CN1787839B - 用于抗医院内感染的免疫的多糖-葡萄球菌表面粘附素载体蛋白缀合物 - Google Patents
用于抗医院内感染的免疫的多糖-葡萄球菌表面粘附素载体蛋白缀合物 Download PDFInfo
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- CN1787839B CN1787839B CN2004800122658A CN200480012265A CN1787839B CN 1787839 B CN1787839 B CN 1787839B CN 2004800122658 A CN2004800122658 A CN 2004800122658A CN 200480012265 A CN200480012265 A CN 200480012265A CN 1787839 B CN1787839 B CN 1787839B
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Abstract
免疫原性多糖-蛋白缀合物,该缀合物具有来自医院病原体的多糖抗原(或者是其具有一个或多个抗原表位的寡糖片段),该抗原与葡萄球菌表面粘附素载体蛋白相缀合,该缀合物用于能同时诱发抗多糖抗原和抗葡萄球菌表面粘附素载体蛋白的抗体应答的免疫原性组合物。这种免疫原性组合物用于对由金黄色葡萄球菌、表皮葡萄球菌或其他医院病原体引起的疾病进行免疫。
Description
技术领域
本发明涉及一种免疫原性多糖-蛋白缀合物,所述缀合物包含来自医院病原体的多糖抗原(或者是其具有一个或多个抗原表位的寡糖片段)和葡萄球菌表面粘附素载体蛋白。本发明还涉及含有所述多糖-蛋白缀合物的免疫原性组合物及其用途。
背景技术
据估计,美国每年有4000万人进入医院,其中约有200万人会罹患医院内感染(Anonyomous 1997)。医院内感染每年导致88,000人死亡,死亡率约为4.4%。据估算,美国每年用于医院内感染的花费为45亿美元(Weinstein 1998)。这种估算不包括在每年3100万次的门诊手术(国家卫生统计中心网站,National Center for Health Statistics’website)、150万疗养院居住者、长期护理机构以及接受流动医疗处置的人中发生的感染。
金黄色葡萄球菌和凝固酶阴性葡萄球菌(CoNS),特别是表皮葡萄球菌,是导致大多数医院内感染的革兰氏阳性医院机会病原体。葡萄球菌感染占全部医院内感染的近25%(约500,000)(Haley,Culveret al.1985)(Boyce 1997)。在有些医院中高达1%的入院者会罹患金黄色葡萄球菌感染(Storch and Rajagopalan 1986)。葡萄球菌(金黄色葡萄球菌和表皮葡萄球菌)造成约47%的医院内血液感染、24%的手术部位感染(SSI)以及17%的医院内肺炎(Anonyomous 1997)。金黄色葡萄球菌和CoNS的医院内感染病人的死亡率差别显著,从5%到68%不等(Nada,Ichiyama et al.1996);(Thylefors,Harbarth etal.1998)。
葡萄球菌感染的范围多种多样,从皮肤感染诸如脓疱病、疖子、伤口感染以及植入装置引起的感染,到严重的危及生命的感染诸如骨髓炎、心内膜炎以及具有转移并发症的菌血症。这种多样性使得设计有效的针对葡萄球菌的免疫原性组合物成为真正的挑战。耐药医院内 细菌的显著增加使得这种设计变得更加困难。耐甲氧西林金黄色葡萄球菌造成医院内感染病人死亡的约40%(Boyce 1997)。近来,万古霉素中度耐药金黄色葡萄球菌(VISA)的出现引起了人们对其传播的更多关注。因而对医院内感染有效的免疫原性组合物的需求正在急速上升。
荚膜多糖
荚膜多糖(CP)参与许多细菌病原体包括流感嗜血杆菌、肺炎链球菌和B族链球菌的毒力,这已经得到了很好的证实。具荚膜细菌能够抵抗白细胞的吞噬作用,并因此能够感染血液和组织。由于抗荚膜多糖的抗体可以中和细菌荚膜的抗吞噬作用(Karakawa,Sutton et al.1988;Thakker,Park et al. 1998),因此在开发预防人类感染葡萄球菌的免疫原性组合物时,葡萄球菌的荚膜成为一个主要的靶点。
在12种已知的金黄色葡萄球菌荚膜的血清型中,5型(CP5)和8型(CP8)占所有临床分离株的约85-90%(Arbeit,Karakawa et al.1984;Karakawa,Fournier et al. 1985;Essawi,Na′was et al. 1998;Na′was,Hawwari et al. 1998)。绝大部分耐甲氧西林金黄色葡萄球菌分离株表达CP5(Sompolinsky,Samra et al. 1985)。抗CP5和CP8的抗体能在体外诱导人类多形核嗜中性粒细胞的类型特异性调理吞噬作用并对动物给予保护(Karakawa,Sutton et al. 1988;Fattom,Sarwaret al.1996)。
绝大部分细菌荚膜多糖在动物或人体内是弱免疫原。但是,如果纯化的多糖与蛋白载体分子缀合,它们即获得免疫原性和T细胞依赖性。一些实验室已经合成了含有与蛋白质共价连接的CP5和CP8的免疫原性缀合物。这些缀合物在小鼠和人体内具有极高的免疫原性并可诱导产生抗体,抗体调理被荚膜包被的金黄色葡萄球菌使其更适于吞噬作用(Fattom,Schneerson et al. 1993;Gilbert et al. 1994;Reynaud-Rondier et al.1991)。含有与铜绿假单胞菌重组外毒素A相缀合的CP5的单价免疫原性组合物具有免疫原性,并且在健康的成年人和晚期肾病患者体内具有良好的耐受性(Welch et al.1996)。在正在接受血液透析的晚期肾病患者上进行的双盲试验中,使用由CP5和CP8共价连接于铜绿假单胞菌重组外毒素A所组成的二价缀合物疫苗,在大约40周后患者对金黄色葡萄球菌所致的菌血症具有了部分免 疫力。此后,随着抗体水平的下降保护力降低(Shinefield et al.2002)。试验结果显示,需要对免疫原性组合物进行改进以有助于更完全的保护。
另一种类型的细胞外多糖,是指多糖粘附素(PS/A;(Tojo,Yamashita et al.1988))、聚-N-琥珀酰β-1-6葡萄糖胺(PNSG;(McKenney,Pouliot et al. 1999))、聚-N-乙酰葡萄糖胺表面多糖(PNAG;(Maira-Litran,Kropec et al.2002)),或者多糖细胞间粘附素(PIA(Mack,Fischer et al.1996))。金黄色葡萄球菌和表皮葡萄球菌均能表达这类多糖。PIA或者PS/A都是线型β-1,6-连接的葡糖胺聚糖。用PS/A(PNSG,PNAG)对小鼠进行免疫后,能够降低肾脏的定植菌群并保护小鼠在受到金黄色葡萄球菌攻击后免于死亡,这种金黄色葡萄球菌在体外极少产生PS/A(PNSG,PNAG)(McKenney,Pouliot et al. 1999)。PIA在与血管内导管相关感染的发病机制中发挥重要的作用(Rupp,Ulphani et al. 1999;Rupp,Ulphaniet al.1999;Rupp and Fey 2001;Rupp,Fey et al.2001)。PIA除了促进单个表皮葡萄球菌细胞之间的粘附外,还与红细胞结合,充当红细胞凝集素(Fey,Ulphani et al.1999)。
葡萄球菌表面粘附素
葡萄球菌表达多种表面粘附素(称为识别粘附的基质分子的微生物表面成分),包括例如纤连蛋白结合蛋白、纤维蛋白原结合蛋白、胶原蛋白结合蛋白和玻连蛋白结合蛋白。这些粘附素能够特异性地识别并结合细胞外基质(ECM)组分,例如纤连蛋白、纤维蛋白原、胶原蛋白和玻连蛋白。这些由金黄色葡萄球菌表达的大量的多种粘附因子参与致病性,它们能够通过各种途径粘附于许多组织并使之感染。抗葡萄球菌粘附素的抗体通过阻止细菌侵入哺乳动物宿主的组织或者通过促进调理吞噬作用,从而减少疾病的发生。用小量金黄色葡萄球菌纤连蛋白结合蛋白A(以融合蛋白形式)免疫大鼠,能够中等程度地保护其免于感染实验性心内膜炎。为诱导生成抗纤连蛋白结合蛋白A的抗体而设计了类似的免疫原性组合物,并在金黄色葡萄球菌所致的小鼠乳腺炎模型上进行了测试。免疫小鼠中发生的严重乳腺炎例数少于对照组小鼠,并且免疫小鼠中从乳腺回收的细菌数少于对照组小鼠。用19和87kDa的纤维蛋白原结合蛋白免疫的小鼠与没有经过免疫 的对照组小鼠相比,乳腺炎的发生率下降,但是用胶原蛋白结合蛋白免疫后显示不具备保护作用(Lee,Pier 1997)。
尽管在缀合多糖抗原与各种蛋白载体方面已经做出了许多努力,但是目前仍然没有能够有效治疗或预防医院内感染的免疫原性组合物。
发明概述
为此,本发明提供一种免疫原性多糖-蛋白缀合物,该缀合物包含至少一种来源于医院病原体的多糖抗原或者具有该至少一种多糖抗原的一个或多个抗原表位的寡糖片段(通过合成或者天然多糖水解制备),其与至少一种葡萄球菌表面粘附素载体蛋白相缀合。本发明所述的缀合物用于免疫原性组合物,该免疫原性组合物能够在受试者体内有效诱发抗医院病原体的多糖抗原和表面粘附素载体蛋白的特异性抗体反应。由此,该缀合物可用于由金黄色葡萄球菌、表皮葡萄球菌以及其他医院病原体所导致的医院内感染的免疫治疗。还可以用在被动免疫中产生免疫球蛋白,从而预防或降低医院内感染的严重性。
一方面,本发明提供了一种免疫原性多糖-蛋白缀合物,该缀合物包含至少一种来源于医院病原体的多糖抗原,该多糖抗原与至少一种葡萄球菌表面粘附素载体蛋白相缀合。该缀合物能够产生抗多糖抗原和抗表面粘附素载体蛋白的特异性抗体。
另一方面,本发明还提供了一种免疫原性多糖-蛋白缀合物,该缀合物包含与至少一种葡萄球菌表面粘附素载体蛋白相缀合的、代表一个或多个抗原表位的寡糖片段,该抗原表位为来源于医院病原体的至少一种多糖抗原的抗原表位。该缀合物能够产生抗多糖抗原和抗表面粘附素载体蛋白的特异性抗体。
又一方面,本发明还提供了一种免疫原性组合物,该组合物含有多糖抗原-表面粘附素蛋白缀合物,并与一种合适的载体或稀释剂相混合。本发明中的免疫原性组合物还可以包含佐剂,例如氢氧化铝或磷酸铝。
又一方面,本发明还提供了诱导哺乳动物对医院内感染产生主动免疫的方法,该方法包括给予罹患此类感染的哺乳动物,包括人类致免疫量本发明的免疫原性组合物。
又一方面,本发明提供了制备抗医院内感染的免疫治疗剂的方法,该方法包括如下步骤:用本发明的免疫原性组合物免疫哺乳动物,收集被免疫的哺乳动物的血浆,从收集的血浆中收获含有抗多糖抗体和抗表面粘附素抗体的高免疫性球蛋白。该高免疫性球蛋白可以用于诱导对医院内感染的被动免疫。
本发明所述的缀合物具有独特的优点:能够诱导产生抗多糖抗原和表面粘附素载体蛋白(二者均为毒力因子)的抗体,并对医院病原体引起的疾病赋予免疫力。也就是说,表面粘附素蛋白本身也能够赋予机体免疫力,而不只是充当多糖抗原的蛋白载体。
附图说明
图1显示了GLC和HPAEC-PAD分析确定的金黄色葡萄球菌CP5和CP8的组成。
图2显示了脱-O-乙酰化金黄色葡萄球菌CP5和CP8的1H-NMR分析。
图3为金黄色葡萄球菌聚集因子-ClfA的示意图。
图4为源自金黄色葡萄球菌ClfA的重组蛋白Clf40和Clf41示意图。
图5为表皮葡萄球菌聚集因子-SdrG的示意图。
图6为源自表皮葡萄球菌SdrG的SdrG(N1N2N3)和SdrG(N2N3)重组蛋白示意图。
图7显示了表面粘附素蛋白的溴乙酰化。
图8显示了金黄色葡萄球菌CP在3-(2-吡啶基二硫代)丙酰肼(PDPH)作用下的活化。
图9显示了硫醇化的金黄色葡萄球菌CP与表面粘附素蛋白的缀合。
图10显示了CP5-和CP8-SdrG(N1N2N3)以及CP5-和CP8-Clf41(N2N3)的缀合物对兔CP特异性抗血清的抗原性分析。
图11显示了CP5-和CP8-Clf41(N2N3)的缀合物对兔ClfA特异性抗血清的抗原性分析。
图12显示了双向免疫扩散法对CP5-和CP8-SdrG(N2N3)6xHis以及CP5-和CP8-Clf40(N1N2N3)6xHis缀合物的抗原性分析。
图13显示了Ouchterlony免疫扩散法对CP5-和CP8-SdrG(N2N3)以及CP5-和CP8-FnbA缀合物的抗原性分析。
图14显示了缀合物的点印迹分析。
图15A-H显示了金黄色葡萄球菌CP8与SdrG(N1N2N3)、SdrG(N2N3)、Clf40(N1N2N3)和Clf41(N2N3)的缀合物产生的免疫应答。
图16A-H显示了金黄色葡萄球菌CP5与SdrG(N1N2N3)、SdrG(N2N3)、Clf40(N1N2N3)和Clf41(N2N3)的缀合物产生的免疫应答。
图17A-F显示了含有或不含佐剂的缀合与非缀合的金黄色葡萄球菌ClfA(N1N2N3)产生的免疫应答。
图18A-F显示了含有或不含佐剂的缀合与非缀合的金黄色葡萄球菌ClfA(N2N3)产生的免疫应答。
图19A-F显示了含有或不含佐剂的缀合与非缀合的表皮葡萄球菌SdrG(N1N2N3)产生的免疫应答。
图20A-F显示了含有或不含佐剂的缀合与非缀合的表皮葡萄球菌SdrG(N2N3)产生的免疫应答。
具体实施方式
有多种毒力因子参与了医院内感染。因此,如将几种毒力决定因子相结合作为免疫原性组合物的组分,则极有可能比仅含有一种毒力决定因子的免疫原性组合物有更强的保护作用。本发明所述的多糖抗原来源于不同的医院内病原微生物,包括但不限于,金黄色葡萄球菌、表皮葡萄球菌及其他凝固酶阴性葡萄球菌(CoNS)、肠球菌、白色念珠菌、肠杆菌、流感嗜血杆菌、肺炎克雷伯菌、大肠杆菌和铜绿假单胞菌。这些抗原都是全身感染的毒力因子,为弱免疫原。它们的免疫原性可通过与载体蛋白缀合而增强。对本发明来说,表面粘附素蛋白为微生物表面用来识别粘附基质分子的成分。它们为购自Inhibitex公司,Alpharetta,GA,USA的MSCRAMM。如下所述,使用葡萄球菌表面粘附素蛋白作为多糖抗原的载体蛋白能够使多糖转变为一种T细胞依赖性抗原,从而诱导抗多糖的IgG免疫应答。而且,缀合物诱导产生抗表面粘附素载体蛋白的抗体,该抗体能够预防感染并且帮助 阻止细菌在哺乳动物宿主组织上的粘附。尽管已经知道,蛋白质-多糖缀合方法中的化学反应可能会损害载体蛋白的抗原表位,但令人惊喜的是在本发明中未发现这种效应,蛋白中的保护性表位仍然具有诱导免疫应答的能力。
细菌细胞表面的表面粘附素蛋白和宿主组织中的配基以锁-钥方式相互作用,导致细菌与宿主的粘附。粘附通常是细菌生存所必需的,并且能帮助细菌逃避宿主防御系统和抗生素的攻击。一旦细菌成功地粘附并定居于宿主组织,它们的生理机能就会发生显著改变,并分泌毒素和酶等有害物质。而且,粘附的细菌通常产生生物被膜并很快能够抵抗大部分抗生素的杀灭作用。
表面粘附素蛋白的代表例包括:纤连蛋白结合蛋白、纤维蛋白原结合蛋白、胶原蛋白结合蛋白和玻连蛋白结合蛋白。这些粘附素特异性地识别并结合细胞外基质组分:纤连蛋白、纤维蛋白原、胶原蛋白和玻连蛋白。
纤连蛋白结合蛋白
纤连蛋白(Fn)是一个440kDa的糖蛋白,存在于ECM和动物的体液中。纤连蛋白基本的生物学功能似乎与它成为表达合适整联蛋白的细胞粘附的底物的能力相关。已经证实有几种细菌特异性地与纤连蛋白结合并粘附于含有纤连蛋白的基质。大部分金黄色葡萄球菌分离株与Fn结合,但是结合的程度有所不同。这反映了在细菌细胞表面表达的表面粘附素分子的数量是不同的。Fn和金黄色葡萄球菌之间的反应具有高度的特异性(Kuusela 1978)。Fn结合是通过称为FnBP-A和FnBP-B的两个蛋白介导的,它们暴露于细胞的表面,分子量为110kDa。Fn的主要结合位点包括一个35-40个氨基酸残基的基序(motif),重复3-5次。表达它们的基因已经被克隆并测序(Jonsson1991)。
WO-A-85/05553中公开了与纤连蛋白、纤维蛋白原、胶原蛋白和/或层粘蛋白具有结合能力的细菌细胞表面蛋白。
Hook等人的美国专利5,320,951和5,571,514中公开了纤连蛋白结合蛋白A(fnbA)的基因序列和产物以及基于该序列的方法。
Hook等人的美国专利5,175,096中公开了fnbB的基因序列,一种杂合的DNA分子(fnbB)及其生物学产物,以及基于该序列的方法。
美国专利5,652,217公开了一种分离并纯化的具有结合活性的蛋白,该蛋白由来源于金黄色葡萄球菌确定序列的杂合DNA分子编码。
美国专利5,440,014公开了一种在金黄色葡萄球菌纤连蛋白结合蛋白的D3同源性单元中的纤连蛋白结合肽,它可以用于反刍动物对由葡萄球菌感染引起的乳腺炎的免疫、创伤治疗、阻断蛋白受体、其他动物的免疫或用于诊断性检测中。
美国专利5,189,015公开了一种对与哺乳动物纤连蛋白具有结合能力的金黄色葡萄球菌菌株的定植进行预防性治疗的方法。这种方法包括给予需要治疗的哺乳动物有效预防剂量的蛋白,该蛋白具有纤连蛋白结合能力,从而预防由与纤连蛋白具有结合能力的金黄色葡萄球菌菌株而引发的感染。其中所述蛋白的分子量在87kDa至165kDa。
美国专利5,416,021公开了编码停乳链球菌中的纤连蛋白结合蛋白的DNA,同时公开的还有:一种大肠杆菌质粒,该质粒含有编码停乳链球菌中的纤连蛋白结合蛋白的DNA;编码停乳链球菌中的纤连蛋白结合蛋白的DNA;用编码停乳链球菌纤连蛋白结合蛋白的DNA所转化的大肠杆菌。
胶原蛋白结合蛋白
胶原蛋白是软骨的主要成分。胶原蛋白(Cn)结合蛋白主要由葡萄球菌菌株表达。金黄色葡萄球菌中与胶原蛋白结合的表面粘附素蛋白粘附于软骨,这个过程构成了葡萄球菌感染发病机制的重要组成部分(Switalski 1993)。已发现,金黄色葡萄球菌与胶原蛋白的结合至少在关节炎和败血症中起作用,但不仅限于此。已经鉴定了分子量为133、110和87kDa的胶原蛋白粘附素(CNAs)(Patti,J.,et al.1992)。表达不同分子量CNAs的菌株在与胶原蛋白结合的能力上没有差异(Switalski 1993)。
从被诊断为脓毒性关节炎或者骨髓炎的患者的关节中获得的葡萄球菌菌株几乎全部表达胶原蛋白结合蛋白,然而从创伤感染获得的表达这种粘附素的分离物少得多(Switalski et al.1993)。类似地,从骨髓炎患者的骨组织分离得到的金黄色葡萄球菌菌株常常具有识别骨特异性蛋白即骨唾液蛋白(BSP)的表面粘附素蛋白(Ryden et al.1987)。金黄色葡萄球菌在关节腔的软骨组织上定植似乎是脓毒性关节炎病理发展的重要因素。
WO 92/07002公开了一种杂合DNA分子,该DNA分子包括金黄色葡萄球菌中编码具有胶原蛋白结合活性的蛋白或多肽的核苷酸序列,还公开了一种包含该核苷酸序列的质粒或噬菌体。
同时公开的还有一种表达胶原蛋白结合蛋白的大肠杆菌菌株,一种由重组DNA转化的微生物,生产胶原蛋白结合蛋白或多肽的方法以及该胶原蛋白结合蛋白或多肽的蛋白序列。
已经报道了一种cna基因的克隆、测序和表达,该基因编码金黄色葡萄球菌胶原蛋白结合蛋白(Patti,J.,et al.1992)。
该cna基因编码一个133kDa的粘附素,该粘附素具有分离自革兰氏阳性菌的表面蛋白的结构特征。
近来,配体结合位点已经被定位于胶原蛋白结合蛋白的N-末端(Patti,J.et al. 1993)。通过分析与表面粘附蛋白不同片段相对应的系列重组蛋白与胶原蛋白的结合活性,鉴定出一个168个氨基酸残基的蛋白质片段(该蛋白质片段对应于151-318氨基酸残基),它具有明显的胶原蛋白结合活性。该蛋白N末端或C末端的短截体导致了配体结合活性下降,而圆二色谱显示该蛋白的构象也发生了改变。
Patti等人(1995)公开了一种来源于金黄色葡萄球菌粘附素的胶原蛋白结合表位,它由cna基因编码。他们在研究中合成了来源于所述蛋白序列的多肽,并用来产生抗体。其中一些抗体抑制蛋白与胶原蛋白的结合。
WO 97/43314披露胶原蛋白结合蛋白中一些已鉴定的表位(M55、M33和M17)可以用于产生保护性抗体。
该申请还公开了胶原蛋白结合蛋白的晶体结构,该晶体结构提供了鉴定干预或完全阻断胶原蛋白与金黄色葡萄球菌胶原蛋白结合蛋白结合的组分所需的关键性信息。已经检定出位于金黄色葡萄球菌胶原蛋白结合蛋白和一个25个氨基酸的多肽上的配体结合位点,它能够直接抑制金黄色葡萄球菌与125I标记的II型胶原蛋白结合。
纤维蛋白原结合蛋白
纤维蛋白是血凝块的主要成分,纤维蛋白原/纤维蛋白是沉积于植入的生物材料上的主要血浆蛋白之一。大量的证据表明细菌与纤维蛋白原/纤维蛋白粘附是引发植入装置相关感染的重要原因。例如,如Vaudaux等人(1989)所指出的,金黄色葡萄球菌与被纤维蛋白原包 被的塑料在体外的粘附以剂量依赖型方式进行。另外,在模拟血液凝集或心脏瓣膜损伤的模型中,Herrmann等人(1993)证实金黄色葡萄球菌倾向于通过一个纤维蛋白原桥梁结合到粘附于表面的血小板上。金黄色葡萄球菌能够直接粘附到体外形成的血凝块中的纤维蛋白原上,并能够以从血浆中沉积的纤维蛋白原作为桥梁,从而粘附于培养的内皮细胞(Moreillon et al.1995;Cheung et al.1991)。正如Vaudaux等人和Moreillon等人所指出的,纤维蛋白原结合蛋白聚集因子(ClfA)缺陷型的突变体与体外纤维蛋白原、移植的导管、血凝块以及心内膜炎大鼠模型中损伤的心脏瓣膜的结合减弱(Vaudaux etal.1995;Moreillon et al.1995)。
结合纤维蛋白原的粘附素通常称为“聚集因子”,它位于金黄色葡萄球菌细胞的表面。细菌和纤维蛋白原在溶液中的相互作用导致细菌细胞的瞬时聚合。纤维蛋白原的结合位点位于纤维蛋白原糖蛋白二聚体的γ链的C-末端。它具有高亲和力,在纤维蛋白原浓度很低时就能发生聚合。科学家们最近指出,聚集因子也可以促进与固相纤维蛋白原、血凝块以及损伤的心脏瓣膜的粘附(McDevitt et al.1994;Vaudaux et al.1995;Moreillon et al.1995)。
已经发现了金黄色葡萄球菌中两个分别编码两种纤维蛋白原结合蛋白即ClfA和ClfB的基因。克隆并测序了clfA基因,发现其编码一个92kDa的多肽。ClfA与纤维蛋白原的γ链结合,ClfB与其α链和β链结合(Eidhin,et al.1998)。ClfB是一个细胞壁相关蛋白,预期分子量为88kDa,而表观分子量为124kDa。它既能与可溶性的也能与固定的纤维蛋白原结合,并充当聚集因子。
聚集因子蛋白即称为ClfA的基因已经被克隆并测序,并在分子水平上对其进行了详细研究(McDevitt et al.1994;McDevitt et al.1995)。该蛋白由933个氨基酸组成。其N-末端有一个39个氨基酸残基的信号肽序列,并与一个520个氨基酸残基的区域(A区)相连,该区含有纤维蛋白原的结合域。接着,连有一个308个氨基酸残基的区域(R区),它由丝氨酸-天冬氨酸二肽的154个重复单元组成。R区由GAYTCN GAY TCN GAY AGY 18个碱基对的重复序列编码,其中Y为嘧啶碱基,N为任意碱基。ClfA的C-末端具备许多革兰氏阳性菌表面蛋白所具有的特征,如LPDTG基序,它负责将蛋白锚定于细胞壁上;膜 锚定蛋白以及C-最末端带有正电荷的氨基酸残基。
血小板整联蛋白αIIbβ3识别纤维蛋白原γ链的C-末端。这是在凝固过程中引发血液凝固的关键步骤。CIfA和αIIbβ3似乎能够精确地识别纤维蛋白原γ链上的相同位点,因为ClfA能够阻断血小板聚集,而一种对应于γ链C-末端(198-411)的多肽既能够阻断整联蛋白也能够阻断ClfA与纤维蛋白原的相互作用(McDevitt et al.1997)。αIIbβ3的纤维蛋白原结合位点与被称为“EF手”的Ca2+结合决定子接近或者重叠。CifA的A区含有几个类“EF手”基序。当Ca2+浓度在3-5mM范围时,能阻断ClfA与纤维蛋白原间的相互作用并改变ClfA蛋白的二级结构。影响ClfA的EF手的突变减弱或阻止ClfA与纤维蛋白原的相互作用。Ca2+与纤维蛋白原γ链同CIfA A区中的结合位点有可能相同或者重叠。
白细胞整联蛋白的α链,即αMβ2中插入了200个氨基酸残基(A或者I结构域),这段氨基酸是负责配体结合活性的区域。I结构域中一个新的金属离子依赖性的粘附位点(MIDAS)基序是配体结合所必需的。纤维蛋白原是其识别的配体之一。纤维蛋白原中的结合位点位于γ链之中(氨基酸残基190-202)。近来报道,白色念珠菌具有一个表面蛋白αIntlp,其性质接近于真核细胞整联蛋白的性质。该表面蛋白在氨基酸序列上与Mβ2的I结构域(包括MIDAS基序)具有同源性。而且Intlp结合纤维蛋白原。
CIfA A区还显示出与αIntlp具有某种程度的序列同源性。检查CIfA A区序列发现一个可能的MIDAS基序。对CIfA中MIDAS基序的DxSxS部分进行突变,突变部位是其中假定的阳离子适配残基,结果发现纤维蛋白原的结合显著减少。O′Connell等人指出与αMβ2(190-202)的γ链结合位点相一致的一段多肽可以阻止CIfA与纤维蛋白原的相互作用(O′Connell 1998)。这样看来,CIfA可能能够与纤维蛋白原γ链上两个不同的位点结合。CIfA上的配体结合位点与真核细胞整联蛋白所使用的位点相似,并与二价阳离子结合的EF手和MIDAS基序有关。
已知的还有纤维蛋白原结合蛋白ClfB,其预测分子量约为88kDa,表观分子量约为124kDa。ClfB是细胞壁相关蛋白,能够结合可溶性的和固定的纤维蛋白原。而且,ClfB结合纤维蛋白原的α链和β 链,并充当聚集因子。
已发现多个与纤维蛋白原结合ClfA和ClfB相关的蛋白,它们结合于细胞外基质。SdrC、SdrD和SdrE蛋白在一级序列和结构构造上与CIfA和ClfB蛋白相关,而且也定位于细胞表面。由于这些蛋白的A区定位于细胞表面,因此它们能够与血浆中的蛋白、细胞外基质以及宿主细胞表面上的分子相互作用。SdrC能够结合到细胞外基质蛋白如玻连蛋白上。SdrE也能结合到细胞外基质上,例如SdrE结合于骨唾液蛋白(BSP)。
已发现SdrC、SdrD、SdrE、CIfA和ClfB的A区有一段高度保守的氨基酸序列,从其中得出了一致序列TYTFTDYVD基序。该基序可以用于多组分疫苗之中,从而对细菌感染具有广谱的免疫。该基序还可以用于生产单克隆或者多克隆抗体,从而获得广谱的被动免疫。在一种可选的实施方案中,来自Sdr和Clf蛋白家族的可变序列基序的任何组合形式,(T/I)(Y/F)(T/V)(F)(T)(D/N)(Y)(V)(D/N),都能够用来赋予免疫力或者诱导保护性抗体的产生。
MHC-II类似蛋白
除了纤维蛋白原、纤连蛋白和胶原蛋白之外,金黄色葡萄球菌菌株还与其它粘附性真核细胞蛋白粘附。这些蛋白中有许多是属于粘附基质蛋白家族的,如玻连蛋白(Chatwal et al.1987)。美国专利5,648,240公开了一个DNA片段,该片段包含一个编码金黄色葡萄球菌广谱粘附素(分子量约70kDa)的基因。该粘附素能够结合纤连蛋白或者玻连蛋白,并且包含一个约30个氨基酸残基的MHC II模拟单位。对这个蛋白的结合特异性进行了更进一步的分析,结果表明它在功能上与MHC II抗原类似,这是因为它可以结合合成的多肽。所以,除了介导细菌粘附到细胞外基质蛋白之外,该粘附素还通过抑制宿主免疫系统从而在葡萄球菌感染中发挥作用。
来源于表皮葡萄球菌的Sdr蛋白
表皮葡萄球菌是一种凝固酶阴性细菌,通常定居在人的皮肤上而且经常是引起异物感染的原因。由于这种细菌能够首先粘附于留置的医疗装置,如人工瓣膜、矫形装置以及静脉内和腹膜透析导管,并随后在其上形成生物被膜,因此使其易于致病。医疗装置相关感染会妨害医疗的成功并显著增加患者的死亡率。所以,开发能够控制或预防 表皮葡萄球菌感染爆发的疫苗的能力非常重要,同样,开发能够预防或治疗同时包括凝固酶阳性和凝固酶阴性细菌在内的广谱细菌感染的缀合物疫苗也非常重要。
三种由表皮葡萄球菌表达的Sdr(丝氨酸-天冬氨酸(SD)重复区)蛋白分别被命名为SdrF、SdrG和SdrH。WO 00/12131给出了这些蛋白的氨基酸序列以及它们的核酸序列,所述文献在此引入本文。
本发明提供了一种用于免疫原性组合物的有效的缀合物,包括至少一种多糖抗原与至少一种上述的表面粘附素蛋白。另外,抗多糖抗原和表面粘附素蛋白的抗体能够使用常规方法获得。同样,包括一种表面粘附素蛋白,如SdrG在内的免疫原性组合物被用于治疗广谱的细菌感染,包括由凝固酶阳性和凝固酶阴性细菌导致的感染。
本发明中的缀合物的其他组分包括至少一种来源于医院病原体的多糖抗原。这些医院病原体包括,但不限于,金黄色葡萄球菌、表皮葡萄球菌、其他凝固酶阴性葡萄球菌(CoNS)、肠球菌、白色念珠菌、肠杆菌、流感嗜血杆菌、肺炎克雷伯氏菌、大肠杆菌以及铜绿假单胞菌。
在本发明的一个实施方案中,所述多糖抗原包括金黄色葡萄球菌的CP5和CP8中的至少一个。
在本发明的另一个事实方案中,所述多糖抗原包括至少一个由金黄色葡萄球菌和/或表皮葡萄球菌表达的PS/A、PNSG、PNAG和PIA。
免疫原性组合物的制备和用途
用本发明所公开的多糖抗原-表面粘附素蛋白缀合物制备免疫原性组合物。该免疫原性组合物引起免疫应答,并产生抗多糖抗原和抗表面粘附素载体蛋白的抗体。
免疫原性组合物还可以由本发明所公开的寡糖抗原-表面粘附素蛋白缀合物制备。该免疫原性组合物引起免疫应答,并产生抗多糖抗原和抗表面粘附素载体蛋白的抗体。
本发明所提供的适于用作免疫原性组合物的缀合物包括,但不限于:
(i)与金黄色葡萄球菌纤维蛋白原结合蛋白或多肽,例如聚集因子A(ClfA)或者其中的有用片段,或者与其具有足够高的同源性的蛋白或片段相缀合的CP5;或者
(ii)与金黄色葡萄球菌纤维蛋白原结合蛋白或多肽,例如聚集因子A(ClfA)或者其中的有用片段,或者与其具有足够高的同源性的蛋白或片段相缀合的CP8;或者
(iii)与金黄色葡萄球菌纤维蛋白原结合蛋白或多肽,例如聚集因子A(ClfA)或者其中的有用片段,或者与其具有足够高的同源性的蛋白或片段相缀合的PIA;或者
(iv)与表皮葡萄球菌纤维蛋白原结合蛋白或多肽,例如SdrG或者其中的有用片段,或者与其具有足够高的同源性的蛋白或片段相缀合的CP5;或者
(v)与表皮葡萄球菌纤维蛋白原结合蛋白或多肽,例如SdrG或者其中的有用片段,或者与其具有足够高的同源性的蛋白或片段相缀合的CP8;或者
(vi)与表皮葡萄球菌纤维蛋白原结合蛋白或多肽,例如SdrG或者其中的有用片段,或者与其具有足够高的同源性的蛋白或片段相缀合的PIA。
在每一种情况下,一种产生于(i)到(vi)中任何一个缀合物的免疫原性组合物都能够有效地对患者进行免疫,以抵抗例如金黄色葡萄球菌的凝固酶阳性菌和例如表皮葡萄球菌的凝固酶阴性菌导致的感染。
除了上述的(i)到(vi)中的缀合物,其中的表面粘附素载体蛋白为纤维蛋白原结合蛋白,本发明的缀合物还可以用任何葡萄球菌表面粘附素蛋白作为表面粘附素载体蛋白,例如纤连蛋白结合蛋白、胶原蛋白结合蛋白以及玻连蛋白结合蛋白。本发明的多糖抗原还可以是PS/A、PNAG或PNSG、或其他来源于医院病原微生物的多糖抗原,如金黄色葡萄球菌、表皮葡萄球菌及其他CoNS、肠球菌、白色念珠菌、肠杆菌、流感嗜血杆菌、肺炎克雷伯氏菌、大肠杆菌以及铜绿假单胞菌。
已知许多将多糖与蛋白相缀合的技术方法且适用于所述的用途。概括来说,多糖应当被激活或者进行处理使得能够缀合,也就是说,至少一部分必须处理成具有与蛋白质或其他分子共价连接的能力。本领域已知有许多这种方法。例如,Jennings的美国专利4,356,170使用高碘酸在多糖中引入醛基,然后用硼氢化氰进行还原性氨基化。Tsay 等人的美国专利4,663,160也使用高碘酸来产生醛基,但其后将多糖与一个以4-12个碳部分衍生处理的(在缩合剂中制备)蛋白相连接,通过还原剂如硼氢化氰存在下的西夫碱反应进行。Gordon的美国专利4,619,828使用溴化氰来激活多糖,然后通过一个4-8个碳原子的间隔桥将其与蛋白缀合。本领域还有其他已知的缀合方法。
本发明的一个实施方案中,CP用3-(2-吡啶基二硫代)丙酰肼(PDPH)活化,藉此CP中碳二亚胺活化的N-乙酰甘露氨糖醛酸的羧化基团与PDPH中的酰肼基团相偶联(图8)。通过用溴乙酸的N-羟基琥珀酰亚胺酯将赖氨酸残基溴乙酰化而使MSCRAMM载体蛋白活化(图7)。然后通过用硫醇取代溴乙酰化的蛋白中的溴,将PDPH硫醇化的CP与活化的表面粘附素蛋白相缀合,并从而产生稳定的硫醚键(图9)。
CP-CONHNHCOCH2CH2SCH2CONH-表面粘附素蛋白
将本发明中含有CP-表面粘附素蛋白缀合物的免疫原性组合物在小鼠中进行试验,结果显示该组合物与未缀合的低免疫原性CP相比,免疫原性增强(图15-20)。此外,由CP-表面粘附素缀合物免疫原性组合物诱导产生的荚膜多糖的特异性抗体和ClfA及SdrG的特异性抗体均能够结合表达相应抗原的活体菌株(表5和6)。根据以上这些结果,我们相信本发明的免疫原性组合物对由诸如金黄色葡萄球菌或表皮葡萄球菌等病原体引起的医院内感染有效。当把由CP-表面粘附素缀合物诱导产生的抗体作为免疫原性组合物,并用在伤口或用于涂层体内或体外医疗装置或聚合生物材料时,该组合物能够阻止或抑制葡萄球菌菌株与伤口部位或生物材料的结合。根据本发明处理的缀合物用于制备免疫原性组合物,从而保护受试者免于医院内感染。这里所说的“受试者”指包括例如人类、灵长类、马、牛、狗和猫在内的温血哺乳动物。
缀合物可以通过常规方法加入到免疫学上可接受的稀释剂或载体中,用于制备可注射的液体溶液或者悬浮液。
本发明的免疫原性组合物代表性形成方法是:将缀合物分散在任何合适的可药用的载体中,例如生理盐水或其他可注射的溶液。这里所用的“可药用的载体”指包括所有与给予的药物具有相容性的溶剂、分散介质、涂层、抗细菌和抗真菌剂以及等渗和吸收延迟剂等。这类 介质和试剂用做药物活性物质上的用途在本领域为大家所熟知。常规的介质或试剂可以用于本发明的组合物,除非它们与该活性化合物不相容。例如,缀合物制品悬浮于磷酸钠缓冲液(PBS)(pH7.0-8.0)中,浓度为每ml含1-100μg多糖。本发明中免疫原性组合物的给药可能会受到任何熟知的方法的影响,包括,但不限于,非胃肠道给药(如皮下给药、腹膜内给药、肌内给药、静脉内给药和皮层内给药),口服及鼻腔给药。免疫原性组合物的优选给药方法是非胃肠道给药。用于非胃肠道给药的溶液或悬浮液包括下列成分:无菌稀释剂如注射用水、盐溶液、不挥发油类、聚乙二醇、甘油、丙二醇、或其他合成的溶剂;抗菌剂如苯甲醇或羟苯甲酸甲酯;抗氧化剂如抗坏血酸或亚硫酸氢钠;螯合剂如乙二胺四乙酸;缓冲剂如醋酸盐、柠檬酸盐或磷酸盐以及张力调节剂如氯化钠或葡萄糖。pH值可以用酸或碱来调节,例如盐酸或氢氧化钠。非胃肠道给药的制剂可以装入安瓿瓶、一次性注射器或者由玻璃或塑料制成的多剂量瓶中。
注射用的免疫原性组合物包括无菌的含水溶液(水溶性)或用于临时配制注射用无菌溶液或分散系的分散系和无菌的粉末。在所有情况下,组合物都必须灭菌并应当是方便注射使用的流体。还必须在生产和储存条件下保持稳定性,并能防止例如细菌和真菌等微生物的污染作用。载体是含有例如水、乙醇、聚(如,甘油、丙二醇以及液体聚乙二醇等)及其适当的混合物的溶剂或分散介质。适当的流动性可通过以下方式维持,例如,使用涂层如卵磷脂,在分散系条件下保持所需的粒度以及使用表面活性剂。通过各种抗细菌和抗真菌剂防止微生物的作用,所述抗细菌剂和抗真菌剂例如对羟基苯甲酸脂类、氯丁醇、苯酚、抗坏血酸以及硫柳汞等。在许多情况下,优选组合物中含有等渗剂,例如:糖、例如甘露醇和山梨醇的多元醇以及氯化钠。组合物中所含有的延缓吸收的试剂导致了注射用组合物的延迟吸收,延缓吸收的试剂例如硬脂酸铝和凝胶。
注射用无菌溶液通过将本发明的缀合物以所需的剂量溶于合适的溶剂中并与所需的以上所提供的成分中的一种或其组合相混合而制备,然后过滤灭菌。一般来讲,分散系通过将活性物质加入无菌的介质而制备,该介质含有基本分散介质和以上所提供的其他所需的成分。对于用来制备注射用无菌溶液的无菌粉末来说,优选的制备方法 是真空干燥法和冷冻干燥法,从而产生活性成分的粉末以及从其预先过滤灭菌的溶液得到的任何另外所需的成分。
在某些实施方案中,免疫原性组合物可以含有一种或者多种佐剂。正如这里所定义的,佐剂指用来增强本发明中免疫原性组合物的免疫原性的物质。因此,佐剂经常被用来促进免疫应答并为有经验的技术人员所熟知。
用来增强所述组合物疗效的佐剂优选,但不限于:
(1)铝盐(明矾),例如氢氧化铝、磷酸铝和硫酸铝等;
(2)水包油型乳剂(含有或不含其他特异性的免疫刺激剂,如胞壁酰肽(如下所示)或者细菌细胞壁成分),例如
(a)含有5%鲨烯、0.5%吐温80和0.5%斯潘85(可任选含有不同量的MTP-PE(如下所示,尽管非必须))的MF59(PCT公布号WO 90/14837)用高压微射流纳米分散仪(microfluidizer)例如M-110Y高压微射流纳米分散仪(Microfluidics公司,Newton,MA)制成亚微米粒子,
(b)含有10%鲨烯、0.4%吐温80、5%聚氧乙烯-聚氧丙烯嵌段共聚物封闭的聚合物L121和苏氨酸-MDP(如下所示)的SAF,被微流体化为亚微米乳剂或者涡旋形成较大粒度的乳剂,
(c)RibiTM佐剂系统(RAS)(Corixa公司,Hamilton,MT),含有2%鲨烯、0.2%吐温80以及一种或多种细菌细胞壁成分,该细胞壁成分来源于美国专利4,912,094(Corixa)所述的3-O-deaylated单磷酰基脂质A(MPLTM)、双分枝菌酸海藻糖(TDM)以及细胞壁骨架(CWS),优选MPL+CWS(DetoxTM);
(3)皂苷佐剂,例如Quil A或STIMULONTM QS-21(Antigenics公司,Framingham,MA)(美国专利5,057,540),或者其中产生的粒子如ISCOMs(免疫刺激复合物);
(4)细菌脂多糖、合成的脂质A类似物例如氨烷基葡萄糖胺磷酸盐化合物(AGP)或者其衍生物或类似物,可从Corixa公司购买并在美国专利6,113,918中有所叙述;这种AGP中的一种是2-[(R)-3-十四酰氧十四酰氨基]乙基2-脱氧-4-O-膦酰基-3-O-[(R)-3-十四酰氧十四酰氨基]-2-[(R)-3-十四酰氧十四酰氨基]-b-D-吡喃葡萄糖苷,也被称为529(原来称作RC529),它被制成含水态或稳定的乳剂,合成的 多聚核苷酸例如含有CpG基序的寡核苷酸(美国专利6,207,646);
(5)细胞因子,例如白细胞介素(如IL-1、IL-2、IL-4、IL-5、IL-6、IL-7、IL-12、IL-15和IL-18等)、干扰素(如γ干扰素)、粒细胞巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)以及肿瘤坏死因子(TNF)等;
(6)ADP-核糖基化的细菌毒素的脱毒突变体,例如野生型的霍乱毒素(CT)或其突变体,例如公布号为WO 00/18434的国际专利申请(也参见WO 02/098368和WO 02/098369)中29位的谷氨酸被其他氨基酸优选组氨酸置换的情形;百日咳毒素(PT)或者大肠杆菌不耐热肠毒素(LT),特别是LT-K63,LT-R72,CT-S109,PT-K9/G129(见如WO 93/13302和WO 92/19265);
(7)其他用做免疫刺激剂以增强组合物疗效的物质。
如上所述,胞壁酰肽包括,但不限于,N-乙酰-胞壁酰-L-苏氨酰基-D-异谷氨酰胺(thr-MDP),N-乙酰-正胞壁酰-L-丙氨酸-2-(1’-2’二棕榈酰-sn-丙三基-3-羟基磷酰氧基)-乙胺(MTP-PE)等。
本发明的免疫原性组合物的给药剂量足以激发免疫应答。剂量可以根据用药个体的身高、体重或年龄调整。个体的抗体应答可以通过测定抗体滴度或杀菌活性进行监测,并可在需要时强化以提高应答。
将本发明的免疫原性组合物给予受试者,以诱导产生体液免疫应答。然后受试者即成为免疫球蛋白(高免疫性的免疫球蛋白)的供体,该免疫球蛋白产生于对免疫原性组合物的免疫应答中。通过常规的血浆分离技术,从被免疫的施药对象的血浆中获取高免疫性球蛋白,并给予其他的受试者以预防或治疗医院感染。
实施例
以上公开的内容从总体上论述了本发明。通过参考以下特定的实施例能够获得更全面的理解。记述这些实施例的目的仅为阐明而非限制本发明。
实施例1
金黄色葡萄球菌CP5和CP8多糖的纯化
金黄色葡萄球菌菌株Lowenstein(ATCC#49521)和Wright(ATCC#49521)分别用于纯化CP5和CP8。将先前公开的方法 (Fournier,Vann et al. 1984;Fournier,Hannon et al. 1987)改良后用于从细胞中纯化多糖。将培养在含有2%NaCl的Columbia肉汤中的细胞,用溶葡萄球菌酶(175U/g细胞)在37℃下消化3小时,再用RNAse和DNAse(均为0.1mg/g)在37℃下消化4小时,然后用链霉蛋白酶(1m/g细胞)在37℃下消化3小时。依次用含有10mM CaCl2的25%和75%的乙醇沉淀酶的消化液,从中制备CP粗品。在Q-琼脂糖凝胶柱上进行阴离子交换色谱,用0.05-0.5M NaCl线性梯度洗脱,使CP得到纯化。残余的磷壁酸用0.05M NaIO4氧化。透析后,在丙烯葡聚糖凝胶S300(Amersham Pharmacia Biotech公司,Piscataway,NJ)柱上进行大小排阻色谱使CP得到进一步纯化。在流份中出现的CP通过分别与金黄色葡萄球菌CP5和CP8特异性抗血清的反应活性来确定。
对静止期的表皮葡萄球菌细胞进行热提取得到纯化的PIA,并将其与含有PIA的培养物上清合并,如Mack等人所述(Mack,Fischeret al.1996)。提取的物质和培养物上清用10K膜浓缩并处理,以除去核苷酸和残余的蛋白。PIA粗品用凝胶过滤或渗滤进行分离。PIA抗原阳性物质进一步用阴离子交换色谱分离,使含有琥珀酸酯的流份得到纯化。含有未琥珀酰化和部分未琥珀酰化PIA的流份,用阳离子交换色谱纯化。PS/A(PNSG,PNAG)用(Maira-Litran,Kropec et al.2002)或(McKenney,Pouliot et al.1999)中所述的方法纯化。
实施例2
金黄色葡萄球菌CP5和CP8的分析
纯化的CP5和CP8的化学性质证明这两种多糖几乎不含核苷酸和残余的蛋白(表1)。
用HPAEC色谱测定的糖成分显示CP5和CP8中有FucpNAc和ManpNAcA存在(图1)。O-去乙酰多糖(图2)的1H NMR谱与先前公开的相似(Vann,Moreau et al. 1987;Moreau,Richards et al.1990),这证明了三种单糖的存在及其结构:2-乙酰氨基-2-脱氧-D-甘露糖醛酸、2-乙酰氨基-2-脱氧-L-岩藻糖以及2-乙酰氨基-2-脱氧-D-岩藻糖。
在双向免疫扩散分析中,当纯化的CP5、CP8以及TA与相应的全细胞抗血清反应时显示出一条沉淀素条带,证明它们在免疫学上是不同的(数据未列出)。
实施例3
表面粘附素蛋白的纯化
被评价的表面粘附素蛋白为-
-金黄色葡萄球菌Clf40(N1N2N3)-聚集因子A结构域的全长(氨基酸(AA)40-559)-图3。
-金黄色葡萄球菌Clf41(N2N3)-Clf40的蛋白酶位点后片段(AA223-559)-图4。
-表皮葡萄球菌SdrG(N1N2N3)-SdrG的A结构域全长(AA50-597)-图5。
-表皮葡萄球菌SdrG(N2N3)-SdrG的蛋白酶位点后片段(AA273-597)-图6。
这些表面粘附素蛋白均购自Inhibitex公司,Alpharetta,GA.,美国。
从大肠杆菌质粒的宿主菌中纯化了去掉组氨酸标记的表面粘附素蛋白。
大肠杆菌pLP1134BL21(DE3)用于金黄色葡萄球菌ClfA41(N2,N3)的纯化,大肠杆菌pLP1135B21(DE3)用于表皮葡萄球菌SdrG(N2,N3)的纯化。将细胞裂解液的可溶性部分先用硫酸胺沉淀,然后在Sephacryl Q-琼脂糖凝胶柱(Amersham Pharmacia Biotech公司,Piscataway,NJ)上进行离子交换色谱,分离得到这两种蛋白。用SDS-PAGE测定最后得到的蛋白的纯度高于90%。
含有过表达的金黄色葡萄球菌Clf40(N1,N2,N3)或Clf41(N2,N3)、表皮葡萄球菌SdrG(N1,N2,N3)或SdrG(N2,N3)的大肠杆菌细胞,一次通过微流体M-110Y高压微射流纳米分散仪,在13000psi下被液化。在4℃,17000rpm下离心30分钟除去细胞残骸。用AKTAexplorer、XK琼脂糖螯合Fast Flow柱和Q琼脂糖HP树脂(Amersham Pharmacia Biotech公司,Piscataway,NJ),从上清中纯化过表达的蛋白。用带有0.1M NiCl2的琼脂糖螯合Fast Flow柱,通过亲和步骤从上清中纯化具有组氨酸标记的蛋白粗品。将粗裂解液上样到用pH 8.0的25mM Tris、0.5M NaCl和5mM咪唑平衡过的柱子上,用5倍柱体积的缓冲液洗脱柱,未结合的蛋白被洗脱下来。随后用pH 8.0的25mM Tris、0.5M NaCl以及500mM咪唑的缓冲液将结合 的蛋白洗脱下来,并大量收集。然后进一步采用在Q琼脂糖HP柱上进行的离子交换色谱从剩余的不纯物中纯化得到蛋白。
实施例4
金黄色葡萄球菌CP5和CP8-表面粘附素载体蛋白缀合物免疫原性组合物的合成
在向多糖中引入一个含有接头的硫醇基,向蛋白载体中引入卤代乙酰基后,金黄色葡萄球菌CP5和CP8多糖通过一个硫醚键分别与上述的表面粘附素载体蛋白连接。通过氨基与溴乙酸的N-羟基琥珀酰亚胺酯反应,将溴乙酰基引入表面粘附素蛋白中(图7)。为了生成硫醇化的CP,将荚膜多糖的N-乙酰甘露氨基糖醛酸中碳二亚胺活化的羧酸基团与巯基活化的酰肼异双功能接头3-(2-吡啶基二硫代)丙酰肼(PDPH,图8)中的酰肼基团反应。用二硫苏糖醇(DTT)还原产生PDPH-硫醇化CP的硫醇化合物,在葡聚糖凝胶G25柱上进行大小排阻色谱使之纯化,并与活化蛋白的溴乙酰基反应,由CP和蛋白之间的溴置换反应形成共价硫醚键连接(图9)。将未反应的溴乙酰基用半胱胺盐酸盐(2-乙胺乙硫醇盐酸盐)加帽。然后反应混合物用Amicon XM100膜浓缩。
实施例5
金黄色葡萄球菌CP5和CP8表面粘附素载体蛋白缀合物免疫原性组合物的鉴定
为了测定缀合的免疫原性组合物中CP和表面粘附素载体蛋白的含量,在用4N三氟乙酸(TFA)将其水解后,通过在Carbo Pac-PA1柱上进行HPAEC-PAD色谱对CP定量。蛋白含量采用Lowry比色法测定。缀合的免疫原性组合物的分子量采用大小排阻色谱结合多角度激光散射法(MALLS)测定。结果参见表2和表3。CP和表面粘附素蛋白缀合物的抗原性采用双向免疫扩散法(图10-13)和点印迹(图14)测定。结果显示CP和表面粘附素蛋白的缀合物既未改变CP也未改变蛋白的抗原性。CP和蛋白的缀合程度通过点印迹法测定缀合物结合硝酸纤维素膜的能力来确定。未缀合的CP不与硝酸纤维素膜结合。
实施例6
CP-表面粘附素载体蛋白缀合物免疫原性组合物在小鼠体内的免疫原性
测定缀合物的免疫原性组合物诱导产生抗CP5、CP8和表面粘附素蛋白载体的IgG的能力。对Swiss-Webster小鼠皮下(SC)免疫三次,每次间隔两周,剂量为1微克(根据CP)。测定含有100微克磷酸铝佐剂以及不含有佐剂的缀合的免疫原性组合物的免疫原性。采用类似的方案评估每种候选的蛋白免疫原性组合物。注射一周后采用标准抗原酶联免疫分析(ELISA)对金黄色葡萄球菌CPs和表面粘附素蛋白的免疫应答进行检测(参见下述实施例7和实施例8)。
实施例7
在以金黄色葡萄球菌CP5和CP8表面粘附素载体蛋白缀合物的免疫原性组合物免疫的小鼠体内的CPs抗体反应
结果(图15和16)显示CPs共价结合到表面粘附素蛋白上导致荚膜多糖(CP)特异性的IgG反应的产生。这表明在CP偶联到表面粘附素载体蛋白上之后,CP T细胞非依赖性的免疫应答转变为T细胞依赖性的免疫应答。缀合的免疫原性组合物吸附到磷酸铝上,可使抗CP的抗体的滴度升高大约10倍。但在小鼠体内给予SdrG(N2N3)作为蛋白载体时不是这种情况。尽管与其他的表面粘附素载体蛋白缀合物与本研究中的佐剂混合(但不吸附)相比,CP5和CP8-SdrG(N2N3)的缀合物吸附到佐剂上所产生的CPs抗体应答一样好,但它并没有导致抗CP的免疫应答增强。去除ClfA和SdrG的N1结构域对于这些蛋白的载体性质没有影响。
实施例8
接种金黄色葡萄球菌CP5和CP8表面粘附素载体蛋白缀合物的小鼠体内的表面粘附素蛋白抗体应答
缀合的表面粘附素蛋白诱导产生的表面粘附素蛋白特异性抗体的滴度与没有结合的表面粘附素蛋白是类似的(图17-20)。这证实了抗原表位没有因表面粘附素蛋白和CP的结合而改变。与没有使用佐剂的同样的免疫原性组合物所免疫的小鼠相比,小鼠体内没有结合的ClfA和CP-ClfA缀合物吸附到磷酸铝上会导致ClfA抗体滴度升高。用未缀合的SdrG免疫的小鼠与用CP-SdrG缀合的免疫原性组合物免疫的小鼠相比产生较低的SdrG抗体滴度。没有结合的SdrG吸附到磷酸铝上,与给予没有明矾的CP-SdrG缀合物相比,会诱导SdrG抗体滴度的升高。CP-SdrG缀合物吸附到明矾上并不会升高SdrG抗体的滴度。
实施例9
CP-表面粘附素蛋白缀合物所诱导产生的抗体对活体菌株表达的CPs和表面粘附素载体蛋白的识别
小鼠体内CP表面粘附素蛋白缀合物诱导产生的抗体与活体菌株的结合可以通过流式细胞术分析来检测。该实验使用的金黄色葡萄球菌见表4。为了分析诱导的抗SdrG缀合物的抗体,使用表达SdrG的乳酸乳杆菌。结果显示(表5和表6),CP5和CP8表面粘附素蛋白缀合物诱导产生的抗荚膜多糖特异性的抗体和抗CIfA或SdrG特异性的抗体都可结合表达相应抗原的活体菌株。这表明对CP和表面粘附素蛋白抗原上天然表达的抗原表位来说,CP结合到表面粘附素蛋白并没有改变免疫应答。
实施例10
流式细胞检测分析方法
使用的金黄色葡萄球菌如下所述:Newman,一种Newman的ClfA敲除突变体,(Newman ClFA∷emr)以及Wright(ATCC 49525)。为了让ClfA表达最大化,使金黄色葡萄球菌在胰蛋白酶大豆肉汤中一直生长到静止期。为了让荚膜表达最大化,使金黄色葡萄球菌在含2%NaCl的Columbia琼脂(BD Microbiology,Sparks,MD)上生长过夜。在存在5μg/ml红霉素的条件下使Newman ClfA∷emr菌株生长以维持敲除突变。用一种表达SdrG的重组乳酸乳杆菌来评价SdrG抗原的识别。使乳酸乳杆菌在含有5g/ml红霉素的M17肉汤中生长至对数生长后期。
收集所有的细菌培养物,在10ml的冷1x PBS(Invitrogen公司,Rockville,MD)中洗涤两次,置于冰上以待分析。以1x PBS将细菌浓度调至OD600nm=2.0,用紫外可见分光光度计检测细菌浓度(Ultrospec 3000,Pharmacia Biotech公司,剑桥,英国)。为了消除小鼠IgG非特异性地以及通过蛋白A介导与细胞表面结合,将兔IgG(Sigma公司,St.Louis,Missouri)用1x PBS(Invitrogen公司,Rockyille,MD)以1∶50稀释(2.32mg IgG),取10ml与所有细菌制备物一起在冰上孵育30分钟。为了评价没有ClfA结合时8型荚膜的识别,将金黄色葡萄球菌Wright菌株用高滴度ClfA特异性的兔抗血清(Inhibitex公司,Alpharetta,GA)(1∶100倍稀释)再孵育30分 钟以封闭ClfA表位。在进行封闭的孵育过程之后,将细菌以10ml的冷1x PBS洗涤两次,3000rpm离心10分钟。细菌沉淀用含有2.5%BSA的1x PBS(Invitrogen公司,Rockville,MD)(PBSA)再次悬浮,并至于冰上。
实验在滴管中进行(BioRad Labs,Hercules,CA)。来自实验动物的未经处理的前血和高滴度抗血清用PBSA稀释,分别取0.5ml的血清稀释液并加入装有20μl的细菌悬浮液的合适的试管中。所有的试管涡旋振荡并在冰上孵育30分钟。接下来,对每个试管进行涡旋振荡并3000RPM离心10分钟。细菌沉淀在0.5ml冷PBSA中洗涤两次。每个沉淀均以0.5ml 1∶200稀释的抗鼠IgG的连有PE的F(ab’)2片段(H&L)(Rockland Labs,Gilbertsville,Pa)的溶液重新悬浮。细菌被悬浮起来并涡旋振荡。这些试管在冰上孵育30分钟,其间每15分钟涡旋振荡一次,共振荡两次。接下来,将细菌洗涤两次,悬浮于PBSA中形成最终的悬浮液。将这些试管置于冰上以待FACS分析。
每个滴管转到12×75mm聚苯乙烯的试管中,以B-DFACSCalibur(BD Biosciences,Mansfield,MA)流式细胞仪进行分析。若给定抗血清的荧光强度大于同样稀释倍数的未经处理的前血的信号强度,则将结果判为阳性。结果如表7所示。
应当理解前述讨论和例子只是某些实施方案的一个详细描述。因此对于本领域技术人员显而易见的是,可以作出各种修改和得出各种等价形式,而不偏离本发明的实质和范围。
本申请引用的所有期刊论文、其它参考文献、专利和专利申请都全文引入本文。
表1 纯化的金黄色葡萄球菌多糖的鉴定
多糖 | 蛋白 (氨基酸分析) (%;w/w) | 核酸 (%;w/w) | 分子量 (g/mol) |
CP5 | 1.1 | 0.05 | 5.1×104 |
CP8 | 0.79 | 0.14 | 4.5×104 |
[0168] 表2 金黄色葡萄球菌CP5和CP8-表面粘附素蛋白(His+)缀合物免疫原性组合物的特征
表3 金黄色葡萄球菌CP5和CP8-表面粘附素蛋白(His)缀合物免疫原性组合物的特征
免疫原性 组合物 | CP (mg/ml) | 表面 粘附素蛋白 (mg/m1) | 比值(w/w) (CP/表面 粘附素蛋白) | 分子量 (g/mol) |
CP5-SdrG (N2N3) (His-) | 0.26 | 0.32 | 0.81∶1 | 1.18+0.1×105 |
CP8-SdrG (N2N3) (His-) | 0.24 | 0.5 | 0.48∶1 | 2.39+0.1×105 |
CP5-FnBPA | 0.085 | 0.135 | 0.63∶1 | 7.73+0.2×105 |
CP8-FnBPA | 0.089 | 0.16 | 0.56∶1 | 9.83+0.3×105 |
表4 用于通过流式细胞术进行的天然抗原的抗血清识别的菌株
表5 采用流式细胞术以CP5-和CP8-CIfA(N2N3)缀合物抗血清对细菌菌株的标记
表6 采用流式细胞术以CP5-和CP8-SdrG(N1N2N3)缀合物抗血清对细菌菌株的标记
表7 流式细胞术分析结果总结
免疫缀合物* | 细菌制备物 | 相关抗原 | 结果 |
CP5-ClfA | Newman | ClfA和CP5 | + |
Newman ClfA∷emr | CP5 | + | |
Wright | ClfA和CP8 | + | |
Wright ClfA封闭 | CP8 | - | |
CP8-ClfA | Newman | ClfA和CP5 | + |
Newman CIfA∷emr | CP5 | - | |
Wright | ClfA和CP8 | + | |
Wright ClfA封闭 | CP8 | + | |
CP5-SdrG | Newman | CP5 | + |
Wright | CP8 | - | |
L.lactis-SdrG | SdrG | + | |
CP8-SdrG | Newman | CP5 | - |
Wright | CP8 | + | |
L.lactis-SdrG | SdrG | + |
*ClfA=ClfA A结构域的N1,N2,N3或者N2,N3区。SdrG=SdrG A结构域的N1,N2,N3或者N2,N3区。
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Claims (7)
1.一种免疫原性多糖-蛋白缀合物,该缀合物包括:至少一种来源于医院病原体的多糖抗原以及至少一种葡萄球菌表面粘附素载体蛋白,
其中所述缀合物包含
(a)与聚集因子A缀合的金黄色葡萄球菌5型荚膜多糖,
(b)与聚集因子A缀合的金黄色葡萄球菌8型荚膜多糖,
(c)与SdrG缀合的金黄色葡萄球菌5型荚膜多糖,或
(d)与SdrG缀合的金黄色葡萄球菌8型荚膜多糖,
其中所述缀合物产生抗多糖抗原和葡萄球菌表面粘附素载体蛋白的特异性抗体。
2.一种免疫原性多糖-蛋白缀合物,所述缀合物包括:代表一个或多个抗原表位的寡糖片段以及至少一种葡萄球菌表面粘附素载体蛋白,该抗原表位为来源于医院病原体的至少一种多糖抗原的抗原表位,
其中所述缀合物包含
(a)与聚集因子A缀合的代表金黄色葡萄球菌5型荚膜多糖的一个或多个抗原表位的寡糖片段,
(b)与聚集因子A缀合的代表金黄色葡萄球菌8型荚膜多糖的一个或多个抗原表位的寡糖片段,
(c)与SdrG缀合的代表金黄色葡萄球菌5型荚膜多糖的一个或多个抗原表位的寡糖片段,
(d)与SdrG缀合的代表金黄色葡萄球菌8型荚膜多糖的一个或多个抗原表位的寡糖片段,
其中所述缀合物产生抗多糖抗原和葡萄球菌表面粘附素载体蛋白的特异性抗体。
3.含有权利要求1或权利要求2所述的缀合物的免疫原性组合物,所述缀合物存在于免疫学上可接受的载体或稀释剂中。
4.权利要求3的免疫原性组合物,所述组合物还包括一种佐剂。
5.权利要求3或权利要求4所述的免疫原性组合物在制备用于诱导罹患医院内感染的哺乳动物受试者对此类感染的主动免疫的药物中的用途。
6.权利要求5的用途,其中所述药物为非胃肠注射制剂。
7.有效量的权利要求1或权利要求2的缀合物在制备用于治疗或者预防医院内感染的组合物中的用途。
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CA2517439A1 (en) | 2004-09-23 |
CN102366630A (zh) | 2012-03-07 |
US20070141077A1 (en) | 2007-06-21 |
WO2004080490A3 (en) | 2004-10-28 |
KR20060035581A (ko) | 2006-04-26 |
CN102366630B (zh) | 2015-04-01 |
ZA200508086B (en) | 2010-04-28 |
JP2006519870A (ja) | 2006-08-31 |
JP2012051922A (ja) | 2012-03-15 |
JP5757841B2 (ja) | 2015-08-05 |
JP5102487B2 (ja) | 2012-12-19 |
US8377451B2 (en) | 2013-02-19 |
NZ561879A (en) | 2009-05-31 |
BRPI0408167A (pt) | 2006-03-21 |
CN1787839A (zh) | 2006-06-14 |
AU2004220590B2 (en) | 2010-02-18 |
US9296795B2 (en) | 2016-03-29 |
BRPI0408167B1 (pt) | 2014-10-21 |
CA2517439C (en) | 2013-07-23 |
US20070087014A1 (en) | 2007-04-19 |
EP1601381A2 (en) | 2005-12-07 |
WO2004080490A2 (en) | 2004-09-23 |
MXPA05009351A (es) | 2006-03-08 |
AU2004220590A1 (en) | 2004-09-23 |
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