WO2022141266A1 - 一种预防手足口病的联合疫苗及其制备方法和应用 - Google Patents

一种预防手足口病的联合疫苗及其制备方法和应用 Download PDF

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WO2022141266A1
WO2022141266A1 PCT/CN2020/141693 CN2020141693W WO2022141266A1 WO 2022141266 A1 WO2022141266 A1 WO 2022141266A1 CN 2020141693 W CN2020141693 W CN 2020141693W WO 2022141266 A1 WO2022141266 A1 WO 2022141266A1
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virus
type
vaccine
combined vaccine
group
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PCT/CN2020/141693
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French (fr)
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李雅静
沈欢
阴彦辉
纪伟
宋萌
高强
尹卫东
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北京科兴生物制品有限公司
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Priority to CN202080108306.2A priority Critical patent/CN116615234A/zh
Priority to EP20967606.3A priority patent/EP4272755A4/en
Priority to PCT/CN2020/141693 priority patent/WO2022141266A1/zh
Publication of WO2022141266A1 publication Critical patent/WO2022141266A1/zh
Priority to ZA2023/07375A priority patent/ZA202307375B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants

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  • the 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.
  • Hand-foot-mouth disease is classified as a Class C infectious disease in my country's "Infectious Disease Prevention and Control Law". It is an acute infectious disease caused by a variety of enteroviruses. . Popular in summer, preschool children have a high incidence, and adults can be an indirect source of infection; it is mainly manifested as rashes on the mouth, hands, and feet, and can be complicated by meningitis, encephalitis, pulmonary edema, circulatory failure and other serious diseases that lead to death.
  • HFMD Hand-foot-mouth disease
  • the viruses that cause HFMD also include Coxsackievirus A (Coxasckievirus A, CA) 2, 4, Types 5, 6, 10, etc., Coxsackievirus B (Coxasckievirus B, CB) types 1, 2, 3, 4, 5, etc., Enterovirus 68 (Enterovirus 68, EV68) and Echovirus (Echovirus) , Echo) etc.
  • EV71 and CA16 are the main pathogens causing HFMD outbreaks. In recent years, reports of HFMD outbreaks caused by CA10 and CA6 have gradually increased, and even become the main circulating serotypes in some areas.
  • HFMD has become one of the public health problems that seriously threaten children's health and social stability. At present, there is no effective antiviral drug for the treatment of hand, foot and mouth disease in clinical practice.
  • the approved EV71 inactivated vaccine is on the market, due to the change of the pathogenic spectrum of HFMD enterovirus and the variety of dominant strains leading to high incidence of HFMD , and there is no cross-immune protection between virus strains, it is impossible to protect the body infected with other types of HFMD, which poses a new challenge to the prevention and control of HFMD. It is difficult to control the outbreak and epidemic of HFMD caused by CA16, CA10 and CA6 with EV71 vaccine.
  • EV71 vaccine has come out. According to clinical research data, the use of the vaccine has a significant effect on the prevention of hand, foot and mouth disease caused by EV71, especially in severe cases. However, the cross protection of EV71 vaccine against Coxsackie virus is weak. In view of the trend of enterovirus circulating serotypes, there is an urgent need for a multi-serotype combination vaccine that can prevent hand, foot and mouth disease in a wider range.
  • the purpose of the present invention is to provide a combined vaccine for preventing hand, foot and mouth disease.
  • the combined vaccine for preventing hand, foot and mouth disease includes inactivated enterovirus type 71 virus, and inactivated Coxsackie virus group A type 16 virus, type 10 virus and type 6 virus .
  • the enterovirus type 71 virus strain is the strain of deposit number CGMCC No.3544.
  • the inactivated Coxsackie virus group A type 16 virus, type 10 virus and type 6 virus described in the present invention are all virus strains newly discovered in the present invention, and have excellent immunogenicity.
  • the above-mentioned newly discovered virus strains of the present invention were all deposited in the General Microorganism Center (CGMCC) of the China Microorganism Culture Collection Management Committee on December 18, 2019.
  • CGMCC General Microorganism Center
  • the deposit number of the coxsackie virus group A type 16 virus strain is: CGMCC NO: 18886.
  • the deposit number of the coxsackie virus group A type 10 virus strain is: CGMCC NO: 18887.
  • the deposit number of the coxsackie virus group A type 6 virus strain is: CGMCC NO: 18888.
  • the antigen content of the inactivated enterovirus type 71 virus is 100-1000 U/mL.
  • the antigen content of the inactivated Coxsackie virus group A type 16 is 200-3000 U/mL, preferably 200-1600 U/mL.
  • the antigen content of the inactivated Coxsackie virus group A type 10 is 200-3000 U/mL, preferably 200-1600 U/mL.
  • the antigen content of the inactivated Coxsackie virus group A type 6 is 1000-3000 U/mL.
  • the combined vaccine further includes an aluminum adjuvant.
  • the aluminum adjuvant is selected from aluminum hydroxide, aluminum phosphate or aluminum sulfate.
  • the final concentration of aluminum content of the aluminum adjuvant is 0.1-1.0 mg/mL in terms of aluminum ions, preferably 0.2-0.8 mg/mL.
  • the combined vaccine is in liquid dosage form.
  • the present invention also provides a method for preparing the combined vaccine.
  • the method includes the following steps: preparing each virus stock solution, respectively adsorbing the stock solution on an aluminum adjuvant, preparing the viral aluminum adsorption product, and then mixing the aluminum adsorption product to prepare the combined vaccine .
  • the present invention also provides that the combined vaccine can prevent or treat enterovirus type 71 virus, coxsackie virus group A type 16 virus, coxsackie virus group A type 10 virus and/or coxsackie virus group A type 6 virus
  • enterovirus type 71 virus coxsackie virus group A type 16 virus
  • coxsackie virus group A type 10 virus coxsackie virus group A type 10 virus
  • coxsackie virus group A type 6 virus The use of medicines for diseases caused by viruses.
  • the vaccine provided by the invention involves the combination of the four most common serotypes (EV71, CA16, CA10, CA6) causing hand, foot and mouth disease, and can protect the hand, foot and mouth disease caused by EV71, CA16, CA10 and CA6 in a wider range.
  • CA16, CA10 and CA6 were all isolated and obtained by the inventor in the laboratory, EV71 was isolated in China CDC (China Center for Disease Control and Prevention), and was obtained by Beijing Kexing Biological Products Co., Ltd. Identification of the spotting library group.
  • the isolation method is as follows: dilution and filtration of specimens collected from clinical patients' throat swabs or feces, inoculation of cells, spotting, purification, screening, identification, and isolation to obtain a pure single virus; inoculation of cells for amplification and establishment of a virus seed bank, according to the following steps: The relevant requirements of the 2015 edition of the Pharmacopoeia of the People's Republic of China are to carry out a complete verification and stability study of the virus species.
  • the combined vaccine provided by the present invention is prepared through the following steps:
  • EV71 virus is inoculated into Vero cells according to a certain MOI (multiplicity of infection), and the microcarrier fermenter is used for step-by-step amplification or cell factory plane culture for 5-9 days; the virus liquid is harvested.
  • the virus liquid is inactivated by formaldehyde, and the ultrafiltration membrane bag of 100-500KD is used for ultrafiltration concentration; the virus liquid after ultrafiltration concentration is purified by sucrose density gradient centrifugation and the empty solid virus particles are separated.
  • the target tube virus was collected and deglycosylated by ultrafiltration; the virus solution after deglycosylation was treated with unrestricted endonuclease, the host cell DNA was removed by ultrafiltration, and the residues of endonuclease and other impurities were removed by molecular sieve chromatography. The steps are to obtain EV71 virus stock solution.
  • CA16 virus is inoculated into Vero cells according to a certain MOI (multiplicity of infection), and the fermenter microcarrier is used for step-by-step amplification or cell factory plane culture for 4-9 days; the virus liquid is harvested.
  • MOI multipleplicity of infection
  • the 100-500KD ultrafiltration membrane package is used for clarification, ultrafiltration and concentration; the virus solution after ultrafiltration concentration is purified by sucrose density gradient centrifugation to remove impurities and separate hollow virus particles.
  • CA10 virus is inoculated into Vero cells according to a certain MOI (multiplicity of infection), and the fermentor microcarrier is used for step-by-step amplification or cell factory plane culture for 4-9 days; the virus liquid is harvested.
  • MOI multipleplicity of infection
  • the 100-500KD ultrafiltration membrane package is used for clarification, ultrafiltration and concentration; the virus solution after ultrafiltration concentration is purified by sucrose density gradient centrifugation to remove impurities and separate hollow virus particles.
  • CA6 virus is inoculated into Vero cells according to a certain MOI (multiplicity of infection), and the fermentor microcarrier is used for step-by-step amplification or cell factory plane culture for 4-9 days; the virus liquid is harvested.
  • MOI multipleplicity of infection
  • the 100-500KD ultrafiltration membrane package is used for clarification, ultrafiltration and concentration; the virus solution after ultrafiltration concentration is purified by sucrose density gradient centrifugation to remove impurities and separate hollow virus particles.
  • the virus liquid purification process is completely carried out by physical methods, which effectively removes the residues of impurities such as host proteins, host DNA, and endonucleases, and provides a guarantee for the safety of the vaccine.
  • the stock solutions of the four serotypes are respectively adsorbed with aluminum adjuvant, mixed according to a certain ratio, and prepared into semi-finished vaccines, which are subpackaged to make finished vaccines.
  • the combined vaccine of the invention has good process consistency and stable process; after a large number of experiments, the best inactivation process parameters have been screened, and the finished product has good stability; animal experiments have proved that it has good safety and effectiveness .
  • the combined vaccine of the invention contains the main pathogenic pathogens of hand, foot and mouth disease, and can well prevent the occurrence of hand, foot and mouth disease caused by EV71, CA16, CA10 and CA6 viruses.
  • the research of the present invention shows that the above-mentioned various antigens do not interfere with each other in antigenicity and immune effect after immunizing the recipient, and have good immunogenicity and safety.
  • the combined vaccine provided by the present invention can prevent the infection of multiple pathogens at the same time, and there is no mutual interference between these antigens, and the corresponding immunogenicity will not decrease compared with the immunogenicity challenged by a single antigen.
  • the use of combined vaccines can significantly simplify vaccination procedures, improve vaccination efficiency and reduce costs. This is the general trend of future technology development and the future market demand.
  • Throat swabs or feces of patients with hand, foot and mouth disease were obtained from the Centers for Disease Control and Prevention, diluted with normal saline, centrifuged, and sterilized by filtration with 0.45 ⁇ m and 0.22 ⁇ m filters.
  • Sensitive cells Very cells or human diploid cells
  • the virus culture medium is a 199 solution containing 2% calf serum, inoculated with the treated throat swab/fecal specimen according to a certain proportion, and placed in a 32.0°C ⁇ 36.5°C5% CO 2 incubator for static culture.
  • CPE enterovirus cytopathic effect
  • CPE enterovirus characteristic lesions
  • the isolated strains were identified from immunological and molecular levels by Elisa enzyme-linked immunosorbent assay and PCR, respectively.
  • EV71 antigen detection system coat EV71 rabbit polyclonal antibody, overnight at 2-8°C, block with 10% calf serum at 37°C for 1-2 hours, add the virus culture to be identified, set a negative control well, warm at 36-37°C Incubate 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.
  • CA16 Antigen Detection System Coat CA16 rabbit polyclonal antibody, overnight at 2-8°C, block with 10% calf serum at 37°C for 1-2 hours, add the virus culture to be identified, set a negative control well, warm at 36-37°C Incubate 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.
  • CA10 antigen detection system Coat CA10 rabbit polyclonal antibody, overnight at 2-8°C, block with 10% calf serum at 37°C for 1-2 hours, add the virus culture to be identified, set a negative control well, warm at 36-37°C Incubate for 1 hour, wash the plate 3-5 times, add CA10-specific monoclonal antibody, incubate at 36-37°C for 1 hour, wash the plate 3-5 times, develop color, and terminate the reaction.
  • CA6 antigen detection system Coat CA6 rabbit polyclonal antibody, overnight at 2-8°C, block with 10% calf serum at 37°C for 1-2 hours, add the virus culture to be identified, set a negative control well, warm at 36-37°C Incubate for 1 hour, wash the plate 3-5 times, add CA6-specific monoclonal antibody, incubate at 36-37°C for 1 hour, wash the plate 3-5 times, develop color, and terminate the reaction.
  • the primer sequences were designed and synthesized by Huada Gene Sequencing Co., Ltd. PCR products were identified by 1% agarose gel electrophoresis.
  • Reverse transcription conditions :
  • the dosage of microcarriers in the 130L fermentor is 2 ⁇ 6g/L, and Vero cells are inoculated according to the number of cells 10 ⁇ 50 ⁇ 10 4 cells/mL, pH 7.0 ⁇ 7.5, dissolved oxygen fluctuates within a small range of 50%; 35 ⁇ 37.5 Cultivate for 3-7 days under the condition of °C, according to the number of cells, inoculate the virus according to the ratio of MOI 0.001-0.1, continue to culture for 3-6 days, and harvest the virus.
  • the 100-500KD ultrafiltration membrane package is clarified and concentrated by ultrafiltration to remove impurity proteins; the virus solution after ultrafiltration concentration is purified by sucrose density gradient centrifugation to remove impurities and separate the hollow virus particles.
  • the ultracentrifugation conditions are 2- 8°C, centrifugation speed 20000-50000rpm, centrifugation for 8-18 hours.
  • the EV71 stock solution is produced by fermenter or cell factory culture. After the virus is harvested, it is first inactivated and then purified.
  • the other processes are equivalent to CA16, CA10, and CA6 types.
  • Aluminum adjuvant was diluted to 3.0 mg/mL (as Al(OH) 3 ) with 0.85% normal saline
  • EV71 antigen was diluted to 400-6000 U/mL with 0.01M PBS
  • CA16 and The CA10 antigen was diluted to 800U/mL-9600U/mL
  • the CA6 antigen was diluted to 4000U/mL-18000U/mL
  • the diluted aluminum adjuvant was added to the diluted EV71, CA16, CA10, and CA6 antigens at room temperature, respectively.
  • the two were adsorbed in equal volumes, stirred while adding, and then continued to mix at room temperature for 30 minutes to obtain EV71, CA16, CA10, and CA6 monovalent aluminum-adsorbed vaccines.
  • the EV71, CA16, CA10, CA6 monovalent aluminum-adsorbed vaccines obtained above were mixed according to a certain proportion, and according to the antigen content of the vaccine stock solution, diluted appropriately with 0.01M PBS (pH 7.2) solution and aluminum hydroxide solution, and then stirred and adsorbed at room temperature for 20 Within ⁇ 10 minutes, the semi-finished product of the vaccine is obtained.
  • the final concentration of aluminum hydroxide is about 1.30 mg/mL (the final concentration converted into aluminum ions is 0.45 mg/mL)
  • the antigen content of EV71 is 960 U/mL
  • the antigen content of CA16 is 200 U.
  • the antigen content of CA10 is 200U/mL ⁇ 1600U/mL
  • the antigen content of CA6 is 1000U/mL ⁇ 3000U/mL
  • the protein dose range corresponding to the above antigen content in the semi-finished product is 1 ⁇ g/mL ⁇ 8 ⁇ g/mL, that is, the total protein dose of the tetravalent semi-finished vaccine is 4 ⁇ g/mL to 32 ⁇ g/mL.
  • syringe filling is performed on the obtained semi-finished product of the vaccine to obtain a finished product of the combined vaccine.
  • the dose of the finished combined vaccine is 0.5 mL/person
  • the protein content of each virus component in the finished combined vaccine is ⁇ 4 ⁇ g/person.
  • the quadrivalent hand-foot-mouth disease combined vaccine was prepared according to the above method, and the specific parameters were shown in Table 5, and three consecutive batches of combined vaccines were prepared for each parameter.
  • Rats and mice were immunized with two injections of the three batches of the tetravalent combined vaccine in the above Example 3, and the neutralizing antibody titers in the serum were detected. At the same time, a monovalent seedling control and a negative control were established. repeat three times. Neutralizing antibody titers were detected by microcytopathic assay. The cells used for the assay were RD cells, and the cells in the neutralization experiment were cultured at 37°C for 7 days, and cytopathic changes were observed. The results of neutralizing antibody titer and positive conversion rate are shown in Table 7. 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 8. According to statistical analysis, there was no significant difference in the GMT value of neutralizing antibody between the combined vaccine and the single vaccine (all P values were greater than 0.05).
  • Example 3 Three batches of EV71/CA16/CA10/CA6 quadrivalent combination vaccines in Example 3 were evaluated for animal safety, including abnormal toxicity test, acute toxicity test in mice and active allergy test in guinea pigs.
  • Abnormal toxicity tests include mouse tests and guinea pig tests.
  • EV71/CA16/CA10/CA6 combined vaccine was injected into 5 mice with 18-22g, 0.5mL/mice, and observed for 7 days. During the observation period, all the mice survived without abnormal reactions, and the mice gained weight when they expired.
  • EV71/CA16/CA10/CA6 combined vaccine was injected into 2 guinea pigs of 250-350 g, 5 mL/pig, and observed for 7 days. During the observation period, all the guinea pigs survived without any abnormal reaction, and the guinea pigs gained weight when they expired.
  • the EV71/CA16/CA10/CA6 combination vaccine was administered to Kunming mice by intramuscular injection at the maximum dosage method, at a dose of 8000U/kg body weight (equivalent to about 120 times the clinical dose of 0.5-year-old children) , no abnormal clinical symptoms and no death occurred in the administered animals.
  • mice were sensitized by intramuscular injection and given the combined vaccine 3 times, with a sensitizing dose of 0.5 mL/pig/time, administered every other day.
  • the mice were administered by intravenous injection, and the challenge dose was 1.0 mL/only/time, administered once. Allergic reactions were continuously observed in the experimental animals after the challenge administration.
  • the test results showed that the EV71/CA16/CA10/CA6 combination vaccine was negative in the guinea pig systemic active allergy test.
  • the three batches of combined seedling products prepared in Example 3 were stored in an environment of 2-8° C., and the antigen content, appearance, loading, pH value, osmolarity, bacterial endotoxin, formaldehyde content, etc. were detected by sampling according to time points. Data were recorded up to 12 months, and the vaccine was tested for abnormal toxicity and immunogenicity in December. The finished joint seedlings were placed at 2-8 °C for 12 months, and the detection indicators did not show a significant decrease. The results are shown in Tables 9 to 11.
  • Example 3 Example 3
  • Examples 1-2 and Examples 4-15 were all tested for immunogenicity, and also for safety evaluation and stability evaluation.
  • 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. Meanwhile, the combination vaccines in these examples were similar to Example 3, and both had the advantages of good safety and high stability.

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Abstract

提供了一种预防手足口病的联合疫苗，包括灭活肠道病毒71型病毒，以及灭活柯萨奇病毒A组16型病毒、10型病毒和6型病毒。还提供了制备所述联合疫苗的方法，所制得的疫苗吸附效果与稳定性均很好。上述多种抗原在免疫对象后互相不干扰抗原性和免疫效果，具有很好的免疫原性和安全性。联合疫苗的使用可以明显简化疫苗接种程序，提高接种效率并降低成本。

Description

一种预防手足口病的联合疫苗及其制备方法和应用 技术领域
本发明属于生物技术领域，具体涉及一种预防手足口病的联合疫苗及其制备方法和应用。
背景技术
手足口病(hand-foot-mouth disease，HFMD)在我国《传染病防治法》中列为丙类传染病，是一种由多种肠道病毒引起的急性传染病，以婴幼儿发病较为常见。夏季流行，学龄前儿童高发，成人可为间接传染源；主要表现为口腔、手、足部位皮疹，可并发脑膜炎、脑炎、肺水肿、循环衰竭等导致死亡的重症。引起手足口病的病毒除肠道病毒71型(Enterovirus 71，EV71)及CA16型(Coxasckievirus A16，CA16)较为常见外，还包括柯萨奇病毒A组(Coxasckievirus A，CA)的2、4、5、6、10型等，柯萨奇病毒B组(Coxasckievirus B，CB)的1、2、3、4、5型等，肠道病毒68型(Enterovirus 68，EV68)以及埃可病毒(Echovirus，Echo)等。EV71和CA16为引起HFMD爆发的主要病原体，近年来由CA10和CA6引起HFMD爆发的报道逐渐增多，甚至在某些地区已经成为主要的流行血清型。
手足口病已成为严重威胁儿童健康和社会稳定的公共卫生问题之一。目前，临床上尚无有效的治疗手足口病的抗病毒药物，虽然现有已获批的EV71灭活疫苗上市，但由于HFMD肠道病毒病原谱的改变以及导致HFMD高发的主导毒株类型多样，且病毒株之间不存在交叉免疫保护作用，无法实现对感染其他型别手足口病毒的机体的保护，对防控HFMD提出了新的挑战。接种EV71疫苗难以控制CA16、CA10和CA6引起的手足口病的爆发和流行。因此，急需制备相应的多价、多联疫苗，以应对HFMD的爆发和重症疾病的流行。目前还 没有EV71、CA16、CA10、CA6四价联合疫苗研制的相关报道，首先，EV71、CA16、CA10与CA6病毒引发的手足口病在近期较短时间内发生了大规模的爆发；其次，病毒引发的手足口病存在一定的地域性分布，对于发生率相对较低的发达国家，投入研发的机构较少；再次，不同病毒抗原之间存在的免疫干扰作用未知，而此干扰作用可能直接降低疫苗单组份的免疫原性，影响免疫效果。因此，为了更大范围地保护机体免受手足口系列病毒的侵害，在我国建立完善的HFMD防控体系，通过制备联苗扩大保护势在必行。
目前已有EV71疫苗问世，根据临床研究数据表明，疫苗的投入使用对由EV71引起的手足口疾病的预防，尤其是对重症的预防效果显著，但EV71疫苗对柯萨奇病毒的交叉保护较弱，鉴于肠道病毒流行血清型的趋势变化，急需一种能够更大范围的预防手足口疾病的多血清型联合疫苗。
发明内容
本发明的目的是提供一种预防手足口病的联合疫苗。
具体而言，本发明提供的预防手足口病的联合疫苗，所述联合疫苗包括灭活肠道病毒71型病毒，以及灭活柯萨奇病毒A组16型病毒、10型病毒和6型病毒。
其中，优选所述肠道病毒71型病毒毒株为保藏编号CGMCC No.3544的毒株。
本发明所述灭活柯萨奇病毒A组16型病毒、10型病毒、6型病毒均为本发明新发现的病毒株，具有优异的免疫原性。本发明上述新发现的病毒株均于2019年12月18日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)。具体而言，所述柯萨奇病毒A组16型病毒毒株的保藏编号为：CGMCC NO:18886。所述柯萨奇病毒A组10型病毒毒株的保藏编号为：CGMCC NO:18887。所述柯萨奇病毒A组6型病毒毒株的保藏编号为：CGMCC NO:18888。
本发明所述联合疫苗中，所述灭活肠道病毒71型病毒的抗原含量为100～1000U/mL。
本发明所述联合疫苗中，所述灭活柯萨奇病毒A组16型的抗原含量为200～3000U/mL，优选为200-1600U/mL。
本发明所述联合疫苗中，所述灭活柯萨奇病毒A组10型的抗原含量为200～3000U/mL，优选为200-1600U/mL。
本发明所述联合疫苗中，所述灭活柯萨奇病毒A组6型的抗原含量为1000～3000U/mL。
作为本发明的一个具体实施方式，所述联合疫苗还包括铝佐剂。
作为本发明的一个具体实施方式，所述铝佐剂选自氢氧化铝、磷酸铝或硫酸铝。
作为本发明的一个具体实施方式，所述铝佐剂的铝含量终浓度以铝离子计为0.1～1.0mg/mL，优选为0.2～0.8mg/mL。
本发明优选所述联合疫苗为液体剂型。
本发明还提供所述联合疫苗的制备方法，该方法包括以下步骤：分别制备各病毒原液，将所述原液分别吸附于铝佐剂，制备病毒铝吸附产物，然后将铝吸附产物混合制备联合疫苗。
本发明还提供所述联合疫苗在制备预防或治疗肠道病毒71型病毒、柯萨奇病毒A组16型病毒、柯萨奇病毒A组10型病毒和/或柯萨奇病毒A组6型病毒引起的疾病的药物中的应用。
本发明提供的疫苗涉及引起手足口疾病的最常见的四种血清型(EV71、CA16、CA10、CA6)组合，能够更大范围的保护由EV71、CA16、CA10、CA6引起的手足口疾病。
本发明所涉及的四种血清型的病毒中，CA16、CA10、CA6均由本发明人于实验室自主分离获得，EV71在中国CDC(中国疾病预防控制中心)分离，由北京科兴生物制品有限公司的挑斑建库组鉴定。分离方法为：将从临床患者咽拭子或粪便中采集的标本稀释、过滤， 接种细胞、挑斑纯化筛选、鉴定，分离获得纯的单一病毒；接种细胞进行扩增、建立毒种库，按照《中华人民共和国药典》2015版相关要求对毒种进行全项检定和稳定性研究。
作为本发明的一个具体实施方式，本发明提供的联合疫苗通过以下步骤制备得到：
EV71原液的制备方法：EV71病毒按照一定的MOI(感染复数)接种Vero细胞，采用微载体发酵罐逐级放大或细胞工厂平面培养5-9天；收获病毒液。对病毒液采用甲醛灭活，100-500KD的超滤膜包对其进行超滤浓缩；对超滤浓缩后的病毒液采用蔗糖密度梯度离心进行纯化和空实心病毒颗粒分离。合并收集目的管病毒，采用超滤方式进行脱糖处理；采用非限制性内切酶处理脱糖后的病毒液，超滤去除宿主细胞DNA，分子筛层析去除内切酶等杂质残留，经过上述步骤即得到EV71病毒原液。
CA16原液的制备方法：CA16病毒按照一定的MOI(感染复数)接种Vero细胞，采用发酵罐微载体逐级放大或细胞工厂平面培养4-9天；收获病毒液。100-500KD的超滤膜包对其进行澄清、超滤浓缩；对超滤浓缩后的病毒液采用蔗糖密度梯度离心进行纯化去除杂质和空实心病毒颗粒分离。合并收集超离目的管病毒，采用超滤方式进行脱糖处理；采用非限制性内切酶处理脱糖后的病毒液，超滤去除宿主细胞DNA；采用甲醛进行灭活，经过上述步骤即得到CA16病毒原液。
CA10原液的制备方法：CA10病毒按照一定的MOI(感染复数)接种Vero细胞，采用发酵罐微载体逐级放大或细胞工厂平面培养4-9天；收获病毒液。100-500KD的超滤膜包对其进行澄清、超滤浓缩；对超滤浓缩后的病毒液采用蔗糖密度梯度离心进行纯化去除杂质和空实心病毒颗粒分离。合并收集超离目的管病毒，采用超滤方式进行脱糖处理；采用非限制性内切酶处理脱糖后的病毒液，超滤去除宿主 细胞DNA；采用甲醛进行灭活，经过上述步骤即得到CA10病毒原液。
CA6原液的制备方法：CA6病毒按照一定的MOI(感染复数)接种Vero细胞，采用发酵罐微载体逐级放大或细胞工厂平面培养4-9天；收获病毒液。100-500KD的超滤膜包对其进行澄清、超滤浓缩；对超滤浓缩后的病毒液采用蔗糖密度梯度离心进行纯化去除杂质和空实心病毒颗粒分离。合并收集超离目的管病毒，采用超滤方式进行脱糖处理；采用非限制性内切酶处理脱糖后的病毒液，超滤去除宿主细胞DNA；采用甲醛进行灭活，经过上述步骤即得到CA6病毒原液。
病毒液纯化过程中完全采用物理方法进行，有效去除了宿主蛋白、宿主DNA、内切酶等杂质残留，为疫苗安全提供了保障。
将四种血清型的原液分别用铝佐剂吸附后，按照一定的比例混合，配制成疫苗半成品，分装后制成成品疫苗。
有益效果
本发明的联合疫苗的工艺一致性良好，工艺稳定；经过大量实验，筛选了最佳的灭活工艺参数，成品具有较好的稳定性；经动物实验证明，有较好的安全性和有效性。
本发明的联合疫苗包含手足口病主要致病病原，能够很好的预防由EV71、CA16、CA10和CA6病毒引起的手足口病的发生。本发明研究表明，上述各种抗原在免疫受种者后互相不干扰抗原性和免疫效果，具有很好的免疫原性和安全性。
本发明提供的联合疫苗可以同时预防多种病原体的侵染，并且这些抗原之间不存在相互干扰的现象，相应的免疫原性较单独抗原激发的免疫原性不会降低。联合疫苗的使用可以明显简化疫苗接种程序，提高接种效率并降低成本，这是未来技术发展的大趋势，也是未来市场需求所在。
具体实施方式
下面通过实施例对本发明作进一步说明，应该理解的是，本发明实施例仅仅是用于说明本发明，而不是本发明的限制，在本发明的构思前提下对本发明的简单改进都属于本发明要求保护的范围。
1.成品联合疫苗的制备
(1)毒株的分离
从各地疾病预防控制中心获得手足口病患者咽拭子或粪便，采用生理盐水进行稀释、离心、0.45μm和0.22μm的滤器过滤除菌。选择细胞生长状态良好，汇合至单层的敏感细胞(Vero细胞或人二倍体细胞)。病毒培养液为含2％小牛血清的199溶液，按照一定比例接种处理好的咽拭子/粪便标本，置32.0℃～36.5℃5％CO 2培养箱中静置培养。每日观察接种后细胞是否有特征性肠道病毒致细胞病变效应(CPE)的出现。如果出现CPE且CPE程度达+++以上时，收获细胞培养物，用细胞培养物继续传代两次；如果无CPE出现，培养至第7天，冻融一次，收获细胞培养物，用细胞培养物继续传代两次。设1支生长状态良好且未接种标本的细胞培养管作为细胞对照。细胞对照应无CPE出现，试验成立。
经3次盲传，细胞出现肠道病毒特征性病变(CPE)的标本判为病毒分离阳性，对病毒分离阳性的细胞培养物继续进行PCR鉴定。
(2)毒株的鉴定
分别采用Elisa酶联免疫法和PCR对分离获得的毒株从免疫学和分子学水平进行鉴定。
EV71抗原检测系统：包被EV71兔多抗，2-8℃过夜，采用10％小牛血清进行37℃封闭1-2小时，加入待鉴定病毒培养物，设置阴性对照孔，36～37℃温育1小时，洗板3～5遍，加入EV71型特异单克隆抗体，36～37℃温育1小时，洗板3～5遍，显色，终止反应。
CA16抗原检测系统：包被CA16兔多抗，2-8℃过夜，采用10％小牛血清进行37℃封闭1-2小时，加入待鉴定病毒培养物，设置阴性 对照孔，36～37℃温育1小时，洗板3～5遍，加入CA16型特异单克隆抗体，36～37℃温育1小时，洗板3～5遍，显色，终止反应。
CA10抗原检测系统：包被CA10兔多抗，2-8℃过夜，采用10％小牛血清进行37℃封闭1-2小时，加入待鉴定病毒培养物，设置阴性对照孔，36～37℃温育1小时，洗板3～5遍，加入CA10型特异单克隆抗体，36～37℃温育1小时，洗板3～5遍，显色，终止反应。
CA6抗原检测系统：包被CA6兔多抗，2-8℃过夜，采用10％小牛血清进行37℃封闭1-2小时，加入待鉴定病毒培养物，设置阴性对照孔，36～37℃温育1小时，洗板3～5遍，加入CA6型特异单克隆抗体，36～37℃温育1小时，洗板3～5遍，显色，终止反应。
分别采用以上四套抗原检测系统对筛选的CA16、CA10、CA6、EV71毒株进行鉴定，结果如表1～表4所示。
表1.CA16毒株Elisa抗原系统鉴定结果
毒株 OD值 结果判断
CA16毒株 2.015 阳性
CA10毒株 0.054 阴性
CA6毒株 0.061 阴性
EV71毒株 0.049 阴性
阴性对照孔 0.068 阴性成立
表2.CA10毒株Elisa抗原系统鉴定结果
毒株 OD值 结果判断
CA16毒株 0.034 阴性
CA10毒株 1.875 阳性
CA6毒株 0.045 阴性
EV71毒株 0.056 阴性
阴性对照孔 0.058 阴性成立
表3.CA6毒株Elisa抗原系统鉴定结果
毒株 OD值 结果判断
CA16毒株 0.042 阴性
CA10毒株 0.059 阴性
CA6毒株 2.154 阳性
EV71毒株 0.061 阴性
阴性对照孔 0.047 阴性成立
表4.EV71毒株Elisa抗原系统鉴定结果
毒株 OD值 结果判断
CA16毒株 0.039 阴性
CA10毒株 0.043 阴性
CA6毒株 0.038 阴性
EV71毒株 2.046 阳性
阴性对照孔 0.050 阴性成立
分子水平鉴定：采用试剂盒(QIAGEN)提取病毒RNA，采用逆转录试剂盒(TAKARA)对提取的RNA进行一步法逆转录和PCR扩增，引物序列自行设计后由华大基因测序有限公司合成，1％琼脂糖凝胶电泳对PCR产物进行鉴定。逆转录条件：
反转录反应：45℃ 10min
反转录酶失活：94℃ 2min
Figure PCTCN2020141693-appb-000001
经PCR扩增，测序，并将测序结果在NCBI数据库中比对，鉴定为相应型别的CA16、CA10、CA6毒株。
(3)单价原液的生产
130L发酵罐内微载体投放量2～6g/L，按照细胞数量10～50×10 4 个/mL接种Vero细胞，pH7.0～7.5，溶氧量在50％小范围内波动；35～37.5℃的条件下培养3～7天，根据细胞计数量，按照MOI 0.001-0.1的比例接种病毒，继续培养3-6天，收获病毒。
100-500KD的超滤膜包对其进行澄清、超滤浓缩，去除杂质蛋白；超滤浓缩后的病毒液采用蔗糖密度梯度离心进行纯化去除杂质和空实心病毒颗粒分离，超速离心条件为2-8℃，离心速度20000-50000rpm，离心8-18小时。合并收集超离目的管病毒，采用超滤方式进行脱糖处理；采用非限制性内切酶于18-30℃的条件下搅拌处理脱糖后的病毒液，超滤去除宿主细胞DNA；采用1:2000～1:8000的甲醛溶液于35-38℃的条件下灭活2-6天，直至病毒完全灭活，经过上述步骤即得到CA16、CA10、CA6型别的病毒原液。
EV71原液的生产采用发酵罐或细胞工厂培养，收获病毒后先灭活再纯化，其他工序段工艺与CA16、CA10、CA6型别相当。
(4)单价铝吸附疫苗的配制
单价铝吸附疫苗的制备：用0.85％生理盐水将铝佐剂稀释至3.0mg/mL(以Al(OH) 3计)，用0.01M PBS将EV71抗原稀释至400-6000U/mL，将CA16和CA10抗原稀释至800U/mL-9600U/mL，将CA6抗原稀释至4000U/mL-18000U/mL，室温下将稀释后的铝佐剂分别加入到稀释后的EV71、CA16、CA10、CA6抗原中，使二者等体积进行吸附，边加边搅拌，完毕后继续室温混合30分钟，获得EV71、CA16、CA10、CA6单价铝吸附疫苗。
(5)四价联合疫苗的配制
将上述四种型别的单价铝吸附产物按照一定的比例进行混合、分装，制备成成品四价手足口病联合疫苗。具体操作过程如下：
将上述获得的EV71、CA16、CA10、CA6单价铝吸附疫苗按照一定的比例混合，根据疫苗原液的抗原含量，用0.01M PBS(pH 7.2)溶液和氢氧化铝溶液适当稀释，然后室温搅拌吸附20±10分钟，即得 到疫苗半成品。在得到的疫苗半成品中，氢氧化铝的含量终浓度约为1.30mg/mL(换算成铝离子的终浓度为0.45mg/mL)，EV71的抗原含量为960U/mL，CA16的抗原含量为200U/mL～1600U/mL，CA10的抗原含量为200U/mL～1600U/mL，CA6的抗原含量为1000U/mL～3000U/mL，半成品中上述各抗原含量对应的蛋白剂量范围分别为1μg/mL～8μg/mL，即四价半成品疫苗的总蛋白剂量为4μg/mL～32μg/mL。之后对得到的疫苗半成品进行注射器灌装，得到联合疫苗成品。联合疫苗成品的剂量为0.5mL/人份，联合疫苗成品中各病毒组分蛋白质含量≤4μg/人份。
按照上述方法配制四价手足口病联合疫苗，具体参数如表5所示，每个参数进行连续三批联合疫苗的制备。
表5 四价联合疫苗的参数设置
Figure PCTCN2020141693-appb-000002
2.联合疫苗的评价
(1)四价联苗成品测试
分别检测上述实施例1～15的四价手足口病联合疫苗成品中的氢氧化铝含量、上清抗原含量、解离后抗原含量，计算吸附率及解离率，结果见表6。
表6 四价联苗成品检测结果
Figure PCTCN2020141693-appb-000003
Figure PCTCN2020141693-appb-000004
(2)免疫原性测试
采用上述实施例3中的三批四价联苗分别两针免疫大鼠和小鼠，检测血清中的中和抗体滴度。同时设立单价苗对照和阴性对照。重复三次。用微量细胞病变法检测中和抗体效价。检定用细胞为RD细胞，中和实验细胞培养在37℃培养7天，观察细胞病变。中和抗体效价及阳转率结果见表7。联合疫苗中各病毒组份免疫原性分别与相对应单价苗进行统计学分析，结果见表8。根据统计分析，联合疫苗与单苗的中和抗体GMT值均无显著性差异(P值均大于0.05)。
表7.中和抗体效价及阳转率
Figure PCTCN2020141693-appb-000005
表8.联合疫苗与单苗免疫原性结果统计学分析
Figure PCTCN2020141693-appb-000006
(3)安全性评价
在评估各联合疫苗免疫原性的整个实验期内，所有动物均健康存活，临床表现无异常。对实施例3中的三批EV71/CA16/CA10/CA6四价联合疫苗进行了动物安全性评价，包括异常毒性试验、小鼠急性毒性试验及豚鼠主动过敏试验。
异常毒性试验包括小鼠试验和豚鼠试验。小鼠试验中，EV71/CA16/CA10/CA6联合疫苗注射18～22g小鼠5只，0.5mL/只，观察7d。观察期内，小鼠全部健存，且无异常反应，到期时小鼠体重增加。豚鼠试验中，EV71/CA16/CA10/CA6联合疫苗注射250～350g豚鼠2只，5mL/只，观察7d。观察期内，豚鼠全部健存，且无异常反应，到期时豚鼠体重增加。
小鼠急性毒性试验中，EV71/CA16/CA10/CA6联合疫苗采用最大给药量法肌肉注射给予昆明小鼠，剂量为8000U/kg体重(相当于体重0.5岁儿童临床使用剂量的约120倍)，给药动物未出现异常临床症状 和死亡。
豚鼠主动过敏试验中，Hartley豚鼠经肌肉注射致敏给予联合疫苗3次，致敏剂量0.5mL/只/次，隔天给药。在末次致敏给药后第14天经静脉注射激发给药，激发剂量1.0mL/只/次，给药1次。试验动物在激发给药后连续观察过敏反应，试验结果显示EV71/CA16/CA10/CA6联合疫苗在豚鼠全身主动过敏试验中过敏反应为阴性。
(4)稳定性评价
将实施例3制得的三批联苗成品储存在2～8℃环境下，按时间点取样检测抗原含量、外观、装量、pH值、渗透压摩尔浓度、细菌内毒素、甲醛含量等。记录数据至12个月，并在12月检测疫苗的异常毒性及免疫原性。联苗成品2-8℃放置12个月，各项检测指标均未见明显下降。结果见表9～表11。
表9. 2～8℃联苗成品抗原含量检测结果(标示量的百分比)
Figure PCTCN2020141693-appb-000007
表10. 2～8℃联苗成品检测结果1
Figure PCTCN2020141693-appb-000008
表11. 2～8℃联苗成品检测结果2
Figure PCTCN2020141693-appb-000009
注：“/”表示未进行此项检测。
除实施例3外，实施例1-2以及实施4-15均进行了免疫原性测试，同时也进行安全性评价和稳定性评价。结果表明这些实施例的联合疫苗与单苗的中和抗体GMT值均无显著性差异，同时这些实施例中的联合疫苗与实施例3类似，均具有安全性好且稳定性高的优势。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (10)

  1. 一种预防手足口病的联合疫苗，其特征在于，所述联合疫苗包括灭活肠道病毒71型病毒，以及灭活柯萨奇病毒A组16型病毒、10型病毒和6型病毒。
  2. 根据权利要求1所述的联合疫苗，其特征在于，所述柯萨奇病毒A组16型病毒毒株的保藏编号为：CGMCC NO:18886。
  3. 根据权利要求1所述的联合疫苗，其特征在于，所述柯萨奇病毒A组10型病毒毒株的保藏编号为：CGMCC NO:18887。
  4. 根据权利要求1所述的联合疫苗，其特征在于，所述柯萨奇病毒A组6型病毒毒株的保藏编号为：CGMCC NO:18888。
  5. 根据权利要求1～4中任一项所述的联合疫苗，其特征在于，
    所述联合疫苗中，所述灭活肠道病毒71型病毒的抗原含量为100～1000U/mL；
    所述联合疫苗中，所述灭活柯萨奇病毒A组16型的抗原含量为200～3000U/mL，优选为200～1600U/mL；
    所述联合疫苗中，所述灭活柯萨奇病毒A组10型的抗原含量为200～3000U/mL，优选为200～1600U/mL；
    所述联合疫苗中，所述灭活柯萨奇病毒A组6型的抗原含量为1000～3000U/mL。
  6. 根据权利要求1～5中任一项所述的联合疫苗，其特征在于，所述联合疫苗还包括铝佐剂；
    优选地，所述铝佐剂选自氢氧化铝、磷酸铝或硫酸铝；
    更优选地，所述铝佐剂为氢氧化铝。
  7. 根据权利要求6所述的联合疫苗，其特征在于，所述铝佐剂的铝含量终浓度以铝离子计为0.1～1.0mg/mL，优选为0.2～0.8mg/mL。
  8. 根据权利要求1～7中任一项所述的联合疫苗，其特征在于，所述联合疫苗为液体剂型。
  9. 权利要求1～8中任一项所述的联合疫苗的制备方法，其特征在于，包括如下步骤：分别制备各病毒原液，分别吸附于铝佐剂，得到病毒铝吸附产物，然后将铝吸附产物混合制备所述联合疫苗。
  10. 权利要求1～8中任一项所述的联合疫苗在制备预防或治疗肠道病毒71型病毒、柯萨奇病毒A组16型病毒、柯萨奇病毒A组10型病毒和/或柯萨奇病毒A组6型病毒引起的疾病的药物中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117106102A (zh) * 2023-10-23 2023-11-24 中国医学科学院医学生物学研究所 肠道病毒多抗原表位融合蛋白、基因、疫苗及其制备方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101695570A (zh) * 2009-11-03 2010-04-21 中国人民解放军军事医学科学院微生物流行病研究所 一种手足口病单、双价灭活疫苗及其制备方法
CN101780278A (zh) * 2009-09-29 2010-07-21 福尔生物制药股份有限公司 二价手足口病灭活疫苗
WO2010139193A1 (zh) * 2009-06-03 2010-12-09 北京科兴生物制品有限公司 手足口病疫苗及其制备方法和应用
CN103386126A (zh) * 2013-06-25 2013-11-13 北京科兴生物制品有限公司 一种含肠道病毒抗原的多价免疫原性组合物
CN105963692A (zh) * 2016-06-23 2016-09-28 北京科兴生物制品有限公司 一种预防手足口病的联合疫苗
CN105999256A (zh) * 2016-06-23 2016-10-12 北京科兴生物制品有限公司 一种预防手足口病的联合疫苗
CN106075423A (zh) * 2016-06-23 2016-11-09 北京科兴生物制品有限公司 一种预防手足口病的联合疫苗
CN107739731A (zh) * 2017-10-12 2018-02-27 泰山医学院 包含柯萨奇病毒a10驯化株的病毒组合物及其应用
CN108853490A (zh) * 2018-06-25 2018-11-23 中国医学科学院医学生物学研究所 一种预防手足口病和甲型肝炎的联合疫苗及其制备方法

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010139193A1 (zh) * 2009-06-03 2010-12-09 北京科兴生物制品有限公司 手足口病疫苗及其制备方法和应用
CN101780278A (zh) * 2009-09-29 2010-07-21 福尔生物制药股份有限公司 二价手足口病灭活疫苗
CN101695570A (zh) * 2009-11-03 2010-04-21 中国人民解放军军事医学科学院微生物流行病研究所 一种手足口病单、双价灭活疫苗及其制备方法
CN103386126A (zh) * 2013-06-25 2013-11-13 北京科兴生物制品有限公司 一种含肠道病毒抗原的多价免疫原性组合物
CN105963692A (zh) * 2016-06-23 2016-09-28 北京科兴生物制品有限公司 一种预防手足口病的联合疫苗
CN105999256A (zh) * 2016-06-23 2016-10-12 北京科兴生物制品有限公司 一种预防手足口病的联合疫苗
CN106075423A (zh) * 2016-06-23 2016-11-09 北京科兴生物制品有限公司 一种预防手足口病的联合疫苗
CN107739731A (zh) * 2017-10-12 2018-02-27 泰山医学院 包含柯萨奇病毒a10驯化株的病毒组合物及其应用
CN108853490A (zh) * 2018-06-25 2018-11-23 中国医学科学院医学生物学研究所 一种预防手足口病和甲型肝炎的联合疫苗及其制备方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Pharmacopoeia of the People's Republic of China", 2015
ELIZABETH CAINE, JEREMY FUCHS, SUBASH DAS, CHARALAMBOS PARTIDOS, JORGE OSORIO: "Efficacy of a Trivalent Hand, Foot, and Mouth Disease Vaccine against Enterovirus 71 and Coxsackieviruses A16 and A6 in Mice", VIRUSES, vol. 7, no. 12, pages 5919 - 5932, XP055450321, DOI: 10.3390/v7112916 *
LIM HEEJI, IN HYUN JU, LEE JUNG-AH, SIK YOO JUNG, LEE SANG-WON, CHUNG GYUNG TAE, CHOI YOUNG KI, CHUNG JAE KEUN, CHO SUN JU, LEE JU: "The immunogenicity and protection effect of an inactivated coxsackievirus A6, A10, and A16 vaccine against hand, foot, and mouth disease", VACCINE, ELSEVIER, AMSTERDAM, NL, vol. 36, no. 24, 1 June 2018 (2018-06-01), AMSTERDAM, NL , pages 3445 - 3452, XP055948899, ISSN: 0264-410X, DOI: 10.1016/j.vaccine.2018.05.005 *
See also references of EP4272755A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117106102A (zh) * 2023-10-23 2023-11-24 中国医学科学院医学生物学研究所 肠道病毒多抗原表位融合蛋白、基因、疫苗及其制备方法
CN117106102B (zh) * 2023-10-23 2024-02-06 中国医学科学院医学生物学研究所 肠道病毒多抗原表位融合蛋白、基因、疫苗及其制备方法

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