CN103316335B - Poliovirus vaccine for oral administration - Google Patents
Poliovirus vaccine for oral administration Download PDFInfo
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- CN103316335B CN103316335B CN201310215041.8A CN201310215041A CN103316335B CN 103316335 B CN103316335 B CN 103316335B CN 201310215041 A CN201310215041 A CN 201310215041A CN 103316335 B CN103316335 B CN 103316335B
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Abstract
The invention provides a poliovirus vaccine for oral administration. The poliovirus vaccine contains poliovirus antigen, carbohydrate, phosphate and carboxylate. The oral poliovirus vaccine provided by the invention can be adopted to raise stability of the vaccine antigen component, has good stability at temperatures of 37 DEG C, 2-8 DEG C and 25 DEG C, has stable antigen activity, can be used to enhance gastric acid resistance after oral administration of the vaccine finished product, and has good immune response stimulation capability and good safety. The poliovirus vaccine for oral administration contains no other humanized or animal-based protein and has better safety. Meanwhile, the poliovirus vaccine requires low cost, and is suitable for industrial production of the product.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to and be suitable for Orally administered poliovirus vaccine formula.
Background technology
Poliomyelitis (Poliomyelitis) is the highly infective disease being caused by Infected With Polioviruses In Vitro people.Poliovirus can infect nerve system of human body, and can within a few hours, cause patient limb paralysis, causes lifelong deformity.Poliomyelitis can occur in all age bracket crowds, but is apt to occur in the infant that is less than 5 years old, so this disease is called again " poliomyelitis ".Poliovirus belongs to the enterovirus genus of Picornaviridae.Observed under electron microscope virus is little spheroidal, and diameter is 24～30nm, rounded graininess.Poliovirus contains sub-thread positive chain RNA, by 3 coat protein, is wrapped up, and is respectively VP1-3, and another one VP4 albumen is interior glutelin.Poliovirus can be divided into I, II and III type according to serum group system, all can cause a disease.
Most people (90%) by Infected With Polioviruses In Vitro there will not be clinical symptoms or only have light symptoms, as fever, tired headache, vomiting and limbs pain etc.In infected crowd, only have about 0.5% people can become lifelong deformity, be mainly because virus is invaded nervous system by blood, destroy the neurocyte of controlling human muscle, cause the imbalance of body paralysis, be called as clinically AFP Cases.In causing disabled crowd, there is people's meeting of about 5-10% dead because respiratory center nervous system is destroyed.In the crowd who survives, have about 40% in 15-40 subsequently, can again occur the symptoms such as muscular paralysis and limbs pain, be called as rear poliomyelitis syndrome.By Infected With Polioviruses In Vitro and bring out and cause the people of lifelong paralysis often to have following sign: immunity defect, pregnancy, excised flat conductor, intramuscular injection, strenuous exercise or be subject to wound etc.Up to the present medical circle is not also treated poliomyelitic specific drug, only can treat patient by relief of symptoms, such as by physiotherapy with take spasmolytic medicine and alleviate muscular paralysis and anxiety.But poliomyelitis can reach good preventive effect by vaccinate.The poliomyelitis vaccine of deactivation (IPV) is succeeded in developing in the fifties in last century by doctor Salk at first.The 1954 Nian U.S. have carried out clinical trial, next year clinical test results proof vaccine there is good safety and protectiveness, in the U.S., carried out large-scale inoculation subsequently movable.The oral poliomyelitis vaccine (OPV) of another attenuation is succeeded in developing in the sixties in last century by doctor Sabin, and worldwide uses rapidly, for control Incidence of Poliomyelitis has been contributed solid strength.China also almost synchronizes with the world and starts to produce OPV vaccine and child has been carried out to large-scale inoculation, and accumulative total has been supplied the trivalent OPV vaccine of approximately 5,000,000,000 person-portions.
Orally administered is a kind of important vaccination mode, and the immunization ways of injection has its unique advantage relatively.To the microbial disease of some gastrointestinal tract cause of disease, adopt oral mode immunoprophylaxis can be more fast, direct stimulating gastrointestinal road mucomembranous immune system, thereby produce corresponding immunne response, make body there is immunoprotection.Orally administered vaccine is because need stand the digestive system of stomach, therefore the stability of its formula components, acid-resisting and safety aspect are had to special requirement.First, vaccine formulation must guarantee the stability of antigenic component, guarantees that vaccine has maximum effectiveness in use.Secondly, as oral vaccine, formula must have good anti-gastric acid ability, to guarantee the activity of antigen.Again, the safety of product is basis, and the safety of product is from the selection of formula components.
The formula components for oral class vaccine of having reported at present (referring to, for example US3783098, US3915794 and EP028563), the first is SPGA, it mainly contains cattle or people's serum albumin, caseinhydrolysate or gelatin; The second is alkali metal salt, sugar (glucose, sucrose or glucosan) and mono phosphoric acid ester salt or two salt of optional glutamic acid, or their mixture.These compositionss have very large defect.First they have potential danger biology, such as protein or the protein hydrolysate in the uncertain animal or human of chemical composition source.In addition, existing these compositionss of the art fail to make attenuated live vaccine to have enough stability and acid-resisting, make the storage requirement of vaccine and effect phase be subject to great restriction, even further affect effectiveness and the safety of vaccine.
The poliomyelitis vaccine,Sabin that China is being used at present, familiar " sugar pill " namely, it by living but the virus that pathogenicity reduces make, preparation is solid form, but this sugar pill is to the infant clothing used time, use not easy, not as liquid preparation is convenient to have child to take, and this sugar pill is when the larger interregional transportation of the temperature difference, and stability is good not.Therefore strong in the urgent need to a kind of stability on market, be convenient to the liquid oral poliovirus vaccine that long-distance transportation, immunogenicity are good, be convenient to infant taking.
Summary of the invention
The object of the present invention is to provide a kind of safe and effective Orally administered poliovirus vaccine that is applicable to.
Another object of the present invention is to provide a kind of method of preparing this vaccine.
Provided by the inventionly be suitable for oral poliovirus vaccine, contain poliovirus antigen, saccharide, phosphate and carboxylate.
Vaccine of the present invention, its every 1mL is containing 10
4～10
8the poliovirus antigen of individual infectious unit.Described infectious unit is CCID
50or PFU or FFU or other can represent to have the unit of infectious antigen.
The saccharide that contains 0.1～0.6g in this vaccine of every 1mL.
The phosphate radical that contains 0.05～0.5mmol in this vaccine of every 1mL.
The carboxylate that contains 0.05～0.5mmol in this vaccine of every 1mL.
Saccharide described in vaccine of the present invention is sucrose, lactose, dextran, trehalose, mannose, galactose, Sorbitol and/or mannitol.
Preferably, saccharide is sucrose.
The contained phosphate of vaccine of the present invention is the mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or the mixture of sodium dihydrogen phosphate and sodium hydrogen phosphate.Be preferably the mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
In above-mentioned phosphate mixt, the molar concentration rate of dihydric phosphate and phosphoric acid hydrogen disalt is 1:10～1:1.Preferred the two molar concentration rate is 1:2.
Described carboxylate has good anti-gastric acid ability, keeps the stable and effectiveness of antigenic component.The preferred carboxylate of the present invention is succinate or citrate.
Oral polio virus vaccine of the present invention, its pH value is 5-8.
Preferably, the vaccine formulation that possesses better antiacid effect and virus stability comprises:
● the final concentration of sucrose solution is 30%(w/v);
● potassium phosphate mole final concentration is 0.1M or 0.2M, and wherein the molar concentration rate of potassium dihydrogen phosphate and dipotassium hydrogen phosphate is 1:2;
● mole final concentration of carboxylate sodium succinate or sodium citrate is 0.1M or 0.2M;
● poliovirus antigen titre is 10
4～10
8individual infectious unit;
● pH value is controlled in pH5-8;
The present invention also provides preparation to be suitable for the method for oral poliovirus vaccine, under aseptic condition, sugar juice, phosphate buffer and carboxylic acid salt solution are mixed to rear filtration sterilization, add poliovirus cell culture fluid to obtain mixed solution, make in every 1ml mixed solution containing 10
4～10
8the poliovirus antigen of individual infectious unit, the sugar of 0.1～0.6g, the carboxylate of the phosphate radical of 0.05～0.5mmol, 0.05～0.5mmol, mixed solution pH value is 5-8, according to specification subpackage; Described phosphate buffered solution is the mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or the mixed solution of sodium dihydrogen phosphate and sodium hydrogen phosphate; Described carboxylate is sodium succinate solution or sodium citrate solution.
Wherein, in above-mentioned phosphate mixt, the molar concentration rate of dihydric phosphate and phosphoric acid hydrogen disalt is 1:1～10.Preferred the two molar concentration rate is 1:2.
The present invention can improve vaccine antigen composition stability, strengthen the oral anti-gastric acid ability entering after stomach of vaccine finished product, and this formula has good immunne response and stimulates ability and good safety.External antiacid test card is bright, and this formula has good acid-resisting.During the immunogenic composition of using this formula can be good at and gastric acid, the effectiveness of antigenic component and activity are protected.Stability test result shows to use the vaccine of this formula, at 37 ℃, 2～8 ℃, 25 ℃, has good stability, and antigen active is stable.Safety testing shows, the vaccine of using this formula has good safety in animal experiment.
As the saccharide of one of component in the present invention's formula, the sugar of certain concentration can improve the stability of bacterin preparation.There is huge challenge in the preparation of stable bacterin preparation.The stability of said preparation depends on the interaction between incorporated excipient in this vaccine combination.Hydrogen bond between the protein and sugar of epitope has the advantage that makes vaccine stable.Sugar must have suitable ratio with effective contact of antigen protein, to keep the stable of this vaccine.At a certain temperature, require sugared concentration to have a marginal value, with the hydrogen bond having between the protein and sugar of some, keep vaccine can stablize the specific time.Sugar is by alternative structure water, can with immobilized artificial membrane and Protein formation hydrogen bond.Sugar and protein and buffer composition appropriately combined, can be within the long storage life preservation viral, and make it have activity.These additives have given structural support to the antigen floating on a liquid.
As the phosphate buffer of one of component in the present invention's formula, the hydrogen phosphate that it contains and dihydrogen phosphate ions have certain buffer capacity to gastric acid, to keep the effective active of antigenic component.Meanwhile, phosphate buffer has certain effect to the stability of antigenic component and effectiveness tool.
Carboxylate as one of component in the present invention formula has good capacity antacid, can maintain the digestive system that antigenic component stands gastric juice, and stable antigen maintains the effectiveness of antigenic component.
Except above-mentioned advantage, the formula that the present invention mentions with prior art is compared also has following advantage: first, phosphate of the present invention is the cocktail buffer of dihydric phosphate and phosphoric acid hydrogen disalt, compares and has better stability and buffer capacity with single kind phosphate; The second, carboxylate of the present invention is selected from citrate and succinate, and above-mentioned two class salt all can reach effect of the present invention, but by test, sodium succinate effect is more excellent aspect acid-resisting and safety; The 3rd, formula of the present invention not containing other people source or animal-based protein, has better safety except composition described in claim, and cost is lower simultaneously, and the industrialization that is suitable for product is produced.
Accompanying drawing explanation
Fig. 1 is that poliovirus vaccine of the present invention and commercially available poliomyelitis sugar pill vaccine are at the stability data of 2-8 ℃ of storage.
Fig. 2 is that poliovirus vaccine of the present invention is at the stability data of 25 ℃ of storages.
Fig. 3 is that poliovirus vaccine of the present invention is at the stability data of 37 ℃ of storages.
The specific embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art; In embodiment, reagent used is commercial goods.
The preparation of embodiment 1 poliovirus antigen
By U.S. ATCC, offered poliovirus (hereinafter to be referred as Polio virus) strain sabin I type, sabin II type, the sabin III type of Xing Zhongwei Bioisystech Co., Ltd of Beijing section, Adaptable growth continuous passage in Vero cell respectively, put in 37.5 ± 1.0 ℃ of incubators and cultivate 3-5 days results virus liquids, after clarification filtration, use CCID
50method detects virus titer value.Above three type Polio virus harvest yields are 10
7-10
8cCID
50within the scope of/ml.
Various unit price Polio virus stock solution is configured for to the antigen component of bacterin preparation.
The preparation of embodiment 2 poliovirus vaccines
(1) phosphate buffer (wherein potassium dihydrogen phosphate and dipotassium hydrogen phosphate molar concentration ratio are 1:2) and the 4M sodium succinate solution storing solution of preparation 3M, filtration sterilization, 4 ℃ save backup;
(2) the 10ml phosphate buffer of step (1) preparation and 15ml sodium succinate solution are mixed;
(3) in (2), add 60% sucrose solution 150ml;
(4) in (3), add the antigenic solution in 100ml embodiment 1, this antigenic solution is the mixed liquor mixed liquor of sabin I type, II type and III type;
(5) do not adopt and supplement volume to 300ml containing viral MEM culture fluid; Vaccine pH value is 7;
(6) filter subpackage, every dose of 3ml, totally 100 doses.
Every dose of the vaccine of the present embodiment preparation is containing following composition: containing above-mentioned 3 kinds of serotype Polio virus each 10
7-10
8cCID
50, 30% sucrose, in phosphate buffer, phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2, phosphate radical total concentration is 0.1M; Sodium succinate solution final concentration is 0.2M.
Embodiment 3 is containing the antiacid effect of poliovirus vaccine of different sucrose formula
(1) preparation of bacterin preparation
Prepare 4 groups of vaccines that contain different sucrose: the phosphate buffer (potassium dihydrogen phosphate and dipotassium hydrogen phosphate molar concentration ratio are 1:2) of 4 groups of 10ml3M of preparation and the mixed liquor of 15ml4M sodium succinate solution, in 4 groups of mixed liquors, add respectively containing 30g, 90g, 150g, the sucrose solution of 180g sucrose is mixed with 4 kinds containing the protective agent (phosphate solution+sodium succinate solution+sucrose solution) of variable concentrations sucrose, respectively to adding in 4 groups of protective agents 100ml antigen component in embodiment 1 (with embodiment 2, be that 3 kinds of serotypes are mixed), adopt and do not contain viral MEM culture fluid polishing volume to 300ml, filtration sterilization, press every dose of subpackage of 3ml, be mixed with 100 vaccinating agent preparations, pH value is 5.In above-mentioned bacterin preparation, every dose contains every serotype poliovirus 10
7～10
8cCID
50.In phosphate buffer, phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2, and phosphate radical total concentration is 0.1M; Sodium succinate solution final concentration is 0.2M; The quality final concentration of sucrose in 4 groups of bacterin preparations (quality g/ volume 100ml) is respectively 10%, 30%, 50%, 60%.
(2) the antiacid effect test of filling a prescription
4 groups of preparations containing different sucrose in (1) are carried out respectively to antiacid effect test.The hydrochloric acid solution of preparing the different pH value of 7 groups of 30ml (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0), is " simulated gastric fluid ".The bacterin preparation of 4 groups of different sucrose is added respectively to above 7 groups of " simulated gastric fluid " antiacid tests, and result of the test is as shown in table 1.
Table 1 is containing the antiacid effect of the formula of different sucrose
Result of the test shows, under phosphate buffer and the certain condition of carboxylate salt concentration, containing the antiacid effect of the formula of 10%-60% different sucrose without significant difference, all in energy and 30ml pH1.8 " simulated gastric fluid ", and result of the test in 2 hours without significant change (another note: the present invention also finds that poliovirus can maintain activity and reach 3 hours in the solution of pH3-5).The present embodiment result shows: the sucrose quality final concentration that 10%-60% is described is all applicable to formula of the present invention.
In embodiment 4 vaccine formulations, phosphate buffering liquid concentration determines
(1) preparation of bacterin preparation
Prepare 8 groups of vaccines that contain different phosphate concns: (wherein potassium phosphate is 4 components, and potassium dihydrogen phosphate and dipotassium hydrogen phosphate molar concentration ratio are 1:2 by the 3M phosphate buffer of 5ml, 10ml, 30ml, 50ml; Sodium ascorbyl phosphate 4 components; sodium dihydrogen phosphate and sodium hydrogen phosphate molar concentration ratio are 1:2) respectively with the mixing of 15ml4M sodium succinate solution; to the sucrose solution that adds respectively the sucrose of 150ml60% in above-mentioned 8 groups of mixed liquors, be mixed with 8 kinds containing the protective agents of different phosphate concns; respectively to adding in 8 groups of protective agents 100ml antigen component in embodiment 1 (with embodiment 2; be that 3 kinds of serotypes are mixed); adopt and do not contain viral MEM culture fluid polishing volume to 300ml; filtration sterilization; press every dose of subpackage of 3ml; be mixed with 100 vaccinating agent preparations, pH value is 6.In above-mentioned bacterin preparation, contain each serotype poliovirus 10
5-10
7cCID
50.Sucrose mass concentration is 30%, and sodium succinate solution final concentration is 0.2M; Phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate in phosphate buffer (or phosphoric acid sodium dihydrogen and sodium hydrogen phosphate) molar concentration ratio is 1:2, and phosphate radical total concentration is respectively 0.05M, 0.1M, 0.3M, 0.5M.
(2) the antiacid effect test of filling a prescription
8 groups of preparations containing variable concentrations phosphate buffer in (1) are carried out respectively to antiacid effect test.The hydrochloric acid solution of preparing the different pH value of 7 groups of 30ml (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0), is " simulated gastric fluid ".The bacterin preparation of 8 groups of (each 4 groups of potassium salt and sodium salts) different phosphate buffer densities is added respectively to above 7 groups of " simulated gastric fluid " antiacid tests, and result of the test as Table 2,3.
Table 2 is containing the antiacid effect of the formula of variable concentrations phosphate buffer (potassium salt)
Table 3 is containing the antiacid effect of the formula of variable concentrations phosphate buffer (sodium salt)
(3) two kinds of antiacid effect tests of phosphate different proportion in phosphate buffer
Adopt the method identical with above-mentioned test, phosphate radical total concentration in phosphate buffer is set as to 0.1M, two kinds of phosphate molar concentration rates are arranged to 4 different proportions, i.e. KH
2pO
4: K
2hPO
4=1:10,1:5,1:2,1:1, detect respectively its antiacid effect.
Result of the test shows; Adopt the bacterin preparation of above-mentioned 4 kinds of phosphate ratios in antiacid effect, all can make " simulated gastric fluid " pH value of 30ml be buffered to more than 5.0 and maintain 2 hours to stablize.
The present embodiment result shows: under sucrose mass concentration and the certain condition of carboxylate salt concentration, containing the antiacid effect of the formula of 0.05M-0.5M variable concentrations phosphate buffer without significant difference, all in energy and 30ml pH1.8 " simulated gastric fluid ", and result of the test is stable in 2 hours; And test discovery, in phosphate buffer, dihydric phosphate and phosphoric acid hydrogen disalt molar concentration ratio are that 1:10-1:1 all can meet preparation demand.Result of the test all meets the requirements, and is suitable for formula of the present invention.
In embodiment 5 vaccine formulations, carboxylate salt concentration determines
(1) preparation of bacterin preparation
Prepare 4 groups of vaccines that contain different sodium succinate concentration: by 3.75ml, 7.5ml, 22.5ml, the 4M sodium succinate solution of 37.5ml respectively with the mixing of 10ml3M phosphate-buffered liquor (potassium dihydrogen phosphate and dipotassium hydrogen phosphate molar concentration ratio are 1:2), to the sucrose solution that adds respectively the sucrose of 150ml60% in above-mentioned 4 groups of mixed liquors, be mixed with 4 kinds containing the protective agents of different sodium succinate concentration, respectively to adding in 4 groups of protective agents 100ml antigen component in embodiment 1 (with embodiment 2, be that 3 kinds of serotypes are mixed), adopt and do not contain viral MEM culture fluid polishing volume to 300ml, filtration sterilization, press every dose of subpackage of 3ml, be mixed with 100 vaccinating agent preparations, pH value is 6.In above-mentioned bacterin preparation, contain each serotype poliovirus 10
7-10
8cCID
50.Sucrose mass concentration is 30%, and in phosphate buffer, phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2, and phosphate radical total concentration is 0.1M; Sodium succinate solution final concentration is respectively 0.05M, 0.1M, 0.3M, 0.5M.
(2) the antiacid effect test of filling a prescription
4 groups of preparations containing different sodium succinate concentration in (1) are carried out respectively to antiacid effect test.The hydrochloric acid solution of preparing the different pH value of 7 groups of 30ml (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0), is " simulated gastric fluid ".The bacterin preparation of 4 groups of different carboxylic acids salinity is added respectively to above 7 groups of " simulated gastric fluid " antiacid tests, and result of the test is as shown in table 4.
Adopt identical method, adopt sodium citrate to replace the sodium succinate in formula, carry out the present embodiment test, result of the test is as shown in table 5.
Table 4 is containing the antiacid effect of different sodium succinate concentration formulas
Table 5 is containing the antiacid effect of different sodium citrate concentration formulas
The present embodiment result shows: under sucrose mass concentration and the certain condition of phosphate buffer density, containing in the equal energy of antiacid effect of the formula of 0.05M-0.5M different carboxylic acids salinity and 30ml pH1.8 " simulated gastric fluid ", meet viral survival condition demand, and result of the test in 2 hours without significant change.Succinate or citric acid salt concentration that 0.05M-0.5M is described are all applicable to formula of the present invention.
The selection of saccharide in embodiment 6 formulas
The present embodiment checking is containing formula stability to antigen at 37 ℃ of temperature of different saccharides.The type Polio virus stock solution of results in embodiment 1 is used for preparing to the vaccine sample of the present embodiment.After sample preparation, in 37 ℃ of placements 2 weeks, for the THERMAL STABILITY of accelerating, adopt CCID50 method to detect sabin I type Polio virus titre value.
The present embodiment is manufactured a batch sample to obtain compositions of the present invention.
(1) sample is divided into 7 groups, and 10 doses every group, every dose containing following common component: 6.2LgCCID
50sabin I type poliovirus, 0.1M phosphate buffer (phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2), 0.2M sodium succinate solution, pH value is respectively between 5～8.7 groups of sample saccharides are respectively 30% sucrose, 30% lactose, 30% dextran, 30% trehalose, 30% galactose, 30% Sorbitol, 30% mannitol.By this sample put 37 ℃ two weeks, detectable antigens content.
(2) result of the test the present embodiment sample preparation is respectively organized sampling respectively and is detected titre value after completing, and result is as shown in table 6.
The stability that contains the sample of different saccharides in table 6 formula
The present embodiment result shows: when in vaccine formulation, saccharide is selected from sucrose, lactose, dextran, trehalose, galactose, Sorbitol or mannitol, this vaccine formulation all can make antigen have good stability at 37 ℃.Visible sucrose, lactose, dextran, trehalose, galactose, Sorbitol or mannitol are all applicable to formula of the present invention.
Embodiment 7 is used the stability of the poliovirus vaccine preparation of this formula
The present embodiment, at different temperature 2-8 ℃, 25 ℃ and 37 ℃, is measured the stability of different serotypes poliovirus in the present invention's formula.
Each serotype poliovirus stock solution of results in embodiment 1 is prepared to the vaccine sample of the present embodiment according to the method for embodiment 2, choose the oral OPV (purchased from China Medical Sciences Academy Medical Biology Institute) of commercially available sugar pill preparation as a control group.This sample is placed 18 weeks, 37 ℃ at 25 ℃ respectively subsequently and is placed 2 weeks, for the stability study accelerating; For real-time stability study, sample is placed 12 months at 2-8 ℃; Regularly adopt fluorescence quantifying PCR method to detect each serotype poliovirus titre value.
The present embodiment is manufactured a batch sample to obtain compositions of the present invention.
(1) sample 1 every dose containing following compositions: 30% sucrose, 0.1M phosphate buffer (phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2), 0.2M sodium succinate solution, unit price Polio virus antigen (sabin I type 8.8LgCCID
50, sabin II type 7.5LgCCID
50, sabin III type 8.2LgCCID50); Matched group is the oral OPV of commercially available sugar pill preparation, contains after testing Polio virus antigen (sabin I type 6.1LgCCID50, sabin II type 5.9LgCCID
50, sabin III type 6.1LgCCID
50), this sample is put respectively to 2-8 ℃, 25 ℃ and 37 ℃ of certain hours, detect respectively three type antigenic contents.
Result: 2-8 ℃ of placement of this sample 12 months, virus titer value in sample 1: sabin I type 8.7LgCCID
50, sabin II type 7.2LgCCID
50, sabin III type 8.0LgCCID
50, matched group virus titer value: sabin I type 5.7LgCCID
50, sabin II type 5.5LgCCID
50, sabin III type 5.6LgCCID
50, three type Polio virus titres are without obvious downward trend.As shown in Figure 1.
This sample 1 is placed 18 weeks at 25 ℃, virus titer value: sabin I type 8.5LgCCID
50, sabin II type 7.0LgCCID
50, sabin III type 7.9LgCCID
50, matched group can't check titre when placing 18 weeks for 25 ℃.Three type Polio virus titres of sample 1 decline not higher than 0.5Lg CCID
50, as shown in Figure 2, the data of matched group do not show in the drawings.
37 ℃ of placements of this sample 2 weeks, virus titer value: sabin I type 8.5LgCCID
50, sabinII type 7.1LgCCID
50, sabin III type 7.8LgCCID
50, matched group can't check titre when placing 2 weeks for 37 ℃.Three type Polio virus titres of sample 1 decline not higher than 0.5LgCCID
50.As shown in Figure 3, the data of matched group do not show in the drawings.
(2) sample 2 every dose containing following compositions: 10% sucrose, 0.05M phosphate buffer (phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2), 0.05M sodium succinate solution, unit price Polio virus antigen (sabin I type 8.8LgCCID
50, sabin II type 7.5LgCCID
50, sabin III type 8.2LgCCID
50).This sample 2-8 ℃ 12 months, 25 ℃ 18 weeks and 37 ℃ after 2 weeks in every dose each serotype virus titer fall all not higher than 0.5LgCCID
50, matched group is the oral OPV of commercially available sugar pill preparation, this sample 2-8 ℃ after 12 months in every dose each serotype virus titer fall all not higher than 0.5LgCCID
50, but 25 ℃ of 18 weeks and 37 ℃ 2 weeks afterwards titres examine and do not measure.
(3) sample 3 every dose containing following compositions: 60% sucrose, 0.5M phosphate buffer (phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2), 0.5M sodium succinate solution, unit price Polio virus antigen (sabin I type 8.8LgCCID
50, sabin II type 7.5LgCCID
50, sabin III type 8.2LgCCID
50).This sample 2-8 ℃ 12 months, 25 ℃ 18 weeks and 37 ℃ after 2 weeks in every dose each serotype virus titer fall all not higher than 0.3LgCCID
50.Matched group is the oral OPV of commercially available sugar pill preparation, this sample 2-8 ℃ after 12 months in every dose each serotype virus titer fall all not higher than 0.5LgCCID
50, but 25 ℃ of 18 weeks and 37 ℃ 2 weeks afterwards titres examine and do not measure.The present embodiment result shows: be of the present inventionly suitable for oral poliovirus vaccine and have good stability, and have more excellent stability than commercially available poliomyelitis sugar pill.
The animal safety test of embodiment 8 vaccine formulations
Vaccine sample in embodiment 7 is carried out in rabbit, mice and Cavia porcellus to safety evaluatio by Orally administered mode.Above-mentioned laboratory animal is respectively purchased from Chinese military medicine academy of science Experimental Animal Center, Beijing Vital River Experimental Animals Technology Co., Ltd..) specification is: the BalB/C mice of the rabbit of 1.5～2.5kg body weight, 18～22g body weight and the Cavia porcellus of 300～400g body weight, male and female half and half.
Get vaccine sample 1,2,3 in embodiment 7 and carry out respectively the present embodiment test, by 3 immunize rabbits of sample, BalB/C mice and Cavia porcellus, just exempt from and immune interval 2 weeks for the second time, immune interval is 1 week for the third time.5 of rabbits, 10 of BalB/C mices, 5 of Cavia porcelluss are used in test.From just exempt to during off-test every day observed and recorded test animal physiological status, comprise amount of survival, the mental status, appetite, feces, body weight, 3 exempt from, after 1 week, it is carried out to anatomic observation organ disease situation.
The present embodiment result of the test shows: it is all qualified aspect undue toxicity and acute toxicity that the vaccine sample three in embodiment 7 is exempted from after rabbit, BalB/c mice and Cavia porcellus, there is not test animal death and organ disease situation, rabbit, BalB/C mice and the Cavia porcellus mental status are good, appetite and feces are normal, Normal-weight rises, without any abnormal.Illustrate and be of the present inventionly suitable for oral Rotavirus Vaccine and there is good safety.
The animal immune originality experiment of embodiment 9 poliovirus vaccines
By to SD rat (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., SD rat specification: 150～170g, male and female half and half.) immune poliomyelitis vaccine assesses the immunogenicity of vaccine.
Rat carries out oral immunity according to the specification of 10 every group.Vaccine carries out 3 times of doubling dilutions according to people with dosage, is made into altogether 5 groups of vaccines.Form 7 has been described experiment grouping situation:
Table 7 rat immunity originality experiment grouping and immunizing dose situation
Within after Oral Administration in Rats immunity the 3rd week, strengthen once, after immunity, within the 2nd week, take a blood sample, with viral residual titration experiment, detect NAT.Neutralization is selected Sabin I, II and III type by virus, and various titre is 100CCID
50.Positive control cell is selected Vero cell.Neutralization test carries out 7 days at 36 ℃, and observation of cell pathological changes.Result shows respectively organizes the neutralization that rat blood serum all has couple sabin I, II and III type, illustrates that this vaccine has good immunogenicity on rat.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (2)
1. be suitable for an oral poliovirus vaccine, it is characterized in that, contain poliovirus antigen, saccharide, phosphate and carboxylate;
In this vaccine of every 1mL, contain 10
4～10
8the poliovirus antigen of individual infectious unit;
The saccharide that contains 0.1～0.6 g in this vaccine of every 1mL;
The phosphate that contains 0.05～0.5mmol in this vaccine of every 1mL;
The carboxylate that contains 0.05～0.5mmol in this vaccine of every 1mL;
Described vaccine pH value is 5-8;
Described phosphate is the mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or the mixture of sodium dihydrogen phosphate and sodium hydrogen phosphate;
In phosphate mixt, the molar concentration rate of dihydric phosphate and phosphoric acid hydrogen disalt is 1:1～10;
Described carboxylate is selected from citrate and succinate;
Described saccharide is sucrose, lactose, dextran, trehalose, galactose, Sorbitol and/or mannitol.
2. the method for preparing poliovirus vaccine described in claim 1, it is characterized in that, under aseptic condition, sugar juice, phosphate buffer and carboxylic acid salt solution are mixed to rear filtration sterilization, add poliovirus cell culture fluid to obtain mixed solution, according to specification subpackage; Described phosphate buffered solution is the mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or the mixed solution of sodium dihydrogen phosphate and sodium hydrogen phosphate; Described carboxylate is sodium succinate solution or sodium citrate solution.
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| CN101300028A (en) * | 2005-02-17 | 2008-11-05 | 葛兰素史密丝克莱恩生物有限公司 | Live attenuated rotavirus vaccine for oral administration |
| CN102876707A (en) * | 2012-10-15 | 2013-01-16 | 北京工业大学 | Preparation method of recombinant poliovirus I-type virus-like particles |
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| CN101300028A (en) * | 2005-02-17 | 2008-11-05 | 葛兰素史密丝克莱恩生物有限公司 | Live attenuated rotavirus vaccine for oral administration |
| CN102876707A (en) * | 2012-10-15 | 2013-01-16 | 北京工业大学 | Preparation method of recombinant poliovirus I-type virus-like particles |
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