CN103316336A - Rotavirus vaccine for oral administration - Google Patents
Rotavirus vaccine for oral administration Download PDFInfo
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- CN103316336A CN103316336A CN2013102150422A CN201310215042A CN103316336A CN 103316336 A CN103316336 A CN 103316336A CN 2013102150422 A CN2013102150422 A CN 2013102150422A CN 201310215042 A CN201310215042 A CN 201310215042A CN 103316336 A CN103316336 A CN 103316336A
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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 prescription.
Background technology
Rotavirus (Rotavirus, RV) be the main diseases that causes the serious diarrhoea of infant and dehydration because of, all be an important public health problem in developing country and developed country, the child in the whole world＜5 years old annual nearly 600, death about 000,85% dead child is in developing country.Rotavirus diarrhea not only causes the disease burden of society, and has brought enormous economic loss.Even if in developed country, rotavirus infection in infants also right and wrong is usually seen.The U.S. discovers that the annual relevant medical expense of rotavirus infection that is used for is up to 3.52～1,000,000,000 dollars.The implementation plan immunity can be saved 0.79 hundred million dollar and is used for health care, and 4.66 hundred million dollars are used for other aspects of society.The investigation of China mainland shows: in the different area of economic level, following rotavirus diarrhea infant outpatient service in 5 years old each required expense of going to a doctor is 64～248 yuan, the each required expense of hospitalization then is 658～1920 yuan, the average out-patient treatment expense of national rotavirus diarrhea is 100 yuan according to a preliminary estimate, and the average time in hospital expense is about 839 yuan.
Up to the present, rotavirus infection does not have the specific treatment medicine, mainly relies on continuous several days fluid infusion and treats.More seriously, infection and the sanitary condition of rotavirus do not have dependency, even sanitary condition improves, still can't effectively control the propagation of virus.Still do not have the specific drug treatment for RV diarrhoea at present, therefore using vaccine prevention is considered to the best preventive measure, and vaccine immunity is unique feasible prevention rotavirus diarrhea higher incidence and the method for mortality rate.In view of this, World Health Organization (WHO) classified the exploitation of Rotavirus Vaccine as first project that global novel vaccine is developed in 1997, and 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 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, make body have immunoprotection thereby produce corresponding immunne response.Orally administered vaccine is owing to standing the digestive system of stomach, so stability, acid-resisting and the safety aspect of its formula components had special requirement.At first, vaccine formulation must guarantee the stability of antigenic component, guarantees that vaccine has maximum effectiveness in use.Secondly, as oral vaccine, prescription must have good anti-gastric acid ability, to guarantee the activity of antigen.Again, security of products is the basis, and security of products is from the selection of formula components.
The formula components of having reported at present at oral class vaccine (referring to, for example US3783098, US3915794 and EP028563), first kind is SPGA, it mainly contains cattle or people's serum albumin, caseinhydrolysate or gelatin; Second kind of alkali metal salt, sugar (glucose, sucrose or glucosan) and mono phosphoric acid ester salt or two salt for optional glutamic acid, or their mixture.These compositionss have very big defective.At 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 subjected to great restriction, even further influence effectiveness and the safety of vaccine.Do not see at present the report that is suitable for Rotavirus Vaccine oral, that stability is strong is arranged.
Summary of the invention
The object of the present invention is to provide a kind of safe and effective Orally administered Rotavirus Vaccine prescription that is fit 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 Rotavirus Vaccine, contain wheel virus antigen, saccharide, phosphate and carboxylate.
Vaccine of the present invention, its every 1mL contains 10
4～10
8The wheel virus 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.
The described saccharide of 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.
The molar concentration rate of dihydric phosphate and phosphoric acid hydrogen disalt is 1:1～10 in the above-mentioned phosphate mixt.Preferred the two molar concentration rate is 1:2.
Described carboxylate has anti-gastric acid ability preferably, keeps the stable and effectiveness of antigenic component.The preferred carboxylate of the present invention is succinate or citrate.
Oral Rotavirus Vaccine of the present invention, its pH value are 5-8.
Preferably, the oral vaccine prescription 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;
● the 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 is controlled in pH5～8;
The present invention also provides preparation to be suitable for the method for oral Rotavirus Vaccine, be under the aseptic condition, sugar juice, phosphate buffer and carboxylic acid salt solution are mixed the after-filtration degerming, add the rotavirus cell culture fluid and obtain mixed solution, make in every 1ml mixed solution to contain 10
4～10
8The phosphate radical of the wheel virus antigen of individual infectious unit, the sugar of 0.1～0.6g, 0.05～0.5mmol, the carboxylate of 0.05～0.5mmol, mixed solution pH value are 5～8, according to the specification packing; 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 the vaccine antigen composition stability, strengthen the oral anti-gastric acid ability of going into behind the stomach of vaccine finished product, and this prescription has good immunne response and stimulates ability and good safety.External antiacid test shows that this prescription has good acid-resisting.During the immunogenic composition of using this prescription can be good at and gastric acid, the effectiveness of antigenic component and activity are protected.The stability test result shows the vaccine of using this prescription, has good stability at 37 ℃, 2～8 ℃, 25 ℃, and antigen active is stable.Safety testing shows that the vaccine of using this prescription has good safety in animal experiment.
As the saccharide of one of component in the present invention's prescription, the sugar of certain concentration can improve the stability of bacterin preparation.There is great challenge in the preparation of stable vaccine preparation.The stability of said preparation depends on the interaction between the incorporated excipient in this vaccine combination.Protein and the hydrogen bond between the sugar of epitope have the advantage that makes vaccine stable.Sugar must have proper proportion with effective contact of antigen protein, to keep the stable of this vaccine.At a certain temperature, require sugared concentration that a marginal value is arranged, keep vaccine can stablize the specific time with protein and the hydrogen bond between the sugar with some.Sugar can form hydrogen bond with immobilized artificial membrane and protein by alternative structure water.The appropriate combination of sugar and protein and buffer composition can be preserved virus, and be made it have activity in the long storage life.These additives have given structural support to the antigen that floats on a liquid.
As the phosphate buffer of one of component in the present invention's prescription, 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.Simultaneously, phosphate buffer has certain effect to stability and the effectiveness tool of antigenic component.
Carboxylate as one of component in the present invention prescription has capacity antacid preferably, can keep the digestive system that antigenic component stands gastric juice, and stable antigen is kept the effectiveness of antigenic component.
Except above-mentioned advantage, the present invention compares with the prescription that prior art is mentioned also has following advantage: at first, phosphate of the present invention is the cocktail buffer of dihydric phosphate and phosphoric acid hydrogen disalt, compares with single kind phosphate to have better stability and buffering ability; 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, the sodium succinate effect is more excellent aspect acid-resisting and safety; The 3rd, prescription of the present invention does not contain other people source or animal-based protein except composition described in the claim, have better safety, and cost is lower simultaneously, is suitable for the industrialization production of product.
Description of drawings
Fig. 1 is that Rotavirus Vaccine of the present invention is 2-8 ℃ of stability of stored data.
Fig. 2 is that Rotavirus Vaccine of the present invention is 25 ℃ of stability of stored data.
Fig. 3 is that Rotavirus Vaccine of the present invention is 37 ℃ of stability of stored data.
The specific embodiment
Following examples further specify 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, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment; Used reagent is the commercial goods among the embodiment.
The preparation of embodiment 1 wheel virus antigen
By (the National Institute of Health of NIH, hereinafter to be referred as NIH) offer Xing Zhongwei Bioisystech Co., Ltd of Beijing section as the Strain UK x D strain (G1) of the initial raw material strain of vaccine, 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]), above strain adapts to growth and continuous passage respectively in Vero cell or MA-104 cell or diploid cell, put to cultivate in 37.5 ± 1.0 ℃ of incubators and gathered in the crops viral liquid in 2～5 days, adopt CCID behind the clarification filtration
50Method or fluorescence quantifying PCR method detect the 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 behind the purification can be used for preparing bacterin preparation.
The preparation of embodiment 2 Rotavirus 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, 2～8 ℃ of preservations are standby;
(2) 10ml phosphate buffer and the 15ml sodium succinate solution with step (1) preparation mixes;
(3) in (2), add 60% sucrose solution 150ml;
(4) add antigenic solution among the 100ml embodiment 1 in (3), this antigenic solution is G1, G2, G3, G4, G9, P1A[8] mixed liquor;
(5) adopt the serum-free MEM culture fluid that does not contain virus to replenish volume to 300ml; Vaccine, pH value are 5-8;
(6) filter packing, every dose of 3ml, totally 100 doses.
The vaccine of present embodiment preparation contains following composition for every dose: contain above-mentioned 6 kinds of serotype rotavirus each 10
5-10
7CCID
50, 30% sucrose, phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2 in the phosphate buffer, the phosphate radical total concentration is 0.1M; Sodium succinate solution final concentration is 0.2M.
Embodiment 3 contains the antiacid effect of Rotavirus Vaccine of different sucrose prescription
(1) preparation of bacterin preparation
Prepare 4 groups of vaccines that contain 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 in 4 groups of mixed liquors and contain 30g; 90g; 150g; the sucrose solution of 180g sucrose is mixed with 4 kinds of protective agents (phosphate solution+sodium succinate solution+sucrose solution) that contain variable concentrations sucrose; add 100ml antigen component among the embodiment 1 respectively in 4 groups of protective agents (with embodiment 2; be that 6 kinds of serotypes are mixed); adopt the serum-free MEM that does not contain virus to keep liquid polishing volume to 300ml; filtration sterilization; press every dose of packing of 3ml, be mixed with 100 vaccinating agent preparations.Every dose contains each serotype rotavirus 10 in the above-mentioned bacterin preparation
5～10
7CCID
50Phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2 in the phosphate buffer, and the phosphate radical total concentration is 0.1M; Sodium succinate solution final concentration is 0.2M; The quality final concentration (quality g/ volume 100ml) of sucrose is respectively 10%, 30%, 50%, 60% in 4 groups of bacterin preparations.
(2) the antiacid effect test of filling a prescription
4 groups of preparations that contain different sucrose in (1) are carried out antiacid effect test respectively.Prepare the hydrochloric acid solution of the different pH value of 7 groups of 30ml (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0), be " simulated gastric fluid ".The bacterin preparation of 4 groups of different sucrose is added above 7 groups of " simulated gastric fluid " antiacid tests respectively, and result of the test is as shown in table 1.
Table 1 contains the antiacid effect of the prescription of different sucrose
Result of the test shows, under the certain condition of phosphate buffer and carboxylate salt concentration, the antiacid effect that contains the prescription of 10%-60% different sucrose does not have significant difference, all in the energy and 30ml pH1.8 " simulated gastric fluid ", and result of the test did not have significant change (annotate in addition: the present invention finds that also rotavirus can keep active in 5 hours in pH is not less than 4.0 solution) in 2 hours.Present embodiment is the result show: the sucrose quality final concentration that 10%-60% is described all is fit to prescription of the present invention.
Phosphate buffering liquid concentration determines in embodiment 4 vaccine formulations
(1) preparation of bacterin preparation
Prepare 8 groups of vaccines that contain different phosphate concns: with 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 components; 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 that adds the sucrose of 150ml60% in above-mentioned 8 groups of mixed liquors respectively is mixed with 8 kinds of protective agents that contain different phosphate concns; add 100ml antigen component among the embodiment 1 respectively in 8 groups of protective agents (with embodiment 2; be that 6 kinds of serotypes are mixed); adopt the culture fluid polishing volume that does not contain virus to 300ml; filtration sterilization; press every dose of packing of 3ml, be mixed with 100 vaccinating agent preparations.Contain each serotype rotavirus 10 in the above-mentioned bacterin preparation
5-10
7CCID
50The sucrose mass concentration is 30%, and sodium succinate solution final concentration is 0.2M; Phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate in the phosphate buffer (or phosphoric acid sodium dihydrogen and sodium hydrogen phosphate) molar concentration ratio is 1:2, and the 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 that contain the variable concentrations phosphate buffer in (1) are carried out antiacid effect test respectively.Prepare the hydrochloric acid solution of the different pH value of 7 groups of 30ml (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0), be " simulated gastric fluid ".The bacterin preparation of 8 groups of (each 4 groups of potassium salt and sodium salt) different phosphate buffer densities is added above 7 groups of " simulated gastric fluid " antiacid tests respectively, and result of the test is shown in table 2,3.
Table 2 contains the antiacid effect of the prescription of variable concentrations phosphate buffer (potassium salt)
Table 3 contains the antiacid effect of the prescription of variable concentrations phosphate buffer (sodium salt)
(3) two kinds of antiacid effect tests of phosphate different proportion in the phosphate buffer
Adopt the method identical with above-mentioned test, phosphate radical total concentration in the phosphate buffer is set at 0.1M, two kinds of phosphate molar concentration rates 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 ratios on antiacid effect, all can make " simulated gastric fluid " pH value of 30ml be buffered to more than 5.0 and keep 2 hours stable.
Present embodiment is the result show: under the certain condition of sucrose mass concentration and carboxylate salt concentration, the antiacid effect that contains the prescription of 0.05M-0.5M variable concentrations phosphate buffer does not have significant difference, all in the energy and 30ml PH1.8 " simulated gastric fluid ", and result of the test is stable in 2 hours; And test finds that dihydric phosphate and phosphoric acid hydrogen disalt molar concentration ratio are that 1:10-1:1 all can satisfy the preparation demand in the phosphate buffer.Result of the test all meets the requirements, and is suitable for prescription of the present invention.
Carboxylate salt concentration determines in embodiment 5 vaccine formulations
(1) preparation of bacterin preparation
Prepare 4 groups of vaccines that contain different sodium succinate concentration: with 3.75ml; 7.5ml; 22.5ml; 37.5ml 4M sodium succinate solution respectively with the mixing of 10ml3M phosphate-buffered liquor (potassium dihydrogen phosphate and dipotassium hydrogen phosphate molar concentration ratio are 1:2); the sucrose solution that adds the sucrose of 150ml60% in above-mentioned 4 groups of mixed liquors respectively is mixed with 4 kinds of protective agents that contain different sodium succinate concentration; add 100ml antigen component among the embodiment 1 respectively in 4 groups of protective agents (with embodiment 2; be that 6 kinds of serotypes are mixed); adopt the MEM culture fluid polishing volume that does not contain virus to 300ml; filtration sterilization; press every dose of packing of 3ml, be mixed with 100 vaccinating agent preparations.Contain each serotype rotavirus 10 in the above-mentioned bacterin preparation
5-10
7CCID
50The sucrose mass concentration is 30%, and phosphoric acid potassium dihydrogen and dipotassium hydrogen phosphate molar concentration ratio are 1:2 in the phosphate buffer, and the 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 that contain different sodium succinate concentration in (1) are carried out antiacid effect test respectively.Prepare the hydrochloric acid solution of the different pH value of 7 groups of 30ml (pH=2.5,2.3,2.0,1.8,1.5,1.3,1.0), be " simulated gastric fluid ".The bacterin preparation of 4 groups of different carboxylic acids salinity is added above 7 groups of " simulated gastric fluid " antiacid tests respectively, and result of the test is as shown in table 4.
Adopt identical method, adopt sodium citrate to replace the sodium succinate in the prescription, carry out the present embodiment test, result of the test is as shown in table 5.
Table 4 contains the antiacid effect of the prescription of different sodium succinate concentration
Table 5 contains the antiacid effect of the prescription of different sodium citrate concentrations
Present embodiment is the result show: under the certain condition of sucrose mass concentration and phosphate buffer density, contain in the equal energy of antiacid effect of prescription of 0.05M-0.5M different carboxylic acids salinity and 30ml pH1.8 " simulated gastric fluid ", satisfy viral survival condition demand, and result of the test there was not significant change in 2 hours.Succinate or citric acid salt concentration that 0.05M-0.5M is described all are fit to prescription of the present invention.
The selection of saccharide in embodiment 6 prescriptions
The following example is the stability for better explanation prescription of the present invention, but the scope of using of this prescription is not limited only to following embodiment for example.
The prescription that present embodiment checking contains different saccharides under 37 ℃ of temperature to the stability of antigen.
The G1 serotype rotavirus stock solution of results among the embodiment 1 is used for preparing the vaccine sample of present embodiment, and method is with reference to embodiment 2.In 37 ℃ of 2 weeks of placement, the THERMAL STABILITY that is used for accelerating adopts CCID after the sample preparation
50Method detects G1 serotype rotavirus titre value.
Present embodiment is made a batch sample to obtain compositions of the present invention.
(1) sample is divided into 7 groups, and 10 doses every group, every dose contains 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 ℃ of two week, detect antigenic content.
(2) result of the test
Each group was taken a sample respectively and is detected the titre value after the preparation of present embodiment sample was finished, and the result is as shown in table 6.
The stability of sample that contains different saccharides in table 6 prescription
Present embodiment is the result show: when saccharide in the vaccine formulation is selected from sucrose, lactose, dextran, trehalose, galactose, Sorbitol or mannitol, 37 ℃ down these vaccine formulations all can make antigen have good stability.As seen sucrose, lactose, dextran, trehalose, galactose, Sorbitol or mannitol all are fit to prescription of the present invention.
Present embodiment is measured the stability of different serotypes rotavirus in the present invention's prescription under different temperature 2-8 ℃, 25 ℃ and 37 ℃.
The vaccine sample that each serotype rotavirus stock solution of results among the embodiment 1 is prepared present embodiment according to the method for embodiment 2.This sample is placed 18 weeks, 37 ℃ of 2 weeks of placement at 25 ℃ respectively subsequently, is used for the stability study that accelerates; For real-time stability study, sample was placed 12 months at 2-8 ℃; Regularly adopt fluorescence quantifying PCR method to detect each serotype rotavirus titre value.
Present embodiment is made a batch sample to obtain compositions of the present invention.
(1) sample 1 every dose contain 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] and 6.0LgCCID
50).This sample is put 2-8 ℃, 25 ℃ and 37 ℃ of certain hours respectively, detect each serotype antigen content.
The result: this sample was placed virus titer value: G15.5LgCCID 12 months for 2-8 ℃
50, G26.5LgCCID
50, G35.6LgCCID
50, G47.2LgCCID
50, G96.4LgCCID
50, P1A[8] and 6.0LgCCID
50, six kinds of serotype rotavirus titres do not have obvious downward trend.As shown in Figure 1.
This sample is placed 18 weeks, virus titer value: G15.5LgCCID for 25 ℃
50, G26.4LgCCID
50, G35.4LgCCID
50, G47.0LgCCID
50, G96.3LgCCID
50, P1A[8] and 5.8LgCCID
50, six kinds of serotype rotavirus titres descend and are not higher than 0.3Lg CCID
50As shown in Figure 2.
This sample is placed 2 weeks, virus titer value: G15.4LgCCID for 37 ℃
50, G26.4LgCCID
50, G35.3LgCCID
50, G47.1LgCCID
50, G96.6LgCCID
50, P1A[8] and 5.8LgCCID
50, six kinds of serotype rotavirus titres descend and are not higher than 0.3Lg CCID
50As shown in Figure 3.
(2) sample 2 every dose contain 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
50This sample in 2-8 ℃ 12 months, 25 ℃ 18 week and 37 ℃ of 2 week back in every dose each serotype virus titer fall all be not higher than 0.3LgCCID
50
(3) sample 3 every dose contain 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
50This sample in 2-8 ℃ 12 months, 25 ℃ 18 week and 37 ℃ of 2 week back in every dose each serotype virus titer fall all be not higher than 0.2LgCCID
50Present embodiment is the result show: of the present inventionly be suitable for oral Rotavirus Vaccine and have good stability.
The animal safety test of embodiment 8 vaccine formulations
Vaccine sample among the embodiment 7 is carried out safety evaluatio by Orally administered mode in rabbit, mice and Cavia porcellus.
Get among the embodiment 7 vaccine sample 1,2,3 and carry out the present embodiment test respectively, with 3 immunize rabbits of sample, BalB/C mice and Cavia porcellus, just exempt from and 2 weeks at interval of immunity for the second time, immunity for the third time is 1 week at interval.5 of rabbits, 10 of BalB/C mices, 5 of Cavia porcelluss are used in test.From just exempting from during the off-test observed and recorded test animal physiological status every day, comprise survival quantity, the mental status, appetite, feces, body weight, 3 exempt from 1 week the back it is carried out 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 among the embodiment 7 is exempted from behind rabbit, BalB/c mice and the Cavia porcellus, dead and the organ disease situation of test animal does not appear, rabbit, BalB/C mice and the Cavia porcellus mental status are good, appetite and feces are normal, body weight normally rises, and is no any unusual.Illustrate and of the present inventionly be suitable for oral Rotavirus Vaccine and have good safety.
The animal immune originality experiment of embodiment 9 Rotavirus Vaccines
By Balb/c neonatal rat immunity Rotavirus Vaccine being assessed the immunogenicity of vaccine.The Balb/c neonatal rat carries out oral immunity according to 10 every group specification.Vaccine carries out according to 1/3rd doubling dilutions of human dosage, is made into 5 groups of vaccines altogether.Table 7 has been described experiment grouping and immunizing dose situation:
The grouping of table 7 neonatal rat immunogenicity experiments and immunizing dose situation
Oral 2 week of initial immunity of neonatal rat, the back was strengthened once, and booster immunization was taken a blood sample after 2 weeks, detected NAT with viral residual titration experiment.Neutralization is selected G1, G2, G3, G4, G9 and P1A[8 respectively for use with virus] type, various titre is 100CCID
50Positive control cell is selected the Vero cell.Neutralization test carries out 7 days at 37 ℃, and the observation of cell pathological changes.The result show respectively organize neonatal rat serum couple G1, G2, G3, G4, G9 and P1A[8 all arranged] neutralization of type rotavirus, illustrate that this vaccine has good immunogenicity neonatal rat.
Though 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 (10)
1. one kind is suitable for oral Rotavirus Vaccine, it is characterized in that, contains wheel virus antigen, saccharide, phosphate and carboxylate.
2. Rotavirus Vaccine as claimed in claim 1 is characterized in that, contains 10 in this vaccine of every 1mL
4～10
8The wheel virus antigen of individual infectious unit.
3. Rotavirus Vaccine as claimed in claim 1 is characterized in that, contains the saccharide of 0.1～0.6g in this vaccine of every 1mL.
4. Rotavirus Vaccine as claimed in claim 1 is characterized in that, contains the phosphate of 0.05～0.5mmol in this vaccine of every 1mL.
5. Rotavirus Vaccine as claimed in claim 1 is characterized in that, contains the carboxylate of 0.05～0.5mmol in this vaccine of every 1mL.
6. as the arbitrary described Rotavirus Vaccine of claim 1～5, it is characterized in that described saccharide is sucrose, lactose, dextran, trehalose, galactose, Sorbitol and/or mannitol.
7. as the arbitrary described Rotavirus Vaccine of claim 1～5, it is characterized in that described phosphate is the mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or the mixture of sodium dihydrogen phosphate and sodium hydrogen phosphate.
8. Rotavirus Vaccine as claimed in claim 7 is characterized in that, the molar concentration rate of dihydric phosphate and phosphoric acid hydrogen disalt is 1:1～10 in the phosphate mixt.
9. as the arbitrary described Rotavirus Vaccine of claim 1～5, it is characterized in that its pH value is 5-8.
10. the method for preparing the arbitrary described Rotavirus Vaccine of claim 1～9, it is characterized in that, under the aseptic condition, sugar juice, phosphate buffer and carboxylic acid salt solution are mixed the after-filtration degerming, add the rotavirus cell culture fluid and obtain mixed solution, according to the specification packing; 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 citrate solution or sodium succinate solution.
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| CN106047821A (en) * | 2016-05-17 | 2016-10-26 | 丽珠集团疫苗工程股份有限公司 | Method for producing rotavirus vaccines in large scale by utilizing bioreactor |
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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 |
| CN102695522A (en) * | 2009-05-12 | 2012-09-26 | 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) | New human rotavirus strains and vaccines |
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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 |
| CN102695522A (en) * | 2009-05-12 | 2012-09-26 | 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) | New human rotavirus strains and vaccines |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047821A (en) * | 2016-05-17 | 2016-10-26 | 丽珠集团疫苗工程股份有限公司 | Method for producing rotavirus vaccines in large scale by utilizing bioreactor |
| 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 |
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