CN101304765A - 链球菌荚膜糖的偶联 - Google Patents
链球菌荚膜糖的偶联 Download PDFInfo
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- CN101304765A CN101304765A CNA200680007342XA CN200680007342A CN101304765A CN 101304765 A CN101304765 A CN 101304765A CN A200680007342X A CNA200680007342X A CN A200680007342XA CN 200680007342 A CN200680007342 A CN 200680007342A CN 101304765 A CN101304765 A CN 101304765A
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- sugar
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- capsular saccharides
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Abstract
用于无乳链球菌荚膜糖的三种偶联方法。在第一种方法中,采用还原胺化已氧化的唾液酸残基侧链,但是先胺化醛基,再通过接头偶联胺与运载体。在第二种方法中,唾液酸残基和/或N-乙酰基-葡糖胺残基脱-N-乙酰化得到胺基,将该胺基通过接头与运载体蛋白偶联。在第三种方法中,利用半乳糖氧化酶方便地通过荚膜糖中的半乳糖残基而非唾液酸残基进行连接。
Description
本文引用的所有文件全文纳入本文作为参考。
技术领域
本发明涉及将细菌荚膜糖与运载体偶联而形成糖偶联物(glycoconjugate)的领域。糖偶联物可用于免疫接种。
背景技术
用细菌荚膜糖疫苗抵御有荚膜细菌已有多年。然而,由于糖是不依赖T细胞的抗原,它们的免疫原性不佳。与运载体偶联(conjugation)能将不依赖T的抗原转变成依赖T的抗原,从而增强记忆应答并产生保护性免疫力。因此,最有效的糖疫苗以糖偶联物为基础,原型偶联疫苗能抵御乙型流感嗜血杆菌(Haemophilusinfluenzae)(“Hib”)[例如,见参考文献78的第14章]。
报道已有偶联疫苗的另一种细菌是无乳链球菌(Streptococcus agalactiae),也称为“B群链球菌”,或简称为“GBS”。Dennis Kasper及同事从事了该疫苗的大部分工作,见例如参考文献1-9。GBS糖转化的Kasper方法通常包括还原胺化纯化的糖与运载体蛋白,例如破伤风类毒素(TT)或CRM197[2]。还原胺化涉及运载体中氨基酸侧链上的胺基与糖中的醛基。如图1所示,由于天然形式的GBS荚膜糖不包含醛基,则应在用过碘酸盐氧化糖的唾液酸残基以产生醛基再偶联[2,10]。
虽然以此方式制备的GBS各血清型(Ia、Ib、II、III和V)的偶联疫苗已显示在人体内安全且有免疫原性[11],但仍需要其它和更好的方法来制备GBS荚膜糖偶联物。
发明内容
本发明依据三种偶联方法,采用这些方法可替代现有技术公开的直接还原胺化,所有这些方法的目的在于(a)与现有技术相比,能使保留的唾液酸残基形式更接近天然多糖所见的形式,和(b)可在偶联反应中用接头来提高与运载体的偶联。
■在第一种方法中,采用还原胺化已氧化的唾液酸残基侧链,但先胺化醛基,再将该胺通过接头与运载体偶联。该方法描述于图2的“途径A”中。
■在第二种方法中,唾液酸残基和/或N-乙酰基-葡糖胺残基脱-N-乙酰化得到胺基,将该胺基通过接头与运载体偶联。该方法描述于图2的“途径B”中。
■第三种方法是通过荚膜糖的半乳糖残基而不是唾液酸残基进行连接。该方法可避免破坏唾液酸残基所形成的关键表位。
因此,在第一方面,本发明提供制备无乳链球菌荚膜糖与运载体分子的偶联物的方法,包括以下步骤:(a)氧化无乳链球菌荚膜糖以在该糖的至少一个末端唾液酸残基中引入醛基;(b)用氨或伯胺还原胺化该醛基得到-CH2-连接的胺;(c)使该-CH2-连接的胺与双功能接头反应得到活化的糖;和(d)使该活化的糖与运载体分子反应,从而得到偶联物。本发明也提供无乳链球菌荚膜糖部分通过接头部分与运载体相连的偶联物,其中所述接头部分与荚膜糖部分中的唾液酸残基相连。
在第二方面,本发明提供制备无乳链球菌荚膜糖和运载体分子的偶联物的方法,包括以下步骤:(a)使荚膜糖脱-N-乙酰化得到脱-N-乙酰化的荚膜糖;(b)使该脱-N-乙酰化的荚膜糖与双功能接头反应得到活化的糖;(c)使该活化的糖与运载体分子反应,从而得到偶联物。该方法可在步骤(a)和(b)之间包括使糖部分再-N-乙酰化的步骤。
在第三方面,本发明提供制备荚膜糖和运载体分子的偶联物的方法,包括以下步骤:(a)氧化荚膜糖在该糖的至少一个半乳糖残基中引入醛基,得到修饰的半乳糖残基;和(b)将该修饰的半乳糖残基与运载体分子偶联。步骤(b)的偶联可以直接进行,或经接头分子进行。本发明也提供荚膜糖部分通过接头部分与运载体相连的偶联物,其中所述接头部分与该荚膜糖部分中的半乳糖残基相连。氧化半乳糖残基特别可用于偶联无乳链球菌荚膜糖,但也适用于具有含半乳糖的荚膜糖的其它细菌,例如脑膜炎奈瑟球菌(Neisseria meningitidis)(血清群W135)、霍乱弧菌(Vibrio cholerae)(包括0139)、肺炎克雷伯菌(Klebsiella pneumoniae)(包括K21)、大肠杆菌(Escherichia coli)(包括K52)、肺炎链球菌(Streptococcuspneumoniae)(包括18C型)等。该方法也可用于含半乳糖的脂多糖和脂寡糖。当半乳糖是糖的末端残基时,该方法特别有用。
荚膜糖
本发明以无乳链球菌的荚膜糖为基础。所述荚膜多糖与GBS的肽聚糖骨架共价相连,其不同于B群抗原,B群抗原是与肽聚糖骨架相连的另一种糖。
GBS的各荚膜多糖在化学上相关,但抗原性极为不同。所有GBS荚膜多糖共有以下三糖核心:β-D-GlcpNAc(1→3)β-D-Galp(1→4)β-D-Glcp。
各种GBS血清型的区别在于该核心的修饰。例如,该核心中是用GlcNAc(Ia)还是用Gal(III)来连接连续的三糖核心造成血清型Ia和III之间的差异(图4)。血清型Ia和Ib均具有与核心中GlcNAc相连的[α-D-NeupNAc(2→3)β-D-Gal(1→)二糖,但连接键是1→4(Ia)或1→3(Ib)。
GBS-相关疾病主要由血清型Ia、Ib、II、III、IV、V、VI、VII和VIII导致,其中超过90%的疾病由五种血清型:Ia、Ib、II、III和V导致。本发明宜利用这五种血清型之一的糖。如图3所示,这五种血清型各自的荚膜糖包含:(a)末端N-乙酰基-神经氨酸(NeuNAc)残基(通常称为唾液酸),在所有五种荚膜糖中该残基均是2→3连接于半乳糖残基;和(b)三糖核心内的N-乙酰基-葡糖胺残基(GlcNAc)。
所有五种糖在三糖核心中包含半乳糖残基,但血清型Ia、Ib、II和III在各自的重复单元中也包含其它半乳糖残基,血清型II糖中每个重复单元含有3个半乳糖残基。在本发明的第三方面,参与偶联反应的半乳糖残基可以是三糖核心中的残基或三糖核心外的残基。当一个糖分子与多个运载体分子相连时,连接键最好涉及各种相连重复单元中相同位置的半乳糖,但在不同重复单元中也可能与不同位置的半乳糖相连。
本发明所用的糖可以是其天然形式,或者可经修饰。例如,糖可以短于天然荚膜糖,或者可以化学修饰。
因此,本发明所用的糖可以是自然界中发现的基本上全长的荚膜多糖,或者可以短于该天然长度。可以解聚全长多糖从而得到本发明所用的较短片段,例如通过温和的酸水解,加热,分子大小排阻层析等。据报道,链的长度可影响GBS糖在家兔体内的免疫原性[4]。
已有报道说可用内切-β-半乳糖苷酶解聚血清型III荚膜糖[参考文献1和4-6],包括用解聚的物质与破伤风类毒素运载体形成偶联物。也用臭氧分解GBS血清型II、III和VIII的荚膜糖来解聚[12]。优选用分子量>30kDa的糖,可以使用基本上全长的荚膜糖。对于血清型Ia,优选用分子量高达约145kDa的多糖。对于血清型Ib,优选用分子量高达约50kDa的多糖。对于血清型III,优选用分子量高达约50kDa的多糖。可根据葡聚糖标准品(例如购自Polymer Standard Service的那些),用凝胶过滤测定这些糖的分子分子量[13]。
与自然界发现的荚膜糖相比,可化学修饰所述糖。例如,糖可以脱-O-乙酰化(部分或全部)、脱-N-乙酰化(部分或全部)、N-丙酸化(部分或全部)等。可以在偶联之前、期间或之后进行脱-乙酰化,但优选在偶联前进行。根据具体的糖,脱-乙酰化可以影响或不影响免疫原性,例如NeisVac-CTM疫苗用脱-O-乙酰化的糖,而MenjugateTM是乙酰化的,但二者均有效。参考文献14讨论了各种血清型的GBS糖上O-乙酰化的相关性,优选能在偶联之前、期间或之后保留7、8和/或9位唾液酸残基的O-乙酰化,例如通过保护/脱保护,再乙酰化等。可用常规试验评估脱-乙酰化等的作用。
如本文参考文献所述,可通过已知技术纯化荚膜糖。常规方法包括碱提取、离心、过滤、RNA酶/DNA酶处理、蛋白酶处理、浓缩、分子大小排阻层析、超滤、阴离子交换层析和进一步超滤。也可用酶变溶菌素处理GBS细胞,该酶切割细菌细胞壁以释放细胞壁组分。
或者,可用参考文献15所述的纯化方法。该方法包括碱提取、乙醇/CaCl2处理、CTAB沉淀和再溶解。
然而,本发明不限于从天然来源纯化的糖,可以通过其它方法,例如全合成或部分合成获得这些糖。
运载体
本发明包括利用通常是蛋白质的运载体分子。共价偶联糖与运载体通常能增强糖的免疫原性,因为将糖从不依赖T的抗原转化为依赖T的抗原,从而能引发免疫记忆。偶联对儿科疫苗特别有用[例如,参考文献16]并且是熟知的技术[例如,参考文献17-25的综述]。
优选的运载体蛋白是细菌毒素或类毒素,例如白喉类毒素或破伤风类毒素。特别优选白喉毒素的CRM197突变体[26-28],因为它是一种白喉类毒素。其它合适的运载体蛋白包括脑膜炎奈瑟球菌(N.meningitidis)外膜蛋白[29]、合成肽[30,31]、热激蛋白[32,33]、百日咳蛋白[34,35]、细胞因子[36]、淋巴因子[36]、激素[36]、生长因子[36]、人血清白蛋白(优选重组体)、含各种病原体衍生抗原的多种人CD4+T细胞表位的人工蛋白[37](例如N19[38])、流感嗜血杆菌的D蛋白[39,40]、肺炎球菌表面蛋白PspA[41]、肺炎球菌溶菌素[42]、离子摄取蛋白[43]、艰难梭菌(C.difficile)的毒素A或B[44]、GBS蛋白(参见下文;特别是GBS67)[195]等。
优选通过-NH2基团(例如运载体蛋白的赖氨酸残基或精氨酸残基侧链中的氨基)与运载体连接。当糖具有游离醛基时,其能通过还原胺化与运载体的胺基反应形成偶联物。本发明的第三方面以还原胺化为基础,涉及糖中已氧化的半乳糖(从中可形成醛)和运载体或接头中的胺基。也可以通过-SH(例如半胱氨酸侧链中的巯基)连接。
可利用多种运载体蛋白来,例如降低运载体抑制的风险。因此,不同GBS血清型可用不同运载体蛋白,例如血清型Ia糖可与CRM197偶联,而血清型Ib糖可与破伤风类毒素偶联。具体的糖抗原也可用多种运载体蛋白,例如血清型III糖可以分成两组,其中一些与CRM197偶联,其它与破伤风类毒素偶联。然而,所有糖通常优选利用相同的运载体蛋白。
一种运载体蛋白可携带多种糖抗原[45,46]。例如,一种运载体蛋白可与血清型Ia和Ib的糖偶联。为实现此目标,可先混合不同的糖再进行偶联反应。然而,一般优选各血清型有不同偶联物,先偶联再混合不同的糖。不同偶联物的运载体可相同。
优选糖∶蛋白质之比(w/w)在1∶5(即,过量的蛋白)和5∶1(即,过量的糖)之间的偶联物。优选1∶2和5∶1之间的比例,例如1∶1.25和1∶2.5之间的比例。也优选1∶1和4∶1之间的比例。糖链较长,则糖的重量通常过量。如实施例所示,不难实现1∶1和4∶1之间的重量比,特别是1∶1和3∶1(之间)的重量比。总体上,本发明提供的偶联物包含的无乳链球菌荚膜糖部分与运载体相连,其中糖:运载体的重量比是至少2∶1。
组合物可包含少量游离运载体[47]。当本发明组合物中给定的运载体蛋白同时存在游离和偶联形式时,未偶联形式优选不超过组合物中所有运载体蛋白总量的5%,更优选低于2%(以重量计)。
偶联后,可以分离游离和偶联的糖。合适的方法有许多,包括疏水层析、正切超滤、渗滤等。[也见参考文献48和49等]。
当本发明组合物包含解聚的寡糖时,优选先解聚再偶联。
引入醛基
本发明第一方面包括氧化唾液酸形成醛,第三方面包括氧化半乳糖形成醛。然后可将醛用于,例如还原胺化的反应。
可通过化学或酶方法氧化羟基得到醛基。这些反应通常在水性条件下进行。
本领域已知氧化GBS糖中的唾液酸来引入醛基的方法[例如,参考文献50]。在唾液酸中产生醛基的常规反应包括用过碘酸盐,特别是偏过碘酸盐(例如,偏过碘酸钠或钾,如NaIO4)氧化邻位羟基[10]。至少血清群II[3,50]、III[2]和V[50]的过碘酸盐氧化已有报道。可用其它氧化条件,例如用四氧化锇等。
在本发明的第三方面,被氧化的-OH优选是与C-6相连的伯-OH(即,不是仲或端基异构-OH基团)。因此,优选将半乳糖转变成半乳己二醛糖(galactohexodialose)。这可用任何合适来源(例如,链孢菌(Fusarium)真菌或树状指孢霉(Dactylium dendroides))的半乳糖氧化酶方便地实现。可利用重组形式的酶,或从其天然来源纯化。半乳糖氧化酶的EC编号1.1.3.9,也称为D-半乳糖:氧6-氧化还原酶。该酶用铜离子辅因子,而氰化物、二乙基二硫代氨基甲酸酯、叠氮化物和羟胺可抑制它,因此,氧化前最好避免使用这些试剂。树状指孢霉的最佳pH约为中性,因此这是氧化的优选pH。该酶反应的产物是H2O2(还原的氧),可以控制该产物的浓度,例如,如果其存在破坏了糖。
因此,对于唾液酸和半乳糖,优选的氧化反应涉及单糖中的末端碳原子,即按照标准命名编号最高的碳。
可采用各种反应条件控制糖链中要转变为醛基的单糖亚单位比例。例如,参考文献50报道通过气相色谱-质谱分析检测到受控过碘酸盐氧化血清型II GBS多糖修饰的唾液酸残基为7%,而在血清型V GBS多糖中观察到更高百分比。参考文献2报道血清型III转变了25%。可初步研究反应条件(例如,时间、温度、浓度等)以找到获得任何所需结果的最佳条件。
总之,通常在糖的5%和50%之间(例如,10-40%,优选15%-30%之间;或5%-20%之间)的所有唾液酸或半乳糖单糖单元中引入醛基。更高的百分比造成糖难以控制,而且免疫原性也不会有任何相应提高。
还原胺化
在本发明的第一方面,采用还原胺化新醛基得到与接头相连的基团。在本发明的第三方面,也可在产生醛基后用还原胺化来连接接头或直接连接运载体。还原胺化是有机化学的标准技术,已广泛应用于生产疫苗用的荚膜糖偶联物。
在第一方面,还原胺化涉及(利用)氨基或伯胺(NH2R)。联用铵盐(例如,氯化铵)和合适的还原剂(例如,氰基硼氢化物,例如氰基硼氢化钠NaBH3CN;硼烷-吡啶;三乙酰氧基硼氢化钠;硼氢化物交换树脂;等等)不难实现还原胺化。还原胺化的结果是唾液酸中的C-8携带-NHR而不是=O。然后可利用该基团与偶联用的双功能接头相连。
在第三方面,氧化的半乳糖在C-6上有一个醛基。该基团以上述相同方式(即,涉及氨基或伯胺)通过还原胺化与双功能接头偶联。或者,可不用接头而是利用运载体上的胺基,采用还原胺化直接连接该醛基与运载体。
还原胺化通常在极性质子溶剂,例如水或醇中进行。
双功能接头
本发明的第一和第二方面(及任选的第三方面)包括利用双功能接头。双功能接头可提供与修饰的荚膜糖中胺基偶联的第一基团以及与运载体偶联的第二基团(通常与运载体中的胺基偶联)。
因此,双功能接头中的第一基团能与修饰的唾液酸(或半乳糖)残基上的胺基(-NHR)反应。该反应通常包括亲电取代该胺基中的氢。双功能接头中的第二基团能与运载体上的胺基反应。该反应通常也包括亲电取代该胺基中的氢。本发明可用异双功能接头和同双功能接头。
当与糖和运载体二者的反应涉及胺时,优选用式X-L-X所示的双功能接头,其中:两个X基团彼此相同,可与所述胺反应;L是接头中的连接部分。优选的X基团是N-氧基琥珀酰亚胺(N-oxysuccinimide)。L优选式-L1-L2-L1-所示,其中L1是羰基。优选的L2基团是1-10个碳原子的直链烷基(例如,C1、C2、C3、C4、C5、C6、C7、C8、C9、C10),例如-(CH2)4-。因此,优选的接头是己二酸N-羟基琥珀酰亚胺二酯(SIDEA):
其它X基团是能与HO-L-OH结合形成酯的那些基团,例如正硼烷、对硝基苯甲酸和磺基-N-羟基琥珀酰亚胺。
本发明所用能与胺反应的其它双功能接头包括丙烯酰卤(例如,丙烯酰氯)[54]、卤酰基卤(haloacylhalide)[55]、戊二酸二琥珀酰亚胺酯(disuccinimidylglutarate)、辛二酸二琥珀酰亚胺酯(disuccinimidyl suberate)、乙二醇二[琥珀酸琥珀酰亚胺酯]等。
通常加入过量的接头(以摩尔计)来修饰糖。
接头/糖反应通常在质子惰性溶剂(例如,DMSO、乙酸乙醇酯等)中进行,因为这种接头通常不溶于水。然而,当使用水溶性接头时,可用的溶剂范围更广,包括质子溶剂,例如水。合适的溶剂包括磺化形式,例如磺化SIDEA:
脱-N-乙酰化和再-N-乙酰化
如三糖核心中的葡糖胺残基一样,GBS荚膜糖中的唾液酸残基被N-乙酰化。虽然本发明第一方面通过醛基中间体在唾液酸的C-8引入了胺基,本发明第二方面利用唾液酸和/或N-乙酰基-葡糖胺残基脱-N-乙酰化产生的胺基。以此方法产生胺的反应方案通常与本发明第一方面所述的一样。
用碱处理糖不难实现GBS糖的脱-N-乙酰化。由于可采用包括碱提取的方法纯化GBS糖[51],则脱-N-乙酰化可以是纯化期间的副反应。
由于N-乙酰化可能是GBS糖重要表位的一部分,不希望完全脱-N-乙酰化,但该过程难以控制。因此,如果脱-N-乙酰化的程度大于所希望的,本发明可包括受控再-N-乙酰化的步骤。可用例如5%碳酸氢铵配制的乙酸酐(CH3CO)2O试剂方便地再-N-乙酰化[52]。然而,本发明人发现如果进行得足够快并且糖的保存时期不长的话,用碱从细菌中提取糖能得到脱-N-乙酰化低于25%的糖,而无需再-N-乙酰化。
脱-N-乙酰化和任选的再-N-乙酰化得到的糖中至少有1个唾液酸残基或葡糖胺脱-N-乙酰化。GBS糖中通常至少有60%,例如>70%、>75%、>80%、>85%、>90%等的唾液酸和葡糖胺残基是N-乙酰化的。其余脱-N-乙酰化的基团(即,-NH2基团)可通过与本发明第一方面所述相同的方法进行偶联,除了最终偶联物中的-NH-源自初始的糖而不是在偶联反应期间加入的。
可在水性条件下进行这些脱-乙酰化和再-乙酰化反应。
在还原胺化醛基的本发明第一和第三方面的实施方式中,优选先将糖基本上完全再-N-乙酰化,然后还原胺化(优选在第三方面的氧化半乳糖之前),从而能避免唾液酸上存在游离胺基,否则会让其它基团连接胺化的醛基。
偶联物
在合适的反应条件下混合修饰的GBS糖与运载体来制备本发明偶联物。如图5所示,总体上可制备两种类型的偶联物:(a)一种糖与一种运载体相连(例如通过其还原性末端)的偶联物;和(b)一种糖与多种运载体相连的偶联物,例如由于几种单糖亚单位均具有反应性。在两种情况中,一个运载体蛋白可与多个糖分子相连,因为运载体具有多个暴露的赖氨酸侧链。
(b)型偶联物在本发明中更常见,因为本发明的修饰的唾液酸或半乳糖残基出现在一个糖(分子)的多个位点[50]。因此,在本发明优选的偶联物中,一个糖分子平均与多个运载体分子偶联。
在本发明的第一种和第三种方法中,当利用氧化的糖偶联时,与糖相连的运载体分子数目取决于所存在的游离醛基数目。如上所述,优选氧化糖中5-50%的唾液酸(第一种方法)或半乳糖(第三种方法)残基。
在本发明的第一和第二方面(及任选的第三方面),偶联物包含接头部分。该接头部分既不源自糖也不源自运载体,而是在偶联物制备期间所用的第三种分子,不难与最终偶联产物中的糖和运载体蛋白相区别。
接头部分包含例如碳、氢、氧和/或氮的原子。优选含有碳和氢的接头,也优选还含有氧和/或氮的接头。包含氮原子的接头可以包含与氮原子键合的碳原子,该氮原子进而可以与第二个碳原子键合(-C-N-C-)。包含氧原子的接头优选该氧原子是羰基的一部分。优选分子量在30-500Da之间的接头部分。优选含有两个羰基的接头。
特别优选的接头部分是-NH-C(O)-(CH2)n-C(O)-,其中n是1、2、3、4、5、6、7、8、9或10。n值优选4。该接头的末端-NH-优选与糖部分的碳原子相连。末端-C(O)-优选与运载体中氨基酸侧链的氮原子相连。通过以下方法不难引入优选的接头部分,包括:还原胺化已氧化的唾液酸中的醛基;得到的-NH2基团与双功能接头(二元酸,例如己二酸,HOOC-(CH2)4-COOH的二酯,例如二琥珀酰亚胺酯)反应;和还原胺化该产物(参见图6[53])。
其它可用于连接接头与糖中已引入的末端-NH2(例如本发明第一方面)或作为脱-N-乙酰化单糖残基一部分(例如本发明第二方面)的末端-NH2的化学方法包括:
-丙烯酰化(例如与丙烯酰氯反应),然后与氨基酸侧链的ε-NH2或半胱氨酸侧链的-SH进行迈克尔型加成反应。如图8所示,得到的接头是-NH-C(O)-(CH2)2-(丙酰胺基),或者如果已有的-NH2参与反应,得到-C(O)-(CH2)2-。
-与卤酰基卤反应,然后与与氨基酸侧链的ε-NH2或半胱氨酸侧链的-SH反应[55]。根据是已有的还是加入的-NH2参与反应,接头是-NH-C(O)-CH2-(如图9所示)或-C(O)-CH2-。
另一优选的接头部分是-C(O)-(CH2)n-C(O)-,其中n是1、2、3、4、5、6、7、8、9或10。n值优选4。该接头一端的-C(O)-优选与糖部分的氮原子相连,另一端-C(O)-优选与运载体中氨基酸侧链的氮原子相连。通过以下方法不难引入优选的接头部分,包括:脱-N-乙酰化单糖亚单元中的-NH2基团与双功能接头(二元酸,例如,己二酸,HOOC-(CH2)4-COOH的二酯,例如,二琥珀酰亚胺酯)反应;和还原胺化该产物(图7)。其它选择包括通过糖的羟基偶联。可先活化(例如,通过CNBr或CDAP)羟基,再偶联。
偶联物与其它抗原的组合
除了提供上述各偶联物,本发明还提供包含本发明偶联物和一种或多种其它抗原的组合物。
其它抗原包括本发明其它偶联物,因此,本发明提供的组合物含有多种本发明偶联物。所述其它抗原可以是通过除本发明那些方法以外的方法制备的GBS糖偶联物,因此本发明提供的组合物包含第一种GBS糖偶联物和第二种GBS糖偶联物,其中所述第一种偶联物是本发明偶联物,所述第二种偶联物不是本发明偶联物。
不同的GBS偶联物可包括同一GBS血清型的不同类型偶联物和/或不同GBS血清型的偶联物。例如,本发明提供的组合物包含血清型Ia、Ib和III中的两种或三种偶联物。可通过先制备不同偶联物(例如,各血清型的不同偶联物),再混合这些偶联物来产生组合物。
如下所述,其它抗原可包含GBS的氨基酸序列。
其它抗原可包括非GBS病原体的抗原。因此,本发明组合物还可包含一种或多种非-GBS抗原,包括其它细菌、病毒或寄生虫抗原。这些抗原可选自以下抗原:
-脑膜炎奈瑟球菌(N.meningitidis)血清群B的蛋白质抗原,例如参考文献56-62所述的那些,其中特别优选蛋白质‘287’(见下文)和衍生物(例如,‘ΔG287’)。
-脑膜炎奈瑟球菌血清群B的外膜囊泡制品(OMV),例如参考文献63、64、65、66等公开的。
-脑膜炎奈瑟球菌血清群A、C、W135和/或Y的糖抗原,如参考文献67公开的血清群C的寡糖或参考文献68的寡糖。
-肺炎链球菌(Streptococcus pneumoniae)的糖抗原[例如,参考文献69-71;参考文献78的第22和23章]。
-甲肝病毒抗原,如灭活病毒[例如,72、73;参考文献78的第15章]。
-乙肝病毒抗原,例如表面和/或核心抗原[例如,73、74;参考文献78的第16章]。
-丙肝病毒抗原[例如,75]。
-百日咳博德特菌(Bordetella pertussis)抗原,例如百日咳博德特菌的百日咳全毒素(PT)和丝状血凝素(FHA),也可任选与百日咳杆菌黏附素(pertactin)和/或凝集原2和3联用[例如,参考文献76和77;参考文献78的第21章]。
-白喉抗原,如白喉类毒素[例如,参考文献78的第13章]。
-破伤风抗原,如破伤风类毒素[例如,参考文献78的第27章]。
-B型流感嗜血杆菌(Haemophilus influenzae B)的糖抗原[例如,参考文献78的第14章]。
-淋病奈瑟菌(N.gonorrhoeae)的抗原[例如,56、57、58]。
-肺炎衣原体(Chlamydia pneumoniae)的抗原[例如,79、80、81、82、83、84、85]。
-砂眼衣原体(Chlamydia trachomatis)的抗原[例如,86]。
-牙龈卟啉单胞菌(Porphyromonas gingivalis)的抗原[例如,87]。
-脊髓灰质炎抗原[例如,88、89;参考文献78的第24章],如IPV。
-狂犬病抗原[例如,90],如冻干的灭活病毒[例如,91,RabAvertTM]。
-麻疹、流行性腮腺炎和/或风疹抗原[例如,参考文献78的第19、20和26章]。
-流感抗原[例如,参考文献78的第17和18章],如血球凝集素和/或神经氨酸酶表面蛋白。
-粘膜炎莫拉菌(Moraxella catarrhalis)抗原[例如,92]。
-酿脓链球菌(Streptococcus pyogenes)(A型链球菌)抗原[例如,93、94、95]。
-金黄色葡萄球菌(Staphylococcus aureus)的抗原[例如,96]。
利用糖或碳水化合物抗原时,优选与运载体偶联以增强免疫原性。熟知乙型流感嗜血杆菌、脑膜炎球菌和肺炎球菌糖抗原的偶联物。
可视需要使有毒的蛋白抗原解毒(例如通过化学和/或遗传方法使百日咳毒素解毒[77])。
组合物包含白喉抗原时,优选也包含破伤风抗原和百日咳抗原。类似地,组合物包含破伤风抗原时,优选也包含白喉和百日咳抗原。类似地,组合物包含百日咳抗原时,优选也包含白喉和破伤风抗原。
可将抗原吸附到铝盐上。
一种类型的优选组合物包含性传播病原体,例如疱疹病毒、淋病奈瑟菌、乳头瘤病毒、砂眼衣原体等其它抗原。另一种类型的优选组合物包含能影响老年人和/或免疫力受损人士的其它抗原,因此,本发明GBS抗原可与以下非GBS病原体的一种或多种抗原混合:流感病毒、粪肠球菌(Enterococcus faecalis)、金黄色葡萄球菌、表皮葡萄球菌(Staphylococcus epidermis)、铜绿假单胞菌(Pseudomonas aeruginosa)、侵肺军团菌(Legionella pneumophila)、单核细胞增生利斯特菌(Listeria monocytogenes)、脑膜炎奈瑟球菌(Neisseria meningitidis)和副流感病毒。
组合物中各抗原的浓度通常是至少1μg/ml。任何给定抗原的浓度通常足以引发针对该抗原的免疫应答。
或者,可用编码抗原的核酸作为本发明组合物所用的蛋白抗原[例如,参考文献97-105]。因此,可用编码该蛋白质的核酸(优选DNA,例如质粒形式)替代本发明组合物的蛋白质组分。
实际上,本发明组合物中抗原的种类数有上限。本发明组合物中抗原(包括GBS抗原)种类数可小于20、小于19、小于18、小于17、小于16、小于15、小于14、小于13、小于12、小于11、小于10、小于9、小于8、小于7、小于6、小于5、小于4或小于3。本发明组合物中GBS抗原种类数可小于6、小于5或小于4。
药物组合物和方法
本发明提供的药物组合物包含:(a)本发明偶联物和(b)药学上可接受的运载体。“药学上可接受的运载体”通常包括本身不诱导产生对接受组合物的个体有害的抗体的任何运载体。合适的运载体一般是大的、代谢缓慢的大分子,例如蛋白质、多糖、多聚乳酸、聚乙醇酸、聚氨基酸、氨基酸共聚物、蔗糖[106]、海藻糖[107]、乳糖和脂质聚集体(例如油滴或脂质体)。本领域普通技术人员熟知这种运载体。疫苗也可含有稀释剂,例如水、盐水、甘油等。此外,可存在辅助物质,例如润湿剂或乳化剂、pH缓冲物质等。灭菌无热原的磷酸盐缓冲生理盐水是典型的运载体。药学上可接受的赋形剂的详尽讨论见参考文献108。
本发明组合物可以是水性形式(即,溶液或混悬液)或干燥形式(例如,冻干的)。如果要用冻干疫苗,则可先用液体介质重建再注射。冻干偶联物疫苗是本领域已知的,例如MenjugateTM产品以冻干形式提供,而NeisVac-CTM和MeningitecTM以水性形式提供。为在冻干期间稳定偶联物,优选在组合物中加入,例如1mg/ml-30mg/ml(如,约25mg/ml)之间的糖醇(例如,甘露醇)或二糖(例如,蔗糖或海藻糖)。
组合物可装在小瓶中,或者可装在预填充的注射器中。注射器可以配或不配针头。注射器含有单剂量的组合物,而小瓶可含有单剂量或多剂量。
本发明水性组合物也适用于重建冻干形式的其它疫苗。本发明组合物用于这种临时重建时,本发明提供一种装有两个小瓶或者装有一个预填充注射器和一个小瓶的试剂盒,先用注射器的内含物活化药瓶的内含物再注射。
本发明组合物可包装成单位剂型或多剂剂型。对于多剂剂型,小瓶优于预填充注射器。可常规设定有效剂量体积,但该组合物的典型人用剂量体积是0.5ml,例如肌肉内注射时。
所述组合物的pH优选6和8之间,更优选约7。可用缓冲液维持稳定的pH。如果组合物含有氢氧化铝盐,则优选使用组氨酸缓冲液[109]。组合物可以是灭菌和/或无热原的。本发明组合物对于人体应是等渗的。
本发明组合物是免疫原性组合物,更优选疫苗组合物。本发明疫苗可以是预防性(即为防止感染)或治疗性(即,为治疗感染)的,但通常是预防性的。用作疫苗的免疫原性组合物包含免疫学有效量的抗原以及所需的任何其它组分。“免疫学有效量”指给予个体能有效治疗或预防的用量(单一剂量或作为系列剂量的一部分)。该用量取决于所治疗个体的健康和身体状况、年龄、待治疗个体的分类学分组(例如非人灵长类、灵长类等)、个体免疫系统合成抗体的能力、所需的保护程度、疫苗制剂、主治医生对医疗状况的评估以及其它相关因素而不同。估计该用量应处于可通过常规试验确定的相对较宽范围内。
在各剂量中,各糖抗原的含量通常在1-50μg之间(以糖的质量检测),例如约1μg、约2.5μg、约4μg、约5μg或约10μg。
GBS影响身体的各部位,因此本发明组合物可以制备成各种形式。例如,这些组合物可以制备成可注射的(液体溶液或混悬液)。对于肺部给药,该组合物可以制备成例如使用细粉或喷雾的吸入剂。该组合物可以制备成栓剂或阴道栓剂。对于鼻、耳或眼睛给药,该组合物可以制备成,例如喷雾剂、滴剂、凝胶或粉末[例如,参考文献110和111]。已有报道说肺炎球菌糖[112,113]、Hib糖[114]、MenC糖[115]和Hib与MenC糖偶联物的混合物[116]的鼻部给药获得成功。
本发明组合物可包含抗微生物剂,特别是当包装成多剂量形式时。
本发明组合物可含有洗涤剂,例如吐温(聚山梨醇酯),如吐温80。洗涤剂通常处于低水平,例如<0.01%。
本发明组合物可含有钠盐(例如,氯化钠)以得到一定张力。NaCl的浓度通常为10±2mg/ml。
本发明组合物通常包含缓冲液。典型的缓冲液是磷酸盐缓冲液。
本发明组合物通常与其它免疫调节剂联合给予。具体地说,组合物通常含有一种或多种佐剂。这种佐剂包括但不限于:
A.含有矿物质的组合物
适合用作本发明佐剂的含矿物质组合物包括矿物盐,例如铝盐和钙盐。本发明包括矿物盐,例如氢氧化物(如过氧化物(oxyhydroxide))、磷酸盐(如羟基磷酸盐、正磷酸盐)、硫酸盐等[如参见参考文献117的第8和9章],或不同矿物质化合物的混合物(如磷酸盐和氢氧化物佐剂的混合物,任选磷酸盐过量),这些化合物可采取任何合适的形式(如凝胶、晶体、无定形等),优选(具有)吸附性。含矿物质的组合物也可配制成金属盐颗粒[118]。
本发明疫苗中可包含铝盐,Al3+的剂量在每剂0.2-1.0mg之间。
典型的磷酸铝佐剂是PO4/Al摩尔比在0.84和0.92之间的无定形羟基磷酸铝,包含0.6mg Al3+/ml。可用低剂量磷酸铝吸附,例如每剂每种偶联物50-100μg Al3+。当使用磷酸铝时,由于溶液中含有游离的磷酸根离子(例如,用磷酸盐缓冲液),无需将抗原吸附到佐剂上。
B.油乳剂
适合用作本发明佐剂的油乳剂组合物包括角鲨烯-水乳剂,例如MF59(5%角鲨烯、0.5%吐温80和0.5%司盘85,用微流化仪配制成亚微米颗粒)[参考文献17的第10章;也可参见参考文献119-121]。MF59用作FLUADTM流感病毒三价亚单位疫苗的佐剂。
特别优选用于组合物的佐剂是亚微米水包油乳剂。本文所用优选的亚微米水包油乳剂是任选含有不同含量的MTP-PE的角鲨烯/水乳剂,例如含4-5%w/v角鲨烯、0.25-1.0%w/v吐温80(聚氧乙烯山梨糖醇酐单油酸酯)和/或0.25-1.0%司盘85(失水山梨醇三油酸酯)和任选的N-乙酰基胞壁酰-L-丙氨酰基-D-异谷氨酰胺基-L-丙氨酸-2-(1′-2′-二棕榈酰基-sn-甘油基-3-羟基磷酰氧基)-乙胺(MTP-PE)的亚微米水包油乳剂。参考文献119和122-123详细描述了用于组合物中的亚微米水包油乳剂、其制备方法和免疫刺激剂,例如胞壁酰肽。完全弗氏佐剂(CFA)和不完全弗氏佐剂(IFA)也可用作本发明佐剂。
C.皂苷制剂[参考文献117的第22章]
皂苷制剂也可用作本发明的佐剂。皂苷是在许多植物种类的树皮、叶、茎、根、甚至花中发现的固醇糖苷和三萜系糖苷的异质混合物(heterologous group)。从皂树(Quillaia saponaria Molina)树皮中分离的皂苷是已广泛研究的佐剂。皂苷也可从丽花菝葜(Smilax ornata)(墨西哥菝葜(sarsaprilla))、锥花丝石竹(Gypsophilla paniculata)(婚纱花(brides veil))和肥皂草(Saponaria officianalis)(皂根(soap root))获得。皂苷佐剂制剂包括纯化的制剂,例如QS21,以及脂质制剂,例如ISCOM。
也可利用HPLC和RP-HPLC纯化皂苷组合物。已经鉴定了用这些技术专门纯化的各组分,包括QS7、QS17、QS18、QS21、QH-A、QH-B和QH-C。皂苷优选QS21。参考文献124公开了QS21的制备方法。皂苷制剂也可含有固醇,例如胆固醇[125]。
可联用皂苷和胆固醇来形成称为免疫刺激复合物(ISCOM)的独特颗粒[参考文献117的第23章]。ISCOM一般也含有磷脂,例如磷脂酰乙醇胺或磷脂酰胆碱。任何已知的皂苷可用在ISCOM中。ISCOM优选含有一种或多种QuilA、QHA和QHC。参考文献125-127进一步描述了ISCOM。ISCOM任选不含其它洗涤剂[128]。
皂苷佐剂开发的综述见参考文献129和130。
D.病毒体和病毒样颗粒
病毒体和病毒样颗粒(VLP)也可用作本发明的佐剂。这些结构通常含有任选与磷脂混合或用磷脂配制的一种或多种病毒蛋白。它们通常无病原性、无复制性,通常不含任何天然病毒基因组。可重组产生或从完整病毒分离病毒蛋白。适用于病毒体或VLP中的这些病毒蛋白包括源自以下病毒的蛋白质:流感病毒(例如HA或NA)、乙肝病毒(例如核心或衣壳蛋白)、戊肝病毒、麻疹病毒、辛德毕斯病毒、轮状病毒、口蹄疫病毒、逆转录病毒、诺瓦克病毒、人乳头瘤病毒、HIV、RNA-噬菌体、Qβ-噬菌体(例如外壳蛋白)、GA-噬菌体、fr-噬菌体、AP205噬菌体和Ty(例如逆转录转座子Ty蛋白p1)。参考文献131-136进一步讨论了VLP。例如,参考文献137进一步讨论了病毒体。
E.细菌或微生物衍生物
适用于本发明的佐剂包括细菌或微生物衍生物,例如肠细菌脂多糖(LPS)的无毒衍生物、脂质A衍生物、免疫刺激性寡核苷酸和ADP-核糖基化毒素及它们的解毒衍生物。LPS的无毒衍生物包括单磷酰基脂质A(MPL)和3-O-脱酰基MPL(3dMPL)。3dMPL是含有4、5或6条酰化链的3脱-O-酰化单磷酰基脂质A的混合物。参考文献138公开了3脱-O-酰化单磷酰基脂质A的优选“小颗粒”形式。这种“小颗粒”的3dMPL足够小从而可经0.22μm膜除菌过滤[138]。其它无毒的LPS衍生物包括单磷酰基脂质A模拟物,例如氨基烷基氨基葡糖苷磷酸酯衍生物,如RC-529[139、140]。
脂质A衍生物包括大肠杆菌的脂质A衍生物,例如OM-174。例如,参考文献141和142描述了OM-174。
适合用作本发明佐剂的免疫刺激性寡核苷酸包括含有CpG基序(含有通过磷酸键与鸟苷相连的未甲基化胞嘧啶的二核苷酸序列)的核苷酸序列。含有回文或聚(dG)序列的双链RNA和寡核苷酸也显示有免疫刺激性。
CpG可含有核苷酸修饰/类似物,例如硫代磷酸酯修饰,可以是双链或单链的。参考文献143、144和145公开了可能的类似物取代,例如用2′-脱氧-7-脱氮鸟苷取代鸟苷。参考文献146-151进一步讨论了CpG寡核苷酸的佐剂效力。
CpG序列可涉及TLR9,例如基序GTCGTT或TTCGTT[152]。CpG序列,例如CpG-A ODN可特异性诱导Th1免疫应答,或者,例如CpG-B ODN能更特异性诱导B细胞应答。参考文献153-155讨论了CpG-A和CpG-B ODN。CpG优选CpG-A ODN。
优选将CpG寡核苷酸构建为5’端易于为受体识别。任选将两条CpG寡核苷酸序列在它们的3’端相连以形成“免疫聚物”(immunomer)。参见,例如参考文献152和156-158。
细菌ADP-核糖基化毒素及其解毒衍生物可用作本发明佐剂。蛋白质优选源自大肠杆菌(大肠杆菌热不稳定肠毒素“LT”)、霍乱(弧菌)(“CT”)或百日咳(杆菌)(“PT”)。参考文献159描述了解毒ADP-核糖基化毒素作为粘膜佐剂的应用,参考文献160描述了其作为胃肠外佐剂的应用。该毒素或类毒素优选含有A和B亚基的全毒素形式。A亚基优选含有解毒性突变;B亚基优选不突变。该佐剂优选解毒的LT突变体,例如LT-K63、LT-R72和LT-G192。ADP-核糖基化毒素及其解毒衍生物,特别是LT-K63和LT-R72作为佐剂的应用见参考文献161-168。氨基酸取代的数字基准优选以参考文献169所述的ADP-核糖基化毒素的A和B亚基比对为基础,该文献专门全文纳入本文作为参考。
F.人免疫调节剂
适合用作本发明佐剂的人免疫调节剂包括细胞因子,例如白介素(如,IL-1、IL-2、IL-4、IL-5、IL-6、IL-7、IL-12[170]等)[171]、干扰素(如,干扰素-γ)、巨噬细胞集落刺激因子和肿瘤坏死因子。
G.生物黏合剂和粘膜黏合剂
生物黏合剂(bioadhesive)和粘膜黏合剂(mucoadhesive)也可用作本发明佐剂。合适的生物黏合剂包括酯化的透明质酸微球[172],或者粘膜黏合剂,例如聚(丙烯酸)的交联衍生物、聚乙烯醇、聚乙烯吡咯烷酮、多糖和羧甲基纤维素。壳多糖及其衍生物也可用作本发明佐剂[173]。
H.微粒
微粒也可用作本发明佐剂。可从生物可降解和无毒的材料(例如,聚(α-羟酸)、聚羟基丁酸、聚原酸酯、聚酐、聚己酸内酯等),优选聚(丙交酯-共-乙交酯)制备微粒(即,直径约100nm到150μm,优选约200nm到约30μm,最优选约500nm到约10μm的颗粒),任选将这些微粒处理成带负电的表面(例如,用SDS)或带正电的表面(例如,用阳离子洗涤剂,如CTAB)。
I.脂质体(参考文献117的第13和14章)
参考文献174-176描述了适合用作佐剂的脂质体制剂的例子。
J.聚氧乙烯醚或聚氧乙烯酯制剂
适用于本发明的佐剂包括聚氧乙烯醚和聚氧乙烯酯[177]。这种制剂还包括与辛苯聚醇联用的聚氧乙烯山梨糖醇酯表面活性剂[178]以及与至少一种其它非离子表面活性剂,例如辛苯聚醇利用的聚氧乙烯烷基醚或酯表面活性剂[179]。优选的聚氧乙烯醚选自:聚氧乙烯-9-月桂基醚(laureth 9)、聚氧乙烯-9-硬脂酰基(steoryl)醚、聚氧乙烯-8-硬脂酰基(steoryl)醚、聚氧乙烯-4-月桂基醚、聚氧乙烯-35-月桂基醚和聚氧乙烯-23-月桂基醚。
K.聚磷腈(PCPP)
PCPP制剂描述于,例如参考文献180和181中。
L.胞壁酰肽
适合用作本发明佐剂的胞壁酰肽的例子包括N-乙酰基-胞壁酰-L-苏氨酰基-D-异谷氨酰胺(thr-MDP)、N-乙酰基-去甲胞壁酰(normuramyl)-L-丙氨酰基-D-异谷氨酰胺(nor-MDP)和N-乙酰基胞壁酰-L-丙氨酰基-D-异谷氨酰胺-L-丙氨酸-2-(1′-2′-二棕榈酰基-sn-甘油基-3-羟基磷酰氧基)-乙胺(MTP-PE)。
M.咪唑并喹诺酮化合物
适合用作本发明佐剂的咪唑并喹诺酮化合物包括参考文献182和183中进一步描述的咪喹莫特(Imiquamod)及其同系物(例如,“瑞喹莫德3M”)。
N.缩氨基硫脲化合物
缩氨基硫脲化合物的例子以及配制、制备和筛选所有适合用作本发明佐剂的这类化合物的方法包括参考文献184所述的那些。缩氨基硫脲能特别有效地刺激人外周血单核的细胞产生细胞因子,例如TNF-α。
O.色胺酮化合物
色胺酮化合物的例子以及配制、制备和筛选所有适合用作本发明佐剂的这类化合物的方法包括参考文献185所述的那些。色胺酮化合物能特别有效地刺激人外周血单核的细胞产生细胞因子,例如TNF-α。
本发明各方面也包括联用一种或多种以上鉴定的佐剂。例如,以下组合物可用作本发明佐剂组合物:(1)皂苷和水包油乳剂[186];(2)皂苷(例如QS21)+无毒的LPS衍生物(例如3dMPL)[187];(3)皂苷(例如QS21)+无毒的LPS衍生物(例如3dMPL)+胆固醇;(4)皂苷(例如QS21)+3dMPL+IL-12(任选+固醇)[188];(5)联用3dMPL与例如QS21和/或水包油乳剂[189];(6)含10%角鲨烯、0.4%吐温80TM、5%普朗尼克嵌段聚合物L121和thr-MDP的SAF,微流化为亚微米乳剂或漩涡振荡产生较大粒度的乳剂;(7)RibiTM佐剂系统(RAS),(Ribi Immunochem),含有2%角鲨烯、0.2%吐温80和由单磷酸酰脂质A(monophosphorylipid A)(MPL)、海藻糖二分枝菌酸酯(TDM)和细胞壁骨架(CWS),优选MPL+CWS(DetoxTM)组成的一种或多种细菌细胞壁组分;和(8)一种或多种矿物盐(例如铝盐)+LPS的无毒衍生物(例如3dMPL)。
参考文献117的第7章公开了用作免疫刺激剂的其它物质。
特别优选使用铝盐佐剂,抗原通常吸附到这种盐上。MenjugateTM和NeisVacTM偶联物利用氢氧化物佐剂,而MeningitecTM利用磷酸盐佐剂。对于本发明组合物,可将一些抗原吸附到氢氧化铝上而使其它抗原与磷酸铝结合。然而通常优选只使用一种盐,例如氢氧化物或磷酸盐,而不是两种。不需要吸附所有偶联物,即一些或所有偶联物可游离在溶液中。
治疗方法
本发明也提供引起哺乳动物免疫应答的方法,包括给予哺乳动物本发明药物组合物。免疫应答优选有保护性并优选涉及抗体。该方法可引起加强应答。
哺乳动物优选人。当疫苗用于预防应用时,人优选儿童(例如,学步儿童或婴儿)或青少年;当疫苗用于治疗应用时,人优选成人。儿童用疫苗也可给予成人,例如用于评价安全性、剂量、免疫原性等。用于治疗的一类人优选育龄妇女(例如,青少年和更年长的)。另一类优选的人是妊娠期妇女。
本发明也提供可用作药物的本发明组合物。所述药物优选能引起哺乳动物的免疫应答(即免疫原性组合物),更优选疫苗。
本发明也提供本发明偶联物在制备用于引起哺乳动物免疫应答的药物中的应用。
这些应用和方法优选用于预防和/或治疗由无乳链球菌引起的疾病,例如新生儿败血症或菌血症、新生儿肺炎、新生儿脑膜炎、子宫内膜炎、骨髓炎、脓毒性关节炎等。
要预防疾病的对象可以与接受本发明偶联物的对象不同。例如,可将偶联物给予妇女(妊娠前或期间)来保护后代(称为“母体免疫接种”[190-192])。
检测治疗性治疗效果的一种方法包括监测给予本发明组合物后的GBS感染。检测预防性治疗效果的一种方法包括监测给予组合物后针对GBS抗原的免疫应答。
本发明优选组合物在可接受百分比的人对象中赋予患者的抗体滴度优于各抗原性组分的血清保护标准。熟知抗原的相关抗体滴度(高于该滴度则认为宿主对该抗原已血清阳转),这种滴度由诸如WHO等组织发布。优选超过80%的对象的样本有统计学有意义的血清阳转,更优选超过90%,还更优选超过93%,最优选96-100%。
本发明组合物通常应对患者直接给药。可通过胃肠外注射(例如皮下、腹膜内、静脉内、肌肉内或组织间隙注射)实现直接递送,或通过直肠、口服、阴道、局部、透皮、鼻内、眼睛、耳部、肺部或其他粘膜给药实现。优选大腿或上臂的肌肉内给予。注射可以通过注射针(例如皮下注射用针),但也可采用无针注射。典型的肌内注射剂量是0.5ml。
可采用本发明引发全身性和/或粘膜免疫力。
剂量疗法可以是单剂量方案或多剂量方案。多剂量可以用于初次免疫方案和/或加强免疫方案。初次剂量方案之后可以是加强剂量方案。可以常规确定致敏剂量之间(例如4-16周之间)以及致敏和加强之间的适当时间安排。
GBS蛋白抗原
如上所述,本发明组合物中可包含GBS蛋白。这些蛋白可用作本发明偶联物的运载体蛋白、其它偶联物的运载体蛋白或未偶联的蛋白抗原。
本发明所用的GBS蛋白抗原包括参考文献93和193-195中公开的那些。本发明所用的5种优选GBS抗原称为:GBS67;GBS80;GBS104;GBS276和GBS322[参见参考文献93]。这5种抗原的其它细节见下文。
这5种GBS蛋白的全长序列见本文的SEQ ID NO 1-5。因此,本发明组合物可包含(a)含有选自SEQ ID NO 1-5的氨基酸序列的多肽,和/或(b)含有(i)与SEQID NO 1-5中一条或多条具有序列相同性的氨基酸序列和/或(ii)SEQ ID NO 1-5的片段的多肽。
根据具体的SEQ ID NO,(i)的序列相同性程度优选大于50%(例如,60%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高)。这些多肽包括同源物、直向同源物、等位变体和功能突变体。通常认为两条多肽序列之间有50%或更高的相同性表明其功能上等同。优选采用MPSRCH程序(Oxford Molecular)执行的Smith-Waterman同源性检索算法测定多肽之间的相同性,该程序利用仿射空位检索,参数是空位开放罚分=12,空位延伸罚分=1。
根据具体的SEQ ID NO,(ii)的片段应含有这些序列的至少n个连续氨基酸,根据具体的序列,n是7或更大(例如,8、10、12、14、16、18、20、22、24、26、28、30、35、40、45、50、60、70、80、90、100或更大)。片段可含有该序列的至少一个T细胞或优选B细胞表位。可以凭经验鉴定T-细胞和B-细胞表位(例如,采用PEPSCAN[196,197]或相似的方法),或者可预测这些表位(例如,采用Jameson-Wolf抗原指数[198]、矩阵方法[199]、TEPITOPE[200]、神经网络[201]、OptiMer & EpiMer[202,203]、ADEPT[204]、Tsites[205]、亲水性[206]、抗原性指数[207]或参考文献208公开的方法等)。其它的优选片段是不含N-末端氨基酸残基或不含N-末端信号肽的SEQ ID NO 1-5。可除去一个或多个结构域,例如前导序列或信号序列区、跨膜区、胞质区或细胞壁锚定基序。优选的片段如下文所示(SEQ ID NO 6-19)。
与SEQ ID NO 1-5相比,这些多肽可包含一个或多个(例如,1、2、3、4、5、6、7、8、9、10等)保守性氨基酸取代,即用具有相似侧链的另一氨基酸取代某一氨基酸。遗传学编码的氨基酸通常可分为4个家族:(1)酸性,即天冬氨酸、谷氨酸;(2)碱性,即赖氨酸、精氨酸、组氨酸;(3)非极性,即丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸;和(4)不荷电极性,即甘氨酸、天冬酰胺、谷氨酰胺、胱氨酸、丝氨酸、苏氨酸、酪氨酸。苯丙氨酸、色氨酸和酪氨酸有时一起分为芳香族氨基酸。这些家族内的单一氨基酸取代不会对生物学活性造成重要影响。与SEQ ID NO 1-5相比,这些多肽也可包含一个或多个(例如,1、2、3、4、5、6、7、8、9、10等)单氨基酸缺失。与SEQ ID NO 1-5相比,这些多肽也可包含一个或多个(例如,1、2、3、4、5、6、7、8、9、10等)插入(例如,各有1、2、3、4或5个氨基酸插入)。
可采用许多方法,例如通过化学合成(全部或部分)、用蛋白酶消化较长的多肽、从RNA翻译、从细胞培养物(例如,重组表达)纯化、从生物自身纯化(例如,细菌培养后或直接从患者纯化)等来制备本发明多肽。产生长度<40个氨基酸的肽的优选方法包括体外化学合成[209,210]。特别优选固相肽合成,例如基于tBoc或Fmoc化学试剂的方法[211]。也可部分或完全采用酶促合成[212]。或者,可采用生物合成替代化学合成,例如可通过翻译产生多肽。这可在体外或体内进行。生物学方法通常局限于产生基于L-氨基酸的多肽,但可利用对翻译机制(例如,氨酰基tRNA分子)的操控来引入D-氨基酸(或非天然氨基酸,例如碘酪氨酸或甲基苯丙氨酸、叠氮基高丙氨酸(azidohomoalanine)等)[213]。然而,当包含D-氨基酸时,优选采用化学合成。本发明多肽可在C-末端和/或N-末端具有共价修饰。
如果本发明组合物中包含这些GBS蛋白,则它们可采取各种形式(例如,天然的、融合体、糖基化的、非糖基化的、脂化的、非脂化的、磷酸化的、非磷酸化的、豆蔻酰化的、非豆蔻酰化的、单体的、多聚的、颗粒化的、变性的等)。它们优选以纯化或基本上纯化的形式使用,即基本上不含其它多肽(例如,不含天然产生的多肽,特别是不含其它GBS或宿主细胞多肽)。
GBS67
测序的血清型V菌株2603 V/R的GBS67核苷酸序列和氨基酸序列见参考文献93的SEQ ID NO 3745和3746。在本文中其氨基酸序列是SEQ ID NO:1:
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELT
GEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKL
EHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDN
SNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKEDDKYYGLQTKFTIQTE
NYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTD
GVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNL
NYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELM
RSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSV
MKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLR
DFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYK
DLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILS
FILIGGAMMSIAGGIYIWKRYKKSSDMSIKKD
GBS67含有C-末端跨膜区,该区域在最接近以上SEQ ID NO:1的C-末端区域以下划线标出。可除去跨膜区的一个或多个氨基酸,或者可截短跨膜区前的氨基酸。这种GBS67片段的一个例子见下文的SEQ ID NO:18。
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELT
GEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKL
EHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDN
SNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKEDDKYYGLQTKFTIQTE
NYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTD
GVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNL
NYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELM
RSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSV
MKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLR
DFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYK
DLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILS
GBS67含有表示细胞壁锚定的氨基酸基序,其在以上SEQ ID NO:1中以斜体字显示。在一些重组宿主细胞系统中,优选除去该基序而有利于重组GBS67蛋白从宿主细胞分泌。因此,在本发明所用一优选GBS67片段中,除去了GBS67的跨膜和细胞壁锚定基序。这种GBS67片段的一个例子见下文的SEQ ID NO:19。
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELT
GEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKL
EHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDN
SNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKEDDKYYGLQTKFTIQTE
NYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTD
GVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNL
NYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELM
RSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSV
MKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLR
DFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYK
DLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITNTHIPPKGI
GBS80
GBS80指推定的细胞壁表面锚定家族蛋白。分离的血清型V菌株2603 V/R测序的GBS80核苷酸序列和氨基酸序列见参考文献93的SEQ ID NO 8779和8780。在本文中其氨基酸序列是SEQ ID NO:2:
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVIS
NYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYV
EDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIP
ANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQD
ALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQT
LGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKA
PEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRRTKD
N
GBS80含有N-末端前导序列区或信号序列区,该区域在以上序列中以下划线标出。可除去GBS80的前导序列区或信号序列区的一个或多个氨基酸。这种GBS80片段的一个例子见下文的SEQ ID NO:6:
AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTV
EAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEIN
IYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRD
EHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTF
ELQYDHTPDKADNPKFSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAV
TGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPD
TIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN
GBS80含有C-末端跨膜区,该区域在靠近以上SEQ ID NO:2的末端以带下划线的序列标出。可除去跨膜区和/或胞质区的一个或多个氨基酸。这种片段的一个例子见下文的SEQ ID NO:7:
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVIS
NYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYV
EDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIP
ANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQD
ALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQT
LGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKA
PEGYVI PDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG
GBS80含有表示细胞壁锚定的氨基酸基序,其在以上SEQ ID NO:2中以斜体字显示。在一些重组宿主细胞系统中,优选除去该基序而有利于重组GBS80蛋白从宿主细胞分泌。因此,可除去GBS80的跨膜区和/或胞质区及细胞壁锚定基序。这种片段的一个例子见下文的SEQ ID NO:8。
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVIS
NYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYV
EDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIP
ANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQD
ALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQT
LGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKA
PEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPS
或者,在一些重组宿主细胞系统中,优选用细胞壁锚定基序将重组表达的蛋白锚定到细胞壁上。可在纯化期间切除所表达蛋白的胞外结构域,或者最终组合物中的重组蛋白可维持连接于灭活的宿主细胞或细胞膜上。
在一个实施方式中,可除去GBS80序列的前导序列区或信号序列区、跨膜区和胞质区及细胞壁锚定基序。这种GBS80片段的一个例子见下文的SEQ ID NO:9:
AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTV
EAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEIN
IYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRD
EHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTF
ELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAV
TGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPD
TIKNNKRPS
尤其(具有)免疫原性的GBS80片段位于该蛋白的N-末端,在本文以SEQ IDNO:10表不:
AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTV
EAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEIN
IYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRD
EHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKG
GBS104
GBS104指推定的细胞壁表面锚定家族蛋白。称为emaA。分离的血清型V菌株2603 V/R测序的GBS104核苷酸序列和氨基酸序列见参考文献93的SEQ ID NO8777和8778。在本文中其氨基酸序列是SEQ ID NO:3:
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGE
ATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVE
GSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNE
RANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNY
SYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINF
NPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNE
GYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFM
QSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGV
ALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDF
PIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQ
DGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIV
YILVGSTFMILTICSFRRKQL
GBS104含有N-末端前导序列区或信号序列区,该区域在以上SEQ IDNO:3的起始处以带下划线的序列标出。可除去GBS104的前导序列区或信号序列区的一个或多个氨基酸。这种GBS104片段的一个例子见下文的SEQ IDNO:11:
GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKT
WKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWL
SKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDN
RVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHING
DRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQE
DFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKK
VSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFK
TIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKI
NHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFI
KVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTI
QNGEVTNLKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL
GBS104含有C-末端跨膜区和/或胞质区,该区域在靠近以上SEQ ID NO:3的末端以带下划线的区域标出。可除去跨膜区和/或胞质区的一个或多个氨基酸。这种GBS 104片段的一个例子见下文的SEQ ID NO:12:
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGE
ATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVE
GSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNE
RANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNY
SYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINF
NPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNE
GYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFM
QSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGV
ALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDF
PIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQ
DGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNT
可除去前导序列区或信号序列区的一个或多个氨基酸和跨膜区或胞质区的一个或多个氨基酸。这种GBS104片段的一个例子见下文的SEQ ID NO:13:
GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKT
WKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWL
SKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDN
RVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHING
DRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQE
DFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKK
VSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFK
TIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKI
NHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFI
KVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTI
QNGEVTNLKADPNANKNQIGYLEGNGKHLITNT
GBS104的其它片段包括GBS104氨基酸28-858(按照SEQ ID NO:3编号)的830个氨基酸的片段,GBS104氨基酸28-387的359个氨基酸的片段,GBS104氨基酸28-609的581个氨基酸的片段或GBS 104氨基酸28-768的740个氨基酸的片段。
GBS276
GBS276指C5a肽酶。GBS276的其它描述见参考文献214-217。分离的血清型V菌株2603 V/R测序的GBS104核苷酸序列和氨基酸序列见参考文献93的SEQID NO 8941和8942。在本文中其氨基酸序列是SEQ ID NO:4:
MRKKQKLPFDKLAIALISTSILLNAQSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADD
ANDLAPQAPAKTADTPATSKATIRDLNDPSHVKTLQEKAGKGAGTVVAVIDAGFDKNHEAWRLTDKTKARYQSKENLEK
AKKEHGITYGEWVNDKVAYYHDYSKDGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLAD
YARNYAQAIRDAVNLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKPRLPLADHPDYGVV
GTPAAADSTLTVASYSPDKQLTETATVKTDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGD
IDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRF
SSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSA
TALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQVTVQTDK
VDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRG
DFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEIT
ETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKN
YNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSRLT
LASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKL
LEGHSNKPEQDGSDQAPDKKPEAKPEQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRALATKAST
RDQLPTTNDKDTNRLHLLKLVMTTFFLG
GBS276含有N-末端前导序列区或信号序列区,该区域在以上SEQ ID NO:4的起始处以带下划线的序列标出。可除去GBS276的前导序列区或信号序列区的一个或多个氨基酸。这种GBS276片段的一个例子见下文的SEQ ID NO:14:
QSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADDANDLAPQAPAKTADTPATSKATIRD
LNDPSHVKTLQEKAGKGAGTVVAVIDAGFDKNHEAWRLTDKTKARYQSKENLEKAKKEHGITYGEWVNDKVAYYHDYSK
DGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMSFG
NAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKPRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETA
TVKTDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQD
KGFPIELPNVDQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSS
VANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAK
KASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQVTVQTDKVDGKHFALAPKALYETSWQKITIPA
NSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEA
NSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANG
KPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKD
GKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSRLTLASKPKTSQPVYRERIAYTYMDEDL
PTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQDGSDQAPDKKPEAKP
EQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRALATKASTRDQLPTTNDKDTNRLHLLKLVMTTF
FLG
GBS276含有C-末端跨膜区和/或胞质区,该区域在靠近以上SEQ ID NO:4的末端以带下划线的序列标出。可除去GBS276的跨膜区或胞质区的一个或多个氨基酸。这种GBS276片段的一个例子见下文的SEQ ID NO:15:
MRKKQKLPFDKLAIALISTSILLNAQSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADD
ANDLAPQAPAKTADTPATSKATIRDLNDPSHVKTLQEKAGKGAGTVVAVIDAGFDKNHEAWRLTDKTKARYQSKENLEK
AKKEHGITYGEWVNDKVAYYHDYSKDGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLAD
YARNYAQAIRDAVNLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKPRLPLADHPDYGVV
GTPAAADSTLTVASYSPDKQLTETATVKTDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGD
IDFKDKIANAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRF
SSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSA
TALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQVTVQTDK
VDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRG
DFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEIT
ETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKN
YNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSRLT
LASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKL
LEGHSNKPEQDGSDQAPDKKPEAKPEQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRALATK
可除去GBS276的前导序列区或信号序列区的一个或多个氨基酸和跨膜区或胞质区的一个或多个氨基酸。这种GBS276片段的一个例子见下文的SEQ IDNO:16:
QSDIKANTVTEDTPATEQAVEPPQPIAVSEESRSSKETKTSQTPSDVGETVADDANDLAPQAPAKTADTPATSKATIRD
LNDPSHVKTLQEKAGKGAGTVVAVI DAGFDKNHEAWRLTDKTKARYQSKENLEKAKKEHGITYGEWVNDKVAYYHDYSK
DGKNAVDQEHGTHVSGILSGNAPSEMKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAVNLGAKVINMSFG
NAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKPRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETA
TVKTDDHQDKEMPVISTNRFEPNKAYDYAYANRGTKEDDFKDVEGKIALIERGDIDFKDKIANAKKAGAVGVLIYDNQD
KGFPIELPNVDQMPAAFISRRDGLLLKDNPPKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSS
VANNKYAKLSGTSMSAPLVAGIMGLLQKQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAK
KASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQVTVQTDKVDGKHFALAPKALYETSWQKITIPA
NSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPTKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEA
NSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANG
KPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKD
GKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDSRLTLASKPKTSQPVYRERIAYTYMDEDL
PTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQDGSDQAPDKKPEAKP
EQDGSGQTPDKKKETKPEKDSSGQTPGKTPQKGQSSRTLEKRSSKRALATK
GBS322
GBS322指表面免疫原性蛋白,也称为“sip”。分离的血清型V菌株2603 V/R测序的GBS322核苷酸序列和氨基酸序列见参考文献93的SEQ ID NO 8539和8540。在本文中其氨基酸序列是SEQ ID NO:5:
MNKKVLLTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNI
ADINLIYPETTLTVTYDQKSHTATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMK
TYSSAPALKSKEVLAQEQAVSQAAANEQVSPAPVKSITSEVPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAP
VRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSPATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAH
PENAGLQPHVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVGTNQALGNKVAQYSTQNMAANNISYVIWQQKF
YSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK
GBS322含有N-末端前导序列区或信号序列区,该区域在以上SEQ ID NO:5的起始处以带下划线的序列标出。可除去GBS322的前导序列区或信号序列区的一个或多个氨基酸。这种GBS322片段的一个例子见下文的SEQ ID NO:17:
DLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSHTATSMKIETPATNAAGQTT
ATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPAPVKSITSE
VPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSP
ATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVNEFSTYRAGDPGDHGKG
LAVDFIVGTNQALGNKVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK
通用术语
术语“含有”包括“包含”以及“由……构成”,例如“含有”X的组合物可仅由X构成或可包含其它物质,例如X+Y。
与数值x相关的术语“约”表示,例如x±10%。
“基本上”不排除“完全”,例如“基本上不含”Y的组合物可完全不含Y。“基本上”可视需要从本发明定义中省去。
当本发明提供的方法包括多个连续步骤时,本发明也提供包括的步骤总数较少的方法。例如,在本发明的第一方面,本发明提供包括以下步骤的方法:(a)氧化GBS荚膜糖以在末端唾液酸残基中引入醛基;和(b)还原胺化该醛基。属于本发明的范围包括无需进行额外步骤(c)和(d),因为步骤(c)和(d)的产物已用作制备偶联物的中间体,并且可在分别和随后的应用(例如用于步骤(c)和(d)中)中使用、保存、运输等。
类似地,当起始糖物质早已部分加工过,则本发明所包括的方法只包括该方法的后几步。例如,在本发明的第三方面,本发明包括的方法包括将修饰的半乳糖残基与运载体分子偶联的步骤,其中该方法的起始物质是已氧化而在半乳糖残基中引入了醛基的糖。
可在不同时间由不同的人在不同地点(例如,在不同国家)进行这些不同的步骤。
应该知道糖环可存在开放和封闭形式,虽然本文结构式显示封闭形式,但开放形式也属于本发明。类似地,应该知道糖可以存在吡喃和呋喃形式,虽然本文结构式显示吡喃形式,但也应包括呋喃形式。也应包括不同端基异构形式的糖。
式NH2R代表伯胺。R基团优选供电子基团,包括C18烃基,更优选C18烷基,特别是甲基。R优选-CH3、-C2H5或-C3H7。烃基可以用一个或多个基团取代,例如:卤素(如Cl、Br、F、I)、三卤甲基、-NO2、-CN、-N+(C16烷基)2O-、-SO3H、-SOC16烷基、-SO2C1-6烷基、-SO3C16烷基、-OC(=O)OC1-6烷基、-C(=O)H、-C(=O)C1-6烷基、-OC(=O)C1-6烷基、-N(C1-6烷基)2、C1-6烷基、-N(C1-6烷基)2、-C(=O)N(C1-6烷基)2、-N(C1-6烷基)C(=O)O(C1-6烷基)、-N(C1-6烷基)C(=O)N(C1-6烷基)2、-CO2H、-OC(=O)N(C1-6烷基)2、-N(C1-6烷基)C(=O)C1-6烷基、-N(C1-6烷基)C(=S)C1-6烷基、-N(C1-6烷基)SO2N(C1-6烷基)2、-CO2C1-6烷基、-SO2N(C1-6烷基)2、-C(=O)NH2、-C(=S)N(C1-6烷基)2、-N(C1-6烷基)SO2C1-6烷基、-N(C1-6烷基)C(=S)N(C1-6烷基)2、-NH-C1-6烷基、-S-C1-6烷基或-O-C1-6烷基。术语“烃基”包括碳和氢构成的线形、支链或环状单价基团。因此,烃基包括烷基、烯基和炔基、环烷基(包括聚环烷基(polycycloalkyl))、环烯基和芳基及它们的组合,例如烷基环烷基、烷基聚环烷基、烷基芳基、烯基芳基、环烷基芳基、环烯基芳基、环烷基烷基、聚环烷基烷基、芳基烷基、芳基烯基、芳基环烷基和芳基环烯基。优选的烃基是C1-14烃基,更优选C1-3烃基。
附图简述
图1显示了用过碘酸盐氧化末端唾液酸残基。
图2显示了本发明的第一和第二方面。
图3显示了GBS血清型Ia、Ib、II、III和V荚膜糖的重复结构。
图4显示了GBS血清型Ia和III的重复结构之间的差异。
图5显示了可以制备的两类偶联物。
图6显示了根据本发明第一方面,使用己二酸的琥珀酰亚胺二酯的优选偶联反应。
图7显示了根据本发明第二方面,使用己二酸的琥珀酰亚胺二酯的优选偶联反应。
图8和9显示了在还原胺化末端唾液酸残基氧化形成的醛基后,利用(8)丙烯酰化和(9)卤代酰卤制备偶联物。
本发明实施方式
制备偶联物和表征
如参考文献15所述纯化GBS血清型Ib的荚膜糖,然后如上所述重乙酰化。糖经脱-N-乙酰化得到用于连接的胺基。利用这些胺基通过直接还原胺化(如现有技术所述,唾液酸的C8上)或通过SIDEA间隔基团(如参考文献218所述的脑膜炎球菌糖)共价偶联糖与单体破伤风类毒素(TT)。
按照参考文献219所述的比色方法测定偶联物中的唾液酸含量。从唾液酸含量外推得到糖总含量(以聚合物的重量计,唾液酸平均为31%)。用Micro BCA蛋白质测定试剂盒(Pierce)测定偶联物中的蛋白质浓度。目标是多糖∶蛋白质重量比在1-4之间,结果如下所示:
偶联 | 糖(mg/ml) | 蛋白质(mg/ml) | 比例 |
还原胺化 | 1.740 | 1.271 | 1.37 |
SUDEA间隔基团 | 0.150 | 0.048 | 3.13 |
为研究如何影响偶联物的交联比,将纯化的GBS Ia和Ib糖氧化至不同程度,然后与CRM197偶联。结果如下所示:
采用相似的实验研究不同的蛋白质运载体。CRM197和破伤风类毒素均用作GBS III型糖的运载体,结果如下所示:
对于GBS II型和V型糖,比较了三种不同的运载体:破伤风类毒素;CRM197和人血清白蛋白。V型糖的氧化程度是15.3%,II型糖是6.9%。结果是
分别检验人血清白蛋白作为Ia型(6.7%氧化)、Ib型(8.2%氧化)和III型(4.1%氧化)糖的运载体:
类型 | 糖浓度(mg/ml) | 蛋白质浓度(mg/ml) | 比例(w/w) |
Ia | 1.112 | 0.784 | 1.42 |
Ib | 3.710 | 3.078 | 1.21 |
III | 3.318 | 2.869 | 1.16 |
利用四种不同的运载体:破伤风类毒素;CRM197;GBS80和GBS67制备Ia、Ib和III型偶联物。利用破伤风和CRM运载体时,Ia的氧化%是9.1%,Ib的是14.2%,III的是13%;利用GBS运载体时,氧化%是8.2%、9.0%和7.9%。然后检验用这些偶联物免疫的动物中对各类型GBS(即,同源攻击)的保护作用,结果如下所示,表示为致命攻击后存活动物%:
TT | CRM197 | GBS80 | GBS67 | PBS对照 | |
Ia | 32 | 48 | 10 | 96 | 5 |
Ib | 52 | 33 | 65 | 92 | 15 |
III | 76 | 60 | 71 | 82 | 0 |
在平行实验中,用V型GBS菌株攻击但没用V型糖免疫接种,结果如下所示:
TT | CRM197 | GBS80 | GBS67 | PBS对照 | |
V | 2 | 0 | 53 | 62 | 0 |
因此,GBS运载体能提供针对V型菌株的一定保护作用,所以利用GBS蛋白作为运载体能赋予背景水平的蛋白质介导保护作用,糖与蛋白质偶联能补充这种保护作用。
检验各种偶联物中的游离糖水平,结果如下所示:
GBS类型 | 运载体 | 游离 |
Ia | CRMGBS80GBS67 | <1.0%3.5%<1% |
CRM | 1.8% |
Ib | GBS80GBS67 | 14.8%<1% |
III | CRMCRMTetToxGBS80GBS67 | 1.6%4.4%3.8%9.1%<1.0% |
应该知道只是通过实施例描述了本发明,可对其作出改进而仍维持在本发明的范围和构思内。
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Claims (27)
1.一种制备无乳链球菌荚膜糖与运载体分子的偶联物的方法,包括以下步骤:(a)氧化无乳链球菌荚膜糖以在该糖的至少一个末端唾液酸残基中引入醛基;(b)用氨或伯胺还原胺化该醛基得到-CH2-连接的胺;(c)使该-CH2-连接的胺与双功能接头反应得到活化的糖;和(d)使该活化的糖与运载体分子反应得到偶联物。
2.一种偶联物,所述偶联物包含通过接头部分与运载体相连的无乳链球菌荚膜糖部分,其中所述接头部分与所述荚膜糖部分中的唾液酸残基相连。
3.如权利要求2所述的偶联物,其特征在于,通过权利要求1所述的方法获得。
4.如以上任一项权利要求所述的偶联物或方法,其特征在于,所述糖是GBS血清型Ia、Ib、II、III或V之一。
5.如以上任一项权利要求所述的偶联物或方法,其特征在于,所述糖是其天然形式。
6.如权利要求1-4中任一项所述的偶联物或方法,其特征在于,所述糖比天然荚膜糖短。
7.如权利要求1-4中任一项所述的偶联物或方法,其特征在于,与天然糖相比,所述糖经过化学修饰。
8.如权利要求7所述的偶联物或方法,其特征在于,所述糖是部分或全部脱-O-乙酰化的。
9.如权利要求7所述的偶联物或方法,其特征在于,所述糖是部分或全部脱-N-乙酰化的。
10.如以上任一项权利要求所述的偶联物或方法,其特征在于,所述运载体是白喉类毒素、破伤风类毒素、CRM197、人血清白蛋白、含各种病原体衍生抗原的多种人CD4+T细胞表位的人工蛋白、流感嗜血杆菌的D蛋白或无乳链球菌蛋白。
11.如以上任一项权利要求所述的偶联物或方法,其特征在于,所述运载体通过该运载体中的-NH2基团与糖相连。
12.如以上任一项权利要求所述的偶联物或方法,其特征在于,所述偶联物中糖∶蛋白质之比(w/w)在1∶5和5∶1之间。
13.如以上任一项权利要求所述的偶联物或方法,其特征在于,在所有唾液酸单糖单元的5%-50%的单元中引入了醛基。
14.如以上任一项权利要求所述的方法,其特征在于,在偶联后分离游离的和偶联的糖。
15.如以上任一项权利要求所述的方法,其特征在于,所述步骤(a)通过化学方法引入醛基。
16.如权利要求15所述的方法,其特征在于,所述步骤(a)包括用过碘酸盐氧化邻位的羟基。
17.如以上任一项权利要求所述的方法,其特征在于,还原胺化涉及氨或伯胺(NH2R)。
18.如权利要求17所述的方法,其特征在于,还原胺化包括联用铵盐与还原剂。
19.如以上任一项权利要求所述的方法,其特征在于,所述双功能接头是异双功能的。
20.如权利要求1-18中任一项所述的方法,其特征在于,所述双功能接头是同双功能的。
21.如权利要求20所述的方法,其特征在于,糖与运载体分子的反应都涉及胺,其中所述接头具有式X-L-X,其中:两个X基团彼此相同并能与所述胺反应;L是接头中的连接部分。
22.如权利要求21所述的方法,其特征在于,X是N-氧基琥珀酰亚胺。
23.如权利要求22所述的方法,其特征在于,所述接头是己二酸N-羟基琥珀酰亚胺二酯。
24.如以上任一项权利要求所述的方法,其特征在于,如果需要,将糖基本上完全再-N-乙酰化,然后还原胺化。
25.如以上任一项权利要求所述的方法或偶联物,其特征在于,各糖与多个运载体相连。
26.一种制备无乳链球菌荚膜糖与运载体分子的偶联物的方法,包括以下步骤:(a)使荚膜糖脱-N-乙酰化以得到脱-N-乙酰化的糖;(b)使该脱-N-乙酰化的糖与双功能接头反应得到活化的糖;和(c)使该活化的糖与运载体分子反应得到偶联物。
27.一种制备荚膜糖与运载体分子的偶联物的方法,包括以下步骤:(a)氧化荚膜糖以在该糖的至少一个半乳糖残基中引入醛基从而得到修饰的半乳糖残基;和(b)将该修饰的半乳糖残基与运载体分子偶联。
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Cited By (7)
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CN102883745A (zh) * | 2010-03-09 | 2013-01-16 | 葛兰素史密丝克莱恩生物有限公司 | 缀合方法 |
CN105307684A (zh) * | 2012-10-02 | 2016-02-03 | 葛兰素史密丝克莱恩生物有限公司 | 非直链糖缀合物 |
CN104717977A (zh) * | 2012-10-03 | 2015-06-17 | 诺华股份有限公司 | 免疫原性组合物 |
CN105377282A (zh) * | 2013-01-15 | 2016-03-02 | 葛兰素史密丝克莱恩生物有限公司 | 环炔衍生化的糖 |
CN105377282B (zh) * | 2013-01-15 | 2020-07-17 | 葛兰素史密丝克莱恩生物有限公司 | 环炔衍生化的糖 |
CN107955079A (zh) * | 2017-11-08 | 2018-04-24 | 江南大学 | 双聚唾液酸仿生材料及其制备方法 |
CN109045292A (zh) * | 2018-11-08 | 2018-12-21 | 山东大学 | 一种a群链球菌寡糖蛋白缀合物及其制备方法与应用 |
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US9675691B2 (en) | 2017-06-13 |
JP2008532930A (ja) | 2008-08-21 |
US20150231231A1 (en) | 2015-08-20 |
US20090043077A1 (en) | 2009-02-12 |
WO2006082530A3 (en) | 2008-07-03 |
CN103349780B (zh) | 2016-05-04 |
NZ560432A (en) | 2010-12-24 |
AU2006211052B2 (en) | 2012-03-29 |
JP5390103B2 (ja) | 2014-01-15 |
CA2596683A1 (en) | 2006-08-10 |
US8513392B2 (en) | 2013-08-20 |
WO2006082530A2 (en) | 2006-08-10 |
ZA200706969B (en) | 2008-11-26 |
US9040055B2 (en) | 2015-05-26 |
US20170246285A1 (en) | 2017-08-31 |
EP1846038A2 (en) | 2007-10-24 |
CA2596683C (en) | 2013-12-31 |
MX2007009277A (es) | 2007-09-19 |
GB0502095D0 (en) | 2005-03-09 |
IL184981A (en) | 2016-06-30 |
US20130295132A1 (en) | 2013-11-07 |
JP2013079285A (ja) | 2013-05-02 |
JP2015147806A (ja) | 2015-08-20 |
CN101304765B (zh) | 2013-03-27 |
IL184981A0 (en) | 2007-12-03 |
EP3498302A1 (en) | 2019-06-19 |
AU2006211052A1 (en) | 2006-08-10 |
CA2830588A1 (en) | 2006-08-10 |
CN103349780A (zh) | 2013-10-16 |
JP5818826B2 (ja) | 2015-11-18 |
US10188719B2 (en) | 2019-01-29 |
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