WO2005037310A1 - Vaccin combine contre l'hepatite a et b et sa preparation lyophilisee - Google Patents

Vaccin combine contre l'hepatite a et b et sa preparation lyophilisee Download PDF

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WO2005037310A1
WO2005037310A1 PCT/CN2004/000874 CN2004000874W WO2005037310A1 WO 2005037310 A1 WO2005037310 A1 WO 2005037310A1 CN 2004000874 W CN2004000874 W CN 2004000874W WO 2005037310 A1 WO2005037310 A1 WO 2005037310A1
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hepatitis
vaccine
combined
virus
lyophilized
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PCT/CN2004/000874
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English (en)
Chinese (zh)
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Pengfu Wang
Jingye Liu
Guifan Guan
Baosheng Xie
Xuan Luo
Lingjiu Liu
Wange Chen
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Changchun Institute Of Biological Products
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    • 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
    • A61K39/29—Hepatitis virus
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32434—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

Definitions

  • the present invention relates to a combined hepatitis B and hepatitis B vaccine and its freeze-dried preparation, in particular to a hepatitis B vaccine virus containing live appropriately attenuated hepatitis B virus and purified hepatitis B expressed by recombinant CH0 cells and / or recombinant yeast Hepatitis A-B combined vaccines for surface antigens, and lyophilized preparations thereof.
  • the invention also relates to a method for preparing the lyophilized hepatitis A-B combined vaccine, and a protective agent for the lyophilized hepatitis A-B combined vaccine.
  • Hepatitis A is a global acute infectious disease caused by the hepatitis A virus (HAV), which is widespread in nature. Because of the prevalence and rapid spread of the hepatitis A epidemic, the prevalence of hepatitis A has become a serious public health problem. Especially in developing countries including China, due to the large population and backward socio-economic and public health conditions, large-scale outbreaks and local epidemics of hepatitis A often occur. Even in the highly developed United States, every year There are also about tens of thousands of patients with hepatitis associated with hepatitis A virus infection.
  • HAV hepatitis A virus
  • Hepatitis B is an infectious disease caused by the hepatitis B virus ⁇ . Hepatitis B has a high morbidity and mortality rate in developing countries and generally has a poor prognosis. To date, there is no effective way to treat hepatitis B. In China, carriers of hepatitis B virus account for about 8-10 of the total population, and as many as 1 million people have chronic hepatitis.
  • Hepatitis A and Hepatitis B Due to the serious damage and high prevalence of Hepatitis A and Hepatitis B, the incidence of these two types of hepatitis in many countries in the world has always accounted for the highest incidence of all viruses' 1. Hepatitis, 70% -80 ° / . about. Facing the severe epidemic situation of hepatitis B virus, China has taken the lead in successfully developing a live attenuated hepatitis B vaccine.
  • hepatitis B vaccine based on hepatitis B surface antigen (HBsAg) was further developed.
  • European Patent No. 0339 667 discloses an A-B containing inactivated hepatitis A virus antigen and hepatitis B virus surface antigen and adjuvant, which can simultaneously prevent hepatitis A and hepatitis B Hepatitis Vaccine.
  • U.S. Patent No. 6,451,320 provides a combination vaccine composition suitable for adolescent vaccination.
  • the combined vaccine composition includes a hepatitis B virus surface antigen and a herpes simplex virus antigen, and one or more viral antigens selected from the hepatitis B virus antigen (HAV) and the EB virus (EBV) antigen, and the HAV is The inactivation originated from the Re-175 virus strain.
  • HAV hepatitis B virus antigen
  • EBV EB virus
  • the hepatitis A virus antigens used in these existing combination vaccines are all inactivated HAV viruses.
  • It is an object of the present invention to provide a ⁇ -Hepatitis B combined vaccine composition comprising a live attenuated hepatitis A vaccine virus and a hepatitis B virus surface antigen (HBsAg).
  • HBsAg hepatitis B virus surface antigen
  • Yet another object of the present invention is to provide a method for preparing a freeze-dried form of a combined hepatitis A-B vaccine composition.
  • the present invention relates to a combined hepatitis A and hepatitis B vaccine, and more particularly to a freeze-dried combined hepatitis A-B vaccine composition containing live hepatitis A vaccine virus and hepatitis B surface antigen protein and a protective agent.
  • the invention further relates to a method for preparing said combination vaccine composition.
  • a combined rubidium-hepatitis B vaccine contains live attenuated hepatitis A vaccine virus and hepatitis B surface antigen protein.
  • the live attenuated hepatitis A vaccine virus used in the present invention may be a known live attenuated hepatitis A vaccine virus.
  • a suitable attenuated hepatitis A vaccine that can be obtained from the stool of a naturally infected hepatitis A patient and obtained through serial dilutions of terminal attenuation, "cloning," purification, or other artificial mutagenesis methods can be used as a seed virus, a cell matrix that is sensitive to the hepatitis A vaccine virus, such as an established human embryo lung diploid cell, is used as the cell matrix according to the method described in detail in Chinese Patent No. 921 14998. 0. Invented live attenuated hepatitis A vaccine virus stock.
  • the vaccine stock solution prepared from the live attenuated hepatitis A vaccine L-A-1 vaccine strain is preferably used.
  • the hepatitis B surface antigen protein used in the present invention may be a known purified hepatitis B virus surface antigen protein.
  • appropriate expression systems can be constructed and selected, such as the expression of hepatitis B virus surface antigen proteins in prokaryotic, eukaryotic, or yeast hosts.
  • the HBsAg gene can be isolated from HBsAg-positive and hepatitis B e-antigen-negative sera of blood donors and cloned into an appropriate plasmid vector such as pBR 322 or a derivative thereof, and then the appropriate recombinant vector is used to transform the appropriate Host cells, such as Chinese hamster ovary (CH0) cells and / or yeast, and culture the recombinant CH0 cells and / or yeast under conditions suitable for expressing HBsAg protein, and then harvest from the recombinant cell culture medium and / or yeast and The desired HBsAg protein was purified.
  • an appropriate plasmid vector such as pBR 322 or a derivative thereof
  • the appropriate recombinant vector is used to transform the appropriate Host cells, such as Chinese hamster ovary (CH0) cells and / or yeast, and culture the recombinant CH0 cells and / or yeast under conditions suitable for expressing HBsAg protein
  • hepatitis B surface antigen can be prepared in accordance with the "Manufacturing and Assay of Recombinant (CH0 Cells) Hepatitis B Vaccine" in the "Chinese Biological Products Regulations” of the 2000 edition.
  • density gradient centrifugation or various ion exchange chromatography methods can be used to isolate and purify the blood-borne hepatitis B surface antigen protein from the serum of HBsAg-positive virus carriers.
  • the live attenuated hepatitis A vaccine virus is wild-type hepatitis A virus isolated from the stool of a naturally infected hepatitis A patient.
  • Hepatitis A LA-1 strain purified by terminal dilution "clone” purified and passaged attenuated in appropriate host cells.
  • the virus titer of the live attenuated hepatitis B vaccine virus contained in the combination vaccine is not less than 6.5 LogCCID50 / ml.
  • hepatitis B virus surface antigen (HBsAg) is obtained from recombinant CH0 cells and / or recombinant yeast and purified.
  • a preferred embodiment of the vaccine composition according to the present invention is characterized in that the protein content of the hepatitis B virus surface antigen (HBsAg) in the combination vaccine is not less than 10 g / ml.
  • HBsAg hepatitis B virus surface antigen
  • the preferred ratio range of the attenuated hepatitis B vaccine virus and hepatitis B surface antigen protein in the combined vaccine is: 6. 5-7. 0LogCCID50 / ml to 12-14 g / ml.
  • a lyophilized protective agent or stabilizer In order to facilitate the transportation, storage, and use of the combined vaccine product of the present invention, to reduce the so-called "cold chain" pressure in these processes, and to ensure the effect of vaccination, the inventors successfully developed a lyophilized protective agent or stabilizer.
  • This lyophilized protective agent or stabilizer can completely guarantee that the liquid combination vaccine product will not be inactivated during the freeze-drying process and during the transportation, storage and use after lyophilization.
  • a freeze-dried tadpole-hepatitis B combined vaccine contains a live attenuated tadpole hepatitis virus and hepatitis B surface antigen protein and a protective agent.
  • the protective agent used in the combination vaccine of the present invention contains trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol, and dextran.
  • Dextran can be high, medium and low molecular weight dextran, such as dextran 70, 40, 20; Low molecular dextran, such as dextran 20 is preferred.
  • the protective agent consists essentially of trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol and dextran.
  • the ⁇ -Hepatitis B combined vaccine of the present invention and its freeze-dried product may further contain one or more adjuvants for improving its immunogenicity without causing any side effects.
  • adjuvants are generally known to those skilled in the art, and include, but are not limited to, aluminum hydroxide or aluminum phosphate gel, 3D-MPL (a ThI cell response stimulant), and the like (see Pharmaceutical Biotechnology, Vol. 61 Vaccine Des ign- the subuni t and adjuvant approch, Powel l and Newman, Plenum Press).
  • the preferred immune adjuvant of the present invention is an aluminum hydroxide solution.
  • the vaccine composition of the present invention may further contain an appropriate amount of an immunological adjuvant, for example, 0.8-1.2 mg / ml aluminum hydroxide solution.
  • an immunological adjuvant for example, 0.8-1.2 mg / ml aluminum hydroxide solution.
  • the adjuvant is provided by a 0.8 to 1.2 mg / ml aluminum hydroxide solution contained in a lyophilized ⁇ hepatitis B combined vaccine dilution.
  • a method for producing a freeze-dried radon-hepatitis B combined vaccine includes:
  • the combination vaccine suspension containing the protective agent obtained in step (2) is lyophilized.
  • the components of the protective agent and their concentration (W / V) in the suspension of the combined vaccine composition are trehalose 3-10%, glutamic acid 0. 5-1. 0 %, Ascorbic acid 0.1-0.3 ° / » urea 0.5-2.0% sorbitol 0.5-2. 03 ⁇ 4, inositol 0.5-1. 0% and dextran 1-6%.
  • the freeze-dried combination vaccine is diluted with the aluminum hydroxide solution to obtain the lyophilized combination vaccine composition obtained in step (3), so that the live content is reduced.
  • the virus titer of the hepatitis B vaccine virus is not less than 6.5 LogCCID 5 o / ml
  • the protein content of the hepatitis B virus surface antigen (HBsAg) is not less than 10 ⁇ g / ml
  • the concentration of aluminum hydroxide is 0.8-1.2 mg / ml.
  • the adjuvant is provided by a solution of aluminum hydroxide contained in a lyophilized hepatitis A-B vaccine dilution.
  • the animal and monkey body experimental observation results show that the combined vaccine composition of the present invention not only has the same good safety as its monovalent vaccine, but also has better immunogenicity than its monovalent vaccine.
  • the combined vaccine composition of the present invention contains a freeze-dried protective agent that can effectively protect hepatitis B vaccine virus and hepatitis B indicating the biological activity of the antigen protein, it can be made into a freeze-dried combined vaccine Preparations, and thus eliminates the "cold chain" stress during vaccine production, transportation, storage and use, which can more effectively ensure the vaccine vaccination effect.
  • the original live hepatitis A vaccine virus Therefore, the production cost and market price of the combined vaccine composition are greatly reduced, thereby facilitating its promotion and universal use in developing countries including China.
  • the following ingredients may be first dissolved in 37 ° C distilled water (1000ml) at the indicated concentrations: sodium glutamate 100. 0 g / L, ascorbic acid 20. 0 g / L, urea 80.0 g / L, sorbitol 80.0 g / L, and inositol 80.0 g / L, combined with 0.1N HCL adjusts the pH to 7.0.
  • the protective agent component A was obtained after filtering and sterilizing through a 0.22 ⁇ m filter membrane. Then, 500.
  • trehalose 0 g was weighed and dissolved in water for injection at a temperature of about 37 ° C to make up to 1000 ml, and the protective agent component B was obtained after filtering and sterilizing through a 0.22 ⁇ m filter membrane. Finally, 300. 0 g of low-molecular-weight dextrose was dissolved in water for injection at a temperature of about 37 ° C to 1000 ml, and the protective agent component C was obtained after autoclaving at 116 ° C for 40 minutes.
  • an appropriate volume of the protective agent components A, B, and C can be added to the above-mentioned mixed suspension of the hepatitis A vaccine stock solution and the hepatitis B vaccine stock solution, so that each component of the protection agent reaches the concentration range as defined above (see Example 2).
  • the adjuvant is not added during the preparation of the vaccine, but is used as a solubilizer and diluent for the freeze-dried product, and is added just before the freeze-dried combination vaccine is used.
  • the content of the added aluminum hydroxide adjuvant is generally limited. When added as a solubilizer and diluent for lyophilized products, it can make hepatitis A vaccine virus and hepatitis B surface antigen protein immunogens in the combined vaccine Sex has increased.
  • the content of adjuvant aluminum hydroxide in the lyophilized tritium-hepatitis B combined vaccine ranges from about 0.8 to 1.2 mg / ml.
  • the combined vaccine for hepatitis B and hepatitis B of the present invention can be prepared by a simple mechanical mixing method.
  • an appropriate volume ratio such as approximately 20: 1 (V / V)
  • the pre-prepared viral titer of the hepatitis A vaccine stock solution with a log concentration of greater than 7.0 Log CCID 50 / ml and the pre-prepared protein concentration is The hepatitis B virus surface antigen stock solution greater than 100 g / ml is mixed, and then an appropriate amount of a pre-made lyophilized protective agent is added and gently mixed.
  • the 199 comprehensive culture solution used as a vaccine diluent is diluted to the required content or concentration of each component in the combined vaccine to obtain a combined vaccine composition in the form of a suspension.
  • the above-mentioned mixed suspension can be freeze-dried using a conventional freeze-drying device.
  • the freeze-drying conditions used are:-30 ° C to-55 ⁇ Pre-freeze at low temperature for 3-7 hours, and then at-30 Vacuum dry at ° C to 35 ° C for 8 to 16 hours.
  • the eutectic temperature of the vaccine lyophilized mixture was below -38 ° C.
  • the lyophilized hepatitis B vaccine virus titer decreased slightly after lyophilization, but the decrease was not greater than about 0.5 LogCCID50 / ml.
  • the lyophilized vaccine product thus obtained can be used at room temperature. It can be stored for a long period of time to 6 months without affecting the viral titer of the hepatitis A vaccine virus and the relative efficacy of the hepatitis B virus surface antigen (using the hepatitis B vaccine expressed by recombinant CH0 cells as a reference vaccine).
  • the lyophilized combination vaccine obtained as above can be dissolved and diluted by using an aluminum hydroxide solution with a content of 0.8-1.2oig / ml to prepare the combination vaccine of the present invention in the form of a suspension.
  • the aluminum hydroxide aqueous solution can be used not only as a solubilizing agent and diluent for a lyophilized combination vaccine, but also to provide an adjuvant with an immune enhancing effect.
  • the combined vaccine of the present invention appears to provide better immunogenicity than its monovalent vaccine compared to the live attenuated hepatitis B vaccine and / or hepatitis B vaccine.
  • the combination vaccine composition of the present invention contains effective protective or stabilizing ingredients, the hepatitis A vaccine virus titer and hepatitis B vaccine are during the freeze-drying process of the vaccine or during long-term storage and transportation after freeze-drying. The relative effectiveness did not decrease significantly.
  • test results of the two components of the pre-prepared combination vaccine showed that the titer of hepatitis V vaccine was not less than 6.5 LogCCID50 / ml, and the content of HBsAg antigen protein was not less than 10 ⁇ g / ml.
  • the results of the combined vaccine immunogenicity test showed that when the rhesus or red-faced monkey was vaccinated with the combined vaccine for 4 weeks, the positive rate of anti-HAV antibody in the animal serum reached 80-100%. At the same time, the positive rate of anti-HBsAg antibody in animal serum was about 20%; the positive rate of anti-HBsAg antibody at 8 weeks was 40-80%; and the positive rate of anti-HBsAg antibody after 2 weeks of inoculation Can reach 80-100%, and Further comparison experiments show that compared with the monovalent vaccine, the primate (rhesus or red-faced monkey) vaccinated with the lyophilized hepatitis A-B hepatitis combined vaccine of the present invention has the same good safety and security as the monovalent vaccine. Seems better immunogenic (see Example 4-1).
  • Example 1 Preparation of hepatitis B vaccine virus stock solution
  • human embryo lung diploid cells were expanded and cultured in a basic medium (MEM) (pH 7. 2-7. 6) supplemented with 10-15% calf serum. After the cells formed a dense monolayer, the growth medium was discarded, and the cells were repeatedly washed with fresh Ear le's solution, and then the seed virus solution prepared according to the method described in Example 1 of Chinese Patent No. 92114998. 0 was inoculated. After 34 ° C-36 ° C adsorption for 3-4 hours, supplement the cell growth maintenance solution (lactalbumin hydrolysate with vitamin C) to the culture and incubate at 34 ° C-36 ° C for about 4 weeks. Change the fluid once a week.
  • MEM basic medium
  • Example 2 Preparation of a freeze-dried hepatitis A-B combined vaccine
  • a 2.0 L stock vaccine virus (L-A-1. Strain with a virus titer of 8.0 LogCCID50 / ml) and 72 ml of recombinant type B expressed from recombinant CH0 cells and / or recombinant yeast were used. Hepatitis virus surface antigen protein stock solution (protein content: 1334 g / ml) was mixed, and then a protective agent stock solution prepared as follows: a protective agent component A550 ml, a protective agent component B 700 ml, and a protective agent component C 450 ml.
  • a lyophilized protective agent stock solution the following ingredients can be first dissolved in distilled water (1000ml) at the indicated concentrations: sodium glutamate 100. 0g / L, ascorbic acid 20. 0 g / L, urea 80. 0 g 0 ⁇ L, sorbitol 80.0 g / L and inositol 80. 0 g / L, and adjusted the pH value to 7.0 with 0.1N HCL.
  • the protective agent component A was obtained after filtering and sterilizing through a 0.22 ⁇ m filter membrane.
  • WHO World Health Organization
  • SDA State Drug Administration
  • hepatitis A vaccine virus is not less than 6.5 LogCCID50 / ml, and the protein content of hepatitis B surface antigen (HBsAg) is not less than 1 (g / ml.)
  • HBsAg hepatitis B surface antigen
  • the venous blood of the immunized animals was collected at 0, 4, 8 and 12 weeks after vaccination, and the serum was separated and the serum alanine aminotransferase (SGPT) or (ALT) was detected by conventional biochemical detection and enzyme-linked immunosorbent assay, respectively.
  • SGPT serum alanine aminotransferase
  • ALT serum alanine aminotransferase
  • the anti-HAV detection kit is used to detect the anti-HAV antibody by an enzyme-linked immunosorbent assay (ELISA); the anti-HSsAg reagent is used to detect the anti-HBsAg antibody by the radioimmunoassay (RIA).
  • HA lyophilized live attenuated hepatitis A virus vaccine
  • HB recombinant CHO vaccine expressed in CH0 cells.
  • Table 1 results shown in Table 1, after monkeys were vaccinated with the lyophilized cricket-hepatitis B combined vaccine and control group No abnormal elevation of serum liver enzymes occurred. Liver biopsy also revealed no abnormal pathological changes in liver tissue in all test animals. It has been shown that the freeze-dried ⁇ hepatitis B combined vaccine has the same good safety as its monovalent vaccine.
  • Example 4 Lyophilized hepatitis A-B vaccine combination immunogenicity test
  • Venous blood was collected from immunized animals at 0, 4, 8 and 12 weeks after vaccination, and serum was isolated Animals were tested for serum anti-HAV and anti-HBsAg antibody levels, and HAV-IgM antibody titers (as indicators of primary infection after vaccination) using conventional enzyme-linked immunosorbent assays. The results are shown in Listings 2, 3, 4, and 5 below.
  • HA lyophilized live attenuated hepatitis A virus vaccine
  • HA980501 is a live monovalent freeze-dried hepatitis A attenuated live vaccine Detection of anti-HBs levels in monkeys vaccinated with freeze-dried hepatitis A-B vaccine
  • HB90401 is a hepatitis B vaccine expressed by recombinant CHO cells From the results shown in Tables 2, 3, 4 and 5, it can be seen that the primate (rhesus or red-faced monkey) was vaccinated with different batches of the ⁇ -Hepatitis B combined vaccine of the present invention at the 4th week. Animal serum anti-HAV antibody positive conversion rate was 80%-100%, and reached 100% after 8 weeks. The positive rate of anti-HBs antibody in animal serum was 20% 4 weeks after the combined vaccine; 40-80% at 8 weeks, and 80-100% at 12 weeks. There was no significant difference in the antibody levels of the combined vaccine compared with its monovalent liver vaccine and hepatitis B vaccine (P> 0.05).
  • the combination vaccine of the present invention has the same good immunogenicity as its monovalent hepatitis A and B vaccines.
  • the serum anti-hepatitis virus IgM antibodies which are indications of primary hepatitis A infection, also turned positive at 2-4 weeks after vaccination, and gradually disappeared after 8 weeks (Table 2) .
  • the animals were sacrificed 10 days after inoculation, and the spleens of the group animals were aseptically removed and single cell suspensions were prepared. The resulting cell suspension was then dispensed into test tubes at a volume of 1 ml per tube. Anti-mouse Nl.1 monoclonal antibody (0.1 ml each) combined with PE (stearylethanolamine) was added to each tube. Flow cytometry (Becton Dickin son, USA) was then used to perform flow cytometry. The results are shown in Table 7 below, where the values are expressed as the mean ⁇ standard deviation of each group of animals.
  • Interleukin 2 (IL-2) was added to the suspension of spleen lymphocytes of the immunized mice at a final concentration of 15 ⁇ l / ml, and the required concentration was obtained after incubation at 37 ° C and 5% C0 2 for 72 hours.
  • Lymphokine activated killer (LAK) cells were used as target cells, and the two cells were mixed in a ratio of 100: 1 effector cell: target cell. The mixed cell suspension was incubated at 37 ° C and 5% CO 2 for 8 hours.
  • mice were injected intraperitoneally with a 2% glycogen aqueous solution. After 24 hours, 5% chicken red blood cell suspension was injected intraperitoneally with 1 ml each. Animals were sacrificed after 30 minutes, and peritoneal macrophages were collected and smeared. The resulting cell smear was then incubated at 37 ° F for 30 minutes.
  • RT-PCR reverse transcription polymerase chain reaction
  • Control group Hepatitis A vaccine group
  • Hepatitis B vaccine group Lianhe vaccine group
  • Control group ⁇ Liver vaccine group HBV vaccine group Hehe vaccine group Control sample 100 72. 33 100 52.1 Experimental sample 0 27. 67 0 47. 9
  • Example 5 Thermal stability test of lyophilized tadpole-hepatitis B combined vaccine The combination vaccine is stored at different temperatures for different times, and then samples are taken for testing of the titer of the liver vaccine virus, the efficacy of the hepatitis B vaccine, and the residual moisture content to evaluate the thermal stability of the freeze-dried hepatitis A-B vaccine of the present invention. .
  • the lyophilized cricket-hepatitis B combined vaccine of the present invention was stored in the dark at a temperature of 2-8 ° C, and samples were taken at 9, 12, 15, 21, and 24 months for hepatitis A vaccine virus titer and hepatitis B virus. Vaccine efficacy, residual moisture content testing.
  • the hepatitis B vaccine expressed by recombinant CH0 cells was used as the reference vaccine. The results are shown in Listings 11 and 12 below. Table 11 Viral titer and relative potency of hepatitis A-B combined vaccine after storage at 2--8 ° C for different times
  • the lyophilized hepatitis A-B vaccine of the present invention was stored for 6 months at room temperature in the dark, and samples were taken at 0, 2, 3, 4, 5, and June to measure the titer of the liver vaccine virus, the efficacy of the hepatitis B vaccine, and Detection of residual moisture content.
  • the hepatitis B vaccine expressed by recombinant CH0 cells was used as a reference vaccine. The results are shown in Tables 1 3 and 15 below.
  • Tibia-hepatitis B combined vaccine titration of tadpole hepatitis virus and relative efficacy of HBV stored at room temperature
  • Vaccine and liver virus titer (LogCCID 5 ./ml) (month) Relative efficacy of hepatitis B (month)
  • the lyophilized cricket-hepatitis B combined vaccine of the present invention Store at 37 ° C. Samples are taken at weeks 0, 1, 2, 3, 4 and 5 for testing of hepatitis A vaccine virus titer, hepatitis B vaccine efficacy, and residual moisture content. The hepatitis B vaccine expressed by recombinant CH0 cells was used as a reference vaccine. The results are shown in Listings 14 and 15 below. Table 14 Hepatitis A-B combined vaccine titer and relative potency of hepatitis B virus stored at 37 ° C
  • Vaccine Hepatitis virus titer (LogCCID50 / ml) (week) Hepatitis B efficacy (ED50) (week) Lot number 0 1 2 3 4 5 0 1 2 3 4
  • the freeze-dried ⁇ -Hepatitis B combined vaccine of the present invention is at room temperature, 2 ° C -8 ° C, 37 ° C.
  • the titer of the hepatitis B vaccine virus and the relative efficacy of the hepatitis B vaccine did not decrease significantly, and even the efficacy of the hepatitis B vaccine was higher than the monovalent vaccine of the hepatitis B vaccine. Also.
  • the lyophilized hepatitis A-B combined vaccine of the present invention is stored at different temperatures for different times, its residual moisture content has increased, but it does not affect the overall lyophilized cricket-Hepatitis B combined vaccine of the present invention. Quality requirements.

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un vaccin combiné contre les hépatites A et B et sa préparation lyophilisée et, plus particulièrement un vaccin combiné contre les hépatites A et B contenant un virus d'hépatite A vivant atténué de façon appropriée et un antigène de surface d'hépatite B purifié, produit par une cellule ovarienne de hamster chinois recombinante (CHO) et/ou par une levure recombinante, ainsi que sa préparation lyophilisée. L'agent de protection utilisé dans cette préparation contient du tréhalose, de l'acide glutamique, de l'acide ascorbique, de l'urée, du sorbitol, de l'inositol et du dextrane. L'inflammation associée aux virus de l'hépatite A et B peut être empêchée par utilisation simultanée d'un vaccin combiné contre les hépatites A et B de cette invention.
PCT/CN2004/000874 2003-07-31 2004-07-28 Vaccin combine contre l'hepatite a et b et sa preparation lyophilisee WO2005037310A1 (fr)

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CNA031499503A CN1513555A (zh) 2003-07-31 2003-07-31 甲-乙型肝炎联合疫苗及其冻干制剂
CN03149950.3 2003-07-31

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WO2005037310A1 true WO2005037310A1 (fr) 2005-04-28

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Publication number Priority date Publication date Assignee Title
CN1887351B (zh) * 2006-07-26 2010-04-21 程菠 一种预防性乙肝疫苗及其制备方法
CN102228687B (zh) * 2011-06-24 2012-10-10 浙江普康生物技术股份有限公司 不含明胶、人血白蛋白保护剂的冷冻干燥甲型肝炎减毒活疫苗及制备方法
CN102988975A (zh) * 2012-11-30 2013-03-27 深圳康泰生物制品股份有限公司 甲型、乙型肝炎联合疫苗及其制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5151023A (en) * 1988-04-28 1992-09-29 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Hepatitis a,b-combined adjuvanted vaccine
CN1299288A (zh) * 1998-03-09 2001-06-13 史密丝克莱恩比彻姆生物有限公司 联合疫苗组合物
CN1308547A (zh) * 1998-05-01 2001-08-15 史密丝克莱恩比彻姆生物有限公司 新型组合物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5151023A (en) * 1988-04-28 1992-09-29 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Hepatitis a,b-combined adjuvanted vaccine
CN1299288A (zh) * 1998-03-09 2001-06-13 史密丝克莱恩比彻姆生物有限公司 联合疫苗组合物
CN1308547A (zh) * 1998-05-01 2001-08-15 史密丝克莱恩比彻姆生物有限公司 新型组合物

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