WO2012100453A1 - 复合疫苗佐剂 - Google Patents

复合疫苗佐剂 Download PDF

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Publication number
WO2012100453A1
WO2012100453A1 PCT/CN2011/072034 CN2011072034W WO2012100453A1 WO 2012100453 A1 WO2012100453 A1 WO 2012100453A1 CN 2011072034 W CN2011072034 W CN 2011072034W WO 2012100453 A1 WO2012100453 A1 WO 2012100453A1
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Prior art keywords
adjuvant
group
vaccine adjuvant
sodium
composite
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PCT/CN2011/072034
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English (en)
French (fr)
Inventor
胡云章
胡凝珠
王海漩
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中国医学科学院医学生物学研究所
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Priority to US13/805,315 priority Critical patent/US8722030B2/en
Publication of WO2012100453A1 publication Critical patent/WO2012100453A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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

Definitions

  • the invention relates to a compound vaccine adjuvant, belonging to the technical field of immunology.
  • Sodium ferulate its chemical name is: 3-decyloxy-4-hydroxycinnamate sodium salt, molecular formula: Ci 0 H 9 NaO 4 , molecular weight: 216.17, structural formula shown in Figure 1, generally in the form of dihydrate Exist, wherein the dihydrate has the formula C 1Q H 9 Na (V2H 2 0, molecular weight: 252.20.
  • Sodium ferulate has various pharmacological effects such as diastolic blood vessels, anti-platelet activity, anti-oxidation and scavenging free radicals.
  • the words are essential trace elements for all organisms.
  • the growth and development of the human body is inseparable from the rhetoric.
  • the level of the body's rhetoric has an impact on the nervous, reproductive and immune systems.
  • the relationship between the words and the body's immune system is particularly complex, mainly in the following four aspects: 1
  • the daily intake and absorption of the words depends on the composition of the food, the age and health of the individual;
  • Cofactors with biologically active enzymes can indirectly affect the immune system;
  • 3 words can have a direct impact on lymphocyte production, maturation and function; 4 words can affect the function of immune promoters.
  • Vaccine adjuvants are a hot spot in vaccine research and development today. Ideal vaccine adjuvants need to be safe, effective, targeted and economical.
  • the current aluminum adjuvant is the longest and most widely used vaccine adjuvant approved for humans. It relies mainly on both the reservoir effect and the immunostimulatory effect to significantly increase the body's immune response.
  • As a vaccine adjuvant it has drawbacks in terms of safety and targeting. Other substances that are being studied or proven to have adjuvant effects have not been approved for use in humans due to safety, efficacy or economic issues.
  • the chemical formula is Zn(OH) 2 and the chemical formula is 99.4046. It has high similarity with aluminum hydroxide in terms of isoelectric point, water solubility, etc., and the preparation process is simple and the cost is low.
  • the present invention provides a safe, effective, stable and inexpensive new compound vaccine adjuvant through extensive experimentation and creative labor.
  • the present invention provides a composite vaccine adjuvant characterized by comprising the following components:
  • the mass ratio of sodium ferulate to the hydroxide is 10:1 to 50:1.
  • the chemical name of sodium ferulate is 3-methoxy-4-hydroxycinnamate sodium salt, the molecular formula is: Ci 0 H 9 NaO 4 , the molecular weight is: 216.17, the structural formula is shown in Figure 1, generally with dihydrate The form of the substance exists, wherein the dihydrate has the formula C ia H 9 Na0 4 *2H 2 0 and the molecular weight is 252.20.
  • the chemical formula of the hydroxide is Zn(OH) 2 and the chemical formula is 99.4046.
  • the dose of sodium ferulate as a vaccine adjuvant in humans is recommended to be: 10-50 mg.
  • the dosage of the hydroxide in the human body as a vaccine adjuvant is recommended to be: 0.25 to 1.5 mg.
  • the immune response induced by the composite vaccine adjuvant is humoral immunity.
  • the sodium ferulate is a commercially available medical product.
  • the hydroxide word is a commercially available medical product.
  • the vaccine adjuvant compounded by sodium ferulate and hydrazine is non-toxic, has no cumulative toxicity, and is safe and reliable in the range of the immunization dose; (2) A vaccine adjuvant compounded with sodium ferulate and hydrazine can effectively induce an antigen-specific humoral immune response, and its induced humoral immune response is superior to a single aluminum adjuvant, and is superior to a single one.
  • the composite vaccine adjuvant of the invention is readily available, all of which are commercially available products, simple preparation process, low cost, stable performance, and can be used as hepatitis B vaccine , genetic engineering vaccines, viral vaccines and other adjuvants.
  • Figure 1 shows the molecular structure of sodium ferulate.
  • Example 2 is a serum anti-HBs IgG antibody level (1:N) of each experimental group after 28 weeks using the composite adjuvant provided in Example 1;
  • Figure 3 is a serum anti-HBs IgG antibody level (1:N) in each experimental group after 28 weeks using the composite adjuvant provided in Example 2;
  • Figure 4 is a graph showing serum anti-HBs IgG antibody levels (1 : N) in each experimental group after 28 weeks using the composite adjuvant provided in Example 3.
  • Example 1 Commercially available sodium fermented sodium lOmg and hydrogenated lmg, fully mixed uniformly, to obtain the composite vaccine adjuvant.
  • sodium ferulate was purchased from Fujian Dongdong Liexun Pharmaceutical Co., Ltd., and the water was delivered by Tianjin Guangfu Fine Chemical Research Institute.
  • the commercially available medical sodium ferulate 25 mg and the hydrogen peroxide lmg were mixed well and uniformly, and the composite vaccine adjuvant was obtained.
  • the commercially available medical sodium ferulate 50 mg and zinc hydroxide lmg were thoroughly mixed to obtain the composite vaccine adjuvant.
  • the commercially available medical sodium ferulate 15 mg and zinc hydroxide lmg were thoroughly mixed to obtain the composite vaccine adjuvant.
  • the commercially available medical sodium ferulate 20 mg and the hydrogenated acid lmg are thoroughly mixed and uniformly, and the compound vaccine adjuvant described above is obtained.
  • the commercially available medical sodium ferulate 35 mg and zinc hydroxide lmg were thoroughly mixed to obtain the composite vaccine adjuvant.
  • Example 8 The commercially available medical sodium ferulate 40 mg and the hydrogenated acid lmg were mixed well and uniformly, and the composite vaccine adjuvant was obtained.
  • the commercially available medical sodium ferulate 45 mg and the hydrogenated acid lmg were mixed well and uniformly, and the composite vaccine adjuvant was obtained.
  • Example 1 demonstrates the immunological effects of the composite vaccine adjuvants provided in Example 1, Example 2, and Example 3 by animal experiments.
  • Test 1 The compound vaccine adjuvant of Example 1 was used.
  • the ICR mice were divided into a composite vaccine adjuvant group, a sodium ferulate single adjuvant group, a hydrazone single adjuvant group, an aluminum adjuvant control group, an adjuvant-free group, and a blank group, and a total of six groups, each group
  • the total volume of 25 injection vaccines and compound vaccine adjuvants was 0.1 ml, namely:
  • the compound vaccine adjuvant group was prepared by mixing the compound vaccine adjuvant of Example 1 (sodium ferulate 10 mg and 1 mg of sodium hydroxide) with 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline, and then subcutaneously injecting into the body of the mouse;
  • the single adjuvant group of sodium ferulate is prepared by dissolving 10 mg of sodium ferulate and 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline, and subcutaneously injecting into the body of the mouse;
  • the single adjuvant group of the hydroxide is prepared by dissolving 1 mg of the hydrazine hydroxide and 5 ug of the hepatitis B surface antigen in 0.1 ml of physiological saline, and subcutaneously injecting into the body of the mouse;
  • the aluminum adjuvant control group was prepared by dissolving 0.4 mg of aluminum hydroxide and 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline water and subcutaneously injecting into the body of the mouse;
  • the adjuvant-free group was prepared by dissolving 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline and subcutaneously into the body of the mouse;
  • Immunization protocol 3 times of total immunization, ICR mice were injected subcutaneously in 0, 1 and 6 months.
  • mice were collected from the tail vein blood, and the serum was separated.
  • ELISA Detection of serum anti-HBs IgG levels according to the protein micropore kit method, the specific method is: 4 ( ⁇ L HBsAg dissolved in 10ml lx coated diluent, mixed, coated 96-well microtiter plate, lOOul / hole The enzyme plate was placed in a wet box at 4 ° C overnight; the liquid in the hole was drained, and the plate was cleaned; I x BSA was added, 200 ul/well, and the plate was placed in a wet box at 37 ° C for 1 h; the liquid in the well was drained, and the film was taken.
  • Table 1 shows the serum anti-HBs IgG antibody levels (1: N) of each experimental group in 28 weeks after the use of the composite adjuvant provided in Example 1.
  • each experimental group can produce anti-HBs IgG antibodies, and generally reached the peak at the 8th week.
  • the composite vaccine adjuvant group achieved the best immune effect, 28 weeks
  • the antibody titer levels were significantly higher than the no adjuvant vaccine group, p ⁇ 0.05, and were able to maintain high levels during the test period. All of the experimental groups added with adjuvant produced significantly higher antibody levels than the no adjuvant group.
  • the antibody levels in the adjuvant adjuvant group and the single adjuvant group were significantly higher than those in the aluminum adjuvant group, P ⁇ 0.05.
  • the antibody level in the compound vaccine adjuvant group was significantly higher than that in the single adjuvant group.
  • the compound vaccine adjuvant has the effect of enhancing the immune effect of hepatitis B surface antigen, and the humoral immunity enhancement effect is higher than that of the aluminum adjuvant and the single hydroxide adjuvant and the single sodium ferulate adjuvant. See Figure 2 for 28 weeks, serum anti-HBs IgG antibody levels (1: N) in each experimental group (sodium ferulate low dose group)
  • the hepatitis B surface antigen used in this test case was provided by Shenzhen Kangtai Biological Products Co., Ltd., sodium ferulate was purchased from Fujian Dongdong Liexun Pharmaceutical Co., Ltd., and the water was delivered by Tianjin Guangfu Fine Chemical Research Institute.
  • the ICR mice were divided into a composite vaccine adjuvant group, a sodium ferulate single adjuvant group, a hydrazone single adjuvant group, an aluminum adjuvant control group, an adjuvant-free group, and a blank group, and a total of six groups, each group
  • the total volume of 25 injection vaccines and compound vaccine adjuvants was 0.1 ml, namely:
  • the compound vaccine adjuvant group was prepared by subcutaneously injecting the compound vaccine adjuvant of Example 2 (sodium ferulate 25 mg + 1 mg of sodium hydroxide) and 5 ug of hepatitis B surface antigen into 0.1 ml of physiological saline, and subcutaneously injecting into the body of the mouse;
  • the sodium sulphate single adjuvant group is prepared by dissolving 25 mg of sodium ferulate and 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline, and subcutaneously injecting into the body of the mouse;
  • the single adjuvant group of the hydroxide is prepared by dissolving 1 mg of the hydrazine hydroxide and 5 ug of the hepatitis B surface antigen in 0.1 ml of physiological saline, and subcutaneously injecting into the body of the mouse;
  • the aluminum adjuvant control group was prepared by dissolving 0.4 mg of aluminum hydroxide and 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline water and subcutaneously injecting into the body of the mouse;
  • the adjuvant-free group was prepared by dissolving 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline and subcutaneously into the body of the mouse;
  • Immunization protocol 3 times of total immunization, ICR mice were injected subcutaneously in 0, 1 and 6 months.
  • mice were collected from the tail vein blood, and the serum was separated.
  • ELISA Detection of serum anti-HBs IgG levels according to the protein micropore kit method, the specific method is: 4 ( ⁇ L HBsAg dissolved in 10ml lx coated diluent, mixed, coated 96-well microtiter plate, lOOul / hole The enzyme plate was placed in a wet box at 4 ° C overnight; the liquid in the hole was drained, and the plate was cleaned; I x BSA was added, 200 ul/well, and the plate was placed in a wet box at 37 ° C for 1 h; the liquid in the well was drained, and the film was taken.
  • the enzyme standard plate After the serum is diluted by the serial ratio, add the enzyme standard plate, lOOul/well, and place it in the wet box at 37 °C for 1 hour; wash the plate with the washing solution prepared by the kit for 4 times, each time for 5 minutes, the last shot of the enzyme plate ; force.
  • Into the kit prepared secondary antibody (peroxidase-conjugated goat anti-mouse IgG), lOOul / well, 37 ° C wet box for lh; wash the plate with washing solution 4 times, each 5min, the last shot of the enzyme plate Add the coloring solution prepared by the kit, lOOul/well, and avoid light for 10-25min. Add stop solution, lOOul per well; insert the enzyme plate into the microplate reader and read the plate at 405nm.
  • Table 2 shows the serum anti-HBs IgG antibody levels (1 : N) of each experimental group in 28 weeks after the use of the composite adjuvant provided in Example 2.
  • each experimental group can produce anti-HBs IgG antibodies, and generally reached the peak at the 8th week.
  • the composite vaccine adjuvant group achieved the best immune effect, and the antibody titer level was significantly higher than that of the unadjuvant vaccine group within 28 weeks, p ⁇ 0.05, and it was able to maintain a high level during the test period.
  • All of the experimental groups added with adjuvant produced significantly higher antibody levels than the no adjuvant group.
  • the antibody levels in the adjuvant adjuvant group and the single adjuvant group were significantly higher than those in the aluminum adjuvant control group.
  • the antibody level in the compound vaccine adjuvant group was significantly higher than that in the single adjuvant group. It is suggested that the compound vaccine adjuvant has the effect of enhancing the immune effect of hepatitis B surface antigen, and the humoral immunity enhancement effect is higher than that of the aluminum adjuvant and the single hydroxide adjuvant and the single sodium ferulate adjuvant. See Figure 3 for 28 weeks, serum anti-HBs IgG antibody levels (1: N) in each experimental group (sodium ferulate sodium dose group)
  • the hepatitis B surface antigen used in this test case was provided by Shenzhen Kangtai Biological Products Co., Ltd., sodium ferulate was purchased from Fujian Dongdong Liexun Pharmaceutical Co., Ltd., and the water was delivered by Tianjin Guangfu Fine Chemical Research Institute.
  • Test 3 Using the compound vaccine adjuvant of Example 3
  • the ICR mice were divided into a composite vaccine adjuvant group, a sodium ferulate single adjuvant group, a hydrazone single adjuvant group, an aluminum adjuvant control group, an adjuvant-free group, and a blank group, and a total of six groups, each group
  • the total volume of 25 injection vaccines and compound vaccine adjuvants was 0.1 ml, namely:
  • the compound vaccine adjuvant group was prepared by dissolving Example 3 (sodium ferulate 50 mg + hydroxide x mg) and hepatitis B surface antigen 5 ug in 0.1 ml of physiological saline, and subcutaneously injected into the body of the mouse;
  • the single adjuvant group of sodium ferulate is prepared by dissolving 50 mg of sodium ferulate and 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline, and subcutaneously injecting into the body of the mouse;
  • the single adjuvant group of the hydroxide is prepared by dissolving 1 mg of the hydrazine hydroxide and 5 ug of the hepatitis B surface antigen in 0.1 ml of physiological saline, and subcutaneously injecting into the body of the mouse;
  • the aluminum adjuvant control group was prepared by dissolving 0.4 mg of aluminum hydroxide and 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline water and subcutaneously injecting into the body of the mouse;
  • the adjuvant-free group was prepared by dissolving 5 ug of hepatitis B surface antigen in 0.1 ml of physiological saline and subcutaneously into the body of the mouse;
  • Immunization protocol 3 times of total immunization, ICR mice were injected subcutaneously in 0, 1 and 6 months.
  • the tail vein blood of the mice was collected, serum was separated, and serum anti-HBs IgG levels were detected by ELISA.
  • the specific method was as follows: 4 ( ⁇ L HBsAg is dissolved in 10ml lx coated diluent, mixed, coated with 96-well microtiter plate, lOOul/well; enzyme Place the target in a wet box, overnight at 4 °C; drop the liquid in the hole, pat the board; add I xBSA, 200 ul / hole, the enzyme plate is placed in the wet box at 37 ° C for 1 h; pour out the liquid in the hole, pat the board After the serum is serially diluted by the ratio, add the enzyme labeling plate, lOOul/well, and place it in the wet box at 37 °C for 1 hour; wash the plate with the washing solution prepared by the kit for 4 times, each time for 5 minutes, the last time to take the net target plate; force .
  • kit prepared secondary antibody (peroxidase-conjugated goat anti-mouse IgG), lOOul / well, 37 ° C wet box for lh; wash the plate with washing solution 4 times, each 5min, the last shot of the enzyme plate; add reagents
  • the coloring solution prepared in the box is lOOul/well, and the color is 10-25 min in the dark.
  • Table 3 shows the serum anti-HBs IgG antibody levels (1 : N) of each experimental group in 28 weeks after the use of the composite adjuvant provided in Example 3.
  • each experimental group can produce anti-HBs IgG antibodies, and generally reached the peak at the 8th week.
  • the composite vaccine adjuvant group achieved the best immune effect, and the antibody titer level was significantly higher than that of the unadjuvant vaccine group within 28 weeks, p ⁇ 0.05, and it was able to maintain a high level during the test period.
  • All of the experimental groups added with adjuvant produced significantly higher antibody levels than the no adjuvant group.
  • the antibody levels in the adjuvant adjuvant group and the single adjuvant group were significantly higher than those in the aluminum adjuvant control group.
  • P ⁇ 0.05 the antibody level in the compound vaccine adjuvant group was significantly higher than that in the single adjuvant group.
  • the compound vaccine adjuvant has the effect of enhancing the immune effect of hepatitis B surface antigen, and the humoral immunity enhancement effect is higher than that of the aluminum adjuvant and the single hydroxide adjuvant and the single sodium ferulate adjuvant.
  • the hepatitis B surface antigen used in this test case was provided by Shenzhen Kangtai Biological Products Co., Ltd., sodium ferulate was purchased from Fujian Dongdong Liexun Pharmaceutical Co., Ltd., and the water was delivered by Tianjin Guangfu Fine Chemical Research Institute.

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Description

复合疫苗佐剂
技术领域
本发明涉及一种复合疫苗佐剂， 属于免疫学技术领域。
背景技术
阿魏酸钠， 其化学名称为： 3-曱氧基 -4-羟基桂皮酸钠盐， 分子式为： Ci0H9NaO4, 分子量为： 216.17 , 结构式如图 1 , 一般以二水合物形式存在， 其 中二水合物的分子式为 C1QH9Na(V2H20, 分子量为： 252.20。 阿魏酸钠具有舒 张血管， 抗血小板活性， 抗氧化和清除自由基等多种药理作用。
辞是所有生物体必需的微量元素。 人体的生长发育离不开辞， 体内辞水平 的高低会对神经、 生殖和免疫系统产生影响。 辞和机体免疫系统之间的关系尤 为复杂， 主要体现在以下四个方面： ①每日辞的摄入与吸收的量取决于食物的 组分、 个体年龄和健康状况； ②辞作为 300多种具有生物活性的酶的辅助因子， 能够间接影响免疫系统； ③辞对淋巴细胞的产生、 成熟和功能都能产生直接影 响； ④辞能够影响免疫促进剂的功能。 利用以上辞的特性， 人们在某些疾病的 治疗中釆用了辞制剂辅助疗法， 即在普通治疗的同时给予患者一定剂量的辞制 剂， 取得了良好的治疗效果。 目前， 已在感染性疾病， 自身免疫病， 疫苗接种 等多项临床实验中观察到了辞的积极作用。
疫苗佐剂是当今疫苗研究开发的热点， 理想疫苗佐剂需要具备安全、 有效、 靶向和经济的特点。 当前的铝佐剂是历史最长且应用最为广泛的被批准用于人 体的疫苗佐剂， 它主要依靠储存库效应和免疫刺激效应两种机制， 显著提高机 体的免疫应答。 但作为疫苗佐剂， 它在安全性和靶向性方面还存在缺陷。 而其 他一些正在研究或已证实有佐剂作用的物质， 由于安全性、 有效性或经济方面 问题， 未能被批准用于人体。
氢氧化辞， 其化学式为 Zn(OH)2, 化学式量为 99.4046。 在等电点、 水溶性 等方面与氢氧化铝具有较高的相似性， 而且制备工艺简单、 成本低廉。
发明内容
为解决现有疫苗佐剂存在的毒副作用、 价格昂贵等问题， 本发明经过大量 实验和创造性劳动， 提供了一种安全、 有效、 稳定、 便宜的新型复合疫苗佐剂。 本发明提供的是这样一种复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 10: 1~50:1。
所述阿魏酸钠的化学名称为 3-曱氧基 -4-羟基桂皮酸钠盐， 分子式为： Ci0H9NaO4, 分子量为： 216.17 , 结构式如图 1所示， 一般以二水合物形式存在， 其中二水合物分子式为 CiaH9Na04*2H20, 分子量为： 252.20。
所述氢氧化辞的化学式为 Zn(OH)2, 化学式量为 99.4046。
所述阿魏酸钠在人体中作为疫苗佐剂的剂量推荐为： 10-50 mg。
所述氢氧化辞在人体中作为疫苗佐剂的剂量推荐为： 0.25~1.5mg。
所述复合疫苗佐剂诱导的免疫反应为体液免疫。
所述阿魏酸钠为市购医用产品。
所述氢氧化辞为市购医用产品。
本发明与现有技术相比具有下列优点和效果： （1 ), 阿魏酸钠与氢氧化辞复 合的疫苗佐剂均无毒， 无累积毒性， 在免疫剂量范围内使用是安全可靠的； （2 ) 阿魏酸钠与氢氧化辞复合的疫苗佐剂， 能够有效诱导抗原特异性的体液免疫应 答， 且其所诱导的体液免疫应答效果优于单一铝佐剂， 同时也优于单一的阿魏 酸钠佐剂和单一的氢氧化辞佐剂； （3 )本发明的复合疫苗佐剂原料易得， 均为 市售产品， 制备工艺简单， 成本低， 性能稳定， 可用作乙肝疫苗、 基因工程疫 苗、 病毒疫苗等的佐剂。
附图说明
图 1为阿魏酸钠分子结构式。
图 2为使用实施例 1提供的复合佐剂后， 28周内， 各实验组小鼠血清抗 - HBs IgG抗体水平（1 :N);
图 3为使用实施例 2提供的复合佐剂后， 28周内， 各实验组小鼠血清抗 - HBs IgG抗体水平（1 :N);
图 4为使用实施例 3提供的复合佐剂后， 28周内， 各实验组小鼠血清抗 - HBs IgG抗体水平（1 :N)。
具体实施方式
下面通过实施例对本发明做进一步描述。
实施例 1 取市购医用阿魏酸钠 lOmg及氢氧化辞 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例例阿魏酸钠购自福建省闽东力捷迅药业有限公司， 氢氧化辞由天 津光复精细化工研究所提供。
实施例 2
取市购医用阿魏酸钠 25mg及氢氧化辞 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例中各个组分的来源、 品质与实施例 1相同。
实施例 3
取市购医用阿魏酸钠 50mg及氢氧化锌 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例中各个组分的来源、 品质与实施例 1相同。
实施例 4
取市购医用阿魏酸钠 15mg及氢氧化锌 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例中各个组分的来源、 品质与实施例 1相同。
实施例 5
取市购医用阿魏酸钠 20mg及氢氧化辞 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例中各个组分的来源、 品质与实施例 1相同。
实施例 6
取市购医用阿魏酸钠 30mg及氢氧化辞 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例中各个组分的来源、 品质与实施例 1相同。
实施例 7
取市购医用阿魏酸钠 35mg及氢氧化锌 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例中各个组分的来源、 品质与实施例 1相同。
实施例 8 取市购医用阿魏酸钠 40mg及氢氧化辞 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例中各个组分的来源、 品质与实施例 1相同。
实施例 9
取市购医用阿魏酸钠 45mg及氢氧化辞 lmg, 充分混合均匀，得所述的复合 疫苗佐剂。
本实施例中各个组分的来源、 品质与实施例 1相同。
实施例 10
本实施例通过动物试验， 对实施例 1、 实施例 2、 实施例 3提供的复合疫苗 佐剂的免疫效果加以证明。
试验 1 : 釆用实施例 1中的复合疫苗佐剂
A、 免疫
将 ICR小鼠分为复合疫苗佐剂组、 阿魏酸钠单一佐剂组、 氢氧化辞单一佐 剂组、 铝佐剂对照组、 无佐剂组和空白组， 共六个组， 每组 25只， 注射用疫苗 及复合疫苗佐剂的总体积均为 0.1ml, 即：
复合疫苗佐剂组是将实施例 1的复合疫苗佐剂（阿魏酸钠 10mg及氢氧化辞 lmg )与乙肝表面抗原 5ug混溶于 0.1ml生理盐水中后，皮下注射到小鼠身体中； 阿魏酸钠单一佐剂组是将阿魏酸钠 10mg和乙肝表面抗原 5ug混溶于 0.1ml 生理盐水中后， 皮下注射到小鼠身体中；
氢氧化辞单一佐剂组是将氢氧化辞 lmg和乙肝表面抗原 5ug混溶于 0.1ml 生理盐水中后， 皮下注射到小鼠身体中；
铝佐剂对照组是将氢氧化铝 0.4mg和乙肝表面抗原 5ug混溶于 0.1ml生理盐 水中后， 皮下注射到小鼠身体中；
无佐剂组是将乙肝表面抗原 5ug溶于 0.1ml生理盐水中后，皮下注射到小鼠 身体中；
空白组是向每只小鼠注射生理盐水 0.1 ml。
免疫方案： 共免疫 3次， 分别在 0、 1、 6月皮下注射 ICR小鼠。
B、 ELISA检测血清抗 - HBs IgG水平
在初次免疫后 4、 8、 16、 24、 32周， 釆集小鼠尾静脉血， 分离血清， ELISA 检测血清抗- HBs IgG水平， 按蛋白微孔试剂盒方法进行检测， 具体方法是： 4(^L HBsAg溶于 10ml l x包被稀释液， 混匀， 包被 96孔酶标板， lOOul/孔； 酶 标板放于湿盒， 4°C过夜； 掉孔内液体， 拍净板子； 加入 I xBSA, 200ul/孔， 酶 标板在 37°C湿盒放置 lh; 倒掉孔内液体， 拍净板子； 血清经倍比系列稀释后， 加入酶标板， lOOul/孔， 37°C湿盒放置 lh; 用试剂盒配制的洗液洗板 4次， 每次 5min,末次拍净酶标板；力。入试剂盒配制的二抗（ peroxidase 结合的羊抗鼠 IgG ), lOOul/孔， 37°C湿盒放置 lh; 用洗液洗板 4次， 每次 5min, 末次拍净酶标板； 加入试剂盒配制的显色液， lOOul/孔，避光显色 10-25min。加终止液，每孔 lOOul; 酶标板放入酶标仪， 在 405nm处读板。
C、 数据分析
对所获得的实验数据以 SPSS11.5 统计软件进行单因素方差分析， P < 0.05 具有统计学意义。
表 1为使用实施例 1提供的复合佐剂后， 28周内， 各实验组小鼠血清抗 - HBs IgG抗体水平（1： N)。
表 1
Figure imgf000006_0001
通过数据分析可以看出，从第四周开始，各实验组都能产生抗 -HBsIgG抗体, 并普遍于第 8周达到峰值。 其中复合疫苗佐剂组所取得的免疫效果最佳， 28周 之内其抗体滴度水平都显著高于无佐剂疫苗组， p < 0.05 , 且能够在检测期间维 持较高的水平。 所有添加佐剂的实验组均产生了显著高于无佐剂组的抗体水平。 其中复合疫苗佐剂组、 氢氧化辞单一佐剂组抗体水平都显著高于铝佐剂对照组 , P < 0.05 , 复合疫苗佐剂组抗体水平显著高于氢氧化辞单一佐剂组。 提示复合疫 苗佐剂具有能够增强乙肝表面抗原的免疫效果的作用， 其体液免疫增强效果高 于铝佐剂和单一氢氧化辞佐剂以及单一阿魏酸钠佐剂。 见图 2 28周内， 各实验 组小鼠血清抗 - HBs IgG抗体水平（1： N) (阿魏酸钠低剂量组 )
本试验例所用乙肝表面抗原由深圳康泰生物制品有限公司提供， 阿魏酸钠 购自福建省闽东力捷迅药业有限公司， 氢氧化辞由天津光复精细化工研究所提 供。
试验 2: 釆用实施例 2中的复合疫苗佐剂
A、 免疫
将 ICR小鼠分为复合疫苗佐剂组、 阿魏酸钠单一佐剂组、 氢氧化辞单一佐 剂组、 铝佐剂对照组、 无佐剂组和空白组， 共六个组， 每组 25只， 注射用疫苗 及复合疫苗佐剂的总体积均为 0.1ml, 即：
复合疫苗佐剂组是将实施例 2的复合疫苗佐剂 (阿魏酸钠 25mg+氢氧化辞 lmg )与乙肝表面抗原 5ug混溶于 0.1ml生理盐水中后，皮下注射到小鼠身体中； 阿魏酸钠单一佐剂组是将阿魏酸钠 25mg和乙肝表面抗原 5ug混溶于 0.1ml 生理盐水中后， 皮下注射到小鼠身体中；
氢氧化辞单一佐剂组是将氢氧化辞 lmg和乙肝表面抗原 5ug混溶于 0.1ml 生理盐水中后， 皮下注射到小鼠身体中；
铝佐剂对照组是将氢氧化铝 0.4mg和乙肝表面抗原 5ug混溶于 0.1ml生理盐 水中后， 皮下注射到小鼠身体中；
无佐剂组是将乙肝表面抗原 5ug溶于 0.1ml生理盐水中后，皮下注射到小鼠 身体中；
空白组是向每只小鼠注射生理盐水 0.1 ml。
免疫方案： 共免疫 3次， 分别在 0、 1、 6月皮下注射 ICR小鼠。
B、 ELISA检测血清抗 - HBs IgG水平
在初次免疫后 4、 8、 16、 24、 32周， 釆集小鼠尾静脉血， 分离血清， ELISA 检测血清抗- HBs IgG水平， 按蛋白微孔试剂盒方法进行检测， 具体方法是： 4(^L HBsAg溶于 10ml l x包被稀释液， 混匀， 包被 96孔酶标板， lOOul/孔； 酶 标板放于湿盒， 4°C过夜； 掉孔内液体， 拍净板子； 加入 I xBSA, 200ul/孔， 酶 标板在 37°C湿盒放置 lh; 倒掉孔内液体， 拍净板子； 血清经倍比系列稀释后， 加入酶标板， lOOul/孔， 37°C湿盒放置 lh; 用试剂盒配制的洗液洗板 4次， 每次 5min,末次拍净酶标板；力。入试剂盒配制的二抗（ peroxidase 结合的羊抗鼠 IgG ), lOOul/孔， 37°C湿盒放置 lh; 用洗液洗板 4次， 每次 5min, 末次拍净酶标板； 加入试剂盒配制的显色液， lOOul/孔，避光显色 10-25min。加终止液，每孔 lOOul; 酶标板放入酶标仪， 在 405nm处读板。
C、 数据分析
对所获得的实验数据以 SPSS11.5 统计软件进行单因素方差分析， P < 0.05 具有统计学意义。
表 2为使用实施例 2提供的复合佐剂后， 28周内， 各实验组小鼠血清抗- HBs IgG抗体水平（1 :N)。
表 2
Figure imgf000008_0001
通过数据分析可以看出，从第四周开始，各实验组都能产生抗 -HBsIgG抗体， 并普遍于第 8周达到峰值。 其中复合疫苗佐剂组所取得的免疫效果最佳， 28周 之内其抗体滴度水平都显著高于无佐剂疫苗组， p < 0.05, 且能够在检测期间维 持较高的水平。 所有添加佐剂的实验组均产生了显著高于无佐剂组的抗体水平。 其中复合疫苗佐剂组、 氢氧化辞单一佐剂组抗体水平都显著高于铝佐剂对照组 ,
P < 0.05 , 复合疫苗佐剂组抗体水平显著高于氢氧化辞单一佐剂组。 提示复合疫 苗佐剂具有能够增强乙肝表面抗原的免疫效果的作用， 其体液免疫增强效果高 于铝佐剂和单一氢氧化辞佐剂以及单一阿魏酸钠佐剂。 见图 3 28周内， 各实验 组小鼠血清抗 - HBs IgG抗体水平（1： N) (阿魏酸钠中剂量组 )
本试验例所用乙肝表面抗原由深圳康泰生物制品有限公司提供， 阿魏酸钠 购自福建省闽东力捷迅药业有限公司， 氢氧化辞由天津光复精细化工研究所提 供。
试验 3: 釆用实施例 3中的复合疫苗佐剂
A、 免疫
将 ICR小鼠分为复合疫苗佐剂组、 阿魏酸钠单一佐剂组、 氢氧化辞单一佐 剂组、 铝佐剂对照组、 无佐剂组和空白组， 共六个组， 每组 25只， 注射用疫苗 及复合疫苗佐剂的总体积均为 0.1ml, 即：
复合疫苗佐剂组是将实施例 3 (阿魏酸钠 50mg+氢氧化辞 lmg )与乙肝表面 抗原 5ug混溶于 0.1ml生理盐水中后， 皮下注射到小鼠身体中；
阿魏酸钠单一佐剂组是将阿魏酸钠 50mg和乙肝表面抗原 5ug混溶于 0.1ml 生理盐水中后， 皮下注射到小鼠身体中；
氢氧化辞单一佐剂组是将氢氧化辞 lmg和乙肝表面抗原 5ug混溶于 0.1ml 生理盐水中后， 皮下注射到小鼠身体中；
铝佐剂对照组是将氢氧化铝 0.4mg和乙肝表面抗原 5ug混溶于 0.1ml生理盐 水中后， 皮下注射到小鼠身体中；
无佐剂组是将乙肝表面抗原 5ug溶于 0.1ml生理盐水中后，皮下注射到小鼠 身体中；
空白组是向每只小鼠注射生理盐水 0.1 ml。
免疫方案： 共免疫 3次， 分别在 0、 1、 6月皮下注射 ICR小鼠。
B、 ELISA检测血清抗 - HBs IgG水平
在初次免疫后 4、 8、 16、 24、 32周， 釆集小鼠尾静脉血， 分离血清， ELISA 检测血清抗- HBs IgG水平， 按蛋白微孔试剂盒方法进行检测， 具体方法是： 4(^L HBsAg溶于 10ml l x包被稀释液， 混匀， 包被 96孔酶标板， lOOul/孔； 酶 标板放于湿盒， 4°C过夜； 掉孔内液体， 拍净板子； 加入 I xBSA, 200ul/孔， 酶 标板在 37°C湿盒放置 lh; 倒掉孔内液体， 拍净板子； 血清经倍比系列稀释后， 加入酶标板， lOOul/孔， 37°C湿盒放置 lh; 用试剂盒配制的洗液洗板 4次， 每次 5min,末次拍净酶标板；力。入试剂盒配制的二抗（ peroxidase 结合的羊抗鼠 IgG ), lOOul/孔， 37°C湿盒放置 lh; 用洗液洗板 4次， 每次 5min, 末次拍净酶标板； 加入试剂盒配制的显色液， lOOul/孔，避光显色 10-25min。加终止液，每孔 lOOul; 酶标板放入酶标仪， 在 405nm处读板。
C、 数据分析
对所获得的实验数据以 SPSS11.5 统计软件进行单因素方差分析， P < 0.05 具有统计学意义。
表 3为使用实施例 3提供的复合佐剂后， 28周内， 各实验组小鼠血清抗 - HBs IgG抗体水平（1 :N)。
表 3
Figure imgf000010_0001
通过数据分析可以看出，从第四周开始，各实验组都能产生抗 -HBsIgG抗体， 并普遍于第 8周达到峰值。 其中复合疫苗佐剂组所取得的免疫效果最佳， 28周 之内其抗体滴度水平都显著高于无佐剂疫苗组， p < 0.05, 且能够在检测期间维 持较高的水平。 所有添加佐剂的实验组均产生了显著高于无佐剂组的抗体水平。 其中复合疫苗佐剂组、 氢氧化辞单一佐剂组抗体水平都显著高于铝佐剂对照组 , P < 0.05 , 复合疫苗佐剂组抗体水平显著高于氢氧化辞单一佐剂组。 提示复合疫 苗佐剂具有能够增强乙肝表面抗原的免疫效果的作用， 其体液免疫增强效果高 于铝佐剂和单一氢氧化辞佐剂以及单一阿魏酸钠佐剂。
见图 4, 28周内， 各实验组小鼠血清抗 - HBs IgG抗体水平（1 :N) (阿魏酸 钠高剂量组）
本试验例所用乙肝表面抗原由深圳康泰生物制品有限公司提供， 阿魏酸钠 购自福建省闽东力捷迅药业有限公司， 氢氧化辞由天津光复精细化工研究所提 供。

Claims

权利要求书
1、 一种复合疫苗佐剂， 其特征在于由下列组分组成： 阿魏酸钠与氢氧化辞 的质量比为 10: 1~50: 1。
2、 根据权利要求 1所述的复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 10:1。
3、 根据权利要求 1所述的复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 25:1。
4、 根据权利要求 1所述的复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 50:1。
5、 根据权利要求 1所述的复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 15:1。
6、 根据权利要求 1所述的复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 20:1。
7、 根据权利要求 1所述的复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 30:1。
8、 根据权利要求 1所述的复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 40:1。
9、 根据权利要求 1所述的复合疫苗佐剂， 其特征在于由下列组分组成： 阿 魏酸钠与氢氧化辞的质量比为 45:1。
10、 一种复合疫苗佐剂的制备方法， 其特征在于： 所述的复合疫苗佐剂由 下列组分组成： 阿魏酸钠与氢氧化辞的质量比为 10: 1~50:1； 制备方法为： 按照 上述配比， 取阿魏酸钠和氢氧化辞， 充分混合均勾， 得所述的复合疫苗佐剂。
PCT/CN2011/072034 2011-01-26 2011-03-22 复合疫苗佐剂 WO2012100453A1 (zh)

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CN101791302A (zh) * 2010-02-24 2010-08-04 王保明 含阿魏酸钠的药物组合物及其制备方法

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