CN103316336B - Rotavirus vaccine for oral administration - Google Patents
Rotavirus vaccine for oral administration Download PDFInfo
- Publication number
- CN103316336B CN103316336B CN201310215042.2A CN201310215042A CN103316336B CN 103316336 B CN103316336 B CN 103316336B CN 201310215042 A CN201310215042 A CN 201310215042A CN 103316336 B CN103316336 B CN 103316336B
- Authority
- CN
- China
- Prior art keywords
- vaccine
- strain
- rotavirus
- phosphate
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention provides a rotavirus vaccine for oral administration. The rotavirus vaccine contains rotavirus antigen, carbohydrate, phosphate and carboxylate. The rotavirus vaccine for oral administration can be adopted to raise stability of vaccine antigenic components, has good stability at 37 DEG C, 2-8 DEG C and 25 DEG C, has stable antigen activity, can enhance stomach acid resistance after oral administration of the vaccine finished product, and has good immune response stimulating capability and good safety. The rotavirus vaccine contains no other humanized or animal-based protein, has better safety, requires lower 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 Rotavirus Vaccine formula.
Background technology
Rotavirus (Rotavirus, RV) be the Etiological causing infant severe diarrhea and dehydration, all an important public health problem in developing country and developed country, the child in whole world <5 year every year nearly 600, the death of about 000, the dead child of 85% is in developing country.Rotavirus diarrhea not only causes the Disease Spectrum of society, and brings huge economic loss.Even if in developed country, rotavirus infection in infants also right and wrong is usually shown in.The research of the U.S. finds that the annual medical expense relevant for rotavirus infection is up to 3.52 ~ 1,000,000,000 dollars.Implementation plan immunity can save 0.79 hundred million dollar for health care, and 4.66 hundred million dollars for other aspects of society.The investigation display of China mainland: in the area that economic level is different, within less than 5 years old, each required expense of rotavirus diarrhea infant outpatient clinic is 64 ~ 248 yuan, the each required expense of hospitalization is then 658 ~ 1920 yuan, the average out-patient treatment expense 100 yuan of national rotavirus diarrhea according to a preliminary estimate, average hospitalization charge is about 839 yuan.
Up to the present, rotavirus infection is without specific treatment medicine, and the main fluid infusion relying on continuous several days is treated.More seriously, infection and the sanitary condition of rotavirus do not have dependency, even if sanitary condition improves, still effectively cannot control the propagation of virus.There is no specific drug treatment for RV diarrhoea at present, therefore apply vaccine prevention and be considered to the best preventive measure, vaccine immunity is the prevention rotavirus diarrhea higher incidence of unique feasible and the method for mortality rate.In view of this, the exploitation of Rotavirus Vaccine was classified as the first project of global novel vaccine development by World Health Organization (WHO) in 1997, the appropriate authority of countries in the world is all in the development work of actively carrying out Rotavirus Vaccine.
Orally administered is a kind of important vaccination mode, and the immunization ways of relative injection formula has the advantage of its uniqueness.To the microbial disease of some gastrointestinal tract cause of disease, adopt oral mode immunoprophylaxis can more fast, direct stimulating gastrointestinal road mucomembranous immune system, thus produce corresponding immunne response and make body have immunoprotection.Orally administered vaccine owing to need stand the digestive system of stomach, therefore has special requirement to the stability of its formula components, acid-resisting and safety aspect.First, vaccine formulation must ensure 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 ensure 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 reported at present (see, such as US3783098, US3915794 and EP028563), the first is SPGA, its serum albumin, caseinhydrolysate or gelatin mainly containing cattle or people; The second is the alkali metal salt of optional glutamic acid, sugar (glucose, sucrose or glucosan) and mono phosphoric acid ester salt or two salt, or their mixture.These compositionss have very large defect.First they have potential biological risk, the protein in the uncertain animal or human source of such as chemical composition or protein hydrolysate.In addition, the art these compositionss existing fail to make attenuated live vaccine have enough stability and acid-resisting, make the storage requirement of vaccine and effect phase be subject to great restriction, affect effectiveness and the safety of vaccine even further.At present there are no the report being suitable for oral, that stability is strong Rotavirus Vaccine.
Summary of the invention
The object of the present invention is to provide a kind of safe and effective applicable Orally administered Rotavirus Vaccine formula.
Another object of the present invention is to provide a kind of method preparing this vaccine.
Provided by the inventionly be suitable for oral Rotavirus Vaccine, containing wheel virus antigen, saccharide, phosphate and carboxylate.
Vaccine of the present invention, its every 1mL is containing 10
4~ 10
8the wheel virus antigen of individual infectious unit.Described infectious unit is CCID
50pFU or FFU or other can represent the unit with infectious antigen.
Saccharide containing 0.1 ~ 0.6g in this vaccine of every 1mL.
Phosphate radical containing 0.05 ~ 0.5mmol in this vaccine of every 1mL.
Carboxylate containing 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.
Phosphate contained by 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 1:1 ~ 10.The two molar concentration rate preferred is 1:2.
Described carboxylate has good anti-gastric acid ability, keeps the stable of antigenic component and effectiveness.The preferred carboxylate of the present invention is succinate or citrate.
Oral Rotavirus vaccine of the present invention, its pH value is 5-8.
Preferably, the oral vaccine formula possessing 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;
● wheel virus antigen 10
5~ 10
7cCID
50;
● pH value controls in pH5 ~ 8;
Present invention also offers the method that preparation is suitable for oral Rotavirus Vaccine, under aseptic condition, sugar juice, phosphate buffer and carboxylic acid salt solution are mixed rear filtration sterilization, adds rotavirus cell culture fluid and obtain mixed solution, make in every 1ml mixed solution containing 10
4~ 10
8the wheel virus antigen of individual infectious unit, the sugar of 0.1 ~ 0.6g, the phosphate radical of 0.05 ~ 0.5mmol, the carboxylate of 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, and the molar concentration rate of dihydric phosphate and phosphoric acid hydrogen disalt is 1:1 ~ 10; Described carboxylate is sodium citrate solution or sodium succinate solution.
The present invention can improve vaccine antigen composition stability, strengthen vaccine finished product oral enter anti-gastric acid ability after stomach, and this formula has good immunne response stimulates ability and good safety.External antiacid test card is bright, and this formula has good acid-resisting.During the immunogenic composition using this formula can be good at and gastric acid, the effectiveness of antigenic component and activity are protected.Stability test result shows the vaccine using this formula, has good stability at 37 DEG C, 2 ~ 8 DEG C, 25 DEG C, and antigen active is stablized.Safety testing shows, the vaccine using this formula has good safety in animal experiment.
As the saccharide of one of component in formula of the present invention, 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 in this vaccine combination between incorporated excipient.Hydrogen bond between the protein and sugar of epitope has the advantage making vaccine stable.Sugar effectively contacts with antigen protein must suitable ratio, to keep the stable of this vaccine.At a certain temperature, require that sugared concentration has a marginal value, the specific time can be stablized with the hydrogen bond between the protein and sugar with some to keep vaccine.Sugar, by alternative structure water, can form hydrogen bond with immobilized artificial membrane and protein.Sugar and protein and buffer composition appropriately combined, can within the long storage life preservation viral, and make it have activity.These additives give structural support to the antigen floated on a liquid.
As the phosphate buffer of one of component in formula of the present invention, its hydrogen phosphate contained 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.
As the carboxylate of one of component in formula of the present invention, there is good capacity antacid, the digestive system that antigenic component stands gastric juice can be maintained, stable antigen, maintain the effectiveness of antigenic component.
Except above-mentioned advantage, the formula that the present invention and prior art are mentioned 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 have better stability and buffer capacity with single 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 in acid-resisting and safety; 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 being suitable for product is produced.
Accompanying drawing explanation
Fig. 1 is the stability data that Rotavirus Vaccine of the present invention stores at 2-8 DEG C.
Fig. 2 is the stability data that Rotavirus Vaccine of the present invention stores at 25 DEG C.
Fig. 3 is the stability data that Rotavirus Vaccine of the present invention stores at 37 DEG C.
Detailed description of the invention
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 amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art; Reagent used in embodiment is commercial goods.
The preparation of embodiment 1 wheel virus antigen
By NIH (National Institute of Health, hereinafter referred to as NIH) be supplied to Strain UK x D strain (G1) of Xing Zhongwei Bioisystech Co., Ltd of Beijing section as vaccine raw starting material strain, UK x DS-1 strain (G2), UK x P strain (G3), UK x ST-3 strain (G4), UK x AU32 strain (G9) and UK x Wa strain (P1A [8]), Adaptable growth the continuous passage in Vero cell or MA-104 cell or diploid cell respectively of above strain, put in 37.5 ± 1.0 DEG C of incubators and cultivate 2 ~ 5 days results virus liquids, CCID is adopted after clarification filtration
50method or fluorescence quantifying PCR method detect virus titer value.Above-mentioned 6 kinds of serotype rotavirus maximum harvest yields can reach 10
6-10
8cCID
50/ ml.Each serotype rotavirus stock solution after purification can be used for preparing bacterin preparation.
The preparation of embodiment 2 Rotavirus Vaccine
(1) prepare 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 3M, filtration sterilization, 2 ~ 8 DEG C save backup;
(2) 10ml phosphate buffer step (1) prepared and the mixing of 15ml sodium succinate solution;
(3) in (2), 60% sucrose solution 150ml is added;
(4) in (3), add the antigenic solution in 100ml embodiment 1, this antigenic solution is the mixed liquor of G1, G2, G3, G4, G9, P1A [8];
(5) the serum-free MEM culture fluid not containing virus is adopted not supplement volume to 300ml; Vaccine, pH value is 5-8;
(6) subpackage is filtered, every agent 3ml, totally 100 doses.
The every agent of vaccine of the present embodiment preparation is containing following composition: containing above-mentioned 6 kinds of serotype rotavirus each 10
5-10
7cCID
50, the sucrose of 30%, 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.
Embodiment 3 is containing the antiacid effect of Rotavirus Vaccine of different sucrose formula
(1) preparation of bacterin preparation
Prepare 4 groups of vaccines containing different sucrose: prepare the phosphate buffer (potassium dihydrogen phosphate and dipotassium hydrogen phosphate molar concentration ratio are 1:2) of 4 groups of 10ml3M and the mixed liquor of 15ml4M sodium succinate solution, add respectively containing 30g in 4 groups of mixed liquors, 90g, 150g, the sucrose solution of 180g sucrose is mixed with 4 kinds of protective agents (phosphate solution+sodium succinate solution+sucrose solution) containing variable concentrations sucrose, respectively to the 100ml antigen component added in 4 groups of protective agents in embodiment 1 (with embodiment 2, be 6 kinds of serotype mixing), adopt the serum-free MEM maintenance medium polishing volume not containing virus to 300ml, filtration sterilization, by the every agent subpackage of 3ml, be mixed with 100 vaccinating agent preparations.In above-mentioned bacterin preparation, every agent is containing each serotype rotavirus 10
5~ 10
7cCID
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; In 4 groups of bacterin preparations, the quality final concentration (quality g/ volume 100ml) of sucrose is respectively 10%, 30%, 50%, 60%.
(2) the antiacid effect test of formula
4 groups of preparations containing different sucrose in (1) are carried out antiacid effect test respectively.Prepare the hydrochloric acid solution of the different pH value (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0) of 7 groups of 30ml, be " simulated gastric fluid ".The bacterin preparation of 4 groups of different sucrose is added respectively above 7 groups of " simulated gastric fluid " antiacid tests, 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, in equal energy and " simulated gastric fluid " of 30ml pH1.8, and result of the test in 2 hours without significant change (another note: the present invention also finds that rotavirus is not less than in the solution of 4.0 at pH can maintain activity more than 5 hours).The present embodiment result shows: illustrate that the sucrose quality final concentration of 10%-60% is all applicable to formula of the present invention.
The determination of phosphate buffering liquid concentration in embodiment 4 vaccine formulation
(1) preparation of bacterin preparation
Prepare 8 groups of vaccines containing different phosphate concn: by 5,10,30, (wherein potassium phosphate is 4 components, and potassium dihydrogen phosphate and dipotassium hydrogen phosphate molar concentration ratio are 1:2 for the 3M phosphate buffer of 50ml; Sodium ascorbyl phosphate 4 component; sodium dihydrogen phosphate and sodium hydrogen phosphate molar concentration ratio are 1:2) respectively with the mixing of 15ml4M sodium succinate solution; the sucrose solution adding the sucrose of 150ml60% in above-mentioned 8 groups of mixed liquors is respectively mixed with 8 kinds of protective agents containing different phosphate concn; respectively to the 100ml antigen component added in 8 groups of protective agents in embodiment 1 (with embodiment 2; be 6 kinds of serotype mixing); adopt the culture fluid polishing volume not containing virus to 300ml; filtration sterilization; by the every agent subpackage of 3ml, be mixed with 100 vaccinating agent preparations.Containing each serotype rotavirus 10 in above-mentioned bacterin preparation
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 formula
8 groups of preparations containing variable concentrations phosphate buffer in (1) are carried out antiacid effect test respectively.Prepare the hydrochloric acid solution of the different pH value (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0) of 7 groups of 30ml, be " simulated gastric fluid ".The bacterin preparation of 8 groups of (potassium salt and each 4 groups of sodium salt) different phosphate buffer densities is added above 7 groups of " simulated gastric fluid " antiacid tests respectively, 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 0.1M, two kinds of phosphate molarities ratios are arranged 4 different proportions, i.e. KH
2pO
4: K
2hPO
4=1:10,1:5,1:2,1:1, detect its antiacid effect respectively.
Result of the test shows; Adopt the bacterin preparation of above-mentioned 4 kinds of phosphate ratio in antiacid effect, " simulated gastric fluid " pH value of 30ml all can be made to be buffered to more than 5.0 and maintain 2 hours and 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, with " simulated gastric fluid " of 30ml PH1.8 in equal energy, and result of the test is stable in 2 hours; And test finds, in phosphate buffer, dihydric phosphate and phosphoric acid hydrogen disalt molar concentration ratio are that 1:10-1:1 all can meet formulation requirements.Result of the test all meets the requirements, and is suitable for formula of the present invention.
The determination of carboxylate salt concentration in embodiment 5 vaccine formulation
(1) preparation of bacterin preparation
Prepare 4 groups of vaccines containing 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 buffer (potassium dihydrogen phosphate and dipotassium hydrogen phosphate molar concentration ratio are 1:2), the sucrose solution adding the sucrose of 150ml60% in above-mentioned 4 groups of mixed liquors is respectively mixed with 4 kinds of protective agents containing different sodium succinate concentration, respectively to the 100ml antigen component added in 4 groups of protective agents in embodiment 1 (with embodiment 2, be 6 kinds of serotype mixing), adopt the MEM culture fluid polishing volume not containing virus to 300ml, filtration sterilization, by the every agent subpackage of 3ml, be mixed with 100 vaccinating agent preparations.Containing each serotype rotavirus 10 in above-mentioned bacterin preparation
5-10
7cCID
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 formula
4 groups of preparations containing different sodium succinate concentration in (1) are carried out antiacid effect test respectively.Prepare the hydrochloric acid solution of the different pH value (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0) of 7 groups of 30ml, be " simulated gastric fluid ".The bacterin preparation of 4 groups of different carboxylic acids salinity is added respectively above 7 groups of " simulated gastric fluid " antiacid tests, 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 the formula of different sodium succinate concentration
Table 5 is containing the antiacid effect of the formula of different sodium citrate concentration
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 " simulated gastric fluid " of 30ml pH1.8, meet viral survival requirement, and result of the test in 2 hours without significant change.Illustrate that succinate or the citric acid salt concentration of 0.05M-0.5M are all applicable to formula of the present invention.
The selection of saccharide in embodiment 6 formula
The following example is the stability in order to formula of the present invention is better described, but the scope of using of this formula is not limited only to following citing embodiment.
The present embodiment checking containing the formula of different saccharide at 37 DEG C of temperature to the stability of antigen.
The G1 serotype rotavirus stock solution of results in embodiment 1 is used for preparing the vaccine sample of the present embodiment, and method is with reference to embodiment 2.Place 2 weeks in 37 DEG C after sample preparation, adopt CCID for the THERMAL STABILITY accelerated
50method detects G1 serotype rotavirus titre value.
The present embodiment manufactures a batch sample to obtain compositions of the present invention.
(1) sample is divided into 7 groups, often organizes 10 doses, and every agent is containing following common component: 6.2LgCCID
50g1 serotype rotavirus, 0.1M phosphate buffer (phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2), 0.2M sodium succinate solution.7 groups of sample saccharides are respectively 30% sucrose, 30% lactose, 30% dextran, 30% trehalose, 30% galactose, 30% Sorbitol, 30% mannitol.
This sample is put 37 DEG C two weeks, detectable antigens content.
(2) result of the test
After the present embodiment sample preparation completes, each group samples detection titre value respectively, and result is as shown in table 6.
Containing the stability of the sample of different saccharide in table 6 formula
The present embodiment result shows: when saccharide in vaccine formulation is selected from sucrose, lactose, dextran, trehalose, galactose, Sorbitol or mannitol, and at 37 DEG C, this vaccine formulation all can make antigen have good stability.Visible sucrose, lactose, dextran, trehalose, galactose, Sorbitol or mannitol are all applicable to formula of the present invention.
Embodiment 7 uses the stability of the rotavirus vaccine formulations of this formula
The present embodiment, at different temperature 2-8 DEG C, 25 DEG C and 37 DEG C, measures the stability of different serotypes rotavirus in formula of the present invention.
Each serotype rotavirus stock solution of results in embodiment 1 is prepared the vaccine sample of the present embodiment according to the method for embodiment 2.This sample is placed 18 weeks, 37 DEG C at 25 DEG C respectively subsequently and is placed 2 weeks, for the stability study accelerated; For real-time stability study, sample is placed 12 months at 2-8 DEG C; Regular employing fluorescence quantifying PCR method detects each serotype rotavirus titre value.
The present embodiment manufactures a batch sample to obtain compositions of the present invention.
(1) the every agent of sample 1 is 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, wheel virus antigen (each serotype rotavirus content: G15.6LgCCID
50, G26.6LgCCID
50, G35.6LgCCID
50, G47.2LgCCID
50, G96.6LgCCID
50, P1A [8] 6.0LgCCID
50).This sample is put respectively 2-8 DEG C, 25 DEG C and 37 DEG C of certain hours, detect each serotype antigen content.
Result: 2-8 DEG C, this sample is placed 12 months, virus titer value: G15.5LgCCID
50, G26.5LgCCID
50, G35.6LgCCID
50, G47.2LgCCID
50, G96.4LgCCID
50, P1A [8] 6.0LgCCID
50, six kinds of serotype rotavirus titres are without obvious downward trend.As shown in Figure 1.
25 DEG C, this sample is placed 18 weeks, virus titer value: G15.5LgCCID
50, G26.4LgCCID
50, G35.4LgCCID
50, G47.0LgCCID
50, G96.3LgCCID
50, P1A [8] 5.8LgCCID
50, six kinds of serotype rotavirus titres decline not higher than 0.3Lg CCID
50.As shown in Figure 2.
37 DEG C, this sample is placed 2 weeks, virus titer value: G15.4LgCCID
50, G26.4LgCCID
50, G35.3LgCCID
50, G47.1LgCCID
50, G96.6LgCCID
50, P1A [8] 5.8LgCCID
50, six kinds of serotype rotavirus titres decline not higher than 0.3Lg CCID
50.As shown in Figure 3.
(2) the every agent of sample 2 is 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, each serotype wheel virus antigen 5-6LgCCID
50.This sample 2-8 DEG C 12 months, 25 DEG C 18 weeks and 37 DEG C after 2 weeks in every agent each serotype titre fall all not higher than 0.3LgCCID
50.
(3) the every agent of sample 3 is 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, each serotype wheel virus antigen 5-6LgCCID
50.This sample 2-8 DEG C 12 months, 25 DEG C 18 weeks and 37 DEG C after 2 weeks in every agent each serotype titre fall all not higher than 0.2LgCCID
50.The present embodiment result shows: be of the present inventionly suitable for oral Rotavirus Vaccine and have good stability.
The animal safety test of embodiment 8 vaccine formulation
Vaccine sample in embodiment 7 is carried out safety evaluatio by Orally administered mode in rabbit, mice and Cavia porcellus.
In Example 7, vaccine sample 1,2,3 carries out the present embodiment test respectively, by sample 3 immunize rabbits, BalB/C mice and Cavia porcellus, just exempts from and second time immunization interval 2 weeks, for the third time immunization interval 1 week.Rabbit 5 is used in test, BalB/C mice 10, Cavia porcellus 5.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 to carry out anatomic observation organ disease situation to it after 1 week.
The present embodiment result of the test shows: the vaccine sample three in embodiment 7 is all qualified in undue toxicity and acute toxicity after exempting from 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 normal, Normal-weight rises, without any exception.Illustrate and be of the present inventionly suitable for oral Rotavirus Vaccine there is good safety.
The animal immune originality experiment of embodiment 9 Rotavirus Vaccine
By assessing the immunogenicity of vaccine to Balb/c neonatal rat immunity Rotavirus Vaccine.Balb/c neonatal rat carries out oral immunity according to 10 specifications often organized.Vaccine carries out according to 1/3rd doubling dilutions of people's dosage, is made into 5 groups of vaccines altogether.Table 7 describes experiment grouping and immunizing dose situation:
The grouping of table 7 neonatal rat immunogenicity experiments and immunizing dose situation
The oral initial immunity of neonatal rat was strengthened once after 2 weeks, and booster immunization was taken a blood sample after 2 weeks, detected NAT with viral residual titration experiment.Neutralization virus selects G1, G2, G3, G4, G9 and P1A [8] type respectively, and various titre is 100CCID
50.Positive control cell selects Vero cell.Neutralization test carries out 7 days at 37 DEG C, and observation of cell pathological changes.Result shows each group of neonatal rat serum all neutralization to G1, G2, G3, G4, G9 and P1A [8] type rotavirus, illustrates that this vaccine has good immunogenicity on neonatal 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 without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (2)
1. be suitable for an oral Rotavirus Vaccine, it is characterized in that,
Every agent is containing following compositions: 30-60% sucrose, 0.1-0.5M phosphate buffer, 0.2-0.5M sodium succinate solution, wheel virus antigen;
Wherein, described wheel virus antigen is the mixture of following Strain: Strain UK x D strain, UK x DS-1 strain, UK x P strain, UK x ST-3 strain, UK x AU32 strain and UK x Wa strain; Adaptable growth the continuous passage in Vero cell or MA-104 cell or diploid cell respectively of described strain, put in 37.5 ± 1.0 DEG C of incubators and cultivate 2 ~ 5 days results virus liquids, adopt CCID50 method or fluorescence quantifying PCR method to detect virus titer value after clarification filtration, each serotype rotavirus stock solution after purification is for preparing bacterin preparation;
Wherein, in described phosphate buffer, phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2;
Containing 5-6 LgCCID in this vaccine of every agent
50each serotype wheel virus antigen or UK x DS-1 strain 6.6LgCCID
50or UK x ST-3 strain 7.2LgCCID
50or UK x AU32 strain 6.6LgCCID
50wheel virus antigen;
Described vaccine pH value is 5-8.
2. prepare the method for Rotavirus Vaccine described in claim 1, it is characterized in that, under aseptic condition, sugar juice, phosphate buffer and carboxylic acid salt solution are mixed rear filtration sterilization, add rotavirus cell culture fluid and obtain mixed solution, according to specification subpackage; Described sugar juice is sucrose solution; Described phosphate buffered solution is the mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate; Described carboxylate is sodium succinate solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310215042.2A CN103316336B (en) | 2013-05-31 | 2013-05-31 | Rotavirus vaccine for oral administration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310215042.2A CN103316336B (en) | 2013-05-31 | 2013-05-31 | Rotavirus vaccine for oral administration |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103316336A CN103316336A (en) | 2013-09-25 |
| CN103316336B true CN103316336B (en) | 2015-01-28 |
Family
ID=49185568
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310215042.2A Active CN103316336B (en) | 2013-05-31 | 2013-05-31 | Rotavirus vaccine for oral administration |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103316336B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047821B (en) * | 2016-05-17 | 2019-07-09 | 丽珠集团疫苗工程股份有限公司 | A method of utilizing bioreactor large-scale production Rotavirus Vaccine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0503337D0 (en) * | 2005-02-17 | 2005-03-23 | Glaxosmithkline Biolog Sa | Compositions |
| EP3427750A1 (en) * | 2009-05-12 | 2019-01-16 | The Government of The United States of America as represented by The Secretary, Department of Health and Human Services | New human rotavirus strains and vaccines |
-
2013
- 2013-05-31 CN CN201310215042.2A patent/CN103316336B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103316336A (en) | 2013-09-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Amlot et al. | Impaired human antibody response to the thymus-independent antigen, DNP-Ficoll, after splenectomy: implications for post-splenectomy infections | |
| CN103893751B (en) | A kind of pneumococal polysaccharide Protein Conjugation vaccine and preparation method thereof | |
| CN101300028B (en) | Live attenuated rotavirus vaccine for oral administration | |
| US5932223A (en) | Rotavirus vaccine formulations | |
| CN100379451C (en) | Vaccine | |
| CN107961367A (en) | Make the stabilized method of effect of LP2086 (fHBP) subtribe B polypeptides in immunogenic composition | |
| CA2266486C (en) | Rotavirus vaccine formulations | |
| CN101259269B (en) | Freeze-drying hepatitis A varicella attenuation combined vaccine and producing method thereof | |
| CN104998255B (en) | New A CYW135 group meningitis cocci combined vaccines and preparation method thereof | |
| CN106999569A (en) | Improved method, adjuvant absorption and its vaccine combination of the dosage reduction obtained of enterovirus inactivation | |
| CN105708829A (en) | Complex vitamin and amino acid oral liquid as well as preparation method and application thereof | |
| CN110227152A (en) | Rotavirus vaccine composition and preparation method thereof | |
| CN103316336B (en) | Rotavirus vaccine for oral administration | |
| RU2378014C2 (en) | Emulsion inactivated associated vaccine against cattle paragrippe-3, rednose and coronoviral infection | |
| Polish Typhoid Committee | Evaluation of typhoid vaccines in the laboratory and in a controlled field trial in Poland: Preliminary report | |
| CN101507816A (en) | Riemerlla anatipestifer bivalent inactivated vaccine and preparation method thereof | |
| CN101708332A (en) | Rabbit triple inactivated vaccine, preparation method and application thereof | |
| CN101618213B (en) | Combined attenuated live vaccine for measles, mumps and encephalitis B and preparation method thereof | |
| CN103316335B (en) | Poliovirus vaccine for oral administration | |
| RU2549434C2 (en) | Method for making brucellosis diagnostic serum | |
| Berlin et al. | Rhesus diploid rabies vaccine (adsorbed), a new rabies vaccine: results of initial clinical studies of preexposure vaccination | |
| CN102112153B (en) | Use of inactivated japanese encephalitis virus particle as adjuvant | |
| RU2378017C2 (en) | Emulsion inactivated associated vaccine against cattle rednose and coronoviral infection | |
| CN103751779A (en) | Poloxamer gel adjuvant for O-typed foot and mouth disease polypeptides | |
| CN103908662A (en) | Biodegradable high-efficiency dengue vaccine, preparation method thereof and pharmaceutical composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |