A combined vaccine for preventing hand-foot-mouth disease and a preparation method and application thereof

Abstract

The application discloses a combined vaccine for preventing hand-foot-mouth disease, which comprises inactivated enterovirus 71 virus, and inactivated coxsackievirus A 16 virus, coxsackievirus A 10 virus and coxsackievirus A 6 virus. The application also discloses a method for preparing the combined vaccine, and the prepared vaccine has good adsorption effect and stability. The multiple antigens do not interfere with antigenicity and immunization effect of each other after immunizing an object, and have good immunogenicity and safety. The use of the combined vaccine can obviously simplify a vaccine inoculation procedure, improve inoculation efficiency and reduce cost.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a combined vaccine for the prevention of hand-foot-mouth disease, its preparation method, and its application. Background Technology

[0002] Hand-foot-mouth disease (HFMD) is classified as a Class C infectious disease in my country's Law on the Prevention and Control of Infectious Diseases. It is an acute infectious disease caused by various enteroviruses, and is most common in infants and young children. It is prevalent in summer, with a high incidence among preschool children; adults can be indirect sources of infection. The main symptoms are rashes on the mouth, hands, and feet, and it can lead to severe complications such as meningitis, encephalitis, pulmonary edema, and circulatory failure, resulting in death. Besides enterovirus 71 (EV71) and Coxasckievirus A16 (CA16), which are relatively common, viruses that cause HFMD include Coxsackievirus A (CA) types 2, 4, 5, 6, and 10; Coxsackievirus B (CB) types 1, 2, 3, 4, and 5; enterovirus 68 (EV68); and echovirus (Echo). EV71 and CA16 are the main pathogens causing HFMD outbreaks. In recent years, there have been increasing reports of HFMD outbreaks caused by CA10 and CA6, and they have even become the main circulating serotypes in some regions.

[0003] Hand, foot, and mouth disease (HFMD) has become a serious public health problem threatening children's health and social stability. Currently, there are no effective antiviral drugs for treating HFMD. Although an approved EV71 inactivated vaccine is available, changes in the enterovirus spectrum causing HFMD and the diversity of dominant strains leading to its prevalence, coupled with the lack of cross-immune protection between strains, mean that it cannot protect against infections with other types of HFMD, posing new challenges to HFMD control. EV71 vaccination is insufficient to control outbreaks and epidemics of HFMD caused by CA16, CA10, and CA6. Therefore, there is an urgent need to develop corresponding multivalent and combination vaccines to address HFMD outbreaks and severe cases. Currently, there are no reports on the development of a quadrivalent combined vaccine using EV71, CA16, CA10, and CA6. Firstly, hand-foot-and-mouth disease (HFMD) caused by EV71, CA16, CA10, and CA6 viruses has experienced a large-scale outbreak in a relatively short period. Secondly, HFMD caused by viruses exhibits a certain geographical distribution, and fewer institutions are investing in research and development in developed countries with relatively low incidence rates. Thirdly, the immune interference between different viral antigens is unknown, and this interference may directly reduce the immunogenicity of individual vaccine components, affecting immunization efficacy. Therefore, to protect the body from HFMD viruses on a wider scale, establishing a comprehensive HFMD prevention and control system in my country and expanding protection through the development of combined vaccines is imperative.

[0004] Currently, EV71 vaccines are available. Clinical research data shows that the use of vaccines is effective in preventing hand-foot-mouth disease caused by EV71, especially in preventing severe cases. However, EV71 vaccines have weak cross-protection against Coxsackieviruses. Given the changing trends of enterovirus serotypes, there is an urgent need for a multi-serotype combined vaccine that can prevent hand-foot-mouth disease on a wider scale. Summary of the Invention

[0005] The purpose of this invention is to provide a combined vaccine for the prevention of hand-foot-mouth disease.

[0006] Specifically, the combined vaccine for the prevention of hand-foot-mouth disease provided by the present invention includes inactivated enterovirus 71, as well as inactivated Coxsackievirus A16, 10 and 6.

[0007] Preferably, the enterovirus 71 strain is the strain with accession number CGMCC No. 3544.

[0008] The inactivated Coxsackievirus A16, A10, and A6 strains described in this invention are all newly discovered virus strains with excellent immunogenicity. All of these newly discovered virus strains were deposited on December 18, 2019, at the China General Microbiological Culture Collection Center (CGMCC). Specifically, the accession number for the Coxsackievirus A16 strain is CGMCC NO:18886. The accession number for the Coxsackievirus A10 strain is CGMCC NO:18887. The accession number for the Coxsackievirus A6 strain is CGMCC NO:18888.

[0009] In the combined vaccine of the present invention, the antigen content of the inactivated enterovirus 71 is 100-1000 U / mL.

[0010] In the combined vaccine of the present invention, the antigen content of the inactivated Coxsackievirus A16 is 200-3000 U / mL, preferably 200-1600 U / mL.

[0011] In the combined vaccine of the present invention, the antigen content of the inactivated Coxsackievirus A10 is 200-3000 U / mL, preferably 200-1600 U / mL.

[0012] In the combined vaccine of the present invention, the antigen content of the inactivated Coxsackievirus A6 is 1000-3000 U / mL.

[0013] As a specific embodiment of the present invention, the combined vaccine further includes an aluminum adjuvant.

[0014] In one specific embodiment of the present invention, the aluminum adjuvant is selected from aluminum hydroxide, aluminum phosphate, or aluminum sulfate.

[0015] As a specific embodiment of the present invention, the final concentration of aluminum content in the aluminum adjuvant, calculated as aluminum ions, is 0.1 to 1.0 mg / mL, preferably 0.2 to 0.8 mg / mL.

[0016] Preferably, the combined vaccine of the present invention is in liquid form.

[0017] The present invention also provides a method for preparing the combined vaccine, the method comprising the following steps: preparing each virus stock solution separately, adsorbing the stock solutions onto aluminum adjuvants to prepare virus aluminum adsorption products, and then mixing the aluminum adsorption products to prepare the combined vaccine.

[0018] The present invention also provides the use of the combined vaccine in the preparation of medicaments for the prevention or treatment of diseases caused by enterovirus 71, Coxsackievirus A16, Coxsackievirus A10 and / or Coxsackievirus A6.

[0019] The vaccine provided by this invention involves a combination of the four most common serotypes (EV71, CA16, CA10, CA6) that cause hand-foot-mouth disease, and can provide broader protection against hand-foot-mouth disease caused by EV71, CA16, CA10, and CA6.

[0020] Of the four serotypes of virus involved in this invention, CA16, CA10, and CA6 were independently isolated by the inventors in the laboratory, while EV71 was isolated at the Chinese CDC (Chinese Center for Disease Control and Prevention) and identified by the speckle sampling and library construction group of Beijing Sinovac Biotech Co., Ltd. The isolation method was as follows: samples collected from throat swabs or feces of clinical patients were diluted, filtered, inoculated into cells, purified and screened using speckle sampling, and identified to obtain pure single viruses; amplification was performed by inoculating cells to establish a virus stock library; and the virus stock underwent comprehensive testing and stability studies according to the relevant requirements of the 2015 edition of the Pharmacopoeia of the People's Republic of China.

[0021] As a specific embodiment of the present invention, the combined vaccine provided by the present invention is prepared through the following steps:

[0022] Preparation method of EV71 stock solution: EV71 virus is inoculated into Vero cells at a certain MOI (Multiple of Infection), and cultured in a microcarrier fermenter for scale-up or in a cell factory for 5-9 days; the viral fluid is then harvested. The viral fluid is inactivated with formaldehyde and concentrated by ultrafiltration using a 100-500 kDa ultrafiltration membrane. The concentrated viral fluid is then purified and separated into hollow and solid viral particles by sucrose density gradient centrifugation. The viruses in the target tubes are collected and desaccharified by ultrafiltration; the desaccharified viral fluid is treated with non-restriction endonucleases, host cell DNA is removed by ultrafiltration, and molecular sieve chromatography is used to remove residual endonucleases and other impurities. The above steps yield the EV71 virus stock solution.

[0023] Preparation method of CA16 stock solution: CA16 virus is inoculated into Vero cells at a certain MOI (Multiple of Infection), and cultured in a fermenter with microcarriers for stepwise scale-up or in a cell factory for 4-9 days; the viral fluid is harvested. It is clarified and concentrated using a 100-500 kDa ultrafiltration membrane; the concentrated viral fluid is purified by sucrose density gradient centrifugation to remove impurities and separate hollow and solid viral particles. The collected viruses in the target tubes are desaccharified by ultrafiltration; the desaccharified viral fluid is treated with non-restriction endonucleases, and host cell DNA is removed by ultrafiltration; it is then inactivated with formaldehyde. The above steps yield the CA16 virus stock solution.

[0024] Preparation method of CA10 stock solution: CA10 virus is inoculated into Vero cells at a certain MOI (Multiple of Infection), and cultured in a fermenter with microcarriers for stepwise scale-up or in a cell factory for 4-9 days; the viral fluid is harvested. It is clarified and concentrated using a 100-500 kDa ultrafiltration membrane; the concentrated viral fluid is purified by sucrose density gradient centrifugation to remove impurities and separate hollow and solid viral particles. The collected viruses in the target tubes are combined and desaccharified by ultrafiltration; the desaccharified viral fluid is treated with non-restriction endonucleases, and host cell DNA is removed by ultrafiltration; it is then inactivated with formaldehyde. The above steps yield the CA10 virus stock solution.

[0025] Preparation method of CA6 stock solution: CA6 virus is inoculated into Vero cells at a certain MOI (Multiple of Infection), and cultured in a fermenter with microcarriers for stepwise scale-up or in a cell factory for 4-9 days; the viral fluid is harvested. It is clarified and concentrated using a 100-500 kDa ultrafiltration membrane; the concentrated viral fluid is purified by sucrose density gradient centrifugation to remove impurities and separate hollow and solid viral particles. The collected viruses in the target tubes are desaccharified by ultrafiltration; the desaccharified viral fluid is treated with non-restriction endonucleases, and host cell DNA is removed by ultrafiltration; it is then inactivated with formaldehyde. The above steps yield the CA6 virus stock solution.

[0026] The virus purification process is carried out entirely using physical methods, which effectively removes impurities such as host proteins, host DNA, and endonucleases, thus ensuring vaccine safety.

[0027] The stock solutions of the four serotypes were adsorbed with aluminum adjuvants, mixed in a certain proportion, and formulated into vaccine semi-finished products. After being packaged, they were made into finished vaccines.

[0028] Beneficial effects

[0029] The combined vaccine of this invention exhibits good process consistency and stability; through extensive experiments, the optimal inactivation process parameters were screened, and the finished product demonstrates good stability; animal experiments have proven its good safety and efficacy.

[0030] The combined vaccine of this invention contains the main pathogens causing hand-foot-mouth disease (HFMD) and can effectively prevent HFMD caused by EV71, CA16, CA10, and CA6 viruses. Research in this invention shows that the various antigens do not interfere with each other's antigenicity and immunizing effect after immunization of recipients, exhibiting excellent immunogenicity and safety.

[0031] The combined vaccine provided by this invention can simultaneously prevent infection by multiple pathogens, and there is no interference between these antigens. The corresponding immunogenicity is not reduced compared to that induced by single antigens. The use of combined vaccines can significantly simplify the vaccination procedure, improve vaccination efficiency, and reduce costs. This represents a major trend in future technological development and reflects future market demand. Detailed Implementation

[0032] The present invention will be further illustrated below by means of embodiments. It should be understood that the embodiments of the present invention are merely for illustrating the present invention and are not intended to limit the present invention. Simple improvements to the present invention under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0033] 1. Preparation of finished combination vaccines

[0034] (1) Isolation of the virus strain

[0035] Throat swabs or feces from patients with hand-foot-mouth disease were obtained from disease control and prevention centers across the country. The samples were diluted with physiological saline, centrifuged, and filtered through 0.45 μm and 0.22 μm filters for sterilization. Sensitive cells (Vero cells or human diploid cells) with good cell growth and confluence into a monolayer were selected. The virus culture medium was a 199 solution containing 2% fetal bovine serum. The treated throat swabs / feces samples were inoculated at a specific ratio and incubated statically in a 32.0℃~36.5℃ 5% CO2 incubator. The cells were observed daily for the presence of characteristic enterovirus-induced cytopathic effect (CPE). If CPE occurred and reached a level of +++ or higher, the cell culture was harvested and passaged twice. If no CPE occurred, the cells were cultured for 7 days, frozen and thawed once, and the cell culture was harvested and passaged twice more. One cell culture tube with good growth and no inoculated samples was used as a cell control. The experiment was considered valid if the cell control showed no CPE.

[0036] After three blind passages, specimens showing enterovirus characteristic lesions (CPE) were identified as positive for virus isolation. Cell cultures that were positive for virus isolation were further identified by PCR.

[0037] (2) Identification of the virus strain

[0038] The isolated strains were identified at the immunological and molecular levels using ELISA and PCR, respectively.

[0039] EV71 antigen detection system: Coat with EV71 rabbit polyclonal antibody and incubate overnight at 2-8℃. Block with 10% fetal bovine serum at 37℃ for 1-2 hours. Add the virus culture to be identified, set up negative control wells, incubate at 36-37℃ for 1 hour, wash 3-5 times, add EV71 type-specific monoclonal antibody, incubate at 36-37℃ for 1 hour, wash 3-5 times, develop color, and terminate the reaction.

[0040] CA16 antigen detection system: Coat with CA16 rabbit polyclonal antibody overnight at 2-8℃, block with 10% fetal bovine serum at 37℃ for 1-2 hours, add the virus culture to be identified, set up negative control wells, incubate at 36-37℃ for 1 hour, wash 3-5 times, add CA16 type-specific monoclonal antibody, incubate at 36-37℃ for 1 hour, wash 3-5 times, develop color, and terminate the reaction.

[0041] CA10 antigen detection system: Coat with CA10 rabbit polyclonal antibody, incubate overnight at 2-8℃, block with 10% fetal bovine serum at 37℃ for 1-2 hours, add the virus culture to be identified, set up negative control wells, incubate at 36-37℃ for 1 hour, wash 3-5 times, add CA10 type-specific monoclonal antibody, incubate at 36-37℃ for 1 hour, wash 3-5 times, develop color, and terminate the reaction.

[0042] CA6 antigen detection system: Coat with CA6 rabbit polyclonal antibody, incubate overnight at 2-8℃, block with 10% fetal bovine serum at 37℃ for 1-2 hours, add the virus culture to be identified, set up negative control wells, incubate at 36-37℃ for 1 hour, wash 3-5 times, add CA6 type-specific monoclonal antibody, incubate at 36-37℃ for 1 hour, wash 3-5 times, develop color, and terminate the reaction.

[0043] The selected CA16, CA10, CA6, and EV71 strains were identified using the four antigen detection systems described above, and the results are shown in Tables 1 to 4.

[0044] Table 1. Results of ELISA antigen identification of CA16 strain

[0045] strain OD value Result judgment CA16 strain 2.015 Positive CA10 strain 0.054 Negative CA6 strain 0.061 Negative EV71 strain 0.049 Negative Negative control well 0.068 negative results

[0046] Table 2. Results of ELISA antigen identification of CA10 strain

[0047] strain OD value Result judgment CA16 strain 0.034 Negative CA10 strain 1.875 Positive CA6 strain 0.045 Negative EV71 strain 0.056 Negative Negative control well 0.058 negative results

[0048] Table 3. Results of ELISA antigen identification of CA6 strain

[0049] strain OD value Result judgment CA16 strain 0.042 Negative CA10 strain 0.059 Negative CA6 strain 2.154 Positive EV71 strain 0.061 Negative Negative control well 0.047 negative results

[0050] Table 4. Results of ELISA antigen identification of EV71 strain

[0051] strain OD value Result judgment CA16 strain 0.039 Negative CA10 strain 0.043 Negative CA6 strain 0.038 Negative EV71 strain 2.046 Positive Negative control well 0.050 negative results

[0052] Molecular-level identification: Viral RNA was extracted using a kit (QIAGEN), and the extracted RNA was subjected to one-step reverse transcription and PCR amplification using a reverse transcription kit (TAKARA). Primer sequences were designed in-house and synthesized by BGI Genomics Co., Ltd. The PCR products were identified by 1% agarose gel electrophoresis. Reverse transcription conditions:

[0053]

[0054] After PCR amplification and sequencing, the sequencing results were compared with the NCBI database and identified as the corresponding CA16, CA10, and CA6 strains.

[0055] (3) Production of unit price stock solution

[0056] The amount of microcarriers added to a 130L fermenter is 2-6 g / L, based on a cell count of 10-50 × 10⁻⁶. 4 Inoculate Vero cells at a concentration of 10 cells / mL, pH 7.0–7.5, with dissolved oxygen fluctuating within a small range of 50%. Culture at 35–37.5°C for 3–7 days. Based on the cell count, inoculate with virus at a ratio of 0.001–0.1 MOI and continue culturing for 3–6 days before harvesting the virus.

[0057] The virus solution is clarified and concentrated using a 100-500 kDa ultrafiltration membrane to remove impurities and proteins. The concentrated virus solution is then purified by sucrose density gradient centrifugation to remove impurities and separate hollow and solid virus particles. The ultracentrifugation conditions are 2-8℃, 20000-50000 rpm, and 8-18 hours. The collected viruses in the target tube are then desaccharified using ultrafiltration. The desaccharified virus solution is then treated with a non-restriction endonuclease at 18-30℃ with stirring, followed by ultrafiltration to remove host cell DNA. Finally, the virus is inactivated using a 1:2000–1:8000 formaldehyde solution at 35-38℃ for 2-6 days until completely inactivated. These steps yield the CA16, CA10, and CA6 virus stock solutions.

[0058] The production of EV71 stock solution is carried out by fermentation tanks or cell factories. After the virus is harvested, it is first inactivated and then purified. Other process steps are similar to those of CA16, CA10 and CA6 types.

[0059] (4) Preparation of monovalent aluminum adsorbed vaccine

[0060] Preparation of monovalent aluminum adsorbed vaccine: The aluminum adjuvant was diluted to 3.0 mg / mL (calculated as Al(OH)3) with 0.85% physiological saline. The EV71 antigen was diluted to 400-6000 U / mL with 0.01M PBS, the CA16 and CA10 antigens were diluted to 800 U / mL-9600 U / mL, and the CA6 antigen was diluted to 4000 U / mL-18000 U / mL. At room temperature, the diluted aluminum adjuvant was added to the diluted EV71, CA16, CA10, and CA6 antigens respectively, so that the two were adsorbed in equal volumes. The mixture was stirred while adding the adjuvant. After the mixture was added, it was mixed at room temperature for 30 minutes to obtain the monovalent aluminum adsorbed vaccine of EV71, CA16, CA10, and CA6.

[0061] (5) Preparation of quadrivalent combined vaccine

[0062] The four types of monovalent aluminum adsorption products mentioned above are mixed and packaged in a certain proportion to prepare a finished tetravalent hand-foot-mouth disease combined vaccine. The specific operation process is as follows:

[0063] The monovalent aluminum-adsorbed vaccines of EV71, CA16, CA10, and CA6 obtained above were mixed in a certain proportion. Based on the antigen content of the vaccine stock solution, they were appropriately diluted with 0.01M PBS (pH 7.2) solution and aluminum hydroxide solution. The mixture was then stirred and adsorbed at room temperature for 20±10 minutes to obtain the vaccine semi-finished product. In the obtained vaccine semi-finished product, the final concentration of aluminum hydroxide was approximately 1.30 mg / mL (equivalent to a final aluminum ion concentration of 0.45 mg / mL), the antigen content of EV71 was 960 U / mL, the antigen content of CA16 was 200 U / mL to 1600 U / mL, the antigen content of CA10 was 200 U / mL to 1600 U / mL, and the antigen content of CA6 was 1000 U / mL to 3000 U / mL. The protein dose range corresponding to each antigen content in the semi-finished product was 1 μg / mL to 8 μg / mL, meaning the total protein dose of the tetravalent semi-finished vaccine was 4 μg / mL to 32 μg / mL. The obtained vaccine semi-finished product was then filled into syringes to obtain the combined vaccine product. The dosage of the combined vaccine product is 0.5 mL / person, and the protein content of each viral component in the combined vaccine product is ≤4 μg / person.

[0064] The quadrivalent hand-foot-mouth disease combined vaccine was prepared according to the above method. The specific parameters are shown in Table 5. Three consecutive batches of combined vaccine were prepared for each parameter.

[0065] Table 5 Parameter settings for quadrivalent combined vaccine

[0066]

[0067] 2. Evaluation of combination vaccines

[0068] (1) Testing of finished tetravalent vaccine

[0069] The content of aluminum hydroxide, supernatant antigen, and antigen content after dissociation in the finished products of the quadrivalent hand-foot-mouth disease combined vaccines of Examples 1 to 15 were tested respectively, and the adsorption rate and dissociation rate were calculated. The results are shown in Table 6.

[0070] Table 6. Test Results of Quadrivalent Vaccine Finished Product

[0071]

[0072]

[0073] (2) Immunogenicity test

[0074] Three batches of the quadrivalent vaccine from Example 3 were used to immunize rats and mice twice each, and the titers of neutralizing antibodies in their serum were detected. A monovalent vaccine control and a negative control were also established. The experiment was repeated three times. The neutralizing antibody titer was detected using the microcytopathic effect method. RD cells were used for the assay; the neutralizing cells were cultured at 37°C for 7 days, and cytopathic effects were observed. The results of neutralizing antibody titers and seroconversion rates are shown in Table 7. The immunogenicity of each viral component in the combined vaccine was statistically analyzed compared with the corresponding monovalent vaccine, and the results are shown in Table 8. According to the statistical analysis, there was no significant difference in the GMT values ​​of neutralizing antibodies between the combined vaccine and the monovalent vaccine (P values ​​were all greater than 0.05).

[0075] Table 7. Neutralizing antibody titer and seroconversion rate

[0076]

[0077] Table 8. Statistical analysis of immunogenicity results of combined vaccines and single vaccines

[0078]

[0079] (3) Safety evaluation

[0080] Throughout the experimental period assessing the immunogenicity of each combined vaccine, all animals survived healthily and showed no abnormal clinical manifestations. Animal safety evaluations were conducted on the three batches of the EV71 / CA16 / CA10 / CA6 quadrivalent combined vaccine from Example 3, including abnormal toxicity tests, acute toxicity tests in mice, and active anaphylaxis tests in guinea pigs.

[0081] The adverse toxicity tests included mouse and guinea pig studies. In the mouse study, five mice weighing 18–22g were injected with the EV71 / CA16 / CA10 / CA6 combined vaccine, 0.5 mL per mouse, and observed for 7 days. All mice survived the observation period without adverse reactions, and their body weight increased at the end of the observation period. In the guinea pig study, two guinea pigs weighing 250–350g were injected with the EV71 / CA16 / CA10 / CA6 combined vaccine, 5 mL per guinea pig, and observed for 7 days. All guinea pigs survived the observation period without adverse reactions, and their body weight increased at the end of the observation period.

[0082] In the acute toxicity test in mice, the EV71 / CA16 / CA10 / CA6 combined vaccine was administered intramuscularly to Kunming mice using the maximum dose method at a dose of 8000 U / kg body weight (equivalent to approximately 120 times the clinical dose for a child with a body weight of 0.5 years). No abnormal clinical symptoms or deaths were observed in the administered animals.

[0083] In the active anaphylaxis test in guinea pigs, Hartley guinea pigs were sensitized by intramuscular injection and given the combined vaccine three times, with a sensitizing dose of 0.5 mL / pig / dose, administered every other day. On day 14 after the last sensitization dose, a challenge dose of 1.0 mL / pig / dose was administered intravenously once. Allergic reactions were continuously observed in the experimental animals after the challenge dose. The results showed that the EV71 / CA16 / CA10 / CA6 combined vaccine produced a negative allergic reaction in the systemic active anaphylaxis test in guinea pigs.

[0084] (4) Stability evaluation

[0085] The three batches of combined vaccine products prepared in Example 3 were stored at 2–8°C. Samples were taken at different time points to test antigen content, appearance, dosage, pH value, osmolality, bacterial endotoxin, and formaldehyde content. Data were recorded for up to 12 months, and abnormal toxicity and immunogenicity of the vaccine were tested in December. No significant decrease was observed in any of the tested indicators after 12 months of storage at 2–8°C. The results are shown in Tables 9–11.

[0086] Table 9. Results of antigen content detection in combined vaccines at 2–8℃ (percentage of labeled amount)

[0087]

[0088] Table 10. Test results of finished combined seedlings at 2~8℃ 1

[0089]

[0090] Table 11.2~8℃ Combined Seedling Finished Product Test Results 2

[0091]

[0092] Note: " / " indicates that this test was not performed.

[0093] Except for Example 3, Examples 1-2 and Examples 4-15 all underwent immunogenicity testing, as well as safety and stability evaluations. The results showed that the neutralizing antibody GMT values ​​of the combined vaccines and single vaccines in these examples were not significantly different. Furthermore, the combined vaccines in these examples, similar to those in Example 3, exhibited good safety and high stability.

[0094] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0095] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0096] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A combination vaccine for the prevention of hand, foot and mouth disease, characterized in that, The antigens of the combined vaccine include inactivated enterovirus 71 virus, and inactivated coxsackievirus A16 virus, coxsackievirus A10 virus and coxsackievirus A6 virus; The strain of the coxsackievirus A16 virus has a preservation number of CGMCC NO: 18886, The strain of the coxsackievirus A10 virus has a preservation number of CGMCC NO: 18887, The strain of the coxsackievirus A6 virus has a preservation number of CGMCC NO: 18888.

2. The combined vaccine according to claim 1, wherein the content of the inactivated enterovirus 71 virus is 100-1000 U / mL; The content of the inactivated coxsackievirus A16 virus in the combined vaccine is 200-3000 U / mL. The content of the inactivated coxsackievirus A10 virus in the combined vaccine is 200-3000 U / mL. The content of the inactivated coxsackievirus A6 virus in the combined vaccine is 1000-3000 U / mL. The content of the inactivated coxsackievirus A16 virus in the combined vaccine is 200-1600 U / mL.

3. The combination vaccine according to claim 2, characterized in that, The content of the inactivated coxsackievirus A10 virus in the combined vaccine is 200-1600 U / mL.

4. The combination vaccine of claim 2, characterized in that, The combined vaccine further comprises an aluminum adjuvant.

5. The combination vaccine according to any one of claims 1 to 4, characterized in that, The aluminum adjuvant is selected from aluminum hydroxide, aluminum phosphate or aluminum sulfate.

6. The combination vaccine of claim 5, characterized in that, The aluminum adjuvant is aluminum hydroxide.

7. The combination vaccine according to claim 6, characterized in that, The aluminum content of the aluminum adjuvant is 0.1-1.0 mg / mL in terms of aluminum ions.

8. The combination vaccine of claim 5, characterized in that, The aluminum content of the aluminum adjuvant is 0.2-0.8 mg / mL in terms of aluminum ions.

9. The combination vaccine according to claim 8, characterized in that, The combined vaccine is in a liquid dosage form.

10. The combination vaccine according to any one of claims 1 to 4, characterized in that, The combined vaccine is prepared by the following steps: preparing each virus stock solution respectively, adsorbing each virus stock solution on an aluminum adjuvant respectively to obtain aluminum adsorption products, and mixing the aluminum adsorption products to prepare the combined vaccine.

11. A method for the preparation of the combination vaccine according to any one of claims 1 to 10, characterized in that, 12. Use of the combined vaccine according to any one of claims 1-10 in the preparation of a medicament for preventing or treating diseases caused by enterovirus 71 virus, coxsackievirus A16 virus, coxsackievirus A10 virus and / or coxsackievirus A6 virus. ​

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