A combined vaccine for preventing hand, foot and mouth disease, and its preparation method and application

Abstract

The present invention discloses a combination vaccine for preventing hand, foot and mouth disease, comprising inactivated enterovirus 71 and inactivated coxsackievirus group A type 16. The present invention also discloses a method for preparing the combination vaccine, which exhibits excellent adsorption efficacy and stability. The multiple antigens described above do not interfere with each other's antigenicity or immune effects after immunization, resulting in excellent immunogenicity and safety. The use of the combination vaccine can significantly simplify the vaccination process, improve vaccination efficiency, and reduce costs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a combined vaccine for preventing hand, foot and mouth disease, and a preparation method and application thereof. Background Art

[0002] Hand, foot, and mouth disease (HFMD), classified as a Category C infectious disease under my country's Infectious Disease Prevention and Control Law, is an acute infectious disease caused by various enteroviruses, most commonly affecting infants and young children. It is prevalent in the summer, with a higher incidence in preschoolers, though adults can be an indirect source of infection. Symptoms primarily include a rash on the mouth, hands, and feet, and can be complicated by meningitis, encephalitis, pulmonary edema, circulatory failure, and other serious complications, leading to death. In addition to the more common enterovirus 71 (EV71) and CA16 (CA16), HFMD is also caused by coxsackievirus A (CA) types 2, 4, 5, 6, and 10; coxsackievirus B (CB) types 1, 2, 3, 4, and 5; enterovirus 68 (EV68); and echoviruses. EV71 and CA16 are the main pathogens causing HFMD outbreaks.

[0003] Hand, foot and mouth disease (HFMD) has become a serious public health issue threatening children's health and social stability. Currently, there are no effective antiviral drugs for the treatment of HFMD. Although an approved inactivated EV71 vaccine is available, the changing pathogenic spectrum of enteroviruses in HFMD, the diversity of the dominant strains responsible for HFMD, and the lack of cross-immunity between viral strains mean that protection against other HFMD strains is ineffective, posing new challenges to HFMD prevention and control. EV71 vaccination is insufficient to control HFMD outbreaks and epidemics caused by CA16. Therefore, the development of corresponding multivalent and combined vaccines is urgently needed to address HFMD outbreaks and the prevalence of severe cases. Currently, the development of a bivalent EV71 / CA16 combination vaccine is progressing slowly, and no related product has yet been successfully marketed. The reasons are as follows: First, hand, foot, and mouth disease (HFMD) caused by the EV71 and CA16 viruses has recently experienced a large-scale outbreak; second, HFMD caused by viruses has a certain regional distribution, and in countries with relatively low incidence rates, fewer institutions have invested in research and development; third, the immune interference between different viral antigens is unknown, and this interference may directly reduce the immunogenicity of a single vaccine component, affecting the immune response. Therefore, in order to protect the body from the HFMD virus series on a larger scale, it is imperative to establish a comprehensive HFMD prevention and control system and expand protection through the preparation of combined vaccines.

[0004] An EV71 vaccine is now available. Clinical research data shows that the vaccine is effective in preventing hand, foot and mouth disease caused by EV71, especially severe cases. However, the EV71 vaccine has weak cross-protection against Coxsackievirus. Given the changing trends in the prevalence of enterovirus serotypes, there is an urgent need for a multi-serotype combined vaccine that can prevent hand, foot and mouth disease on a larger scale. Summary of the Invention

[0005] The purpose of the present invention is to provide a combined vaccine for preventing hand, foot and mouth disease.

[0006] Specifically, the present invention provides a combined vaccine for preventing hand, foot and mouth disease, which comprises inactivated enterovirus 71 virus and inactivated coxsackievirus group A type 16 virus.

[0007] Among them, the enterovirus 71 virus strain is preferably the strain with the deposit number CGMCC No.3544.

[0008] The inactivated Coxsackievirus A16 strain described in the present invention is a newly discovered strain with excellent immunogenicity. All of the newly discovered strains were deposited with the China General Microbiology Center (CGMCC) of the China Microorganism Culture Collection on December 18, 2019. Specifically, the deposit number of the Coxsackievirus A16 strain is CGMCC NO: 18886.

[0009] In the present invention, the combined vaccine is preferably a two-in-one vaccine comprising the inactivated enterovirus 71 virus and the inactivated coxsackievirus group A type 16 virus with the above-mentioned specific deposit number.

[0010] In the combined vaccine of the present invention, the antigen content of the inactivated enterovirus 71 virus is 100 to 1000 U / mL.

[0011] In the combined vaccine of the present invention, the content of the inactivated Coxsackievirus group A type 16 antigen is 200-3000 U / mL, preferably 200-1600 U / mL.

[0012] As a specific embodiment of the present invention, the combination vaccine further comprises an aluminum adjuvant.

[0013] As a specific embodiment of the present invention, the aluminum adjuvant is selected from aluminum hydroxide, aluminum phosphate or aluminum sulfate.

[0014] As a specific embodiment of the present invention, the final concentration of aluminum content in the aluminum adjuvant is 0.1 to 1.0 mg / mL, preferably 0.2 to 0.8 mg / mL, calculated as aluminum ions.

[0015] In the present invention, the combination vaccine is preferably in liquid dosage form.

[0016] The present invention also provides a method for preparing the combined vaccine, which comprises the following steps: preparing each virus stock solution separately, adsorbing the stock solution onto an aluminum adjuvant respectively to prepare a virus aluminum adsorption product, and then mixing the aluminum adsorption products to prepare the combined vaccine.

[0017] The present invention also provides use of the combined vaccine in preparing a drug for preventing or treating diseases caused by enterovirus 71 and / or coxsackievirus group A type 16.

[0018] The vaccine provided by the present invention involves a combination of the two most common serotypes (EV71 and CA16) that cause hand, foot and mouth disease, and can protect against hand, foot and mouth disease caused by EV71 and CA16 on a larger scale.

[0019] Of the two serotypes of viruses covered by this invention, CA16 was isolated independently by the inventors in their laboratory, while EV71 was isolated at the China CDC (China Center for Disease Control and Prevention) and identified by the plaque selection and library construction team of Beijing Kexing Biological Products Co., Ltd. The isolation method involves diluting and filtering specimens collected from clinical patient throat swabs or feces, inoculating cells, and performing plaque selection, purification, screening, and identification to isolate a pure single virus. The inoculated cells are then amplified and a virus seed library is established. The virus seeds are then fully tested and stability studied in accordance with the relevant requirements of the 2015 edition of the Pharmacopoeia of the People's Republic of China.

[0020] As a specific embodiment of the present invention, the combined vaccine provided by the present invention is prepared by the following steps:

[0021] Preparation of EV71 stock solution: EV71 virus is inoculated into Vero cells at a specific MOI (multiplicity of infection) and amplified in microcarrier fermenters or cultured in a cell factory for 5-9 days. The virus solution is harvested. The virus solution is inactivated with formaldehyde and concentrated by ultrafiltration using a 100-500 kD ultrafiltration membrane. The concentrated virus solution is purified and separated into solid and empty virus particles using sucrose density gradient centrifugation. The virus collected from the target tube is combined and deglycosylated by ultrafiltration. The deglycosylated virus solution is treated with a non-restriction endonuclease, ultrafiltration is used to remove host cell DNA, and molecular sieve chromatography is used to remove impurities such as the endonuclease. The EV71 virus stock solution is obtained after these steps.

[0022] Preparation of CA16 stock solution: CA16 virus is inoculated into Vero cells at a specific MOI (multiplicity of infection), amplified in microcarrier fermentation tanks or cultured on a flat surface in a cell factory for 4-9 days; the virus solution is harvested. It is clarified and concentrated by ultrafiltration using a 100-500 kD ultrafiltration membrane; the ultrafiltered and concentrated virus solution is purified by sucrose density gradient centrifugation to remove impurities and separate empty and solid virus particles. The viruses collected from the ultrafiltration tube are combined and deglycosylated by ultrafiltration; the deglycosylated virus solution is treated with a non-restriction endonuclease and ultrafiltered to remove host cell DNA; and the virus is inactivated with formaldehyde. After these steps, the CA16 virus stock solution is obtained.

[0023] The virus liquid purification process is carried out entirely by physical methods, which effectively removes residual impurities such as host proteins, host DNA, and endonucleases, providing a guarantee for vaccine safety.

[0024] The two serum types' original solutions are adsorbed with aluminum adjuvant respectively, mixed in a certain proportion, and prepared into vaccine semi-finished products, which are then packaged to make finished vaccines.

[0025] Beneficial effects

[0026] The combined vaccine of the present invention has good process consistency and stable process; after a large number of experiments, the optimal inactivation process parameters have been screened, and the finished product has good stability; and animal experiments have proved that it has good safety and effectiveness.

[0027] The combined vaccine of the present invention contains the main pathogens of hand, foot and mouth disease (HFMD), effectively preventing the occurrence of HFMD caused by EV71 and / or CA16 viruses. Studies have shown that the various antigens mentioned above do not interfere with each other's antigenicity and immune efficacy after immunization of the recipient, demonstrating excellent immunogenicity and safety.

[0028] The combination vaccine provided by the present invention can simultaneously prevent infection from multiple pathogens, without interference between these antigens, and the corresponding immunogenicity is not reduced compared to the immunogenicity stimulated by each antigen alone. The use of combination vaccines can significantly simplify vaccination procedures, improve vaccination efficiency, and reduce costs. This is a major trend in future technological development and a future market demand. DETAILED DESCRIPTION

[0029] The present invention is further described below by way of examples. It should be understood that the examples of the present invention are merely for illustrating the present invention, rather than limiting the present invention. Simple improvements to the present invention based on the concept of the present invention fall within the scope of protection claimed in the present invention.

[0030] 1. Preparation of finished combination vaccines

[0031] (1) Isolation of virus strains

[0032] Throat swabs or stool samples from patients with hand, foot and mouth disease (HFMD) were obtained from local Centers for Disease Control and Prevention. The samples were diluted with normal saline, centrifuged, and sterilized by filtration through 0.45μm and 0.22μm filters. Sensitive cells (Vero cells or human diploid cells) with good growth and confluence to a monolayer were selected. Virus culture medium, a 199 solution containing 2% calf serum, was inoculated with the treated pharyngeal swab / stool specimen in a specific ratio and incubated in a 32.0°C–36.5°C, 5% CO2 incubator. The cells were observed daily for the development of characteristic enteroviral cytopathic effect (CPE). If CPE was present and reached a CPE level of +++ or higher, the cell culture was harvested and subcultured twice. If CPE was absent, the cells were cultured until day 7, frozen and thawed once, and the cell culture was harvested and subcultured twice. A well-grown, uninoculated cell culture tube was maintained as a control. The control should show no CPE, indicating a successful test.

[0033] After three blind passages, specimens with enterovirus-characteristic lesions (CPE) in cells were judged to be positive for virus isolation, and PCR identification was further performed on the cell cultures that were positive for virus isolation.

[0034] (2) Identification of virus strains

[0035] The isolated strains were identified at the immunological and molecular levels using Elisa enzyme-linked immunosorbent assay and PCR, respectively.

[0036] EV71 antigen detection system: Coat with EV71 rabbit polyclonal antibody at 2-8°C overnight, block with 10% calf serum at 37°C for 1-2 hours, add the virus culture to be identified, set up negative control wells, incubate at 36-37°C for 1 hour, wash the plate 3-5 times, add EV71-specific monoclonal antibody, incubate at 36-37°C for 1 hour, wash the plate 3-5 times, develop color, and terminate the reaction.

[0037] CA16 antigen detection system: Coat with CA16 rabbit polyclonal antibody at 2-8°C overnight, block with 10% calf serum at 37°C for 1-2 hours, add the virus culture to be identified, set up negative control wells, incubate at 36-37°C for 1 hour, wash the plate 3-5 times, add CA16-specific monoclonal antibody, incubate at 36-37°C for 1 hour, wash the plate 3-5 times, develop color, and terminate the reaction.

[0038] The above two antigen detection systems were used to identify the screened CA16 and EV71 strains, respectively. The results are shown in Tables 1 and 2.

[0039] Table 1. Results of Elisa antigen identification system for CA16 strains

[0040] strain OD value Result judgment CA16 strain 2.015 Positive EV71 strain 0.054 Negative Negative control wells 0.068 Negative

[0041] Table 2. Results of Elisa antigen identification system for EV71 strains

[0042] strain OD value Result judgment CA16 strain 0.039 Negative EV71 strain 1.865 Positive Negative control wells 0.050 Negative

[0043] Molecular identification: Viral RNA was extracted using a kit (QIAGEN), and the extracted RNA was reverse transcribed and amplified by PCR using a reverse transcription kit (TAKARA). The primer sequences were designed and synthesized by BGI Sequencing Co., Ltd. The PCR products were identified by 1% agarose gel electrophoresis. Reverse transcription conditions:

[0044] Reverse transcription reaction: 45℃ for 10 min

[0045] Reverse transcriptase inactivation: 94°C for 2 minutes

[0046]

[0047] After PCR amplification and sequencing, the sequencing results were compared with the NCBI database and identified as the corresponding type of CA16 strain.

[0048] (3) Production of monovalent stock solution

[0049] The dosage of microcarriers in a 130L fermenter is 2-6 g / L, and the number of cells is 10-50×10 4 Vero cells were inoculated with 100 μg / mL of culture medium at a pH of 7.0-7.5 and a dissolved oxygen content fluctuating within a small range of 50%. The cells were cultured at 35-37.5°C for 3-7 days. The virus was inoculated at an MOI of 0.001-0.1 based on the cell count. The cells were cultured for another 3-6 days and the virus was harvested.

[0050] The virus is clarified and concentrated using a 100-500 kD ultrafiltration membrane to remove impurity proteins. The ultrafiltered and concentrated virus solution is purified using sucrose density gradient centrifugation to remove impurities and separate empty and solid virus particles. Ultracentrifugation conditions are 2-8°C, a centrifugal speed of 20,000-50,000 rpm, and centrifugation for 8-18 hours. The viruses in the ultrafiltration target tube are combined and collected and desugared using ultrafiltration. The desugared virus solution is treated with a non-restriction endonuclease at 18-30°C with stirring and ultrafiltration to remove host cell DNA. The virus is inactivated using a 1:2000-1:8000 formaldehyde solution at 35-38°C for 2-6 days until the virus is completely inactivated. After the above steps, a CA16 virus stock solution is obtained.

[0051] The production of EV71 concentrate uses fermentation tanks or cell factories for culture. After the virus is harvested, it is first inactivated and then purified. The other process steps are equivalent to those of the CA16 type.

[0052] (4) Preparation of monovalent aluminum adsorption vaccine

[0053] Preparation of monovalent aluminum adsorption vaccine: dilute the aluminum adjuvant to 3.0 mg / mL (calculated as Al(OH)3) with 0.85% saline, dilute the EV71 antigen to 400-6000 U / mL with 0.01 M PBS, and dilute the CA16 antigen to 800 U / mL-9600 U / mL. Add the diluted aluminum adjuvant to the diluted EV71 and CA16 antigens at room temperature to allow equal volumes of adsorption, stirring while adding. After completion, continue mixing at room temperature for 30 minutes to obtain EV71 and CA16 monovalent aluminum adsorption vaccines.

[0054] (5) Preparation of multivalent combination vaccines

[0055] The two types of monovalent aluminum adsorption products are mixed and packaged in a certain proportion to prepare a bivalent hand, foot and mouth disease combined vaccine. The specific operation process is as follows:

[0056] The EV71 and CA16 monovalent aluminum adsorption vaccines obtained above are mixed in a certain proportion, appropriately diluted with 0.01M PBS (pH 7.2) solution and aluminum hydroxide solution according to the antigen content of the vaccine stock solution, and then stirred and adsorbed at room temperature for 20±10 minutes to obtain a vaccine semi-finished product. In the obtained vaccine semi-finished product, the final concentration of aluminum hydroxide is about 1.30mg / mL (the final concentration converted to aluminum ions is 0.45mg / mL), the antigen content of EV71 is 100U / mL~1000U / mL, and the antigen content of CA16 is 200U / mL~1600U / mL. The protein dosage range corresponding to the above-mentioned antigen content in the semi-finished product is 1μg / mL~8μg / mL, that is, the total protein dosage of the bivalent semi-finished vaccine is 2μg / mL~16μg / mL. The obtained vaccine semi-finished product is then syringe-filled to obtain a combined vaccine finished product. The dosage of the finished combined vaccine is 0.5 mL / person, and the protein content of each viral component in the finished combined vaccine is ≤4 μg / person.

[0057] The bivalent hand, foot and mouth disease combined vaccine was prepared according to the above method. The specific parameters are shown in Table 3. Three consecutive batches of combined vaccine were prepared for each parameter.

[0058] Table 3 Parameter settings for bivalent combination vaccines

[0059]

[0060] 2. Evaluation of combination vaccines

[0061] (1) Combined vaccine product testing

[0062] The aluminum hydroxide content, supernatant antigen content, and dissociated antigen content in the finished product of the above-mentioned bivalent hand, foot and mouth disease combined vaccine were respectively tested, and the adsorption rate and dissociation rate were calculated. The results are shown in Table 4.

[0063] Table 4 Test results of finished bivalent vaccine products

[0064]

[0065] (2) Immunogenicity test

[0066] The three batches of bivalent vaccines in Example 5 above were used to immunize rats and mice with two injections, respectively, and the neutralizing antibody titer in the serum was detected. At the same time, a monovalent vaccine control and a negative control were set up. Repeat three times. The neutralizing antibody titer was detected by microcytopathic method. The cells used for the test were RD cells, and the neutralization experimental cells were cultured at 37°C for 7 days to observe the cytopathic effect. The results of the neutralizing antibody titer and the positive conversion rate are shown in Table 5. The immunogenicity of each virus component in the combined vaccine was statistically analyzed with the corresponding monovalent vaccine, and the results are shown in Table 6. According to the statistical analysis, there was no significant difference in the neutralizing antibody GMT values ​​between the combined vaccine and the single vaccine (P values ​​were all greater than 0.05).

[0067] Table 5. Neutralizing antibody titer and positive conversion rate

[0068]

[0069] Table 6. Statistical analysis of immunogenicity results of combined vaccines and single vaccines

[0070]

[0071] (3) Safety evaluation

[0072] All animals survived healthy and showed no abnormal clinical manifestations throughout the experimental period for evaluating the immunogenicity of each combination vaccine. The three batches of EV71 / CA16 bivalent combination vaccine in Example 5 were subjected to animal safety evaluation, including abnormal toxicity tests, acute toxicity tests in mice, and active allergy tests in guinea pigs.

[0073] Abnormal toxicity testing included both mouse and guinea pig studies. In the mouse study, five mice weighing 18-22 g were injected with the EV71 / CA16 combination vaccine at 0.5 mL per mouse and observed for 7 days. All mice survived the observation period without any adverse reactions, and their weight increased at the end of the study. In the guinea pig study, two guinea pigs weighing 250-350 g were injected with the EV71 / CA16 combination vaccine at 5 mL per mouse and observed for 7 days. All guinea pigs survived the observation period without any adverse reactions, and their weight increased at the end of the study.

[0074] In the acute toxicity test in mice, the EV71 / CA16 combination vaccine was administered intramuscularly to Kunming mice using the maximum dose method at a dose of 8000U / kg body weight (equivalent to approximately 120 times the clinical dose for a 0.5-year-old child). No abnormal clinical symptoms or death occurred in the administered animals.

[0075] In the active allergy test in guinea pigs, Hartley guinea pigs were sensitized with the combination vaccine three times via intramuscular injection at a sensitization dose of 0.5 mL per animal per dose, administered every other day. Fourteen days after the final sensitization dose, a challenge dose of 1.0 mL per animal per dose was administered intravenously once. The animals were continuously observed for allergic reactions following the challenge. The results showed that the EV71 / CA16 combination vaccine was negative for allergic reactions in the systemic active allergy test in guinea pigs.

[0076] (4) Stability evaluation

[0077] The three batches of finished combination vaccines prepared in Example 5 were stored at 2-8°C. Samples were taken at regular intervals to test for antigen content, appearance, volume, pH, osmolarity, bacterial endotoxins, and formaldehyde content. Data were recorded for 12 months, and the vaccine was tested for abnormal toxicity and immunogenicity in December. After 12 months of storage at 2-8°C, no significant decrease in any of the tested parameters was observed. The results are shown in Tables 7-9.

[0078] Table 7. Antigen content test results of combined vaccine products at 2-8°C (percentage of labeled amount)

[0079]

[0080] Table 8. Test results of finished combined vaccine products at 2-8°C 1

[0081]

[0082] Table 9. Test results of finished combined vaccine products at 2-8℃2

[0083]

[0084] Note: “ / ” indicates that this test was not performed.

[0085] Except for Example 5, Examples 1-4 and Example 6 were all tested for immunogenicity, and safety and stability evaluations were also performed. The results showed that there was no significant difference in the neutralizing antibody GMT values ​​between the combination vaccines of these examples and the single vaccines. At the same time, the combination vaccines in these examples, similar to Example 5, all had the advantages of good safety and high stability.

[0086] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within 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 scope of protection of the present invention.

[0087] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0088] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A combined vaccine for preventing hand, foot and mouth disease, characterized in that: The antigens of the combined vaccine include inactivated enterovirus 71 virus and inactivated coxsackievirus A group 16 virus; wherein the deposit number of the coxsackievirus A group 16 virus strain is: CGMCC NO:18886.

2. The combined vaccine according to claim 1, characterized in that In the combined vaccine, the antigen content of the inactivated enterovirus 71 virus is 100 to 1000 U / mL.

3. The combined vaccine according to claim 1, characterized in that In the combined vaccine, the antigen content of the inactivated Coxsackievirus group A type 16 is 200 to 3000 U / mL.

4. The combined vaccine according to claim 3, characterized in that In the combined vaccine, the antigen content of the inactivated Coxsackievirus group A type 16 is 200 to 1600 U / mL.

5. The combined vaccine according to any one of claims 1 to 4, characterized in that The combination vaccine also includes an aluminum adjuvant.

6. The combined vaccine according to claim 5, characterized in that The aluminum adjuvant is selected from aluminum hydroxide, aluminum phosphate or aluminum sulfate.

7. The combined vaccine according to claim 6, characterized in that The aluminum adjuvant is aluminum hydroxide.

8. The combined vaccine according to claim 5, characterized in that The final concentration of aluminum content in the aluminum adjuvant is 0.1-1.0 mg / mL in terms of aluminum ions.

9. The combined vaccine according to claim 8, characterized in that The final aluminum content concentration of the aluminum adjuvant is 0.2-0.8 mg / mL in terms of aluminum ions.

10. The combined vaccine according to any one of claims 1 to 4, characterized in that The combined vaccine is in liquid dosage form.

11. The method for preparing the combined vaccine according to any one of claims 1 to 10, characterized in that: The method comprises the following steps: preparing virus stock solutions respectively, adsorbing virus stock solutions on aluminum adjuvants respectively to obtain virus aluminum adsorption products, and then mixing the aluminum adsorption products to prepare the combined vaccine.

12. Use of the combined vaccine according to any one of claims 1 to 10 in the preparation of a medicament for preventing or treating diseases caused by enterovirus 71 and / or coxsackievirus group A type 16.

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