion liquid dissolving in phosphate buffer,
The pH value of mixed solution is adjusted to 7.0-8.0, is added in vaccine antigen aqueous solution, makes the final concentration of of choline type ionic liquid
0.001-4mol/L is uniformly mixed, obtains vaccine antigen composition.
The third aspect, the present invention provide a kind of vaccine, and the vaccine includes vaccine antigen combination as described in relation to the first aspect
Object.
Compared with prior art, the invention has the following beneficial effects:
(1) vaccine antigen composition of the invention by addition there is the choline type ionic liquid of good biocompatibility to make
For additive, thermal stability, multigelation stability, accelerated stability and the long term storage for significantly improving vaccine antigen are stablized
Property significantly improves stability of the antigen in solution and adjuvant environment and does not influence antigen active；
(2) vaccine antigen composition of the invention can inhibit to occur between virus, recombinant virus sample particle polymerized methylene
Cracking or aggregation, avoid the variation of vaccine antigen grain structure and the reduction of antigen active；
(3) vaccine antigen composition of the invention uses choline type ionic liquid as additive, easily prepared, dissolubility
It is good and easy to operate, it has a vast market foreground.
Detailed description of the invention
Antibody titer (P < 0.05 *) in Fig. 1 test case 4 after the FMDV antigen of the mouse injection ionic liquid of type containing choline；
Fig. 2 is the gel high-efficient liquid phase chromatogram of HBsAg-VLP after embodiment 12 is placed 2 months at 4 DEG C in test case 7
Spectrum；
Fig. 3 is the gel high-efficient liquid phase chromatogram of HBsAg-VLP after comparative example 9 is placed 2 months at 4 DEG C in test case 7
Spectrum；
Fig. 4 is the transmission electron microscope picture of HBsAg-VLP after embodiment 13 is placed 1 day at 4 DEG C in test case 8；
Fig. 5 is the transmission electron microscope picture of HBsAg-VLP after comparative example 9 is placed 1 day at 4 DEG C in test case 8.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below by way of specific embodiment come into
One step illustrates technical solution of the present invention, but the present invention is not limited in scope of embodiments.
Embodiment 1
A kind of FMDV solution, preparation method are as follows:
1) by a certain amount of choline type ionic liquid [cho] [Cl] and [cho] [SO4] it is dissolved separately in 20mM, pH7.5's
In phosphate buffer, and its pH is adjusted to pH 7.5 with the HCl of the NaOH of 4mol/L or 2mol/L, making concentration is
The choline type ionic liquid of 1mol/L；
2) respectively added into 100 μ g/mLFMDV solution (pH 7.5) of 1mL the above-mentioned ionic liquid of 0.5mL [cho] [Cl] and
[cho][SO4], it mixes, obtains the FMDV solution added with the compound choline type ionic liquid of 0.5mol/L.
Embodiment 2
Compared with Example 1, in addition to the in vitro liquid of choline type is [cho] [Cl] and [cho] [HPO4] except, other conditions
With embodiment 1.
Embodiment 3
Compared with Example 1, in addition to the in vitro liquid of choline type is [cho] [SO4] and [cho] [Br] except, other conditions
With embodiment 1.
Embodiment 4
A kind of FMDV solution, preparation method are as follows:
1) a certain amount of choline type ionic liquid [cho] [Cl] is dissolved in 20mM, in the phosphate buffer of pH 7.5,
And its pH is adjusted to pH 7.5 with the HCl of the NaOH of 4mol/L or 2mol/L, make concentration be 1mol/L choline type from
Sub- liquid；
2) the above-mentioned choline type ionic liquid of 1mL is added into 100 μ g/mL FMDV solution (pH 7.5) of 1mL, is mixed, is obtained
To the FMDV solution for being added with 0.5mol/L choline type ionic liquid.
Embodiment 5
Compared with Example 4, in addition to choline type ionic liquid is [cho] [SO4] except, other conditions are the same as embodiment 4.
Embodiment 6
Compared with Example 4, other than choline type ionic liquid is [cho] [Br], other conditions are the same as embodiment 4.
Embodiment 7
Compared with Example 4, in addition to choline type ionic liquid is [cho] [HPO4] except, other conditions are the same as embodiment 4.
Comparative example 1
Compared with Example 4, in addition to ionic liquid is non-choline type ionic liquid TMAP (trimethylammonium
Dihydrogen phosphate, TMAP) except, other conditions are the same as embodiment 4.
Comparative example 2
Compared with Example 4, in addition to ionic liquid is non-choline type ionic liquid TEAA (triethylammonium
Acetate, TEAA) except, other conditions are the same as embodiment 4.
Comparative example 3
Compared with Example 4, in addition to ionic liquid is non-choline type ionic liquid TEAP (triethylammonium
Dihydrogen phosphate, TEAP) except, other conditions are the same as embodiment 4.
Comparative example 4
1mLpH 7.5,20mM phosphate buffer are added into the FMDV solution of 1mL100 μ g/mL.
Influence of the 1 different type ionic liquid of test case to FMDV thermal stability
In 96- orifice plate, the diluted Sypro Orange dyestuff of 0.2 μ L25 times is added in each hole, addition is implemented respectively
The 19.8 μ L of FMDV solution of example 1-7 and comparative example 1-4, using the thermal stability of differential scanning fluorescence (DSF) method evaluation FMDV.
Temperature range in DSF experiment is 25 DEG C to 95 DEG C, and heating rate is 1 DEG C/min, equilibration time 10min, with first heat
Denaturation temperature (that is: Tm1) evaluates the thermal stability of FMDV particle, the results are shown in Table 1.
Table 1 adds influence of the different type ionic liquid to FMDV thermal stability
| Tm1/℃ | Tm1/℃ | ||
| Embodiment 1 | 56.5 | Embodiment 7 | 54.0 |
| Embodiment 2 | 56.1 | Comparative example 1 | 49.7 |
| Embodiment 3 | 56.0 | Comparative example 2 | 53.2 |
| Embodiment 4 | 55.8 | Comparative example 3 | 53.4 |
| Embodiment 5 | 54.4 | Comparative example 4 | 53.0 |
| Embodiment 6 | 53.9 |
As shown in Table 1, comparing embodiment 1 and embodiment 2-7 are it is found that be applied in combination [cho] [Cl], [cho] [SO4] choline
Type ionic liquid is more conducive to improve choline type ionic liquid to FMDV thermal stability；Comparing embodiment 1 and comparative example 1-3 can
Know, the more non-choline type ionic liquid of choline type ionic liquid improves the stability of antigen vaccine significant；Comparing embodiment 1 with it is right
Ratio 4 is it is found that choline type ionic liquid can significantly improve 3.5 DEG C of thermal stability of antigen.
2 multigelation stability test of test case
Research object is used as using optimum embodiment 1, embodiment 1 and comparative example 4 are respectively placed in -80 DEG C of ultra low temperature freezers
In, after 3 days take out be placed in 4 DEG C completely melt after, sampling, using gel high performance liquid chromatography (HPSEC, Chinese patent:
201510025994.7) measure remaining FMDV content in sample.According to said method multigelation 5 times remain after measuring each freeze thawing
Remaining FMDV content.Sample buffer is the phosphate buffer of pH 7.5, and the results are shown in Table 2.
Antigenic content after 2 multigelation of table 5 times
As shown in Table 2, addition choline type ionic liquid is remarkably improved the multigelation stability of FMDV, 5 ultralow temperature
After multigelation, FMDV antigen residue content and initial value are very nearly the same, and the comparative example 4 without adding choline type ionic liquid exists
After freeze thawing 1 time, antigenic content is significantly reduced, and only remains 24 μ g/mL.
3 37 DEG C of acceleration of test case crack stability test
Test choline type ionic liquid is usually used in the stabilizer of albumen, the influence for FMDV accelerated stability with other.
Selection example 1, comparative example 4 and common stabilizer include final concentration of 1% BSA, 10% sorbierite, 10% sugarcane
Sugar, pH value of solution are 7.5；FMDV antigen concentration is 200 μ g/mL.Sample is placed in 37 DEG C of Preservation in sterile condition.Using the efficient liquid of gel
Phase chromatography measures the situation of change of complete FMDV antigenic content in solution.With initial FMDV antigenic content for 100%, meter
It calculates after placing different time, remaining antigenic content.
Granule content percentage in 3 37 DEG C of placement process of table
| Time (h) | Comparative example 4 | 1%BSA | 10% sucrose | 10% sorbierite | Embodiment 1 |
| 0 | 100 | 100 | 100 | 100 | 100 |
| 2 | 70 | 97.8 | 93.9 | 96.4 | 99.7 |
| 4 | 55.3 | 87.9 | 88 | 80.9 | 98.1 |
| 9 | — | 75.8 | 70 | 72.1 | 98.5 |
| 17 | — | — | 67.4 | 69.8 | 93 |
| 22 | — | — | 58.5 | 67.9 | 87.6 |
| 30 | — | — | — | — | 76.3 |
| 40 | — | — | — | — | 71.3 |
| 48 | — | — | — | — | 68.7 |
As shown in Table 3, [cho] [Cl] and [cho] [SO4] combination can to significantly improve acceleration of the FMDV at 37 DEG C steady
Qualitative, significant effect is usually used in the additive of protein, 48h under the conditions of 37 DEG C of choline type ionic liquid of the invention better than other
Antigen particles content is still up to 68.7% afterwards, and other control group antigen particles sharply decline, and can not detect.
Comparative example 5
The PBS buffer solution of pH8.0 is added to FMDV solution, makes the final concentration of 50 μ g/mL of FMDV.
Comparative example 6
By FMDV solution by 56 DEG C of heating, it is cracked into FMDV 12S completely.
4 choline type ionic liquid of test case is to FMDV antigen active
24 Balb/c mouse are divided into 4 groups, every group 6.Embodiment 1, embodiment 4, comparative example 5 and comparative example 6 are divided
Not and after ISA206 adjuvant in equal volume uniform mixing and emulsifying, using immune mouse is injected intraperitoneally, injection dosage is 100 μ L.First
Secondary immune rear 14 days progress secondary immunities.Using ELISA kit, the serum for exempting from rear 14 days mouse to two carries out antibody content
Measurement.As shown in Figure 1, comparative example 6 is the FMDV particle after cracking completely, the experimental results showed that basic immunologic incompetence.Addition
The embodiment 1 and 4 of choline type ionic liquid does not reduce the immunocompetence of FMDV, and antibody titer has been also compared with comparative example 5
It is promoted, furthermore the antibody titer of embodiment 1 is slightly above embodiment 4, illustrates that compound choline type ionic liquid proposes FMDV stability
High effect is better than single choline type ionic liquid.
In this embodiment, while influence of the ionic liquid to FMDV stability in ISA206 adjuvant has been monitored.Pass through
Using gel high performance liquid chromatography, comparative example 5, embodiment 1 and 4 antigenic content of embodiment after measurement and ISA206 emulsification are at any time
Between situation of change, as the result is shown 4 DEG C place 14 days after, antigenic content is respectively 60%, 95%, the 95% of initial value.Show
Stability of the FMDV antigen in IS206 adjuvant can be improved in addition ionic liquid.
Embodiment 8
A kind of HPV-VLP solution, preparation method are as follows:
1) a certain amount of choline type ionic liquid [cho] [Cl] is dissolved in 20mM, in the phosphate buffer of pH 7.5,
And its pH is adjusted to pH 7.5 with the HCl of the NaOH of 4mol/L or 2mol/L, make concentration be 2mol/L choline type from
Sub- liquid；
2) the above-mentioned ionic liquid of 1mL [cho] [Cl] is added into 200 μ g/mL HPV-VLP solution (pH 7.5) of 1mL, mixed
It is even, obtain the HPV-VLP solution added with 1mol/L choline type ionic liquid.
Embodiment 9
Compared with Example 8, other than the concentration of choline type ionic liquid is 0.1mol/L, the same embodiment of other conditions
8。
Embodiment 10
Compared with Example 8, other than the concentration of choline type ionic liquid is 0.5mol/L, the same embodiment of other conditions
8。
Comparative example 7
PH 7.5,20mM phosphate buffer, the final concentration of 100 μ g/mL of HPV-VLP are added into HPV-VLP solution.
Influence of the 5 choline type ionic liquid of test case to the stability of HPV-VLP vaccine antigen
It in human papillomavirus virus-like particles (HPV-VLP) vaccine antigen, adds [cho] [Cl] of various concentration, tests
It influences the thermal stability of vaccine antigen, and wherein HPV-VLP is the antigen of vaccine of cervical cancer.Thermal stability determination is using poor
Show that scanning calorimetric (DSC) method carries out, embodiment 8-10 and comparative example 7 are tested, the temperature range of DSC is 25 DEG C to 95
DEG C, heating rate is 1 DEG C/min, equilibration time 10min, adds the thermostabilization of various concentration [cho] [Cl] to HPV-VLPs
The influence of property is shown in Table 4.
HPV-VLP thermal denaturation temperature changes [cho] [Cl] of 4 various concentration of table afterwards
As shown in Table 4, the thermal stability of HPV-VLP, and stabilization can be improved in choline type ionic liquid [cho] [Cl]
At concentration dependent within the scope of additive amount of the present invention.
Embodiment 11
The HPV-VLP solution and aluminium adjuvant of embodiment 8 are mixed, concentration containing HPV-VLPs is 100 μ g/mL in sample.
Comparative example 8
The HPV-VLP solution and aluminium adjuvant of comparative example 7 are mixed, concentration containing HPV-VLPs is 100 μ g/mL in sample.
Influence of the 6 choline type ionic liquid of test case to HPV-VLP stability in aluminium adjuvant
Test choline type ionic liquid pair with and the mixed HPV-VLP stability of aluminium adjuvant influence.By embodiment 11
With the sample of comparative example 8 be respectively placed in 4 DEG C it is sterile be kept in dark place, measurement place 0,2,4,8,12,16,20,24 day when HPV-VLP
The situation of change of partial size, particle size determination are carried out using dynamic light scattering (Dynamic Light Scattering, DLS), knot
Fruit is shown in Table 5.
The variation (nm) of HPV-VLP partial size in 54 DEG C of placement process of table
| Time (day) | Embodiment 11 | Comparative example 8 |
| 0 | 95 | 95 |
| 2 | 95 | 94 |
| 4 | 94 | 92 |
| 8 | 92 | 90 |
| 12 | 92 | 85 |
| 16 | 90 | 81 |
| 20 | 88 | 78 |
| 24 | 88 | 72 |
As shown in Table 5, addition choline type ionic liquid can inhibit the cracking of HPV-VLP particle, improve its stability.
Embodiment 12
A kind of HBsAg-VLP solution, preparation method are as follows:
1) a certain amount of choline type ionic liquid [cho] [Cl] is dissolved in 20mM, in the PBS buffer solution of pH 7.5, and
Its pH is adjusted to pH 7.5 with the HCl of the NaOH of 4mol or 2mol, makes the choline type ionic liquid that concentration is 1mol/L；
2) the above-mentioned ionic liquid of 1mL is added into 1mL 100g/mL HBsAg-VLP solution (pH 7.5), is mixed, is obtained
HBsAg-VLP solution added with 0.5mol/L choline type ionic liquid.
Comparative example 9
Compared with embodiment 12, other than not adding choline type ionic liquid, other conditions are compared with embodiment 12
Example 9 only contains HBsAg-VLP and PBS buffer solution.
Influence of the 7 choline type ionic liquid of test case to preventing HBsAg-VLP from cracking
Using embodiment 12 and comparative example 9, after being placed 2 months by the measurement of HPSEC method, complete HBsAg-VLP particle
Situation of change.The results show that the HBsAg-VLP in 12 choline type ionic liquid of embodiment do not occur significantly crack (see
Fig. 2), and the HBsAg-VLP in comparative example 9PBS then observes apparent cracking (see Fig. 3).By Fig. 2 and Fig. 3 it is found that addition gallbladder
HBsAg-VLP cracking can be effectively prevented in alkaline ionic liquid.
Embodiment 13
A kind of HBsAg-VLP solution, preparation method are as follows:
In (the NH containing 0.3mol/L4)2SO4HBsAg-VLP solution in be added 0.005mol/mL [cho] [AOT].
Influence of the 8 choline type ionic liquid of test case to preventing HBsAg-VLP from assembling
The influence that this test description ionic liquid [cho] [AOT] assembles HBsAg-VLP under high salt concn.It will implement
Example 13 and comparative example 9 are placed 1 day under the conditions of 4 DEG C, using the metamorphosis situation of transmission electron microscope observing particle.The results show that
HBsAg-VLP particle in [cho] [AOT] environment still keeps the state (see Fig. 4) relatively dispersed, and then sees in PBS environment
Observe the case where particle is assembled (see Fig. 5).By Fig. 4-5 it is found that choline type ionic liquid can effectively prevent HBsAg-VLP and gather
Collection.
To sum up, the present invention improves vaccine as additive using the choline type ionic liquid with good biocompatibility
The stability of antigen.This kind of ionic liquid stabilizer has many advantages, such as property stability, solubility height, good biocompatibility, energy
Stability of the antigen in solution and adjuvant environment is significantly improved, prevents it from assembling or cracking, and do not influence the activity of antigen, it should
Kind of additive is also easy to that preparation, dissolubility is good and is easy to add.Therefore, stabilizer of the choline type ionic liquid as vaccine antigen
Addition is easy, and stablizing effect is good, has biggish practical value.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of vaccine antigen composition, which is characterized in that the composition includes vaccine antigen and choline type ionic liquid.
2. composition according to claim 1, which is characterized in that the vaccine antigen includes virus type vaccine antigen and again
Group virus sample particle vaccines antigen.
3. composition according to claim 1 or 2, which is characterized in that the virus type vaccine antigen includes hoof-and-mouth disease
Malicious antigen, Polio virus antigens, japanese encephalitis virus antigen, pig circular ring virus antigen or rabies virus antigen；
Preferably, the recombinant virus sample particle vaccines antigen includes recombination hepatitis B surface antigen virus-like particle, human papilloma
Virus-like particle or Hepatitis E virus sample particle.
4. composition according to claim 1-3, which is characterized in that the anion of the choline type ionic liquid
Including trimethylamine ion, lactic acid ion, propionate, caproic acid ion, hydrogen phosphate, dihydrogen phosphate, chloride ion, bromide ion,
Any one of iodide ion, acetate ion, sulfate ion, trifluoro methylsulfonimide ion or more library ester acid ions or extremely
Few two kinds of combination, the preferably combination of chloride ion and sulfate ion；
Preferably, the molar concentration rate of the chloride ion and sulfate ion is 1:10-10:1.
5. composition according to claim 1-4, which is characterized in that the pH value of the choline type ionic liquid is
7.0-8.0 preferably 7.5.
6. composition according to any one of claims 1-5, which is characterized in that the choline type ionic liquid is added to
Final concentration of 0.001-4mol/L in vaccine antigen solution, preferably 0.005-1mol/L.
7. composition according to claim 1-6, which is characterized in that the composition further includes adjuvant.
8. composition according to claim 1-7, which is characterized in that the adjuvant includes oil emulsion adjuvant, aluminium assistant
Any one of agent or particulate adjuvants or at least two combination；
Preferably, the particulate adjuvants include particles of polylactic acid or chitosan particle.
9. a kind of prepare the method such as the described in any item vaccine antigen compositions of claim 1-8, which is characterized in that the side
Method includes the following steps: choline type ion liquid dissolving in buffer, adjusts the pH value of mixed solution, and vaccine antigen is added
It is uniformly mixed in aqueous solution, obtains vaccine antigen composition；
Preferably, the buffer includes phosphate buffer；
Preferably, the temperature of the vaccine antigen aqueous solution is 4-25 DEG C；
Preferably, the pH value be 7.0-8.0, preferably 7.5；
Preferably, final concentration of 0.001-4mol/L of the choline type ionic liquid in vaccine antigen composition.
10. a kind of vaccine, which is characterized in that the vaccine includes such as the described in any item vaccine antigen combinations of claim 1-8
Object.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111548395A (en) * | 2020-05-25 | 2020-08-18 | 中国农业科学院兰州兽医研究所 | Bivalent multi-epitope recombinant virus-like particle of foot-and-mouth disease virus and application thereof |
| CN111588846A (en) * | 2020-05-21 | 2020-08-28 | 中国科学院过程工程研究所 | Vaccine composition and preparation method and application thereof |
| CN112500838A (en) * | 2020-12-31 | 2021-03-16 | 舟山冰池新能源科技发展有限公司 | Novel choline eutectic solvent with adjustable viscosity and reversible absorption |
| WO2021222196A1 (en) * | 2020-04-28 | 2021-11-04 | President And Fellows Of Harvard College | Methods and compositions relating to ionic liquid adjuvants |
| CN115572236A (en) * | 2022-11-10 | 2023-01-06 | 辽宁大学 | Choline lactic acid ionic liquid, preparation method thereof and application thereof in iodine extraction |
| WO2023014853A1 (en) * | 2021-08-06 | 2023-02-09 | Merck Sharp & Dohme Llc | Hpv vaccine |
| US11684594B2 (en) | 2020-05-12 | 2023-06-27 | President And Fellows Of Harvard College | Antifungal prophylaxis for cornea |
-
2018
- 2018-12-14 CN CN201811534732.3A patent/CN109464661B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| DAVID M.等: ""In Vitro Assessment of Choline Dihydrogen Phosphate (CDHP) as a Vehicle for Recombinant Human Interleukin-2 (rhIL-2)"", 《CELLULAR AND MOLECULAR BIOENGINEERING》 * |
| KATHERINE D.等: ""Structure and function of proteins in hydrated choline dihydrogen phosphate ionic liquid"", 《PHYS. CHEM. CHEM. PHYS.》 * |
| RESLAN 等: ""Choline ionic liquid enhances the stability of Herceptins (trastuzumab)"", 《CHEMICAL COMMUNICATIONS》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021222196A1 (en) * | 2020-04-28 | 2021-11-04 | President And Fellows Of Harvard College | Methods and compositions relating to ionic liquid adjuvants |
| US11684594B2 (en) | 2020-05-12 | 2023-06-27 | President And Fellows Of Harvard College | Antifungal prophylaxis for cornea |
| CN111588846A (en) * | 2020-05-21 | 2020-08-28 | 中国科学院过程工程研究所 | Vaccine composition and preparation method and application thereof |
| CN111548395A (en) * | 2020-05-25 | 2020-08-18 | 中国农业科学院兰州兽医研究所 | Bivalent multi-epitope recombinant virus-like particle of foot-and-mouth disease virus and application thereof |
| CN112500838A (en) * | 2020-12-31 | 2021-03-16 | 舟山冰池新能源科技发展有限公司 | Novel choline eutectic solvent with adjustable viscosity and reversible absorption |
| WO2023014853A1 (en) * | 2021-08-06 | 2023-02-09 | Merck Sharp & Dohme Llc | Hpv vaccine |
| CN115572236A (en) * | 2022-11-10 | 2023-01-06 | 辽宁大学 | Choline lactic acid ionic liquid, preparation method thereof and application thereof in iodine extraction |
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