CN111053898B

CN111053898B - Vaccine composition and application thereof

Info

Publication number: CN111053898B
Application number: CN201911367533.2A
Authority: CN
Inventors: 王治伟; 范鑫; 徐树利; 隋晓斌; 吴慕胜; 李海涛; 高强; 尹卫东
Original assignee: Sinovac Research & Development Co ltd
Current assignee: Sinovac Research & Development Co ltd
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2023-05-16
Anticipated expiration: 2039-12-26
Also published as: CN111053898A

Abstract

The present invention relates to a liquid vaccine composition comprising diphtheria toxoid, tetanus toxoid, pertussis toxin, filaggrin, pertactin, inactivated type I poliovirus Sabin strain, inactivated type II poliovirus Sabin strain and inactivated type III poliovirus Sabin strain; wherein, the antigen proportion of Sabin strains I, II and III is (4-15): (11-45): (11-45). The vaccine composition provided by the invention can be used for basic immunity or booster immunity, and can improve the titer of antibodies in serum of an immunized person on the basis of ensuring the safety.

Description

Vaccine composition and application thereof

Technical Field

The invention relates to the field of vaccines, in particular to a vaccine composition capable of simultaneously preventing pertussis, diphtheria, tetanus and poliomyelitis.

Background

Timely vaccination is an effective preventive and protective measure. Pertussis, diphtheria and tetanus combined vaccines (DTP) have played an important role in the prevention and control of these three infectious diseases as the earliest vaccine to be incorporated into the WHO extended immunization program (EPI). In the 70 s of the 20 th century, serious adverse reactions appear after the whole cell pertussis vaccine (DTwP) is inoculated, and the phenomenon of resisting vaccination and stopping use appears in the countries such as Japan, the Netherlands and the like, so that the inoculation rate is reduced, and the incidence of pertussis is rebound. Currently, most developed countries produce cell-free pertussis vaccine (DTaP) with less side reaction by co-purification or chromatographic purification, as a basic combination vaccine and a conventional immunization vaccine. Acellular pertussis vaccines can be divided into co-purified vaccines and chromatographic purified vaccines from the point of view of production technology. At present, partial enterprises in China and Japan adopt a co-purification process, namely, after bacteria are cultured, protective antigens such as PT, FHA, PRN and the like are salted out and precipitated, then sucrose density gradient centrifugation is used for removing impurities, and meanwhile active ingredients rich in PT, FHA and PRN are collected. The chromatographic purification is to purify different protective antigens by column chromatography, and then quantitatively mix the antigens into vaccine. The separation and purification method has the advantages of high cost, definite components, easy quality control and less side effect. At present, most developed countries adopt column chromatography to separate and purify each component to produce acellular pertussis vaccines.

Poliomyelitis (WPV) is an acute infectious disease that is widely transmitted and extremely harmful caused by polioviruses I, II, III, and affects people of any age group, but mainly children under 3 years old (50% of all cases), severely causing irreversible paralysis of limbs and even death. Before the vaccine emerged, the disease was prevalent worldwide. In the middle and later 50 s, two scientists Salk and Sabin in the United states successively developed inactivated poliomyelitis vaccine (Inactivated Poliovirus Vaccine, IPV for short) and attenuated live oral poliomyelitis vaccine (Oral Poliovirus Vaccine, OPV for short). From this point on, there are powerful weapons to prevent and eliminate poliomyelitis, and both vaccines have been practically proven to be effective. Worldwide health organization (World Health Organization, WHO) initiated a worldwide initiative to eradicate poliomyelitis in 1988, after which the act of eradicating poliomyelitis has made significant progress in both reducing the incidence of poliomyelitis and reducing the spread of poliovirus. From 1 month 2007 to 6 months 2008, 16 countries detected wild strains (WPV) from stool specimens of AFP cases, only WPVI and WPVIII were detected, no WPVII was detected worldwide since 1999, and these advances were due to the poliomyelitis elimination strategy implemented jointly worldwide. In global poliomyelitis (WPV) eradication activities, oral live attenuated poliomyelitis vaccines (OPV) are widely used, and the incidence of global poliomyelitis has been greatly reduced. However, due to the risk of Vaccine-related paralytic poliomyelitis (Vaccine Associated Paralytic Poliomyelitis, VAPP), and Vaccine-derived poliovirus (Vaccine-derived Poliovirus, VDPV), the use of oral poliomyelitis attenuated live Vaccine OPV must be stopped in the future to eradicate poliomyelitis entirely. Only using polio inactivated vaccine IPV can VAPP and VDPV risk be avoided. The inactivated poliomyelitis vaccine can prevent poliomyelitis IPV outbreak and generate group immunity, and is the best choice for eliminating the subsequent stage of poliomyelitis. At present, manufacturers worldwide produce IPV, but all used production strains are wild strains. The requirement of biological safety level 3 (BSL-3) is met when the wild strain is used for producing IPV, and even the condition of BSL-4 is not absolutely ensured after poliomyelitis is eliminated worldwide. In the combined vaccine approved in China at present, the strain selected by the IPV vaccine is Salk strain, but Salk strain belongs to a strong wild strain and has stronger toxicity, so WHO encourages vaccine manufacturers to research and develop attenuated strain polio inactivated vaccine Sabin IPV (Inactivated Poliovirus Vaccine, sabin strain, sIPV for short).

At present, the development of the DTaP-sIPV combined vaccine with higher safety and stability has important significance.

Disclosure of Invention

The invention aims to provide a vaccine composition capable of simultaneously preventing pertussis, diphtheria, tetanus and poliomyelitis.

Specifically, the invention provides a liquid vaccine composition which comprises diphtheria toxoid, tetanus toxoid, pertussis toxin, filaggrin, pertussis adhesion, an inactivated type I poliovirus Sabin strain, an inactivated type II poliovirus Sabin strain and an inactivated type III poliovirus Sabin strain.

The invention also provides a liquid vaccine composition, the antigen of which consists of the following substances: diphtheria toxoid, tetanus toxoid, pertussis toxin, filiform hemagglutinin, pertactin, inactivated type I poliovirus Sabin strain, inactivated type II poliovirus Sabin strain, inactivated type III poliovirus Sabin strain.

According to the advantage of high safety of the Sabin strain, the composition of the invention has the advantages that the antigen ratio of the Sabin strain poliovirus of type I, type II and type III is preferably (4-15) in order to ensure the immunogenicity of the Sabin strain and avoid the interference effect of the Sabin strain with other antigens in the combined vaccine as much as possible: (11-45): (11-45).

As a specific preferred embodiment of the present invention, the liquid vaccine composition comprises the following components per 0.5 mL: 4-15 DU of type I Sabin strain poliovirus, 11-45 DU of type II Sabin strain poliovirus, and 11-45 DU of type III Sabin strain poliovirus.

The invention finds through experiments that when the antigen proportion of Sabin strains I, II and III in the liquid vaccine composition is 1: (2.5-3.5): (2.5-3.5), the liquid vaccine composition as a whole has an optimal immunization effect.

As a preferred embodiment of the present invention, the vaccine composition comprises the following components:

more preferably, the antigen ratio of the Sabin strains type I, type II and type III polioviruses is 1: (2.5-3.5): (2.5-3.5).

The vaccine composition provided by the invention can be used for basic immunity and can also be used for enhancing immunity.

The invention is found through a great deal of practice that when the proportion and the dosage of pertussis toxin, filiform hemagglutinin and pertactin contained in the composition are optimally adjusted, the vaccine composition can further meet the requirement of basic immunity or enhanced immunity on the basis of ensuring the safety.

When the liquid vaccine composition is used for preparing a basic immunity vaccine, the mass ratio of pertussis toxin, filiform hemagglutinin and pertactin contained in the composition is preferably (20-25): (20-25): (3-8).

As a preferred embodiment of the present invention, the vaccine composition is used for basic immunization, and each 0.5ml single dose of the composition contains 20-25 mug pertussis toxin, 20-25 mug filamentous hemagglutinin and 3-8 mug pertussis adhesion element.

As a preferred embodiment of the present invention, the vaccine composition is used for basic immunization, and 25. Mu.g of pertussis toxin, 25. Mu.g of filamentous hemagglutinin, and 8. Mu.g of pertussis adhesion element are contained in the composition per 0.5ml single dose.

As a preferred embodiment of the invention, the vaccine composition is for basic immunization, comprising the following components per 0.5ml single dose:

when the liquid vaccine composition is used for preparing the booster vaccine, the mass ratio of pertussis toxin, filiform hemagglutinin and pertactin contained in the composition is preferably (2.5-8): (5-8): (2.5-3).

As a preferred embodiment of the present invention, the vaccine composition is used for enhancing immunity, and each 0.5ml single dose of the composition contains 2.5-8 mug pertussis toxin, 5-8 mug filamentous hemagglutinin and 2.5-3 mug pertussis adhesion element.

As a preferred embodiment of the present invention, the vaccine composition is used for booster immunization, and the composition contains pertussis toxin 8. Mu.g, filamentous hemagglutinin 8. Mu.g, and pertactin 2.5. Mu.g per 0.5ml single dose.

As a preferred embodiment of the present invention, the liquid vaccine composition is used for booster immunization, and the composition contains the following components per 0.5ml single dose:

the vaccine composition provided by the invention can also comprise an M199 culture medium on the basis of containing the antigen stock solution. The invention has been found through practice that when M199 culture medium is added into the combined vaccine, the stability of poliovirus antigen is obviously improved, thereby having an important effect on the overall stability of the liquid vaccine composition. The amount of M199 medium is preferably 5 to 20% of 10X concentration of M199 medium added to the composition.

The vaccine composition provided by the invention can further comprise an adjuvant on the basis of the antigen stock solution. The adjuvant can be aluminum hydroxide and/or aluminum phosphate. According to the invention, when the aluminum content in the adjuvant is 0.3-1.25 mg/ml, preferably 0.4-0.8 mg/ml, the potency of each antigen can be marked with higher quality while the safety of the liquid vaccine composition is ensured.

The vaccine composition provided by the invention can also contain a soluble phosphate buffer solution on the basis of the antigen stock solution, and the soluble phosphate buffer solution is used for adjusting the pH value of the vaccine composition.

The vaccine composition provided by the invention can also contain a certain amount of sodium chloride or equivalent substances on the basis of the antigen stock solution so as to maintain osmotic pressure.

The invention also provides a method for preparing the vaccine composition.

As a preferred embodiment of the present invention, the method specifically includes the steps of: mixing liquid raw materials including pertussis toxin, filiform hemagglutinin, pertactin, diphtheria toxoid stock solution, tetanus toxoid stock solution, inactivated Sabin strain I poliovirus stock solution, inactivated Sabin strain II poliovirus stock solution and inactivated Sabin strain III poliovirus stock solution. Wherein the various raw materials are prepared in liquid form and then directly mixed.

After the liquid raw materials are mixed, substances such as an adjuvant and the like are further added, so that a stable liquid vaccine composition suitable for human body use can be obtained. Specifically, an aluminum salt adjuvant diluted with an aqueous sodium chloride solution may be further added, followed by adding a phosphate buffer solution, and adjusting the pH to 5.8 to 7.2.

The invention protects the application of the vaccine composition in preparing basic immunity vaccines.

The invention protects the application of the vaccine composition in preparing an immunity-enhancing vaccine.

The invention provides a vaccine kit, comprising a first chamber and a second chamber; the first chamber contains a basic immunity vaccine prepared from the vaccine composition; the second chamber contains a booster vaccine prepared from the vaccine composition described above.

The invention provides a vaccine kit, comprising a first chamber, a second chamber and a third chamber; each of the first and second chambers contains a basic immune vaccine prepared from the vaccine composition; the third chamber contains a booster vaccine prepared from the vaccine composition.

The invention provides a vaccine kit, which comprises a first chamber, a second chamber, a third chamber and a fourth chamber; each of the first chamber, the second chamber and the third chamber contains a basic immunity vaccine prepared from the vaccine composition; the fourth chamber contains a booster vaccine prepared from the vaccine composition described above.

When in actual inoculation, the vaccine kit provided by the invention can be used for efficiently completing the vaccination and avoiding wrong inoculation or missed inoculation. For example: the basic immunization is carried out by using the basic immunization vaccine in the first chamber (or the first chamber and the second chamber in sequence or the first chamber, the second chamber and the third chamber in sequence) in the kit, and then the booster immunization is carried out by using the booster immunization vaccine in the second chamber (or the third chamber or the fourth chamber) in the kit.

Compared with the prior art, the vaccine composition provided by the invention is a liquid preparation, and can maintain the stability of the property of the liquid preparation in various links of preparation, transportation, storage and use. Furthermore, the vaccine composition provided by the invention can be used for basic immunity or reinforcing immunity, and can improve the titer of antibodies in serum of an immunized person on the basis of ensuring the safety.

Drawings

FIG. 1 shows the results of type I, type II, type III neutralizing antibody titres for sIPV of example 10;

FIG. 2 is a graph showing the results of the pertussis (aP) antibody titer assay described in example 10; wherein aP-PT represents pertussis toxin, aP-FHA represents filiform hemagglutinin, and aP-PRN represents pertussis bacillus adhesin;

FIG. 3 shows the results of the antibody titre assay for Diphtheria (DT) and tetanus (TT) of the immunization program described in example 10.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example 1

The present example provides a liquid vaccine formulation comprising the following components per 1 human dose:

pertussis toxin 8 mug, filiform hemagglutinin 8 mug, pertussis bacillus adhension 2.5 mug, diphtheria toxoid 2Lf, tetanus toxoid 5Lf, inactivated poliovirus type I stock solution 4DU, inactivated poliovirus type II stock solution 11DU, inactivated poliovirus type III stock solution 11DU, aluminum content 0.6mg/ml and proper amount of sodium chloride aqueous solution.

The combined vaccine is mixed uniformly before use and then used for intramuscular injection.

The liquid vaccine formulation provided in this example can be used to boost immunity.

Example 2

pertussis toxin 8 mug, filiform hemagglutinin 8 mug, pertussis bacillus adhension 2.5 mug, diphtheria toxoid 2Lf, tetanus toxoid 5Lf, inactivated poliovirus type I stock solution 15DU, inactivated poliovirus type II stock solution 45DU, inactivated poliovirus type III stock solution 45DU, aluminum content 0.8mg/ml and proper amount of sodium chloride aqueous solution.

Example 3

25 mug of pertussis toxin, 25 mug of filiform hemagglutinin, 8 mug of pertussis bacillus adhension, 12.5Lf of diphtheria toxoid, 3.5Lf of tetanus toxoid, 15DU of inactivated poliovirus type I stock solution, 45DU of inactivated poliovirus type II stock solution, 45DU of inactivated poliovirus type III stock solution, 0.8mg/ml of aluminum content and a proper amount of sodium chloride aqueous solution.

The liquid vaccine preparation provided in this example can be used for basic immunization.

Example 4

25 mug of pertussis toxin, 25 mug of filiform hemagglutinin, 8 mug of pertussis bacillus adhension, 12.5Lf of diphtheria toxoid, 3.5Lf of tetanus toxoid, 7.5DU of inactivated poliovirus type I stock solution, 22.5DU of inactivated poliovirus type II stock solution, 22.5DU of inactivated poliovirus type III stock solution, 0.8mg/ml of aluminum content and a proper amount of sodium chloride aqueous solution.

Example 5

This example provides a method for preparing each monovalent vaccine stock solution referred to in examples 1-4.

1. Diphtheria toxoid stock solution

Starting diphtheria bacillus, transferring 1-3 generations into a seed tube of a toxigenic culture medium, transferring to the toxigenic culture medium to prepare seeds for production, culturing the seeds in a culture tank liquid, culturing the seeds in a maltose comprehensive culture medium at 34-36 ℃ for 45-52 hours, adding formaldehyde for sterilization after culturing, adding ammonium sulfate to precipitate hybrid protein into the culture, centrifugally collecting supernatant, ultrafiltering and concentrating, adding ammonium sulfate to precipitate diphtheria toxin protein, centrifugally collecting precipitate, adding PB buffer solution for dissolving, ultrafiltering to remove ammonium sulfate in toxin, adding formaldehyde, placing the mixture at 35-37 ℃ for detoxification for 30 days, ultrafiltering to remove formaldehyde in toxoid, and sterilizing and filtering to obtain diphtheria toxoid stock solution.

2. Tetanus toxoid stock solution

Starting clostridium tetani, transferring 1-3 generations in a seed tube of a toxigenic culture medium, transferring to the toxigenic culture medium to prepare production seeds, culturing the production seeds by adopting a culture tank liquid, adopting a double Chen Peiyang base as the culture medium, culturing at 34-36 ℃ for 62-72 hours, adding formaldehyde for sterilization after culturing, filtering the culture solution to remove bacteria, adding ammonium sulfate to precipitate toxin protein, centrifugally collecting precipitate, adding formaldehyde after removing ammonium sulfate in toxin by ultrafiltration, placing the solution at 35-37 ℃ for detoxification for 30 days, removing formaldehyde in toxoid by ultrafiltration, and performing sterilization filtration to obtain tetanus toxoid stock solution.

3. Acellular pertussis vaccine stock solution

1) Starting pertussis bacillus, inoculating the pertussis bacillus into an improved bag-ginger culture medium or an active carbon semi-comprehensive culture medium, culturing for no more than 48 hours at 35-37 ℃, inoculating the pertussis bacillus into a CPB culture medium, transferring for two generations, culturing for 18-26 hours each generation, performing amplification culture to prepare enough seeds for production, inoculating the pertussis bacillus into a fermentation tank for liquid culture, culturing for two days at 37 ℃, adding merosal or formaldehyde into the culture after the culture is finished for sterilization, centrifuging the harvested culture, and preserving the cells and supernatant at 2-8 ℃ for the next purification.

2) Purification of pertussis toxin and filamentous hemagglutinin: loading the supernatant onto Capto SP chromatographic column, eluting with phosphate buffer solution containing urea and sodium chloride gradient, collecting the eluting components containing pertussis toxin and filiform hemagglutinin, loading the eluting components containing pertussis toxin onto Capto MMC chromatographic column, eluting with Tris-HCL buffer solution and sodium chloride gradient, and collecting pertussis toxin eluting peak; loading the elution peak containing the filiform hemagglutinin on a hydroxyapatite chromatographic column for purification, and collecting the elution peak.

3) Separating and purifying pertussis adhesion element: and (3) thermally releasing the thalli at 60 ℃, ultrafiltering and concentrating, loading the thalli on a Capto sphere chromatographic column, taking acetic acid buffer solution as a mobile phase, collecting an elution component containing pertussis adhesion, loading the thalli on a Capto SP chromatographic column, taking acetic acid buffer solution as a mobile phase, performing gradient elution with sodium chloride, and collecting an elution peak to obtain the separated and purified pertussis adhesion.

4) Preparing acellular pertussis stock solution: the pertussis toxin is detoxified by glutaraldehyde, the filiform hemagglutinin and the pertussis adhesion element are treated by formaldehyde, and the acellular pertussis stock solution is obtained after sterilization and filtration.

4. Poliovirus stock solution

Poliovirus type I stock:

1) Cell preparation: taking 1 or more cell tubes in a working cell bank, resuscitating, and culturing in a first-stage cell reactor at 36.5+/-0.5 ℃ until the cell concentration is 1-5 multiplied by 106 cells/ml; the cells in the first-stage cell reactor are digested by trypsin and inoculated into the second-stage cell reactor, and are cultured at 36.5+/-0.5 ℃ until the cell concentration is 2-10 multiplied by 106 cells/ml; the cells of the second-stage cell reactor are digested by trypsin, inoculated into the third-stage cell reactor, and cultured at 36.5+/-0.5 ℃ until the cell concentration is 1-5 multiplied by 106 cells/ml, and then inoculated with viruses.

2) Virus culture and harvesting: the cell culture solution in the third-stage cell reactor is replaced by a maintenance solution, the I-type poliovirus working seed batch virus seed is inoculated with Vero cells according to MOI=0.002-0.02, and the Vero cells are cultured at 32.5+/-0.5 ℃. Culturing the virus for 2-4 days, and obtaining cell supernatant, namely the poliovirus Sabin strain monovalent harvest liquid.

3) Clarifying and purifying virus harvest liquid: the harvest liquid is concentrated by 50-200 times by a 300-500kDa ultrafiltration membrane bag after centrifugal clarification. After concentration, the virus is purified and separated by adopting a sucrose density gradient centrifugation, wherein the sucrose gradient is specifically that a first gradient of sucrose density gradient liquid is 30% of sucrose, a second gradient of sucrose density gradient liquid is 55% of sucrose, and the virus is collected in a zone where the virus is located after centrifugation at 30000rpm for 10 hours. And carrying out anion exchange chromatography on the virus liquid collected by density gradient centrifugation, monitoring the wavelength to be 280nm and 254nm, and collecting the flow-through liquid to obtain the virus chromatographic liquid. Concentrating the virus chromatographic liquid to obtain the purified liquid. The anion exchange chromatography may specifically be DEAE Sepharose Fast Flow, Q Sepharose Fast Flow or other anion exchange chromatography. In one embodiment of the invention, anion exchange chromatography is used as DEAE Sepharose Fast Flow.

4) Virus inactivation: adding formaldehyde solution into the purified solution after 0.22 mu m sterilization and filtration to make the final concentration of free formaldehyde 90 mu g/ml, inactivating the solution for 6 days at 37.0+/-1.0 ℃, and continuously inactivating the solution for 6 days at 37.0+/-1.0 ℃ after sterilization and filtration to obtain the monovalent stock solution.

Poliovirus type II stock:

2) Virus culture and harvesting: the cell culture solution in the third-stage cell reactor is replaced by a maintenance solution, the virus seed batch of the type II poliovirus working seeds is inoculated with Vero cells according to MOI=0.01-0.1, and the culture is carried out at 32.5+/-0.5 ℃. Culturing the virus for 2-4 days, and obtaining cell supernatant, namely the poliovirus Sabin strain monovalent harvest liquid.

Poliovirus type III stock:

2) Virus culture and harvesting: the cell culture solution in the third-stage cell reactor is replaced by a maintenance solution, the virus seed batch MOI=0.05-0.5 of the poliovirus type III working seeds is inoculated with Vero cells, and the Vero cells are cultured at 32.5+/-0.5 ℃. Culturing the virus for 2-4 days, and obtaining cell supernatant, namely the poliovirus Sabin strain monovalent harvest liquid.

5) Virus inactivation: adding formaldehyde solution into the purified solution after 0.22 mu m sterilization and filtration to make the final concentration of free formaldehyde 90 mu g/ml, inactivating the solution for 6 days at 37.0+/-1.0 ℃, and continuously inactivating the solution for 6 days at 37.0+/-1.0 ℃ after sterilization and filtration to obtain the monovalent stock solution.

Example 6

This example provides a method of preparing the liquid vaccine composition of examples 1-4, comprising the steps of:

1) Mixing pertussis toxin, filiform hemagglutinin, pertussis adhesion agent, diphtheria toxoid stock solution, tetanus toxoid stock solution, inactivated type I poliovirus stock solution, inactivated type II poliovirus stock solution and inactivated type III poliovirus stock solution provided in example 5, adding an aluminum salt adjuvant diluted by sodium chloride aqueous solution, adding phosphate buffer solution, and adjusting the pH to 5.8-7.2 to obtain a semi-finished product;

2) And (5) sub-packaging the semi-finished product into penicillin bottles or pre-filling syringes.

Example 7

This example demonstrates the effect of different Sabin strain antigen content on the IPV immune effect of a combination vaccine.

In this example, DTaP-sIPV combination vaccine test samples (test samples 1, 2 and 3) containing different I, II and III type D antigens were prepared, the antigen contents of the three combination vaccines are shown in Table 1, and commercial IPV vaccines were used as controls. Three test substances and controls were immunized with Wistar rats, 10 animals were injected with 0.5ml each, serum was collected and isolated 21 days after immunization, and the in vivo poliomyelitis neutralizing antibody titer of the rats was detected by cell lines to calculate ED50 values.

Table 1: antigen content of three combination vaccines

The results of the detection of the titers of the type I, type II and type III poliomyelitis neutralizing antibodies are shown in tables 2, 3 and 4, and the results show that the type I, type II and type III poliomyelitis neutralizing antibodies in rats immunized by using the three DTaP-sIPV combined vaccines are not lower than those of the control commercial IPV vaccine.

Table 2: rat sIPV-I neutralization potency assay

Sample name	Test article 1	Sample 2	Sample 3	Control
					ED50 dilution factor	7.8	5.2	5.5	4.9

Table 3: rat sIPV-II neutralization potency assay

Sample name	Test article 1	Sample 2	Sample 3	Control
					ED50 dilution factor	6.8	5.1	5.6	4.7

Table 4: rat sIPV-III neutralization potency assay

Sample name	Test article 1	Sample 2	Sample 3	Control
					ED50 dilution factor	3.8	2.7	2.8	1.9

Example 8

This example investigated the effect of M199 medium on the stability of the conjugate vaccine.

Specifically, in this example, M199 culture mediums with different concentrations were added to the mixed sIPV adsorption solution of type I, type II and type III Sabin strains prepared in example 5, and the effect on the stability of viral antigens was examined. Different volumes of M199 medium concentrate (10 XM 199 medium, i.e., concentrated M199 medium having a final concentration of all components 10 times that of the conventional M199 medium) were added, a control group to which no M199 was added and a test group to which 5%, 10% and 20% (by volume) of M199 concentrate were added for each 0.5ml of sIPV adsorption solution were set, respectively, and sIPV adsorption solutions containing different concentrations of M199 were placed at 37℃for 0 day, 7 day, and 14 day periods of investigation, respectively, to determine the D antigen content in the sIPV adsorption solutions.

The test results show that the content of the D antigen is continuously reduced along with the extension of the standing time when M199 is not added, and the reduction amplitude of the D antigen in the stock solutions of the poliovirus type I, type II and type III is between 20 and 30 percent after the sIPV adsorption solution is placed for 14 days at 37 ℃. When the concentration M199 addition amount in the IPV adsorption solution is 5%, 10% and 20%, the IPV D antigen content of the I type, the II type and the III type is basically stable in 0-1 day, the D antigen content of the I type, the II type and the III type is not obviously reduced after the sIPV adsorption solution is placed for 14 days at 37 ℃, and the result shows that the addition of the M199 culture medium plays an important role in the stability of poliovirus antigens.

Example 9

This example provides a method of testing the finished product of the liquid vaccine formulations provided in examples 1-4.

1 authentication test

The detection of PT, FHA, PRN antigen by ELISA should contain corresponding antigen (appendix in "adsorption Baibai broken combined vaccine" of three parts of Chinese pharmacopoeia 2015 edition). By adopting an immune double diffusion method (three parts of Chinese pharmacopoeia 2015 edition), the product forms an obvious precipitation line with diphtheria toxoid, tetanus toxoid and tetanus toxoid immune serum.

2 physical examination

2.1 appearance: after shaking, the suspension should be in a uniform milky suspension without shaking or undispersed clot or foreign matter

2.2, the filling amount is as follows: the standard quantity (more than or equal to 0.5 ml) is not lower than the standard quantity according to the legal examination.

3 chemical examination

3.1pH value: should be 5.8-7.2.

3.2 aluminium content: should not be higher than 0.8 mg/dose.

3.3 free formaldehyde content: should not be higher than 0.2g/L.

3.4 glutaraldehyde content: should be less than 0.01g/L.

4 potency assay

4.1 cell-free pertussis vaccine: diluted to the first immunization dose at the appropriate dilution factor, and serially diluted by 5-fold. Immunization time was 21 days. The immunological titer of each 1 human dose should be not less than 4.0IU and the lower limit of the 95% confidence limit should be not less than 2.0IU. If the above requirements are not met, a retry can be performed, but all valid test results must be calculated as geometric averages (e.g., using weighted geometric averages when using probability analysis). And judging the product to be qualified when the requirements are met.

4.2 diphtheria vaccine: the immunological titer of diphtheria toxoid should be not less than 30IU per 1 human dose.

4.3 tetanus vaccine: the immune titer of tetanus toxoid should be no less than 40IU per 1 human dose.

5 efficacy experiment

Adsorption of Sabin strain poliomyelitis inactivated vaccine: after serial gradient multiple dilution of each batch of vaccine, 180-220 g of Wistar rats with more than a clean grade are immunized respectively, each group of vaccine has 10 dilutions, male and female halves,each animal was immunized with 0.5ml of the test sample. Blood was collected for 21 days, serum was isolated and stored at-20 ℃. And (3) detecting the neutralizing antibody titer of the anti-type I/II/III 3 viruses in the serum respectively, and calculating the serum positive transfer rate. Efficacy testing was performed on internal references in the same manner. The immunogenicity of the test sample should be comparable to the internal reference, i.e.: test article ED ₅₀ The value is not higher than 400% of the reference.

6, sterile inspection: according to legal examination, the bacteria should grow aseptically.

7 pyrogen examination

According to the law examination, the injection dose is 1.0 mug polysaccharide per lkg according to the body weight of the rabbits, the body temperature rise of three rabbits in the initial test is lower than 0.6 ℃, and the body temperature rise of 3 rabbits is comprehensively lower than 1.3 ℃; or in the retested 5 rabbits, the body temperature of the rabbits with the temperature rise of 0.6 ℃ or higher than 0.6 ℃ is not more than 1, and the pyrogen examination of the test sample is judged to be in accordance with the rule when the total body temperature rise of the primary test and the retested 8 rabbits is 3.5 ℃ or lower than 3.5 ℃.

8 specific toxicity test

8.1 cell-free pertussis vaccine: toxicity references were diluted per lot designation. One group of at least 10 animals per dilution of the toxicity reference and test was used with NIH mice (female or male half) weighing 14-16 g. Each mouse was intraperitoneally injected with 0.5ml and tested as a-b.

a) Mouse leukocytosis test

Mice peripheral blood was taken for white blood cell count at 3 days post injection, respectively. The test results were statistically treated, and the activity of leukocytosis toxicity of the mice injected with the test sample should be not higher than 0.5LPU/ml.

b) Mouse histamine sensitization assay

At 4 days post injection, each mouse was intraperitoneally injected with 0.5ml of a solution containing 4mg of histamine dihydrochloride or 2mg of histamine diphosphate, and after 30 minutes the anal temperature of the mouse was measured separately. The test results were statistically treated so that the test samples had a histamine-sensitized toxicity of not more than 0.8HSU/ml and no animal death.

8.2 diphtheria, tetanus vaccine

With guinea pigs weighing 250-350 g, each batch of products is not less than 4, each abdomen is subcutaneously injected with 2.5ml, each side is injected with 1.25ml, and observation is carried out for 30 days. The injection site may become wet, becoming hard after 5-10 days, and may be incompletely absorbed for 30 days. Weighing at 10 days, 20 days and 30 days, and increasing the weight ratio before injection, and judging that patients with local suppuration, necrosis, tetanus and advanced paralysis are qualified.

9 toxicity reversal experiments: each batch was left at 37℃for 4 weeks, as per 8.1 b) in the protocol.

10 bacterial endotoxin examination: the examination according to the general rule 1143 of three parts in Chinese pharmacopoeia should not be higher than 100 EU/dose.

The detection results are shown in Table 5.

Table 5: vaccine detection results

Example 10

The embodiment provides a method for carrying out mixed immunization by utilizing the liquid vaccine preparation provided by the invention. The method comprises the following steps:

the vaccine liquid preparation provided in example 4 was used as a basic immune type vaccine, and was subcutaneously immunized twice on days 1 and 14, respectively. IgG antibody titers in mouse serum were determined by indirect ELISA, by orbital blood collection on day 28. The IgG titer in the serum was assayed again 3 months later (i.e., day 90) and the vaccine liquid preparation provided in example 1 was immunized by subcutaneous injection as boost, and the IgG titer in the serum was assayed by blood sampling 14 days after boost (i.e., day 104). The results are shown in Table 6 and FIGS. 1 to 3.

Table 6: antibody titer assay results

	For 28 days	90 days	104 days
				Type I sIPV neutralizing antibody titres (log) ₂ )	6.778	6.111	7.222
Type II sIPV neutralizing antibody titres (log) ₂ )	4.667	3.889	5.111
				Type III sIPV neutralizing antibody titres (log) ₂ )	6.667	6.000	7.222
aP-PT antibody titer (lg)	5.077	4.595	5.468
				aP-FHA antibody titer (lg)	5.890	5.860	6.372
aP-PRN antibody titre (lg)	4.475	4.054	4.656
				DT antibody titer (lg)	5.649	3.249	6.200
TT antibody titer (lg)	5.408	5.499	6.191

In fig. 1-3, P < 0.05; * Represents P < 0.01; * Represents P < 0.001.

The experimental results show that the vaccine liquid preparation provided in example 4 can induce mice to produce significant neutralizing antibodies against type I/II/III poliovirus, antibodies (IgG) against pertussis component (PT, FHA, PRN), antibodies (IgG) against DT and antibodies (IgG) against TT after two immunizations with the vaccine liquid preparation as a basic immunizing agent. After 3 months the antibody levels in mice were examined, we found that a significant decrease in neutralizing antibodies against polioviruses type I/II/III, antibodies (IgG) against pertussis components (PT, PRN), antibodies (IgG) against DT, was all present. However, the vaccine liquid preparation provided in example 1 was used as a booster immunization, and was able to significantly improve neutralizing antibodies against type I/II/III polioviruses, antibodies against pertussis component (PT, FHA, PRN), antibodies against DT (IgG), and antibodies against TT (IgG) in mice.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A liquid vaccine composition, wherein the antigen consists of: diphtheria toxoid, tetanus toxoid, pertussis toxin, filiform hemagglutinin, pertactin, inactivated type I poliovirus Sabin strain, inactivated type II poliovirus Sabin strain and inactivated type III poliovirus Sabin strain;

wherein, the antigen ratio of the Sabin strains I, II and III is 1: (2.5 to 3.5): (2.5-3.5);

each 0.5ml of single dose of the composition contains 7.5-15 DU of type I Sabin strain poliovirus, 22.5-45 DU of type II Sabin strain poliovirus, 22.5-45 DU of type III Sabin strain poliovirus, 2-12.5 Lf of diphtheria toxoid and 2.5-5 Lf of tetanus toxoid;

each 0.5ml single dose of the composition contains 20-25 mug of pertussis toxin, 20-25 mug of filiform hemagglutinin and 3-8 mug of pertussis bacillus adherents; or each 0.5ml single dose of the composition contains 2.5-8 mug of pertussis toxin, 5-8 mug of filiform hemagglutinin and 2.5-3 mug of pertussis bacillus adherents;

the composition also comprises 5% -20% of M199 culture medium concentrate of 10X.

2. The composition of claim 1, further comprising one or more of an aluminum adjuvant, a soluble phosphate buffer, and sodium chloride.

3. The composition according to claim 1, wherein the composition comprises the following components per 0.5ml single dose:

diphtheria toxoid 2-12.5 Lf;

tetanus toxoid 2.5-5 lf;

20-25 mug of pertussis toxin;

20-25 mug of filiform hemagglutinin;

3-8 mug of pertussis adhesion element;

inactivated Sabin strain I poliovirus 7.5-15 DU;

inactivated Sabin strain II poliovirus 22.5-45 DU;

inactivated Sabin strain III poliovirus 22.5-45 DU.

4. The composition according to claim 1, wherein the composition comprises the following components per 0.5ml single dose:

diphtheria toxoid 2-12.5 Lf;

tetanus toxoid 2.5-5 lf;

pertussis toxin 2.5-8 mug;

5-8 mug of filiform hemagglutinin;

2.5-3 mug of pertussis adhesion element;

inactivated Sabin strain I poliovirus 7.5-15 DU;

inactivated Sabin strain II poliovirus 22.5-45 DU;

inactivated Sabin strain III poliovirus 22.5-45 DU.

5. Use of the vaccine composition of any one of claims 1-3 in the preparation of a basic immune vaccine.

6. Use of the vaccine composition according to any one of claims 1-2, 4 for the preparation of a booster vaccine.

7. A vaccine kit comprising a first chamber and a second chamber; the first chamber contains a basic immunity vaccine prepared from the vaccine composition of any one of claims 1-3; the second chamber contains a booster vaccine prepared from the vaccine composition of any one of claims 1-2 and 4;

alternatively, a first chamber, a second chamber, and a third chamber are included; the first chamber and the second chamber each contain a basic immunity vaccine prepared from the vaccine composition of any one of claims 1-3; the third chamber contains an enhanced immunity vaccine prepared from the vaccine composition of any one of claims 1-2 and 4;

alternatively, the first chamber, the second chamber, the third chamber, and the fourth chamber are included; each of the first, second and third chambers contains a basic immune vaccine prepared from the vaccine composition of any one of claims 1-3; the fourth chamber contains a booster vaccine prepared from the vaccine composition of any one of claims 1-2 and 4.