CN103893751B - Pneumococcal polysaccharide-protein conjugate vaccine and its preparation method - Google Patents

Pneumococcal polysaccharide-protein conjugate vaccine and its preparation method Download PDF

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CN103893751B
CN103893751B CN201410114934.8A CN201410114934A CN103893751B CN 103893751 B CN103893751 B CN 103893751B CN 201410114934 A CN201410114934 A CN 201410114934A CN 103893751 B CN103893751 B CN 103893751B
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protein
polysaccharide
pneumococcal
vaccine
carrier
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CN103893751A (en
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朱涛
宇学峰
邱东旭
毛慧华
邵忠琦
刘正
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天津康希诺生物技术有限公司
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092Streptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines

Abstract

本发明提供了一种肺炎球菌多糖蛋白缀合疫苗及其制备方法,所述肺炎球菌多糖蛋白缀合疫苗是包括一种或多种肺炎链球菌荚膜多糖与蛋白质耦联结合的免疫缀合物,所述免疫缀合物中至少有一种免疫缀合物是一种单一的肺炎链球菌荚膜多糖与两种或两种以上的蛋白质耦联而形成的,与现有的肺炎球菌结合疫苗相比,其免疫原性更强,可以在更广阔的人群中引起免疫应答;同时由于2种载体蛋白通过多糖共价耦联,其中具有保护性的蛋白抗原表位也会诱导比两种蛋白混合注射时更高的免疫反应,相互协同作用,进一步增进了载体蛋白的免疫原性,增加了机体对多糖的免疫反应;其制备方法简单,适合规模化工业生产的需要。 The present invention provides a pneumococcal polysaccharide-protein conjugate vaccine and method of preparation, a pneumococcal polysaccharide protein conjugate vaccine comprising one or more S. pneumoniae capsular polysaccharide coupled to the protein binding immunoconjugate the immunoconjugate at least one immunoconjugate is a single S. pneumoniae capsular polysaccharide of two or more proteins formed by coupling, in conjunction with the conventional vaccine pneumococcal phase ratio, which is more immunogenic, may cause the immune response in a broader population; and because the two kinds of the polysaccharide to a carrier protein by covalent coupling, wherein a protective protein epitopes will induce mixing ratio of the two proteins higher injection immune response, interact synergistically to further enhance the immunogenic carrier protein, increase the immune response to polysaccharide; the preparation method is simple, the need for large-scale industrial production.

Description

一种肺炎球菌多糖蛋白缀合疫苗及其制备方法 Pneumococcal polysaccharide-protein conjugate vaccine and its preparation method

技术领域 FIELD

[0001] 本发明涉及免疫学领域,尤其是肺炎球菌多糖蛋白缀合疫苗及其制备方法。 [0001] The present invention relates to the field of immunology, in particular pneumococcal polysaccharide-protein conjugate vaccine and its preparation method.

背景技术 Background technique

[0002] 由肺炎球菌(肺链)所引起的感染是全世界发病率和死亡率的主要起因。 [0002] is a major cause of morbidity and mortality worldwide by the pneumococcal infection (lungs chain) caused. 肺炎、发热性菌血症和脑膜炎是侵入性肺炎球菌性疾病的最常见表现形式,而呼吸道内的细菌扩散可导致中耳感染、窦炎或复发性支气管炎。 Pneumonia, fever, bacteremia and meningitis is the most common form of invasive pneumococcal disease, and the spread of bacteria in the respiratory tract can cause middle ear infections, sinusitis or recurrent bronchitis. 与侵入性疾病相比,非侵入性的表现形式通常不那么严重,但更多见。 Compared with invasive disease, non-invasive manifestations are usually less severe, but more common. 由于抗生素耐药性传染病的扩散,以及肺炎球菌肺炎经常在流感感染之后出现,肺炎球菌疾病在流感期间发作的可能性进一步增加。 As the proliferation of antibiotic-resistant infectious diseases, as well as pneumococcal pneumonia frequently after influenza infection, further increasing the likelihood of pneumococcal disease during an influenza attack. 由肺炎链球菌引发的疾病已成为全球一个重要的公共卫生问题。 Caused by pneumococcal disease has become a major global public health problem. 肺炎球菌已成为全球儿童的头号杀手。 Pneumococcal has become the number one killer of children around the world.

[0003] 我国肺炎的病死率为16.4%,其中50岁以上中老年人及1岁以下婴幼儿分别高达28.6%和22.0%。 Mortality rate [0003] of pneumonia was 16.4%, including the elderly over 50 years of age and infants up to 1 year 28.6% and 22.0%, respectively. 我国肺炎球菌在健康儿童中的携带率较高,资料统计显示,在北方地区健康儿童中的携带率为24.2%,南方地区为31.3%。 Of pneumococcal carriage rates higher in healthy children, the statistics show that in the northern region of healthy children carry 24.2%, 31.3% in the south. 而该病是导致5岁以下儿童死亡的重要原因。 The disease is an important cause of death in children under five years of age. 主要原因在于婴幼儿免疫系统的发育尚不完善,免疫力较弱。 The main reason is the development of the infant's immune system is not perfect, the immune system is weak. 并且年龄越小的婴儿,免疫力越弱。 And the younger the baby, the weaker the immune system.

[0004] 肺炎球菌大约有90种血清型(菌株),我国的统计资料显示肺炎球菌感染菌株前几个血清型依次为:5、6、19、23、14、2、4型。 [0004] About 90 pneumococcal serotypes (strains), China's statistics show that the first few strains of pneumococcal infection serotypes were: 5,6,19,23,14,2,4 type. 据一项收集了860株肺炎球菌分离株的研究显示, 有109株(12.7%)表现为血清群6,在中国肺炎球菌血清型6的红霉素耐药性达100%,其中的6A、6B和6C型分别为62株(56.9%)、38株(34.9%)和9株(8.2%)。 According to a study collection of pneumococcal isolates 860 showed that 109 (12.7%) showed serogroup 6, in China pneumococcal serotypes 6 of erythromycin resistance up to 100%, which 6A, 6B and 6C are type 62 (56.9%), 38 (34.9%), and 9 (8.2%).

[0005] 中国生物技术集团成都生物制品研究所生产的23价肺炎球菌疫苗是选取了23种最常见的致病菌(1,2,3,4,5,6B,7F,8,9N,9V,10A,11A,12F,14,15B,17F,18C,19A,19F,20, 22F,23F,33F),分别发酵培养并分离提纯肺炎球菌荚膜上的各型多糖,按等比例混合制成疫苗。 [0005] China National Biotec Group Chengdu Institute of Biological Products 23-valent pneumococcal vaccine is to select the 23 most common pathogens (1,2,3,4,5,6B, 7F, 8,9N, 9V , 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F), respectively, fermentation and separation and purification of each pneumococcal polysaccharide capsule on, made by mixing equal proportions vaccine.

[0006] 细菌的多糖是一种胸腺非依赖性抗原,这种抗原与胸腺依赖性抗原的主要区别在于前者不需要T淋巴细胞的辅助来产生抗体。 [0006] Bacterial polysaccharides is a thymus-independent antigens, and the main difference between this antigen is thymus-dependent antigens is that the former does not require helper T-lymphocytes to produce antibodies. 因此多糖疫苗存在以下问题:(1)在幼小动物或婴幼儿体内只能产生微弱的免疫反应,甚至不产生免疫反应,免疫反应随年龄的增长而增强;(2)产生低亲和力的抗体;(3)只产生短暂的免疫反应,不具备反复接种时的免疫记忆和免疫增强效应;(4)容易产生免疫耐受;(5)普通的佐剂对这种抗原不易起到免疫增强的作用。 So exist polysaccharide vaccine the following questions: (1) in infants or young animal body can only produce a weak immune response, not even generate an immune response, the immune response is enhanced with age; (2) the production of antibodies of low affinity; ( 3) produce only temporary immune response, do not have an immunological memory in repeated vaccination and immune enhancement effect; (4) easy to produce immune tolerance; (5) common to this antigen adjuvant immune enhancement effect is not easy to play. 23价多糖疫苗对于侵入型肺链感染的保护率为50-70%。 23-valent polysaccharide vaccine for invasive pulmonary infection protection chain was 50-70%. 而且只能用于2岁以上的人群接种,而肺炎的高峰发病年龄为6-12月龄。 And it can only be used to inoculate the population over the age of 2, and the peak age of onset of pneumonia in 6 - 12 months of age.

[0007] 多糖蛋白缀合疫苗(结合疫苗)是目前最先进的疫苗技术,在特异抗原上加上蛋白质载体,可增加其免疫原性。 [0007] The polysaccharide-protein conjugate vaccines (combined vaccines) is the most advanced vaccine technology, coupled with the carrier proteins on specific antigen, may increase its immunogenicity. 蛋白质载体具有T细胞依赖特性,多糖蛋白缀合疫苗可将非T细胞依赖性质的多糖抗原转变为T细胞依赖性质的抗原,激发机体的T辅助细胞产生一系列的免疫增强效应。 A protein having T cell-dependent characteristic vector, polysaccharide-protein conjugate vaccine polysaccharide antigens can be converted to non-dependent nature of T cell-dependent nature of the T-cell antigen, T helper cells stimulate the body's immune enhancing effect produce a series. 荚膜多糖结合疫苗,在多糖上加蛋白载体,由非T细胞依赖性抗原变为T细胞依赖性抗原,增加其免疫原性。 Capsular polysaccharide conjugate vaccine, add the protein carrier in a polysaccharide, a T-independent antigen becomes T cell-dependent antigen, increasing its immunogenicity. 结合疫苗接种后产生的抗体在质量和数量上是一代疫苗的400-1000倍,产生免疫保护更广更强,保护时间更长久,达到高效保护。 Antibody production after vaccination is 400-1000 times the combined generation of vaccines in terms of quality and quantity, resulting in a wider stronger immune protection, longer protection time, to achieve efficient protection.

[0008] 2000年,美国辉瑞公司第一个7价肺炎球菌结合疫苗上市;美国辉瑞公司开发婴幼儿更有针对性的7(4、68、利、14、18(:、19?和23?)价肺炎球菌蛋白疫苗,对5岁以下儿童也有效。荚膜多糖蛋白结合疫苗,在多糖上加蛋白载体,由非T细胞依赖性抗原变为T细胞依赖性抗原,可增加其免疫原性,可用于6周龄以上的儿童。目前辉瑞公司正在中国注册13价结合疫苗,增加了6个血清型(1,3,5,7F,6A,19A)。 [0008] 2000, Pfizer first 7-valent pneumococcal conjugate vaccine on the market; the US company Pfizer to develop more targeted infants 7 (4,68, Lee, 14, 18 (: 19 and 23?? ) -valent pneumococcal protein vaccine for children under 5 years is also effective. capsular polysaccharide-protein conjugate vaccine, add the protein carrier in a polysaccharide, a T-independent antigen becomes T cell-dependent antigen, increase its immunogenicity can be used for children over 6 weeks of age. Pfizer is currently China registered 13-valent conjugate vaccine, an increase of six serotypes (1,3,5,7F, 6A, 19A).

[0009] 但是我国的资料显示肺炎球菌感染菌株血清型依次为:5、6、1、19、23、14、2、4,辉瑞7价肺炎球菌结合疫苗对我国常见致病菌型覆盖率只有50%左右,而13价结合疫苗的覆盖率也仅有70%。 [0009] However, our data show that serotype strains of pneumococcal infection were: 5,6,1,19,23,14,2,4, Pfizer 7-valent pneumococcal conjugate vaccine coverage for our common pathogens type only 50%, while the 13-valent conjugate vaccine coverage is also only 70%. 惠氏7价肺炎球菌结合疫苗需要接种4针剂,每针860元,价格昂贵,不利于推广。 Wyeth's 7-valent pneumococcal conjugate vaccine is given as 4 injections, 860 yuan per pin, expensive and not conducive to the promotion. 13价疫苗估计其价格将更为昂贵。 13-valent vaccine is estimated that the price will be more expensive. 因此美国辉瑞公司的7和13价结合疫苗不太适合我国大范围的儿童肺炎预防。 So Pfizer 7 and 13-valent conjugate vaccine is not suitable for the prevention of pneumonia in children a wide range of our country.

[0010]尽管13价肺炎球菌结合疫苗已经上市,但是其中有几个血清型的免疫效果相对其他血清型较低,如3型。 [0010] While the 13-valent pneumococcal conjugate vaccine have been listed, but there are several immune effect serotypes low relative to other serotypes, such as 3 type. 且随着不同血清型的增加和同种载体蛋白的重复使用,使得载体蛋白用量增多,其免疫原性反而降低。 And with different serotypes of the same kind to increase reuse and a carrier protein, such that the increased amount of the carrier protein, but reduced immunogenicity.

发明内容 SUMMARY

[0011] 本发明所要解决的技术问题在于提供一种肺炎球菌多糖蛋白缀合疫苗。 [0011] The present invention solves the technical problem is to provide a pneumococcal polysaccharide-protein conjugate vaccine.

[0012] 本发明所要解决的另一技术问题在于提供上述肺炎球菌多糖蛋白缀合疫苗的制备方法。 [0012] The present invention solves the technical problem is to provide the above-described other pneumococcal polysaccharide-protein vaccine production method of conjugation.

[0013 ]为解决上述技术问题,本发明的技术方案是: [0013] To solve the above technical problem, the technical solution of the present invention is:

[0014] -种肺炎球菌多糖蛋白缀合疫苗,是包括一种或多种肺炎链球菌荚膜多糖与蛋白质耦联结合的免疫缀合物,所述免疫缀合物中至少有一种免疫缀合物是一种单一的肺炎链球菌荚膜多糖与两种或两种以上的蛋白质耦联而形成的。 [0014] - pneumococcal polysaccharide-protein conjugate vaccine, comprising one or more immunoconjugates Streptococcus pneumoniae capsular polysaccharide coupled to binding protein, the immunoconjugate conjugated to at least one immune was a single, Streptococcus pneumoniae capsular polysaccharide of two or more proteins formed by the coupling.

[0015] 优选的,上述肺炎球菌多糖蛋白缀合疫苗,包含多价肺炎球菌缀合物的免疫组合物,是分离提纯多种血清型肺炎球菌荚膜上的荚膜多糖与蛋白质(载体蛋白)偶联结合后混合而成的。 [0015] Preferably, in the pneumococcal polysaccharide protein conjugate vaccine, comprising a multivalent pneumococcal conjugate immunogenic composition is the separation and purification of various proteins on the capsular polysaccharide pneumococcal capsular serotypes (carrier protein) after coupling the mixture of binding.

[0016] 优选的,上述肺炎球菌多糖蛋白缀合疫苗,所述用于与荚膜多糖耦联结合的蛋白质是能够用于和荚膜多糖直接耦联或者通过化学连接头进行耦联的蛋白质。 [0016] Preferably, in the pneumococcal polysaccharide protein conjugate vaccine, in combination with means for coupling the capsular polysaccharide and protein can be utilized for direct coupling capsular polysaccharides or proteins for coupling by chemical connector.

[0017] 优选的,上述肺炎球菌多糖蛋白缀合疫苗,所述用于与荚膜多糖耦联结合的蛋白质为白喉类毒素,破伤风类毒素,载体蛋白CRM197,嗜血流感杆菌表面蛋白HiD,百日咳Prn 表面蛋白,百日咳Fha抗原和/或肺炎链球菌表面蛋白PspA。 [0017] Preferably, in the pneumococcal polysaccharide conjugate vaccine proteins, for binding to the capsular polysaccharide coupled to the protein is diphtheria toxoid, tetanus toxoid, the CRM197 carrier protein, the surface protein of Haemophilus influenzae HiD , Prn pertussis surface protein, Fha pertussis antigen and / or pneumococcal surface protein PspA.

[0018] 优选的,上述肺炎球菌多糖蛋白缀合疫苗,所述用于与荚膜多糖耦联结合的两种或两种以上的蛋白质中,其中一种蛋白质是具有保护性的抗原,而另一个蛋白是没有限制, 可以任意选择。 [0018] Preferably, in the pneumococcal polysaccharide protein conjugate vaccine, the combination of the two for the coupling of two or more capsular polysaccharides or proteins, wherein a protein is a protective antigen, and the other a protein is not limited and may be arbitrarily selected.

[0019]优选的,上述肺炎球菌多糖蛋白缀合疫苗,所述用于与荚膜多糖耦联结合的两种或两种以上的蛋白质中,其中一种是嗜血流感杆菌表面蛋白HiD或者是PspA蛋白。 [0019] Preferably, in the pneumococcal polysaccharide protein conjugate vaccine, the combination of the two for the coupling of two or more capsular polysaccharides or proteins, one of which is a surface protein of Haemophilus influenzae or HiD It is PspA protein.

[0020] 优选的,上述肺炎球菌多糖蛋白缀合疫苗,所述肺炎链球菌荚膜多糖是分离提纯血清型肺炎球菌荚膜上的荚膜多糖,所述血清型肺炎球菌的血清型包括1,2,3,4,5,6A,6B, 7F,8,9N,9V,10A,11A,12F,14,15B,17F,18C,19A,19F,20,22F,23F^P/^^33F。 [0020] Preferably, in the pneumococcal polysaccharide protein conjugate vaccine, a Streptococcus pneumoniae capsular polysaccharide serotype is isolated and purified pneumococcal capsular polysaccharide capsule on the pneumococcal serotypes comprises serotypes 1, 2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F ^ P / ^^ 33F.

[0021] 上述肺炎球菌蛋白疫苗的制备方法,具体步骤为: [0021] The method for preparing the vaccine pneumococcal proteins, the specific steps:

[0022] (1)分别分离提纯多种血清型肺炎球菌荚膜上的荚膜多糖; [0022] (1) are isolated and purified capsular polysaccharide on multiple serotypes of pneumococcus;

[0023] (2)分别分离提纯所选用的载体蛋白; [0023] (2) were purified by the selected carrier protein isolate;

[0024] (3)分别将各种荚膜多糖与载体蛋白偶联结合,其中,至少一种荚膜多糖与两种或两种以上的蛋白质耦联结合; [0024] (3) respectively, binding with various capsular polysaccharide conjugated to a carrier protein, wherein at least one capsular polysaccharide in combination with two or more proteins or coupling;

[0025] (4)将各种偶联结合混合形成疫苗原液,其中,至少一种荚膜多糖与两种或两种以上的蛋白质耦联结合。 [0025] (4) mixing various binding conjugate vaccine stock solution is formed, wherein at least one capsular polysaccharide in combination with two or more coupling or two proteins.

[0026] 所述荚膜多糖的分离纯化的具体步骤是一般技术人员已经掌握的现有技术;载体蛋白的生产也是一般技术人员已经掌握的现有技术。 [0026] The isolated and purified capsular polysaccharide specific steps prior art generally have mastered the art; production of a carrier protein prior art also have the general skill mastered.

[0027] 优选的,上述肺炎球菌蛋白疫苗的制备方法,所述步骤(3)中荚膜多糖与载体蛋白的偶联结合方式包括: [0027] Preferably, the preparation method of the pneumococcal vaccine protein, said step (3) of the capsular polysaccharide conjugated to a carrier protein binding include:

[0028] (1)荚膜多糖用⑶AP(1-氨基-4-二甲基-吡啶-四氟化硼)和三乙胺活化法偶联结合载体蛋白。 [0028] (1) a capsular polysaccharide ⑶AP (1- amino-4-dimethylamino - pyridine - tetrafluoroborate) and triethylamine activation method conjugate bound to a carrier protein. 该反应为异脲键连接反应。 This reaction is a reaction isourea bond. 在高pH时,CDAP与多糖残基上的羟基反应,转化成氰酸酯,载体蛋白上的氨基与其迅速反应,形成异脲键。 When the pH high, the reaction on the hydroxyl CDAP and polysaccharide residue, is converted into cyanate, amino group on the carrier protein with its rapid response, isourea bond formed. 该方法操作简单,容易重复;或[0029] (2)先对多糖进行104活化形成自由醛基并和载体蛋白上的氨基进行连接结合;或 This method is simple, easy to repeat; or [0029] (2) of the first activated polysaccharide 104 formed on the free amino and aldehyde groups and the connection carrier protein binding; or

[0030] (3)用己二酸二肼酰(ADH)和酸性多糖中的羧基(或者在多糖中氧化所获得的羧基)连接,然后再在EDAC存在的条件下和蛋白进行连接;或 [0030] (3) with adipic acid dihydrazide (ADH) and acidic polysaccharides carboxyl group (or a carboxyl group in the obtained oxidized polysaccharide) is connected, and then connected at the EDAC and the presence of protein; or

[0031] (4)荚膜多糖与载体蛋白偶联结合还可以通过对多糖进行片段,衍生加入链接头后再和载体蛋白上的氨基或羧基进行连接。 [0031] (4) a capsular polysaccharide conjugated to a carrier protein can also bind to the polysaccharide fragment derived added before connecting link head and the amino or carboxyl groups on the carrier protein.

[0032] 优选的,上述肺炎球菌多糖蛋白缀合疫苗的制备方法,所述步骤(3)中荚膜多糖与载体蛋白的偶联结合的5种方式均可以将肺链多糖链接的表面蛋白上,本发明以上述第(2) 种为优选。 The [0032] Preferably, the above-described method for preparing protein conjugated pneumococcal polysaccharide vaccine, said step (3) in five ways capsular polysaccharide conjugated to a carrier protein can be bound to the surface of lung protein linked polysaccharide chains The present invention in the above (2) types are preferred.

[0033] 优选的,上述肺炎球菌多糖蛋白缀合疫苗的制备方法,所述步骤(3)中一种荚膜多糖与两种或两种以上的蛋白质耦联结合的连接方式包括: [0033] Preferably, said protein is conjugated pneumococcal polysaccharide vaccine preparation method, the step of connection (3) of one capsular polysaccharide and two or more binding proteins of coupling comprises:

[0034] (1)单步反应法,将两种载体蛋白和荚膜多糖同时加入到反应容器中进行反应,这种方法较为简单,但是缀合物中不同蛋白的比例并不容易准确控制;或 [0034] (1) a single step reaction method, the capsular polysaccharide of two carrier proteins and added simultaneously to the reaction vessel reaction, this method is simple, but the proportions of the different protein conjugate is not easy to accurately control; or

[0035] (2)顺序法,先将荚膜多糖与其中一种载体蛋白进行连接,然后经过纯化,去除未结合的游离蛋白和游离多糖,再加入第二种载体蛋白进行连接,可以产生具有稳定结合比例的结合产品。 [0035] (2) sequential method, the capsular polysaccharide first with one of the carrier protein, and then purified to remove free protein and free unbound polysaccharide, the carrier protein and then adding a second connection, can be produced having stable combined ratio of combination products.

[0036] 优选的,上述肺炎球菌多糖蛋白缀合疫苗的制备方法,具体步骤为: [0036] Preferably, said protein is conjugated pneumococcal polysaccharide vaccine preparation, the specific steps:

[0037] (1)选取24种血清型的肺炎球菌培养,该24种血清型为1,2,3,4,5,6A,6B,7F,8, 91叭,1(^,11厶,12卩,14,158,17卩,18(:,19厶,19卩,20,22卩,23卩和33卩; [0037] (1) Select the 24 kinds of pneumococcal serotypes culture, the 24 kinds of serotype 1,2,3,4,5,6A, 6B, 7F, 8, 91 pair of, 1 (^, 11 Si, Jie 12, 14,158,17 Jie, 18 (:, 19 Si, 19 Jie, Jie 20, 22, 23 and 33 Jie Jie;

[0038] (2)分别提纯以上各种血清型肺炎球菌中抗原性强的荚膜多糖:肺炎球菌,灭活后离心收集上清液,经超滤浓缩,根据各肺炎球菌血清型特性分别加入适量(体积分数为70% 的)预冷乙醇,离心收集,得粗制多糖;将粗制多糖溶于乙酸钠溶液中,然后按1:2比例以冷酚混匀,离心去除蛋白,反复酚提5-6次,收集上清,用蒸馏水透析,透析后液体加2mol/L氯化钙溶液,加入乙醇搅拌,离心去除核酸,收集上清,补加乙醇(终浓度80%搅拌),离心收集沉淀,用乙醇、丙酮洗涤沉淀,脱水干燥后即为精制荚膜多糖,置-20°C保存备用,具体方法和参数可以参见美国专利US2007/0231340 [ 1 ](惠氏13价专利); [0038] (2) above were purified pneumococcal serotypes Strongly antigenic capsular polysaccharide: pneumococcus, inactivated supernatant was collected by centrifugation, concentrated by ultrafiltration, are added in accordance with various characteristics of pneumococcal serotypes amount (70% volume fraction) of pre-cooled ethanol, collected by centrifugation, to obtain the crude polysaccharides; crude polysaccharide was dissolved in sodium acetate solution and then 1: 2 ratio of phenol to cold mix, protein is removed by centrifugation, repeated phenol mention 5-6 times, the supernatant was collected, dialyzed against distilled water, dialysis liquid added 2mol / L calcium chloride solution, ethanol was added with stirring, the nucleic acid is removed by centrifugation, the supernatant was collected, supplemented with ethanol (final 80% concentration with stirring), centrifuged The precipitate was collected, washed with ethanol and acetone precipitation, after dried was purified capsular polysaccharides alternate set at -20 ° C, the specific methods and parameters may be found in U.S. Patent No. US2007 / 0231340 [1] (13 monovalent Patent Wyeth);

[0039] (3)通过现有的疫苗工艺生产CRM197(参照中国专利CN102766647),白喉类毒素(参照美国专利US2005/0002957),破伤风类毒素(参照中国专利CN102961741),百日咳的各种抗原(参照中国专利CN102793915),或将HiD(又称Protein D,参照欧洲专利EP0594610及国际专利W02007/071710),PspA蛋白(参照中国专利CN103288936)克隆至大肠杆菌进行表达并分离纯化; [0039] (3) by a conventional process for producing the CRM197 vaccine (see Chinese patent CN102766647), diphtheria toxoid (see U.S. Patent No. US2005 / 0002957), tetanus toxoid (see Chinese patent CN102961741), various pertussis antigens ( Referring to Chinese patent CN102793915), or HiD (also known as protein D, see European patent EP0594610 and the international Patent W02007 / 071710), PspA protein (see Chinese patent CN103288936) cloned into E. coli and expressed isolated and purified;

[0040] (4)取检测合格的各种血清型肺炎球菌的荚膜多糖,用高碘酸钠活化法获得醛基然后和两种蛋白同时结合,得到目标缀合物;或将其中一种蛋白和多糖先进行耦联,在不封闭活性基团的情况下进行纯化,将得到的纯化产物与第二种蛋白进一步进行耦联得到目标缀合物。 [0040] (4) take the serotypes tested and pneumococcal capsular polysaccharides, obtained aldehyde with sodium periodate activation method and then binding the two proteins simultaneously, to give the title conjugate; or one for coupling to proteins and polysaccharides, purified without blocking the active groups, the resulting product was purified with a second target protein is further coupled to give the conjugate.

[0041] 上述肺炎球菌蛋白疫苗在制备用于诱导对肺炎链球菌荚膜多糖缀合物和载体蛋白的免疫应答的药物方面的应用。 [0041] Application of the above pneumococcal proteins in the preparation of a vaccine medicament for inducing an immune Streptococcus pneumoniae capsular polysaccharide conjugate and the carrier protein response.

[0042]本发明的有益效果: [0042] Advantageous effects of the invention:

[0043]上述肺炎球菌多糖蛋白缀合疫苗,是含两种或两种以上不同载体蛋白的免疫缀合物,与现有的肺炎球菌结合疫苗相比,其免疫原性更强,可以在更广阔的人群中引起免疫应答;同时由于2种载体蛋白通过多糖共价耦联,其中具有保护性的蛋白抗原表位也会诱导比两种蛋白混合注射时更高的免疫反应,相互协同作用,进一步增进了载体蛋白的免疫原性, 增加了机体对多糖的免疫反应;其制备方法简单,适合规模化工业生产的需要。 [0043] The pneumococcal polysaccharide-protein conjugate vaccine, a immunoconjugate comprising two or more different carrier proteins, as compared with the conventional conjugate vaccine pneumococcal which more immunogenic, may be more broad population elicit an immune response; at the same time since the two kinds of the polysaccharide to a carrier protein by covalent coupling, wherein a protective protein epitopes will induce higher immune responses when injected mixed ratio of the two proteins, interact synergistically, further enhance the immunogenic carrier protein, increase the immune response to polysaccharide; the preparation method is simple, the need for large-scale industrial production.

具体实施方式 Detailed ways

[0044]下面结合具体实施例对本发明所述技术方案作进一步的说明。 [0044] The following specific examples in conjunction with the aspect of the present invention will be further described.

[0045] 获得荚膜多糖和载体蛋白: [0045] The capsular polysaccharide and a carrier protein obtained:

[0046] (1)选取24种血清型(1,2,3,4,5,6厶,68,7卩,8,91利,1(^,11厶,12卩,14,158,17卩, 18C,19A,19F,20,22F,23F,33F)的肺炎球菌培养; [0046] (1) Select the 24 kinds of serotypes (Si 1,2,3,4,5,6, 68,7 Jie, 8,91 Lee, 1 (^, 11 Si, 12 Jie, 14,158,17 Jie, 18C, 19A, 19F, 20,22F, 23F, 33F) of pneumococcal culture;

[0047] (2)分别提纯以上各种血清型肺炎球菌中抗原性强的荚膜多糖:肺炎球菌,灭活后离心收集上清液,经超滤浓缩,根据各肺炎球菌血清型特性分别加入适量(体积分数为70% 的)预冷乙醇,离心收集,得粗制多糖;将粗制多糖溶于乙酸钠溶液中,然后按1:2比例以冷酚混匀,离心去除蛋白,反复酚提5-6次,收集上清,用蒸馏水透析,透析后液体加2mol/L氯化钙溶液,加入乙醇搅拌,离心去除核酸,收集上清,补加乙醇(终浓度80%搅拌),离心收集沉淀,用乙醇、丙酮洗涤沉淀,脱水干燥后即为精制荚膜多糖,置-20°C保存备用; [0047] (2) above were purified pneumococcal serotypes Strongly antigenic capsular polysaccharide: pneumococcus, inactivated supernatant was collected by centrifugation, concentrated by ultrafiltration, are added in accordance with various characteristics of pneumococcal serotypes amount (70% volume fraction) of pre-cooled ethanol, collected by centrifugation, to obtain the crude polysaccharides; crude polysaccharide was dissolved in sodium acetate solution and then 1: 2 ratio of phenol to cold mix, protein is removed by centrifugation, repeated phenol mention 5-6 times, the supernatant was collected, dialyzed against distilled water, dialysis liquid added 2mol / L calcium chloride solution, ethanol was added with stirring, the nucleic acid is removed by centrifugation, the supernatant was collected, supplemented with ethanol (final 80% concentration with stirring), centrifuged The precipitate was collected, washed with ethanol and acetone precipitation, after dried was purified capsular polysaccharides backup set at -20 ° C;

[0048] (3)通过上述技术方案中记载的现有疫苗工艺生产CRM197,白喉类毒素,破伤风类毒素,百日咳的各种抗原,也可以将HiD,PspA蛋白克隆至大肠杆菌进行表达并分离纯化。 [0048] (3) produced by a conventional process vaccines described in the above technical solution the CRM197, diphtheria toxoid, tetanus toxoid, pertussis various antigens, may be HiD, PspA protein was cloned into E. coli expressed and isolated purification.

[0049] 实施例1 一种7F型荚膜多糖与两种不同载体蛋白缀合物的制备 Preparation Example 1 A 7F capsular polysaccharide of two different carrier protein conjugate [0049] Embodiment

[0050] 将5g肺链7F型多糖溶解于1L乙酸钠缓冲液(50mM pH4.5)中,在室温下,用带有磁子的磁力搅拌器搅拌20分钟,以使多糖充分的溶解,加入0.2g NaI〇4,并室温下反应过夜, 使多糖上相邻的双羟基被氧化成醛基。 [0050] 5g of lung chain 7F polysaccharide was dissolved in 1L of sodium acetate buffer (50mM pH4.5), the mixture was stirred with a magnetic stirrer with a magnetic at room temperature for 20 minutes to fully dissolve the polysaccharide, was added 0.2g NaI〇4, and at room temperature overnight, the adjacent hydroxyl groups on the polysaccharide are oxidized to the bis aldehyde. 用纯化水进行20次等体积超滤换液,将反应中剩余的NaI0 4及反应过程中所生成的小分子物质滤除,得到引入了活化度为6.2的7F活化多糖的水溶液;并可将活化好的多糖浓缩冻干。 20 for purified water with ultrafiltration volume change was inferior, and the reaction NaI0 4 remaining in the reaction of small molecules generated was filtered off, the solution is introduced to give a degree of activation of the activated polysaccharide 7F 6.2; and may good activated polysaccharide concentrate lyophilized. 活化多糖的分子大小的确定利用凝胶过滤层析柱及利用多角度激光光散射来进行,利用葡聚糖分子大小标准来校准。 Activated polysaccharide molecular size determined by gel filtration chromatography and using a multi-angle laser light scattering is performed using dextran molecular size standards to calibrate. 多糖的含量通过蒽酮法确定,引入的醛基含量利用Park,JT和Johnson,MJ(1949)生物化学杂志(Journal of Biological Chemistry),181,149-151页所述方法确定。 Polysaccharide content determined by the anthrone method, the content of aldehyde groups introduced by Park, JT and Johnson, MJ (1949) J. Biol. (Journal of Biological Chemistry), pages 181,149-151 the determination method. 活化多糖的结构完整性由质子1H 和13C NMR确定。 Structural integrity of the activated polysaccharide is determined by proton 1H and 13C NMR. 活化多糖的纯度通过测量LAL(内毒素)含量来确定。 The purity of the activated polysaccharide is determined by measuring the LAL (endotoxin) content.

[0051 ]将各个活化的7F多糖分别放入三角瓶中,用纯化水稀释多糖至llg/L,再加入1/9 体积的无菌的磷酸缓冲液(500mM),按表2所加入5g/L的CRM197蛋白粉末和5g/L的肺炎表面蛋白PspA粉末,并用带有磁子的磁力搅拌进行搅拌,加入2g/L氰基硼氢化钠,搅拌溶解,反应18小时后加入1. lg/L硼氢化钠,混合4小时后加入硫酸铵至lmol/L,用1M硫酸铵溶液+ 50mM磷酸缓冲液(pH7 · 5)平衡疏水层析柱(填料为Fractogel Propyl (S),Merck),上样后, 再用上述缓冲液洗5个柱体积,最终用50mM磷酸缓冲液洗脱并收集多糖-蛋白质缀合物。 [0051] The respective activated 7F polysaccharide were placed in an Erlenmeyer flask, dilute the polysaccharide to llg / L with purified water and then sterile phosphate buffer was added 1/9 volume (500 mM), is added in Table 2 5g / L of CRM197 protein powder and 5g / L pneumoniae surface protein PspA powder, and with a magnetic stirring with a magnetic stirring, addition of 2g / L of sodium cyanoborohydride, dissolved with stirring. after 18 hours the reaction was added 1. lg / L sodium borohydride, ammonium sulfate was added 4 hours after mixing to lmol / L, using 1M ammonium sulfate solution + 50mM phosphate buffer (pH7 · 5) balanced by a hydrophobic chromatography column (filler Fractogel Propyl (S), Merck), the sample after the above-described wash buffer and then 5 column volumes, the final eluted with 50mM phosphate buffer, and collecting the polysaccharide - protein conjugate. 在装备50KDa滤膜的超滤系统中用生理盐水对多糖-蛋白质缀合物溶液进行15次等体积超滤, 收集回流并用囊式滤器过滤除菌,保存于4°C。 50KDa ultrafiltration system equipped with the membrane of saline polysaccharide - protein conjugate solution was inferior 15 ultrafiltration volume was collected and filter sterilized with a reflux filter capsule and stored at 4 ° C.

[0052] 通过CL-4B即SEC-MALLS方法检测多糖蛋白缀合物的分子量分布情况;通过免疫双扩的方法使用不同的抗体血清确定多糖蛋白缀合物中的蛋白和多糖种类;通过蒽酮法检测多糖蛋白缀合物的多糖含量;Lowry法蛋白含量检测多糖蛋白缀合物的总蛋白含量,再通过计算得到缀合物的多糖蛋白结合比(Ratio) ; CRM197、PspA蛋白浓度通过酶联免疫法检测。 [0052] i.e., the molecular weight distribution by SEC-MALLS method of detecting protein polysaccharide conjugates CL-4B; determining polysaccharide protein conjugates of polysaccharide and protein using different types of antibodies by immune sera double-spread; by anthrone detection polysaccharide content of polysaccharide-protein conjugates; total protein content Lowry protein assay polysaccharide-protein conjugates, and then obtain a polysaccharide-protein conjugates binding ratio (ratio) by calculation; CRM197, PspA protein concentration by enzyme-linked detection immunoassay. 结果见表1,为实施例1所述7F型荚膜多糖与两种不同载体蛋白缀合物的多糖蛋白浓度。 The results are shown in Table, for the embodiment in Example 1 Protein concentration 7F capsular polysaccharide to the carrier protein conjugate two different polysaccharides 1. [0053]表17F型多糖蛋白缀合物原液的多糖蛋白浓度 [0053] Table 17F polysaccharide protein concentration polysaccharide protein conjugate stock solution

[0054] [0054]

Figure CN103893751BD00081

[0055] 实施例2-种3型荚膜多糖与两种不同载体蛋白缀合物的制备 Preparation Example 2 isoform 3 capsular polysaccharide of two different carrier protein conjugate [0055] Embodiment

[0056] 将2g肺链3型多糖溶解于1L磷酸钠缓冲液(50mM pH7.2)中,在室温下,用带有磁子的磁力搅拌器搅拌20分钟,以使多糖充分的溶解,加入0.2g NaI〇4,并室温下反应过夜,使多糖上相邻的双羟基被氧化成醛基。 [0056] 2g of 3 lung chain polysaccharide was dissolved in 1L phosphate buffer (50mM pH7.2), the mixture was stirred with a magnetic stirrer with a magnetic at room temperature for 20 minutes to fully dissolve the polysaccharide, was added 0.2g NaI〇4, and at room temperature overnight, the adjacent hydroxyl groups on the polysaccharide are oxidized to the bis aldehyde. 用纯化水进行20次等体积超滤换液,将反应中剩余的NaI04及反应过程中所生成的小分子物质滤除,得到引入了活化度为9.1的3型活化多糖的水溶液;并且将活化好的多糖浓缩冻干。 20 for purified water with a volume inferior ultrafiltration was changed, the reaction and the remaining NaI04 generated during the reaction was filtered off small molecules, to obtain an aqueous solution is introduced 3 activation of the activation of the polysaccharide was 9.1; and the activated good lyophilized polysaccharide concentrated. 活化多糖的分子大小的确定利用凝胶过滤层析柱及利用多角度激光光散射来进行,利用葡聚糖分子大小标准来校准。 Activated polysaccharide molecular size determined by gel filtration chromatography and using a multi-angle laser light scattering is performed using dextran molecular size standards to calibrate. 多糖的含量通过蒽酮法确定,引入的醛基含量利用Park,JT和Johnson,MJ(1949)生物化学杂志(Journal of Biological Chemistry),181,149-151页所述方法确定。 Polysaccharide content determined by the anthrone method, the content of aldehyde groups introduced by Park, JT and Johnson, MJ (1949) J. Biol. (Journal of Biological Chemistry), pages 181,149-151 the determination method. 活化多糖的结构完整性由质子1H 和13C NMR确定。 Structural integrity of the activated polysaccharide is determined by proton 1H and 13C NMR. 活化多糖的纯度通过测量LAL(内毒素)含量来确定。 The purity of the activated polysaccharide is determined by measuring the LAL (endotoxin) content.

[0057]将各个活化的3型多糖分别放入三角瓶中,用纯化水稀释多糖至llg/L,再加入1/9 体积的无菌的磷酸缓冲液(500mM,pH7.2),再加入5g/L的CRM197蛋白粉末,并用带有磁子的磁力搅拌进行搅拌,加入2g/L氰基硼氢化钠,搅拌溶解,反应18小时后加入硫酸铵至1 · 5mol/L,用1 · 5M硫酸铵溶液+50mM磷酸缓冲液(pH7 · 5)平衡疏水层析柱(填料为Fractogel Propyl(S),Merck),上样后,再用上述缓冲液洗5个柱体积,最终用50mM磷酸缓冲液洗脱并收集多糖-蛋白质单载体缀合物。 [0057] The respective activated polysaccharide were placed in 3 Erlenmeyer flask, dilute the polysaccharide to llg / L with purified water, then add 1/9 volume of sterile phosphate buffer (500mM, pH7.2), was added 5g / L of CRM197 protein powder, and with a magnetic stirring with a magnetic stirring, addition of 2g / L of sodium cyanoborohydride, dissolved with stirring, was added ammonium sulfate to 1 · 5mol / L after 18 hours, with 1 · 5M + 50mM ammonium phosphate buffer solution (pH7 · 5) balanced by a hydrophobic chromatography column (filler Fractogel Propyl (S), Merck), after loading, the buffer solution and then washed 5 column volumes, finally with 50mM phosphate buffer and collecting the eluted polysaccharide - protein conjugates single carrier. 在装备l〇〇KDa滤膜的超滤系统中用生理盐水对多糖-蛋白质缀合物溶液进行5次等体积超滤,然后进行浓缩至多糖浓度为5g/L,加入5g/L的HiD蛋白, lg/L氰基硼氢化钠,搅拌溶解,反应48小时后加入0.7g/L硼氢化钠,混合4小时后硫酸铵至1 · 5mol/L,用1 · 5M硫酸铵溶液+50mM磷酸缓冲液(pH7 · 5)平衡疏水层析柱(填料为Fractogel Propyl(S),Merck),上样后,再用上述缓冲液洗5个柱体积,最终用50mM磷酸缓冲液洗脱并收集多糖-蛋白质双载体缀合物。 Physiological saline by ultrafiltration system equipped l〇〇KDa Membrane polysaccharide - protein conjugate solution for 5 volume ultrafiltration inferior, and concentrated to a polysaccharide concentration of 5g / L, was added 5g / L of protein HiD , lg / L of sodium cyanoborohydride, dissolved with stirring. after 48 hours the reaction was added 0.7g / L of sodium borohydride, 4 hours after mixing of ammonium sulfate to 1 · 5mol / L, ammonium sulfate with 1 · 5M phosphate buffer solution + 50mM solution (pH7 · 5) balanced by a hydrophobic chromatography column (filler Fractogel Propyl (S), Merck), after loading, the buffer solution and then washed 5 column volumes, finally eluting with a 50mM phosphate buffer solution and collected polysaccharide - bis protein carrier conjugate. 在装备lOOKDa滤膜的超滤系统中用生理盐水对多糖-蛋白质缀合物溶液进行15次等体积超滤,收集回流并用囊式滤器过滤除菌,保存于4°C。 In the system equipped with an ultrafiltration membrane lOOKDa saline polysaccharide - protein conjugate solution was inferior 15 ultrafiltration volume was collected and filter sterilized with a reflux filter capsule and stored at 4 ° C.

[0058] 通过CL-4B即SEC-MALLS方法检测多糖蛋白缀合物的分子量分布情况;通过免疫双扩的方法使用不同的抗体血清确定多糖蛋白缀合物中的蛋白和多糖种类;通过蒽酮法检测多糖蛋白缀合物的多糖含量;Lowry法蛋白含量检测多糖蛋白缀合物的总蛋白含量,再通过计算得到缀合物的多糖蛋白结合比(Ratio) ;CRM197、HiD蛋白浓度通过酶联免疫法检测。 [0058] i.e., the molecular weight distribution by SEC-MALLS method of detecting protein polysaccharide conjugates CL-4B; determining polysaccharide protein conjugates of polysaccharide and protein using different types of antibodies by immune sera double-spread; by anthrone detection polysaccharide content of polysaccharide-protein conjugates; total protein content Lowry protein assay polysaccharide-protein conjugates, and then obtain a polysaccharide-protein conjugates binding ratio (ratio) by calculation; CRM197, HiD protein concentration by enzyme-linked detection immunoassay. 结果见表2,为实施 The results are shown in Table 2, for the implementation of

[0059] 例2所述3型荚膜多糖与两种不同载体蛋白缀合物的多糖蛋白浓度。 Polysaccharide protein concentration [0059] Example 2 of the type 3 capsular polysaccharide of two different carrier proteins in conjugates.

[0060] 表23型多糖蛋白缀合物原液的多糖蛋白浓度 [0060] Table 23 concentration of proteoglycan polysaccharide protein conjugate stock solution

[0061] [0061]

Figure CN103893751BD00091

[0062]实施例3-种13价肺炎球菌多糖蛋白缀合疫苗的配制 [0062] Example 3 - Species 13 valent pneumococcal polysaccharide-protein conjugate vaccine formulated together

[0063]按照背景技术中公知方法制备4,6B,9V,14,18C,19F,23F等单载体缀合物,其中载体蛋白均为CRM197。 [0063] Preparation 4,6B, 9V, 14,18C, 19F, 23F and other single-carrier conjugate according to methods well known in the background art, wherein both the carrier protein CRM197. 按照实施例1或2描述的方法,制备1,3,5,6A,7F,19A型多糖的双载体结合物,其中1,5,7F型多糖采用PspA为第二载体,3,6A,19A采用HiD为第二载体。 The method described in Example 1 or 2, prepared 1,3,5,6A, 7F, bis 19A polysaccharide carrier conjugate, wherein the polysaccharide 1,5,7F PspA using a second carrier, 3, 6A, 19A HiD using the second carrier. 将各种结合产物按照下表3进行疫苗配制。 The product was subjected to various combinations of vaccine formulated according to Table 3 below. 这些单价组分在混合后,加入最终浓度为0.5mg/L的磷酸铝佐剂。 These monovalent components after mixing, a final concentration of 0.5mg / L of aluminum phosphate adjuvant.

[0064]表3各种抗原的载体蛋白类型以及制剂中最终含量 [0064] Table 3 various antigens to a carrier protein and the type of content in the final formulation

Figure CN103893751BD00092

Figure CN103893751BD00101

[0067]实施例4 一种13价肺炎球菌多糖蛋白缀合疫苗的药效评价,以及与现有单载体结合疫苗的比较 4 one kind of the 13-valent pneumococcal polysaccharide-protein Pharmacological evaluation conjugated vaccine, combined vaccine and comparing with the conventional single-carrier [0067] Example

[0068] 12-14g雌性3周龄BALB/c.幼鼠,随机分为4组,每组10只,分别腹腔注射按照实施例3配制的结合疫苗,市售13价肺链结合疫苗,空白对照(磷酸错)以及载体蛋白混合物。 [0068] 12-14g 3-week-old female BALB / c. Rats, were randomly divided into 4 groups, 10 per group, were injected intraperitoneally combined vaccine according to Example 3, a commercially available 13-valent vaccine formulation lung chain binding, blank control (phosphate wrong) and a carrier protein mixture. 其中载体蛋白混合物的成分为:CRM19730ug,HiD蛋白3. lug,PspA蛋白4.2ug,这些蛋白在混合后加入最终浓度为0.5mg/L的磷酸铝佐剂。 Wherein the component carrier protein mixture was: CRM19730ug, HiD protein 3. lug, PspA protein 4.2ug, these proteins were added to a final concentration after mixing of 0.5mg / L of aluminum phosphate adjuvant.

[0069] 于第0、2、4周程序注射3次,每只幼鼠0.5ml含(0.5ug偶联物)。 [0069] 3 injections on weeks 0,2,4 first program, each containing 0.5ml rats (0.5 ug conjugate). 每个实验组分别于第三针注射后的7天采血,分离血清,置-20°C保存备用。 Each experimental group were 7 days after the third injection needle blood, serum was separated, the standby counter at -20 ° C.

[0070]对于荚膜多糖和载体蛋白抗体滴度检测分别采用间接ELISA法,将各型肺炎多糖, 载体蛋白溶解于0.05mol/L pH9.6的碳酸盐缓冲溶液中,以20ug/ml浓度包被酶标板,2%的BSA液封闭。 [0070] capsular polysaccharide and a carrier protein for antibody titer by indirect ELISA were used, each pneumoniae polysaccharide, the carrier protein was dissolved in a carbonate buffer solution 0.05mol / L pH9.6 in, ml concentration of 20ug / ELISA plates were coated, blocked 2% BSA solution. 实验时将待测血清100,200,400,800,1600,3200,6400,12800,25600,51200, 102400,204800,409600倍稀释后加入酶标板,置37°C反应40min,常规洗涤后加入辣根过氧化物酶标记的羊抗鼠二抗。 The experiment measured the serum 100,200,400,800,1600,3200,6400,12800,25600,51200, 102400,204800,409600 fold diluted ELISA plate was added, the reaction is set 37 ° C 40min, was added after conventional washing horseradish peroxidase-labeled goat anti-mouse secondary antibody. 同时加入不含血清的缓冲液作为阴性对照。 While adding serum-free buffer was used as a negative control. 用四氨基联苯胺显色,酶标仪450nm波长处读取A值。 With four diaminobenzidine chromogenic microplate reader at a wavelength of 450nm A value read. 计算每孔中A值与阴性孔A值得比值,但比值大于2.1时代最大的稀释倍数为血清中的抗体滴度,对10只小鼠的滴度进行几何平均,并以10为底计算对数值,结果见表4。 A value in each well was calculated with the negative wells worth ratio A, but the maximum dilution ratio is greater than 2.1 era serum antibody titers, titers of 10 mice geometric mean, and computing the base 10 logarithm The results are shown in Table 4.

[0071 ]表4两种剂型疫苗及载体蛋白混合物小鼠免疫原性比较 [0071] The two formulations in Table 4 Vaccine and immunogenic carrier protein mixture mice Comparison

Figure CN103893751BD00111

[0074]从数据看出,在采取了双载体后(加*标注,1,3,5,6A,7F,19A),小鼠对肺链多糖的免疫反应显著增强,其中3型尤其显著。 [0074] From the data, after taking a binary vector (marked with *, 1,3,5,6A, 7F, 19A), in mice with immune responses of the lung significantly enhanced polysaccharide chain wherein 3 type particularly remarkable. 另外,与同剂量的蛋白混合物对比,双载体缀合物也显示了其载体蛋白具有更优的免疫原性(加*标注)。 Further, in contrast to the same dose of protein mixtures, dual carrier conjugate also shows a carrier protein which has better immunogenic (marked with *).

[0075]上述参照实施例对该肺炎球菌蛋白疫苗及其制备方法进行的详细描述,是说明性的而不是限定性的,可按照所限定范围列举出若干个实施例,因此在不脱离本发明总体构思下的变化和修改,应属本发明的保护范围之内。 Detailed description of the pneumococcal protein vaccine and method of preparation [0075] Referring to the above-described embodiments are illustrative and not limiting, may include a number of embodiments according to the defined range, without departing from the present invention variations and modifications of the general inventive concept, should be within the scope of the present invention.

Claims (8)

1. 一种肺炎球菌多糖蛋白缀合疫苗,其特征在于:是包括一种或多种肺炎链球菌荚膜多糖与蛋白质耦联结合的免疫缀合物,所述免疫缀合物中至少有一种免疫缀合物是一种单一的肺炎链球菌荚膜多糖与两种蛋白质耦联而形成的双载体缀合物,该两种蛋白质选自: 嗜血流感杆菌表面蛋白HiD、PspA蛋白、载体蛋白CRM197。 A pneumococcal polysaccharide protein conjugate vaccine, characterized by: comprising one or more immunoconjugates Streptococcus pneumoniae capsular polysaccharide coupled to binding protein, the immunoconjugate in at least one immunoconjugates is a single, dual-carrier conjugates of Streptococcus pneumoniae capsular polysaccharide coupled to the formation of two proteins, the two proteins are selected from: Haemophilus influenzae surface protein HiD, PspA protein, vector protein CRM197.
2. 根据权利要求1所述的肺炎球菌多糖蛋白缀合疫苗,其特征在于:包含多价肺炎球菌缀合物的免疫组合物,是分离提纯多种血清型肺炎球菌荚膜上的荚膜多糖与蛋白质耦联结合后混合而成的。 According to claim 1 pneumococcal polysaccharide protein conjugate vaccine, as claimed in claim wherein: the multivalent immunogenic composition comprising a pneumococcal conjugate, the capsular polysaccharide is isolated and purified pneumococcal capsular serotypes of after binding to the protein coupling are mixed.
3. 根据权利要求1所述的肺炎球菌多糖蛋白缀合疫苗,其特征在于:所述肺炎链球菌荚膜多糖是分离提纯血清型肺炎球菌荚膜上的荚膜多糖,所述血清型肺炎球菌的血清型包括1,2,3,4,5,6A,6B,7F,8,9N,9V,10A,11A,12F,14,15B,17F,18C,19A,19F,20,22F,23F^P/^^ 33F〇 According to claim 1 pneumococcal polysaccharide protein conjugate vaccine, characterized in that: said capsular polysaccharide is Streptococcus pneumoniae capsular polysaccharide serotype in the separation and purification of pneumococcal capsular, the pneumococcal serotypes serotypes include 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F ^ P / ^^ 33F〇
4. 权利要求1-3之一所述的肺炎球菌蛋白疫苗的制备方法,其特征在于:具体步骤为: (1) 分别分离提纯多种血清型肺炎球菌荚膜上的荚膜多糖; (2) 分别分离提纯所选用的载体蛋白; (3) 分别将各种荚膜多糖与载体蛋白耦联结合,其中,至少一种荚膜多糖与两种蛋白质耦联结合成双载体缀合物; (4) 将各种耦联结合混合形成疫苗原液。 Pneumococcal protein vaccine preparation according to claim one of 1 to 3, wherein: the specific steps: (1) are isolated and purified capsular polysaccharide on multiple serotypes of pneumococcus; (2 ) were purified by separating the selected carrier protein; (3) respectively, the binding of various capsular polysaccharide coupled to a carrier protein, wherein at least one capsular polysaccharide and coupling the two proteins bind dual carrier conjugate; (4 ) in conjunction with various hybrid coupling is formed of vaccine.
5. 根据权利要求4所述的肺炎球菌蛋白疫苗的制备方法,其特征在于:所述步骤(3)中荚膜多糖与载体蛋白的耦联结合方式包括: (1) 荚膜多糖用CDAP和三乙胺活化法耦联结合载体蛋白,该反应为异脲键连接反应,在高pH时,CDAP与多糖残基上的羟基反应,转化成氰酸酯,载体蛋白上的氨基与其迅速反应, 形成异脲键,或(2) 先对多糖进行104活化形成自由醛基并和载体蛋白上的氨基进行连接结合;或(3) 用己二酸二肼酰和酸性多糖中的羧基或者在多糖中氧化所获得的羧基连接,然后再在EDAC存在的条件下和蛋白进行连接。 The production method of pneumococcal protein vaccine according to claim 4, wherein: said step (3) described in conjunction with coupling the capsular polysaccharide to carrier protein comprises: (a) capsular polysaccharides with CDAP and activation triethylamine coupled carrier protein binding, the reaction is isourea linkage reaction, the pH at high, and react with hydroxyl groups on the polysaccharide residue CDAP converted into cyanate, amino group on the carrier protein with its rapid response, isourea bond formed, or (2) prior to the formation of free polysaccharide aldehyde activated 104 and an amino group on the carrier and connected binding protein; or (3) a carboxyl group with adipic acid dihydrazide and acidic polysaccharides or polysaccharide carboxy oxidation obtained connection, then the connection is the presence of EDAC and proteins.
6. 根据权利要求4所述的肺炎球菌多糖蛋白缀合疫苗的制备方法,其特征在于:所述步骤(3)中荚膜多糖与载体蛋白的耦联结合的连接方式包括: (1) 单步反应法,将两种载体蛋白和荚膜多糖同时加入到反应容器中进行反应,或(2) 顺序法,先将荚膜多糖与其中一种载体蛋白进行连接,然后经过纯化,去除未结合的游离蛋白和游离多糖,再加入第二种载体蛋白进行连接。 The pneumococcal polysaccharide protein as claimed in claim 4, wherein the vaccine production method of conjugation, wherein: said step (3) is coupled in connection capsular polysaccharide to carrier protein binding include: (1) Single reaction-step method, the two carrier proteins and capsular polysaccharides added simultaneously to the reaction vessel reaction, or (2) sequential method, wherein the first capsular polysaccharide to a carrier protein to connect, and then purified, to remove unbound free protein-free polysaccharide and then adding a second connection carrier protein.
7. 根据权利要求4所述的肺炎球菌蛋白疫苗的制备方法,其特征在于:具体步骤为: (1) 选取24种血清型的肺炎球菌培养,该24种血清型为1,2,3,4,5,6A,6B,7F,8,9N,9V, 10八,11厶,12卩,14,158,17卩,18(:,19厶,19卩,20,22卩,23卩和33卩; (2) 分别提纯以上各种血清型肺炎球菌中抗原性强的荚膜多糖:肺炎球菌,灭活后离心收集上清液,经超滤浓缩,根据各肺炎球菌血清型特性分别加入体积分数为70 %的预冷乙醇,离心收集,得粗制多糖;将粗制多糖溶于乙酸钠溶液中,然后按1:2比例以冷酚混匀,离心去除蛋白,反复酚提5-6次,收集上清,用蒸馏水透析,透析后液体加2mol/L氯化钙溶液, 加入乙醇搅拌,离心去除核酸,收集上清,补加乙醇,终浓度80 %搅拌,离心收集沉淀,用乙醇、丙酮洗涤沉淀,脱水干燥后即为精制荚膜多糖,置_20°C保存备用; (3 7. A method for preparing a protein pneumococcal vaccine according to claim 4, wherein: the specific steps: (1) selecting 24 kinds of pneumococcal serotypes culture, the 24 kinds of serotypes 1,2,3, 4,5,6A, 6B, 7F, 8,9N, 9V, 10 VIII, 11 Si, 12 Jie, Jie 14,158,17, 18 (:, 19 Si, 19 Jie, Jie 20, 22, 23, and Jie Jie 33; (2) above were purified pneumococcal serotypes strongly antigenic capsular polysaccharide: pneumococcus, the supernatant was collected by centrifugation, inactivated, concentrated by ultrafiltration, according to the characteristics of each pneumococcal serotypes were added volume fraction of 70% of pre-cooled ethanol, collected by centrifugation, to obtain the crude polysaccharides; crude polysaccharide was dissolved in sodium acetate solution and then 1: 2 ratio of phenol to cold mix, protein is removed by centrifugation, repeatedly mention phenol 5- 6, the supernatant was collected, dialyzed against distilled water, dialysis liquid added 2mol / L calcium chloride solution, ethanol was added with stirring, the nucleic acid is removed by centrifugation, the supernatant was collected, supplemented with ethanol at a final concentration of 80% was stirred, the precipitate was collected by centrifugation, dried ethanol, the precipitate was washed with acetone, is dried after purified capsular polysaccharides were stored set _20 ° C; (3 ) 生产CRM197,或将HiD,PspA蛋白克隆至大肠杆菌进行表达并分离纯化; (4) 取检测合格的各种血清型肺炎球菌的荚膜多糖,用高碘酸钠活化法获得醛基然后和两种蛋白同时结合,得到目标缀合物;或将其中一种蛋白和多糖先进行耦联,在不封闭活性基团的情况下进行纯化,将得到的纯化产物与第二种蛋白进一步进行耦联得到目标缀合物。 ) The CRM197 production, or HiD, PspA protein was cloned into Escherichia coli and expressed isolated and purified; capsular polysaccharide (4) takes tested and pneumococcus serotypes to obtain an aldehyde with sodium periodate activation method and then both proteins bind the same time, to give the title conjugate; or one for coupling to the protein and polysaccharide, purified without blocking the active groups, the resulting product was purified protein is further coupled with a second linked to afford the title conjugate.
8.权利要求1-3之一所述的肺炎球菌多糖蛋白缀合疫苗在制备用于诱导对肺炎链球菌荚膜多糖缀合物和载体蛋白的免疫应答的药物方面的应用。 Pneumococcal polysaccharide protein according to one of claim 1-3 conjugated in the manufacture of a vaccine medicament for inducing an immune conjugates of Streptococcus pneumoniae capsular polysaccharide and a carrier protein response area.
CN201410114934.8A 2014-03-26 2014-03-26 Pneumococcal polysaccharide-protein conjugate vaccine and its preparation method CN103893751B (en)

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