CN116350765A - Vaccine for hand-foot-mouth disease and preparation method and application thereof - Google Patents
Vaccine for hand-foot-mouth disease and preparation method and application thereof Download PDFInfo
- Publication number
- CN116350765A CN116350765A CN202111620464.9A CN202111620464A CN116350765A CN 116350765 A CN116350765 A CN 116350765A CN 202111620464 A CN202111620464 A CN 202111620464A CN 116350765 A CN116350765 A CN 116350765A
- Authority
- CN
- China
- Prior art keywords
- virus
- vaccine
- coxsackievirus
- group
- inactivated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 88
- 208000030194 mouth disease Diseases 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 241000700605 Viruses Species 0.000 claims abstract description 113
- 241001529459 Enterovirus A71 Species 0.000 claims abstract description 43
- 241000988559 Enterovirus A Species 0.000 claims abstract description 19
- 241000709687 Coxsackievirus Species 0.000 claims abstract description 17
- 208000020061 Hand, Foot and Mouth Disease Diseases 0.000 claims description 25
- 208000025713 Hand-foot-and-mouth disease Diseases 0.000 claims description 25
- 229910052782 aluminium Inorganic materials 0.000 claims description 21
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 18
- 238000004321 preservation Methods 0.000 claims description 12
- 239000002671 adjuvant Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 239000004411 aluminium Substances 0.000 claims description 6
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims description 5
- -1 aluminium ions Chemical class 0.000 claims description 5
- 230000002265 prevention Effects 0.000 claims description 3
- 229910021502 aluminium hydroxide Inorganic materials 0.000 claims description 2
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 claims description 2
- 229940001007 aluminium phosphate Drugs 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 235000011128 aluminium sulphate Nutrition 0.000 claims description 2
- 239000001164 aluminium sulphate Substances 0.000 claims description 2
- BUACSMWVFUNQET-UHFFFAOYSA-H dialuminum;trisulfate;hydrate Chemical compound O.[Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O BUACSMWVFUNQET-UHFFFAOYSA-H 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000000427 antigen Substances 0.000 description 39
- 102000036639 antigens Human genes 0.000 description 39
- 108091007433 antigens Proteins 0.000 description 39
- 102100036369 Carbonic anhydrase 6 Human genes 0.000 description 32
- 102100032566 Carbonic anhydrase-related protein 10 Human genes 0.000 description 32
- 101000867836 Homo sapiens Carbonic anhydrase-related protein 10 Proteins 0.000 description 32
- 238000000108 ultra-filtration Methods 0.000 description 30
- 239000007788 liquid Substances 0.000 description 28
- 210000004027 cell Anatomy 0.000 description 23
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 22
- 239000000047 product Substances 0.000 description 15
- 241000709661 Enterovirus Species 0.000 description 14
- 238000001514 detection method Methods 0.000 description 14
- 102000004533 Endonucleases Human genes 0.000 description 13
- 108010042407 Endonucleases Proteins 0.000 description 13
- 239000012535 impurity Substances 0.000 description 13
- 239000011550 stock solution Substances 0.000 description 12
- 230000000120 cytopathologic effect Effects 0.000 description 11
- 239000013642 negative control Substances 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 230000005847 immunogenicity Effects 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 229930006000 Sucrose Natural products 0.000 description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 7
- 238000000432 density-gradient centrifugation Methods 0.000 description 7
- 238000000855 fermentation Methods 0.000 description 7
- 230000004151 fermentation Effects 0.000 description 7
- 230000000415 inactivating effect Effects 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 238000001179 sorption measurement Methods 0.000 description 7
- 239000005720 sucrose Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 210000003501 vero cell Anatomy 0.000 description 7
- 244000309466 calf Species 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 238000003306 harvesting Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 239000002808 molecular sieve Substances 0.000 description 6
- 230000003472 neutralizing effect Effects 0.000 description 6
- 239000011265 semifinished product Substances 0.000 description 6
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 238000007865 diluting Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229940031346 monovalent vaccine Drugs 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 241000700198 Cavia Species 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241001466953 Echovirus Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000005678 Rhabdomyoma Diseases 0.000 description 2
- 230000001594 aberrant effect Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 229940001442 combination vaccine Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 210000000605 viral structure Anatomy 0.000 description 2
- 206010009192 Circulatory collapse Diseases 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010037423 Pulmonary oedema Diseases 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 231100000409 cytocidal Toxicity 0.000 description 1
- 230000000445 cytocidal effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000001840 diploid cell Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008297 liquid dosage form Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 229940031348 multivalent vaccine Drugs 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 208000005333 pulmonary edema Diseases 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 206010040560 shock Diseases 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- 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
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a novel coxsackievirus A group 4 virus strain. The invention also discloses a vaccine for preventing or treating hand-foot-mouth disease, which comprises the coxsackievirus A group 4 virus strain. The vaccine of the present invention may also include inactivated enterovirus type 71 virus, inactivated coxsackievirus type a group 16 virus, type 10 virus and/or type 6 virus. The invention also discloses a preparation method and application of the vaccine.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a novel coxsackievirus A group 4 virus strain, a vaccine containing the strain for preventing or treating hand-foot-mouth disease, and a preparation method and application thereof.
Background
Hand-foot-and-mouth disease (HFMD) is an acute infectious disease caused by various enteroviruses, and is common in infant morbidity. Hand-foot-mouth disease is mainly manifested by rash in the mouth, hands and feet, and can be complicated with severe death symptoms caused by meningitis, encephalitis, pulmonary edema, circulatory failure and the like.
Viruses causing hand-foot-and-mouth disease include, in addition to Enterovirus type 71 (Enterovirus 71, EV71) and Coxakievirus type 16 (Coxakievirus A16, CA 16), types 2, 4, 5, 6, 10, 24, etc., coxakievirus type B (CB), types 1, 2, 3, 4, 5, etc., enterovirus type 68 (EV 68), and types 6, 25, 30, etc., of the Epstein-Barr virus (Echovirus). EV71 and CA16 are the major pathogens responsible for HFMD outbreaks, and recent reports of HFMD outbreaks by CA10, CA24 and CA6 have increased, even in some areas, to become the major prevalent serotypes.
Hand-foot-and-mouth disease has become one of the public health problems that seriously threaten children's health and social stability. At present, no effective antiviral drug for treating hand-foot-mouth disease exists clinically. Although the EV71 inactivated vaccine obtained in the prior art is marketed, the main strain type which causes high incidence of HFMD is various due to the change of the pathogenic spectrum of the HFMD enterovirus, and the cross immunity protection effect does not exist among the virus strains, so that the protection of organisms infected with other hand-foot-mouth viruses cannot be realized, and a new challenge is provided for preventing and controlling HFMD. It is difficult to control the outbreak and prevalence of hand-foot-and-mouth disease caused by CA16, CA10, CA6, CA4, etc. by inoculating EV71 inactivated vaccines.
Thus, there is a need for a multi-serotype combination vaccine that is capable of preventing a greater range of hand-foot-and-mouth disease to address outbreaks of HFMD and the prevalence of severe disease.
Disclosure of Invention
In order to solve the problems, the invention screens out a new virus strain with good immune effect, which can be used for preventing, treating or resisting hand-foot-mouth disease. On this basis, vaccines or medicaments for preventing or treating hand-foot-and-mouth disease have been developed.
According to one aspect of the invention, there is provided a coxsackievirus group a type 4 virus (CA 4) strain. The strain is preserved in China general microbiological culture collection center (CGMCC) (No. 3 of North Chen West Lu No. 1 of the Korean area of Beijing city) at 2021, 12 months and 02 days, and the preservation number is CGMCC NO 45051.
According to another aspect of the present invention there is provided a vaccine for the prevention of hand-foot-and-mouth disease comprising an inactivated strain of the above-described deposited coxsackievirus group a type 4 virus (CA 4).
According to some embodiments of the invention, the vaccine further comprises an inactivated enterovirus 71 type virus (EV 71), an inactivated coxsackievirus a group 16 type virus (CA 16), an inactivated coxsackievirus a group 10 type virus (CA 10) and/or an inactivated coxsackievirus a group 6 type virus (CA 6). According to some embodiments of the invention, the vaccine comprises inactivated CA4, EV71, CA16, CA10 and CA6.
According to some embodiments of the invention, the enterovirus 71 type virus (EV 71) strain has a preservation number of CGMCC No.3544. According to some embodiments of the invention, the coxsackievirus A group 16 virus (CA 16) strain has a preservation number of CGMCC NO:18886. According to some embodiments of the invention, the coxsackievirus A group 10 virus (CA 10) strain has a preservation number of CGMCC NO:18887. According to some embodiments of the invention, the coxsackievirus A group 6 virus (CA 6) strain has a preservation number of CGMCC NO:18888.
According to some embodiments of the invention, the antigen content of the inactivated enterovirus type 71 virus in the vaccine is 100-1000U/mL, preferably 500-1000U/mL.
According to some embodiments of the invention, the antigen content of inactivated coxsackievirus type 16 a in the vaccine is 200-3000U/mL, preferably 200-1600U/mL, more preferably 800-1200U/mL.
According to some embodiments of the invention, the antigen content of inactivated coxsackievirus type 10 a in the vaccine is 200-3000U/mL, preferably 200-1600U/mL, more preferably 800-1200U/mL.
According to some embodiments of the invention, the antigen content of inactivated coxsackievirus type 6 a in the vaccine is 1000 to 3000U/mL, preferably 1500 to 3000U/mL, more preferably 1600 to 2400U/mL.
According to some embodiments of the invention, the antigen content of inactivated coxsackievirus type 4 in the vaccine is 200-3000U/mL, preferably 1000-3000U/mL, more preferably 1600-2400U/mL.
According to a specific embodiment of the invention, the vaccine further comprises an aluminium adjuvant. According to a specific embodiment of the invention, the aluminium adjuvant is selected from aluminium hydroxide, aluminium phosphate or aluminium sulphate. According to a specific embodiment of the invention, the final concentration of aluminum content of the aluminum adjuvant is 0.1-1.0 mg/mL, preferably 0.2-0.8 mg/mL, calculated as aluminum ions.
According to some embodiments of the invention, the vaccine is in liquid dosage form.
According to a further aspect of the invention, there is also provided a method of preparing said vaccine. According to some embodiments of the invention, the method comprises the steps of: preparing each virus stock solution respectively, adsorbing the stock solutions to an aluminum adjuvant respectively to prepare virus aluminum adsorption products, and then mixing the aluminum adsorption products to prepare the vaccine.
According to a specific embodiment of the present invention, a vaccine virus purified solution can be prepared by the steps of:
(1) Inactivating EV71, CA16, CA10, CA6 and CA4 viruses, and concentrating by ultrafiltration;
(2) Subjecting the concentrated EV71, CA16, CA10, CA6 and CA4 virus concentrates to sucrose density gradient centrifugation;
(3) The centrifugations were collected and combined.
According to an embodiment of the present invention, a virus purified solution of EV71, CA16, CA10, CA6, CA4 may be prepared in advance.
According to a specific embodiment of the present invention, the EV71 stock solution can be prepared by the steps of: inoculating EV71 virus into Vero cells according to a certain MOI (multiplicity of infection), and adopting a microcarrier fermentation tank to enlarge step by step or perform cell factory plane culture for 5-9 days; harvesting virus liquid; inactivating virus liquid by formaldehyde; ultrafiltration concentration is carried out by adopting an ultrafiltration membrane with the thickness of 100-500 KD; purifying the virus liquid after ultrafiltration and concentration by adopting sucrose density gradient centrifugation; combining the collected target virus liquid, and performing desugaring treatment in an ultrafiltration mode; treating the desugared virus liquid by adopting non-limiting endonuclease; removing host cell DNA by ultrafiltration; and removing impurities such as endonuclease by molecular sieve chromatography to obtain EV71 virus stock solution.
According to a specific embodiment of the invention, the CA16 stock may be prepared by the steps of: inoculating the CA16 virus into Vero cells according to a certain MOI (multiplicity of infection), and adopting a fermentation tank microcarrier to enlarge step by step or adopting a cell factory to culture for 4-9 days; harvesting virus liquid; clarifying the virus liquid by adopting an ultrafiltration membrane with the thickness of 100-500 KD, and carrying out ultrafiltration concentration; the virus liquid after ultrafiltration concentration is subjected to sucrose density gradient centrifugation to remove impurities; combining the collected target virus liquid, and performing desugaring treatment in an ultrafiltration mode; treating the desugared virus liquid by adopting non-limiting endonuclease; removing host cell DNA by ultrafiltration; removing impurities such as endonuclease by molecular sieve chromatography; and inactivating by formaldehyde to obtain the CA16 virus stock solution.
According to a specific embodiment of the invention, the CA10 stock may be prepared by the steps of: inoculating the CA10 virus into Vero cells according to a certain MOI (multiplicity of infection), and adopting a fermentation tank microcarrier to enlarge step by step or cell factory plane culture for 2-9 days; harvesting virus liquid; clarifying the virus liquid by adopting an ultrafiltration membrane with the thickness of 100-500 KD, and carrying out ultrafiltration concentration; the virus liquid after ultrafiltration concentration is subjected to sucrose density gradient centrifugation to remove impurities; combining the collected target virus liquid, and performing desugaring treatment in an ultrafiltration mode; treating the desugared virus liquid by adopting non-limiting endonuclease, and removing host cell DNA by ultrafiltration; removing impurities such as endonuclease by molecular sieve chromatography; and inactivating by formaldehyde to obtain the CA10 virus stock solution.
According to a specific embodiment of the invention, the CA6 stock may be prepared by the steps of: inoculating the CA6 virus into Vero cells according to a certain MOI (multiplicity of infection), and adopting a fermentation tank microcarrier to enlarge step by step or adopting a cell factory to culture for 3-9 days; harvesting virus liquid; clarifying the virus liquid by adopting an ultrafiltration membrane with the thickness of 100-500 KD, and carrying out ultrafiltration concentration; the virus liquid after ultrafiltration concentration is subjected to sucrose density gradient centrifugation to remove impurities; combining the collected target virus liquid, and performing desugaring treatment in an ultrafiltration mode; treating the desugared virus liquid by adopting non-limiting endonuclease, and removing host cell DNA by ultrafiltration; removing impurities such as endonuclease by molecular sieve chromatography; and inactivating by formaldehyde to obtain the CA6 virus stock solution.
According to a specific embodiment of the invention, the CA4 stock may be prepared by the steps of: inoculating the CA4 virus into Vero cells according to a certain MOI (multiplicity of infection), and adopting a fermentation tank microcarrier to enlarge step by step or adopting a cell factory to culture for 2-9 days; harvesting virus liquid; clarifying the virus liquid by adopting an ultrafiltration membrane with the thickness of 100-500 KD, and carrying out ultrafiltration concentration; purifying the virus liquid after ultrafiltration and concentration by adopting sucrose density gradient centrifugation to remove impurities; combining the collected target virus liquid, and performing desugaring treatment in an ultrafiltration mode; treating the desugared virus liquid by adopting non-limiting endonuclease, and removing host cell DNA by ultrafiltration; removing impurity residues such as endonuclease by molecular sieve chromatography; and inactivating by formaldehyde to obtain the CA4 virus stock solution.
The virus liquid purification process is carried out by adopting a physical method, so that impurity residues such as host proteins, host DNA, endonuclease and the like are effectively removed, and the safety of the vaccine is ensured.
The stock solutions of the five serotypes are respectively adsorbed by an aluminum adjuvant and then mixed according to a certain proportion to prepare a vaccine semi-finished product, and the vaccine semi-finished product is prepared after split charging.
According to a further aspect of the invention there is provided the use of a vaccine of the invention in the prevention or treatment of hand-foot-and-mouth disease.
The term "hand-foot-and-mouth disease" or "HFMD" as used herein is an acute infectious disease caused by a variety of enteroviruses. Viruses causing hand-foot-and-mouth disease include, for example, enteroviruses (EV) and coxsackieviruses, of which EV71 and CA16 are more common. In addition, coxsackieviruses include coxsackieviruses type 2, 4, 5, 6, 10, 24, etc. of coxsackieviruses group a (CA), coxsackieviruses type 1, 2, 3, 4, 5, etc. of coxsackieviruses group B (CB), enteroviruses type 68 (EV 68), and types 6, 25, 30 of Echo viruses (Echo).
According to specific embodiments of the present invention, the vaccine may prevent or treat hand-foot-and-mouth disease caused by EV71, CA16, CA10, CA6 and/or CA 4. Besides the hand-foot-and-mouth disease caused by the viruses, the vaccine of the invention can also prevent or treat the hand-foot-and-mouth disease caused by other enteroviruses to a larger extent.
The term "cytopathic effect" or "CPE" as used herein refers to a phenomenon called cytopathic effect in which cells are observed microscopically to become round, necrotic, shed from the wall of a bottle after a certain period of culture by cell culture and inoculation of a cytocidal virus in an in vitro experiment. Characteristic enterovirus cytopathic effects can generally be manifested as cell rounding, refractive enhancement, shedding from the bottle wall, and the like.
There is no report on the development of five-valent EV71, CA16, CA10, CA6 and CA4 vaccines. The invention provides EV71, CA16, CA10, CA6 and CA4 pentavalent vaccine for the first time, which can well prevent hand-foot-mouth disease caused by EV71, CA16, CA10, CA6 and/or CA4 virus. Researches show that the vaccine provided by the invention can prevent infection of various pathogens at the same time, the mutual interference phenomenon does not exist among the antigens, the corresponding immunogenicity is not reduced compared with the immunogenicity excited by the independent antigens, the vaccine has good immunogenicity and safety, and the vaccine is good in stability and can be stored for a long time. The use of multivalent vaccines can significantly simplify vaccination procedures, improve vaccination efficiency and reduce costs. The vaccine preparation process disclosed by the invention has good consistency and stable process, and a large number of experiments prove that the optimal inactivation process parameters are screened, so that the safety performance of the vaccine can be improved, the complete inactivation is realized, the using amount of formaldehyde is controlled to be minimum, and the cost is saved.
Detailed Description
The invention is further illustrated by the following examples. It should be understood that these embodiments of the invention are merely illustrative of the invention and are not to be construed as limiting the invention.
EXAMPLE 1 isolation of strains
The throat swab or the excrement of the hand-foot-mouth disease patients is obtained from a multi-place disease prevention control center, diluted by normal saline, centrifuged, and filtered and sterilized by a filter of 0.45 mu m and a filter of 0.22 mu m respectively. The sensitive cells (Vero cells or human diploid cells) were selected to have good cell growth status and confluent to monolayers. The virus medium was M199 medium (GIBCO) containing 2% calf serum. Inoculating the processed throat swab/fecal specimen at a certain ratio, placing at 32.0-36.5 deg.C and 5% CO 2 And (5) standing and culturing in an incubator. Cells after inoculation were observed daily for the appearance of characteristic enterovirus cytopathic effects (CPE). If CPE is present and the CPE level is up to++ (1+, < 25%; and when the ratio of 2+,25% -50%, 3+,50% -75% and 4+,75% -100% is more than 2%, harvesting the cell culture, and continuing to passaging twice by using the cell culture. If no CPE was present, cultures were grown to day 7, freeze-thawed once, cell cultures were harvested and passaged twice with cell cultures. A cell culture tube with good growth state and without specimen was used as a cell control. If no CPE was present in the cell control, the test was established.
After 3 blind passes, the sample with CPE on the cells is judged to be positive in virus separation, and the PCR identification is continued on the cell culture positive in virus separation.
EXAMPLE 2 identification of strains
Strains isolated in example 1 were identified from an immunological and molecular level using Elisa enzyme-linked immunosorbent assay and PCR, respectively.
(1) Elisa enzyme-linked immunosorbent assay identification
EV71 antigen detection system: coating EV71 rabbit polyclonal antibody (Beijing Kexing), blocking at 37 ℃ for 1-2 hours by adopting 10% calf serum (), adding virus culture to be identified, setting a negative control hole, incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, adding EV71 type specific monoclonal antibody (Beijing Kexing), incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, developing color, and stopping reaction.
CA16 antigen detection System: coating CA16 rabbit polyclonal antibody (Beijing Kexing), sealing at 37 ℃ for 1-2 hours by adopting 10% calf serum overnight at 2-8 ℃, adding virus culture to be identified, setting a negative control hole, incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, adding CA16 type specific monoclonal antibody (Beijing Kexing), incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, developing color, and stopping reaction.
CA10 antigen detection System: coating CA10 rabbit polyclonal antibody (Beijing Kexing), sealing at 37 ℃ for 1-2 hours by adopting 10% calf serum overnight at 2-8 ℃, adding virus culture to be identified, setting a negative control hole, incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, adding CA10 type specific monoclonal antibody (Beijing Kexing), incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, developing color, and stopping reaction.
CA6 antigen detection System: coating CA6 polyclonal antibody (Beijing Kexing), sealing at 37 ℃ for 1-2 hours by adopting 10% calf serum overnight at 2-8 ℃, adding virus culture to be identified, setting a negative control hole, incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, adding CA6 type specific monoclonal antibody (Beijing Kexing), incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, developing color, and stopping reaction.
CA4 antigen detection System: coating CA4 polyclonal antibody (Beijing Kexing), sealing at 37 ℃ for 1-2 hours by adopting 10% calf serum overnight at 2-8 ℃, adding virus culture to be identified, setting a negative control hole, incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, adding CA4 type specific monoclonal antibody (Beijing Kexing), incubating at 36-37 ℃ for 1 hour, washing the plate for 3-5 times, developing color, and stopping reaction.
The strains isolated in example 1, and the CA16, CA10, CA6, and EV71 strains used were identified using the above five antigen detection systems, and the results are shown in tables 1 to 5.
TABLE 1 identification of the Elisa antigen System of the CA16 strain
Antigen detection system | OD value | Result judgment |
CA16 virus | 2.010 | Positive and negative |
CA10 virus | 0.044 | Negative of |
CA6 virus | 0.067 | Negative of |
CA4 virus | 0.051 | Negative of |
EV71 virus | 0.049 | Negative of |
Negative control well | 0.062 | Negative establishment |
TABLE 2 identification of the Elisa antigen System of the CA10 Strain
TABLE 3 identification of the Elisa antigen System of the CA6 Strain
Antigen detection system | OD value | Result judgment |
CA16 virus | 0.069 | Negative of |
CA10 virus | 0.048 | Negative of |
CA6 virus | 2.005 | Positive and negative |
CA4 virus | 0.054 | Negative of |
EV71 virus | 0.068 | Negative of |
Negative control well | 0.051 | Negative establishment |
TABLE 4 identification of the Elisa antigen System of the strains isolated in example 1
Antigen detection system | OD value | Result judgment |
CA16 virus | 0.050 | Negative of |
CA10 virus | 0.048 | Negative of |
CA6 virus | 0.071 | Negative of |
CA4 virus | 2.032 | Positive and negative |
EV71 virus | 0.046 | Negative of |
Negative control well | 0.060 | Negative establishment |
TABLE 5 identification of the Elisa antigen System of the EV71 strain
Antigen detection system | OD value | Result judgment |
CA16 virus | 0.065 | Negative of |
CA10 virus | 0.052 | Negative of |
CA6 virus | 0.048 | Negative of |
CA4 virus | 0.050 | Negative of |
EV71 virus | 2.101 | Positive and negative |
Negative control well | 0.059 | Negative of |
(2) Identification of molecular level
Extracting viral RNA by using an RNA extraction kit (QIAGEN), carrying out one-step reverse transcription and PCR amplification on the extracted RNA by using a reverse transcription kit (TAKARA), synthesizing by Huada gene sequencing Limited company after the primer sequence is self-designed, and identifying a PCR product by using 1% agarose gel electrophoresis. Reverse transcription conditions:
amplified by PCR and sequenced. Comparing the sequencing results in NCBI database, identifying the strain separated in the example 1 as Coxsackie virus A group 4 virus strain, and delivering to China general microbiological culture collection center (CGMCC) for preservation, wherein the preservation number is CGMCC NO:45051.
Example 3 production of monovalent stock solutions
The adding amount of microcarrier (GE Healthcare, cytodex) in a 130L fermentation tank is 2-6 g/L, and the quantity of cells is 10-50 multiplied by 10 4 Inoculating Vero cells per mL, and controlling pH to 7.0-7.5, wherein the dissolved oxygen content fluctuates within a small range of about 50%; culturing at 35-37.5 deg.c for 3-7 days, inoculating virus according to the cell count and MOI 0.001-0.1 proportion, culturing for 3-6 days and collecting culture supernatant containing virus.
Clarifying, ultrafiltering and concentrating the supernatant by using an ultrafiltration membrane bag with the thickness of 100-500 KD, and removing impurity proteins. Purifying the virus liquid after ultrafiltration concentration by adopting sucrose density gradient centrifugation, removing impurities and separating empty and solid virus particles, wherein the ultracentrifugation condition is 2-8 ℃, the centrifugation speed is 20000-50000 rpm, and the centrifugation is carried out for 8-18 hours. Combining and collecting the target virus of ultracentrifugation, and performing desugaring treatment by ultrafiltration. And (3) stirring the desugared virus liquid at 18-30 ℃ by adopting non-limiting endonuclease. Then, host cell DNA is removed by ultrafiltration, and impurity residues such as endonucleases are removed by molecular sieve (GE Healthcare) chromatography. Adopting formaldehyde solution of 1:2000-1:8000, inactivating for 2-6 days at 35-38 ℃ until the virus is completely inactivated. The virus stock of the inactivated CA6, CA16, CA10 and CA4 strains is obtained through the steps.
The EV71 stock solution is produced by adopting a fermentation tank or a cell factory for culture, virus is harvested, inactivated and purified, and other process steps are equivalent to those of CA6, CA16, CA10 and CA4 strains.
EXAMPLE 4 preparation of monovalent aluminum adsorbed vaccine
Preparation of monovalent aluminum adsorption vaccine: the aluminum adjuvant was diluted to 3.0mg/mL with 0.85% physiological saline (as Al (OH) 3 Diluting EV71 antigen to 400-6000U/mL with 0.01M PBS, diluting CA16 and CA10 antigen to 800-9600U/mL, diluting CA6 antigen to 4000-18000U/mL, diluting CA4 antigen to 3000-18000U/mL, respectively adding diluted aluminum adjuvant into diluted EV71, CA16, CA10, CA6 and CA4 antigens at room temperature, allowing the two antigens to be adsorbed in equal volumes, stirring while adding, and continuing mixing at room temperature for 30 minutes after the completion to obtain EV71, CA16, CA10, CA6 and CA4 monovalent aluminum adsorption vaccine.
EXAMPLE 5 preparation of pentavalent vaccine
Mixing and subpackaging the five types of monovalent aluminum adsorption products according to a certain proportion to prepare the finished product of the vaccine for the pentavalent hand-foot-and-mouth disease. The specific operation process is as follows:
mixing the EV71, CA16, CA10, CA6 and CA4 monovalent aluminum adsorption vaccine obtained in the above way according to a certain proportion, properly diluting with 0.01M PBS (pH 7.2) solution and aluminum hydroxide solution according to the antigen content of vaccine stock solution, and stirring and adsorbing for 20+/-10 minutes at room temperature to obtain a vaccine semi-finished product. In the obtained vaccine semi-finished product, the final concentration of the content of aluminum hydroxide is about 1.30mg/mL (the final concentration converted into aluminum ions is 0.45 mg/mL), the antigen content of EV71 is 960U/mL, the antigen content of CA16 is 200-1600U/mL, the antigen content of CA10 is 200-1600U/mL, the antigen content of CA6 is 1000-3000U/mL, the antigen content of CA4 is 1000-3000U/mL, the protein dose ranges corresponding to the antigen contents in the semi-finished product are 1-8 mug/mL respectively, namely the total protein dose of the pentavalent semi-finished vaccine is 5-40 mug/mL. And then filling the obtained vaccine semi-finished product by using a syringe to obtain a vaccine finished product. The dosage of the vaccine finished product is 0.5 mL/human part, and the protein content of each virus component in the vaccine finished product is less than or equal to 4 mug/human part.
The vaccine for the pentavalent hand-foot-and-mouth disease is prepared according to the method, specific parameters are shown in table 6, and each parameter is used for preparing three batches of vaccine continuously.
TABLE 6 parameter settings for pentavalent combination vaccine
Example 6 evaluation of pentavalent vaccine
(1) Testing of pentavalent vaccine finished products
The aluminum hydroxide content, the supernatant antigen content and the dissociated antigen content in the above pentavalent hand-foot-and-mouth disease vaccines 1 to 15 were detected respectively, and the adsorption rate and the dissociation rate were calculated, and the results are shown in table 7.
TABLE 7 detection results of vaccine finished products for pentavalent hand-foot-mouth disease
(2) Immunogenicity testing
Three batches of the pentavalent vaccine 1 were used to immunize rats and mice with two needles, respectively, and the neutralizing antibody titer in serum was detected. Monovalent vaccine controls and negative controls were set up simultaneously. Repeated three times. Neutralizing antibody titers were detected by a minicytopathic assay. Assay cells were human malignant embryonic Rhabdomyoma (RD) cells, which were cultured at 37℃for 7 days to observe cytopathy. The neutralizing antibody titers and the cation transfer rate results are shown in Table 8. The immunogenicity of each viral component in the vaccine was statistically analyzed against the corresponding monovalent vaccine, and the results are shown in table 9. According to statistical analysis, there was no significant difference in GMT values (P values were greater than 0.05) between the pentavalent vaccine and the neutralizing antibody of the monovalent vaccine.
TABLE 8 titers and conversion of neutralizing antibodies to yang
TABLE 9 statistical analysis of the results of immunogenicity of pentavalent and monovalent vaccines
(3) Safety evaluation
All animals survived healthily throughout the experimental period in which immunogenicity of each vaccine was assessed, with no abnormalities in clinical presentation. Three batches of EV71/CA16/CA10/CA6/CA4 pentad vaccine for vaccine 1 were subjected to an aberrant toxicity test to verify their animal safety assessment.
Abnormal toxicity tests include mouse tests and guinea pig tests. In the mouse test, 18-22 g of EV71/CA16/CA10/CA6/CA4 pentad vaccine was injected into 5 mice, 0.5 mL/mouse, and observed for 7 days. During the observation period, the mice were all healthy and free of abnormal reactions, and the mice gained weight at the expiration. In guinea pig experiments, 250-350 g of the EV71/CA16/CA10/CA6/CA4 vaccine was injected into 2, 5 mL/mouse and observed for 7 days. During the observation period, guinea pigs were all healthy and there was no abnormal reaction, and the body weight of guinea pigs increased at the expiration.
(4) Stability evaluation
And storing the three batches of vaccine finished products of the vaccine 1 in an environment of 2-8 ℃, and sampling and detecting the antigen content, appearance, loading, pH value, osmotic molar concentration, bacterial endotoxin, formaldehyde content and the like according to time points. Data were recorded for 12 months and the aberrant toxicity and immunogenicity of the vaccine was detected at 12 months. The vaccine finished product is placed for 12 months at the temperature of 2-8 ℃, and all detection indexes are not obviously reduced. The results are shown in tables 10 to 12.
Table 10. Antigen content detection results after the pentavalent vaccine product is stored at 2-8deg.C for a period of time.
Table 11. PH, osmolality and sterility test of the pentavalent vaccine product after storage at 2-8deg.C for a period of time.
Table 12. Toxicity and neutralizing antibody GMT after the pentavalent vaccine product is stored at 2-8deg.C for a period of time.
Note that: "/" indicates that this detection was not performed.
In addition to vaccine 1, vaccines 2 to 15 were also tested for immunogenicity, as well as safety and stability. The results show that the neutral antibody GMT values of the vaccines 2-15 and the monovalent vaccine are not significantly different, and meanwhile, the vaccines 2-15 are similar to the vaccine 1, and have the advantages of good safety and high stability.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention. In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further. Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (10)
1. A coxsackievirus A group 4 virus strain has a preservation number of CGMCC NO:45051.
2. A vaccine for preventing or treating hand-foot-mouth disease is characterized by comprising inactivated coxsackievirus A group 4 virus, wherein the preservation number of the coxsackievirus A group 4 virus strain is CGMCC NO:45051.
3. The vaccine of claim 2, further comprising an inactivated enterovirus type 71 virus, an inactivated coxsackievirus type a group 16 virus, an inactivated coxsackievirus type a group 10 virus, and/or an inactivated coxsackievirus type a group 6 virus.
4. The vaccine of claim 3, wherein the vaccine is administered in a vaccine composition,
the preservation number of the enterovirus 71 type virus strain is CGMCC No.3544;
the preservation number of the coxsackievirus A group 16 virus strain is CGMCC NO 18886;
the preservation number of the coxsackievirus A group 10 virus strain is CGMCC NO 18887;
the collection number of the coxsackievirus A group 6 virus strain is CGMCC NO 18888.
5. Vaccine according to any one of claims 2 to 4, characterized in that in the vaccine the inactivated coxsackievirus group a type 4 virus is present in an amount of 200 to 3000U/mL, preferably 1000 to 3000U/mL.
6. The vaccine according to any one of the claims 3 to 5, characterized in that in the vaccine,
the content of the inactivated enterovirus 71 type virus is 100-1000U/mL, preferably 500-1000U/mL;
the content of the inactivated coxsackievirus A group 16 virus is 200-3000U/mL, preferably 200-1600U/mL;
the content of the inactivated coxsackievirus A group 10 virus is 200-3000U/mL, preferably 200-1600U/mL;
the content of the inactivated coxsackievirus A group 6 virus is 1000-3000U/mL, preferably 1500-3000U/mL.
7. Vaccine according to any one of claims 2 to 6, characterized in that the vaccine further comprises an aluminium adjuvant, preferably selected from aluminium hydroxide, aluminium phosphate or aluminium sulphate.
8. Vaccine according to claim 7, characterized in that the aluminium adjuvant has an aluminium content final concentration of 0.1-1.0 mg/mL, preferably 0.2-0.8 mg/mL calculated as aluminium ions.
9. A method of preparing the vaccine of any one of claims 2 to 8.
10. Use of the vaccine of any one of claims 2 to 8 in the manufacture of a medicament for the prevention or treatment of hand-foot-and-mouth disease.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111620464.9A CN116350765A (en) | 2021-12-27 | 2021-12-27 | Vaccine for hand-foot-mouth disease and preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111620464.9A CN116350765A (en) | 2021-12-27 | 2021-12-27 | Vaccine for hand-foot-mouth disease and preparation method and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116350765A true CN116350765A (en) | 2023-06-30 |
Family
ID=86922442
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111620464.9A Pending CN116350765A (en) | 2021-12-27 | 2021-12-27 | Vaccine for hand-foot-mouth disease and preparation method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116350765A (en) |
-
2021
- 2021-12-27 CN CN202111620464.9A patent/CN116350765A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104099301B (en) | Coxsackie virus A16 type virus strain, application, vaccine and preparation method thereof | |
CN101402944A (en) | EV-71 virus seed, inactivated vaccine for human and method of producing the same | |
CN105963692B (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
CN101695570A (en) | Univalent and bivalent inactivated vaccine for hand-foot-and-mouth disease and preparation method thereof | |
CN109680026A (en) | The purifying of recombinant C A16 virus-like particle, the application in vaccine and vaccine | |
CN109689862B (en) | Purification of recombinant EV71 virus-like particle and vaccine preparation method thereof | |
CN103436583A (en) | Method for producing therapeutic monoclonal antibody of canine distemper virus, product thereof and hybridoma cell | |
WO2023045426A1 (en) | Coxsackie virus a16 strain and immunogenic composition and application thereof | |
CN104928260A (en) | Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof | |
CN112717128B (en) | Combined vaccine for preventing hand-foot-mouth disease and preparation method and application thereof | |
CN106075423B (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
CN105999256B (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
CN116769733A (en) | Coxsackie virus B group 4 strain and application thereof | |
CN108949700B (en) | Goat parainfluenza virus 3 type JS14-2 strain and application thereof | |
CN116615234A (en) | Combined vaccine for preventing hand-foot-mouth disease and preparation method and application thereof | |
CN103160475B (en) | Enterovirus 71 type viral strain, its application, vaccine and preparation method | |
CN116350765A (en) | Vaccine for hand-foot-mouth disease and preparation method and application thereof | |
CN116350773A (en) | Vaccine for hand-foot-mouth disease and preparation method and application thereof | |
CN116350772A (en) | Vaccine for hand-foot-mouth disease and preparation method and application thereof | |
CN116350774A (en) | Vaccine for hand-foot-mouth disease and preparation method and application thereof | |
CN116350766A (en) | Multivalent vaccine for hand-foot-and-mouth disease, and preparation method and application thereof | |
CN111686246B (en) | Antigen-antibody complex vaccine for porcine epidemic diarrhea virus and preparation method thereof | |
CN112791179B (en) | Combined vaccine for preventing hand-foot-and-mouth disease and preparation method and application thereof | |
CN103160474B (en) | Enterovirus 71 type virus strain, vaccine, animal model establishment method | |
CN102210858B (en) | Combined EV71 (enterovirus 71)-HA (hepatitis A) vaccine |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |