TWI711700B - Improved methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof - Google Patents

Abstract

The present invention is directed to improved methods of Enterovirus inactivation by formaldehyde in presence of tromethamine buffer resulting in maximum recovery of D-antigen. Subsequent adsorption of said sIPV on aluminium hydroxide provides significantly dose reduced sIPV compositions.

Description

Improved method of inactivating enterovirus, adjuvant adsorption and vaccine composition with reduced dose obtained

The present invention relates to an improved method for inactivating enterovirus, adjuvant adsorption and a vaccine composition with reduced dose obtained.

The prevalence of poliovirus has been greatly reduced by using an oral polio vaccine (OPV) based on live attenuated Sabin poliovirus strain. However, OPV has limitations for the post-eradication era. Therefore, the development of Sabin inactivated polio vaccine (Sabin IPV; sIPV) plays an important role in the World Health Organization (WHO) polio eradication strategy. The use of attenuated sabine instead of the wild-type Salk poliovirus strain will provide additional safety during vaccine production. In addition, in order to prevent the emergence of transmissible vaccine-derived polioviruses (cVDPVs), OPV should be stopped and replaced with IPV after eradication of polio. These cVDPVs are transmissible and can become neurotoxic (similar to wild Type poliovirus) and cause paralytic poliomyelitis associated with the vaccine. Such virus strains could potentially re-spread poliovirus in the world and invalidate the eradication efforts.

IPV is delivered by intramuscular (IM) or deep subcutaneous (SC) injection. IPV can currently be used in a non-adjuvanted independent formulation or in different combinations, including DT-IPV (with diphtheria and tetanus toxoid) and the hexavalent DTPHepB-Hib-IPV vaccine (with pertussis, type B) Hepatitis and Haemophilus influenzae b). The standard dose of currently accepted polio vaccine contains D antigen, such as 40 units of inactivated poliovirus type 1 (Mahoney), 8 units of inactivated poliovirus type 2 (MEF-1) and 32 units of inactivated poliovirus type 3 (Saukett) (e.g. Infanrix-IPV ^™ ). The existing stand-alone IPV preparation does not contain adjuvants.

Most experts agree that the global use of IPV is preferable because of its proven protective tracking records and security. However, when compared with OPV, the cost price of IPV is significantly higher. This is mainly due to the following requirements: (i) more viruses per dose; (ii) additional downstream processing (ie concentration, purification and inactivation) and related quality control (QC) inspections; (iii) loss of antigen or Poor downstream recovery and (iv) containment. So far, financial challenges in low- and middle-income countries have been the main disadvantages of IPV innovation and implementation. The production cost of SIPV is currently estimated to be equivalent to that of IPV, which is about 20 times higher than that of OPV. After eradication of poliovirus, the global demand for IPV in the future may increase from the current level of 80 million doses per year to 450 million doses per year. Therefore, methods to "expand" the supply of IPV will be needed.

Insufficient supply of conventional vaccines to meet global needs or production of conventional vaccines The cost of preventing vaccines from being sold at affordable prices in developing countries requires effective vaccine formulations with reduced doses, which use lower doses of IPV antigen to provide protection against infection. Compared with the existing market-sold preparations, it may be safer to receive lower doses of IPV. Therefore, multiple strategies for preparing IPV at a more affordable price need to be evaluated.

In the case of pandemic influenza vaccines, adjuvants are used to allow dose reduction, increase availability, and reduce vaccine costs. Therefore, it is speculated that adjuvanted vaccine formulations of sIPV will reduce costs and increase the number of sIPV agents available worldwide.

Different research teams around the world have evaluated the use of several adjuvants to reduce the dose of vaccines (especially influenza vaccines). These adjuvants include aluminum, emulsions, and TLR agonists (MPL, CpG, poly-IC). , Imiquimod), dmLT, 1,25-dihydroxyvitamin D3, CAF01, poly[bis(carboxyphenoxy)-phosphazene] (PCPP) and Venezuelan equine encephalitis (VEE) replicon particles. Most types of adjuvants being studied encounter the following obstacles: i) unknown safety or classified as toxic by regulatory agencies, ii) restrictions on the route of administration, iii) lack of manufacturing reproducibility, and iv) adjuvant stability .

It has been previously reported that emulsion adjuvants (MF-59, AS03, AF3) provide strong dose reduction effects (>30 times) for influenza and hepatitis B vaccines. These adjuvants work by forming a reservoir at the injection site, allowing the distribution and release of antigenic substances and stimulating plasma cells that produce antibodies. However, these adjuvants are considered to be too toxic for the use of widely used human preventive vaccines and are generally reserved for severe and/or advanced conditions that have a high tolerance for side effects, such as cancer.

In addition, aluminum salts are considered safe and have been used in combination vaccines containing sIPV, and have the lowest development barriers and can be produced inexpensively. However, aluminum adjuvants are not known to allow a significant dose reduction.

One of the most critical steps to produce vaccines against pathogens (especially viral vaccines) is virus inactivation. In the case of virus inactivation, formalin is the most commonly used inactivator in vaccine production. Formaldehyde irreversibly cross-links the primary amine group of the surface protein with other adjacent nitrogen atoms in the protein or DNA through the -CH ₂ -bond to inactivate the virus. The potential problem of using formalin for virus inactivation involves a series of chemical reactions. The reaction products produced by these chemical reactions can induce cross-linking of viral proteins and aggregation of viral particles. This may hinder the inactivation efficiency of formalin and may also lead to partial destruction of the immunogenicity of the antigen in the vaccine. Therefore, it has previously been reported that formalin inactivation of polio virus may affect the immunogenicity and antigenicity of the virus. See the literature Morag Ferguson et al., Journal of General Virology (1993), 74, 685-690. Most importantly, the previously disclosed formaldehyde inactivation method is especially implemented in the presence of phosphate buffer, in which a significant loss of D-antigen (inactivation) is observed with the epitope modification of Sabin I/II/III. D-antigen recovery after live: 22% for Sabin I, 15% for Sabin II and 25% for Sabin III), thus failing to maintain the epitope conformation. Therefore, antibodies produced by recipients of formalin-inactivated poliovirus (in the presence of phosphate buffered saline) may not contribute to a protective immune response.

When inactivating the polio virus, which is particularly capable of recovery, through the combination of formalin and ultraviolet light, scientists try to overcome the limitation of ultraviolet inactivation alone or formalin inactivation. See literature e.g. McLean, et al., "Experiences in the Production of Poliovirus Vaccines," Prog. Med. Virol., vol 1, pp. 122-164 (1958). The document Taylor et al., J. Immunol. (1957) 79: 265-75 describes the use of a combination of formalin and ultraviolet light to inactivate polio virus. The document Molner et al., Am.J.Pub.Health (1958) 48:590-8 describes the generation of measurable levels of circulation in the blood of subjects vaccinated with ultraviolet-formalin inactivated polio vaccine Antibody. The document Truffelli et al., Appl. Microbiol. (1967) 15: 516-27 reported the inactivation of adenovirus and simian virus by a three-stage inactivation method consisting of formalin, ultraviolet light and β-propiolactone (BPL) 40 Tumorogenicity in hamsters. The document Miyamae, Microbiol. Immunol. (1986) 30: 213-23 describes the preparation of Sendai virus immunogens by treatment with ultraviolet light and formalin. However, the promising formaldehyde substitutes discussed previously, such as β-propiolactone (BPL), have been reported to produce immune complex reactions when combined with other components of the rabies vaccine. In addition, it has been shown to produce squamous cell carcinoma, lymphoma and hepatocellular carcinoma in mice.

Therefore, it is particularly desirable to adopt favorable formaldehyde inactivation conditions that can maintain the structural integrity of the antigenic structure of the Sabin virus strain and to utilize a sIPV (Sabin IPV) vaccine composition that can lead to a significant dose reduction (ie 8 to 10 times reduction) The safe and cost-effective adjuvants that can reduce production costs, increase vaccine supply and provide affordable vaccines to developing countries.

The inventors of this case unexpectedly discovered that the loss of D-antigen after formaldehyde inactivation may be due to the presence of phosphate buffer, which unexpectedly caused undesired aggregation of poliovirus. The present invention provides an improved method of inactivation of formaldehyde in the presence of TRIS buffer so as to ensure minimal epitope modification and subsequent Minimize the loss of D-antigen. Subsequently, a Sabin IPV vaccine composition with a significantly reduced dose can be obtained, wherein the dose is reduced by at least 8 times for Sabin type 1 and at least 3 times for Sabin type 3.

An important aspect of the present invention is an improved method for inactivating and adsorbing formalin on aluminum salts. The method includes the following steps: a) adding purified sabin IPV product to a TRIS buffer with a pH between 6.8 and 7.2 (30 to 50mM), b) add M-199 medium containing glycine (5gm/l) to the mixture of (a), c) add 0.025% formaldehyde while mixing, d) place the step on a magnetic stirrer (c) The resulting mixture was incubated at 37°C for 5 to 13 days, e) The incubated mixture was subjected to moderate 0.22μ filtration on the 7th day and the final filtration on the 13th day, f) the step (e ) Store the resulting product at 2 to 8°C, g) Perform D-Ag ELISA to determine D-antigen unit, h) Take the required volume of autoclaved Al(OH) ₃ to obtain aluminum in a 50 ml container The final concentration of (Al ⁺⁺⁺ ) is 0.8 to 1.2 mg/dose, i) Add sIPV product with adjusted D-Ag unit and use diluent (10x M-199+0.5 glycine%) to make up the volume, j) Adjust the pH of the final formulation and obtain a final formulation with a pH between 6 and 6.5, k) The formulation product is magnetically stirred overnight at 2 to 8°C, and the formalin inactivation in step (a) is not Carry out in the presence of phosphate buffer.

The first embodiment of the present invention is that the buffer used during formaldehyde inactivation can be selected from the group consisting of TRIS, TBS, MOPS, HEPES and bicarbonate buffers.

The preferred aspect of this first embodiment is that the formaldehyde inactivation can be carried out in the presence of TRIS buffer or TBS (TRIS buffered saline), wherein the concentration of the TRIS buffer or TBS is selected from 30mM, 40mM and 50mM, preferably 40mM And the formaldehyde inactivation can be carried out at a pH selected from 6.8, 6.9, 7, 7.1 and 7.2 (preferably between 6.8 to 7.2), wherein the inactivation does not use any phosphate buffer.

The second embodiment of the present invention is that the adsorption of sIPV inactivated by formalin can be completed on aluminum hydroxide, wherein the concentration of the aluminum hydroxide is selected from 1.5 mg/dose, 1.8 mg/dose, 2.2 mg/dose, Preferably, it is 2 mg/dose to 2.4 mg/dose, and the adsorption pH is selected from 6.2, 6.3, 6.4 and 6.5, preferably the pH is 6.5.

The third embodiment of the present invention is that the improved method of formalin inactivation and aluminum hydroxide adsorption can lead to the recovery of D-antigen after inactivation between 50% and 80% and the adsorption percentage of aluminum hydroxide can be Between 85 and 99%.

One aspect of this third embodiment is that the present invention provides an improved method of formalin inactivation and aluminum hydroxide adsorption, which results in at least 8 times the sabin type I compared to the standard dose of 40 DU-8 DU-32 DU The dose of Sabin III was reduced and the dose of Sabin III was reduced by at least 3 times.

The second aspect of this third embodiment is that the present invention provides an improved method of formaldehyde inactivation and aluminum hydroxide adsorption, which results in a vaccine composition comprising i) at least a 5 D-antigen unit dose of inactivated poliovirus type 1 , Ii) at least 8 D-antigen unit dose of inactivated type 2 poliovirus, and iii) at least 10 D-antigen unit dose of inactivated type 3 poliovirus.

The fourth embodiment of the present invention is that the aluminum salt adjuvant is aluminum hydroxide, and its concentration at a pH of about 6.5 is between 1.5mg/0.5ml agent to 2.5mg/0.5ml agent, preferably Between 2.100mg/0.5ml dose to 2.4mg/0.5ml dose.

An aspect of this fourth embodiment is that the total aluminum content in the trivalent vaccine (type 1, type 2 and type 3) can be 800 to 1000 μg (preferably 800 μg) Al ³⁺ per 0.5 ml dose, characterized by at least 400 μg Al ^{3+ is} used for type 1, at least 200 μg Al ^{3+ is} used for type 2 and at least 200 μg Al ^{3+ is} used for type 3.

Another aspect of this fourth embodiment is that the reduced-dose poliovirus vaccine composition may consist of type 1 and type 3 without type 2, wherein the dose volume may be between 0.1 and 0.4 ml.

The reduced-dose vaccine composition prepared by the method of the present invention may be i) "independent sIPV", wherein the antigen may include type 1 sIPV or type 2 sIPV or type 3 sIPV, or type 1 and type 2 sIPV, or Type 1 and Type 3 sIPV, or Type 2 and Type 3 sIPV, or Type 1, Type 2 and Type 3 SIPV, or ii) "combination vaccine containing sIPV", wherein the non-IPV antigen of the combination vaccine can be selected from but Not limited to diphtheria toxoid, tetanus toxoid, whole cell pertussis antigen, acellular pertussis antigen, hepatitis B surface antigen, Haemophilus influenzae b antigen, Neisseria meningitidis A antigen, Neisseria meningitidis C antigen, Neisseria meningitidis W-135 antigen, Neisseria meningitidis Y antigen, Neisseria meningitidis X antigen, Neisseria meningitidis B vesicle or purified antigen , Hepatitis A antigen, Salmonella typhi ( Salmonella typhi ) antigen, Streptococcus pneumoniae ( Streptococcus pneumoniae ) antigen.

The non-IPV antigens may be adsorbed on aluminum salts (such as aluminum hydroxide), aluminum salts (such as aluminum phosphate), or a mixture of both aluminum hydroxide and aluminum phosphate, or may not be adsorbed.

The polio virus can be grown in cell culture. The cell culture may be a VERO cell line or PMKC derived from a continuous cell line of monkey kidney. VERO cells can be conveniently cultured in microcarriers. After growth, ultrafiltration, diafiltration, and chromatography can be used to purify virus particles. Before administration to the patient, the virus must be inactivated, which can be achieved by formaldehyde treatment.

The composition may be present in a vial or may be present in a ready filled syringe. The syringe can be equipped with or without a needle. The syringe contains a single dose of the composition, while the vial may contain a single dose or multiple doses (e.g., 2 doses). In one embodiment, the agent is used in humans. In another embodiment, the agent is for adults, adolescents, young children, infants, or people less than one year old and can be administered by injection.

The vaccine of the present invention can be packaged in unit dose form or multiple dose form (for example, 2 doses). The multi-dose composition may be selected from 2 doses, 5 doses and 10 doses. For multiple dose forms, vials are preferred to pre-filled syringes. An effective dose volume can be established conventionally, but the volume of a conventional dose composition for injection into a human is 0.5 ml.

Figure 1: Aluminum phosphate gel prepared in 0.9% NaCl (relationship between pH and zeta potential under different concentrations of aluminum phosphate gel).

Figure 2: Aluminum phosphate gel prepared in WFI (relationship between pH and zeta potential under different concentrations of aluminum phosphate gel).

Figure 3: Aluminum hydroxide gel prepared in 0.9% NaCl (relationship between pH and zeta potential under different concentrations of aluminum hydroxide gel).

Figure 4: Aluminum hydroxide gel prepared in WFI (relationship between pH and zeta potential under different concentrations of aluminum hydroxide gel).

Example 1 Purification of Sabine IPV (sIPV)

1) Tangential flow filtration (TFF): Use a tangential flow filtration system with a 100Kda box (0.5m ² ) to concentrate the clarified harvest tank 10 times, and then use phosphate buffer (40mM, pH 7.0) to percolate the harvest solution 3 times.

2) Column chromatography: Purification by ion exchange chromatography (IEC). Using an Akta detector (GE Healthcare), the 10-fold TFF concentrate was passed through DEAE Sepharose fast flow (weak anion exchanger) packed in a chromatography column xk-26. It was found that the negatively charged impurities were bound to the chromatography column, and 40mM phosphate buffer was used to recover Collect the polio virus in the fluid.

3) TRIS buffer exchange: In order to minimize the loss of antigen in the very troublesome inactivation method (13 days), the purified virus pool was exchanged from phosphate buffer to TRIS buffer via a TFF system (100KDa, 0.1m ² ) Solution (40mM, pH 7). The purified virus pool was exchanged with 3 times volume of TRIS buffer.

Example 2 A) Inactivation of sIPV

A 10-fold concentrated M-199 with 0.5% glycine was added to obtain a final 1-fold concentration. While continuing to stir, the inactivator formalin (0.025%) was added to the purified virus product. The inactivation was carried out at 37°C under continuous stirring for 13 days, which included 0.22 u filtration on the 7th and 13th days.

B) Inactivation of sIPV in TRIS buffer and phosphate buffer

Inactivate with 0.025% formaldehyde at 37°C for 13 days.

When the formaldehyde inactivation method was specifically performed in the presence of phosphate buffer, significant D-antigen loss was observed for Sabin type 1. However, it was found that inactivation of formaldehyde in the presence of TRIS buffer resulted in minimal loss of D-antigen.

For the preservation of D-antigen content of sIPV 1, 2 and 3, it was found that TRIS buffer at a concentration of 40 mM was the most effective.

C) Determination of D-antigen content by ELISA Day 1: Flat coating

1. Pipet 100 μl of specific bovine anti-polio antibody to PBS in each well.

2. Seal the microtiter plate and incubate overnight at room temperature.

Day 2: Closed

1. Wash the plate 3 times (washing/dilution buffer 0.05% Tween 20 in 1x PBS).

2. Add 300μl blocking buffer (1% BSA in PBS) to each well.

3. Seal the plate and incubate at 37±1°C for 45 minutes.

Sample addition:

1. Wash the plate 3 times.

2. Add 100μl of sample diluent to all wells except the wells of row A.

3. Add 100μl of standard to the first two wells in columns 2 and 3.

4. Add 100μl of sample to the first two wells in columns 4 to 12.

5. Dilute the sample in advance to an appropriate concentration.

6. Add 100μl of sample diluent to the first two wells in column 1.

7. By transferring 100 μl from each well to the adjacent well in the same column and discarding 100 μl from the last well, the column is serially two-fold diluted.

8. Incubate at 37°C for 2 hours.

9. Keep the plate at 4°C overnight.

Day 3: Add monoclonal antibody

1. Wash the plate 3 times.

2. Add 100 μl of diluted (1:240) specific monoclonal antibody.

3. Seal the plate and incubate at 37°C for 2 hours.

Conjugate:

1. Wash the plate 3 times.

2. Add 100 μl of the diluted conjugate (1 type 1: 2400, type 2 1: 1500, type 3 1: 4800).

3. Seal the plate and incubate at 37°C for 1 hour.

Join the pledge:

1. Add 100μl TMB substrate to all wells.

2. Incubate the mixture for 10 minutes at room temperature.

3. Stop the reaction by adding 100 μl 2M H ₂ SO ₄ .

4. Read the plate at 450/630nm.

5. Use KC4 software to calculate the D-antigen concentration.

Example 3 sIPV adsorption

1. Use autoclaved 1% Al(OH) ₃ and AlPO ₄ stock solutions to prepare formulations.

2. Take the required volume of Al(OH) ₃ /AlPO ₄ to obtain the required aluminum concentration in a 100 ml glass bottle.

3. Add inactivated poliovirus samples with known D-Ag units and use diluents for volume supplementation.

4. Adjust the pH of the final formulation to 6.5 using 1N HCl/NaOH.

5. Keep the formulation product on a magnetic stirrer at 2 to 8°C overnight.

Example 4 Pre-formulation studies

Different concentrations of Al(OH) ₃ and AlPO ₄ were prepared in 0.9% brine and WFI to examine the changes in size and zeta potential with pH.

It was observed that in the presence of WFI and brine, as the pH increased from 5 to 7.5, the zeta potential of AlPO ₄ decreased (negative charge) (see Figures 1 and 2).

However, the zeta potential of Al(OH) ₃ in brine remains unchanged and is independent of pH and the salt concentration of Al(OH) ₃ (see Figures 3 and 4).

Example 5 Study on the adsorption of sIPV on aluminum phosphate and aluminum hydroxide

It was found that Sabin 3 poliovirus uses aluminum phosphate (AlPO ₄ ) to adsorb only 50 to 60%. However, Sabin 3 poliovirus using Al(OH) ₃ shows at least 90% adsorption. Therefore, compared with aluminum phosphate, aluminum hydroxide was found to be more effective for the adsorption of Sabin 1, 2 and 3, respectively.

Example 6 Study on the immunogenicity of aluminum-adsorbed sIPV

In order to detect the immune response of adjuvanted sIPV in rats, an SNT test (serum neutralization test) was performed. The serum is separated and used to test the presence of neutralizing antibodies against specific poliovirus. The control serum was used to validate the test. Virus anti-titration was also performed to obtain the number of challenge virus particles added.

Animal model: 50% male and 50% female Wistar rats (8 weeks old, about 200gm) in each group.

Vaccination route: intramuscular.

Volume: 0.5ml

Blood draw: 21st day.

Bleeding site: posterior orbital plexus

Surprisingly, it was found that, compared to Sabine IPV with 40 DU/dose of Shaq IPV vaccine and 5 DU/dose of aluminum phosphate adjuvant, Sabin 1 IPV with 5 DU/dose of aluminum hydroxide adjuvant Has better seroconversion.

Compared with 8 DU/dose of Shaq IPV vaccine, 8 DU/dose of adjuvanted type 2 sIPV resulted in equivalent seroconversion.

Compared with the Shaq IPV vaccine with 32 DU/dose, it was found that 10 DU/dose of type 3 sIPV with adjuvant produced equivalent seroconversion.

Immune response to adjuvanted Sabin poliovirus

We have observed that if the virus is adjuvanted with Al(OH) ₃ , it shows an excellent dose reduction.

If we consider a single-dose regimen for immunization, 5-16-10 D-Ag is the best combination for Sabin polio 1, 2 and 3, respectively.

If we consider using two doses for immunization, 2.5-8-5 gives excellent immunity.

Supporting experimental data for Sabine

Immune response to adjuvanted Shaq poliovirus

We have observed that if the virus is adjuvanted with Al(OH) ₃ , it shows an excellent dose reduction.

If we consider a single-dose regimen for immunization, 8-2-5 D-Ag is the best combination for Shaq polio types 1, 2 and 3, respectively.

If we consider using two doses for immunization, 5-2-5 gives excellent immunity.

Shaq's supporting experimental data

Supporting experimental data of Shaq (10-2-5) single dose

Supporting experimental data of Shaq (10-2-12) single dose

Supporting experimental data of Shaq (5-8-10) single dose

Supporting experimental data of Shaq (7.5-16-10) single dose

In view of the many possible implementations to which the principles of the present invention can be applied, the described implementations should be regarded as only preferred examples of the present invention, and should not be regarded as limiting the scope of the present invention. On the contrary, the scope of the present invention is defined by the scope of the attached patent application. Therefore, the present invention includes all contents within the scope and spirit of the patent application.

Claims (10)

A method for preparing a vaccine composition containing enterovirus particles, wherein the method comprises the following steps: a) preparing a medium containing enterovirus particles; b) purifying the enterovirus particles from the medium; c) having a pH of 6.8 to 7.2 Collect the enterovirus particles with a buffer with a concentration range of 30mM to 70mM; d) Stabilize the purification by adding M-199 medium containing glycine to a final concentration of 1X M-199 containing 0.05% glycine E) Inactivate the enterovirus particles by using 0.025% formaldehyde at 37°C for 5 to 13 days; and f) adsorb the enterovirus particles on an aluminum salt adjuvant, where the adsorption percentage on aluminum is At least 95%, the vaccine composition containing the enterovirus particles is a reduced-dose inactivated polio vaccine (IPV), and the reduced-dose IPV contains the following Salk serotype poliovirus: i) Inactivated type 1 poliovirus with a dose of less than 15D-antigen units (DU) instead of the standard dose of 40DU; and/or ii) inactivated type 2 poliovirus with a dose of less than 18DU; and/ Or iii) Inactivated poliovirus type 3 with a dose of less than 17 DU instead of the standard dose of 32 DU. The method according to claim 1, wherein the buffer in step c) is selected from the group consisting of TRIS, TBS, MOPS, HEPES, bicarbonate buffer, and combinations thereof. The method according to claim 2, wherein the buffer in step c) is a TRIS buffer. The method according to claim 1, wherein the aluminum salt adjuvant in step f) is selected from aluminum hydroxide or aluminum phosphate or a mixture of the two. The method according to claim 4, wherein the aluminum salt adjuvant is aluminum hydroxide, and its concentration at a pH of about 6.5 is between 1.5 mg/0.5 ml agent to 2.5 mg/0.5 ml agent. The method according to claim 5, wherein the total aluminum content in the vaccine composition is less than 1.2 mg Al ³⁺ per 0.5 ml dose. The method according to claim 1, wherein the Salk serotype 1 IPV is a Mahoney strain, the Salk serotype 2 IPV is a MEF-1 strain, and the Salk Serotype 3 IPV is the Saukett strain. The method according to claim 1, wherein the reduced-dose inactivated polio vaccine (IPV) comprises: i) Shaker single-dose composition, which has a combination of Shaker 1, 2 and 3 selected from 8-2-5; ii) Shaker two-dose composition, which has a Shaker selected from 5-2-5 Shaker type 1, type 2 and type 3 combinations; iii) Shaker single-dose composition having a combination of Shaker type 1, type 2 and type 3 selected from 10-2-5; iv) Shaker single-dose combination A combination of Shaker type 1, type 2 and type 3 selected from 10-2-12; v) a single-dose composition of Shaker having type 1 and type 2 selected from 7.5-16-10 And type 3 combination; vi) a single-dose composition of Shak, which has a combination of Shak 1, 2 and 3 selected from 10-2-10; or vii) a single-dose composition of Shak, which has a combination selected from 10-2-16 combination of Shaq 1, 2 and 3 types. The method according to any one of claims 1 to 8, wherein the method for preparing an inactivated polio vaccine (IPV) containing a reduced dose of Saq polio virus comprises the following steps: a) Preparing a medium containing the polio virus; b) purifying the polio virus from the medium; c) collecting the polio virus in a TRIS buffer with a pH of 6.8 to 7.2 and a concentration range of 30 mM to 70 mM; d ) Stabilize the purified polio by adding M-199 medium containing glycine to a final concentration of 1X M-199 containing 0.05% glycine Virus; e) inactivate the poliovirus by using 0.025% formaldehyde at 37°C for 5 to 13 days; and f) in aluminum hydroxide with a concentration of 1.5mg/0.5ml agent to 2.5mg/0.5ml agent The poliovirus is adsorbed on the adjuvant, and the adsorption percentage on the aluminum hydroxide adjuvant is at least 95% for Shaq serotypes IPV type 1, IPV type 2, and IPV type 3. The method according to any one of claims 1 to 8, wherein the multivalent vaccine composed of the reduced dose of IPV further comprises one or more antigens derived from a pathogen selected from the group consisting of Haemophilus influenzae ) b, Neisseria meningitidis type A, Neisseria meningitidis type C, Neisseria meningitidis type W, Neisseria meningitidis type Y, Neisseria meningitidis type X Type, Neisseria meningitidis type B, Streptococcus pneumoniae , Salmonella typhi , hepatitis A, hepatitis B, diphtheria toxoid, tetanus toxoid, whole cell pertussis and acellular pertussis.

Images