WO2000023104A1 - Vaccin lyophilise a virus vivant modifie d'hepatite a et son stabilisant - Google Patents

Vaccin lyophilise a virus vivant modifie d'hepatite a et son stabilisant Download PDF

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Publication number
WO2000023104A1
WO2000023104A1 PCT/CN1999/000157 CN9900157W WO0023104A1 WO 2000023104 A1 WO2000023104 A1 WO 2000023104A1 CN 9900157 W CN9900157 W CN 9900157W WO 0023104 A1 WO0023104 A1 WO 0023104A1
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Prior art keywords
vaccine
live
virus
hepatitis
attenuated
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PCT/CN1999/000157
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English (en)
French (fr)
Inventor
Jingye Liu
Pengfu Wang
Guangpu Li
Baosheng Xie
Zongming Song
Shuyan Li
Jing Liu
Ying Yu
Xizhen Zhang
Ben Liang
Lingjiu Liu
Wei Wang
Ling Zhang
Yong Xue
Jing Li
Yuhong Li
Hui Lin
Zongju Wan
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Changchun Institute Of Biological Products Ministry Of Public Health
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Application filed by Changchun Institute Of Biological Products Ministry Of Public Health filed Critical Changchun Institute Of Biological Products Ministry Of Public Health
Priority to DE69926522T priority Critical patent/DE69926522D1/de
Priority to EP99947187A priority patent/EP1123710B1/en
Priority to AU60762/99A priority patent/AU6076299A/en
Priority to US09/807,783 priority patent/US6884422B1/en
Priority to AT99947187T priority patent/ATE300955T1/de
Publication of WO2000023104A1 publication Critical patent/WO2000023104A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • 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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18434—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 hepatitis B vaccine, in particular to a freeze-dried live attenuated hepatitis B vaccine that can be stored for a long time at normal temperature and its industrial production method; the invention further relates to a freeze-dried live vaccine virus protection agent and Its application in the production of live freeze-dried attenuated live vaccines, in particular to its application in the production of live freeze-dried hepatitis A live attenuated vaccine.
  • Hepatitis A is an acute infectious disease with a high global incidence caused by the hepatitis A virus (HAV), which is widely present in nature. Because of the widespread and rapid spread of hepatitis A, the epidemic and prevention of liver disease has become a serious public health and epidemic prevention social problem. According to statistics, about 4 billion people around the world are threatened by the disease. Even in the highly developed United States, about 10,000 hepatitis patients are associated with hepatitis A virus infection each year. Especially in developing countries including China, because of its large population and relatively backward socio-economic and public health conditions, large-scale outbreaks and epidemics of hepatitis A often occur in some local areas, especially after natural disasters.
  • HAV hepatitis A virus
  • U.S. Patent Nos. 4,532,215 and 4,636,469 respectively disclose methods for preparing wild virus strains from the feces of patients with hepatitis A through at least 5 passages to prepare HM-175 virus strains that can be used for vaccine production.
  • U.S. Patent No. 4,506,016 discloses a method for adapting hepatitis virus to monkey kidney cell lines first, and then to human embryo lung fibroblasts to prepare an attenuated hepatitis B vaccine.
  • Chinese patents ZL 85107525 and ZL 92114998.0 respectively disclose the preparation methods of hepatitis A virus and eight-1 virus strains, and methods for preparing live attenuated hepatitis A vaccines using these virus strains as virus strains, respectively.
  • Chinese Patent Application No. 94101257.3 discloses a technology for multiplying hepatitis A virus by a suspension adsorption method.
  • the live attenuated hepatitis B vaccine vaccines prepared in the prior art mentioned above are all in the form of a suspension liquid. In a suspension liquid medium in which no effective protective agent exists, the free live virus cannot survive for a long time at normal temperature, even at 2 ⁇ 8. Under the temperature condition of C, the storage period is only 3 to 6 months.
  • live attenuated vaccines used to induce or increase the production of specific antibodies in mammals, especially humans, namely live attenuated vaccines, inactivated vaccines, subunit vaccines and genetically engineered vaccines.
  • live attenuated vaccines result in a stronger immune response in the host.
  • live attenuated hepatitis A vaccines used so far are in the form of aqueous suspensions.
  • One common disadvantage of these liquid preparations with other existing viral or bacterial vaccine liquid preparations is the lack of sufficiently high storage stability.
  • These liquid preparations are usually very unstable when stored at room temperature.
  • live attenuated hepatitis B vaccine in suspension state can usually only be stored for about 7 days at room temperature, even at 2 to 8 ° C. It also has a shelf life of only 3 to 6 months. Therefore, these vaccines must be stored, transported, and used under very low temperature conditions, resulting in high costs, which has led to the large-scale promotion and application of the vaccine, especially in economically inefficient conditions. The promotion and application of developed regions and tropical and subtropical regions are greatly restricted.
  • the present inventors prepared a freeze-dried live attenuated hepatitis A vaccine preparation, and developed a freeze-dried protection agent for protecting live vaccine viruses, especially live hepatitis A vaccine viruses, thereby completing This invention.
  • An object of the present invention is to provide a freeze-dried live attenuated hepatitis B vaccine.
  • the vaccine is composed of an effective preventive dose of a live hepatitis A attenuated live vaccine vaccine and a live vaccine virus protection agent.
  • Live attenuated hepatitis A vaccine can be stored for a long time at normal temperature, which overcomes the "cold chain", pressure and expense of vaccine during storage, transportation and use, reduces vaccine loss and related costs, and guarantees vaccine quality. Improved vaccination efficiency.
  • the live attenuated hepatitis A vaccine vaccine stock solution is prepared according to a known method.
  • the live vaccine virus protection agent is composed of human serum albumin and / or gelatin, an appropriate amount of trehalose, and is selected from glutamic acid, arginine, and aspartic acid. And one or two of lysine and its alkali metal salt, and ascorbic acid, urea, mannitol and / or sorbitol, inositol, etc., wherein the amino acid metal-lowering salt includes sodium glutamate.
  • the content of each main component in the lyophilized live vaccine virus protection agent is human serum albumin 0-20 g L, gelatin 5-10 g / L, and trehalose 50- 100g / L, sodium glutamate 7.5 ⁇ 15g / L, ascorbic acid 0.5 ⁇ 5.5g / L, urea 5 ⁇ 28g / L, sorbitol and / or mannitol 2 ⁇ 10g / L, inositol 4 ⁇ 10g / L and the rest Measure the water.
  • the live vaccine virus protecting agent may also be free of human serum albumin.
  • Another object of the present invention is to provide a method for preparing a live attenuated hepatitis V vaccine in freeze-dried form as described above, the method comprising:
  • a live vaccine virus protecting agent is added at a ratio of 1: 1 (v / v), and the protecting agent comprises human serum albumin and / or gelatin and trehalose , Ascorbic acid, one or two selected from glutamic acid, arginine, aspartic acid and lysine and their alkali metal salts, and urea, mannitol and / or sorbitol, inositol, etc., and Get mixed with the vaccine composition;
  • step (3) freeze-drying the vaccine composition obtained in step (2).
  • the drying step includes first pre-freezing the vaccine composition at a temperature of about -20 ° C to -50 ° C for 3 to 6 hours. It is then vacuum dried in a suitable lyophilizer for 10 to 20 hours, with a drying temperature range of -38 to 35 ° C.
  • each of the main components and their contents in the protective agent is: human serum albumin 0-20g / L, gelatin 5-10g / L, trehalose-, 50 ⁇ 100g / L, sodium arginine or arginine or sodium glutamate 7.5 ⁇ 15g / L, ascorbic acid 0.5 ⁇ 5.5g L, urea 5 ⁇ 28g / L, sorbitol and / or mannitol 2 ⁇ 10g / L and Organic alcohol 4 ⁇ 10g / L, the balance is water.
  • said protective agent is suitable for protecting vaccine virus activity such as enterovirus, paramyxovirus, arbovirus and herpes virus during its lyophilization process and lyophilized state
  • the lyophilized live vaccine virus protection agent is also suitable for protecting the live vaccine virus including enteric disease virus 15, paramyxovirus, arbovirus, virus or herpes virus during and after freeze-drying, Used as a related protective agent.
  • a live attenuated hepatitis B vaccine vaccine (vaccine stock) can be prepared according to a method disclosed in the prior art, for example, the method disclosed in Chinese Patent ZL 92114998.0, and then the live vaccine virus protection agent of the present invention is in an appropriate ratio ( For example, the ratio of 1: 1 (v / v) is mixed with the original liquid phase of the obtained 20 seedlings. After being packaged under aseptic conditions, it is placed in a freeze-drying device and subjected to freeze-drying treatment to obtain the freeze-dried live hepatitis A attenuated vaccine of the present invention.
  • ZL 92114998.0 describes in detail the method for preparing the attenuated hepatitis A strain LA-1, and the production of a live attenuated hepatitis B vaccine using the hepatitis A LA-1 attenuated lotion as a virus species. Methods. To put it simply, the method includes first dividing human embryo lung diploid cells at a seeding rate of 1: 2 ⁇ 1: 4, using a cell culture solution (MEM) (pH 7.2 with 10 ⁇ 15% newborn bovine serum) -7.6) Expansion passage.
  • MEM cell culture solution
  • the vaccine virus seed solution prepared according to the method described in Example 1 of Chinese Patent ZL 92114998.0, and supplemented with a cell culture maintenance solution was cultured at 34 ⁇ 36 ° C, and the solution was changed once a week, after about 3 to 4 weeks Discard the maintenance solution and the remaining calf serum, directly add the 199 comprehensive culture solution with or without phenol red, and continue the culture (at 34 ⁇ 36 ° C) for 4 to 6 days, then collect the cells. After three repeated freeze-thaw cycles, the cells were disrupted by ultrasound, combined with centrifugation to remove cell debris, and the supernatant was collected to obtain a live attenuated hepatitis A vaccine stock solution.
  • a live hepatitis A attenuated live vaccine vaccine can also be prepared according to the method described in Chinese Patent ZL 85107525.
  • the present invention further provides a lyophilized live vaccine virus protectant that enables the live-dried vaccine virus in freeze-dried form to maintain its biological activity for a long time.
  • the protectant is basically composed of human serum albumin and / or gelatin, trehalose, ascorbic acid, It is selected from the group consisting of one or two of glutamic acid, arginine, aspartic acid and lysine, and alkali metal salts thereof, and urea, mannitol and / or sorbitol, inositol, etc .;
  • the content of each basic component in the protective agent is human serum albumin 0 ⁇ 20g L, gelatin 5 ⁇ 10g / L, trehalose 50 ⁇ 100g / L, sodium glutamate 7.5 ⁇ 15g / L, ascorbic acid 0.5 ⁇ 5.5g / L , Urea 5 ⁇ 28g / L, sorbitol and / or mannitol 2 ⁇ 10
  • Hepatitis A virus is a picornavirus that does not have an envelope or lipids. Like most enteroviruses, hepatitis B virus is easily inactivated at room temperature without effective protective agents when it exists in liquid form, losing its ability to regenerate, proliferate, and infect its sensitive host cells. However, once live vaccines are due to lack of effective protective agents or due to inappropriate transportation or storage, etc. If the conditions are inactivated, it will be difficult to achieve the necessary immune protection ability.
  • human serum albumin and gelatin mainly function as protein and colloid scaffolds, and provide space support and partial nutrition protection for live or lyophilized live viruses.
  • Trehalose has the function of stabilizing cell and protein structure and resisting high temperature damage. Studies have shown that many biologically active materials such as antibodies, enzymes, viruses, etc. that have been dried in the presence of trehalose can regain their vitality after rehydration (Roser B., Food Sci & Technol, 2 (7): 166-169 , 1991; Roser B., Biopharm. 4 (8): 47-53, 1991; Roser B., Colace C, New Scientist, 138: 24-28, 1993), Polio vaccine using trehalose drying has been obtained success.
  • Basic amino acid salts such as glutamic acid, urea, mannitol and / or sorbitol and inositol, and ascorbic acid, etc., in the lyophilized protection agent of the present invention mainly play a role in adjusting the pH, stabilizing the hydration state or the osmotic pressure of the dehydration process, And antioxidant effects.
  • the live vaccine virus protection agent of the present invention can be prepared in a suitable container according to the conventional reagent preparation method, but before mixing trehalose, gelatin, sorbitol and / or mannitol and inositol, these materials should be stored at 37 ° C. Pre-heat for 24 to 48 hours, and mix the protective agent and the vaccine to be lyophilized at a ratio of about 1: 1 (v / v) evenly within 0.5 to 2 hours before the vaccine to be lyophilized.
  • the live attenuated hepatitis A vaccine for lyophilization is prepared according to a conventional method (such as the method described in Chinese Patent ZL 92114998.0).
  • a conventional freeze-drying device can be used to aliquot the vaccine composition as described above. Pre-freeze at -20 ° C ⁇ -50 ° C for 3 ⁇ 6 hours, and then vacuum-dry at -38 ° C to 35 ° C for 10 ⁇ 20 hours. During freezing, the eutectic temperature should be lower than -30 ° C. .
  • a live attenuated hepatitis B vaccine is used as an example, with and without the freeze-dried protection agent of the present invention, before and after lyophilization.
  • Virus (active) titers, and the effect of lyophilized protection on storage stability of vaccine viruses Xiang conducted a series of comparative experiments.
  • the test results show that the live vaccine virus protective agent of the present invention not only has excellent protective effect on the activity of sting liver virus during its freeze-drying treatment, but also can significantly improve hepatitis A during long-term storage after freeze-drying. Storage stability of live attenuated vaccines at 2-8 ° C, normal temperature and higher temperature (37 ° C).
  • the inventors also tested the protective effect of the freeze-dried protective agent of the present invention on the virus activity of the live attenuated measles vaccine and the hepatitis A-measles combined vaccine by the same method, and performed the same virus (active) drops before and after lyophilization.
  • Comparison test and storage stability test of lyophilized products under different temperature and storage time conditions The test results show that the live vaccine virus protection agent of the present invention can also effectively protect the virus activity of the live attenuated live attenuated vaccine and the combined hepatitis A-measles vaccine during the freeze-drying process, and that these vaccine viruses are at 2 ⁇ 8 ° C, Storage stability at room temperature (25 ° C) and higher (37 ° C).
  • the lyophilized live vaccine virus protectant (I) containing ⁇ albumin is formulated according to the following formula:
  • 5.5 g of medical gelatin is dissolved in about 300 ml of distilled water, and it is boiled to dissolve by heating. It is sterilized by autoclaving at about 116 ° C for 40 minutes, and then cooled to room temperature to 30-35 ° C, and then lO is added to the gelatin solution. .Og use a filter membrane or filter column (0.1, 0.2, 0.5, 1.0 ⁇ m) to continuously filter the human serum albumin obtained by sterilization, and stir well before mixing.
  • the lyophilized live vaccine virus protection agent (II) was prepared according to the following formulation, basically according to the method described in Example 1 (II):
  • the lyophilized protective agent (II) prepared in this embodiment does not contain It is more expensive and may cause hepatitis or even HIV virus contaminated human serum albumin, and L-arginine or its sodium salt is used instead of sodium glutamate in the protective agent (I).
  • L-arginine or its sodium salt is used instead of sodium glutamate in the protective agent (I).
  • a small amount of mannitol is added to the protective agent (II), which can further improve the protective effect.
  • a live attenuated hepatitis A vaccine stock solution was prepared according to the method described in Chinese Patent ZL 92114998.0. Briefly, human embryo lung diploid cells proliferated at an appropriate seeding rate were infected with the hepatitis I LA-1 virus strain established by the applicant, and cultured continuously at 35 ° C for 4 weeks. Change the fluid once a week. The positive rate of infected cells was detected by indirect immunofluorescence (IF) at about 3 weeks. If it reached 90% or more, discard the maintenance solution and the remaining newborn bovine serum, and add a low-concentration salt but no phenol red. 199 comprehensive culture solution, and continue to culture for 4 to 6 days to collect cells.
  • IF indirect immunofluorescence
  • the protective agent (I) prepared according to the method described in Example 1 was added to the above hepatitis A live attenuated vaccine stock solution at a ratio of 1: 1 (v / v). After mixing, they were aseptically packed and placed in a closed vacuum. Freeze-drying is performed in a freeze dryer (FS150-SS20C, Hull Co., USA). First, the vaccine composition is pre-frozen at about -40 ° C for 4 hours, and then gradually warmed up at -38 ° C to 32. Under vacuum conditions for 15 hours, the live freeze-dried hepatitis A vaccine is obtained.
  • Example 4 Preparation of a freeze-dried live-attenuated measles vaccine
  • the vaccine stock solution was mixed with the live vaccine virus protection agent ( ⁇ ) prepared according to the method described in Example 2 at a ratio of 1: 1 (v / v), and the vaccine composition was pre-frozen at about -40 ° C. 5 Hours, and then vacuum-dried at about -35 ° C to 34 ° C for about 14 hours to obtain the desired lyophilized live-attenuated measles vaccine.
  • ⁇ live vaccine virus protection agent
  • Example 5 Stability of a freeze-dried live-wrapped hepatitis vaccine
  • a vaccine Yue prepared as described in the procedure of Example 3 for 10-fold dilution series, then take 10 ⁇ 27 dilution of virus dilution was titrated virus, and to The same batch of vaccine stocks having the same thousand-tit titer without adding the lyophilized protective agent provided by the present invention was freeze-dried under the same conditions, and the lyophilized sample was used as a control. The two vaccine samples were titrated at the same time.
  • virus infectivity titers of 5 batches of vaccine samples before freezing and after lyophilization were all lower than 0.5 LogCCID 5 . / ml.
  • the present example further tested the stability of the freeze-dried live-attenuated hepatitis B vaccine at 2 to 8 ° C in the presence of a protective agent in the same manner as described above.
  • the stability comparison of the liquid preparation and lyophilized preparation of live attenuated hepatitis B vaccine at 2-8 ° C, room temperature and 37 ° C was compared. The results are shown in Table 1 and Table 2 respectively. Show.
  • the live vaccine virus protective agent of the present invention has stability of viral protein and nucleic acid structure under dry, high temperature and osmotic pressure conditions, and effectively maintains the vitality of the vaccine virus, and significantly The effect of extending its validity.
  • Example 6 The immunogenicity and safety test of a freeze-dried live attenuated hepatitis virus vaccine showed that healthy rhesus monkeys (average weight 4.5Kg) with negative anti-HAV and normal serum alanine aminotransferase (SGPT) levels were The experimental subjects were vaccinated with a lyophilized live attenuated hepatitis A vaccine prepared according to the method described in Example 3 and stored at room temperature for one month as the experimental group. A liquid dose of a live attenuated hepatitis A vaccine with the same virus titer was used as a control.
  • SGPT normal serum alanine aminotransferase
  • mice in the experimental group were vaccinated with 1.0 ml of lyophilized vaccine samples intravenously, and animals in the group 4 (control group) were vaccinated with control (liquid) vaccine samples of the same dose.
  • the venous blood of the animals was collected at 0, 2, 4, 6, and 8 weeks after the inoculation, and the animal serum SGPT value and anti-HAV antibody titer were measured.
  • the liver tissues of each group of animals were punctured and biopsied. The results are shown in Listing 3 below. Table 3 Serum SGPT and anti-HAV test results before and after vaccinated monkeys
  • freeze-dried live measles attenuated live vaccine using the live vaccine virus lyophilization protectant of the present invention showed a decrease in titer of 0.5 Log CCID 5 in the 5 batches of lyophilized vaccine samples. / ml. Frozen one thousand live attenuated vaccine measles stored for 15 months at virus titers 2 ⁇ 8 ° C to reduce ⁇ 0. 5 Log CCID 50 / ml , stored 4 weeks viral titer decreased temperature of 37 ° C for ⁇ l.OLog CCID 50 / ml.

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Description

冻干甲型肝炎减毒活疫苗及其保护剂 技术领域
本发明涉及曱型肝炎疫苗， 特别是涉及一种可在常温下长期保存的冷 冻干燥形式的曱型肝炎减毒活疫苗及其工业化生产制备方法； 本发明进一 步涉及冻干活疫苗病毒保护剂及其在生产冻干减毒活疫苗中的应用， 特别 是涉及其在生产冻干甲型肝炎减毒活疫苗中的应用。 技术背景
曱型肝炎是一种由自然界广泛存在的甲型肝炎病毒（HAV ) 引起的全 球性高发病率的急性传染病。 由于曱型肝炎具有流行普遍而且漫延迅速的 特点， 所以曱肝的流行和预防已成为一个严重的公共健康和防疫的社会问 题。 据统计， 全世界约有 40 亿人口受到该疾病的威胁， 即使在经济高度 发达的美国， 每年也约有 10，000例以上的肝炎病人与曱型肝炎病毒感染有 关。 特别是在包括中国在内的发展中国家， 由于人口众多， 社会经济和公 共卫生条件相对落后， 所以常常在某些局部地区发生甲型肝炎的大规模暴 发和流行， 特别是在自然灾害之后。 据粗略统计， 在中国有近 4亿人口受 到曱型肝炎的威胁， 每 10 万人口中约有 200至 300人遭受曱型肝炎疾病 的侵害。 因此， 发展高特异性和安全性的曱肝减毒活疫苗是临床病毒学研 究中的一个重要课题， 自本世纪七十年代末以来， 许多研究者致力于曱型 肝炎灭活疫苗或减毒活疫苗的研究， 不断有关于这一研究领域的成果和专 利被公开。 例如， 美国专利 4,164,566号公开了在亚人类灵长目的动物（如 狨猴） 细胞培养物中连续传代选育曱型肝炎病毒株， 并以所得到的曱型肝 炎病毒抹 CR326F抹在多种细胞系内传代增殖， 制备甲型肝炎疫苗的方法。 美国专利 4,532,215号和 4，636,469号分别公开了从曱型肝炎病人粪便中分 离野毒株， 至少经 5代次传代， 制备可用作疫苗生产的 HM-175病毒株的 方法。 美国专利 4,506,016 号公开了使曱肝病毒首先适应于猴肾细胞系， 然后再适应于人胚肺成纤维细胞， 以制备减毒的曱型肝炎疫苗的方法。 中 国专利 ZL 85107525号和 ZL 92114998.0号分别公开了曱型肝炎 和 八-1 两病毒株的制备方法 , 以及分别以这些病毒株作为毒种制备甲型肝炎减毒 活疫苗的方法。 另外， 中国专利申请 94101257.3号公开了以悬浮吸附法增 殖甲型肝炎病毒的技术。 上述现有技术制备的曱型肝炎减毒活疫苗制品都 是悬浮液体剂型的， 在无有效保护剂存在的悬浮液体介质中， 游离的活病 毒不能在常温下长时间存活， 既使在 2~8。C的温度条件下也只有 3~6个月 的贮存有效期。 因此， 必需在低温冷藏条件下即通过所谓 "冷链" 运输、 贮存和使用， 从而使曱型肝炎减毒活疫苗的大规模推广应用， 特别是在经 济不发达地区及热带和亚热带地区的推广应用受到很大的限制。
一般说来， 用于诱导或增加哺乳动物， 特别是人体内特异性抗体产生 的疫苗主要有四类， 即減毒活疫苗、 灭活疫苗、 亚单位疫苗及基因工程疫 苗等。 通常， 减毒活疫苗可以使宿主产生更强的免疫应答。
就甲型肝炎减毒活疫苗而言， 目前主要是仍处于实验室研究阶段的基 于甲型肝炎 CR326F病毒^ ^朱的减毒活疫苗 （Merck Co. ) 和已在中国境内普 遍应用的、 分别基于曱型肝炎病毒株 H2和曱型肝炎 L-A-1 病毒株的减毒 活疫苗 （分别由浙江医科院 〔中国浙江〕、 中国医学科学院医学生物学研 究所 〔中国昆明〕和中国卫生部长春生物制品研究所 〔中国长春〕 生产）。 大规模人群观察结果表明， 这些疫苗， 特别是基于曱型肝炎 L-A-1 病毒株 的减毒活疫苗， 现已免疫接种易感人群达 8,000 万人以上， 目前尚未发现 任何与疫苗接种有关的较严重的临床副反应。 血清学试验结果显示， 接种 这种减毒活疫苗后绝大多数受试者都能产生良好的抗体反应。 仅一次接种 抗体阳转率即可达 95%左右， 并且在首次接种后的 1-12 个月之间如再加 强免疫， 则抗体阳转率可达 100%, 而且抗体滴度均可达到相当高的水平 (参见中国专利 ZL 92114998.0 )。
然而， 迄今使用的曱型肝炎减毒活疫苗都是水悬浮液形式的， 这些液 体制剂与现有的其他病毒或细菌疫苗液体制剂的一个共同缺点是缺乏足够 高的保存稳定性， 一般说来， 这些液体制剂在常温下保存通常很不稳定， 例如悬浮液状态的曱型肝炎减毒活疫苗在常温下通常只能保存大约 7天的 时间， 既使在 2〜8 °C的温度条件下也仅有 3~6个月的保存有效期， 因此， 这些疫苗必需在很低的温度条件下贮存、 运输和使用， 导致高成本， 从而 使该疫苗的大规模推广和应用， 特别是在经济不发达地区及热带和亚热带 地区的推广应用受到很大的限制。
鉴于上述这些技术现状， 本发明人通过长期的研究和生产实践发现， 在按已公开的方法 （例如按中国专利 ZL 92114998.0 号中所述的方法）生 产的甲型肝炎减毒活疫苗原液中加入一种适当的保护剂， 将所得曱型肝炎 疫苗组合物制成冻干形式的疫苗制剂， 将使疫苗的贮存有效期限至少延长 三倍， 从而大大减少了疫苗在贮存、 运输和使用过程中的 "冷链，， 压力和 费用消耗， 降低了疫苗的损失及相关成本， 提高了疫苗的接种效率， 为疫 苗的大规模推广应用提供了重要条件。 发明内容
本发明人经过长期的反复研究， 制备出冷冻干燥形式的甲型肝炎减毒 活疫苗制剂， 并研制了用于保护活疫苗病毒， 特别是甲型肝炎活疫苗病毒 的冻干保护剂， 从而完成了本发明。
本发明的一个目的是提供一种冻干曱型肝炎减毒活疫苗， 该疫苗由含 有有效预防剂量的甲型肝炎减毒活疫苗原液和活疫苗病毒保护剂组成， 使 曱型肝炎减毒活疫苗可在常温下长期保存， 克服了疫苗在贮存、 运输和使 用过程中的 "冷链，， 压力和费用消耗， 降低了疫苗的损失及相关成本， 保 证了疫苗质量， 提高了疫苗的接种效率。
根据本发明的这一目的， 其中所说的曱型肝炎减毒活疫苗原液是按已 知方法制备的。
根据本发明这一目的一个优选实施方案， 其中所说的活疫苗病毒保护 剂是由人血清白蛋白和 /或明胶、 适量的海藻糖、 选自谷氨酸、 精氨酸、 天 冬氨酸和赖氨酸的一种或两种及其碱金属盐， 以及抗坏血酸、 尿素、 甘露 醇和 /或山梨醇、 肌醇等组成， 其中所说的氨基酸减金属盐包括谷氨酸钠。
根据本发明这一目的的一个优选实施方案， 其中所说的冻干活疫苗病 毒保护剂中各主要成分的含量分别为人血清白蛋白 0~20g L、 明胶 5~10g/L、 海藻糖 50〜100g/L、 谷氨酸钠 7.5〜15g/L、 抗坏血酸 0.5〜5.5g/L、 尿素 5〜28g/L、 山梨醇和 /或甘露醇 2〜10g/L、 肌醇 4〜10g/L及余量水。
在上述优选实施方案中， 所说的活疫苗病毒保护剂也可以不含有人血 清白蛋白。
本发明的另一个目的是提供一种制备如上文所述的冷冻干燥形式的曱 型肝炎减毒活疫苗的方法， 该方法包括：
( 1 )制备曱型肝炎减毒活疫苗原液；
( 2 )在步骤（ 1 )所述的病毒活疫苗原液中， 按 1 ： 1 ( v/v ) 的比例加 入活疫苗病毒保护剂， 该保护剂由人血清白蛋白和 /或明胶、 海藻糖、 抗坏 血酸、 选自谷氨酸、 精氨酸、 天冬氨酸和赖氨酸中的一种或两种及其碱金 属盐、 以及尿素、 甘露醇和 /或山梨醇、 肌醇等组成， 并混勾之得到疫苗组 合物；
( 3 )冷冻干燥由步骤（2 ) 中得到的疫苗组合物。
根据本发明这一目的的一个优选实施方案， 其中步骤（3 ) 所说的冷冻 干燥步骤包括首先将所说的疫苗组合物于大约- 20 °C ~ -50 °C的温度下预冷 冻 3~6小时。 然后在适当的冻干装置中真空干燥 10〜20小时， 干燥温度范 围为 -38〜35 °C。
本发明的再一个目的是提供一种冻干活疫苗病毒保护剂， 该保护剂基 本上由人血清白蛋白和 /或明胶、 海藻糖、 抗坏血酸、 选自谷氨酸、 精氨 酸、 天冬氨酸和赖氨酸中的一种或两种及其碱金属盐、 以及尿素、 甘露醇 和 /或山梨醇、 肌醇等组成。
根据本发明这一目的的一个优选实施方案， 其中所说的保护剂中各主 要成分及其含量分别为： 人血清白蛋白 0~20g/L、 明胶 5〜10g/L、 海藻糖 -、 50~100g/L、精氨酸钠或精氨酸或谷氨酸钠 7.5~15g/L、抗坏血酸 0.5〜5.5g L、 尿素 5〜28g/L、 山梨醇和 /或甘露醇 2〜10g/L和机醇 4~10g/L， 余量为水。
根据本发明这一目的的优选实施方案， 其中所说的保护剂适用于保护 如肠道病毒、 副粘病毒、 虫媒病毒和疱疹病毒在其冻干过程及冻干状态下 的疫苗病毒活性， 即所述的冻干活疫苗病毒保护剂也适应于包括肠道病 15 毒、 副粘病毒、 虫媒、病毒或疱疹病毒在其冷冻干燥过程中及冻干后对活疫 苗病毒的保护作用， 用作相关的保护剂。
本发明可以按照已公开的现有技术方法， 例如中国专利 ZL 92114998.0 中公开的方法制备曱型肝炎减毒活疫苗原液 （疫苗原液）， 然后将本发明 的活疫苗病毒保护剂按适当的比例 （例如 1 ： 1 ( v/v ) 的比例） 与所得疫 20 苗原液相混合。 无菌条件下分装后， 置于冷冻干燥装置内进行冻干处理， 得到本发明的冻干形式的甲型肝炎减毒活疫苗。 冻干时， 首先将此曱型肝 炎减毒活疫苗组合物于大约 -20~-5(TC , 最好在大约 -40 °C温度下预冷冻 3 至 6小时， 然后逐渐升温， 于大约 -38至 35 °C的温度下真空干燥约 10〜20 小时， 该温度范围内， 冻结状态的混合物会逐渐升华而被干燥， 由此制成 25 本发明的冻干曱型肝炎减毒活疫苗。 中国专利 ZL 92114998.0 号中详细描述了制备曱型肝炎减毒株 L-A-1 的方法， 以及以所说的曱型肝炎 L-A-1 减毒抹为毒种， 工业化规模生产曱 型肝炎减毒活疫苗的方法。 筒单地说， 该方法包括首先将人胚肺二倍体细 胞按 1 :2〜1 :4的分种率，使用加有 10〜15%新生牛血清的细胞培养液（MEM ) ( pH 7.2-7.6 )扩增传代。 将细胞培养瓶置于 37°C下旋转培养 5〜8天， 使 之生长成致密的细胞单层， 然后弃去细胞生长液， 并以平衡盐溶液如 Earle 氏液反复冲洗细胞 3〜5次后接种按中国专利 ZL 92114998.0实施例 1 中所 述方法制备的疫苗病毒种子液， 并补加细胞培养维持液， 于 34〜36°C下培 养， 每周换液一次， 约 3至 4周后弃去维持液和残留的小牛血清， 直接加 入含有或不含有酚红的 199综合培养液， 继续培养（ 34~36°C下） 4至 6天 后， 收集细胞。 经三次反复冻融后超声破碎细胞， 合并离心除去细胞碎片， 收集上清液即得到甲型肝炎减毒活疫苗原液。
另外， 也可按照中国专利 ZL 85107525号中所述的方法制备用于本发 明的甲型肝炎减毒活疫苗原液。
本发明进一步提供了可使冷冻干燥形式的活疫苗病毒长时间保持其生 物学活性的冻干活疫苗病毒保护剂， 该保护剂基本上由人血清白蛋白和 /或 明胶、 海藻糖、 抗坏血酸、 选自谷氨酸、 精氨酸、 天冬氨酸和赖氨酸中的 一种或两种及其碱金属盐、 以及尿素、 甘露醇和 /或山梨醇、 肌醇等组成； 其中所说的保护剂中各基本成分的含量分别为人血清白蛋白 0〜20g L、 明 胶 5〜10g/L、 海藻糖 50~100g/L、谷氨酸钠 7.5〜15g/L、抗坏血酸 0.5〜5.5g/L、 尿素 5〜28g/L、 山梨醇和 /或甘露醇 2〜10g/L和肌醇 4~10g/L, 余量为水。
甲型肝炎病毒是一种不带有包膜， 也不含脂类的微小核糖核酸病毒。 曱型肝炎病毒与大多数肠道病毒一样， 在以液态形式存在时， 如无有效保 护剂， 常温下均很容易被失活， 失去其再生、 增殖和对其敏感宿主细胞感 染的能力。 但活疫苗一旦由于无有效保护剂或由于不适宜的运输或储存等 条件被失活， 便难于达到所应具有的免疫保护能力。 因此， 研究并使用具 有有效保护作用的活疫苗病毒保护剂， 制备出可以在常温甚至更高温度下 可长期保存的冻干减毒活疫苗， 特别是冻干曱型肝炎减毒活疫苗是本发明 的另一重要特征。
在如上文所述的活疫苗病毒保护剂中， 人血清白蛋白和明胶主要起蛋 白质和胶体支架作用， 为悬液或冻干后的活病毒提供空间支持和部分营养 保护作用。 海藻糖具有稳定细胞和蛋白质结构、 抵御高温破坏的功能。 研 究证明， 在海藻糖存在下干燥处理后的许多抗体、 酶、 病毒等生物活性材 料， 当重新水合后均能恢复其活力 （Roser B.， Food Sci & Technol,2(7): 166-169,1991 ; Roser B.,Biopharm.4(8): 47-53,1991 ; Roser B., Colace C, New Scientist,138: 24-28,1993 ), 利用海藻糖干燥处理小儿麻痹疫苗已获得了成 功。 谷氨酸等碱性氨基酸盐、 尿素、 甘露醇和 /或山梨醇和肌醇、 以及抗坏 血酸等， 在本发明的冻干保护剂中则主要起到调整 pH, 稳定水合状态或 脱水过程的渗透压， 以及抗氧化等作用。
本发明的活疫苗病毒保护剂可以在适当的容器内， 按常规试剂配制方 法配制， 但在混入海藻糖、 明胶、 山梨醇和 /或甘露醇及肌醇之前， 应将这 些物质于 37°C下预加热 24~48小时， 并且在待冻干的疫苗分装前 0.5〜2小 时内将保护剂与待冻干疫苗以大约 1 ： 1 ( v/v ) 的比例混合均匀。 用于冷 冻干燥处理的曱型肝炎减毒活疫苗原液是按常规方法 （如按中国专利 ZL 92114998.0 所述的方法） 制备的， 可使用常规冷冻干燥装置将已分装好的 上述疫苗组合物于 -20°C~-50°C下预冷冻 3〜6小时， 然后于 -38°C至 35 °C下 真空干燥 10〜20小时， 冻 过程中， 其共熔点温度应低于 -30°C。
为了证实本发明提供的活疫苗病毒保护剂的保护作用， 以曱型肝炎减 毒活疫苗为例， 在加入和不加入本发明冻干保护剂条件下， 对其冻干前和 冻干后的病毒（活性） 滴度， 以及冻干保护剂对疫苗病毒之储存稳定的影 响进行了一系列的比较试验。 试验结果显示， 本发明的活疫苗病毒保护剂 不仅在其冷冻干燥处理过程中对曱肝病毒的活性具有极好的保护作用， 而 且在冻干后的长期放置中， 可以显著地提高甲型肝炎减毒活疫苗在 2-8 °C、 常温和较高温度下 （37°C ) 的保存稳定性。
在此基础上， 发明人还以同样方法检测了本发明的冻干保护剂对麻疹 减毒活疫苗以及甲肝一麻疹联合疫苗病毒活性的保护效果， 进行了同样的 冻干前后病毒（活性） 滴度比较试验和冻干制品在不同温度与贮存时间条 件下的贮存稳定性试验。 试验结果显示， 本发明的活疫苗病毒保护剂同样 可以有效地保护麻疹减毒活疫苗及甲肝一麻疹联合疫苗在其冷冻干燥处理 过程中的病毒活性， 以及这些疫苗病毒在 2〜8 °C、 常温 （25 °C ) 以及更高 温度下 （37。C ) 的保存稳定性。
我们的试验还发现， 当本发明所提供的冻干保护剂用于曱型肝炎减毒 活疫苗、 麻疹减毒活疫苗及曱型肝炎一麻疹联合疫苗时， 在如前所述的冻 干保护剂中加入极少量或完全不加入人血清白蛋白， 如适当调整冻干曲 线 ， 同样可以实现良好的冻干保护效果。 本发明的实施方案 实施例 1 冻干活疫苗病毒保护剂 （I ) 的制备
按下列配方配制含有^ k清白蛋白的冻干活疫苗病毒保护剂 （ I ):
成 分 含量 (g U
人血清白蛋白 10.0
明胶 5.5
海藻糖 65.0
谷氨酸钠 10.0
尿素 20.0 抗坏血酸 5.5
山梨醇 6.5
肌醇 7.5
首先将医用明胶 5.5g溶解于大约 300ml蒸馏水中，加热煮沸使之溶解， , 于大约 116°C下高压蒸汽灭菌 40分钟后室温冷却至 30~35 °C , 然后向该明 胶溶液中加入 lO.Og使用滤膜或滤柱（0.1、 0.2、 0.5、 Ι.Ο μ ιη ) 连续过滤 除菌所获得的人血清白蛋白， 并充分搅拌混匀后备用。
按上述配方称取海藻糖、 尿素、 抗坏血酸、 山梨醇和肌醇， 并依次加 入到大约 500ml蒸馏水中， 在振荡搅拌均勾后将混合物于 37°C预加热约 24 ：" 小时， 冷却至室温 （22〜26°C )后将所得混合物溶液与上述的明胶-人血清 白蛋白溶液混合均匀，以蒸馏水补足体积至 1L,并用 0.1N HC1调至 pH7.0， 再次过滤除菌后即得到本发明的冻干活疫苗病毒保护剂 （1 )。 实施例 2 冻干活疫苗病毒保护剂 （II ) 的制备
按下述配方， 基本上按照实施例 1 所述的方法配制冻干活疫苗病毒保 15 护剂 （II ):
成 分 含 量（ 2/L )
明胶 8.5
海 糖 75.0
尿素 15.5
L-精氨酸 (或 L-精氨酸钠） 10.1
抗坏血酸 3.0
山梨醇 5.0
甘露醇 5.0
肌醇 4.0
与保护剂 （I ) 不同的是， 本实施例制备的冻干保护剂 （II ) 不含有 格较昂贵并且有可能导致肝炎甚或 HIV病毒污染的人血清白蛋白， 并且其 中以 L-精氨酸或其钠盐代替保护剂 （I ) 中的谷氨酸钠。 另外， 该保护剂 ( II ) 中添加了少量的可以进一步提高保护效果的甘露醇。 实施例 3 冻干甲型肝炎减毒活疫苗的制备
按照中国专利 ZL 92114998.0 号所述方法制备甲型肝炎减毒活疫苗原 液。 简单地说， 首先用申请人所建立的曱型肝炎 L-A-1 病毒株感染按适当 分种率传代增殖的人胚肺二倍体细胞， 并且于 35°C培养条件下连续培养 4 周， 每周换液一次。 约 3周时以间接免疫萤光法 （IF )检测被感染细胞的 阳性率， 如达 90%以上时弃去维持液及残留的新生牛血清， 并加入含低浓 度盐但不含酚红的 199综合培养液， 继续培养 4〜6天后收集细胞。 经三次 反复冻融并超声破碎细胞后， 离心除去细胞碎片， 收集上清液即得到悬液 形式的曱型肝炎减毒活疫苗原液。
按照实施例 1 中所述方法制备的保护剂 （I ) 以 1 ： 1 ( v/v ) 的比例加 入到上述甲肝减毒活疫苗原液中， 混匀后无菌分装并置于密闭的真空冷冻 干燥器 （FS150-SS20C型， Hul l Co. , USA ) 内进行冻干处理， 首先将疫 苗组合物于大约 -40°C下预冷冻 4小时， 然后逐渐升温， 在 -38°C至 32°C条 件下真空干燥约 15小时即得到所需的冻干甲型肝炎减毒活疫苗。 实施例 4 冻干麻疹减毒活疫苗的制备
按照现行的中国生物制品规程中 《麻疹减毒活疫苗制造与检定规程》 的要求制备原液形式的麻疹减毒活疫苗。 将该疫苗原液与按实施例 2 中所 述方法制备的活疫苗病毒保护剂 （Π ) 以 1： 1 ( v/v ) 的比例混合， 将此疫 苗组合物于大约 -40°C预冷冻 5 小时， 然后于约 -35 °C至 34°C下真空于燥约 14小时， 得到所需的冻干麻疹减毒活疫苗。
实施例 5 冻干曱型肝炎减毒活疫苗的稳定性 首先用无菌水对按实施例 3 所述方法制备的多批冻干曱型肝炎减毒活 疫苗进行 10倍系列稀释，然后取 10·2 7稀释度稀释的病毒液进行病毒滴定， 并以不加本发明所提供的冻干保护剂的具有同样千前滴度的同一批疫苗原 液， 在相同条件下冷冻干燥之， 以此冻干样品作为对照。 两组疫苗样品同 时进行病毒滴定， 35 °C培养 28 天后收集培养物并经超声、 离心等后处理， 以酶联免疫吸附法 （ELISA ) 和间接免疫萤光（IF ) 法检测甲型肝炎病毒 滴度。 结果显示， 加入本发明冻干保护剂冷冻干燥的样品， 冻干后 5份实 验组样品的感染滴度为 6.33至 6.50, 而不加本发明冻干保护剂冷冻干燥的 样品， 冻干后 5份对照组样品的感染滴度则下降到 1.33至 2.33。
在另一项试验中， 则观察到在加入本发明保护剂的情况下， 5 批疫苗 样品， 在冻千前和冻干后的病毒感染性滴度下降值均 < 0.5LogCCID5。/ml。
在此基础上， 本实施例进一步按上述相同的方法试验了在有保护剂存 在时， 冻干曱型肝炎减毒活疫苗于 2~8°C温度下， 分别保存 3至 12个月的 稳定性， 并比较了曱型肝炎减毒活疫苗液体制剂和冻干制剂在 2〜8°C、 室 温及 37°C条件下保存不同时间的稳定性比较，结杲分别见表 1和表 2所示。
加保护剂冻干的曱肝减毒活疫苗于 2~8°C的稳定性 样品批号 时间 (月）
0 3 6 9 12
1 6.50 6.67 6.67 6.50 6.50
2 6.67 6.50 6.67 6.50 6.67
6.50 6.50 6.33 6.50 6.50
4 6.50 6.67 6.67 6.50 6.33
5 6.33 6.50 6.50 6.50 6.33 说明： 表中不同保存时间 （月数）后检测的病毒感染性滴度 （ LogCCID5。/ml )„ 曱型肝炎减毒活疫苗液体剂型和冻干剂型的稳定性比较 * 有 效 期
保存温度 液体剂型 冻千剂型
2 ~ 8 °C 3个月 12个月
室温 ( 25 V ) 7天 3个月
37。C 1天 7天
* 所给数据为 5批样品的最低保存期限
从上列表 1 和表 2所示的结果可以看出， 本发明的活疫苗病毒保护剂 具有在干燥、 高温和渗透压条件下稳定病毒蛋白质及核酸结构, 有效地维 持疫苗病毒的活力， 并显著延长其有效期的作用。
实施例 6 冻干曱型肝炎减毒活疫苗的免疫原性和安全性试验 以每 5 只血清抗 HAV 阴性， 血清谷丙转氨酶（SGPT ) 水平正常的健 康恒河猴（平均体重 4.5Kg ) 为实验对象， 以接种按实施例 3 所述方法制 备并于室温下贮存 1 个月的冻干甲型肝炎减毒活疫苗为实验组， 并以未经 冻干处理也不加冻干保护剂的同样病毒滴度的甲型肝炎减毒活疫苗液体剂 型作为对照。 分别给实验组 （1-3组）动物静脉接种冻干疫苗样品 1.0ml , 第 4组（对照组）动物则静脉接种同样剂量的对照 （液体）疫苗样品。 接 种后 0、 2、 4、 6和 8周采集动物静脉血液， 检测动物血清 SGPT值和抗 HAV抗体滴度， 同时， 对各組动物进行肝组织穿刺并进行活体组织病理学 检查。 结果如下列表 3所示。 表 3 冻干曱型肝炎减毒活疫苗接种猴体前、 后血清 SGPT和抗 HAV 检测结果
Figure imgf000015_0001
*采用赖氏法检测 SGPT, 结果〉 25U / ml为转氨酶异常升高;
* *所给数值为各组 5只动物的阳转率。 从表 3 所示结果可以看出， 本发明的冻干曱型肝炎减毒活疫苗在经过 冷冻干燥处理并于室温 （25。C ) 下贮存 30 天后， 仍保留其初始状态下的 免疫原性和使用安全性。 冻干过程和室温贮存 30 天期间基本上没有导致 疫苗生物学功能的丢失， 具有同样良好的使用安全性及免疫原性。 实施例 7 冻干麻疹减毒活疫苗的保存稳定性
基本上按照实施例 5 中所述方法检测冻干麻疹减毒活疫苗在冻干前和 冻干后病毒感染性滴度的改变， 以及冻干后的麻疹活疫苗分别在 2〜8°C和 37 °C温度下的保存稳定性。
结杲显示， 使用本发明的活疫苗病毒冻干保护剂冷冻干燥麻疹减毒活 疫苗， 冻干处理过的 5批疫苗样品的滴度下降幅度均 0.5Log CCID5。/ml。 冻千麻疹减毒活疫苗在 2~8 °C下保存 15 个月病毒滴度降低 < 0.5Log CCID50/ml , 37°C温度下保存 4周病毒滴度下降 < l.OLog CCID50/ml。

Claims

权利要求
1、 一种冻干曱型肝炎减毒活疫苗， 其特征在于由含有有效预防剂量的 曱型肝炎减毒活疫苗原液和活疫苗病毒保护剂组成。
2、 根据权利要求 1所述的冻干甲型肝炎减毒活疫苗， 其中所说的曱型 肝炎减毒活疫苗原液是按已知方法制备的。
3、 根据权利要求 1所述的冻干曱型肝炎减毒活疫苗， 其中所说的活疫 苗病毒保护剂基本上由人血清白蛋白和 /或明胶、 海藻糖、 选自谷氨酸、 精 氨酸、 天冬氨酸和赖氨酸中的一种或两种氨基酸或其碱金属盐， 以及抗坏 血酸、 尿素、 甘露醇和 /或山梨醇和肌醇组成。
4、 根据权利要求 3所述的冻干曱型肝炎减毒活疫苗， 其中所说的氨基 酸碱金属盐包括谷氨酸钠。
5、 根据权利要求 3或 4所述的冻干曱型肝炎减毒活疫苗， 其中所说的 活疫苗病毒冻干保护剂中各主要成分的含量分别为人血清白蛋白 0~20g/L、 明胶 5~10g/L、 海藻糖 50~100g/L、 谷氨酸钠 7.5~15g/L、 抗坏血 酸 0.5~5.5g/L、 尿素 5~28g/L、 甘露醇和 /或山梨醇 2~10g/L、 肌醇 4~10g/L 及余量水。
6、 根据权利要求 3所述的冻干曱型肝炎减毒活疫苗， 其中所说的活疫 苗病毒冻干保护剂也可以不含有人血清白蛋白。
7、 制备如权利要求 1至 6中任何一项所限定的冻干曱型肝炎减毒活疫 苗的方法， 该方法包括：
( 1 )制备甲型肝炎减毒活疫苗原液；
( 2 )在步骤（1 )所述的病毒疫苗原液中， 按 1： l(v/v)的比例加入活 疫苗病毒保护剂， 该保护剂由人血清白蛋白和 /或明胶、 海藻糖、 抗坏血酸、 选自谷氨酸、 精氨酸、 天冬氨酸和赖氨酸的一种或两种氨基酸或其碱金属 盐、 尿素、 甘露醇和 /或山梨醇和肌醇组成， 并混勾之得到疫苗组合物；― ( 3 )冷冻干燥由步骤（2 )得到的疫苗组合物。
8、 根据权利要求 7的制备方法， 其中步骤（3 ) 所说的冷冻干燥步骤 包括首先将所说的疫苗组合物于大约 -20°C〜- 50 °C的温度下预冷冻 3~6 小 时， 然后真空干燥 10〜20小时， 干燥温度的范围为 -38~35 。
9、 一种减毒活疫苗病毒冻干保护剂， 该保护剂基本上由人血清白蛋 白和 /或明胶、 海藻糖、 抗坏血酸， 选自谷氨酸、 精氨酸、 天冬氨酸和赖 氨酸中的一种或二种氨基酸或其碱金属盐、 尿素以及甘露醇和 /或山梨醇 和月 il醇组成。
10、 根据权力要求 9 的减毒活疫苗病毒冻干保护剂， 其中各组成成份 的含量分别为： 人血清白蛋白 0〜20g/L、 明胶 5~10 g/L、海藻糖 50〜100 g/L、 谷氨酸钠 7.5~15 g/L、 抗坏血酸 0.5~5.5 g/L、 尿素 5〜28 g/L、 山梨醇和 /或 甘露醇 2〜10 g/L和月几醇 4~10 g/L, 余量为水。
11、 根据权利要求 9 所述的减毒活疫苗病毒冻干保护剂， 其适用范围 也包括肠道病毒、 副粘病毒、 虫媒病毒或疱疹病毒等的活疫苗病毒的冻干 保护。
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