CN100556459C - 多价脑膜炎球菌多糖-蛋白质缀合疫苗 - Google Patents
多价脑膜炎球菌多糖-蛋白质缀合疫苗 Download PDFInfo
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- CN100556459C CN100556459C CNB028053982A CN02805398A CN100556459C CN 100556459 C CN100556459 C CN 100556459C CN B028053982 A CNB028053982 A CN B028053982A CN 02805398 A CN02805398 A CN 02805398A CN 100556459 C CN100556459 C CN 100556459C
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- polysaccharide
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- neisseria meningitidis
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Abstract
本发明描述了可以对脑膜炎球菌疾病提供广泛保护的联合疫苗,所述脑膜炎球菌疾病由病原性细菌脑膜炎奈瑟氏球菌(Neisseria meningitides)所致。疫苗由4种不同的多糖-蛋白质缀合物组成,这些缀合物被配制成单一剂量形式的疫苗。将来源于脑膜炎奈瑟氏球菌血清型A、C、W-135和Y的纯化荚膜多糖化学活化,并通过共价化学键选择性连接至载体蛋白质上,形成多糖-蛋白质缀合物,所述缀合物能够在儿童以及成人中引起对多种脑膜炎奈瑟氏球菌株系的长效免疫。
Description
本申请要求美国临时专利申请号60/263,435(2001年1月23日提交)的权利。
发明背景
发明领域
本发明总的来说涉及医学领域,并且更具体地,本发明涉及微生物学、免疫学、疫苗以及通过免疫对细菌病原体感染的预防。
相关领域概述
脑膜炎奈瑟氏球菌(Neisseria meningitides)是全球细菌性脑膜炎和脓毒症的主要原因。在最近的30年中,地方性脑膜炎球菌疾病的发病率在发达国家为每100,000人中有1到5人,而在发展中国家为每100,000人中10到25人(Reido,F.X.等人,1995)。流行期间脑膜炎球菌疾病的发病率可达每1000,000人中1,000人。在美国每年大约有2,600例细菌性脑膜炎病例,而在发展中国家平均有330,000例。病例致死率在10到20%之间。
病原脑膜炎球菌被多糖荚膜所包被,附着在生物体外膜表面。基于荚膜多糖的免疫学特异性,已经鉴定出13种不同的脑膜炎球菌血清型(Frasch,C.E.等人,1985)。这13个血清型中的5个引起了大部分脑膜炎球菌疾病;它们包括血清型A、B、C、W135和Y。血清型A引起大部分流行性疾病。血清型B、C和Y引起大多数地方性疾病和疾病的区域性爆发。
人类鼻口咽粘膜是唯一已知的脑膜炎奈瑟氏球菌的天然贮库。细菌在粘膜细胞的外表面以及鼻咽的上皮下组织定居。脑膜炎球菌可被持续携带几个月,脑膜炎球菌通过直接接触或通过空气飞沫传播。脑膜炎球菌是以吞噬泡的形式通过胞吞作用经由粘膜上皮侵入的。对入侵的脑膜炎球菌的宿主防御依赖于补体介导的溶菌作用。在补体介导的溶菌作用中发挥作用的血清抗体相当大部分直接针对外部荚膜多糖。
基于脑膜炎球菌多糖的疫苗已经被描述,其引起针对荚膜多糖的免疫应答。这些抗体可以对血清型特异性脑膜炎球菌引起补体介导的溶菌作用。脑膜炎球菌多糖疫苗显示对儿童和成人有效(Peltola,H.等人,1977以及Artenstein,M.S.等人,1970),但对于婴儿和幼儿其效能有限(Reingold,A.L.等人,1985)。对年幼群体,此多糖的后继给药产生弱加强应答或无加强应答(Goldschneider,I.等人,1973以及Gold,R.等人,1977)。脑膜炎球菌多糖疫苗的保护期不能持续很长,并且据估计对成人和大于4岁的儿童而言为3到5年(Brandt,B.等人,1975,H.等人,1980以及Ceesay,S.J.等人,1993)。对1至4岁的儿童而言保护期少于3年(Reingold,A.L.等人,1985)。
多糖不能与主要组织相容性复合体分子结合,而这是抗原呈递和刺激T-辅助淋巴细胞的前提条件,也即它们是非T-细胞依赖性抗原。多糖能够不需T-辅助淋巴细胞的辅助而刺激B淋巴细胞产生抗体。由于对B淋巴细胞的非T-细胞依赖性刺激,这些抗原免疫后缺乏记忆诱导。在成人体内,多糖抗原可以产生非常有效的非T-细胞依赖性应答,但在婴儿和幼儿的不成熟免疫系统中这些非T-细胞依赖性应答却很弱。
非T-细胞依赖性多糖抗原可以通过将多糖共价连接至蛋白质分子(“载体”或“载体蛋白质”)上而转变为T-细胞依赖性抗原。与缀合疫苗的多糖组分结合的B细胞可以被肽特异的T辅助细胞激活,所述肽为缀合的载体蛋白质的一部分。对载体蛋白质的T-辅助细胞应答可以增加针对多糖的抗体的产生。
已证明,血清型B多糖在人群中具有极弱的免疫原性至无免疫原性(Wyle,F.A.等人,1972)。这一血清型多糖与蛋白质的化学连接在实验动物中没有显著改变免疫应答(Jennings,H.J.等人,1981)。据认为,对这一血清型多糖缺乏免疫应答的原因是血清型B多糖与多唾液酸化的宿主糖蛋白之间的结构相似性,所述宿主糖蛋白例如神经细胞粘附分子。
一种基于血清型C多糖的脑膜炎球菌缀合疫苗已有叙述。这种单价疫苗可引起针对相应于血清型C的脑膜炎奈瑟氏球菌株系上存在的荚膜多糖的强功能性抗体应答。这种疫苗仅仅能够防止由血清型C细菌引起的疾病。
现有的基于脑膜炎球菌多糖的疫苗对幼儿的用途有限,而且不能为成人提供长时间保护。已被证实能够对所有有危险感染脑膜炎球菌的群体(包括儿童)产生长时间保护的唯一脑膜炎球菌疫苗是基于来源于单一脑膜炎奈瑟氏球菌血清型的多糖的疫苗,其不能提供针对其它血清型感染的保护。因此,就需要一种能够在脑膜炎球菌感染的易感儿童和成人中对脑膜炎球菌疾病产生广谱、长效保护的脑膜炎球菌缀合疫苗。本发明的多价脑膜炎球菌多糖通过提供疫苗制剂解决了这一需要,其中在所述制剂中来源于脑膜炎奈瑟氏球菌主要致病血清型的免疫原性多糖通过与载体蛋白质缀合而转变成T-细胞依赖性抗原。
发明概述
本发明提供由病原性脑膜炎奈瑟氏球菌制备的脑膜炎球菌多糖-蛋白质缀合物的免疫学组合物,其可用于治疗疾病。
本发明提供包含有两种或多种蛋白质-多糖缀合物的免疫学组合物,其中每一缀合物各包含有连接至载体蛋白质的来源于脑膜炎奈瑟氏球菌的荚膜多糖。
本发明提供包含有两种或多种不同蛋白质-多糖缀合物的免疫学组合物,其中每一缀合物各包含有连接至载体蛋白质的来源于脑膜炎奈瑟氏球菌不同血清型的荚膜多糖。
本发明提供由病原性脑膜炎奈瑟氏球菌制备的脑膜炎球菌多糖-蛋白质缀合物的疫苗。本发明提供多价脑膜炎球菌疫苗,其由免疫学有效量的2到4种不同的蛋白质-多糖缀合物组成,其中每一缀合物各包含有不同的连接至载体蛋白质的荚膜多糖,并且其中每一荚膜多糖选自血清型A、C、W135和Y的荚膜多糖。
本发明也提供制备多价脑膜炎球菌多糖-蛋白质组合物的方法,其包括从病原性脑膜炎奈瑟氏球菌中纯化两种或多种荚膜多糖;将纯化的多糖与一种或多种载体蛋白质缀合,以及混合这些缀合物以产生多价脑膜炎球菌多糖-蛋白质组合物。
本发明还提供诱导对脑膜炎奈瑟氏球菌荚膜多糖的免疫学应答的方法,其包括给人类或动物施用免疫学有效量的本发明免疫学组合物。
本发明提供多价脑膜炎球菌疫苗,其由免疫学有效量的2到4种不同的蛋白质-多糖缀合物组成,其中每一缀合物各含有不同的连接至载体蛋白质的荚膜多糖,并且其中每一荚膜多糖选自血清型A、C、W135和Y的荚膜多糖。
本发明提供保护脑膜炎奈瑟氏球菌易感人群或动物的方法,其包括向人类或动物施用免疫学有效剂量的本发明疫苗。
于此引用的所有专利、专利申请以及其它出版物特此全面引入作为参考。
发明详述
本发明包括具有两种或多种不同的蛋白质-多糖缀合物的免疫学组合物,其中每一缀合物各包含有一种连接至载体蛋白质的荚膜多糖。因此,本发明包括含有两种或多种不同荚膜多糖的组合物,其中所述荚膜多糖被连接至一种或多种载体蛋白质上。
荚膜多糖可以通过本领域技术人员所公知的标准技术制备(参考文献)。在本发明中荚膜多糖优选制备自脑膜炎奈瑟氏球菌血清型A、C、W135和Y。
在优选的实施方案中,这些脑膜炎球菌血清型缀合物分别制备然后配制成单一剂量形式。例如,从脑膜炎奈瑟氏球菌血清型A、C、W135和Y分别纯化荚膜多糖。
在本发明的优选实施方案中,纯化的多糖在连接至载体蛋白质前被解聚并活化。在本发明的优选实施方案中利用温和氧化条件将来源于脑膜炎奈瑟氏球菌血清型A、C、W135和Y的荚膜多糖部分解聚。
在解聚或部分解聚多糖后可以接着进行活化步骤,“活化”是指对多糖进行化学处理以提供能够与载体蛋白质反应的化学基团。优选的活化方法包括在15到30℃、pH 5.0±0.1下于生理盐水中利用己二酸二酰肼处理约2小时。在美国专利5,965,714中描述了一种活化方法。
一经活化,荚膜多糖就可以被连接至一种或多种载体蛋白质上。在本发明优选的实施方案中,每一种荚膜多糖分别与单一载体蛋白质种类连接。在优选的实施方案中将来源于脑膜炎奈瑟氏球菌血清型A、C、W135和Y的荚膜多糖分别连接至同一种载体蛋白质上。
载体蛋白质可以包括失活的细菌毒素,例如白喉类毒素、CRM197、破伤风类毒素、百日咳类毒素、大肠杆菌LT、大肠杆菌ST以及来源于铜绿假单胞杆菌(Pseudomonas aeruginosa)的外毒素A。也可以使用细菌外膜蛋白,例如外膜复合体c(OMPC)、孔蛋白、转铁蛋白结合蛋白、肺炎球菌溶血素(pneumolysis)、肺炎球菌表面蛋白A(PspA)或肺炎球菌粘附素蛋白(PsaA)。其它蛋白质如卵清蛋白、匙孔虫戚血蓝蛋白(KLH)、牛血清白蛋白或纯化的结核菌素蛋白质衍生物(PPD)也可以作为载体蛋白质使用。载体蛋白质优选无毒、无反应原性,并且可得到足够的量和纯度。载体蛋白质应易于接受标准连接程序的处理。在本发明优选实施方案中,纯化自白喉棒杆菌(Corynebacteria diphtheriae)培养物并利用甲醛化学去毒的白喉毒素被用作载体蛋白质。
荚膜多糖与载体蛋白质缀合后,可以利用多种技术对多糖-蛋白质缀合物进行纯化(富集,就多糖-蛋白质缀合物的量而言)。纯化步骤的一个目的是从多糖-蛋白质缀合物中去除未结合的多糖。美国专利6,146,902上描述了一种涉及在硫酸铵存在下进行超滤的纯化方法。或者,可以通过任意数目的标准技术从未反应的蛋白质和多糖中将缀合物纯化出来,所述标准技术包括大小排阻层析、密度梯度离心、疏水相互作用层析或硫酸铵分级分离等。参见,例如P.W.Anderson等人(1986),免疫学杂志(J.Immunol.)137:1181-1186。也见于H.J.Jennings和C.Lugowski(1981)免疫学杂志(J.Immunol.)127:1011-1018。
在将多糖与载体蛋白质缀合后,本发明免疫学组合物可以通过混合多种多糖-蛋白质缀合物而制成。本发明免疫学组合物包含两种或多种连接至1种或多种载体蛋白质上的不同荚膜多糖。本发明优选实施方案为二价免疫学组合物,其包含分别连接至白喉类毒素上的来源于脑膜炎奈瑟氏球菌血清型A和C的荚膜多糖。更优选地,本发明为四价免疫学组合物,其包含分别连接至白喉类毒素上的来源于脑膜炎奈瑟氏球菌血清型A、C、W-135和Y的荚膜多糖。
载体蛋白质的制备和使用,以及多种潜在连接方法为本领域技术人员所公知。本发明缀合物可以通过这些技术人员利用本发明所讲授以及在一般文献上可轻易得到的信息来制备。也可自以下任一或全部美国专利中获得指导,其中这些专利的教导在此全部引用作为参考:U.S.4,356,170;U.S.4,619,828;U.S.5,153,312;U.S.5,422,427和U.S.5,445,817。
本发明免疫学组合物通过从不同的脑膜炎球菌血清型分别制备多糖-蛋白质缀合物然后将缀合物混合而制得。本发明免疫学组合物可以作为疫苗使用。本发明疫苗的配制可以利用本领域已知方法完成。本发明疫苗组合物还可以包含一种或多种佐剂。佐剂包括(作为例子而不限于此)铝佐剂、弗氏佐剂、BAY、DC-chol、pcpp、单磷酰脂A、CpG、QS-21、霍乱毒素和甲酰甲硫氨酰肽。参见例如:疫苗设计,亚单位与佐剂方法(Vaccine Design,the Subunit and Adjuvant Approach),1995(M.F.Powell和M.J.Newman编辑,Plenum出版社,纽约)。佐剂优选为铝佐剂,如氢氧化铝或磷酸铝。
如下所述,本发明疫苗和免疫学组合物在多种动物模型中引起类似T-细胞依赖性的免疫应答,而多糖疫苗引起类似非T-细胞依赖性的免疫应答。因此,本发明组合物也可用于研究在类似T-细胞依赖性的脑膜炎奈瑟氏球菌抗原免疫应答中涉及的生物学路径和过程。
施用于人或动物的本发明疫苗的量以及施用方案可以按照药学和兽医领域一般技术人员所公知的标准技术确定,并在确定时考虑到诸如所用具体抗原、佐剂(如果存在)、年龄、性别、体重、物种和特定动物或患者的状况以及施用路径等因素。在本发明中,对于接种以对抗脑膜炎奈瑟氏球菌,可提供有效剂量的多糖-蛋白质载体量可以从每公斤体重约0.02μg到约5μg。在本发明优选的组合物和方法中,剂量为每公斤体重约0.1μg到3μg。例如,如果感染后时间不长,由于细菌增殖时间较短,有效剂量可以需要较少的抗体。同样,有效剂量依赖于诊断时的细菌负载量。对于治疗使用,可考虑在一段时期内施与多次注射。
本发明的多价缀合物可以以单一剂量或以系列(即以“加强”或“几次加强”)形式施用。例如,就象目前针对预防儿童疾病的其它疫苗所推荐的那样,儿童可以在生命早期接受单一剂量,此后不超过10年接受加强剂量。
加强剂量可使致敏B细胞产生抗体,即回忆应答。也就是说,多价缀合物疫苗与得到批准的多糖疫苗相比,可在年幼群体中引起高的初次(即在疫苗的单次施用后)功能抗体应答,而且还能够引起回忆应答(即在加强施用后),这表明由本发明的多价缀合疫苗所引起的保护性免疫应答具长效性。
本发明组合物包括经孔道例如口腔、鼻腔、肛门、阴道、经口、胃内、粘膜(包括经舌、肺泡、牙龈、鼻或呼吸道粘膜)等等施用的液体制剂,诸如混悬液、糖浆或酏剂;以及经肠胃外、皮下、皮内、肌内、腹膜内或静脉施用(例如注射施用)的制剂,诸如无菌混悬剂或乳剂。优选静脉内和肠胃外施用。这些组合物可以与适当的载体、稀释剂或赋形剂诸如无菌水、生理盐水、葡萄糖等等混和。组合物也可以被冻干。按照所期望的施用路径和制剂形式,组合物可以包含诸如润湿剂或乳化剂、pH缓冲剂、胶凝剂或粘度增强添加剂、防腐剂、调味剂、色素等等辅料。可以在不需过多实验的前提下参考标准教科书(例如在此引用作为参考的Remington药物科学,17版,1985)来制备适宜的制剂。
本发明组合物可以以液体制剂的形式方便地提供,所述液体制剂例如等渗水溶液、混悬液、乳液或粘性组合物,其可以被缓冲至选定的pH。如果优选消化道吸收,本发明组合物可以为药丸、片剂、胶囊、囊片等等“固体”形式,包括缓释或液体填充型“固体”制剂(例如以明胶包裹液体,由此明胶在胃中溶解用于在消化道中实现递送)。如果希望鼻腔或呼吸道(粘膜)施用,组合物可以通过压力喷雾给药器、泵给药器或气雾给药器施用。气溶胶通常借助碳氢化合物在压力下形成。泵给药器优选按计量分配剂量或分配具有特定微粒大小的剂量。
液体制剂通常较凝胶、其它粘性组合物和固体组合物更易制备。另外,液体组合物多少可以更方便地在动物、儿童(尤其是幼儿)和其它可能在吞咽药丸、片剂、胶囊等等方面有困难的个体上施用,尤其是通过注射或口服方式。在多剂量给药情况下液体组合物也更方便。另一方面,可以将粘性组合物配制在适当的粘性范围内以提供与粘膜(诸如胃或鼻粘膜的内衬)的更长接触时间。
明显地,适当的载体和其它添加剂的选择依赖于确切的施用路径和特定剂型的性质,例如液体剂型(例如是否组合物被配制成溶液、悬液、凝胶或其它液体形式)或固体剂型(例如是否组合物被配制成药丸、片剂、胶囊、囊片、缓释剂或液体填充形式)。
溶液、悬液和凝胶除含有活性成分之外通常含有大量的水(优选纯化的水)。也可以存在小量的其它成分,如pH调节剂(例如碱,诸如NaOH)、乳化剂或分散剂、缓冲剂、防腐剂、湿润剂、胶凝剂(例如甲基纤维素)、色素和/或调味剂。组合物可以为等渗的,即它可以具有与血液和泪液相同的渗透压。
本发明组合物所期望的等渗性可以利用酒石酸钠、丙二醇或其它无机或有机溶质实现。尤其对含有钠离子的缓冲剂而言,优选使用氯化钠。
组合物粘度可以利用药学可接受的增稠剂来维持选定水平。优选甲基纤维素,因为其可以容易并经济的得到,而且应用简单。其它合适的增稠剂包括,例如黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等等。增稠剂的优选浓度依赖于所选试剂。要点是使用可达到所选粘度的量。粘性组合物通常通过向溶液中添加这些增稠剂制备。
药学可接受防腐剂可被利用以延长组合物的保存期。苯甲醇是适宜的,但也可以使用各种防腐剂,包括例如对羟苯甲酸酯、硫柳汞、氯代丁醇或苯扎氯铵。防腐剂的合适浓度为基于总重的0.02%到2%,但根据所选试剂可以有明显变动。
本领域技术人员将理解,本组合物所选组分与脑膜炎奈瑟氏球菌多糖-蛋白质载体缀合物之间必须为化学惰性的。
本发明将通过参考下列举例说明性但非限制性实施例进一步描述,所述实施例详细阐明本发明概念的几个优选实施方案。本发明的其它实施例在不背离本发明精神的前提下为本领域技术人员所显见。
实施例
实施例1
脑膜炎奈瑟氏球菌血清型A、C、W135和Y的纯化荚膜多糖粉末的制备
制备粗糊状物
分别地,将脑膜炎奈瑟氏球菌血清型A、C、W135和Y的冻存湿种子培养物融化并在液体Watson Scherp培养基的帮助下重构,而后接种至含有Mueller Hinton琼脂培养基的Blake瓶中。将Blake在CO2气氛中35至37℃孵育15至19小时。孵育后,将生长物从Blake瓶中移出并加至4L含有Watson Scherp培养基的摇瓶中。将摇瓶在水平摇床上35至37℃孵育3至7小时。将4L瓶中的内容物转移至含有Watson Scherp培养基的发酵容器中。发酵容器在35至37℃孵育7至12小时,其间加入补料和消泡剂并控制溶解氧含量和pH。孵育后,将发酵容器内容物转移至500L大罐中,加入塞太弗伦(Cetavlon),并且混和1小时。将塞太弗伦处理的生长物以每小时约30至70升的流速以约15,000至17,000g离心。利用第二次塞太弗伦沉淀将多糖粗品从上清中沉淀出来。向上清中加入塞太弗伦并将材料在室温下混和至少1小时。将材料在1至5℃贮存8至12小时。以每分钟300至400ml的流速,以约45,000至50,000g离心收集沉淀的多糖。收集的糊状物在-60℃或更低的温度下贮存直至进一步处理。
纯化多糖粉末的制备
将未活化的糊状物融化并转移至搅拌器中。将糊状物于0.9M氯化钙中搅拌以产生均一悬液。将悬液以约10,000g离心15分钟。将上清通过无棉绒垫滗析入容器作为第一提取物。向糊状物加入第二体积的0.9M氯化钙,并搅拌以产生均一悬液。悬液如上所述离心,且将上清与来自第一次提取的上清混和。总共进行4次提取,并汇集上清。汇集的提取物通过利用10-30 kDA MWCO螺旋形超滤装置进行超滤浓缩。
向浓缩液中加入氯化镁,并用氢氧化钠调节pH值至7.2到7.5之间。向浓缩物中加入DNase和RNase,并于25到28℃混合孵育4小时。加乙醇至浓度为30到50%。通过10,000g离心2小时除去沉淀的核酸和蛋白质。回收上清并通过将乙醇加至80%且于1-5℃静置过夜使多糖沉淀。虹吸除去乙醇,并将沉淀的多糖10,000g离心5分钟。用乙醇洗涤沉淀的多糖。用丙酮洗涤多糖,10,000g离心15至20分钟。将多糖真空干燥。将初始多糖粉末溶入乙酸钠溶液。向其中加入氯化镁,并用氢氧化钠溶液调节pH值至7.2到7.5之间。向溶液中加入DNase和RNase,并于25到28℃混合孵育4小时以去除残余的核酸。在与这些酶孵育后,向多糖-酶混合物中加入等体积乙酸钠-苯酚溶液,并置于1至5℃水平摇床中约30分钟。混合物10,000g离心15至20分钟。将上层水相回收并保存。向水相中加入等体积乙酸钠-苯酚溶液,并按照如上所述提取。总共进行4次提取以从多糖溶液中去除蛋白质和内毒素。合并的水相提取物用注射用水稀释多达10倍,并对10倍体积注射用水超滤透析(diafilter)。向超滤透析后的多糖中加入氯化钙。多糖通过加入乙醇至80%并于1至5℃过夜得以沉淀。弃掉乙醇上清,并通过10,000g离心15分钟收集多糖。纯化的多糖用乙醇洗涤两次,并用丙酮洗涤一次。将洗涤后的粉末在干燥器中进行真空干燥。干粉在-30℃或更低的温度下贮存直至进行缀合。
实施例2
从脑膜炎奈瑟氏球菌血清型A、C、W135和Y中纯化的荚膜多糖粉末的解聚
本制备所用材料包括来自脑膜炎奈瑟氏球菌血清型A、C、W-135和Y的纯化的荚膜多糖粉末(按照实施例1制备)、无菌50mM乙酸钠缓冲液(pH6.0)、无菌1N盐酸、无菌1N氢氧化钠、30%过氧化氢以及无菌生理盐水(0.85%氯化钠)。
对每一血清型多糖分别进行反应解聚。向一不锈钢罐中装入纯化的荚膜多糖粉末多达60克。向多糖中加入无菌50 mM乙酸钠缓冲液(pH6.0)至浓度为每升2.5g多糖。多糖溶液在1至5℃下混和12至24小时以实现溶解。将反应罐与热交换器相连。再加入一些50mM乙酸钠缓冲液(pH6.0)以稀释多糖至每升1.25 g的反应浓度。将多糖溶液加热至55℃±0.1。向反应混合物中加入30%过氧化氢至其反应浓度为1%。
反应进程通过随反应时间跟踪多糖分子大小的变化来监控。每隔15至20分钟,取出等份反应混和物试样并注射至HPSEC柱以测量多糖的分子大小。当多糖的分子大小到达目标分子大小时,关闭加热器并通过冰水浴循环将多糖溶液迅速冷却至5℃。通过将反应罐连接至装备有3000MWCO再生纤维素柱的超滤器上,将解聚多糖溶液浓缩至每升15g。浓缩后的解聚多糖溶液对10倍体积无菌生理盐水(0.85%氯化钠)超滤透析。将解聚多糖在1至5℃贮存直至下一工序。
解聚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,所述凝胶过滤层析柱的商品名为“UltahydrogelTM250”,其利用葡聚糖分子大小标准来校准。多糖的量对于血清型A通过磷含量而对于血清型C、W135和Y通过唾液酸含量来确定,其中所述磷含量测定利用Bartlet,G.R.J(1959)生物化学杂志(Journal of Biological Chemistry),234,466-468页的方法进行,所述唾液酸含量测定利用Svennerholm,L.(1955)生物化学生物物理学报(Biochimica Biophysica Acta)24,604-611页的方法进行。O-乙酰基含量的确定利用Hesterin,S.(1949)生物化学杂志(Journal of Biological Chemistry),180,249页的方法进行。还原活性利用Park,J.T.和Johnson,M.J.(1949)生物化学杂志(Journal ofBiological Chemistry),181,149-151页所述方法确定。解聚多糖的结构完整性由质子1H和13C NMR确定。解聚多糖的纯度通过测量LAL(内毒素)含量和残余过氧化氢含量来确定。
实施例3
脑膜炎奈瑟氏球菌血清型A、C、W135和Y解聚多糖的衍生化
本制备所用材料包括来源于脑膜炎奈瑟氏球菌的过氧化氢解聚的血清型A、C、W-135和Y荚膜多糖(按照实施例2制备)、己二酸二酰肼、仅用于血清型A的1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDAC)、氰基硼氢钠、无菌1N盐酸、无菌1N氢氧化钠、无菌1M氯化钠以及无菌生理盐水(0.85%氯化钠)。
每一血清型多糖在分开的反应中进行衍生。向一不锈钢罐中装入纯化的解聚多糖,并用无菌0.85%生理盐水稀释至终浓度为每升6g多糖。向此溶液中加入溶于无菌0.85%生理盐水的浓缩的己二酸二酰肼至其反应浓度为每升1g。仅对血清型A而言,加入溶于无菌0.85%生理盐水的浓缩EDAC至其反应浓度为每升1g。调节pH值至5.0±0.1,并利用无菌1N盐酸和无菌1N氢氧化钠在室温下(15-30℃)维持此pH值2小时。2小时后,向此反应混合物中加入溶于无菌0.85%生理盐水的浓缩氰基硼氢钠至其反应浓度为每升2g。反应在室温下(15-30℃)搅拌44小时±4小时,并维持pH值5.5±0.5。此反应期后,将pH值调至6.0±0.1,并且通过将反应罐连接至装备有3000MWCO再生纤维素柱的超滤器上,将衍生的多糖浓缩至每升12g多糖。浓缩后的衍生多糖对30倍体积的1M氯化钠超滤透析,并紧接着对10倍体积的0.15M氯化钠超滤透析。将反应罐与超滤器分离,并在1至5℃贮存7天。将反应罐与装备有3000 MWCO再生纤维素柱的超滤器重新连接,并对30倍体积的1M氯化钠超滤透析,并紧接着对10倍体积的0.15M氯化钠超滤透析。
使用在解聚多糖中所用的相同方法对衍生多糖的分子大小、多糖的量以及O-乙酰基含量进行测定。酰肼含量测定利用Snyder,S.L.和Sobocinski,P.Z.(1975)分析生物化学(Analytical Biochemistry)64,282-288页的2,4,6-三硝基苯磺酸方法进行。衍生多糖的结构完整性由质子1H和13C NMR确定。衍生多糖的纯度通过测量未结合的酰肼的水平、LAL(内毒素)含量和残余氰基硼氢化物含量来确定。
实施例4
载体蛋白质的制备
粗白喉类毒素蛋白质的制备
将冻干的种子培养物进行重构并孵育16到18小时。取一份培养物转移至含有生长培养基的0.5升摇瓶中,并将培养瓶在旋转摇床上34.5-36.5℃孵育7至9小时,从培养瓶取一份培养物转移至含有生长培养基的4升摇瓶中,并将培养瓶在旋转摇床上34.5-36.5℃孵育14至22小时。用此4升摇瓶的培养物接种含有生长培养基的发酵罐。将发酵罐在34.5-36.5℃孵育70至144小时。发酵罐内容物通过深层滤器过滤至收集器中。向收获物中加入37%甲醛溶液至其终浓度为0.2%。调节pH值到7.4-7.6。将收获物通过0.2微米芯形滤器过滤至无菌20升瓶中。将瓶子在34.5-36.5℃条件下孵育7天。向每一20升瓶中加入37%甲醛溶液至其终浓度为0.4%。调节混合物pH值到7.4-7.6。将瓶子在摇床上、34.5-36.5℃条件下孵育7天。向每一20升瓶子中加入37%甲醛溶液至其终浓度为0.5%。调节混合物pH值到7.4-7.6。将瓶子在34.5-36.5℃条件下孵育8周。对此类毒素粗品进行去毒测试。在测试期间瓶子于1至5℃贮存。
粗白喉类毒素蛋白质的纯化
将类毒素粗品升至室温,并将这些20升瓶的内容物合并装入纯化罐。调节类毒素pH值至7.2-7.4,并向类毒素粗品中加入活性炭并混和2分钟。将活性炭类毒素混合物静置1小时,然后用芯形滤器过滤至第二个纯化罐中。向滤液中加入固体硫酸铵至70%饱和度。调节pH值到6.8-7.2,并将溶液静置16小时。过滤收集沉淀的蛋白质,并用70%饱和硫酸铵溶液(pH 7.0)洗涤。沉淀溶解于无菌蒸馏水,并将蛋白质溶液过滤至不锈钢收集器中。调节pH值到6.8-7.2,并加入硫酸铵至40%饱和度。调节pH值到7.0-7.2,并将溶液静置16小时。通过过滤去除并丢弃沉淀。将硫酸铵加入滤液至60%饱和度,并调节pH值到7.0-7.2。将混合物静置16小时,并利用过滤收集沉淀的蛋白质。沉淀溶解于无菌蒸馏水,过滤去除不溶的蛋白质,并对0.85%生理盐水超滤透析。
纯化的白喉类毒素蛋白质的浓缩和无菌过滤
利用10,000 MWCO再生纤维素滤柱,将蛋白质溶液浓缩至15克/升并对10倍体积0.85%生理盐水超滤透析。浓缩的蛋白质溶液通过0.2微米滤膜过滤。蛋白质溶液在1至5℃贮存直至进行缀合。
蛋白质浓度通过Lowry,O.H.等人(1951)生物化学杂志(Journal ofBiological Chemistry)193,265-275页的方法确定。蛋白质纯度通过测定无菌度、LAL(内毒素)含量和残余甲醛含量确定。
实施例5
制备脑膜炎奈瑟氏球菌血清型A、C、W-135和Y多糖与白喉类毒素蛋白质的单价缀合物
本制备所用材料包括来源于脑膜炎奈瑟氏球菌血清型A、C、W-135和Y的己二酸衍生多糖(按照实施例3制备)、无菌白喉类毒素蛋白质(按照实施例4制备)、EDAC、硫酸铵、无菌1N盐酸、无菌1N氢氧化钠以及无菌生理盐水(0.85%)。
每一血清型多糖缀合物分别通过反应制备。所有的四种缀合物都通过下列方法制备。向一不锈钢罐中装入纯化的己二酸衍生多糖(反应浓度为700-1000μmol/L活性酰肼)以及纯化的白喉类毒素蛋白质(反应浓度为3.8-4.0g/L蛋白质)。利用0.85%生理盐水稀释这些原料至目标反应浓度,并将pH调至5.0±0.1。向多糖蛋白质混合物中加入EDAC至其反应浓度为2.28-2.4g/L。反应pH在15-30℃于5.0±0.1保持2小时。2小时后,利用无菌1N氢氧化钠调节pH至7.0±0.1,并将反应物在1至5℃贮存16-20小时。
将反应混合物升温至15-30℃,并将反应罐连接至装备有30,000MWCO再生纤维素柱的超滤器上。加入固体硫酸铵至60%饱和度(对血清型A、W-135和Y而言)和50%饱和度(对血清型C而言)。缀合反应混合物对20倍体积60%饱和度硫酸铵溶液(对血清型A、W-135和Y而言)和50%饱和度硫酸铵溶液(对血清型C而言)超滤透析,并紧接着对20倍体积0.85%生理盐水超滤透析。超滤透析后的缀合物先通过含有1.2微米和0.45微米滤器的滤囊(filter capsule)过滤,然后再通过含有0.22微米滤器的滤囊过滤。
多糖的量和O-乙酰基含量利用与在解聚和衍生多糖中所用相同的方法测定。蛋白质的量通过Lowry方法确定。缀合物的分子大小利用凝胶过滤层析柱来确定,所述凝胶过滤层析柱的商品名为“TSK6000PW”,其利用DNA作为空体积标记物、ATP作为总体积标记物以及牛甲状腺球蛋白作为参考标记物。另外,从TSK6000PW柱上洗脱的缀合物的分子大小通过多角度激光光散射测定。缀合物的抗原特性利用双夹心ELISA方法,通过与抗多糖血清型特异性抗体的结合来测定。缀合物纯度通过测定未结合(未缀合)多糖的量(通过在疏水相互作用层析柱上洗脱)、未缀合蛋白质的量(利用毛细管电泳)、无菌度、LAL(内毒素)含量、残余EDAC含量以及残余铵离子含量来确定。
实施例6
多价脑膜炎球菌A、C、W-135和Y多糖白喉类毒素缀合疫苗的配制
本制备所用材料包括血清型A、C、W-135和Y多糖-白喉类毒素缀合物(按照实施例5制备)、无菌100mM磷酸钠缓冲的生理盐水(0.85%氯化钠)。
向不锈钢积聚罐(bulking tank)中的生理盐水(0.85%)中加入一份无菌100-500mM磷酸钠缓冲的生理盐水以产生10mM磷酸钠的最终疫苗浓度。向含有10mM无菌磷酸钠生理盐水的积聚罐中加入2到4种无菌单价脑膜炎球菌多糖-白喉类毒素缀合物各一份,生成每毫升缓冲液中含有每种血清型多糖各8μg的终浓度。将配制的四价缀合物混和并通过0.2μm滤器过滤至第二个积聚罐中。
多价制剂中每一种血清型多糖的量通过糖成分分析,使用脉冲电流检测及高pH阴离子交换层析来确定。蛋白质的量通过Lowry方法测定。疫苗的pH值利用连接至pH计的复合电极测定。多价缀合疫苗的抗原特性利用双夹心ELISA方法,通过结合抗多糖血清型特异性抗体来测定。多价缀合疫苗的免疫原性通过测定在动物模型中疫苗的每一缀合物引起初次和加强抗多糖Ig G免疫应答的能力来确定。多价缀合疫苗的纯度通过测定未结合(未缀合)多糖的量(利用脉冲电流检测及高pH阴离子交换层析),无菌度、LAL(内毒素)含量、致热原含量和一般安全性来确定。
实施例7
氢氧化铝佐剂辅助的多价脑膜炎球菌多糖白喉类毒素蛋白质缀合物的制备
吸附至氢氧化铝的缀合物的制备。本制备所用材料包括实施例5描述的血清型A、C、W-135和Y多糖白喉类毒素缀合物制备物、无菌生理盐水(0.85%氯化钠)以及无菌的溶于生理盐水(0.85%氯化钠)的氢氧化铝。
向含有生理盐水的积聚罐中加入每一无菌单价脑膜炎球菌多糖白喉类毒素缀合物各一份,生成每毫升缓冲液中每一血清型多糖各8μg的终浓度。向多价缀合疫苗中加入溶于生理盐水(0.85%氯化钠)的氢氧化铝,以获得每毫升疫苗0.44mg铝离子的终浓度。
实施例8
磷酸铝佐剂辅助的缀合物的制备
本制备所用材料包括实施例5描述的血清型A、C、W-135和Y多糖白喉类毒素缀合物制备物、无菌生理盐水(0.85%氯化钠)以及无菌的溶于生理盐水(0.85%氯化钠)的磷酸铝。
向含有生理盐水的积聚罐中加入每一无菌单价脑膜炎球菌多糖-白喉类毒素缀合物各一份,生成每毫升缓冲液中每一血清型多糖各8μg的终浓度。向多价缀合疫苗中加入溶于生理盐水(0.85%氯化钠)的无菌磷酸铝,以获得每毫升疫苗0.44mg铝离子的终浓度。
实施例9
四价缀合疫苗的免疫原性
在临床评价之前,研究了四价缀合疫苗在小实验动物上引起免疫应答的能力。利用小鼠、大鼠和兔研究缀合疫苗相对于多糖疫苗的免疫原性。由于这些动物模型能够通过免疫应答模式区分缀合疫苗与相应的多糖,因此它们是有用的。缀合疫苗引起类似T-细胞依赖性的免疫应答,而多糖疫苗引起类似非T-细胞依赖性的免疫应答。
在小鼠免疫原性研究中,将缀合物用生理盐水(0.85%氯化钠)稀释,按人剂量的1/4到1/16施用。对小鼠施用1或2次疫苗,即缀合物或多糖疫苗,并在接种后2周采集其血液样本。以一组小鼠作为未免疫对照组。利用ELISA方法测定每一血清型多糖的抗体。血清样本与结合在ELISA微量滴定板孔上的过量的每一种荚膜多糖孵育。利用甲基化人血清白蛋白将每一血清型多糖连接至滴定孔上。孵育后,用缓冲液洗涤滴定孔,并将能够结合抗脑膜炎球菌多糖抗体的二级抗体-酶缀合物加至抗体-多糖复合体中。洗涤滴定板,并将化学底物加至多糖-脑膜炎球菌抗体-二级抗体-酶缀合物中。经酶水解化学底物的一部分后导致颜色形成。颜色形成的量与结合在滴定孔中的多糖-脑膜炎球菌抗体-二级抗体-酶缀合物的量成正比。疫苗的效价通过与针对每一血清型的参考抗血清作比较而确定,所述参考抗血清在同一滴定板上测定,其间使用4参数拟合进行平行计算。小鼠参考抗血清由同一株系小鼠产生,所述小鼠分别用各血清型缀合疫苗进行了3次免疫。小鼠参考抗血清基于产生1.0光密度的稀释度的倒数来确定效价。
表1总结了针对每一血清型的抗多糖Ig G效价,所述效价从接种2次四价缀合疫苗(液体和氢氧化铝制剂)或相应四价多糖疫苗的Swiss-Webster小鼠获得。此Ig G效价在一组十只小鼠的合并血清上进行测定。利用2组各十只小鼠来测定每一种疫苗制剂的免疫应答。两组都在第一天接种。在第15天(接种后两周)从第一组十只小鼠采集血液样本,而在第15天对第二组十只小鼠进行第二次疫苗接种。至两周后的第29天,从第二组十只小鼠以及未免疫对照组采集血液样本。所有的抗体同时进行滴定,即15天和29天血液样本连同未免疫对照以及小鼠参考血清同时测定。
四价缀合疫苗(未用佐剂和以氢氧化铝为佐剂)在这一小鼠模型中可以引起强的抗多糖Ig G免疫应答。氢氧化铝佐剂能够提高对这四种血清型多糖缀合物任一种的初次和加强应答。与未免疫对照相比较,在这一小鼠模型中四价多糖疫苗引起微弱的对血清型A、C和W135的免疫应答,而血清型Y虽引起可观的免疫应答,但无加强应答。在此模型中四价多糖疫苗未能引起对所有四种血清型多糖的加强应答。根据对每一血清型缀合疫苗的免疫应答强度和加强应答模式,这一模型可以很容易地区分多糖疫苗和缀合疫苗。
在年轻健康成人和健康幼儿中已经临床研究了未用佐剂的四价缀合疫苗的安全性和免疫原性。在成人研究中,给受试者接种单一剂量疫苗,所述疫苗经配制含有四种缀合物各4μg(按多糖计)。血液样本在恰好接种前和接种后28天采集。每一血清型缀合物的抗体通过ELISA测定法来测定,所述测定法可以确定抗多糖Ig G的量。此ELISA方法与在小鼠血清中存在Ig G抗体时测定其数量的方法非常相似。
简言之,血清样本在包被有过量脑膜炎球菌多糖的ELISA微量滴定孔中孵育,所述脑膜炎球菌多糖通过甲基化的人血清白蛋白结合至滴定板。结合的抗体的量通过与过氧化物酶标记的小鼠抗人Ig G特异性单克隆抗体反应来确定。使用过氧化物酶底物在随后的反应中生成显色产物,其可以通过分光光度法测定。产生的生色团的光密度与血清中与微量滴定板上的脑膜炎球菌多糖结合的Ig G抗体的量相关。抗体的量利用4-参数逻辑斯谛曲线方法,通过与具有指定值的人参考血清(CDC1922)进行比较而计算。另外,还测定了抗体溶解血清型特异细菌的能力。血清样本首先热失活以破坏补体。将血清样本在无菌96孔微量滴定板中进行2倍系列稀释。将血清型特异细菌连同幼兔补体加至血清稀释物中并孵育。孵育后,向血清/补体/细菌混合物中加入琼脂覆盖培养基。待琼脂覆盖物变硬,而后在5%二氧化碳、37℃条件下孵育过夜。第二天,计数孔中出现的细菌菌落。终点效价通过与补体对照孔的平均值相比产生大于50%的杀死率的血清稀释度的倒数来确定。
表2所示总结了四价缀合疫苗接种前后的成年人血清中针对每一血清型的抗多糖Ig G平均浓度和血清杀菌抗体(SBA)平均效价,其中所述缀合疫苗配制成每剂量4μg多糖。对所有四种血清型缀合物的免疫应答令人满意,其在Ig G抗体和功能性杀菌抗体应答上比得上得到批准的多糖疫苗引起的免疫应答。疫苗对这一年龄组来说是安全的,并且发现其安全性曲线与得到批准的多糖疫苗的相似。
表2
接种配制成每剂量4μg多糖的四价脑膜炎球菌缀合疫苗的年轻健康成人的抗多糖Ig G GMC(组平均浓度)和血清杀菌抗体GMT(组平均效价)
在幼年组(小于2岁的幼儿),对多糖疫苗的免疫应答很弱并且据估计免疫力在一年后消退。给12至15个月大小的幼儿施用单一剂量的配制成每剂量每一血清型多糖4μg的四价缀合疫苗,并且在第一次剂量后两个月给他们施用第二剂的四价缀合疫苗。在第一次和第二次接种前以及第二次接种后一个月采集血液样本。表3总结了对4种血清型缀合物的抗体应答。在第二次给予四价缀合物后,观察到对每一血清型的Ig G抗体和杀菌功能性抗体的加强应答。对本年龄组而言,由缀合疫苗引起的Ig G抗体水平比得上由得到批准的多糖引起的Ig G抗体水平;对血清型C多糖而言,6周后的应答为3.64μg/mg(2.96-4.49)Ig G抗体应答。然而,对本年龄组而言,由缀合疫苗引起的杀菌抗体水平大大高于由得到批准的多糖疫苗所正常引起的水平;6周后SBA效价为7.2(5.0-10.4)。在年幼群体中这种Ig G抗体和杀菌抗体之间不一致性的原因据认为是年幼群体中多糖引起高比例低亲合性抗体的结果。相反,缀合物似乎可引起更高比例的高亲合性抗体。高亲合性抗体被认为负责此杀菌活性。
表3
接种2次配制成每剂量4μg多糖的四价脑膜炎球菌缀合疫苗的健康幼儿的抗多糖Ig G GMC(组平均浓度)和血清杀菌抗体GMT(组平均效价)
与得到批准的多糖疫苗相比,四价缀合疫苗除能在幼小群体中引起高的功能性抗体应答外,还能够引起回忆应答,这显示由本发明四价缀合疫苗引起的保护是长效的。在四价缀合疫苗的研发中,首先进行的是二价AC缀合制剂的研究。此疫苗比目前批准的单价C缀合物有更广的覆盖度,但其并不对血清型W135和Y引起的疾病起保护作用。
一项比较二价AC多糖疫苗与二价AC缀合疫苗的免疫应答的临床研究在婴儿受试者中进行。在这项研究中,编入第三组婴儿作为对照组并使他们接受流感嗜血杆菌(Haemophilus influenzae)b型缀合物。所有三个疫苗组接受相同的儿科疫苗。二价AC缀合物组在第6、10和第14周龄接受三次缀合疫苗(每剂量4μg多糖)。二价AC多糖组在第10和第14周龄接受两次二价AC多糖疫苗(每剂量50μg多糖)。流感嗜血杆菌(Haemophilus influenzae)b型缀合物组在第6、10和第14周龄接受三次缀合疫苗。在第6周(接种前)以及在第18周(接种后4周)采集血液样本。当婴儿到11至12月龄时采集血液样本,并且已经接受二价AC缀合疫苗或二价AC多糖疫苗的婴儿再接受一次AC多糖的加强接种。加强接种多糖的原因是为了评价受试者是否可引起回忆应答。
本研究的结果(包括初次和多糖加强免疫应答)见于表4的Ig G抗体应答和表5的SBA抗体应答。对多糖和缀合疫苗而言,初次系列免疫接种后的Ig G抗体应答大致相同。然而,缀合物接种的受试者的杀菌抗体应答大大高于多糖接种的受试者。据观察一岁受试者,接种多糖的婴儿引起非常少的杀菌功能性抗体。据推测,接种多糖疫苗的婴儿产生的抗体为低亲合性抗体,而接种缀合疫苗似乎引起高亲合性抗体,这样即解释了高得多的杀菌抗体效价。在初次接种系列中接受缀合疫苗的受试者中,由加强接种多糖疫苗引起高水平功能性抗体,这显示:这些受试者已经被致敏而具有记忆或T-细胞依赖性抗体应答。在初次接种系列中接受多糖疫苗的受试者中,由多糖加强接种产生了中等水平的应答,这显示其为非T-细胞依赖性应答。
表4
在初次系列免疫(6、10和14周龄)及利用二价AC多糖在第11至12月龄加强接种之前和之后婴儿抗血清型A和C的抗多糖Ig G GMC(组平均浓度)
表5
在初次系列免疫(6、10和14周龄)及利用二价AC多糖在第11至12月龄加强接种之前和之后婴儿抗血清型A和C的SBA抗体GMT(组平均效价)
本发明的益处除了为年幼群体提供了对抗脑膜炎球菌疾病的更好保护以及提供了对血清型A、C、W-135和Y的更为广泛的保护外,四价缀合物还提供了对其它病原体的保护,这通过引起对载体蛋白质的抗体应答完成。当使用与白喉类毒素缀合的四价缀合疫苗施用给婴儿时,这些受试者也接受了常规的儿科免疫,包括白喉类毒素。因此,在这些受试者中对白喉类毒素的抗体应答无明显提高。但是,当将此白喉类毒素缀合物施用给没有相伴接受含白喉类毒素的疫苗的受试者时,观察到对白喉类毒素产生强的加强应答。这些受试者已经在2、3和4月龄接受了3次DTP给药方案。在本研究中,受试者在2至3岁接受单一剂量的二价AC缀合物或单一剂量的二价AC多糖疫苗。血清样本在接种时和接种后30天采集。所述二价AC缀合物利用白喉类毒素作为载体蛋白质。
两个疫苗组的白喉类毒素免疫应答见于表6。正如所预望的,多糖未在受试者中刺激产生抗白喉免疫应答,然而在接受AC缀合物的受试者中观察到了强的抗白喉免疫应答。因此,脑膜炎球菌缀合疫苗可以提供额外的益处,即刺激产生对载体蛋白质的免疫应答,这样当以白喉类毒素作为载体蛋白质时就提供了对白喉棒杆菌(Corynebacteria diphtheriae)所致疾病的保护。
表6
接种二价AC白喉类毒素缀合疫苗(配制成每剂量4μg多糖)或二价AC多糖疫苗(配制成每剂量50μg多糖)的健康幼儿中产生的抗白喉类毒素抗体GMT(组平均效价)(通过ELISA方法测定,单位IU/ml)
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Claims (12)
1.含有4种不同的且分别制备的纯化的蛋白质-荚膜多糖缀合物的组合的免疫学组合物,其中第一缀合物包含缀合至载体蛋白质的纯化的脑膜炎奈瑟氏球菌血清型W-135荚膜多糖,第二缀合物包含缀合至载体蛋白质的纯化的脑膜炎奈瑟氏球菌血清型Y荚膜多糖,第三缀合物包含缀合至载体蛋白质的纯化的脑膜炎奈瑟氏球菌血清型A荚膜多糖,第四缀合物包含缀合至载体蛋白质的纯化的脑膜炎奈瑟氏球菌血清型C荚膜多糖。
2.权利要求1所述的免疫学组合物,其还包含有佐剂。
3.权利要求2所述的免疫学组合物,其中所述佐剂选自铝佐剂、弗氏佐剂、BAY、DC-chol、pcpp、单磷酰脂A、CpG、QS-21、霍乱毒素和甲酰甲硫氨酰肽。
4.权利要求3所述的免疫学组合物,其中所述铝佐剂为氢氧化铝。
5.权利要求2所述的免疫学组合物,其中所述载体蛋白质为单一载体蛋白质种类。
6.权利要求5所述的免疫学组合物,其中所述单一载体蛋白质种类包含选自白喉类毒素、CRM197、破伤风类毒素、百日咳类毒素、大肠杆菌LT、大肠杆菌ST以及铜绿假单胞杆菌外毒素A的失活的细菌毒素。
7.权利要求6所述的免疫学组合物,其中所述失活的细菌毒素为白喉类毒素。
8.权利要求6所述的免疫学组合物,其中所述失活的细菌毒素为CRM197。
9.权利要求1所述的免疫学组合物,其中所述组合物被配制为无菌液体。
10.权利要求1所述的免疫学组合物,其还包含药学可接受的防腐剂。
11.权利要求10所述的免疫学组合物,其中所述药学可接受的防腐剂选自苄醇、对羟苯甲酸酯、硫柳汞、氯代丁醇或苯扎氯铵。
12.权利要求1所述的免疫性组合物,其中第一缀合物包含缀合至载体蛋白质的纯化的4μg脑膜炎奈瑟氏球菌血清型W-135荚膜多糖,第二缀合物包含缀合至载体蛋白质的纯化的4μg脑膜炎奈瑟氏球菌血清型Y荚膜多糖,第三缀合物包含缀合至载体蛋白质的纯化的4μg脑膜炎奈瑟氏球菌血清型A荚膜多糖,第四缀合物包含缀合至载体蛋白质的纯化的4μg脑膜炎奈瑟氏球菌血清型C荚膜多糖。
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